High Performance Liquid Chromatography (HPLC)

Introduction.

High performance liquid chromatography (HPLC) is a powerful tool in analysis. It is basically a highly improved form of column chromatography (Clark, 2007). 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 you 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. The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive.

High Performance Liquid Chromatography is one mode of chromatography, one of the most used analytical techniques. Chromatographic process can be defined as separation technique involving mass-transfer between stationary and mobile phase. HPLC utilises a liquid mobile phase to separate the components of a mixture. The stationary phase can be a liquid or a solid phase. These components are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture separates into its components. The amount of resolution is important, and is dependent upon the extent of interaction between the solute components and the stationary phase. The stationary phase is defined as the immobile packing material in the column. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.

Figure 1: Retention time

HPLC as compared with the classical technique is characterized by:

o o

Small diameter (2-5 mm), reusable stainless steel columns Column packing with very small (3, 5 and 10 mm) particles and the continual development of new substances to be used as stationary phases

o o o

Relatively high inlet pressures and controlled flow of the mobile phase Precise sample introduction without the need for large samples Special continuous flow detectors capable of handling small flow rates and detecting very small amounts

o o o

Automated standardized instruments Rapid analysis High resolution

Plate 1: HPLC

Initially, pressure was selected as the principal criterion of modern liquid chromatography and thus the name was "high pressure liquid chromatography" or HPLC. This was, however, an unfortunate term because it seems to indicate that the improved performance is primarily due to the high pressure. This is, however, not true. In fact high performance is the result of many factors: very small particles of narrow distribution range and uniform pore size and distribution, high pressure column slurry packing techniques, accurate low volume sample injectors, sensitive low volume detectors and of course, good pumping systems. Naturally, pressure is needed to permit a given flow rate of the mobile phase, otherwise, pressure is a negative factor not contributing to the improvement in separation. Recognizing this, most experienced chromatographers today, refer to the technique as high performance liquid chromatography still permitting the use of the acronym HPLC.

Theory of HPLC.

HPLC is a dynamic adsorption process. Analyte molecules, while moving through the porous packing beads, tend to interact with the surface adsorption sites. Depending on the HPLC mode, the different types of the adsorption forces may be included in the retention process: 1. Hydrophobic (non-specific) interactions are the main ones in reversed-phase (RP) separations. 2. Dipole-dipole (polar) interactions are dominant in normal phase (NP) (mode. 3. Ionic interactions are responsible for the retention in ion-exchange chromatography. (Kazakevich, 2007)

All these interactions are competitive. Analyte molecules are competing with the eluent molecules for the adsorption sites. So, the stronger analyte molecules interact with the surface. The weaker the eluent interaction, the longer the analyte will be retained on the surface. Size exclusion chromatography (SEC) is another case. It is the separation of the mixture by the molecular size of its components. The basic principle of SEC separation is that the bigger the molecule, the less possibility there is for it to penetrate into the adsorbent pore space. So, the bigger the molecule the less it will be retained (Kazakevich, 2007).

Figure 2: Flow Scheme for HPLC (Source: High Performance Liquid Chromatography, (Clark, 2007))

Application of HPLC

Preparative HPLC refers to the process of isolation and purification of compounds. Important is the degree of solute purity and the throughput, which is the amount of compound produced per unit time. This differs from analytical HPLC, where the focus is to obtain information about the sample compound. The information that can be obtained includes identification, quantification, and resolution of a compound.

Chemical Separations can be accomplished using HPLC by utilizing the fact that certain compounds have different migration rates given a particular column and mobile phase. Thus, the chromatographer can separate compounds (more on chiral separations) from each other using HPLC; the extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase.

Purification refers to the process of separating or extracting the target compound from other (possibly structurally related) compounds or contaminants. Each compound should have a characteristic peak under certain chromatographic conditions. Depending on what needs to be separated and how closely related the samples are, the chromatographer may choose the conditions, such as the proper mobile phase, to allow adequate separation in order to collect or extract the desired compound as it elutes from the stationary phase. The migration of the compounds and contaminants through the column need to differ enough so that the pure desired compound can be collected or extracted without incurring any other undesired compound.

Identification of compounds by HPLC is a crucial part of any HPLC assay. In order to identify any compound by HPLC a detector must first be selected. Once the detector is selected and is set to optimal detection settings, a separation assay must be developed. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph. The identifying peak should have a reasonable retention time and should be well separated from extraneous peaks at the detection levels which the assay will be performed. To alter the retention time of a compound, several parameters can be manipulated. The first is the choice of column, another is the choice of mobile phase, and last is the choice in flow rate. All of these topics are reviewed in detail in this document.

Identifying a compound by HPLC is accomplished by researching the literature and by trial and error. A sample of a known compound must be utilized in order to assure identification of the unknown compound. Identification of compounds can be assured by combining two or more detection methods.

Quantification of compounds by HPLC is the process of determining the unknown concentration of a compound in a known solution. It involves injecting a series of known concentrations of the standard compound solution onto the HPLC for detection. The chromatograph of these known concentrations will give a series of peaks that correlate to the concentration of the compound injected.

