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HPLC Theory

I) EFFECT OF VARIABLES ON COLUMN EFFICIENCY

A mathematical approximation of the behavior of chromatographic column efficiency is obtained from the van Deemter equation:

H = A + B/u + Cu, where H is the plate height, u is the linear velocity of the mobile phase, A is the eddy diffusion term, B is the longitudinal diffusion coefficient, and C is the coefficient of the mass transfer term. The lower the value of H, the more efficient the column.

The eddy diffusion term A, represents the multitude of pathways by which a component finds its way through the column. In a poorly packed column, the retention time for molecules of the same component can vary significantly depending on the numerous flow paths that could be taken. This effect results in band-broadening. The mobile phase velocity also affects the eddy diffusion parameter, and at moderate to high flow rates the zone-broadening is greater. At low flow rates, the molecules of a component are not significantly dispersed by multi-channeling; diffusion averaging results in this case.

The longitudinal diffusion term, B/u, describes a band-broadening process that is inversely related to the mobile phase velocity. The analyte is in the column for a shorter time when the flow rate is high; hence the diffusion term is less. In HPLC, this term is negligible since diffusion coefficients of liquids are very small relative to gases (as in GC).

The mass transfer term, Cu, describes the time available for equilibrium of an analyte to be established between the mobile and stationary phases. At high mobile flow rates there is less time for this equilibrium to take place and a contribution to the broadening effect is observed.

II) SEPARATION MECHANISMS

A useful classification of the various LC techniques is based on the type of distribution (or equilibrium) that is responsible for the separation. The common interaction mechanisms encountered in LC are classified as adsorption, partition, ion-exchange, gel permeation or size exclusion, and chiral interaction. In practice, most LC separations are the result of mixed mechanisms. A brief description of the separation mechanisms is presented below.

Adsorption: When the stationary phase in HPLC is a solid, the type of equilibrium between this phase and the liquid mobile phase is termed ‘adsorption’. All of the pioneering work in chromatography was based upon adsorption methods, in which the stationary phase is a finely divided polar solid that contains surface sites for retention of analytes. The composition of the mobile phase is the main variable that affects the partitioning of analytes. Silica and alumina are the only stationary phases used, the former being preferred for most applications. Applications of adsorption chromatography include the separation of relatively non-polar water-insoluble organic compounds. Because of the polar nature of the stationary phase and the impact of subtle variations in mobile phase composition on the retention time, adsorption chromatography is very useful for the separation of isomers in a mixture.

Partition: The equilibrium between the mobile phase and a stationary phase comprising of either a liquid adsorbed on a solid or an organic species bonded to a solid is described as ‘partition’. The predominant type of separation in HPLC today is based on partition using bonded stationary phases. Bonded stationary phases are prepared by reaction of organochlorosilane with the reactive hydroxyl groups on silica. The organic functional group is often a straight chain octyl (C-8) or octyldecyl (C-18); in some cases a polar functional group such as cyano, diol, or amino may be part of the siloxane structure. Two types of partition chromatography may be distinguished, based on the relative polarities of the phases.

When the stationary phase is polar and the mobile phase relatively less polar (n-hexane, ethyl ether, chloroform), this type of chromatography is referred to as ‘normal-phase chromatography’. For this reason, the use of silica as the stationary phase (as in adsorption chromatography) is also considered to be a normal phase separation method.

When the mobile phase is more polar than the stationary phase (which may be a C-8 or C-18 bonded phase), this type of chromatography is called ‘reversed-phase chromatography’. Reversed-phase chromatography separations are carried out using a polar aqueous-based mobile phase mixture that contains an organic polar solvent such as methanol or acetonitrile. Because of its versatility and wide range of applicability, reversed-phased chromatography is the most frequently used HPLC method. Applications include non-ionic compounds, polar compounds, and in certain cases ionic compounds.

Ion-exchange: Ion-exchange separations are carried out using a stationary phase that is an ion-exchange resin. Packing materials are based either on chemically modified silica or on styrene-divinylbenzene copolymers, onto which ionic side groups are introduced. Examples of the ionic groups include (a) sulfonic acid (strong cation exchanger), (b) carboxylic acid (weak cation exchanger), (c) quaternary ammonium groups (strong anion exchanger), and (d) tertiary amine group (weak anion exchanger). The most important parameters that govern the retention are the type of counter-ion, the ionic strength, pH of the mobile phase, and temperature. Ion chromatography is the term applied for the chromatographic separation of inorganic anions/cations, low molecular weight organic acids, drugs, serums, preservatives, vitamins, sugars, ionic chelates, and certain organometallic compounds.

The separation can be based on ion-exchange, ion-exclusion effects, or ion pairing. Conductivity detectors in ion chromatography provide universal and sensitive detection of charged species. The employment of some form of ion-suppression immediately after the analytical column eliminates the limitation of high background signal from the mobile phase in conductivity detection.

