HPLC: High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) is an advanced form of liquid chromatography used in separating the complex mixture of molecules encountered in chemical and biological systems, in order to understand better the role of individual molecules. In liquid chromatography, a mixture of molecules dissolved in a solution (mobile phase) is separated into its constituent parts by passing through a column of tightly packed solid particles (stationary phase). The separation occurs because each component in the mixture interacts differently with the stationary phase. Molecules that interact strongly with the stationary phase will move slowly through the column, while the molecules that interact less strongly will move rapidly through the column. This differential rate of migration facilitates the separation of the molecules.

The advantages of HPLC over other forms of liquid chromatography are several. It allows analysis to be done in a shorter time and achieves a higher degree of resolution, that is, the separation of constituents is more complete. In addition, it allows stationary columns to be reused a number of times without requiring that they be regenerated, and the results of analysis are more highly reproducible. A further advantage of HPLC is that it permits both instrumentation and quantitation to be automated.

Components of HPLC Analysis

HPLC has four basic components: a solvent delivery system to provide the driving force for the mobile phase; a means by which samples can be introduced into the solvent; the column; and some type of detector. A recorder is used to display the results and an integrator performs the calculations.

The column used for a specific separation is based on the type of the molecules to be analyzed. Various types of chromatographic modes can be used for the separation of the molecules. For example, ion exchange columns separate charged molecules such as amino acids, proteins, or nucleotides . Size exclusion columns separate organic polymers such as polyvinyls and silicones or biopolymers such as proteins, nucleic acids, or sugars. Adsorption columns separate molecules based on their interaction with the stationary phase. This mode is useful for the separation of vitamins, dyes, lipids, phenols, and antioxidants. Partition columns are used to separate molecules based on the way that the solvent becomes partitioned into stationary and mobile layers, and is useful in analyzing steroids, aromatics, vitamins, and antibiotics. The molecules eluting from any one of these different types of column are then analyzed by various types of detectors, measuring absorbance, fluorescence, or electrochemical or radiochemical properties. Other types of detectors include mass spectroscopy and refractive index.

HPLC Applications

A recent advancement of HPLC has been the development of the denaturing HPLC method (DHPLC). This procedure can separate double-stranded DNA molecules that differ by as little as one base pair . The speed of analysis (approximately 5 minutes per sample) and the size of DNA fragment that can be analyzed (up to 2.0 kilobytes) has made it a preferred method for a variety of applications in the field of molecular biology. Applications of DHPLC include the detection of single nucleotide polymorphisms (SNPs). These are single base-pair variations in DNA that can give valuable information on genetic variation within a population. They can also help to identify the genes that cause certain human diseases.

To determine if the two genes of interest differ, they are first amplified by the polymerase chain reaction and then injected together into a so-called reversed-phase column. In this type of column, the stationary phase is less polar than the mobile phase, which is the opposite of the arrangement found in standard columns. Once the genes are injected, their DNAs bind to the stationary phase. Increasing the temperature causes each gene to separate into its two strands (a phenomenon called denaturing). Once denatured, the strands can enter the mobile phase and move through the column.

Cooling the column causes the strands of the genes to rejoin, and the DNAs reattach to the column. The temperature is manipulated to make the strands constantly separate and rejoin, with the balance determined by the strength of the attraction that exists between the strands. If the two genes are exactly identical, they will spend more time in the stationary phase, and elute from the column more slowly. If the genes differ even by a single nucleotide, however, they will spend more time in the mobile phase, and leave the column more quickly.

Y chromosome analysis is one of the most powerful molecular tools for tracing human evolution. Polymorphisms in the human Y chromosome, detected by DHPLC, can be used as markers for tracing human evolution. This will eventually help to elucidate the patterns of human origins, migration, and mixture. The ability to rapidly and efficiently genotype SNPs by use of DHPLC is also useful in medicine, through the identification of mutations that result in susceptibility to certain diseases or that affect physical responses to certain drugs.

see also DNA; Polymerase Chain Reaction; Polymorphisms; Y Chromosome.

Prema Rapuri

Bibliography

Bidlingmeyer, Brian A. Practical HPLC Methodology and Applications. New York: John Wiley & Sons, 1992.

Underhill, Peter A., Peidong Shen, Alice A. Lin, et al. "Y Chromosome Sequence Variation and the History of Human Populations." Nature Genetics 26 (2000): 358-361.