HPLC | High performance liquid chromatography

hey everyone quick back chemistry Basics here let's talk about hlc hlc is also known as high performance liquid chromatography or high pressure liquid chromatography hplc is usually a modified column chromatography in column chromatography a column is specked with an absorbent material like silica and the mobile pH is passed down the column because of gravity but in hlc a high press pump is attached with the column the high press pump can generate a high pressure up to 40 map pascals the column is filled with an absorbent material which has a very small particle size the small

particle size gives a very large surface area for the sample molecules to interact as a result the efficiency of Separation increases giving high resolution let's see the components of hlc the column the column is made up of stainless steel which can withstand a very high pressure up to 50 map pascals the length of the column can vary from 5 to 25 cm and have an internal diameter of 4.5 mm the flow rate of mobile pH through the colum is usually 1 to 3 ml per minute the stationary phase as discussed earlier the stationary phase is

made up of an absorbent material that has a very small particle size the particle size is kept uniform to obtain a better performance usually chemically modified silica D venile Benzene Etc are commonly used as a stationary phase the mobile phase the mixture of different solvents can be used as a mobile phase the solvent use depends on the type of sample molecules which are to be separated the mobile phas solvent can be polar or non-polar the mobile phas is usually kept in the solvent Reservoir the solvent Reservoir is attached with the pump which pumps the mobile

face in the column with a high pressure just before hplc column there's an injector which allows introduction of the sample in the column the detector in order to detect the uite that is sample coming out of the column the hlc column is attached with a detector different types of detector such as UV detector IR detector fluoresence detector refractive index detector Mass spectrometer electrochemical detectors Etc can be used the detectors are connected with the computer which collects the information now let's see the working of hlc as the sample molecules get separated they are detected by the

detector and a peak is obtained on the computer this peak is plotted with respect to the retention time to identify the components we need to have standards let's understand this with an example let's see we run glucose as our sample and the peak of glucose is obtained at 5 minutes next we run sucrose as our sample and the peak of sucrose is obtained at 8 minutes now we analyze the unknown sample for the detection of sugars present in it many Peaks are obtained in the chromatogram the peak obtained at 5 minutes indicates the presence of

glucose however there's no Peak at 8 minutes which indicates that sucrose was absent in our sample the remaining two peaks are still unknown as we don't have any standards to compare the retention time this is how the detection of sample is done in hlc and for the detection we must have the standards using hplc it is also possible to find the concentration of sample but before doing so we need to have standard curve let's say we take different conc conentration of glucose and run through hlc the peaks of glucose are obtained at 5 minutes and

with an increase in the concentration of glucose the area under the curve increases once the standard information about the area under the curl and the concentration of glucose is obtained just by measuring the area of glucose peak in the unknown sample the concentration of the glucose can be estimated now let's see different types of hlc normal phase hlc in this type of hlc the stationary face is polar while the mobile face is non-polar usually silica is preferred as a stationary phase as silica has s ioh Group which gives its polar nature during normal phase hlc

polar molecules are retained on the stationary phase while the non-polar molecues move fast down the column with the mobile phase reverse phase hlc in this method the stationary face is nonpolar while the mobile face is polar the non-polar molecules are retained on the stationary phase while the polar molecules moves fast down the column size exclusion hlc this method is based on size exclusion graphy or gel permeation chromatography in this method the stationary phase particles are porous as a result small molecules gets inside the pores and takes a long time to move while the large molecules

easily pass down the column hence separation occurs based on the molecular size ion Exchange hlc in this method the stationary phase has an ionic charge if the molecules of Interest has a positive charge then a stationary phase is kept negative and if the molecule of Interest has a negative charge then the stationary phase is kept positive hence separation occurs based on the molecular charge [Music]