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Rezaul Karim

Environmental Science and Technology

Jessore University of Science and Technology

Chapter 7 High performance liquid chromatography

Instrumental Technique for Environmental Analysis

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Chapter content

Liquid Chromatography (LC);

Scope;

Instrumentation;

Applications

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Reference

Daniel C. Harris , 2010, QuantitativeChemical Analysis, 8th edition, W. H.

Freeman and Company , Madison Avenue

New York, NY 10010 S. Ahuja and N. Jespersen (Eds), 2006,

Comprehensive Analytical Chemistry,

Volume 47, Elsevier B.V. Robinson, 1995. Undergraduate instrumental

analysis, Marcel Dekker, Inc. NY, USA.

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High performance liquid

chromatography High performance liquid chromatography is basically

a highly improved form of column chromatography.

Instead of a solvent being allowed to drip through acolumn under gravity, it is forced through underhigh pressures of up to 400 atmospheres. Thatmakes it much faster.

It also allows us to use a very much smallerparticle size for the column packing materialwhich gives a much greater surface area for

interactions between the stationary phase and themolecules flowing past it.

This allows a much better separation of thecomponents of the mixture.

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RANGE OF APPLICATIONS

It is often suitable for organic compounds that are too unstableor insufficiently volatile to be amenable to gaschromatographyanalysis without prior derivatisation.

It is suited for the separation of a wide range of chemicals,including pharmaceuticals, foods, heavy industrials andbiochemicals.

is likely to provide a set of chromatographic conditions foralmost any type of compounds.

Alteration of these conditions to suit the matrix involvedmight be the only required step for the food scientist to performa HPLC analysis of a new compound.

In food science, HPLC has been applied to several categories ofsubstances: carbohydrates, lipids, vitamins, additives, synthetic

colourings, natural pigments, contaminants (degradationproducts, pesticides, or naturally occurring substances), as

well as amino acids and others

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Scope of HPLC

LC is the most widely used of all of the analytical separationtechniques.

The reasons for the popularity of the method are its sensitivity;

its ready adaptability to accurate quantitative

determinations; its ease of automation;

its suitability for separating non-volatile species orthermally fragile ones, and

above all, its widespread applicability to substances that areimportant to industry, to many fields of science, and to the public.

Examples of such materials include amino acids, proteins, nucleicacids, hydrocarbons, carbohydrates, drugs, terpenoids, pesticides,antibiotics, steroids, metal-organic species, and a variety ofinorganic substances.

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Basic Principles

HPLC is a form of liquid chromatography,where separation (or partition) occursbetween a mobile phase (the solvent)and a stationary phase (the column

packing). It is the ability with which the sample

constituents will distribute themselvesbetween the two phases that will effect the

separation. Depending on the nature of the stationary

phase, the separation process can be of fourdifferent modes:

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In several basic type of chromatography, themobile phase is a liquid.

These phase type often classified byseparation mechanism or by the type of

stationary phase. The varieties include:

Partition chromatography

adsorption or liquid-solid chromatography

Ion exchange or ion chromatography Size exclusive chromatography

Affinity chromatography and

chiral chromatography.

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adsorption chromatography, where theseparation is based upon repeated

adsorption-desorption steps; partition chromatography where the

separation is based on partition between themobile and the stationary phase;

ion-exchange chromatography where thestationary phase is made up of an ionicsurface of opposite charge to that of thesample; and

size exclusion chromatography wherethe sample is separated according to itsmolecular size through a column filled with amaterial having precisely controlled pore size.

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two modes of action, depending on

the polarity of the two phases Normal phase chromatography, where the

stationary phase is polar in nature (e.g., silica oralumina) and the mobile phase is non polar (e.g.,hexane). In this mode, polar samples are retained

more strongly by the column therefore allowingelution of non polar compounds first.

Reversed-phase chromatography, where thestationary phase is non-polar in nature (e.g.,

hydrocarbon) and the elution solvent (or mobile phase) ispolar (e.g., water or methanol). This being the exactreverse of normal phase chromatography, non-polarcompounds will be retained longer on the column.

