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Pigh
erformance
L iquid
C hromatography
HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution HPLC instruments consist of a reservoir of mobile phase a pump an injector a separation column and a detector
Compounds are separated by injecting a sample mixture onto the column The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase The mobile phase must be degassed to eliminate the formation of air bubbles
This unit is equipped
with two pump units
and a UVVis
detector
The gradient is
controlled via the
pump controllers
LC
methods are not
as sensitive to
temperature
Columns are
commonly
mounted outside
the instrument
Solvents
bull All solvents should be lsquoHPLCrsquo grade
ndash This is a type of reagent grade material
ndash It has been filtered using a 02 μm filter
bull Filtered solvent helps extend pump life by preventing scoring It also reduces the chances of a column plugging
Solvent degassing
All solvents should be degassed prior to use
This reduces the chances of bubbles being formed in the column or detector Oxygen present at high pressure can also cause a problem
Methods that can be used
Displacement with a less soluble gas
Applying a vacuum
Heating the solvent
HPLC Injectors
In order to introduce a sample onto the column for analysis a special valve called the injector must be used to transfer the sample into the pressurized system
Injectors may look different from the outside but internally most are 6-port rotary valves These valves consist of a fixed body (the stator) plus an internal seal that rotates (the rotor)
Three internal passages connect alternate pairs of external ports The valves can switch between two positions referred to as the inject and load positions respectively In the load position the pump is connected to the column and the sample inlet is connected to one end of a piece of tubing called the sample loop
The other end of the sample loop is connected to the waste port Rotation results in reconnecting the various lines that enter the valve so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet
The column
HPLC has seen significant improvement over
the last 20 years primarily due to improved
column technology
Packings are more uniform and smaller
Phases are commonly chemically bound to
the packing
Packing methods have improved
Pump types
bullIsocratic pump - delivers constant mobile phase composition
bullsolvent must be pre-mixed
bulllowest cost pump
bullGradient pump -delivers variable mobile phase composition
bullcan be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution HPLC instruments consist of a reservoir of mobile phase a pump an injector a separation column and a detector
Compounds are separated by injecting a sample mixture onto the column The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase The mobile phase must be degassed to eliminate the formation of air bubbles
This unit is equipped
with two pump units
and a UVVis
detector
The gradient is
controlled via the
pump controllers
LC
methods are not
as sensitive to
temperature
Columns are
commonly
mounted outside
the instrument
Solvents
bull All solvents should be lsquoHPLCrsquo grade
ndash This is a type of reagent grade material
ndash It has been filtered using a 02 μm filter
bull Filtered solvent helps extend pump life by preventing scoring It also reduces the chances of a column plugging
Solvent degassing
All solvents should be degassed prior to use
This reduces the chances of bubbles being formed in the column or detector Oxygen present at high pressure can also cause a problem
Methods that can be used
Displacement with a less soluble gas
Applying a vacuum
Heating the solvent
HPLC Injectors
In order to introduce a sample onto the column for analysis a special valve called the injector must be used to transfer the sample into the pressurized system
Injectors may look different from the outside but internally most are 6-port rotary valves These valves consist of a fixed body (the stator) plus an internal seal that rotates (the rotor)
Three internal passages connect alternate pairs of external ports The valves can switch between two positions referred to as the inject and load positions respectively In the load position the pump is connected to the column and the sample inlet is connected to one end of a piece of tubing called the sample loop
The other end of the sample loop is connected to the waste port Rotation results in reconnecting the various lines that enter the valve so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet
The column
HPLC has seen significant improvement over
the last 20 years primarily due to improved
column technology
Packings are more uniform and smaller
Phases are commonly chemically bound to
the packing
Packing methods have improved
Pump types
bullIsocratic pump - delivers constant mobile phase composition
bullsolvent must be pre-mixed
bulllowest cost pump
bullGradient pump -delivers variable mobile phase composition
bullcan be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
This unit is equipped
with two pump units
and a UVVis
detector
The gradient is
controlled via the
pump controllers
LC
methods are not
as sensitive to
temperature
Columns are
commonly
mounted outside
the instrument
Solvents
bull All solvents should be lsquoHPLCrsquo grade
ndash This is a type of reagent grade material
ndash It has been filtered using a 02 μm filter
bull Filtered solvent helps extend pump life by preventing scoring It also reduces the chances of a column plugging
Solvent degassing
All solvents should be degassed prior to use
This reduces the chances of bubbles being formed in the column or detector Oxygen present at high pressure can also cause a problem
Methods that can be used
Displacement with a less soluble gas
Applying a vacuum
Heating the solvent
HPLC Injectors
In order to introduce a sample onto the column for analysis a special valve called the injector must be used to transfer the sample into the pressurized system
Injectors may look different from the outside but internally most are 6-port rotary valves These valves consist of a fixed body (the stator) plus an internal seal that rotates (the rotor)
Three internal passages connect alternate pairs of external ports The valves can switch between two positions referred to as the inject and load positions respectively In the load position the pump is connected to the column and the sample inlet is connected to one end of a piece of tubing called the sample loop
