What is HPLC Basic Overview_HPLC_PM_BF

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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.


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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.


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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


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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.


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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.


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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.


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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.


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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


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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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?


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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


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


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.


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.


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.


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.


66/172


67/172


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.


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.


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

What is HPLC Basic Overview_HPLC_PM_BF

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