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High Pressure Liquid Chromatography procedure steps

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Quantitative Analysis: In order to determine the amount/ concentration of a given component present in a mixture, quantitative analysis is carried out. High Performance Liquid Chromatography (HPLC) is a vastly superior system of liquid chromatography where separation and determination of components within mixtures of non-volatile substances are done very sensitively, efficiently and at a very fast rate compared to Liquid Chromatography. This is due to a very high pressure of up to 400 atmospheres being applied into the HPLC system for the mobile phase to move through the column and carry the components within the mixture along with it. Here are the stages taken in determining the concentration of a component within a mixture using HPLC: 1. Firstly, many standard solutions (pure samples) are prepared with various range of concentrations, using the actual sample component which is being examined to determine its concentration within the mixture. The mixture will also be examined finally before drawing a calibration curve to identify the concentration of the specific component in the mixture. ...read more.


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. ...read more.


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. 1. 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. ...read more.

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