The other end of the sample loop is connected to the waste port Rotation results in reconnecting the various lines that enter the valve so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet
The column
HPLC has seen significant improvement over
the last 20 years primarily due to improved
column technology
Packings are more uniform and smaller
Phases are commonly chemically bound to
the packing
Packing methods have improved
Pump types
bullIsocratic pump - delivers constant mobile phase composition
bullsolvent must be pre-mixed
bulllowest cost pump
bullGradient pump -delivers variable mobile phase composition
bullcan be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
LC
methods are not
as sensitive to
temperature
Columns are
commonly
mounted outside
the instrument
Solvents
bull All solvents should be lsquoHPLCrsquo grade
ndash This is a type of reagent grade material
ndash It has been filtered using a 02 μm filter
bull Filtered solvent helps extend pump life by preventing scoring It also reduces the chances of a column plugging
Solvent degassing
All solvents should be degassed prior to use
This reduces the chances of bubbles being formed in the column or detector Oxygen present at high pressure can also cause a problem
Methods that can be used
Displacement with a less soluble gas
Applying a vacuum
Heating the solvent
HPLC Injectors
In order to introduce a sample onto the column for analysis a special valve called the injector must be used to transfer the sample into the pressurized system
Injectors may look different from the outside but internally most are 6-port rotary valves These valves consist of a fixed body (the stator) plus an internal seal that rotates (the rotor)
Three internal passages connect alternate pairs of external ports The valves can switch between two positions referred to as the inject and load positions respectively In the load position the pump is connected to the column and the sample inlet is connected to one end of a piece of tubing called the sample loop
The other end of the sample loop is connected to the waste port Rotation results in reconnecting the various lines that enter the valve so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet
The column
HPLC has seen significant improvement over
the last 20 years primarily due to improved
column technology
Packings are more uniform and smaller
Phases are commonly chemically bound to
the packing
Packing methods have improved
Pump types
bullIsocratic pump - delivers constant mobile phase composition
bullsolvent must be pre-mixed
bulllowest cost pump
bullGradient pump -delivers variable mobile phase composition
bullcan be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Solvents
bull All solvents should be lsquoHPLCrsquo grade
ndash This is a type of reagent grade material
ndash It has been filtered using a 02 μm filter
bull Filtered solvent helps extend pump life by preventing scoring It also reduces the chances of a column plugging
Solvent degassing
All solvents should be degassed prior to use
This reduces the chances of bubbles being formed in the column or detector Oxygen present at high pressure can also cause a problem
Methods that can be used
Displacement with a less soluble gas
Applying a vacuum
Heating the solvent
HPLC Injectors
In order to introduce a sample onto the column for analysis a special valve called the injector must be used to transfer the sample into the pressurized system
Injectors may look different from the outside but internally most are 6-port rotary valves These valves consist of a fixed body (the stator) plus an internal seal that rotates (the rotor)
Three internal passages connect alternate pairs of external ports The valves can switch between two positions referred to as the inject and load positions respectively In the load position the pump is connected to the column and the sample inlet is connected to one end of a piece of tubing called the sample loop
The other end of the sample loop is connected to the waste port Rotation results in reconnecting the various lines that enter the valve so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet
The column
HPLC has seen significant improvement over
the last 20 years primarily due to improved
column technology
Packings are more uniform and smaller
Phases are commonly chemically bound to
the packing
Packing methods have improved
Pump types
bullIsocratic pump - delivers constant mobile phase composition
bullsolvent must be pre-mixed
bulllowest cost pump
bullGradient pump -delivers variable mobile phase composition
bullcan be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Solvent degassing
All solvents should be degassed prior to use
This reduces the chances of bubbles being formed in the column or detector Oxygen present at high pressure can also cause a problem
Methods that can be used
Displacement with a less soluble gas
Applying a vacuum
Heating the solvent
HPLC Injectors
In order to introduce a sample onto the column for analysis a special valve called the injector must be used to transfer the sample into the pressurized system
Injectors may look different from the outside but internally most are 6-port rotary valves These valves consist of a fixed body (the stator) plus an internal seal that rotates (the rotor)
Three internal passages connect alternate pairs of external ports The valves can switch between two positions referred to as the inject and load positions respectively In the load position the pump is connected to the column and the sample inlet is connected to one end of a piece of tubing called the sample loop
The other end of the sample loop is connected to the waste port Rotation results in reconnecting the various lines that enter the valve so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet
The column
HPLC has seen significant improvement over
the last 20 years primarily due to improved
column technology
Packings are more uniform and smaller
Phases are commonly chemically bound to
the packing
Packing methods have improved
Pump types
bullIsocratic pump - delivers constant mobile phase composition
bullsolvent must be pre-mixed
bulllowest cost pump
bullGradient pump -delivers variable mobile phase composition
