High Performance Liquid Chromatography Lab Report

Introduction:

High Performance Liquid Chromatography or also known as High Performance Liquid Chromatography is one of the most powerful and most commonly used analytical separation technique. HPLC is a form of liquid chromatography that separated solutes/compounds dissolved in the solution (High-performance liquid chromatography, 2012). It is an improved form of column chromatography, where the solvent is passed through under high pressure instead of letting it drip down due to gravity. The sample is injected into the column to separate the sample of interest. The two different phases in HPLC are mobile and stationary phase. Separation of compounds is based on the stationary and mobile phase. Mobile phase is the solvent and the stationary

It responds to all of the compounds eluted (universal detector) but does not respond to the compounds with low concentrations; therefore it is not widely used. The refractive index measure the ability of mobile phase to bend light. The light is passed through a flow cell and through the fluid in the cell, refraction takes place and the direction of the light changes slightly. The mobile phase background signal is substracted from the sample signal (Dolan, 2012). A light emitting diode is used as the light source in most RI detectors. A pair of photo diodes detects the light emitted. As the refractive index varies the photo diode light beam position changes so that more or less light shines on each diode. More light is struck on the upper diode. If there is a change in the refractive index, the light beam position may move down causing less light on the upper and more on the lower diode. The RI detectors are contained in an insulated compartment as the temperature change may affect the RI detectors. RI is mainly used for saccharides and sugars which absorb only little but refracts a lot (Laboratory training courses on HPLC, GC, AAS, lab safety, spectroscopy).

2. UV/Vis absorbance detector:

It is the most commonly used detector. UV detectors use light to analyse samples such as nucleic acids, proteins and to perform toxic and therapeutic drug testing. This is easy to measure. The three types of UV detectors are fixed, variable

Mass spectrometry detectors are possibly the best for selectivity. It works with the positive ion, a positive ion is achieved by knocking off two or more electrons of the atom or molecule (The mass spectrometer - how it works, no date.) To have the same kinetic energy the positive ions are accelerated. The magnetic field deflects the ions according to their masses. The deflection depends on how many electrons were removed in the first stage. The higher the charge of ion, the more it is deflected. In other words, the lighter they are the more they are deflected. The ion passing through the machine is detected electrically. The size of the magnetic field is related to the mass of each ion being detected as the magnetic field is used to bind the ions to the detectors (Laboratory training courses on HPLC, GC, AAS, lab safety,