What is HPLC Basic Overview_HPLC_PM_BF

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LAAQ-B-LC001B 1

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 components contained in mixtures.

An overview of HPLC, from the basic principles of chromatography to the characteristics of HPLC itself, is presented here.

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LAAQ-B-LC001B 2

Invention of Chromatography by

M.

M. Tswett

Tswett

Ether CaCO3 Chlorophyll Chromato Chromato Chromato Chromato

graphy

Colors

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

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LAAQ-B-LC001B 3

Comparing Chromatography to the

Flow of a River...

Base

Water flow

Light 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 by the 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

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LAAQ-B-LC001B 4

Mobile 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.

→

Separation

occurs due to differences in the speed of motion.

Strong Weak Mobile Mobile phase phase Stationary Stationary phase phase

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 water corresponds 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.

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LAAQ-B-LC001B 5

Chromato

-

graphy

/

-

graph /

-

gram /

--

grapher



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).

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LAAQ-B-LC001B 6

Three States of Matter and

Chromatography Types

Mobile phase

Gas Liquid Solid

Stationary phase Gas Liquid Solid Gas Gas chromatography chromatography Liquid Liquid chromatography chromatography

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 be used.

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.

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LAAQ-B-LC001B 7

Liquid Chromatography



Chromatography in which the mobile phase

is a

liquid

.



The liquid used as the mobile phase is

called the

“eluent”

.



The stationary phase is usually a solid or a

liquid.



In general, it is possible to analyze any

substance 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

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LAAQ-B-LC001B 8

Interaction Between Solutes, Stationary

Phase, and Mobile Phase



Differences in the interactions between the solutes and

stationary and mobile phases enable separation.

Solute

Stationary

phase

Mobile 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

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LAAQ-B-LC001B 9

Column Chromatography and

Planar 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, capillary chromatography 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.

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LAAQ-B-LC001B 10

Separation Process and Chromatogram

for Column Chromatography

O u t p u t c o n c e n t r a t i o n Time

Chromatogram

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 descend slowly, 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”.

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LAAQ-B-LC001B 11

Chromatogram

t

_R_R

t

00 I n t e n s i t y o f d e t e c t o r s i g n a l Time

Peak

t

RR

: Retention time

h

A

t

00

_{: 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, tRand t0are sometimes multiplied by the eluent flow rate and handled as volumes. The volume corresponding 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

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LAAQ-B-LC001B 12

From Liquid Chromatography to High

Performance Liquid Chromatography



Higher degree of separation!

→

Refinement of packing material (3 to 10 µm)



Reduction of analysis time!

→

Delivery of eluent by pump

→

Demand for special equipment that can

withstand high pressures

The arrival of

high 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 to increase. 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.

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LAAQ-B-LC001B 13

Pump

Sample injection unit (injector) Column Column oven (thermostatic column chamber) Detector Eluent (mobile phase) Drain Data processor Degasser

Flow Channel Diagram for High

Performance Liquid Chromatograph

A high performance liquid chromatograph differs from a column chromatograph in that it is subject to the following performance requirements.

Solvent Delivery Pump

A solvent delivery pump that can maintain a constant, non-pulsating flow of solvent at a high pressure against the resistance of the column is required.

Sample Injection Unit

There is a high level of pressure between the pump and the column; a device that can inject specific amounts of sample under such conditions is required. Column

The technology for filling the column evenly with refined packing material is required. Also, a material that can withstand high pressures, such as stainless steel, is required for the housing.

Detector

Higher degrees of separation have increased the need for high-sensitivity detection, and levels of sensitivity and stability that can respond to this need are required in the detector.

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LAAQ-B-LC001B 14

Advantages of High Performance

Liquid 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 of

samples

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 of reproducibility 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 µg and pg levels is usually possible and, in some cases, even smaller quantities can be

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LAAQ-B-LC001B 15

Fields in Which High Performance

Liquid 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. 

Environmental

samples

Inorganic ions Hazardous organic substances, etc. 

Organic industrial

products

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 is particularly high, it is also quite high for drug discovery and R&D departments.

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LAAQ-B-LC001B 16

HPLC 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.

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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 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 are essential. 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 a constant, 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.

Detector

This 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,

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LAAQ-B-LC001B 18

Solvent 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 srcinating 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.

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LAAQ-B-LC001B 19

Solvent Delivery Pump:

Representative 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 by microcomputers 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.

