High performance liquid chromatography (HPLC) separates the components of a liquid mixture quickly and precisely.The stationary phase is made of very small solid particles, often silica, tightly packed into a narrow steel column. The small particles give a very large surface area, which improves separation. The mobile phase is a liquid solvent, such as a mixture of methanol and water. Because the particles are packed so tightly, the solvent must be pumped through the column at high pressure.A small volume of the sample is injected into the solvent stream at the start of the column. Components with more affinity for the stationary phase are held back and take longer to come out. As each component leaves the column it passes a detector, often one that measures ultraviolet absorption, and a peak appears on the chromatogram.
The time from injection to detection is the component's retention time. A component is identified by comparing its retention time with that of a known substance run under exactly the same conditions: the same column, solvent, flow rate and temperature. The area under each peak shows the relative amount of that component.HPLC can be linked to a mass spectrometer, so each separated component can be identified from its mass spectrum. Uses include checking the purity of medicines, detecting drugs in urine and testing food for additives.Worked example: a two-component mixture gives peak areas of 1.2 and 3.6 units. Assuming the detector responds equally to both, the first component makes up 1.2 ÷ (1.2 + 3.6) × 100 = 25% of the mixture.
The time from injection to detection is the component's retention time. A component is identified by comparing its retention time with that of a known substance run under exactly the same conditions: the same column, solvent, flow rate and temperature. The area under each peak shows the relative amount of that component.HPLC can be linked to a mass spectrometer, so each separated component can be identified from its mass spectrum. Uses include checking the purity of medicines, detecting drugs in urine and testing food for additives.Worked example: a two-component mixture gives peak areas of 1.2 and 3.6 units. Assuming the detector responds equally to both, the first component makes up 1.2 ÷ (1.2 + 3.6) × 100 = 25% of the mixture.