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THE MOBILE PHASE- The

solvent The choice of the solvent will depend onthe nature of the

operation mode, i.e., isocratic or gradient elution(and, ofcourse, on the solubility of the sample in the chosen elutionmedium).

The polarity for such an elution medium can, therefore, varyfrom buffered aqueous solutions to hydrocarbons.

The chosen medium (including water) needs to always be verypure.

HPLC grade solvents are commercially available and shouldalways be used.

The choice of a gradient eluent is always done by trial and error,

usually starting with a single solvent and increasing theconcentration of the second mobile phase component by usuallyusing an initial mixing rate of 2% per minute to achieve thedesired conditions in the mobile phase.

In some circumstances, up to 3 different solvents can be used.

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The column and the solvent

Normal phase HPLC This is essentially just the same as you will already have read about in

thin layer chromatography or column chromatography. Although it isdescribed as "normal", it isn't the most commonly used form of HPLC.

The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for example.

A typical column has an internal diameter of 4.6 mm (and may be lessthan that), and a length of 150 to 250 mm.

Reversed phase HPLC

In this case, the column size is the same, but the silica is modified tomake it non-polar by attaching long hydrocarbon chains to its surface -typically with either 8 or 18 carbon atoms in them.

A polar solvent is used - for example, a mixture of water and analcohol such as methanol.

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INSTRUMENTATION

These are the reservoir(s) for the mobile phase(s),

the pump(s),

a solvent gradient and solvent flow programmer,

a sample inlet fitted with an appropriate filter and aloop,

a column, sometimes a column oven,

a detector,

a recorder, and almost invariably, an integrator.

Automatic injectors, sample carousels andcollectors are also commonly used.

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Looking at the whole process

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The Injector

A few methods are used to inject the sampleonto the column, the simplest one being adirect injection with a micro-syringe.

But most systems will use sampling devices.

The most common way of injecting the sampleon the column is an injection valve in which thesample is injected into a holding loop.

Loops are designed to inject a specific volumeonto the column, usually of the order of 10 to20 pL for an analytical column.

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The Stationary Phase - The

column Two of the most important steps in the

development of an analytical method are theselection of the separation mode and theappropriate column packing.

The columns most commonly encountered have aninternal diameter (i.d.) of 4.5 to 5 mm and are 10 to25 cm in length; they are packed with stationaryphases having 5 to 10 Tm in diameter.

Usually made of stainless steel, the compressing endfittings of the columns are of various designs.

Column packing is very important forchromatography resolution.

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Even then, only short columns can be used,otherwise resistance to solvent flow increases andpressure rises too high.

There is also a limit to the number of theoreticalplates (N) one can achieve.

Another difficulty is that it is more difficult topack a long column efficiently. Joining columns

together does not appear to produce an additiveeffect in terms of plate numbers.

This is due to problems associated with thermalgradients which develop across the columndiameter as a result of the increased pressure

required (and diffusion problems occurringbetween the columns, no matter how small theinter-column volume is).

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The chemical composition of the stationary phaseencountered in columns are of the following types:

silica,

styrene-divinylbenzene,

polysaccharides and

other polymers, as well as diatomaceous earths.

The Cls, NH2, SugarPak TM

and silica column of packing of 5 pm and 20-25 cm inlength are the ones

most commonly used in food laboratories, performingabout 95% of the work.

The choice of the column type will naturally varywith the application.

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Injection of the sample Injection of the sample is entirely automated, and you wouldn't

be expected to know how this is done at this introductorylevel.

Because of the pressures involved, it is notthe same as in gas

chromatography (if you have already studied that). Retention time

The time taken for a particular compound to travel through thecolumn to the detector is known as its retention time.

This time is measured from the time at which the sample isinjected to the point at which the display shows a maximumpeak height for that compound.

Different compounds have different retention times

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The detector

There are several ways of detecting when a

substance has passed through the column.

A common method which is easy to explainuses ultra-violet absorption

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Applications

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