Upload
magicianchemist
View
223
Download
0
Embed Size (px)
Citation preview
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
1/172
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
2/1721
LAAQ-B-LC001B 1
What Is HPLC?What Is HPLC?
Basic Principles
Fundamentally, chromatography is a technique used to separate the components contained
in a sample.
Above all, high performance liquid chromatography (HPLC) is a type of chromatography that,
because of its wide application range and quantitative accuracy, is regarded as an
indispensable analytical technique, particularly in the field of organic chemistry. It is also
widely used as a preparation technique for the isolation and purification of target componentscontained in mixtures.
An overview of HPLC, from the basic principles of chromatography to the characteristics of
HPLC itself, is presented here.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
3/1722
LAAQ-B-LC001B 2
Invention of Chromatography byInvention of Chromatography by
M.M. TswettTswett
Ether
CaCO3
Chlorophyll
ChromatoChromatography
ColorsColors
The Russian-Polish botanist M. Tswett is generally recognized as the first person to
establish the principles of chromatography.
In a paper he presented in 1906, Tswett described how he filled a glass tube with chalk
powder (CaCO3) and, by allowing an ether solution of chlorophyll to flow through the chalk,
separated the chlorophyll into layers of different colors. He called this technique
chromatography.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
4/1723
LAAQ-B-LC001B 3
Comparing Chromatography to theComparing Chromatography to the
Flow of a River...Flow of a River...
Base
Water flowLight leaf
Heavy stone
Chromatography can be often compared to the flow of a river.
A river consists of a stationary riverbed and water that continuously moves in one direction.
What happens if a leaf and a stone are thrown into the river? The relatively light leaf does
not sink to the bottom, and is carried downstream by the current. On the other hand, the
relatively heavy stone sinks to the bottom, and although it is gradually pulled downstream bythe current, it moves much more slowly than the leaf.
If you stand watch at the mouth of the river, you will eventually be able to observe the arrival
of the leaf and the stone. However, although the leaf will arrive in an extremely short time,
the stone will take much longer to arrive.
This analogy represents the components of chromatography in the following way:
River: Separation field
Leaf and stone: Target components of sample
Standing watch at the river mouth: Detector
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
5/1724
LAAQ-B-LC001B 4
Mobile Phase / Stationary PhaseMobile Phase / Stationary Phase
A site in which a moving
phase (mobile phase) and
a non-moving phase
(stationary phase) make
contact via an interface
that is set up.
The affinity with the mobile
phase and stationary
phase varies with the
solute. Separationoccurs due to differences
in the speed of motion.
Strong Weak
MobileMobile
phasephase
StationaryStationary
phasephase
In chromatography, the field of separation is divided into two phases. One phase, called the
stationary phase, does not move. The other phase, called the mobile phase, moves at a
constant speed in one direction.
The stationary phase and mobile phase make contact via an interface. They do not
intermingle, and are kept in a steady state of equilibrium.
In the river analogy, the riverbed corresponds to the stationary phase and the flowing watercorresponds to the mobile phase.
Let us suppose that some substance has been introduced into the flow of the mobile phase
and led to the separation site. If this substance contains a component that is only weakly
attracted by the stationary phase and a component that is strongly attracted by the stationary
phase, the former component will be pulled along quickly by the flow of the mobile phase
whereas the latter component will stick to the stationary phase and only move slowly.
In this way, differences in the properties of the various components contained in the sample
being analyzed give rise to differences in speed. This makes it possible to separate
components from each other.
Incidentally, in the river analogy, the interaction that determines the speed of motion isbased on gravity (and buoyancy in water). In chromatography, various physical and
chemical properties, such as solubility and the degree of adsorption, determine the
dynamics of separation.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
6/1725
LAAQ-B-LC001B 5
ChromatoChromato--graphygraphy // --graph /graph / --gram /gram /
--graphergrapher
Chromatography: Analytical technique
Chromatograph: Instrument
Chromatogram: Obtained picture
Chromatographer: Person
There are many similar terms in this field and so let us clarify some of them.
Chromatography is the name of the analytical technique itself.
A chromatograph is an analytical instrument that is used to perform chromatography. The
product names of the chromatographs given in the catalogs of analytical instrument
manufacturers should all include this word.
A chromatogram is produced by recording the results obtained with chromatography on
recording paper (or some other medium).
A chromatographer is a person who carries out a chromatography experiment.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
7/1726
LAAQ-B-LC001B 6
Three States of Matter andThree States of Matter and
Chromatography TypesChromatography Types
Mobile phase
Gas Liquid Solid
Stationary
phase
Gas
Liquid
Solid
GasGas
chromatographychromatographyLiquidLiquid
chromatographychromatography
There are various ways of categorizing chromatography. Here, let us categorize it in terms of
the three states of matter.
There are generally three states of matter: gas, liquid, and solid. If we could use stationary
phases and mobile phases of any state, this would give a total of nine different types of
chromatography. Using a gas as the stationary phase or a solid as the mobile phase,
however, is not practical (even if it is possible) and this restricts the combinations that can beused.
Chromatography performed using a gas as the mobile phase and a liquid or a solid as the
stationary phase is called gas chromatography (GC). Chromatography performed using a
liquid as the mobile phase and a liquid or a solid as the stationary phase is called liquid
chromatography (LC). Both of these techniques are indispensable, particularly in the field of
organic chemistry.
In addition to these, there is a technique called supercritical fluid chromatography (SFC), in
which a supercritical fluid kept at a high temperature and high pressure is used as the mobile
phase.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
8/1727
LAAQ-B-LC001B 7
Liquid ChromatographyLiquid Chromatography
Chromatography in which the mobile phaseis a liquid.
The liquid used as the mobile phase iscalled the eluent.
The stationary phase is usually a solid or aliquid.
In general, it is possible to analyze anysubstance that can be stably dissolved in
the mobile phase.
Liquid chromatography (LC) is chromatography in which the mobile phase is a liquid.
Stationary Phase
Usually a solid or a liquid is used as the mobile phase. (This includes the case
where a substance regarded as a liquid is chemically bonded, or applied, to the
surface of a solid.)
The most common form of stationary phase consists of fine particles of, for
example, silica gel or resin packed into a cylindrical tube. These packed particles
are called packing material or packing and the separation tube into which they
are packed is called the separation column or simply the column. In day-to-day
analysis work, column is sometimes used to refer to the stationary phase and
stationary phase is sometimes used to refer to the column.
Mobile Phase
Various solvents are used as mobile phases. The mobile phase conveys the
components of the dissolved sample through the separation field, and facilitates the
repeated three-way interactions that take place between the phases and the
sample, thereby leading to separation.
The solvent used for the mobile phase is called the eluent or eluant. (In LC, the
term mobile phase is also used to refer to this solvent. In this text, however, we
shall use the term eluent.)
Sample
In general, it is possible to analyze any substance that can be stably dissolved in the
eluent. This is one advantage that LC has over GC, which cannot be used to
analyze substances that do not vaporize or that are thermally decomposed easily.
The sample is generally converted to liquid form before being introduced to the
system. It contains various solutes. The target substances (the analytes) are
separated and detected.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
9/1728
LAAQ-B-LC001B 8
Interaction Between Solutes, StationaryInteraction Between Solutes, Stationary
Phase, and Mobile PhasePhase, and Mobile Phase
Differences in the interactions between the solutes and
stationary and mobile phases enable separation.
Solute
Stationary
phaseMobile phase
Degree of adsorption,
solubility, ionicity, etc.
The solutes interact with the stationary and mobile phases. These interactions are the most
important contributing factor behind separation.
Representative examples of the types of interactions that take place in liquid
chromatography are given below. (They are not based on strict classifications.)
Adsorption
Distribution
Hydrophobic interaction
Ion exchange
Ion pair formation
Osmosis and exclusion
Affinity
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
10/1729
LAAQ-B-LC001B 9
Column Chromatography andColumn Chromatography and
Planar ChromatographyPlanar Chromatography
Separation column
Packing material
Column Chromatography
Paper or a
substrate coated
with particles
Paper Chromatography
Thin Layer Chromatography (TLC)
Liquid chromatography can be categorized by shape of separation field into column-shaped
and planar types.