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LAAQ-B-LC001B 20

Solvent Delivery Pump:

Schematic Diagram of Plunger Pump

Motor and cam

Plunger Plunger seal Check valves Pump head

10 -100µL

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.

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LAAQ-B-LC001B 21

Solvent Delivery Pump:

Single 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.

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LAAQ-B-LC001B 22

Solvent Delivery Pump:

Dual 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 180º phase difference so that they perform aspiration and discharge alternately.

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LAAQ-B-LC001B 23

Gradient 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”.

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LAAQ-B-LC001B 24

Aim of Gradient System (1)



In isocratic mode

Long analysis time!!

Poor

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?

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LAAQ-B-LC001B 25

Aim of Gradient System (2)



If the eluent composition is changed gradually during

analysis...

95% 30% C o n c e n t r a t i o n o f m e t h a n o l i n e l u e n t

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.

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LAAQ-B-LC001B 26

High

-

/ Low

-

Pressure 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-high-pressure gradient. The terms “high high-pressure” and “low high-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.

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LAAQ-B-LC001B 27

Advantages and Disadvantages of

High

-

/ Low

-

Pressure 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 of solutions 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,

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LAAQ-B-LC001B 28

Degasser



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 srcinating 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 delivery may 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.

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LAAQ-B-LC001B 29

Online Degasser

Gas-liquid separation membrane method Helium purge method

Helium cylinder To draft To pump Eluent container Regulator Drain valve To pump Eluent container

Polymeric film tubeVacuum chamber

An online degasser is used to degas the eluent continuously, even during analysis. It is not absolutely essential for HPLC, but it is required to fully attain the basic performance specifications of an HPLC system.

There are two types of online degasser: those that use the helium purge method and those that use the gas-liquid separation membrane method.

Helium Purge Method

With this method, helium gas is bubbled into the eluent, and air is thereby replaced with helium. The solubility of helium in liquids is generally low, and does not cause the formation of bubbles when water and organic solvents are mixed.

The greatest advantage of this method is that, in comparison with other methods, it has a large degassing effect. It does, however, have some disadvantages. For example, a helium gas cylinder is required, and when using mixtures of solvents, highly volatile solvents may vaporize, causing a change in the composition. Gas-Liquid Separation Membrane Method (Vacuum Degassing Method)

With this method, the eluent is passed through a tube made of polymeric film, and the outside of the tube is decompressed (i.e., in a vacuum). Because the tube is made of a material that allows the permeation of gases but not liquids, by the time the eluent reaches the tube outlet, some degree of degassing has been achieved. All this method requires is the electricity to run a vacuum pump. Hardly any

maintenance is necessary. For this reason, it is currently the most commonly used method. One disadvantage of this method, though, is that sufficient degassing may not be achieved for high eluent flow rates.

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LAAQ-B-LC001B 30

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.

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LAAQ-B-LC001B 31

Manual Injector

INJECT position

LOAD 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.

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LAAQ-B-LC001B 32

Manual Injector:

Operating Principle of Sample Injection

LOAD

INJECT

To column From pump To column From pump L o o p L o o p

The injection principle is as follows:

First, the sample is injected in the LOAD state with the microsyringe. The injected sample solution is stored in the sample loop.

Next, the knob is turned to switch to the INJECT state. This causes the eluent delivered from the pump to flow through the loop, thereby conveying the sample to the column.

The injection procedure can be summarized as follows: 1. Insert the microsyringe in the INJECT position. 2. Turn the knob to switch to the LOAD position. 3. Push the microsyringe plunger to inject the sample. 4. Turn the knob to switch to the INJECT position.

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LAAQ-B-LC001B 33

Manual Injector:

Injection 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 microsyringe’s measuring capacity. A disadvantage is that it is easy for inconsistencies in the volume to occur due to variations 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 disadvantage is 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.

(35)

LAAQ-B-LC001B 34

Autosampler

(Pressure Injection Method)

To column

From pump From pump To column

Sample Loop

LOAD

INJECT

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 is switched 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.

(36)

LAAQ-B-LC001B 35

Autosampler

(Total

-

Volume Injection Method)

From pump From pump To column

Sample vial Needle

Measuring pump

To column

LOAD

INJECT

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 is unlikely 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.

