HPLC Methods

HPLC or High Performance Liquid Chromatography is the process of separating, identifying and quantifying compounds. This is done based on the compounds’ idiosyncratic polarities. Before the 1970′s there were not many reliable chromatography methods available. By the 1980′s a much better method was devised. In the past few decades methods have vastly improved and today HPLC has evolved producing results that are much more accurate and precise.

A small quantity of a sample sent for analysis is integrated into the mobile phase’s stream. The motion of the Analyte is made to slow down using a specific chemical and travels the column’s length via its physical interactions during the stationary phase. The Analyte is slowed according to its nature and composition. The retention time is when the Analyte exits at the other end of the column and under specific conditions it is viewed as unique in pointing out an identifying characteristic of the said Analyte. If a smaller size column is used, higher back pressure is created which causes an increase in the linear velocity. This gives the said components much less time to disperse inside the column thus resulting in much better resolution in the chromatogram. The solvents commonly used are water or other various liquids which are organic such as methanol and acetonitrile.

A further modification of HPLC is a variation of the mobile phase during the process of the analysis also known as gradient elution. Normally the gradient for the reversed phase begins at %5 methanol and progresses steadily up to %50 methanol over a period of 25 minutes, dependent on how hydrophobic an Analyte is, the incline or gradient is chosen. The aforementioned gradient takes apart the mixtures of Analyte. In a process similar to liquid-liquid extraction, the partitioning process goes underway.

The HLPC method may seem complicated but upon closer scrutiny is not at all that difficult to follow.