A representative type of chromatography that uses a column-shaped field is column
chromatography, which is performed using a separation column consisting of a cylindrical
tube filled with packing material. Another type is capillary chromatography, which is
performed using a narrow hollow tube. Unlike column chromatography, however, capillarychromatography has yet to attain general acceptance. (In the field of GC, however, capillary
chromatography is a commonly used technique.)
Types of chromatography that use a planar (or plate layer) field include thin layer
chromatography, in which the stationary phase consists of a substrate of glass or some
other material to which minute particles are applied, and paper chromatography, in which
the stationary phase consists of cellulose filter paper.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
11/17210
LAAQ-B-LC001B 10
Separation Process and ChromatogramSeparation Process and Chromatogramfor Column Chromatographyfor Column Chromatography
Outp
ut
conc
entration
Time
ChromatogramChromatogram
The separation process for column chromatography is shown in the above diagram.
After the eluent is allowed to flow into the top of the column, it flows down through the
spaces in the packing material due to gravity and capillary action. In this state, a sample
mixture is placed at the top of the column. The solutes in the sample undergo various
interactions with the solid and mobile phases, splitting up into solutes that descend quickly
together with the mobile phase and solutes that adsorb to the stationary phase and descendslowly, so differences in the speed of motion emerge. At the outlet, the elution of the various
solutes at different times is observed.
A detector that can measure the concentrations of the solutes in the eluate is set up at the
column outlet, and variations in the concentration are monitored. The graph representing the
results using the horizontal axis for times and the vertical axis for solute concentrations (or
more accurately, output values of detector signals proportional to solute concentrations) is
called a chromatogram.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
12/17211
LAAQ-B-LC001B 11
ChromatogramChromatogram
tR
t0
Intensityofdetectorsignal
Time
Peak tR : Retention time
h
A
t0 : Non-retention time
A : Peak area
h : Peak height
Usually, during the time period in which the sample components are not eluted, a straight
line running parallel to the time axis is drawn. This is called the baseline.
When a component is eluted, a response is obtained from the detector, and a raised section
appears on the baseline. This is called a peak. The components in the sample are
dispersed by the repeated interactions with the stationary and mobile phases, so the peaks
generally take the bell-shape form of a Gaussian distribution.
The time that elapses between sample injection and the appearance of the top of the peak is
called the retention time. If the analytical conditions are the same, the same substance
always gives the same retention time. Therefore, the retention time provides a means to
perform the qualitative analysis of substances.
The time taken for solutes in the sample to go straight through the column together with the
mobile phase, without interacting with the stationary phase, and to be eluted is denoted as
t0. There is no specific name for this parameter, but terms such as non-retention time and
hold-up time seem to be commonly used.
Because the eluent usually passes through the column at a constant flow rate, t R and t0 are
sometimes multiplied by the eluent flow rate and handled as volumes. The volumecorresponding to the retention time is called the retention volume and is notated as VR.
The length of a straight line drawn from the top of a peak down to the baseline is called the
peak height, and the area of the raised section above the baseline is called the peak area.
If the intensities of the detector signals are proportional to the concentrations or absolute
quantities of the peak components, then the peak areas and heights are proportional to the
concentrations of the peak components. Therefore, the peak areas and heights provide a
means to perform the quantitative analysis of sample components. It is generally said that
using the peak areas gives greater accuracy.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
13/17212
LAAQ-B-LC001B 12
From Liquid Chromatography to HighFrom Liquid Chromatography to High
Performance Liquid ChromatographyPerformance Liquid Chromatography
Higher degree of separation!
Refinement of packing material (3 to 10 m)
Reduction of analysis time!
Delivery of eluent by pump Demand for special equipment that can
withstand high pressures
The arrival ofhigh performance liquid chromatography!
In order to increase the separation capability of column chromatography, in addition to
increasing the surface area of the stationary phase so that the interaction efficiency is
increased, it is also necessary to homogenize the separation field as much as possible so
that dispersion in the mobile and stationary phases is minimized. The most effective way of
achieving this is to refine the packing material.
Refining the packing material, however, causes resistance to the delivery of the eluent toincrease. This is similar to the way that water drains easily through sand, which has
relatively large particles, whereas it does not drain easily through clay-rich soil, which has
relatively fine particles.
Depending on gravity and capillary action would cause analysis to take a very long time to
be completed, and the idea of delivering the eluent forcibly using a high-pressure pump was
proposed. This was the start of high performance liquid chromatography.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
14/172
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
15/17214
LAAQ-B-LC001B 14
Advantages of High PerformanceAdvantages of High Performance
Liquid ChromatographyLiquid Chromatography
High separation capacity, enabling the batch
analysis of multiple components
Superior quantitative capability and reproducibility
Moderate analytical conditions
Unlike GC, the sample does not need to be vaporized.
Generally high sensitivity
Low sample consumption
Easy preparative separation and purification ofsamples
HPLC is a type of separation analysis, and this is the most important aspect of this analytical
technique. Even if the sample consists of a mixture, it allows the target components to be
separated, detected, and quantified. It also allows simultaneous analysis of multiple
components.
It could be said that HPLC is more suited to quantitative analysis than it is to qualitative
analysis. Under the appropriate conditions, it is possible to attain a high level ofreproducibility with a coefficient of variation not exceeding 1%.
One advantage that HPLC has over GC is that, in general, analysis is possible for any
sample that can be stably dissolved in the eluent. With GC, gas is used as the mobile phase,
so substances that are difficult to vaporize or that decompose easily when heated cannot be
analyzed. For this reason, particularly in the fields of pharmaceutical science and
biochemistry, HPLC is used much more frequently than GC.
The level of sensitivity that can be attained varies with the detector, but detection down to
the g and pg levels is usually possible and, in some cases, even smaller quantities can be
detected.
The amount of sample used is very small, and is usually in the range of 1 to 100 L.
The components contained in the sample are eluted from the column separately. So, if anon-destructive detector is used, the preparative separation and purification of specific
components is possible. In fact, liquid chromatographs specially designed for preparative
separation are commercially available.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
16/17215
LAAQ-B-LC001B 15
Fields in Which High PerformanceFields in Which High Performance
Liquid Chromatography Is UsedLiquid Chromatography Is Used
Biogenic substances Sugars, lipids, nucleic
acids, amino acids,
proteins, peptides, steroids,
amines, etc.
Medical products Drugs, antibiotics, etc.
Food products Vitamins, food additives,
sugars, organic acids,amino acids, etc.
Environmentalsamples Inorganic ions
Hazardous organicsubstances, etc.
Organic industrialproducts Synthetic polymers,
additives, surfactants, etc.
HPLC is currently being used in a broad range of fields. In particular, in the field of
biochemistry, it is widely used as an indispensable analytical technique.
From the perspective of an analytical instrument manufacturer, we observe that the industry
that purchases the highest number of high performance liquid chromatographs is the
pharmaceutical industry. It is said that the number of deliveries for this industry accounts for
about 40% of the total. Although the number of deliveries to quality control departments isparticularly high, it is also quite high for drug discovery and R&D departments.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
17/17216
LAAQ-B-LC001B 16
HPLC Hardware: Part 1HPLC Hardware: Part 1
Solvent Delivery System,
Degasser, Sample Injection Unit,
Column Oven
The analytical instrument used to perform high performance liquid chromatography is called
a high performance liquid chromatograph.
A high performance liquid chromatograph consists of several units. Here, in Part 1, I shall
present all the units except the detector and data processor.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
18/17217
LAAQ-B-LC001B 17
Pump
Sample injection unit
(injector)
Column
Column Oven
(thermostatic
column chamber)
Detector
Eluent
(mobile phase)
Drain
Data processor
Flow Channel Diagram for HPLCFlow Channel Diagram for HPLC
Degasser
The configuration of a high performance liquid chromatograph includes a solvent delivery pump, a
sample injection unit, a column chamber, a detector, and a data processor (recorder). These units areessential. Accessories are added as necessary.
The roles of these five basic units are as follows:
Solvent Delivery Pump
This unit delivers the eluent to the column. It incorporates features that allow it to maintain aconstant, non-pulsating flow of solvent at a high pressure against the resistance of the
column.
Sample Injection Unit
This unit introduces the sample to the column by injecting a specific quantity of sample
solution. Types include a manual injector, which performs injection using a microsyringe, and
an autosampler, which automatically injects a series of samples.
