High Pressure Liquid Chromatography procedure steps

2. A pump is used to force a liquid mobile phase from the solvent reservoir attached to the system, to flow through the liquid chromatograph. The mobile phase is generally a polar solvent- for example a mixture of water and methanol. (high performance liquid chromatography - hplc, no date)

 A very high pressure is applied through the liquid chromatograph; therefore the separation of the component will be at a very fast rate. This step is taken prior to injecting a pure sample. The pure sample will be introduced into the flow stream of the mobile phase. Once every pure sample is analysed, the mixture will also be analysed finally.

3.  The mobile phase carries the mixture into a column to be separated into its components. The column generally consists of a modified silica stationary phase; the silica is made non-polar by attaching “eight or eighteen carbon atoms” (high performance liquid chromatography - hplc, no date) to its surface. The silica particles are very small in size and this allows a much greater surface area; hence the stationary phase and the molecules flowing past it, can have a better interaction and the components within the mixture can have a better separation. The interactions take place in the column; the polar mobile phase will interact more with the polar molecules of components in the mixture. Whereas the stationary phase will interact less with the polar components due to the hydrocarbon chain attached to silica which makes it non-polar.

There will be equilibrium for each component in the mixture; if the component is polar, it will spend more time dissolved in the mobile phase than being adsorbed on the silica with hydrocarbons attached to it. Therefore polar components will be carried away in the mobile phase and will be much faster at exiting the column than less polar or non-polar components.

4. Retention time is what is known as the time which a specific component travels through the column and reaches the detector. Retention time is measured beginning from the time the sample mixture enters the liquid chromatogram from injection port till the point where the specific compound has its maximum peak height displayed on the display screen which is attached to the liquid chromatogram. Each particular component will have their own retention time; therefore it could be used to identify each individual component in the mixture. However, the conditions that the process is carried out must remain the same; otherwise the components will have different rates of reactions measured in different analyses. These conditions are: the pressure used when forcing the mobile phase into the system because the flow rate of the solvent can differ according to the pressure which will differ the retention time for the same component in different analyses; the polarity and the particle size of the stationary phase; the polarity and composition of the liquid mobile phase; the temperature within the column.

5. One of the ways to detect when the component has passed through the column is using a UV detector. The component will absorb the UV light to a certain extent in accordance to the amount of the specific compound that is passing through the beam of UV light at that moment. The component that absorbs the light energy, will change the amount of light the sensor detects and this change is directed to a computer data system. Different components and mobile phases absorb the UV light at different wavelengths. If for example a water-methanol mixture was used as a mobile phase, a wavelength greater than 205 nm would have to be set; methanol, individually, absorbs below 205 nm of wavelength and water absorbs below 190 nm of wavelength in the UV spectrum. Setting the wavelength greater than 205 nm will prevent any false readings introduced by the solvent from being detected by the sensor.

6. The output of the components is a series of peaks which is also known as a liquid chromatogram.  Each peak represents an individual component; the desired component could be identified from its retention time in the previous analysis where pure samples were being examined; the retention times for the same components is either the same or fairly close. The pure samples have different concentrations; therefore the areas under the peaks are directly proportional to the concentrations of the pure samples according to Beer’s law. Finally a calibration curve graph could be drawn in order to determine the concentration of the desired component within the mixture by finding the corresponding concentration value to its peak area analysed in the mixture.