Liquid Chromatography (High Performance)

HPLC – High Performance Liquid Chromatography, better known as LC, is used in analytical laboratories as drug testing kits since the last 4 decades as a separating and identifying technique. Conventionally, HPLC has used different types of detectors such as Fluorescence detectors, UV spectrophotometers and electrochemical detectors. Quite recently, HPLC has been coupled with MS in order to be used as a popular instrument in forensic laboratories.

The fundamental HPLC system has mobile phase reservoir, degasser, pump, inlet unit, columns and detectors. Different tubing and valves are involved which connect different parts of the HPLC system together. The pumps are used to propel the mobile phase through the system, which can be binary, having 2 solvents or having 4 solvents i.e. quaternary. It operates under quite high pressure of up to 6000 psi. A characteristic system has flow rates from 0.1 to 10 mL/min. The inlet part for the sample has injecting needle and tray which are connected to a loop of 100 micro liters. The column part is temperature controlled, which is really essential in the laboratories whose temperatures tend to fluctuate. Temperature control enables the mobile phase to remain constantly fluid, which ensures reproducible results.

When the sample is put through high pressure liquid i.e. the mobile phase, it is carried to the analytical column where it separates. Then the sample is carried forward into the detector. The separation of the sample is done when it is divided into the 2 phases of the analyte i.e. the mobile and stationary phase. HPLC uses different modes to separate the compound which includes reverse phase, normal phase, affinity, ion exchange, chiral and size exclusion chromatography techniques. The commonly used techniques in toxicology and drug tests are ion exchange, normal and reverse phase chromatography. In the normal phase technique, polar stationary and non polar mobile phases are used. The normal phase technique is typical for non polar isomers and compounds. On the other hand, the reverse phase technique is opposite of normal phase such that it uses polar mobile phase and non polar stationary phase. Being very practical, this is then commonly used to analyze drugs and their metabolites. The reverse phase is best for polar compound that are soluble in water, which generally involves majority of the drugs and their metabolites. The ion exchange chromatography divides by the action of analytes with their counter ions attached with the stationary phase. These stationary phases either come in cationic mode – benzyl sulfonic acid or their anionic modes – quaternary ammonium, which depends on its usage.

The mobile phase of this technique contains buffers and solvents which transfer the analytes through the columns. This phase can be either isocratic elution, fixed or modified at different rates while the elution of the gradients takes place. The gradient elution is then used for separating the constituents in a better way especially when the sample has multiple parts which have different affinity for stationary phase. The gradient elution process can improve the peak shape but also need re-equilibration time after the normal run. This ensures that the system returns back to the starting mobile phase before starting the remaining analysis.

The main component of liquid separation chromatography is the separating column. These columns are in the market in different size and shapes but have the same general structuring. The columns need a strong backbone for the bonded phase. This backbone is made of silica or particles that range from 1.5 to 10 micro meters. Generally seen, if the particle is smaller, it is more quickly separated. The functional part that is linked to backbone is known as the bonded phase. Examples of bonded phase include: C8, CN, C18 and phenyl hexyl. When the bonded phase interacts with the analytes, separation occurs. The analytes having the maximum affinity for bonded phase would separate at a slow rate to the mobile phase, thereby being in the column for longer time period. Those having the least affinity will elute quickly while having lesser interaction. Every part would elute as a band or a peak from the column. The time taken by a peak to leave the column is known as the retention time which is used to determine the species of the component and the quantity of the compound is determined by the peak height or area.

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