Chapter 7 High performance liquid chromatography Instrumental Technique for Environmental Analysis

Rezaul Karim Environmental Science and Technology Jessore University of Science and Technology

Chapter content
Liquid Chromatography (LC); Scope; Instrumentation; Applications

Reference
Daniel C. Harris , 2010, Quantitative Chemical 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.

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 a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. It also allows us to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture.
4

RANGE OF APPLICATIONS

It is often suitable for organic compounds that are too unstable or insufficiently volatile to be amenable to gas chromatography analysis without prior derivatisation. It is suited for the separation of a wide range of chemicals, including pharmaceuticals, foods, heavy industrials and biochemicals. is likely to provide a set of chromatographic conditions for almost any type of compounds. Alteration of these conditions to suit the matrix involved might be the only required step for the food scientist to perform a HPLC analysis of a new compound. In food science, HPLC has been applied to several categories of substances:
carbohydrates, lipids, vitamins, additives, synthetic colourings, natural pigments, contaminants (degradation products, pesticides, or naturally occurring substances), as well as amino acids and others
5

Scope of HPLC

LC is the most widely used of all of the analytical separation techniques. 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 or thermally fragile ones, and above all, its widespread applicability to substances that are important to industry, to many fields of science, and to the public.

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

Basic Principles

HPLC is a form of liquid chromatography, where separation (or partition) occurs between a mobile phase (the solvent) and a stationary phase (the column packing). It is the ability with which the sample constituents will distribute themselves between the two phases that will effect the separation. Depending on the nature of the stationary phase, the separation process can be of four different modes:
7

In several basic type of chromatography, the mobile phase is a liquid. These phase type often classified by separation 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.
8

adsorption chromatography, where the separation is based upon repeated adsorption-desorption steps; partition chromatography where the separation is based on partition between the mobile and the stationary phase; ion-exchange chromatography where the stationary phase is made up of an ionic surface of opposite charge to that of the sample; and size exclusion chromatography where the sample is separated according to its molecular size through a column filled with a material having precisely controlled pore size.
9

10

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 or alumina) and the mobile phase is non polar (e.g., hexane). In this mode, polar samples are retained more strongly by the column therefore allowing elution of non polar compounds first. Reversed-phase chromatography, where the stationary phase is non-polar in nature (e.g., hydrocarbon) and the elution solvent (or mobile phase) is polar (e.g., water or methanol). This being the exact reverse of normal phase chromatography, non-polar compounds will be retained longer on the column.

11

The choice of the solvent will depend on the nature of the operation mode, i.e., isocratic or gradient elution (and, of course, on the solubility of the sample in the chosen elution medium). The polarity for such an elution medium can, therefore, vary from buffered aqueous solutions to hydrocarbons. The chosen medium (including water) needs to always be very pure. HPLC grade solvents are commercially available and should always be used. The choice of a gradient eluent is always done by trial and error, usually starting with a single solvent and increasing the concentration of the second mobile phase component by usually using an initial mixing rate of 2% per minute to achieve the desired conditions in the mobile phase. In some circumstances, up to 3 different solvents can be used.
12

THE MOBILE PHASE- The solvent

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 is described as "normal", it isn't the most commonly used form of HPLC. The column is filled with tiny silica particles, and the solvent is nonpolar - hexane, for example. A typical column has an internal diameter of 4.6 mm (and may be less than 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 to make 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 an alcohol such as methanol.

13

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 a loop, a column, sometimes a column oven, a detector, a recorder, and almost invariably, an integrator.

Automatic injectors, sample carousels and collectors are also commonly used.

14

Looking at the whole process

15

The Injector

A few methods are used to inject the sample onto the column, the simplest one being a direct injection with a micro-syringe. But most systems will use sampling devices. The most common way of injecting the sample on the column is an injection valve in which the sample is injected into a holding loop. Loops are designed to inject a specific volume onto the column, usually of the order of 10 to 20 pL for an analytical column.
16

17

The Stationary Phase - The column

Two of the most important steps in the development of an analytical method are the selection of the separation mode and the appropriate column packing. The columns most commonly encountered have an internal diameter (i.d.) of 4.5 to 5 mm and are 10 to 25 cm in length; they are packed with stationary phases having 5 to 10 Tm in diameter. Usually made of stainless steel, the compressing end fittings of the columns are of various designs. Column packing is very important for chromatography resolution.

18

Even then, only short columns can be used, otherwise resistance to solvent flow increases and pressure rises too high. There is also a limit to the number of theoretical plates (N) one can achieve. Another difficulty is that it is more difficult to pack a long column efficiently. Joining columns together does not appear to produce an additive effect in terms of plate numbers. This is due to problems associated with thermal gradients which develop across the column diameter as a result of the increased pressure required (and diffusion problems occurring between the columns, no matter how small the inter-column volume is).

19

The chemical composition of the stationary phase encountered 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 in length are the ones most commonly used in food laboratories, performing about 95% of the work. The choice of the column type will naturally vary with the application.
20

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 introductory level. Because of the pressures involved, it is not the same as in gas chromatography (if you have already studied that). Retention time The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. Different compounds have different retention times

21

The detector There are several ways of detecting when a substance has passed through the column. A common method which is easy to explain uses ultra-violet absorption

22

Applications

23