What is HPLC?

What is HPLC?
HPLC is best decribed as high performance liquid chromatography since the essence of the technique is the highly resolutive separations which can be achieved by teh use of uniform microparticulate chromatographic supports and well designed equipment. Other acronyms such as high pressure or high speed liquid chromatography are often used but do not reflect the essential features of the technique.

The use of liquid chromatography for the analysis of biological materials has many advantages over teh classical technique of gas chromatography, since the liquid mobile phase allows the separation and recovery of substances which are not readily volatilized. In addition, liquid chromatography is to be preferred for molecules which have high polarity, high molecular weight, a number of ionic groups or thermal instability. Such as features are characteristic of most biological macromolecules, and have caused many difficulties in other chromatographic studies.

The use of a liquid instead of a gaseous phase introduces several important constraints on the chromatographic system. Since liquids are more viscous (20 to 200 times) and exhibit lower diffusion rates (3,000 to 30, 000 times) than gases, the separation columns must be operated at higher pressures in high performance liquid chromatography (500 -5000 psi) than in gas chromatography. The analysis time is kept short in liquid chromatography by the use of small columns (typically 4 mm by 30 cm) and small particle sizes (<10u). Recently analysis times have been further improved by the introduction of 5-6u particles. HPLC columns are usually 25 to 50 cm long and have internal diamters from 2 to 4 mm with linear flow velocities of teh mobile phase of typically 20 cm/min; conventional columns are much larger and have usual elution velocities of 0.1 cm/min. Another advantage in HPLC is that closed, reusable columns are used, thus hundreds of samples can be run through an individual coumn without repacking.

Biological macromolecules are usually structurally complex. For example, nucleic acids often exhibit molecular weights in excess of 1x10^6, while proteins, although smaller, exhibit great diversity due to teh occurrence of twenty different amino acids in protein sequences. Polysaccharides often contain an enormous number of branch points in the carbohydrate backbone and therefore can present a tremendous separation challenge. In many classes of lipid the nature of any esterified fatty acid is highly heterogeneous, with saturated and unsaturated acids of different chain lengths present in many purified lipid preparations.

It is clear, therefore, that the selectivity available with microparticulate silica-based columns will be fully utilized in biological analysis. Already several published seaprations suggest that the resolving power of this new chromatographic technique is excellent, eg. porcine insulin is well separated from the monodesamido derivative (charge difference of -1), ovine and porcine endorphins (difference of a single methyl group) can be completely resolved.