(37)

LAAQ-B-LC001B 36

Column Oven



Air circulation heating type



Block heating type



A

luminum 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 (40

°

C to 60

°

C).

(38)

LAAQ-B-LC001B 37

Tubing and Preparation for

Solvent 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 this time, care is required to ensure that the flow channels are not blocked. Care is also required regarding the purity of the solvents delivered.

(39)

LAAQ-B-LC001B 38

Tubing



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 pressures of 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.

(40)

LAAQ-B-LC001B 39

Connectors



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 where the 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.

(41)

LAAQ-B-LC001B 40

Dead Volume

(Extra

-

column volume)



Dead volume can cause peaks broadening.

Tube

Male nut

_{Dead 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 minimal dead 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.

(42)

LAAQ-B-LC001B 41

Mobile 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 high a 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.

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 more stable, 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.

(43)

LAAQ-B-LC001B 42

Replacement of

Eluent

 Mutually insolublesolvents 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.

(44)

LAAQ-B-LC001B 43

Mixing, Filtration, and Offline

Degassing of the

Eluent

Decompression by aspirator Ultrasonic cleaning unit Decompression by aspirator Membrane filter with pore

size of approx. 0.45 µm

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 recommended that 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 µm.

Filtration under reduced pressure takes care of both filtration and degassing at the same time.

(45)

LAAQ-B-LC001B 44

Reversed Phase Chromatography

Part 1

Part 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.

(46)

LAAQ-B-LC001B 45

Polarity 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 of water and oil.

O

H

– +

C

H

H H

H

Water

Methane Acetic acid

C

H

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.

(47)

LAAQ-B-LC001B 46

Nonpolar

(Hydrophobic) Functional Groups

and Polar (Hydrophilic) Functional Groups



Nonpolar Functional

Groups



-(CH

2

)

n

CH

3  Alkyl groups 

-C

6

H

5 Phenyl groups 

Polar Functional

Groups



-COOH

Carboxyl groups 

-NH

2  Amino 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.

(48)

LAAQ-B-LC001B 47

Partition 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 phases are 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.

(49)

LAAQ-B-LC001B 48

Normal Phase / Reversed Phase

Stationary

phase

Mobile 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 HPLC perform separation with reversed phase chromatography, and it can fairly be described as a standard separation mode.

(50)

LAAQ-B-LC001B 49

Reversed 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 an octadecyl 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.

(51)

LAAQ-B-LC001B 50

Separation Column for Reversed

Phase Chromatography



C

18

(ODS) type



C

8

(octyl) type



C

4

(butyl) type



Phenyl type



TMS type



Cyano type

Si

-O-Si-O-Si

C

₁₈

(ODS)

CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH22 CH CH33

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 short aliphatic 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.

(52)

LAAQ-B-LC001B 51

Effect of Chain Length of

Stationary Phase

C

18

(ODS)

Strong

C

8

C

4

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.

(53)

LAAQ-B-LC001B 52

Hydrophobic Interaction

H

22

O

H

22

O

H

22

O

H

22

O

H

22

O

H

22

O

H

22

O

Network of hydrogen bonds

H

22

O

H

22

O

H

22

O

H

22

O

H

22

O

_H

2 2

O

H

22

O

Nonpolar solute If a nonpolar substance is added...

…the network is broken and...

H

22

O

H

22

O

H

₂₂

O

H

22

O

H

22

O

H

22

O

H

22

O

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 the retention 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.

(54)

LAAQ-B-LC001B 53

Relationship Between Retention

Time and Polarity

C

18

(ODS)

CH

33

Strong

Weak

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.

(55)

LAAQ-B-LC001B 54

Basic Settings for

Eluent

Used in

Reversed 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.

(56)

LAAQ-B-LC001B 55

Difference in Solute Retention Strengths

for Water and Water

-

Soluble Organic

Solvents

H

22

O

H

22

O

_H

2 2

O

H

22

O

H

22

O

_H

2 2

O

H

22

O

Tightly packed network

CH

33

OH

Nonpolar solute Nonpolar solute

Nonpolar stationary phase

Loose network

CH

33

OH

CH

33

OH

CH

33

OH

CH

33

OH

CH

33

OH

CH

33

OH

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

(57)

LAAQ-B-LC001B 56

Relationship between Polarity of

Eluent

and

Retention 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 overall retention time decreases.

(58)

LAAQ-B-LC001B 57

Chromatogram 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,R S