High-Performance Liquid Chromatography (HPLC(

HPLC is a modern high-resolution liquid chromatographic technique best described as high-performance liquid chromatography because the essence of the technique is the high resolution of the separations achieved by using uniform microparticulate, chromatographic supports and well-designed equipment. HPLC has also been called high-pressure or high-speed liquid chromatography but these acronyms do not reflect the essential features of the technique.

The development of HPLC has been in several areas including theory, special columns, and equipment. Though the theoretical principles of HPLC were firmly established by Martin and Synge (1) in the early 1950s, HPLC in practice did not appear until the late 1960s because of instrumental problems. Horváth et al. (2) constructed one of the first practical HPLC apparatuses for use in their research on nucleotides . Subsequently, dramatic developments in packing materials, particle sizes (as small as 3 µm), narrow-bore columns (as small as 0.5 mm inner diameter for microbore columns(   and high column inlet pressures (up to 12,000 psi) have been achieved for high resolution and efficiency.

 HPLC is carried out in all classical modes of column chromatography involving a liquid mobile phase. The most important are liquid-solid adsorption, liquid-liquid and organo-bonded partition, ion-exchange, size-exclusion, and affinity chromatography . HPLC has the advantages over classical liquid chromatography that the columns are reusable, the sample introduction can be automated, flow rates can be controlled precisely, and detection and quantification can be achieved by using continuous flow detectors. These features have led to improved analytical accuracy and precision.

The widespread application of HPLC in biochemical studies is evidenced by countless publications in the field. For example, HPLC is widely used by molecular biologists to isolate nucleic acids from synthetic oligonucleotides to natural plasmids, and by protein biochemists to purify peptides and proteins. Those requiring more detailed practical information are directed to the reviews cited previously and also to a number of excellent monographs (5), (11-13).

References

1. A. J. P. Martin and R. L. M. Synge (1941) Biochem. J. 35, 1358–1368. 

2. C. Horváth, B. A. Preiss, and S. R. Lipsky (1967) Anal. Chem. 39, 1422–1428. 

3. M. Colpan and D. Riesner (1988) in Modern Physical Methods in Biochemistry, Part B (A. Neuberger and L. L. M. Van Deenen, eds.), Elsevier, Amsterdam, pp. 85–105. 

4. P. R. Brown (ed.) (1984) HPLC in Nucleic Acid Research ( Chromatographic Science, Vol.28), Marcel Dekker, New York. 

5. A. Fallon, R. F. G. Booth, and L. D. Bell (1987) Applications of HPLC in Biochemistry, Elsevier, Amsterdam. 

6. W. S. Hancock (1984) Handbook of HPLC for the Separation of Amino Acids, Peptides, and Proteins, Vols.I and II, CRC Press, Boca Raton.  

7. M. T. W. Hearn and M. I. Aguilar (1988) in Modern Physical Methods in Biochemistry, Part B (A. Neuberger and L. L. M. Van Deenen, eds.), Elsevier, Amsterdam, pp. 107–142. 

8. A. R. Kerlavage (ed.) (1989) The Use of HPLC in Receptor Biochemistry, Alan R. Liss, New York. 

9. C. T. Mant and R. S. Hodges (eds.) (1991) High-Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis, and Conformation, CRC Press, Boca Raton. 

10. R. M. Chicz and F. E. Regnier (1990) in Guide to Protein Purification (M. P. Deutscher, ed.), Methods in Enzymology 182, Academic Press, New York, pp. 392–421. 

11. H. Engelhardt (ed.) (1986) Practice of High Performance Liquid Chromatography: Applications, Equipment and Quantitative Analysis, Springer-Verlag, Berlin. 

12. U. D. Neue (1997) HPLC Columns: Theory, Technology, and Practice, Wiley-VCH, New York.

13. A. Weston and P. R. Brown (1997) HPLC and CE: Principles and Practice, Academic Press, San Diego.