High Performance Liquid Chromatography (HPLC) Testing

In a previous article we discussed and reviewed the use of Gas Chromatography as it relates to USP raw material testing. In this article, we will address the other commonly used chromatograph method – High Performance Liquid Chromatography otherwise known as HPLC testing.

HPLC relies on pump systems that work to pass a high pressured, sample based liquid solvent through a specially designed column that is filled with absorbent material.  Very commonly used in the testing of raw materials as it relates to specific USP and EP test methods, the components in a sample  mixture are separated based upon their absorption capacity in relation to the column material thus enabling the various individual components to be clearly identified and quantified. The active component of the column, the sorbent, is typically a granular material made of solid particles like silica or polymers that vary in size from 2–50 micrometers. The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile and/or methanol) and is referred to as a “mobile phase”. Its composition and  temperature play a major role in the separation process by influencing the interactions taking place between sample components and sorbent. The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature the separation is performed at.

Over the years many various types of HPLC have been developed and used in the analysis of raw material samples. Partition chromatography was the first kind of chromatography that chemists developed. The partition coefficient principle has been applied in paper chromatography, thin layer chromatography, gas phase and liquid–liquid applications. Normal–phase chromatography was also one of the first kinds of HPLC that chemists developed. Commonly known as normal-phase HPLC (NP-HPLC) this method separates components based on their affinity for a polar stationary surface such as silica, hence it is based on a component’s ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface.  Displacement chromatography works on the principle that a molecule with a high affinity for the chromatography matrix will compete effectively for binding sites, and thus displace all molecules with lesser affinities enabling the user to determine the various analytes in a samples. Ion-exchange chromatography works on the principle that  retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Solute ions of the same charge as the charged sites on the column are excluded from binding, while solute ions of the opposite charge of the charged sites of the column are retained on the column.

While this overview article will not make you an expert in HPLC testing, it hopefully provides a lay person’s overview of the basic tenets of HPLC testing and a brief history of the various types of liquid chromatography that have been developed and used over the past century. As it expected, the complexity of features of the HPLC instruments used in the laboratory setting today are truly specialized and effective in identifying components in sample materials. This is why the use of HPLC testing in the qualification testing of raw materials is commonly used in the pharmaceutical industry.