Types of HPLC

There are many ways to classify liquid column chromatography. If this classification is based on the nature of the stationary phase and the separation process, three modes can be specified.

a) b) c) d)

Partition Adsorption Chromatography Ion exchange Chromatography Size exclusion Chromatography

Figure 3: Application of Liquid Chromatography

Adsorption Chromatography

Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes. The stationary phase is an adsorbent like silica gel or any other silica based packing and the separation is based on repeated adsorption-desorption steps (Yip, 1997).

Figure 4: Adsorption Chromatography

In gel permeation chromatography proteins do not bind significantly to the matrix. Sorting does not occur as a result of differential binding but of differential exclusion from pores in the beads. On the other hand many sorbents are available that bind proteins with different affinities. At equilibrium the behaviour of a specific protein can be described by a partition coefficient a, the fraction of the protein that is adsorbed (Scott, 2008). Therefore a can have values between 0, no binding whatever, and 1.0 complete adsorption without any desorption. Differences in partition coefficients can be utilized for fractionation by allowing proteins to choose between a stationary, adsorbed and a mobile, unadsorbed phase. In this case the completely unadsorbed protein will move with the buffer front, its mobility compared

to the buffer front, Rf, will be unity. In contrast, the completely absorbed protein will not move at all, and Rf will be 0. In other words: a + Rf = 1.0. Binding of protein to the matrix is most simply achieved by mixing them. Such socalled batch adsorption is especially popular with hydroxyapatite, but can also be performed with ion exchange resins. One of the great advantages of adsorptive methods is the ability to use high volumes of sample, obviating concentration steps. This saves time and generally leads to higher overall yields in protein preparations. For example, one can load ion exchange resins in batch mode by stirring a large-volume sample with the resin. Subsequently, the resin is poured into a column for gradient elution, or batch eluted on a sintered glass funnel. Thus, one can move rapidly through large volume steps at the early stages of the preparation (McNaught & Wilkinson, 1997).

Purposes of adsorption. o o o o o o Removal of impurities (impurities adsorb to adsorbent) Isolation of product (volume reduction) Invariably also involves purification In ion exchange, it also can be used to Demineralization Metathesis, example Na+ with H+ to convert Na- citrate to citric acid

Uses of absorption. o o o o o o Dehumidification Odour/colour/taste removal Gas pollutant removal (H2S) Water softening and deionization Hydrocarbon fractionation Pharmaceutical purification

Types of adsorption

a) Physical adsorption

Result of intermolecular forces causing preferential binding of certain substances to certain adsorbents

o o

Reversible by addition of heat via steam, hot inert gas or oven Attachment to the outer layer of adsorbent material

b) Chemisorption

o o o

Result of chemical interaction Large amount heat released irreversible Mainly found in catalysis

In adsorption chromatography there are two types of forces: o o Forces attracting solutes to adsorbent (Stationary Phase). Forces tending to remove solutes from adsorbent to move with the mobile phase.

i.

Force of attraction.

They may be classified into according to their strength:

Dipoledipole attraction: It is a force takes place between polar adsorbent and polar solutes.

Hydrogen bonding:

It is a type of bond weaker than covalent bonds.

Hydrogen bonds are formed between the OH group hydrogen (as in silica) and electronegative atoms such as Oxygen, nitrogen in solutes.

Polarizability forces: A force occurs between polar adsorbents and solutes that can polarize such as aromatic compounds.

o o

Weak covalent bonds: As those take place during complex formation. Van der Waals forces: Non polar attraction forces occur between the atoms of nuclei and electrons of other atoms.

ii.

Forces cause solutes movements

Displacement: In this case solvent molecules compete with the solutes for the adsorption sites of the stationary phase. This competition makes solutes move in different speeds.

o -

Elutropic Series of solvents: Solvent are arranged in this series according to their strength in ascending (increasing) order.

Elution: It is the tendency of solutes to dissolve and move with the mobile phase. The solvent used as mobile phase must be just good enough to dissolve the solutes to allow competition with the adsorption power of the stationary phase. If very strong solvents are used they will wash out all solutes together without separation. Ether / hydrocarbons / carbonyl solvents are of common use.

Liquid Solid (Adsorption) Chromatography

Adsorption chromatography is used for the separation of non polar or fairly polar organic molecules. In this technique the stationary phase is the surface of a finely divided polar solid and the analyte competes with the mobile phase for sites on the surface of the packing. Retention of the analyte occurs as a result of adsorption forces. Finely divided silica and alumina are used as stationary phases with organic solvents such as hexane acting as the mobile phase. The only variable that can be altered to affect the partition coefficient of the analytes is the composition of the mobile phase (Latif, 2010). A particular advantage of adsorption chromatography is its ability to resolve isomeric mixtures.

f c

a b d&e g&h j

A basic liquid chromatography consists of a: a) A solvent inlet filter b) Pump c) Inline solvent filter d) Injection valve e) Pre column filter f) Column

g) Detector h) Recorder i) j) Back pressure regulator Waste reservoir

The schematic above is of the basic instrumentation of a liquid-solid chromatograph. The solvent inlet brings in the mobile phase which is then pumped through the inline solvent filter and passed through the injection valve. This is where the mobile phase will mix with the injected sample. It then gets passed through another filter and then passed through the column where the sample will be separated into its components. The detector detects the separation of the analytes and the recorder, or usually a computer will record this information. The sample then goes through a backpressure filter and into waste.

