HPLC Lecture Notes PDF

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Dr. Thukaa Z. Abdul-Jalil Lec 11&12 High performance liquid chromatography (HPLC) is now one of the most powerful tools in analytical chemistry. It has the ability to separate, identify, and quantitate the compounds that are present in any sample that ca...

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Dr. Thukaa Z. Abdul-Jalil Lec 11&12 High performance liquid chromatography (HPLC) is now one of the most powerful tools in analytical chemistry. It has the ability to separate, identify, and quantitate the compounds that are present in any sample that can be dissolved in a liquid. Today, compounds in trace concentrations as low as parts per trillion (ppt) may easily be identified. HPLC can be, and has been, applied to just about any sample, such as pharmaceuticals, food, nutraceuticals, cosmetics, environmental matrices, forensic samples, and industrial chemicals. The instrument of HPLC is composed of the following parts : A reservoir (Solvent Delivery): A reservoir (Solvent Delivery) holds the solvent (called the mobile phase, because it moves). Two basic elution modes are used in HPLC. The first is called isocratic elution. In this mode, the mobile phase, either a pure solvent or a mixture, remains the same throughout the run. A typical system is outlined in Figure bellow The second type is called gradient elution, wherein, as its name implies, the mobile phase composition changes during the separation. This mode is useful for samples that contain compounds that span a wide range of chromatographic polarity. As the separation proceeds, the elution strength of the mobile phase is increased to elute the more strongly retained sample components HPLC PUMPS A high-pressure pump solvent manager is used to generate and meter a specified flow rate of mobile phase, typically milliliters per minute. For analytical purposes, HPLC pumps should have flow rates that range from 0 to 10 ml/min., but for preparative HPLC, flow rates in excess of 100 ml/min may be required. HPLC SAMPLE VALVES Since sample valves come between the pump and the column it follows that HPLC sample valves must also tolerate pressures up to 10,000 psi. For analytical HPLC, the sample volume should be selectable from sub micro liter to a few micro liters, whereas in preparative HPLC the sample volume may be even greater than 10 ml. HPLC COLUMNS The column contains the chromatographic packing material needed to effect the separation. This packing material is called the stationary phase because it is held in place by the column hardware.. The apparatus is suitable for all types of column chromatography (adsorption, partition, gel filtration, ion exchange, etc.) HPLC DETECTORS UV/Vis spectrophotometers, including diode array detectors, are the most commonly employed detectors. Fluorescence spectrophotometers, differential refractometers, electrochemical detectors, mass spectrometers, light scattering detectors, radioactivity detectors or other special detectors may also be used. HPLC is analogous to GLC in its sensitivity and ability to provide both quantitative and qualitative data in the single operation. It differs in that the stationary phase bonded to a porous polymer is held in a narrow stainless steel column and the liquid mobile phase is forced through under considerable pressure. The apparatus for HPLC is more expensive than GLC, mainly because a suitable pumping system is required and all connections have to be screw-jointed to withstand the pressure involved. The mobile phase is a miscible solvent mixture, which either remains constant (isocratic separation) or may be changed continuously in its proportions, by including a mixing chamber ( gradient elution).The major differences between HPLC and GLC is that the separation in HPLC normally operates at ambient temperature, so that the HPLC is mainly used for those classes of compounds which are non-volatile, e.g. higher terpenoinds, phenolics of all types, alkaloids, lipids and sugars. It works best for compounds which can be detected in the ultra- violet or visible regions of the spectrum.