Size Exclusion: High molecular weight solutes (>10,000) are typically separated using size exclusion chromatography – gel filtration or gel permeation. In size-exclusion LC, the components of a mixture are separated according to their ability to penetrate into the pores of the stationary phase material. Packing materials used are wide-pore silica gel, polysaccharides, and synthetic polymers like polyacrylamide or styrene-divinylbenzene copolymer. In gel filtration the mobile phase is aqueous and the packing material is hydrophilic, while in gel permeation an organic mobile phase is used and the stationary phase is hydrophobic. Size-exclusion applications include the separation of large molecular weight biomolecules, and molecular weight distribution studies of large polymers and natural products. For a homologous series of oligomers, the retention time (volume) can be related to the logarithm of the molecular mass.

Chiral Interaction: Chiral compounds or enantiomers have identical molecular structures that are mirror images of each other. Rapid and accurate stereochemical resolution of enantiomers is a challenge in the field of pharmaceuticals and drug discovery. A chiral stationary phase contains one form of an enantiomeric compound immobilized on the surface of the support material. Typically, derivatives of optically active polysaccharides that are chemically bonded to silica form the packing material. A chiral separation is based on differing degrees of stereochemical interaction between the components of an enantiomeric sample mixture and the stationary phase.

III) METHOD DEVEOPMENT IN PARTITION CHROMATOGRAPHY

Successful chromatography requires a proper balance of the intermolecular forces between the solute, the mobile phase, and the stationary phase. Method development tends to be more complex in HPLC relative to GC because in the latter the mobile phase is inert and makes no contribution to the separation process. The important criteria to consider for method development are resolution, sensitivity, precision, accuracy, limit of detection, limit of quantitation, linearity, reproducibility, time of analysis and robustness of the method. In all of these, the column quality plays an important role since the peak shape affects all criteria required for optimum separation. The factors that affect the column efficiency have already been described above.

Column dimensions and particle size affect the speed of analysis, resolution, column backpressure, detection limit, and solvent consumption. HPLC methods have traditionally been developed using columns measuring 10, 15 or 25 cm in length and 4.6 mm ID. Short columns of 5 cm or less in length and 1 or 2 mm ID are now available; when packed with particles of size 5 micron or less, very high efficiency columns are obtained. The advantages of using shorter columns are lower backpressures, dramatic solvent savings, greater sensitivity, reduced analysis time, and applicability to small sample quantities - all achieved without compromising resolution. Using these columns, gradient methods may be used to achieve very rapid analyses of samples that contain a wide polarity range of analytes. The future of reversed-phase HPLC method development will involve a significant increase in the use of use narrow-bore and micro-bore columns.

Often in choosing a column for partition chromatography, the polarity of the stationary phase is matched roughly to that of the analytes in the sample; a mobile phase of different polarity is used for elution. The analytes must be soluble in the mobile phase and the solvent must be compatible with the analytical method.

As a general guide, use normal phase chromatography for the separation of polar compounds and reversed-phase chromatography for components that are in the moderately polar to non-polar range.

Normal phase chromatography commonly involves the use of silica, aminopropyl, diol, and cyanopropyl stationary phases. These columns may be used to separate polar compounds such as amines, anilines, nitroaromatics, phenols, and pesticides.

Isocratic elution in reversed-phase chromatography is typically accomplished using a mobile phase mixture of water and another solvent of lower eluting strength (acetonitrile, methanol). In cases where the time of analysis is compromised or when the resolution is poor, gradient elution using 2 or 3 different solvents is recommended.

The relative polarity of a solvent is a useful guide to solvent selection in partition chromatography. The relative polarities of the listed solvents may differ slightly depending on the literature source, since the scale used to measure polarity may be different. The following should suffice as a general reference for relative solvent polarity.

There is a strong dependence of the retention time on the mobile phase composition, and the retention parameter may be easily altered by variation of solvent polarity. This is the easiest way to improve chromatographic resolution of two overlapping species or to decrease overall separation time for components with widely differing retention values. A good starting point is a mixture of water and a polar organic solvent (methanol or acetonitrile). The effect of mobile phase polarity on elution time can be tested at a few different solvent proportions. If greater selectivity is required, a mobile phase comprising of 3-4 solvents may be used. Theoretical calculations have indicated that a mobile phase mixture of water, THF, methanol, and acetonitrile may be used to resolve most reversed-phase applications within a reasonable length of time.

The various analytes to be separated may also be arranged based on the polarities of their functional groups. A general guide to relative solute polarity going from non-polar to the most polar group is as follows:

Unfortunately, theoretical predictions of mobile phase and stationary phase interactions with a given set of sample components are not always accurate, but they do help to narrow down the choices for method development. The separation scientist must usually perform a series of trial-and-error experiments with different mobile phase compositions until a satisfactory separation is achieved.

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