HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

HPLC

1. High Performance Liquid Chromatography

2. HPLC is a technique used to separate, identify and separate the components in a mixture of compounds. High performance and high speed compared with traditional column chromatography because of the forcibly pumped mobile phase. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column. The stationary phase of the column, the sorbent, is typically a granular material made of solid particles like silica, polymers with 2–50 micrometers in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the sorbent particles. The mobile phase is the mixture of solvents like water/acetonitrile/methanol.

3. The composition and the temperature of the mobile phase plays a major role in the separation process Its composition and temperature play a major role in the separation process by influencing the interactions taking place between sample components and stationary phase. The interactions includes hydrophobic (dispersive), dipole–dipole and ionic or a combination of all the types. Traditional liquid chromatography relies on the force of gravity to pass the mobile phase through the column In HPLC 50-350 bar pressure is employed, with the column dimensions of 2.1-4.6 mm diameter and 30-250 mm length. The stationary phase particles are 2-50 µm, which offers an efficient separation.

4. Components of HPLC  Pump Injector Column Detector Recorder Degasser Column Heater

5. Pump Most important part of the system positioned in the upper stream of the chromatograph. Generates a flow of eluent from the solvent reservoir to the system. Most LC pumps generates the flow by back-and-forth motion of a motor-driven piston. This piston motion produces pulses. Recent pumps generate much less pulse compared to the older ones. These pumps are very important, since a minor change in the flow rate can influence the analysis. Injector Placed next to the pump, place for injection of sample. Hamilton syringe is used for sample injection. The use of auto sampler (auto-injector) system is also widely used that allows repeated injections in a set scheduled-timing.

6. Column Depending on the type of application, the column varies in size and make. Generally made of stainless steel and packed with high pressure to ensure good resolution and separation. For biomolecules, polyether ether ketone is employed as column material instead of stainless steel. Pre-columns and guard columns – for protecting the main column Pre-column – To filter the solvents Guard column – TO filter the sample Detectors Used to analyze the column eluents Five different types UV-visible adsorption detector Refractive index detector Electrochemical detector Fluorescent detector Mass spectrometer

7. Recorder The change in eluent detected by a detector is in the form of electronic signal, to read those signals computer based data processor is attached with the system. Different types are available. Simple system consisting of in-built printer and word processor PC type consisting of display monitor, keyboard and printer. Software are also available for data acquisition, peak-fitting, baseline correction, automatic concentration calculation, molecular weight determination etc. Column heater The LC separation is often largely influenced by the column temperature. In order to obtain repeatable results, it is important to keep the consistent temperature conditions. For sugar and organic acids, better resolutions can be obtained at elevated temperature (50~80  o C ). It is also important to keep stable temperature to obtain repeatable results even it is analyzed at around room temperature. There are possibilities that small different of temperature causes different separation results.  Thus columns are generally kept inside the column oven (column heater).

8. Applications Medical field - detecting vitamin D levels in blood serum Legal issues - detecting performance enhancement drugs in urine Research - separating the components of a complex biological sample or similar synthetic chemicals from each other Manufacturing units - production process of pharmaceutical and biological products