Column Oven
This unit maintains the column at a constant temperature. Temperature is an important factor
that influences separation, so maintaining the column at a constant temperature makes it
possible to improve the quality of separation and the reproducibility. This unit is also called a
thermostatic column chamber.
DetectorThis unit detects the components eluted from the column. There are many different types of
detectors, based on various operating principles, and the detector used is selected according
to the properties of the target compounds and the objective of analysis. UV-VIS absorbance
detectors are the most commonly used.
Data Processor (Recorder)
This unit draws chromatograms by recording the signals received from the detector on charts
or magnetic media. Data processors that, in addition to recording, have functions for adding
peak areas and performing quantitative calculations are commonly used. Furthermore,
systems that perform both instrument control and data processing using PCs are
widespread.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
19/17218
LAAQ-B-LC001B 18
Solvent Delivery PumpSolvent Delivery Pump
Performance Requirements
Capacity to withstand high load pressures.
Pulsations that accompany pressure
fluctuations are small.
Flow rate does not fluctuate.
Solvent replacement is easy.
The flow rate setting range is wide and the
flow rate is accurate.
The solvent delivery pump is the most important part of a high performance liquid
chromatograph. The basic performance requirements are as follows:
1. High-pressure discharge that is easily capable of overcoming the increase in
column load pressure that results from the refinement of the packing material.
2. The pulsating flow caused by pressure fluctuations originating in aspiration /
discharge operation only give rise to a small amount of noise in the detector.3. The eluent flow rate does not fluctuate.
4. When replacing solution, operations such as rinsing are easy and solution
consumption is relatively low.
5. The flow rate setting range is wide, and the flow rate can be set accurately.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
20/17219
LAAQ-B-LC001B 19
Solvent Delivery Pump:Solvent Delivery Pump:
Representative Pumping MethodsRepresentative Pumping Methods
Syringe pump
Plunger pump
Diaphragm pump
In order to satisfy performance requirements, pumps based on a wide variety of
mechanisms have been devised and used.
Pumps can be categorized according to the driving mechanism into a variety of types,
including gas-driven pumps, motor-driven pumps, and peristaltic pumps. At present,
because of their ability to maintain stable solvent delivery for long periods and to deliver
solvent at high pressures, motor-driven pumps based on step motors controlled bymicrocomputers are widely used.
Pumps can also be categorized according to the mechanism used to discharge the solution.
Syringe pumps push out solvent at a constant speed using a large syringe. Plunger pumps
use a reciprocating piston called a plunger. Diaphragm pumps push and pull an inflecting
plate called a diaphragm. At present, plunger pumps are mainly used.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
21/17220
LAAQ-B-LC001B 20
Solvent Delivery Pump:Solvent Delivery Pump:
Schematic Diagram of Plunger PumpSchematic Diagram of Plunger Pump
Motor and cam
Plunger
Plunger seal
Check
valves
Pump head
10 -100L
In a motor-driven plunger pump, a process consisting of the aspiration, compression, and
discharge of solution is repeated. The operating principle is shown in the above diagram.
The operation of the step motor is converted, through a cam, to reciprocating motion of the
plunger. A material such as sapphire or a special ceramic is usually used for the plunger.
The eluent is aspirated and discharged by the motion of this plunger.
Check valves ensure that solution flows only in one direction. The valves contain balls,typically made of ruby, which become wedged in the seats of the valves, thereby blocking
the flow channels, if the solution starts to flow in the opposite direction. Some pumps use a
mechanism in which the check valves are forcibly opened and closed by a magnetic force.
The plunger seal prevents the solution drawn into the pump head from leaking into the drive
unit. The seal is continuously being worn away by the action of the plunger, so it is a
consumable item that must be replaced at regular intervals. It is typically made of fluororesin
or polyethylene.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
22/17221
LAAQ-B-LC001B 21
Solvent Delivery Pump:Solvent Delivery Pump:
Single Plunger TypeSingle Plunger Type
Check valves
Plunger head
With a single-plunger pump, the plunger moves slowly at a constant speed during solution
discharge and moves at a high speed during aspiration. This movement minimizes the
reduction in pressure that occurs when the solution is aspirated, and reduces pulsation. This
mechanism is called constant displacement with quick return (CDQR).
Because of their simple structure, single-plunger pumps are easy to maintain and have come
to be widely used. There is a limit, however, to the extent by which pulsation can be reduced,and increasing demands for greater sensitivity have led to a decrease in their use.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
23/17222
LAAQ-B-LC001B 22
Solvent Delivery Pump:Solvent Delivery Pump:
Dual Plunger TypeDual Plunger Type
Check valves
Plunger heads
Type Type
Dual-plunger pumps are based on the idea that pulsation can be reduced by having one
plunger perform aspiration while the other performs discharge. At present, this type of
delivery pump is mainly used with HPLC.
There are two types of dual-plunger pumps: a parallel type, in which the plungers are
arranged in parallel, and a serial type, in which the plungers are arranged in series. With
both types, pulsation is minimized by operating the plungers with a 180 phase difference sothat they perform aspiration and discharge alternately.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
24/17223
LAAQ-B-LC001B 23
Gradient SystemGradient System
Isocratic system
Constant eluent composition
Gradient system
Varying eluent composition
HPGE (High Pressure Gradient)
LPGE (Low Pressure Gradient)
The technique of delivering solution with a constant composition as the eluent is called
isocratic elution. The technique of varying the eluent composition during a single analysis
is called gradient elution.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
25/17224
LAAQ-B-LC001B 24
Aim of Gradient System (1)Aim of Gradient System (1)
In isocratic mode
Long analysis time!!Long analysis time!!
PoorPoor
separation!!separation!!
CH3OH / H2O = 6 / 4
CH3OH / H2O = 8 / 2
(Column: ODS type)
In the analysis of multiple components using HPLC, attempting to clearly separate every
single component results in an extremely long analysis time. On the other hand, attempting
to reduce the analysis time by changing the eluent composition has an adverse effect on
separation among components with relatively short retention times. Is there no way of
reducing the analysis time while maintaining a good level of separation?
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
26/17225
LAAQ-B-LC001B 25
Aim of Gradient System (2)Aim of Gradient System (2)
If the eluent composition is changed gradually duringanalysis...
95%
30%
Concentrationofmethanolineluent
In order to separate components with short retention times, an eluent composition with a low
elution strength is used immediately after sample injection. After most of these components
have been eluted, the eluent composition is changed so that components with long retention
times are eluted relatively quickly. This technique is called gradient elution.
Using gradient elution in this way makes it possible to maintain good separation and reduce
the analysis time.
The chromatograms obtained with gradient elution contain many peaks so there is a
tendency to think of gradient elution as a technique that achieves an extremely high level of
separation. As can be seen above, however, the main objective of gradient elution is the
batch analysis of multiple components.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
27/17226
LAAQ-B-LC001B 26
HighHigh-- / Low/ Low--Pressure Gradient SystemPressure Gradient System
High-pressure gradient
Mixer
Low-pressure
gradient unit
Low-pressure gradient
Mixer
Gradient types can be categorized according to hardware configuration as either high-
pressure gradient or low-pressure gradient. The terms high pressure and low pressure
indicate whether the location at which solutions with different compositions meet is at a high
pressure or normal pressure.
A high-pressure gradient system uses multiple solvent delivery pumps. The mixing ratio is
regulated by the independent control of the solvent delivery flow rate for each pump.In a low-pressure gradient system, a low-pressure gradient unit that mixes the solutions is
installed at a point upstream from the pump. This unit generally incorporates solenoid valves
at the inlets for each solution, and the mixing ratio is regulated by controlling the opening
and closing times of the valves.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
28/17227
LAAQ-B-LC001B 27
Advantages and Disadvantages ofAdvantages and Disadvantages of
HighHigh-- / Low/ Low--Pressure Gradient SystemsPressure Gradient Systems
High-pressure gradient system
High gradient accuracy
Complex system configuration (multiple
pumps required)
Low-pressure gradient system
Simple system configuration
Degasser required
An advantage of the high-pressure gradient system is that, because the accuracy of the
mixing ratio depends on the solvent delivery performance of the pumps, using high-
performance pumps makes it easy to obtain a high level of accuracy. High accuracy helps
improve the reproducibility of retention times and peak area values. It also makes this
system suitable for semi-micro analysis.
