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TECHNICAL REVIEW:
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Prepared by: Shariffah Huzaimah Al-Junid
10 october 2008
INTRODUCTION
� HPLC terms:
� High performance liquid chromatography
� High pressure liquid chromatography
� High price liquid chromatography
� Important analytical tool for separating and � Important analytical tool for separating and
quantifying components in complex liquid mixture
� Common method use for analysis of:
� Biological compound
� Pharmaceuticals
� Environmental toxicant
BASIC PRINCIPLES OF
CHROMATOGRAPHY
� Definition- separation process (distribute sample
mixture between two phases in chromatographic bed)
� Stationary phase
� Column packing material
� Stronger interaction with stationary phase than mobile phase – elute less quickly (longer retention time)
� Mobile phase
� Liquid media – continuously flow through the column and carry the analytes
� Mixture of solvents
CHROMATOGRAPHY
PLANAR
PAPER
THIN LAYER
GAS CHROMATOGRAPHY ADSORPTION
COLUMN
GAS CHROMATOGRAPHY
LIQUID CHROMATOGRAPHY
ADSORPTION
ION EXCHANGE
SIZE EXCLUSIONSOLID PHASE CHROMATOGRAPHY
HPLC INSTRUMENT
Quaternary Pump
Solvent Degasser
Solvent Reservoirs
Column Compartment
Autosampler
Detector
Quaternary Pump
• To hold the solvents used to make up the mobile phase
SOLVENT RESERVOIR
• To prevent bubbles in the mobile phaseDEGASSER
• Quaternary pump that mixes the solvents
• Pumps them through column and detector
PUMP
• To hold column and control column temperature using thermostats
COLUMN COMPARTMENT
• Draws prescribed volumes from sample vials and injects them onto the columnAUTOSAMPLER
• Detector that monitors the entire spectrum of the column effluent at regular intervals
DETECTOR
Flow chart….
MOBILE PHASE
PUMP INJECTION
COLUMNDETECTORRECORDER
DATA SYSTEM
INSTRUMENT- SOLVENT
RESERVOIR
� Several reservoirs suited to pump type
� Degassing : remove of dissolved gas
� Sparging line bubble or gas
� Vacuum pumping
� Dust removal – interference with detection, column clogging, damage pumping system
� Millipore filter under vacuum
� Solvent containers – enclosed to protects users from toxic solvent vapors (chloroform, aromatic hydrocarbons and etc.)
Cont..
Isocratic Elution Gradient ElutionGradient ElutionGradient ElutionGradient Elution
� Eluent composition constant during the
whole analysis
� Eluent composition
changed during the
analysis
� increase separation � increase separation
efficiency
� decrease the retention
time
� Peak shape is improved (Less tailing)
Isocratic Elution (B : Acetonitrile) Gradient Elution
� Column : 0.46 * 25cm Hypersil ODS
� Flowrate : 1.0 mL/min
� Eluent : Aqueous Buffer (pH 3.5) and Acetonitrile
(1) benzyl alcohol, (2) Phenol, (3) 3’, 4’- dimethoxy-toluene, (4) benzoin
(5) ethyl benzoate, (6) toluene, (7) 2, 6 -dimethoxytoluene, (8) o-methoxybiphenyl
INSTRUMENT - PUMP
� Requirement
� High pressure up to 6000psi – force solvents
through stationary phase beds
� Pulse free, prevents remixing of solutes
� Flow rate range: 0.1 to 10ml/min� Flow rate range: 0.1 to 10ml/min
� Integrated degassing system – vacuum degasser
� Resistant to corrosion
� acidic, basic and strong eluent in mobile phase
Cont..
� Reciprocation pumps (most use) � advantages: small internal volume, high output pressures,
constant flow rates
� disadvantages: produce a pulsed flow, cause baseline noise
� Displacement pumps
advantages: output is pulse free � advantages: output is pulse free
� disadvantages: limited solvent capacity (<250 mL) inconvenience when change solvents
� Pneumatic pumps
� advantages: inexpensive, output is pulse free
� disadvantages: limited solvent capacity, not amenable to gradient elution and limit pressures <2000 psi
INSTRUMENT - COLUMN
� Stainless steel
� Resistant to the high pressure
� Inert to chemical corrosion
� Criteria for a column
� Length: 10, 15 and 25cm� Length: 10, 15 and 25cm
� Particle diameter: 3, 5 or 10um
� Internal diameter: 4 or 4.6um
� Column packing
� silica, alumina, a polystyrene-divinyl-benzene
synthetic or an ion-exchange resin
Cont..
� Silica � Particle shape, surface properties, and pore structure
help to get a good separation
� Inert to most compounds
� High surface activity
� Used to separate a wide variety of chemical compounds� Used to separate a wide variety of chemical compounds
� Guard column� Anterior to the separating
� Prevent column contamination
� Filter or remove:� Particulate matter
� Compounds and ions
� Protect analytical column
Cont..Cont..Cont..Cont..
