LIQUID CHROMATOGRAPHY

LIQUID CHROMATOGRAPHY M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar.

LIQUID CHROMATOGRAPHY 1. Introduction 2. Retention Mechanisms in Liquid Chromatography 3. Method Development

Liquid Chromatography Liquid Chromatography (LC) is a chromatographic technique in which the mobile phase is a liquid. LC is a much older technique than GC, but was overshadowed by the rapid development of GC in the 1950’s and 1960’s. LC is currently the dominate type of chromatography and is even replacing GC in its more traditional applications. Advantages of LC compared to GC: LC can be applied to the separation of any compound that is soluble in a liquid phase. LC more useful in the separation of biological compounds, synthetic or natural polymers, and inorganic compounds Liquid mobile phase allows LC to be used at lower temperatures than required by GC LC better suited than GC for separating compounds that may be thermally labile

Advantages of LC compared to GC (continued): Retention of solutes in LC depend on their interaction with both the mobile phase and stationary phase. GC retention based on volatility and interaction with stationary phase LC is more flexible in optimizing separations  change either stationary or mobile phase Most LC detectors are non-destructive most GC detectors are destructive LC is better suited for preparative or process-scale separations Disadvantage of LC compared to GC: LC is subject to greater peak or band-broadening. RESOLUTION!!!! much larger diffusion coefficients of solutes in gases vs. liquids Low- and High-performance Liquid Chromatography: Many types of liquid chromatography are available, based on different stationary phase and mobile phase combinations. - each type may be further characterized based on its overall efficiency or performance

Low-performance liquid chromatography – LC methods that use large, non-rigid support material particles > 40 mm in diameter – poor system efficiencies and large plate heights – such systems have the following characteristics: broad peaks poor limits of detection long separation times columns can only tolerate low operating pressures

Column chromatography – an example of the equipment used in low-performance liquid chromatography Solvent reservoir Column head Column Column packing Porous glass plate Sample is usually applied directly to the top of the column. Detection is by fraction collection with later analysis of each fraction

High-performance liquid chromatography (HPLC) – LC methods that use small, uniform, rigid support material particles < 40 mm in diameter usually 3-10 mm in practice – good system efficiencies and small plate heights (H!) – such systems have the following characteristics: narrow peaks shorter separation times

A typical HPLC system: • Higher operating pressures • need for mobile phase • delivery requires special • pumps and other system • components • Sample applied using • closed system (i.e., • injection valve) • detection uses a flow-through detector

Similar to GC, solutes can be eluted from a column by using either a constant column conditions or gradient elution Isocratic elution: use of a constant mobile phase composition to elute solutes simple, inexpensive difficult to elute all solutes with good resolution in a reasonable amount of time  general elution problem Gradient elution: changing the composition of the mobile phase with time  solvent programming going from a weak mobile phase to a strong one. weak mobile phase  solvent A strong mobile phase  solvent B solvent change can be stepwise, linear or non-linear

Gradient elution of mixture of 30 amino-acids In choosing a mobile phase for LC, several factors need to be considered – type of stationary phase used determines what will be a strong or weak mobile phase – solubility of the solutes – viscosity of the mobile phase – type of detector used and solvent's background signal – purity of the solvents – miscibility of the solvents (for gradient elution)

Types of Liquid Chromatography (Retention Mechanisms ): Techniques in LC are classified according to the method of solute separation Adsorption chromatography Affinity chromatography Partition chromatography Size-exclusion chromatography Ion-exchange chromatography 1. Adsorption chromatography (Liquid-solid Chromatography) • A LC technique which separates solutes based on their adsorption to an underivatized solid particles is known as adsorption chromatography, or liquid-solid chromatography. • This technique is suited for non-polar small compounds (MW<5000).

b. One advantages of adsorption chromatography, as is also true for GSC, is that it is able to retain and separate some compounds that can not be separated by other methods. One such application is in the separation of geometrical isomers. c. Mechanism (a) Retention of solute in adsorption chromatography can be viewed as solute A displacing n moles of solvent M from a surface. Amp + nMsp Asp + nMmp

Phase 2 Phase 2 Phase 1 Phase 1 2. Partition chromatography (Liquid-liquid Chromatography) a. Partition chromatography, or liquid-liquid chromatography is a Chromatographic technique in which solute are separated based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support. b. The support material used in partition chromatography is usually silica. Un-bonded and banded stationary phase. c. Mechanism: The retention of solute in partition chromatography is given by: k = KD (Vs/Vm)

d. Applications of partition Chromatography Normal Phase LC and Reversed-phase LC Stationary phase: polar non-polar How can ions be separated by using partition Chromatography?

e. Ion-pair Chromatography (a) Ion-pair chromatography is used for the separation of ionic and ionizable compounds and mixtures of neutral and ionic compounds. (b) In this method, counterions (species of opposite charge to the solutes) thereby regulate the retention. Typically alkyl amines or tetra alkyl amines are added to ion pair with acids whereas alkyl sulfates, sulfonates, or phosphates are used to ion pair with bases A- + B+ A-B+

3. Ion-Exchange Chromatography a. Ion-exchange chromatography is a liquid chromatography technique inwhich solutes are separated by their adsorption onto a support containing fixed charge onto a support containing fixed charges on its surface. b. Ion-exchange is a fairly common technique used in water softeners and in the industrial removal or replacement of ionic compounds for products. Ion-exchange is used in chromatography for separation of a wide variety of charged compounds, including inorganic ions, organic ions, and biological compounds (such as amino acids, proteins and nucleic acids) c. Mechanism n(support—A-B+) + Cn+ (support—A-)nCn+ + nB+ K = ([B+]n[Cn+]/[B+]n[Cn+])

Antibody Enantiomer with Low affinity to the antibody Insert matrix Enantiomer with high affinity to the antibody 4. Affinity chromatography For biomolecule separation Antibody-antigen

Standard entropy effect 2 2a 1- dc K = 5. Size-exclusion Chromatography for polymer and bio-polymer Retention of a solute is dependent on Standard entropy effect. Diameter of the pore is very important for solute selectivity. Multiple pore sizes should be used for separate solutes with different sizes. GPC: Gel permeation Chromatography (polymer scientists) GFC: Gel filtration Chromatography (biochemists)

LC Method Development Problem Definition Mode selection What type of LC should be used? Stationary phase and mobile phase, Temperature Selectivity Optimization Column length, particle size, flow rate, instrument configuration, sample injection… System optimization Method Validation Accuracy, sensitivity, specificity, detection Limit, quantification limit, linearity

Selection of a mobile phase for a particular LC application can be done by using various tables that summarize properties for common LC solvents: LIKE DISSOLVES LIKE!

Mobile-phase selection in partition Chromatography ‘ ‘ (P’1 – P’2)/2 For NPLC (capacity factors): k2/k1 = 10 ‘ (P’2 – P’1)/2 ‘ For RPLC (capacity factors): k2/k1 = 10 Example 28-1: In a reversed-phase column, a solution was found to have A retention time of 31.3 min, while an un-retained species required 0.48 min For elution when the mobile phase was 30% (by volume) methanol and 70% water. Calculate (a) k’ , (b) a water/methanol composition that should bring k’ to a value of about 5, and (c) the retention time of this solution under this new mobile phase (assuming that the retention time of the un-retained species is not changed).

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LIQUID CHROMATOGRAPHY