A disadvantage is that, because one pump is required for each solution, as the number ofsolutions increases, so does the complexity and cost of the required HPLC system.
An advantage of the low-pressure gradient system is that, because a low-pressure gradient
unit can generally handle four solutions, the equipment cost per solution is relatively low.
A disadvantage is that, because different solutions are mixed under normal pressure,
bubbles are easily formed and a degasser is therefore required.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
29/17228
LAAQ-B-LC001B 28
DegasserDegasser
Problems caused by dissolved air in the eluent
Unstable delivery by pump
More noise and large baseline drift in detector cell
In order to avoid these problems, the eluent
must be degassed.
Since analysis is performed in air at a pressure of one atmosphere, air bubbles are always
dissolved in the eluent. If the eluent is passed through the HPLC system in this state,
problems originating in the dissolved air may occur.
The most serious problems occur if the bubbles enter the solvent delivery pump. The
compression and expansion of the bubbles by the plunger is enough to disrupt the delivery of
the all-important eluent. The flow rate may drop, pulsation may occur, or solvent deliverymay stop completely.
Problems also occur if bubbles enter the flow cell in the detector. Noise may be produced
when the bubbles pass through, and baseline fluctuations may occur if bubbles accumulate
in the cell. Furthermore, even if there are no bubbles, depending on the quantity of dissolved
gas, the detected background level may be affected, and the peak response itself may even
change.
In order to prevent these problems, the eluent is degassed beforehand.
Degassing methods consist of offline degassing, in which the eluent is degassed before it
is set in the instrument, and online degassing, in which the eluent is continuously
maintained in a degassed state after being set in the instrument. The device used to performcontinuous online degassing is called a degasser.
A method in which the pressure is reduced with an aspirator while the eluent is either
subjected to ultrasound or agitated with a magnetic stirrer is commonly used to perform
offline degassing. Although this method is simple and relatively inexpensive, air is gradually
dissolved back into the eluent afterwards, and not only is the effect of degassing diminished
during analysis, baseline fluctuations may also occur. It is recommended that the following
online degasser is used.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
30/172
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
31/17230
LAAQ-B-LC001B 30
Sample Injection Unit (Injector)Sample Injection Unit (Injector)
Performance Requirements
No sample remaining in unit
Minimal broadening of sample band
Free adjustment of injection volume
Minimal loss
Superior durability and pressure resistance
The performance specifications required of the sample injection unit used in HPLC are as
follows:
1. It must have a structure that does not allow the sample to remain in the unit.
2. It must have a structure that minimizes spread of the sample band.
3. It must be possible to freely set the sample injection volume.
4. Sample loss must be minimal.
5. It must have superior durability and pressure resistance.
In order to satisfy these requirements, nearly all commercially available sample injection
units for HPLC, whether they are manual injectors or autosamplers, are based on
mechanisms that allow flow channel selection using 6-port valves.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
32/17231
LAAQ-B-LC001B 31
Manual InjectorManual Injector
INJECT positionINJECT position
LOAD positionLOAD position
From pump
To column
From pump
To column
With a manual injector, the sample is injected manually using a microsyringe.
Regardless of the manufacturer of the HPLC system itself, a large number of currently used
products incorporate Rheodyne 6-port valves.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
33/172
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
34/17233
LAAQ-B-LC001B 33
Manual Injector:Manual Injector:
Injection MethodInjection Method
Syringe measurement method
It is desirable that no more than half the loop
volume is injected.
Loop measurement method
It is desirable that at least 3 times the loop
volume is injected.
Syringe Measurement Method
With this method, the volume is measured with the microsyringe, and this volume is
injected into the manual injector.
An important advantage of this method is that the injection volume can be changed
freely as long as it is within the range of the microsyringes measuring capacity. A
disadvantage is that it is easy for inconsistencies in the volume to occur due tovariations in the skill level and personal style of the measurer.
The speed of the injected sample band is higher near the center of the tube and
lower near the internal wall of the tube. For this reason, if a volume almost equal to
the loop volume is injected, there is a possibility that some of it may leave the loop.
If possible, do not inject more than half the loop volume.
Loop Measurement Method
With this method, using the way that the sample leaves the loop if more than the
loop volume is injected, an amount equal to the loop volume is conveyed to the
column by deliberately injecting more than the loop volume.
An advantage of this method is that there is little chance of inconsistencies
occurring between analysts, so the injection reproducibility is high. A disadvantageis that the loop must be replaced in order to change the sample injection volume.
As previously mentioned, the injected sample band does not flow through the loop
evenly, so if an amount only slightly larger than the loop volume is injected, eluent
may remain on the inner wall. Therefore, inject at least 3 times the loop volume.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
35/17234
LAAQ-B-LC001B 34
AutosamplerAutosampler
(Pressure Injection Method)(Pressure Injection Method)
To columnFrom pump From pump To column
Sample Loop
LOADLOAD INJECTINJECT
Injection by an autosampler consists of the automatic execution of the operations performed
by a manual injector using a microsyringe based on control by a computer program.
Pressure Injection Method
With this method, after a specific amount of sample is aspirated from the sample
vial and conveyed to the sample loop attached to a 6-port valve, the valve isswitched so that eluent is delivered into the loop and the sample is consequently
conveyed to the column. In other words, some of the operations performed by an
analyst using a manual injector are performed by a machine.
An advantage of this method is that, as with a manual injector, injection based on
the loop measurement method is possible, and a large-volume injection can be
facilitated by replacement of the sample loop.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
36/17235
LAAQ-B-LC001B 35
AutosamplerAutosampler
(Total(Total--Volume Injection Method)Volume Injection Method)
From pump From pump To column
Sample vial
Needle
Measuring pump
To column
LOADLOAD INJECTINJECT
Total-Volume Injection Method
With this method, a specific amount of sample is measured from the sample vial
into a tube, and eluent is delivered directly into this tube, thereby conveying the
sample to the column.
The most important advantage of this method is that, because eluent flows around
inside the needle at all times except during injection operation, sample carryover isunlikely to occur. Another advantage is that, because it is not necessary to aspirate
a volume greater than that conveyed to the column, there is little sample loss.
With this method, even if there are only very small bubbles in the section of tube
leading from the measuring pump to the tip of the needle, it can become impossible
to aspirate accurate amounts. For this reason, the rinsing fluid that fills the
autosampler interior must also be degassed.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
37/17236
LAAQ-B-LC001B 36
Column OvenColumn Oven
Air circulation heating type
Block heating type
Aluminum block heater
Insulated column jacket type
Water bath
In HPLC, particularly in the widely used techniques of reversed phase chromatography,
normal phase chromatography, and ion exchange chromatography, temperature control is
an extremely important consideration. For example, the following are generally observed if
the column temperature increases. (There are some exceptions.)
The retention time becomes shorter.
The load pressure decreases due to a decrease in the viscosity of the eluent. The number of theoretical plates improves due to an increase in the diffusion
coefficient.
For this reason, it can be said that within a range in which possible deterioration of the
column can be ignored, analysis is often performed at as high a temperature as possible
(40C to 60C).
A wide variety of heating mechanisms, such as air circulation heating and block heating, are
employed in column chambers. In general, air circulation heating is most commonly used,
although the use of block heating, which requires relatively little space, has increased in
recent years.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
38/17237
LAAQ-B-LC001B 37
Tubing and Preparation forTubing and Preparation for
Solvent DeliverySolvent Delivery
Prior to Analysis
When first setting up an HPLC system or when changing the flow-channel configuration in
accordance with the type of analysis, the units and columns must be connected with tubing.
When connecting the units with tubing, in order to prevent the spread of sample inside the
tubing, short tubes with narrow diameters must be used to the extent possible without
hampering execution of the experiment.
When the tubing is connected, rinsing fluid and eluent are delivered from the pump. At thistime, care is required to ensure that the flow channels are not blocked. Care is also required
regarding the purity of the solvents delivered.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
39/17238
LAAQ-B-LC001B 38
TubingTubing
Material
Stainless steel (SUS)
PEEK (polyether
ether ketone)
Fluororesin
O.D. (outer diameter)
1.6 mm
I.D. (inner diameter)
0.1 mm
0.3 mm
0.5 mm
0.8 mm etc.