Of all the methods of separation possible in the liquid phase, adsorption chromatography is probably the widest used and has been practiced for the longest time. The original work considered to be the earliest application of liquid chromatography, involved separation of substances in a column filled with powdered chalk which acts as a weak adsorbent. Most applications of classical column chromatography are based on the use of packing materials such as silica gel, alumina, charcoal and florisil, all of which possesses very definite, yet often quite different, adsorptive properties.

A great broadening of the application of this application method came about with the advent of Thin Layer Chromatography (TLC). In this technique a thin layer of adsorbent, most often silica gel or alumina, is used as the medium on which a sample is applied as a spot and developed by the action of a liquid phase rising up through the adsorptive layer. In this report it is not important to describe TLC in detail, but only to show the fact that through its use come the realisation that quite complex separations could be achieved on adsorbent was selected carefully. Modern liquid solid chromatography (LSC) offers the same style of separation mechanism, only with greater resolving power, speed and ease of quantitation.

Although adsorption chromatography has been widely used over a considerable number of years, it is apparent that there are many points of detail which need to be considered if highly reproducible results are to be obtained. Provided care is taken with the selection of appropriate operating conditions, adsorption chromatography has one of the widest ranges of applicability of any LC method for the high resolution separation of non ionic species of low molecular weight.

Figure 5: Silica Gel

Figure 6: Silica Gel mechanism

The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent. In normal phase type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more than the mobile phase with respect to the sample. In reverse phase packing material is relatively non polar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the non polar components of the solutes and the non polar stationary phase. Typical stationary phases are non polar hydrocarbons and the solvents are polar aqueous organic mixtures such as methanol water or acetonitrile water.

Figure 7: The relationship between polarity and elution times for normal phase and reverse phase chromatography.

Adsorbability of a compound increases with

o o o o o

Increasing molecular weight up to certain extent. A higher number of functional groups such double bonds Halogen compounds Increasing polirisability od the molecule Electron clouds of the molecule

Applications of Adsorption Chromatography

LSC is best suited for the separation of non polar compounds with molecular weights below 5000. Solutes must be soluble in non polar solvents and should have a limited solubility in aqueous solvents. It should be remembered that the mobile phase in LSC should be non-polar modified with a polar solvent (Latif, 2010). However, the solvent polarity must not be very large since irreversible adsorption on the stationary phase can occur precluding the use of LSC. Therefore, water is usually excluded from mobile phases to be used in LSC. Separations of difficult to separate isomers were possible with LSC (Latif, 2010).

Advantages/Disadvantages

Liquid solid column chromatography is an effective separation technique when all appropriate parameters and equipment are used. This method is especially effective when the compounds within the mixture are coloured, as this gives the scientist the ability to see the separation of the bands for the components in the sample solution. Even if the bands are not visible, certain components can be observed by other visualization methods. One method that may work for some compounds is irradiation with ultraviolet light. This makes it relatively easy to collect samples one after another. However, if the components within the solution are not visible by any of these methods, it can be difficult to determine the efficacy of the separation that was performed. In this case, separate collections from the column are taken at specified time intervals. Since the human eye is the primary detector for this procedure, it is most effective when the bands of the distinct compounds are visible.

Liquid-solid column chromatography is also a less expensive procedure than other methods of separation, like HPLC and GC. This is because the most basic forms of column chromatography do not require the help of expensive machinery like high pressure solvent pumps used in HPLC. In methods besides flash chromatography, the flow of the mobile phase, the detection of each separation band, and the collection of each component, are all done manually by the scientist. Although this introduces many potential instances of experimental error, this method of separation can be very effective when done correctly. Also, the glass wear used for liquid-solid column chromatography is relatively inexpensive and readily available in many laboratories. Burets are commonly used as the separating column, which in many cases will work just as well as an expensive pre-prepared column. For smaller scale chromatography, Pasteur pipettes are often used.

References

Clark, J. (2007). High Performance Liquid Chromatography. Retrieved 28 August, 2011, from http://www.chemguide.co.uk/analysis/chromatography/hplc.html Kazakevich, Y. (2007). Types of HPLC. In Y. Kazakevich, & H. McNair, HPLC for Pharmaceutical Scientists. Latif, M. A. (2010). Analytical Chemistry. Retrieved 29 August, 2011, from Islamic University of Gaza : http://www.monzir-pal.net/ McNaught, A., & Wilkinson, A. (1997). adsorption chromatography. In A. McNaught, & A. Wilkinson, Compendium of Chemical Terminology. Scott, R. P. (2008). Principles Introduction. In R. P. Scott, Principles and Practice of Chromatography . Yip, K. (1997). Types of Chromatography. Retrieved 29 August, 2011, from Biochemical Engineering: http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm