High Performance Liquid Chromatography (HPLC) was developed in the late 1960s and early 1970s. Today it is widely applied for separations and purifications in a variety of areas including pharmaceuticals, biotechnology, environmental, polymer and food industries.

HPLC has over the past decade become the method of choice for the analysis of a wide variety of compounds. Its main advantage over gas chromatography is that the analytes do not have to be volatile, so macromolecules are suitable for HPLC analysis.

HPLC is accomplished by injection of a small amount of liquid sample into a moving stream of liquid (called the mobile phase) that passes through a column packed with particles of stationary phase. Separation of a mixture into its components depends on different degrees of retention of each component in the column. The extent to which a component is retained in the column is determined by its partitioning between the liquid mobile phase and the stationary phase. In HPLC this partitioning is affected by the relative solute/stationary phase and solute/mobile phase interactions. Types of HPLC include partition, displacement, normal-phase, reversed-phase, size-exclusion, ion-exchange, and bio-affinity. Changes in mobile and stationary phase composition can have an enormous impact on sample separation. Since the compounds have different mobilities, they exit the column at different times (i.e., they have different retention times). It is important to remember that any changes in operating conditions will affect the retention time which will affect the accuracy of identification.

The retention time is the time between injection and detection. There are numerous detectors which can be used in liquid chromatography, depending on the species of interest. It is a device that senses the presence of components different from the liquid mobile phase and converts that information to an electrical signal. For qualitative identification one must rely on matching retention times of known compounds with the retention times of components in the unknown mixture.

Quantitative analysis is often accomplished through analytical HPLC. An automatic injector providing reproducible injection volumes is standard on modern commercial systems. Other components include a mobile phase reservoir (generally a clean solvent bottle), a pump, a sample inlet, a column, a detector with associated electronics, and computer interface. The pump, which is used to deliver precise, reproducible, and constant amount of mobile phase, often operates at pressures ranging from 500 – 5000 psi. These high pressures are needed because the stationary phase column packing consists of small, tightly packed particles. It takes significant pressure to push the mobile phase through this stationary phase at a reasonable flow rate.

Figure 1: Illustrative representation of the utility of HPLC for nucleic acid purification and downstream possibilities.

The purification columns and buffers available from ADS Biotec are utilized in reverse-phase, ion-pairing mode, HPLC. Briefly, a non-polar stationary phase is used in conjunction with an ion-pairing polar, mobile phase to separate nucleic acids. The columns are packed with non-porous polystyrene divinylbenzene (PS-DVB), which provide superior separation when compared to C-18 silica columns. The mobile phase is composed of triethylammonium acetate (TEAA) or hexylammonium acetate (HAA), with and without acetonitrile. The negatively charged nucleic acids pair with the positively charged amine salt in the mobile phase to form a hydrophobic complex, which interacts with the non-polar column [2]. As the concentration of acetonitrile increases, the mobile phase becomes increasingly non-polar until the nucleic acid/amine salt complexes forced from the column and are captured in elution fractions. This effect is directly correlated to the length of the nucleic acids, therefore the longer a sequence is, the longer it will remain on the column and the higher concentration of acetonitrile it will take to elute it from the column.

Figure 2: Graphical representation of nucleic acids complexing with amine salts, prior to binding with the PS-DVB sphere (purple, left).

References:

1. “Handbook of Instrumental Techniques for Analytical Chemistry” Frank Settle, Editor: “High Performance Liquid Chromatography”, Phyllis Brown, Kathryn DeAntonois, Prentice Hall, 1997, pp. 147-164.
2. Dickman, M. J. Ion Pair Reverse-Phase Chromatography: A Versatile Platform for the Analysis of RNA. Chromatography Today. 2011.