Materials used for tubing include stainless steel (SUS316) and PEEK (polyether ether
ketone).
Stainless steel can withstand pressures of 100 MPa or more, making it particularly suitable
for tubing in places subject to high load pressures (e.g., flow channels upstream from the
separation column). A disadvantage is that it is prone to corrosion by acids or halogens.
PEEK is a type of engineering plastic, and despite being a resin, it can withstand pressuresof up to around 25 MPa. It can be used across the entire pH range (i.e., pH 1 to 14), but it
does not allow the use of organic solvents with a high solvency, such as chloroform and/or
THF.
Various types of fluororesin tubes are also used for tubing. In general, they have a high
resistance to organic solvents and are easy to handle but there seem to be many types that
have a relatively low pressure resistance. They are suitable for the tubing situated
downstream from the column and drain tubes.
The outer diameter of the tubing used for HPLC is 1.6 mm (1/16 inches). This seems to be
an accepted standard almost everywhere in the world.
The inner diameter of the tubing is determined in accordance with the purpose of use. Ingeneral, tubing with an inner diameter of 0.25 to 0.3 mm is used in standard HPLC.
Tubing with an inner diameter of 0.1 mm is used for semi-micro HPLC. Although resistance
occurs at the standard flow rate (approx. 1 mL/min), this property is utilized in resistance
tubes.
Tubing with an inner diameter of 0.5 mm or more is used in preparative LC. It is also used
for sample loops and post-column reaction tubes in cases where a significant degree of
volume is required.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
40/17239
LAAQ-B-LC001B 39
ConnectorsConnectors
Male nut (SUS)
Ferrule (SUS) Sealing possible up to 40
MPa
Male nut (PEEK) Can be connected without
any tools
Resists pressures of up to
approx. 25 MPa
Male nut
Ferrule
Male nut (PEEK)
The tubing is attached to connection ports using connectors.
A stainless steel connector consists of two parts: a ferrule and a male nut. When these are
threaded onto a stainless steel tube and the male nut is tightened, the ferrule is pressed into
the tube and thereby secured.
A spanner is required for tightening. Tighten the nut as far as possible by hand, and then
turn it about half a rotation using the spanner. (Tighten it approx. 45 from the point wherethe ferrule is secured.) Excessive tightening may result in damage to the thread of the nut.
Although stainless steel connectors have a high pressure resistance, they require a spanner,
and for the connection of columns that are frequently detached and reattached, something
easier to use is more suitable. The PEEK male nut was developed in response to this need.
No tools are required for this connector. It can withstand pressures of up to 25 MPa when
tightened with fingers. Also, because the ferrule is not secured to the tube, connection is
always in a position that suits the connection port.
Although the type of connector shown in the above diagram consists of a ferrule and nut
combined into one, types, like stainless steel connectors, that consist of separable ferrules
and nuts are also widely used.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
41/17240
LAAQ-B-LC001B 40
Dead VolumeDead Volume
(Extra(Extra--column volume)column volume)
Dead volume can cause peaks broadening.
Tube
Male nut Dead volumeDead volume
Excellent connection Poor connection
The volume of the space outside the column that has no direct relationship with separation is
called the dead volume. If the dead volume is large, it can cause peaks to spread.
Therefore, care is required to ensure that the dead volume is minimized, especially with
respect to parts of the flow channel that the sample passes through (i.e., between the
injector, column, and detector).
As much as possible, use injectors and detector flow cells of structures that have minimaldead volume. Also, for the tubing that connects these parts, use tubes that are as short and
have as small an inner diameter as possible without creating an undue level of resistance or
causing handling problems.
Care is also required when connecting the tubing. As shown in the above diagram, if the
tube is inserted to the inside end of the connection port, there is no problem. If, however, it is
not inserted to the end, dead volume is created. In this case, the peaks may be broadened
or they may have shoulders.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
42/17241
LAAQ-B-LC001B 41
Mobile PhaseMobile Phase
Water Ultrapure water can be
used with confidence.
Commercial distilled
water for HPLC is also
acceptable.
Organic Solvent HPLC-grade solvent can
be used with confidence.
Special-grade solvent is
acceptable depending on
the detection conditions.
Care is required regarding
solvents containing
stabilizers (e.g.,
tetrahydrofuran and
chloroform)
Sometimes, powdered cuttings and organic dirts are present on new tubing. After connecting
new tubing, be sure to rinse the flow channels. It is generally advisable to use alcohol-based
solvents (e.g., 2-propanol) for rinsing. It is not necessary to use solvents of a particularly
high purity for this purpose.
After rinsing the flow channels, prepare eluent and deliver it. For this, use solvent of as higha purity as possible.
The water prepared by an ultrapure water system can be used with confidence. In general,
however, this level of purity is not always necessary. Purified water that has undergone a
purification process consisting of at least two stages, such as reverse osmosis and ion
exchange or ion exchange and distillation, is usually acceptable. Of course, commercial
purified water specifically intended for HPLC may also be used.
HPLC-grade organic solvents can be used with confidence. In general, however, HPLC-
grade solvents are a little expensive, and depending on the analytical conditions, it may be
acceptable to use special-grade solvent.
Solvents such as tetrahydrofuran and chloroform contain additives, and this may cause
problems in detection or separation. Of course, solvents containing additives are morestable, so, in some cases, it may be better to use such solvents as long as there are no
problems with analysis. Therefore, decide whether or not to use solvents containing
additives in accordance with the specific details of the analytical process.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
43/17242
LAAQ-B-LC001B 42
Replacement ofReplacement ofEluentEluent
Mutually insoluble solvents
must not be exchanged
directly.
Aqueous solutions containing
salt and organic solvents
must not be exchanged
directly.
Water
Hexane
2-Propanol
Buffer solution
Water-soluble
organic solvent
Water
When replacing the solution in the flow channels, exercise care regarding the mutual
solubility of the pre- and post-replacement solutions.
When exchanging solvents that do not mix together, such as water and hexane, do not
exchange them directly. First replace one with a solvent that dissolves in both (e.g., 2-
propanol).
Some inorganic salts dissolve easily in water but do not dissolve easily in organic solvents.Therefore, when replacing a buffer solution with an organic solvent, first deliver water
through the flow channels to rid them of salt and organic solvent.
Also, care is required to ensure that none of the pre-replacement solution is mixed with the
post-replacement solution. Pour some of the solution about to be delivered into a small
beaker and rinse the suction filters and tubes in this solution before setting the solution vial.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
44/17243
LAAQ-B-LC001B 43
Mixing, Filtration, and OfflineMixing, Filtration, and Offline
Degassing of theDegassing of the EluentEluent
Decompression
by aspirator
Ultrasonic
cleaning unit
Decompression
by aspirator
Membrane filter with pore
size of approx. 0.45 m
After preparing the eluent, mix it well. This is to homogenize the solution and to prevent
problems related to bubbles occurring during delivery by expelling supersaturated dissolved
air.
Even if an online degasser is used, in some cases it is easy for bubbles to be produced
immediately after the start of delivery. In order to start delivery smoothly, it is recommendedthat a moderate amount of degassing is performed beforehand.
More specifically, connect the inlet of an aspirator to the mouth of the bottle and, while
applying ultrasonic waves to the solution, decompress the bottle. If an ultrasonic cleaning
unit is not available, perform decompression while agitating the solution intensely with a
magnetic stirrer.
This operation need not be performed for a long time. A few tens of seconds is sufficient. As
long as the production of bubbles at the start of delivery is prevented, the online degasser
will handle degassing in subsequent operation.
In the case of an eluent that contains a relatively high concentration of salt, such as a buffer
solution, it is recommended that the technique of filtration under reduced pressure is used.Use a membrane filter with a pore size of approx. 0.45 m.
Filtration under reduced pressure takes care of both filtration and degassing at the same
time.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
45/17244
LAAQ-B-LC001B 44
Reversed Phase ChromatographyReversed Phase Chromatography
Part 1Part 1
Basic Principles
A large number of separation modes are used in high performance liquid chromatography,
but the most widely used mode by far is reversed phase (distribution) chromatography. The
principle and characteristics of this separation mode are described here.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
46/17245
LAAQ-B-LC001B 45
Polarity of SubstancesPolarity of Substances
Polarity Property of a substance
whereby the positions of the
electrons give rise to
positive and negative poles
Water: Polar
Methane: Nonpolar
Miscibility of solvents Solvents of similar
polarities can be easily
dissolved together.