� Flow rate
� Depends on column internal diameter
� Also depends on type of analysis
Internal diameter Standard Flow RateInternal diameter (mm)
Standard Flow Rate(µl/min)
4.6 1000
2.1 200
1.0 50
0.30 4
0.15 1
INSTRUMENT - AUTOSAMPLER
� Sample injection:
� With syringe and septum injector
� With a loop valve
� With an automated injection system (autosampler)
Autosampler
INSTRUMENT - DETECTOR
� Most commonly use detectors:� UV Detector
� Refractive Index Detector
� Fluorescence Detector
� Others detectors� Others detectors
� Electrochemical Detector
� Light Scattering Detector
� Conductivity Detector
� Photoconductivity Detector
� Infrared Detector
� Radioactivity Detector
� Mass Spectrometer
UV detector
� General detector
� Useful for wide range of analytes
� Record compounds that adsorb ultraviolet or visible light (most organic solvents)
� Unaffected by temperature fluctuations and � Unaffected by temperature fluctuations and suitable for gradient elution
� Moderate sensitivity
� Deuterium lamps (340 - 600nm) and Tungsten lamp (340 – 850nm)
Fluorescence detector
� Sensitive and selective
� Very sensitive to a few analytes which do fluoresce
and fluorescing derivatives
� Lambda 280-305 nm and emission at lambda 340-
500 nm500 nm
Refractive Index
• Mobile phase need to stay same
• Sensitive to changes in pressure and temperature
• Useless in gradient elution
• Not useful for trace analysis
• Poor sensitivity• Poor sensitivity
• Problem with baseline stability
COMPARISON OF HPLC
DETECTORS
MODES OF SEPARATION
Adsorption chromatographyAdsorption chromatographyAdsorption chromatographyAdsorption chromatography
� Normal Phase Chromatography
� Mobile phase – nonpolar solvents (n-hexane or tetrahydrofuran)
� Stationary phase – polar (silica gel-OH, NH )
Cover almost 90% of all
chromatographic applications
� Stationary phase – polar (silica gel-OH, NH2)
� Retention mechanism – dipole-dipole
� Reverse Phase Chromatography
� Mobile phase – polar solvents (Water, Methanol, Acetonitrile)
� Stationary phase – nonpolar (modified silica – C8, C18, Phenyl)
� Retention mechanism – hydrophobic
�
�
� Ion exchange chromatography�Mobile phase – aqueous buffer
� pH and ionic strength use to control elution time
�Stationary phase – ionically charge surface� opposite charge to sample ions
�Retention mechanism – ionic interaction
� Size exclusion chromatography�Stationary phase – material control pore size �Sample screened or filtered according to size� Larger molecule rapidly washed� Smaller molecules penetrate inside porous
packing material
PREPARATION OF EQUIPMENT TO
SAMPLE
SELECTION OF MOBILE PHASE
PREPARATION OF INTERPRETATION
OF RESULT SAMPLE INJECTION
PREPARATION OF MOBILE PHASE
SAMPLE SOLUTION AND SAMPLE VOLUME
OF RESULT
SELECTION OF MOBILE PHASE
� Factors to consider : must interact with a suitable
stationary phase – separate mixture as fast and
efficiently.
� Purity – HPLC grade
� UV transparency – detector compatibility� UV transparency – detector compatibility
�Refractive index
� Solubility
� Viscosity
�Chemical inertness with sample compounds
�Corrosive resistance
�Toxicity
� Price
Increasing Increasing polarity
PREPARATION OF MOBILE PHASE
� Solvent and reagent - HPLC grade
� Mixed mobile phase or buffer
� Volume contraction effects – mixing water-miscible
solvents
� pH adjustment, addition of non-ionic additives� pH adjustment, addition of non-ionic additives
� Filter immediately before use
� Pore size 0.45um to 0.8um
� Types of filter:
� Nylon – hydrophilic (high content of
water/aqueous)
� PTFE – hydrophobic, chemically resistant
� Regenerated cellulose – hydrophilic, (aqueous
solution, sample filtration)
Cont..
� Degassed
� Polar solvents (dissolve high amount of air)
� Sonicate for 5 to 10 minutes
� Fresh prepared every analysis
� Good practice to prepare only as much will be � Good practice to prepare only as much will be
within short time
SAMPLE PREPARATION AND
SAMPLE VOLUME
SAMPLE PREPARATIONSAMPLE PREPARATIONSAMPLE PREPARATIONSAMPLE PREPARATION SAMPLE VOLUMESAMPLE VOLUMESAMPLE VOLUMESAMPLE VOLUME
� To extract target
compound
� Dilute in mobile phase
� Volume of solvent
required for dissolution
may vary
� Filter to ensure that it
contain no solid
� Types of filtration
� Filtration by membrane
� Solid phase extraction with disposable cartridges
� Protein precipitation
� Desalting
� Avoid band broadening
Signals – peaksSignals – peaks
INTERPRETATION OF RESULT
Whole entity - chromatogramWhole entity - chromatogram
f
W0.05
RETENTION TIME
� Time for analyte to reach detector� Retention time (tR) – the period between sample injection and recording of peak maximum
� Ideal tracer� Dead time (tM) / breakthrough time– retention time of unretained solute (time that require by the mobile phase to pass unretained solute (time that require by the mobile phase to pass through the column)
tR
Height and area under curve –
amount of sample
ADVANTAGES AND DISADVANTAGESADVANTAGES AND DISADVANTAGESADVANTAGES AND DISADVANTAGESADVANTAGES AND DISADVANTAGES
ADVANTAGESADVANTAGESADVANTAGESADVANTAGES DISADVANTAGESDISADVANTAGESDISADVANTAGESDISADVANTAGES
� High speed
� High resolution
� High sensitivity
� Cost
� Complexity
� Low sensitivity for some � High sensitivity
� Reproducibility of ±1%
� Accuracy
� Automation
� Low sensitivity for some
compounds
THANK YOUTHANK YOU