bullcan be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
HPLC Injectors
In order to introduce a sample onto the column for analysis a special valve called the injector must be used to transfer the sample into the pressurized system
Injectors may look different from the outside but internally most are 6-port rotary valves These valves consist of a fixed body (the stator) plus an internal seal that rotates (the rotor)
Three internal passages connect alternate pairs of external ports The valves can switch between two positions referred to as the inject and load positions respectively In the load position the pump is connected to the column and the sample inlet is connected to one end of a piece of tubing called the sample loop
The other end of the sample loop is connected to the waste port Rotation results in reconnecting the various lines that enter the valve so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet
The column
HPLC has seen significant improvement over
the last 20 years primarily due to improved
column technology
Packings are more uniform and smaller
Phases are commonly chemically bound to
the packing
Packing methods have improved
Pump types
bullIsocratic pump - delivers constant mobile phase composition
bullsolvent must be pre-mixed
bulllowest cost pump
bullGradient pump -delivers variable mobile phase composition
bullcan be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Injectors may look different from the outside but internally most are 6-port rotary valves These valves consist of a fixed body (the stator) plus an internal seal that rotates (the rotor)
Three internal passages connect alternate pairs of external ports The valves can switch between two positions referred to as the inject and load positions respectively In the load position the pump is connected to the column and the sample inlet is connected to one end of a piece of tubing called the sample loop
The other end of the sample loop is connected to the waste port Rotation results in reconnecting the various lines that enter the valve so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet
The column
HPLC has seen significant improvement over
the last 20 years primarily due to improved
column technology
Packings are more uniform and smaller
Phases are commonly chemically bound to
the packing
Packing methods have improved
Pump types
bullIsocratic pump - delivers constant mobile phase composition
bullsolvent must be pre-mixed
bulllowest cost pump
bullGradient pump -delivers variable mobile phase composition
bullcan be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
The column
HPLC has seen significant improvement over
the last 20 years primarily due to improved
column technology
Packings are more uniform and smaller
Phases are commonly chemically bound to
the packing
Packing methods have improved
Pump types
bullIsocratic pump - delivers constant mobile phase composition
bullsolvent must be pre-mixed
bulllowest cost pump
bullGradient pump -delivers variable mobile phase composition
bullcan be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Pump types
bullIsocratic pump - delivers constant mobile phase composition
bullsolvent must be pre-mixed
bulllowest cost pump
bullGradient pump -delivers variable mobile phase composition
bullcan be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
1048729 Reciprocation pumps
--- advantages small internal volume high output pressures
ready adaptability to gradient elution and constant flow rates
--- disadvantages produce a pulsed flow cause baseline noise
1048729 Displacement pumps
--- advantages output is pulse free
--- disadvantages limited solvent capacity (lt250 mL)
inconvenience when change solvents
1048729 Pneumatic pumps
--- advantages inexpensive output is pulse free
--- disadvantages limited solvent capacity not amenable to
gradient elution and limit pressures lt2000 psi
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Packings
Originally these were irregular silica and
alumina A range of synthetic regularly
shaped packings are now available
Porous - channels through packing
Superficially porous - rough surface
Smooth - bead like
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Packing size
As packing size is decreased efficiency and
pressure requirements are increased
Common diameters for analytical work
diameter plates
10 μm 5000
5 μm 9000
3 μm 15000
All are for a 15 cm x 46 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Column body
Typically consist of stainless steel with a high
precision internal bore
Some manufacturers offer column inserts
- donrsquot need to repurchase the column
fittings
Others offer columns where the external
body can be compressed to improve packing
efficiency
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
HPLC column examples
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Column stationary phases
Today most packing fall into four classes
Silica or alumina
Bound phases on either alumina or silica
Gels
Controlled-pore glass or silica
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Absorption phases - alumina
common mobile phases
hexane chloroform 2-propanol
example application - amines
silica
common mobile phases
hexane chloroform 2-propanol
example applications - ethers esters
porphyrins fat-soluble vitamins
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Partition phases
Can be broken down into
Normal phase - polar materials bound to the support
Reverse phase - non-polar materials bound to the support
Mixed phase - may have some of each
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Partition phases
Normal
Amino (-NH2)
Cyano (-CN)
Diol (glycidoxy-ethylmethoxysilane)
Reverse
C-2 or RP-2 (-Si-CH2CH3)
C-8 or RP-8 (-Si-(CH2)7CH3)
C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker more retentive phase
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Ion exchange phases
Strong cation - sulfonic acid group
Strong anionic - quarternary amine
Weak anion - primary amine
Weak cation - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Capillary and Microbore columns
Several companies have begun offering
columns with smaller ID
Microbore column - 1 mm ID packed
column
Capillary column - lt 1 mm ID internal
bound phase
These columns require smaller solvent flows
reduced sample size and improved detector
design