Polar and nonpolar
molecules have a similar
relationship to that ofwater
and oil.
O
H H
+
C
H H
H
H
WaterMethane Acetic acid
CCH
H
O
O
H
Two atoms share an electron cloud to form a covalent bond, and this whole structure
constitutes a molecule. However, even though the electron cloud is shared, it is not
necessarily evenly distributed between the bonded atoms, and the electrons may be located
more closely to the atom that exerts greater pull on them. Electrons are negatively charged,
so the atom to which the electrons are pulled becomes a negative pole, and the other atom
becomes a positive pole. This type of bonded state is described as polar.
The strength with which a bonded atom pulls electrons is called electronegativity.
Comparing the electronegativities of some commonly encountered atoms gives the following:
F > O > Cl, N > Br > C, H
If the center of the negative charge and the center of the positive charge in a molecule do not
coincide, that molecule is polar. Water molecules are typical polar molecules. In methane,
however, although there is polarity in the individual C-H bonds, overall the molecule has a
regular tetrahedral structure, so there is no polarity.
In general, it is said that substances that are either both polar or both nonpolar have a high
mutual solubility. On the other hand, polar and nonpolar substances have a low mutual
solubility.
Using water to represent polar solvents and oil to represent nonpolar solvents, the
relationship between a polar and nonpolar solvent can be likened to the relationship between
oil and water.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
47/17246
LAAQ-B-LC001B 46
NonpolarNonpolar(Hydrophobic) Functional Groups(Hydrophobic) Functional Groups
and Polar (Hydrophilic) Functional Groupsand Polar (Hydrophilic) Functional Groups
Nonpolar Functional
Groups
-(CH2)nCH3Alkyl groups
-C6H5 Phenyl groups
Polar Functional
Groups
-COOH
Carboxyl groups
-NH2Amino groups
-OH
Hydroxyl groups
In some cases, molecules with complex structures contain both nonpolar and polar parts.
The overall polarity of such a molecule is determined by the functional groups that are
bonded.
Representative examples of nonpolar functional groups include alkyl groups and phenyl
groups, which are composed entirely of weakly electronegative carbon and hydrogen atoms.
The longer the alkyl group chain, the lower the polarity.Polar functional groups include molecules composed of strongly electronegative halogen
and nitrogen atoms. Representative examples include carboxyl groups, amino groups, and
hydroxyl groups.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
48/17247
LAAQ-B-LC001B 47
Partition ChromatographyPartition Chromatography
A liquid (or a substance regarded as a
liquid) is used as the stationary phase,
and the solute is separated according to
whether it dissolves more readily in the
stationary or mobile phase.
Liquid-liquid chromatography
Depending on how readily a solute dissolves in two solvents that are not mutually soluble, a
difference may emerge between the concentration of solute in each solvent. The technique
of using this property to transfer a component dissolved in one solvent to another solvent, or
to concentrate or clean up the component, is called solvent extraction.
The type of chromatography that directly applies the principle of solvent extraction is called
partition chromatography. In partition chromatography, the stationary and mobile phasesare both thought of as liquids, and the strength of retention of a solute is determined
according to whether the solute dissolves more readily in the stationary or mobile phase. Of
course, the liquids used for the stationary and mobile phases must not be mutually soluble.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
49/17248
LAAQ-B-LC001B 48
Normal Phase / Reversed PhaseNormal Phase / Reversed Phase
Stationary
phaseMobile phase
Normal
phase
High polarity
(hydrophilic)
Low polarity
(hydrophobic)
Reversed
phase
Low polarity
(hydrophobic)
High polarity
(hydrophilic)
Partition chromatography can be performed in one of two modes: normal phase and
reversed phase. The combination of a stationary phase with a high polarity and a mobile
phase with a low polarity is called normal phase and the opposite combination is called
reversed phase.
Reversed phase chromatography is described here. The term reversed phase gives the
impression that this technique is somewhat unorthodox. In fact, most people that use HPLCperform separation with reversed phase chromatography, and it can fairly be described as a
standard separation mode.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
50/17249
LAAQ-B-LC001B 49
Reversed Phase ChromatographyReversed Phase Chromatography
Stationary phase: Low polarity
Octadecyl group-bonded silical gel (ODS)
Mobile phase: High polarity
Water, methanol, acetonitrile
Salt is sometimes added.
Stationary Phase
Compounds with a low polarity, such as those composed of aliphatic chains without
localized electrons, are used. However, to be used as packing material for HPLC,
the substance used must be chemically stable and capable of withstanding high
pressures, so it is not true to say that any substance with a low polarity is sufficient.
The most commonly used substance is produced by chemically bonding anoctadecyl group (-C18H37) to the surface of silica gel. This type of packing material
is commonly known as ODS, and an ODS column, into which ODS is packed, is
almost synonymous with a column for reversed phase chromatography.
Mobile Phase
The most commonly used solvents are water, methanol, and acetonitrile. Water is
the solvent with the highest polarity, and by mixing it with methanol or acetonitrile,
which have lower polarities, the overall polarity of the solution can be adjusted.
Salts and acids are also added sometimes in order, for example, to adjust the pH
value or form ion pairs.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
51/17250
LAAQ-B-LC001B 50
Separation Column for ReversedSeparation Column for Reversed
Phase ChromatographyPhase Chromatography
C18 (ODS) type
C8 (octyl) type
C4 (butyl) type
Phenyl type
TMS type
Cyano type
Si -O-Si
C18 (ODS)
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
In general, packing material produced by chemically bonding hydrophobic (low-polarity)
functional groups to a silica gel substrate is used as the stationary phase.
The most widespread of such packing materials is a type called ODS, which is formed by
bonding octadecyl groups (-C18H37) to the surface of silica gel. The structure of this material
is illustrated above.
In addition to ODS, packing materials produced by bonding octyl groups, which have a shortaliphatic chain, phenyl groups, and cyanopropyl groups are commercially available, and are
used in cases where a different separation selectivity from that of ODS is required. Also, the
support material is not limited to silica gel. For example, materials formed by bonding
octadecyl groups to the surface of a resin are also available.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
52/17251
LAAQ-B-LC001B 51
Effect of Chain Length ofEffect of Chain Length of
Stationary PhaseStationary Phase
C18 (ODS)
Strong
C8
C4
Medium
Weak
If a stationary phase produced by chemically bonding an aliphatic chain to silica gel is used,
the length of the aliphatic chain influences the retention strength for the solute.
It is said that, in general, longer chains have a greater retention strength. Beyond a certain
length, however, the retention strength does not change significantly.
To effect an overall increase or decrease in the speed with which a component is eluted,rather than replacing the stationary phase, changing the composition of the mobile phase, as
described later, is significantly simpler and cheaper. Therefore, as long as an ODS column
is used as the separation column, there is unlikely to be any problem deciding on the
separation conditions.
The analysis of a protein is an example of a situation necessitating the use of a stationary
phase produced by bonding octyl groups or groups with shorter aliphatic chains.
In general, proteins are denatured and precipitated in organic solvents, so there cannot be a
high concentration of organic solvent in the mobile phase. Therefore, a stationary phase
produced by bonding a short aliphatic chain is used, thereby decreasing the overall retention
strength, and the amount of organic solvent added to the mobile phase is decreased.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
53/17252
LAAQ-B-LC001B 52
Hydrophobic InteractionHydrophobic Interaction
H2O
H2OH2O
H2O
H2O H2O
H2O
Network of hydrogen bonds
H2O
H2OH2O
H2O
H2O H2O
H2O
Nonpolar solute
If a nonpolar
substance is added...
the network is broken and...
H2OH2O H2O
H2OH2O H2O
H2O
Nonpolar solute
Nonpolar stationary phase
the nonpolar substance
is pushed to a nonpolar
location.
Although reversed phase chromatography is regarded as a type of partition mode, it is said
that the retention mechanism is difficult to explain in terms of partition. On the other hand,
because the force that acts between the nonpolar solute and the nonpolar stationary phase
is only a weak dispersion force (van der Waals force), it is impossible to explain the
mechanism simply in terms of the theory of adsorption.
