APPLICATION OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

1. Analysis of peptides

High performance liquid chromatography can separate peptide into their sources, quantity and complexity. Peptides containing 50 amino acid residues (Mant & Hodges, 2002). Development in improving in terms of scale, instrumentation and column packings make separation of peptides become easier. Mant & Hodges (2002) state that purification of a single peptide from a complex mixture will require a different approach from that necessary for separating all components of a mixture and whereas the former approach may require only the application of a single HPLC mode, the latter will require a combination of separation modes (multistep or multidimensional HPLC) for efficient resolution of all desired peptides. Modes of HPLC used in peptide separations are size exclusion HPLC (SEC), ion-exchange HPLC (IEC) and reversed-phase HPLC (RPC). The maximum separation potential of a particular HPLC may be achieved by manipulating mobile phase.

RPC technique is used to separate peptides of nearly related structures. RPC is suitable to separate peptide from a complex mixture. RPC containing octyl (C 8) or octadecyl (C18)

functionalities, although C3 sorbents have been used for more hydrophobic peptides and proteins (Lau et al.,(1984), Taneja et al.,(1984) & Glajch (1986)). Column size in terms internal diameter range is 2-3 mm (narrow bore) and 4-4.6 mm (analytical) depends on the HPLC mode (RPC or IEC) (Burke et al.,1991). The smaller internal column gives better detection sensitivity and speed (Mant & Hodges, 2002). In RPC require the peptide standard to monitor column performance. Alkyphenone series is used for peptide standard monitoring (Kikta Jr & Stange, 1977).

SEC is performed for peptide-protein separation and/or molecular weight determinations. According to Regnier (1983), peptides are separated based only on peptide size i.e ideal SCE occurs only when there is no interaction between the solutes and the column matrix. However, in modern SEC columns are weakly anionic (negatively charged and slightly hydrophobic, resulting in deviation from ideal size exclusion behavior (Mant et al.,1987). For best compromise between separation time and efficiency of resolution, optimum flow rates range is 0.2-1.0 mL/min and the sample volume must be small as possible (Mant & Hodges, 2002). Peptide standards for monitoring both nonideal and ideal SEC is a series of synthetic peptide standards (Ac-[G-L-G-AK-G-A-G-V-G]n amide, where n = 1-5) (Mant et al.,2002). IEC commonly used together with RCP for separation of peptides and sample desalting before further analysis. Buffer pH, the nature and ionic strength of and anion or cation employed for displacement of acidic, respectively (McDermott & Kidd, 1984). Sodium and chloride ion used most in IEC. Volatile pyridine-acetic acid buffers used for resolution of peptide mixture on IEC (Dizdaroglu, 1985). Peptide may be removed from an ion-exchange sorbent by either gradient or isocratic elution. When separating mixtures of peptides with wide range of net charges the linear gradient elution mode is used. Salt gradient of either sodium chloride in phosphate, tris, or citrate mobile phase buffer used for gradient elution (Garipey et al.,1985). In IEC, peptide standard to asses the effect of pH variation on the resolving capability and load capacity of an ion-exchange (Mant & Hodges, 2002).