HPLC | High Performance Liquid Chromatography | Application of HPLC

hello everyone in this video we'll talk about high performance liquid chromatography let's just analyze the name first high performance can tell us that this particular chromatographic technique have high resolution power second of all liquid means here we use a mobile phase which is liquid so hplc chromatography is just a chromatographic technique which has a very high resolution where the mobile phase is liquid and the mobile phase is passed through a column under very high pressure so this is the overall principle of high performance liquid chromatography so in this video we would understand why this is

high performance and how exactly this chromatographic technique works what is the advantage and disadvantage of these chromatography system so just like any other chromatographic technique hplc also works on the same principle so in a column chromatography which we have seen earlier and if you don't know about these videos the link would be available in the i button so the principle starts with equilibration where the column is equilibrated with buffer followed by sample binding then wash and ultimately illusion so hplc has a similar operation paradigm but here the difference lies in the column and the column

is pretty special in hplc the column is generally tightly packed and these column materials are small in size the particle size varies from 2 to 50 micron in average and in hplc the pressure can range from 50 to 350 bar and this is pretty high pressure and this high pressure ensures the high performance of this chromatographic technique now hplc columns could be of different type there could be affinity columns there could be gel filtration columns there could be ion exchange columns all of these columns are available in hplc so we can understand hplc to be

a mode where all these column materials are same instead the column materials are more sturdy in this case which can withstand the high pressure for example the gfc column would be porous but here the material would be way sturdy than a conventional gfc column now all of these column would be operating under very high pressure and that is the key point about hplc so let's just look at the column in bit more details so the hplc column includes silica or hydroxyapatite media or any kind of polystyrene kind of beads which can withstand very high pressure

now usage of smaller diameter beads generally results in improved separation and this this reason is due to increased surface area now the column pressure increases as the bead diameter is reduced so always there is a cut-off limit that how much the pressure can increase so we have a lower limit of these bead size so the first step of this whole procedure is sample injection where sample a very small amount of sample is sufficient enough for this technique so sample is injected into the system via a specialized sample injector generally the sample injector is connected with

the auto sampler which can load multiple different samples in a semi automated fashion in this machine now let us look at the simplified version of the inner working of hplc and try to understand the components first of all there would be a pump which can pump a mobile phase in this system now the pump is connected with a de-gasser system which can remove all the dissolved gas in these fluids ultimately there is a sample injector connected with a rotatable valve which can allow the injection of the sample under high pressure then the sample along with

the mobile phase is passed through this column from this column it goes to a detector system in the detector the sample annihilates are actually detected the samples that are separated are actually detected and the data is displayed in a attached computer software and the rest amount of the fluid is either collected or collected in a waste bottle so let us understand the flow of liquid in this setup so first of all the mobile phase would be moving through several tubings and these tubings are very sturdy such that it can with withstand high pressure now via

degasser it would pass through the pump while it is passing through the dsr system dissolved gases would be removed now the pump can able to move the liquid to the sample injector from the sample injector there would be a mixing of the mobile phase with the injected sample and it would move through the column and ultimately reach the detector while in the detector the samples would be detected so there are two type of hplc columns one is known as normal phase hplc in the normal phase hplc what happens is the stationary phase that means the

column material is polar in nature often it has hydroxyl groups so hydroxyl group groups are attached to the silica beads so when we put sample in this column they start separating and the substances which are polar would be left behind what are substances which are nonpolar or hydrophobic in nature they would be eluted first and that is how the separation works now there is another kind of operation regime which is known as reverse phase hplc in this reverse phase hplc the column material or the stationary phase has hydrophobic alkyl groups now it can form hydrophobic

bond with nonpolar or hydrophobic and nylon so obviously in this case when we inject the sample the particular samples which are hydrophobic in nature or highly non-polar they would be retained in the column and their retention time would increase whereas the polar substances would be eluted first so overall we have seen that hplc can function in two different regimes reverse phase or in normal phase and the column materials can be different in these two different operational regime ultimately the separated annihilates would enter the detection system the detector the detector works via pier lambert law there

would be a led light which passed through a filter and ultimately it passes through the sample the absorbance value is detected and this detector is generally a ccd camera or photo multiplier tube ultimately from the absorbance the sample concentration and purity can be monitored or can be calculated and ultimately the sample would flow through these tubes and while it is flowing through this detector the light from the led can pass through the sample and provide us the necessary information about that particular annihilate and that particular information is converted to a digital signal which is displayed

in the monitor so in the monitor absorbance versus retention time graph is plotted and it plots the chromatogram here the number of peak in this chromatogram depicts how many annihilate molecules are there also with the help of retention time we would understand the sequence of illusion of these particular annihilate molecules so let us quickly review the advantage and disadvantage of this hplc system coming to the advantage part hplc have very high resolving power because it used high pressure and very small particle size of the bead which increase the surface area and enhance the separation procedure

the speed of separation is unprecedented and it is very much reproducible so it can be used under laboratory conditions or in industrial settings the automated sampler makes it easy to use and it can be adapted for industrial purposes and high throughput works then disadvantage includes it could be expensive because the hplc setup is expensive at the first run but it is very efficient i would say in summary we have looked at working principle of hplc advantages versus disadvantages of hplc and we also had a quick idea that how the hplc data is analyzed how the

chromatogram looked like which gave us an overall understanding of this procedure so i hope you enjoyed this video if you like this video give it a quick thumbs up don't forget to like share and subscribe and you can join my courses in an academy using my code ap 10 you can get a 10 discount on my courses thank you guys thanks for listening and do let me know in the comment how you like my videos thank you