Therefore, the concept of hydrophobic interaction is used as a model to explain theretention mechanism of reversed phase chromatography.
The polar mobile phase molecules are formed by a network of hydrogen bonds. Although
polar and ionic solutes can participate in this network, a nonpolar solute cannot form
hydrogen bonds easily. So, in order to dissolve, it must break the network, consequently
creating an energy imbalance.
A simple way for the entire solution to regain a stable energy balance would be to push out
the nonpolar solute. If the solution is in contact with a nonpolar stationary phase, pushing the
solute toward this stationary phase would reduce the number of breaks in the network, and
improve the energy balance.
The retention mechanism of reversed phase chromatography, then, can be understood byconsidering a model in which the solute is repelled by the mobile phase and pushed onto the
stationary phase, rather than one in which the solute and stationary phase are positively
attracting each other.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
54/17253
LAAQ-B-LC001B 53
Relationship Between RetentionRelationship Between Retention
Time and PolarityTime and Polarity
C18 (ODS)
CH3
StrongStrongWeakWeak
OH
In reversed phase chromatography, strongly hydrophobic substances (i.e., substances with
a relatively low polarity) are strongly retained by the stationary phase, and therefore have
relatively long retention times. Therefore, in a chromatogram containing multiple peaks, the
substances are eluted, broadly speaking, in descending order of polarity.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
55/17254
LAAQ-B-LC001B 54
Basic Settings forBasic Settings forEluentEluent Used inUsed in
Reversed Phase ModeReversed Phase Mode
Water (buffer solution) + water-soluble organic
solvent
Water-soluble organic solvent: Methanol
Acetonitrile
Tetrahydrofuran etc.
The mixing ratio of the water (buffer solution) and
organic solvent has the greatest influence on
separation.
If a buffer solution is used, its pH value is an
important separation parameter.
In general, a solution of the following composition is used as the eluent in reversed phase
mode:
Water (buffer solution) + water-soluble organic solvent
In many cases, separation adjustment is performed by changing the composition of this
eluent. In gas chromatography, the composition of the carrier gas, which acts as the mobile
phase, is hardly ever changed. In liquid chromatography, however, the composition of the
mobile phase is a key aspect of separation adjustment.
The most commonly used water-soluble organic solvents are methanol and acetonitrile.
Other solvents, such as tetrahydrofuran (THF) are also used.
The factor that has the greatest influence over the retention and separation of the solute is
the ratio with which the water (buffer solution) and water-soluble organic solvent are mixed.
In many cases, the mixing ratio of the organic solvent with respect to water has a greater
influence on solute retention than the type of organic solvent used.
Instead of just using water, sometimes salt or another substance is added in order to create
a pH buffer solution. In this case, the pH has a great influence over separation. Although the
type of buffer salt used and its concentration influence separation, it is the pH that needs
foremost consideration.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
56/17255
LAAQ-B-LC001B 55
Difference in Solute Retention StrengthsDifference in Solute Retention Strengths
for Water and Waterfor Water and Water--Soluble OrganicSoluble Organic
SolventsSolvents
H2O
H2OH2O
H2O
H2O H2O
H2O
Tightly packed network
CH3OH
Nonpolar solute
Nonpolar solute
Nonpolar stationary phase
Loose network
CH3OHCH3OH
CH3OHCH3OH
CH3OH
CH3OH
Let us review some of the points made about hydrophobic interaction.
Polar mobile phase molecules are formed by a network of hydrogen bonds. If a nonpolar
solute enters this network, hydrogen bonds are broken, and this creates an energy
imbalance. In order to minimize this imbalance, the solute is pushed onto the nonpolar
stationary phase. This is the basic principle behind solute retention due to hydrophobic
interaction.Because water has a very high polarity, its network of hydrogen bonds is believed to be
extremely tightly packed. Solvents such as methanol and acetonitrile, however, despite
having some level of polarity, are not as polar as water, so their hydrogen bonds are
believed to be much weaker. In solvents that form loose networks like this, the force with
which a nonpolar solute is pushed onto the nonpolar stationary phase is not that strong.
The above gives rise to the following basic rule concerning reversed phase mode:
The greater the proportion of water in the eluent, the greater the solute retention strength.
Or
The greater the polarity of the eluent, the greater the solute retention strength.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
57/17256
LAAQ-B-LC001B 56
Relationship between Polarity ofRelationship between Polarity ofEluentEluent andand
Retention Time in Reversed Phase ModeRetention Time in Reversed Phase Mode
60/40
Eluent: Methanol / Water
80/20
70/30
In practice, using single solvents such as water or methanol as the eluent is quite rare.
Usually, mixtures of these solvents are used. This makes it possible to control the overall
solute retention strength.
The above diagram illustrates how differences in the eluent affect the chromatogram. As the
polarity of the eluent decreases (i.e., as the proportion of methanol increases), the overallretention time decreases.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
58/17257
LAAQ-B-LC001B 57
Chromatogram ParametersChromatogram Parameters
Methods for Expressing Separation
and Column Performance
The parameters that can be obtained from chromatograms are explained here.
Retention Factor, k
Sometimes called the capacity factor or the capacity ratio, this parameter
expresses the solute retention strength of the stationary phase.
Theoretical Plate Number, N
This parameter is an indicator of the performance of the separation column.
Separation Factor, a
This parameter is equal to the ratio of the retention factors for two peaks.
Resolution, RS
This parameter expresses the degree of separation between two peaks.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
59/17258
LAAQ-B-LC001B 58
Retention Factor,Retention Factor, kk
tR
t0
Strengthofdetectorsignal
Time
tR: Retention time
t0: Non-retention time
0
0R
t
ttk
=
If we hypothesize that the solute does not interact with the stationary phase at all, and
remains within the eluent the whole time, then the corresponding peak would appear at t0.
This means that the time obtained by subtracting t0 from the retention time, tR, can be
regarded as time for which the solute stayed in the stationary phase.
If a solute remains in the stationary phase for a relatively long time, it indicates that the
retention strength for that solute is relatively high. It is therefore possible to express the
strength with which a solute is retained by calculating the ratio of times that the solute
remains in the stationary and mobile phases. This is called the retention factor.
If the retention factor (k) is 1, it indicates that the solute remains in the stationary and mobile
phases for the same time. If it is less than 1, it indicates that the solute is not retained to a
significant degree before elution. If it is 3 or greater, it indicates that the solute undergoes
significant interaction with the stationary phase before elution.
One problem is the calculation of t0.
In theory, the volume of the eluent inside the column can be calculated by multiplying the
internal volume of the column (i.e., the volume of the cylinder) by the porosity of the packing
material. Dividing this by the eluent flow rate gives t0. For example, if the inner diameter and
length of the column are 0.46 cm and 15 cm respectively, the porosity is 0.6, and the eluentflow rate is 0.8 mL/min, then t0 can be calculated as follows:
t0 = (0.232 15) 0.6 0.8 = 1.87 [min]
In practice, however, because the porosity inside the column is hardly ever known, and the
volume of other parts, such as tubing, affects the calculation, it is difficult to obtain an
accurate value for t0.
A working value can be obtained, however, by actually measuring the retention time for a
solute that is known not to be retained by the stationary phase. With the reversed phase
mode, substances such as nitrite ion and urea are often used.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
60/17259
One theoretical way of handling chromatography is the plate theory model. This is based
on the concept of handling the process of chromatography as repeated solvent extraction in
a flask.
The solute that enters the separation site is partitioned between the stationary and mobile
phases according to a specific ratio. The mobile phase moves, so the solute partitioned in
the mobile phase also moves, and is partitioned again. As this behavior is repeated againand again, substances with different partition coefficients are separated in a way that can be
thought of as repeated solvent extraction performed to increase the degree of refinement.
In this model, if one occurrence of solvent extraction is denoted as one plate, then the
theoretical plate number is the number of plates corresponding to the extraction performed
by the separation column. If the theoretical plate number is large, this means that extraction
is performed a correspondingly large number of times, and indicates a relatively high level of
separation performance.