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Capillary and Microbore columns Capillary and Microbore columns
Aromatic Compounds
mobile phase2 ethylacetate in
hexane
flow rate 4 μlmin
column Fusica II 300μm ID x
25 cm silica
sample
1 toluene
2 nitrobenzene
3 acetophenone
4 26-dinitrobenzene
injection 60 nl
detection UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Silica based columns
Silica is the ideal support for HPLC columns It offers a large mechanical stability excellent
physicochemical surface properties a wide range of bonding chemistries and is compatible
with a broad range of organic solvents
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
pH stability
In general HPLC columns are stable within a pH range of 2 to 8 If you are measuring a pH value the measurement must be done in the aqueous media before mixing the eluent with organic solvents
Modern HPLC columns can be used outside that pH range The new bonding chemistries allow use down to pH 1 for some stationary phases However please check vendorrsquos product information before using silica based column outside the pH range of 2 to 8 However best lifetimes are obtained between pH 20 and pH 68
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Mechanical stability
Stationary phases based on silica are mechanically very stable The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem However please avoid pressure shocks on the column Pressure shocks lead to channelling in the column which results in peak splitting in the corresponding chromatogram
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Mobile phases (Eluents)
Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range
bull Filter all prepared buffer through a 05 μm filter before using them in your HPLC system
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
The use of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head These impurities block adsorption sites change the selectivity of the column and lead to peak splitting in the chromatogram In gradient elution impurities cause so called ldquoGhost Peaksrdquo
Ghost peaks are peaks that always appear in the same position on the chromatogram Their origin is not the sample but the impurities from the solvents or solvent additives Therefore it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
To avoid irreversible adsorption at the column head you should always use a pre-column
The use of a pre-column increases the life time of a column dramatically In addition a pre-column can filter solid parts stemming from pump seals or injection rotors An alternative to a pre-column is an in-line filter These filters are attached directly to the column These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at
Detectors fall roughly into two classes
Bulk property - measures an overall change in the mobile phase
Solute property - measures a solute specific property
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Properties of a good detector
A detector must provide
bull high sensitivity low detection limits
bull linearity
bullReproducibility
This is true for any detector
Each detector will have specific advantages and will vary as to peak shape and spread noise and flowtemperature dependence they have
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
UVVis detector
A solute property detector
Sample must exhibit absorption in UVVis range Solvent must not absorb significantly at the measured wavelength
Types Filter photometer - single
bull Variable wavelength
bull Multiwavelength
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Refractive index detector
Bulk property detector - general purpose
Based on refraction of light as it passes from
one media to another Presence of a solute
changes the refractive index of the solvent
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Heat of absorption detector
A small amount of heat is released when a sample absorbs on a suitable surface
This detector can measure this
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Electrochemical detectors
A number of properties have been evaluated
Detector types
Dielectic constant
Amperometric
Conductometric
Polarographic
Potentiometric
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Dielectric constant detector
Bulk property detector
Measures changes in polarity of the liquid phase passing through the cell
Conductometric detector
Measures conductivity of the solvent Useful for
solutions of ions
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Amperometric detectors
Most frequently applied type of electrochemical
detector
A known potential is applied across a set of
electrodes - typically a glassy carbon type
Ability to oxidize or reduce a species can be
measured
Typically limited to working with a specific class
of materials per analysis
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Separation is based on the analytersquos relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Normal Phase- Polar stationary phase and non-polar solvent
bull Reverse Phase- Non-polar stationary phase and a polar solvent
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Methan0l CH3OH
bull Acetonitrile CH3CN
bull Tetrahydrofuran
bull Water H2O
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Solid Support - Backbone for bonded phases Usually 10micro 5micro or 3micro silica or polymeric
particles Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support Extremely stable Reproducible
Guard - Protects the analytical column Particles Interferences Prolongs the life of the analytical column
bull Analytical - Performs the separation
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
C-2 Ethyl Silyl -Si-CH2-CH3
bullCN Cyanopropyl Silyl -Si-(CH2)3-CN
bull C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
bullC-8 Octyl Silyl -Si-(CH2)7-CH3
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
bull
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
UV Single wavelength (filter) [610 8330] Variable wavelength (monochromator)
[8316 8325] Multiple wavelengths (PDA) [555]
Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Restekreg ULTRA C-18 and CN Columns (250mm x 46mm 5micro)
Mobile Phase (11 MethanolWater) 15 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
Supelcosil LC-PAH Columns A BConditions A 150mm x 46mm 5micro
Flow Rate 15 mLmin
Conditions B 50mm x 46mm 3micro
Flow Rate 30 mLmin