Although the formulas defining the theoretical plate number are given above, the reason why
these formulas are used is not given here. (In fact, textbooks on the fundamentals and
practical application of HPLC usually do not give the derivation of these formulas.) For more
details, refer to specialized literature on the subject. For the purposes of this text, remember
that the theoretical plate number is an indicator of the efficiency (performance) of the
separation column.
LAAQ-B-LC001B 59
Theoretical Plate Number,Theoretical Plate Number, NN
W
W1/2H1/2
H
2
.21
R
R
/
R
W
t
W
2
2
2
545
16
=
=
=
Area
Ht
tN
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
61/17260
In one of the formulas given for the theoretical plate number, the retention time appears in
the numerator and the peak width appears in the denominator. This shows that these
quantities are important factors in the evaluation of column performance.
If peaks are sharp, they can be completely separated from nearby peaks. Therefore, a high-
performance column can be thought of as one that gives small peak widths.The sample band is diffused inside the column, so peaks with a short retention time are
relatively sharp, whereas peaks with a long retention time are relatively broad (in the case of
an isocratic system). Therefore, if two columns give the same peak width for a given solute,
the column that gives a longer retention time can be evaluated as having a higher lever of
performance.
If a separation column is used repeatedly, the peaks gradually become broader and the
retention times gradually become shorter. In other words, the column performance
deteriorates. Appropriate management can be performed by regularly obtaining the
theoretical plate number.
There is no specific value, however, for the theoretical plate number below which the columnmust be replaced. Each case must be evaluated independently according to whether or not
the desired separation can be achieved and whether or not the decreases in sensitivity and
area reproducibility caused by the broadening of peaks are within acceptable limits.
LAAQ-B-LC001B 60
Evaluation of Column Efficiency Based onEvaluation of Column Efficiency Based on
Theoretical Plate NumberTheoretical Plate Number
If the retention times are
the same, the peak width
is smaller for the one with
the larger theoretical plate
number.
If the peak width is the
same, the retention time is
longer for the one with the
larger theoretical plate
number.
N: Large
N: Small
N: SmallN: Large
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
62/17261
LAAQ-B-LC001B 61
Separation Factor,Separation Factor, aa
Separation factor: Ratio ofks of two peaks
)( 12
1
2
kk
k
k
>
=k1 k2
The separation factor for two peaks is the ratio of their retention factors. The relationship
between the elution positions of two peaks is expressed using this parameter.
It can also be said that the separation factor expresses the separation selectivity. This is
because the size of indicates whether the two peaks are in closely neighboring positions
or separated positions.
The separation factor only expresses the positional relationship between two peaks. It
provides no information about peak separation (i.e., the degree of overlap). Even if the
separation factor is large, if the peaks are broad, they may not be well separated. Even if the
separation factor is small, if the theoretical plate number for the column is high and the
peaks are sharp, they may be sufficiently separated.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
63/17262
LAAQ-B-LC001B 62
Resolution,Resolution, RRSS
2,2/11,2/1
RR
21
RR
S
12
12
18.1
)(2
1
hh WW
tt
WW
ttR
+
=
+
=
tR1 tR2
W1 W2
W1/2h,1 W1/2h,2 h1/2
The resolution indicates the extent to which two peaks are separated or, from a different
perspective, the extent to which they overlap.
While the separation factor indicates only the positional relationship between two peaks, and
does not indicate the degree of overlap, the resolution does, to a certain extent, indicate the
degree of separation.
The formulas used to obtain the resolution are given above.
It can be seen that this parameter is equal to the ratio of the difference between the retention
times of the two peaks and the average value of the two peak widths. If the distance between
the peaks is large compared to the peak widths, they are well separated, whereas if the
opposite is true, they are overlapping.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
64/17263
LAAQ-B-LC001B 63
Resolution Required for CompleteResolution Required for Complete
SeparationSeparation
If the peaks are isosceles triangles,
they are completely separated.
tR2 -tR1 = W1 = W2
RS = 1
(tR2 -tR1)
W1 W2 W1 W2
If the peaks are Gaussian distributions,
RS > 1.5 is necessary for complete separation.
tR2 -tR1 = W1 = W2
RS = 1
(tR2 -tR1)
What level of resolution is required for two peaks to be completely separated?
Let us suppose that the two peaks are isosceles triangles. If the two triangles are standing
alongside each other with their bases making contact, the difference between the retention
times and the average value of the peak widths are equal, and the resolution equals 1. This
means that if the resolution is greater than 1, the bases of the two triangles do not make
contact, and complete separation is attained.This does not apply, however, to peaks shaped like Gaussian distributions. At a resolution of
1, the peak skirts overlap. To be able to say that complete separation is attained, a resolution
of at least 1.5 is probably required.
The above reasoning is based on the assumption that the two peaks are almost the same
height. If the heights are different, sufficient separation may not be attained at the levels of
resolution specified above. In particular, the smaller peak may be partially covered by the
larger peak, making it impossible to identify the peak top.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
65/17264
LAAQ-B-LC001B 64
Relationship Between ResolutionRelationship Between Resolution
and Other Parametersand Other Parameters
The resolution is a
function of the
separation factor, the
theoretical plate
number, and the
retention factor.
The separation can be
improved by improving
these 3 parameters!
+
=
+
=
1
1
4
1
)(2
1
2
2
21
1R2RS
k
kN
WW
ttR
By manipulating the formula previously given for the resolution in the way shown above, it
can be demonstrated that this parameter is a function of the theoretical plate number (N),
the separation factor (), and the capacity factor (k).
The above formula indicates that an increase in the theoretical plate number, an increase in
the separation factor, or an increase in the retention factor translates to an increase in the
resolution. Therefore, the separation can be improved by improving these 3 parameters.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
66/172
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
67/172
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
68/17267
LAAQ-B-LC001B 67
To Improve Separation...To Improve Separation...
kincreased
Nincreased
increased
Before
adjustment
Eluent replaced with one
of lower elution strength.
Column replaced with one of
superior performance.
Column lengthened.
Column (packing material) replaced.Eluent composition changed.
Column temperature changed.
The specific measures that can be used to improve the resolution can be summarized as
follows:
Increase the Capacity Factor (k)
Change the eluent composition so that elution is generally slower. In reversed
phase mode, reduce the proportion of organic solvent in the eluent.
There are also methods that involve changing the stationary phase. For example, in
ion exchange mode, replace the column with one filled with packing material that
has a large exchange capacity.
Increase the Theoretical Plate Number (N)
In general, replace the column with one of superior performance. For example, use
a column filled with packing material of a smaller pore size. It is also effective to
either use a longer column or multiple connected columns.
In some cases, the theoretical plate number can be improved by increasing the
column temperature or changing the solvent composition so that eluent viscositydecreases.
Increase the Separation Factor ( )
Change separation conditions such as the column used, the eluent composition,
and the temperature.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
69/17268
LAAQ-B-LC001B 68
pH Buffer Solution Used for EluentpH Buffer Solution Used for Eluent
Selection and Preparation of
Buffer Solution
Sometimes, for reasons related to separation or detection, a pH buffer solution must be used
as the eluent. Here, the basic concepts behind the pH buffer solution used for the eluent are
explained.
8/13/2019 What is HPLC Basic Overview_HPLC_PM_BF
70/17269
LAAQ-B-LC001B 69
Acid Dissociation EquilibriumAcid Dissociation Equilibrium
HA A- H++
H+
OH-
If an acid is added...
...the equilibrium shifts to
the left to offset the
increase in H+.
the equilibrium shifts
to the right to offset the
decrease in H+.
If an alkali is
added...
The equilibrium always shiftsThe equilibrium always shifts
in a way that offsets changes.in a way that offsets changes.
A typical type of acid dissociation equilibrium is shown above.
Let us suppose that an acid, HA, which is in an undissociated state, and its A ions, which
are in a dissociated state, are in equilibrium at a certain ratio. If a small quantity of another
acid or an alkali is added to this solution, although the H+ concentration temporarily
increases or decreases, the above equilibrium shifts in a way that offsets this change, so the
H+ concentration does not change significantly. A solution like this, whose pH value onlychanges slightly when a small quantity of an acid or alkali is added, is called a pH buffer
solution.
The pH buffering power is first exhibited when HA and A are in a complementary state. If
the equilibrium shifts greatly to the left or right, there is unlike