Lecture 49

Añadido: 2026-05-09

[00:00:21]Welcome to this massive open online course on advanced analytical technique.

[00:00:28]And today we are on the last part of unit four, that is we are on the verge of finishing this course and today we are going to explore the last and the best separation technique, that is HPLC.

[00:00:43]That is high performance liquid chromatography.

[00:00:47]Sometimes this is also been referred as high pressure liquid chromatography. So before taking up the main course, let us explore that what will be the our objectives, which we are governing in our coming slides. So a brief outline is given here. So initially we will be introducing the HPLC and then we are going to see the need and importance of HPLC, then the principles and theory of separation via HPLC, then what are the different components which are involved in HPLC, then we are going to study the column and the stationary phase materials used in HPLC, then the some factors we are going to discuss which affect the separation efficiency of HPLC, then again instrumentation and detectors, type of HPLC methods, then the method development and of course the optimization which is the most necessary step of any separation process and then we are going to study few applications of HPLC and finally we are going to see some of the modern advances in the area of separation technology and we also look for certain troubleshooting. So let us begin our course the of HPLC, that is high performance liquid chromatography. So have you ever wondered that how the scientists are able to separate, to identify and to analyze components of a mixture up to a very small or up to a very trace value, sometimes of the order of 10 raised to the power minus 12 g up to 10 raised to the power minus 15 g.

[00:02:41]So how we are going to identify such minute quantities? And how, you know, by just testing the breath of a person we can identify the nature of poisoning it has undergone. So all these have the same answer, that is the most versatile separation technique that is called as the high performance liquid chromatography is the answer to all these intriguing questions.

[00:03:08]So this HPLC is actually revolutionized the separation process.

[00:03:14]It gives you ability to detect ultra trace amount of substances in a mixture. And the beauty of using HPLC is that now you are free from certain uh limitations that now you can analyze any of your compound.

[00:03:35]That is the restriction of being a volatile compound is removed in HPLC.

[00:03:40]So the beauty of HPLC is that you can analyze all your compounds, whether you can vaporize them or you cannot, you can analyze them.

[00:03:50]You can analyze thermally labile compounds. You can analyze certain pesticides. You can analyze you can analyze ion exchangers. You can analyze complex compounds. So the beauty of HPLC is that now you actually analyze any sort of compound, organic compound, inorganic compound, polymers, etc. etc. So it is the most advanced form of liquid chromatography. Ideally speaking, it is the most advanced form because you know, we have covered, we have started our journey from chromatography, then we have discussed elution chromatography, then comes the GC and now the best one which is HPLC. So here actually this like the previous technique, this technique is also based on the principle of differential partitioning of analytes between a stationary and mobile phase.

[00:04:51]So here the main difference is that since the column is highly in a in a highly packed form and this packing is because that particles, stationary phase particles are very uniformly packed and that is why we need high pressure to push our solvent through that tightly packed column. And that is why sometime it is referred as high pressure liquid chromatography because of the extensive pressure. Sometimes, you know, sometimes a pressure of 300 psi is is required to successfully push your mobile phase through the packed column.

[00:05:35]So here the main difference between this technique, between HPLC and GC is that GC technique of separation is only valid for separating volatile compounds.

[00:05:50]But HPLC is valid for separating any compound.

[00:05:55]And moreover, it is said that if you can dissolve then HPLC can resolve.

[00:06:02]So that is actually the main thrust of HPLC.

[00:06:07]So why HPLC? Now the question arises, why we are, you know, why we are discussing HPLC.

[00:06:15]We are discussing HPLC because of a number of superior factors as compared to our traditional liquid chromatography. The first and the foremost is the speed. So if you compare it with liquid chromatography, HPLC has a very fast speed of analysis of your mixture.

[00:06:38]Similarly, it gives you the best possible resolution.

[00:06:43]So it is very good in terms of running of your sample and at the same time it is saving your time and it is giving you the best analyzed, best resolved results of different components present in that mixture.

[00:07:00]And moreover with regard to another difference between GC and HPLC is that that all even if a compound is vaporizable then there are chances that you cannot fully vaporize it.

[00:07:12]So again, HPLC comes to your help. So you can analyze that compound which is not fully volatile by using HPLC. So HPLC gives you fast, accurate and high resolution separation of different components of a given mixture. It is used extensively for thermally unstable, thermally labile compounds, non-volatile compounds, polar compounds, inorganic compounds, polymers, biomolecules. So you just name the you just think of a compound and HPLC is the answer. So if you can dissolve, HPLC can resolve. This is like this. So it is very beneficial for the analysis of pharmaceuticals, in food analysis, in environmental testing and of course do HPLC analysis with regard to certain issues relating to some theft or some, you can say, murder or some other issues where you need extensive and specific results.

[00:08:29]So historical evolution. Let us, you know, get a just get a brief idea that how this HPLC evolved.

[00:08:38]So the basic idea of chromatography was envisaged by Mikhail Tswett who actually termed chromatography from the two Greek words chroma and graphein.

[00:08:55]Chroma of course means color and graphein means study, that is the study of color.

[00:09:00]This Mikhail Tswett was actually a Russian botanist who invented this chromatographic technique to separate various plant pigments, particularly the chlorophylls and xanthophylls. And we have discussed it in quite detail in our lectures when we were discussing the elution chromatography. So Mikhail Tswett was the person who actually laid the foundation of this versatile separation branch of chromatography. Then comes the Martin Synge who actually gave a theoretical idea about the separation of components from column.

[00:09:42]So Mikhail Tswett actually introduced the concept of plate theory.

[00:09:48]And on the basis of that the first chromatograph was developed and in 1950s the partition chromatography comes into play.

[00:09:58]Then in '70s, HPLC was developed where actually we have you where we have employed very high pressure pumps from ranging from 100 psi to 300 and sometimes more than that.

[00:10:13]So, nowadays, the modern era actually using the advanced version of HPLC that is called as UHPLC, that is the ultra high performance liquid chromatography.

[00:10:24]And now even micro HPLC, that is your HPLC is very very simple, very sophisticated, very sturdy equipment. So, modern day we are using micro HPLC. And actually by using this, you are now getting very faster separation sometimes in minutes.

[00:10:48]So, this branch of separation is continuously developing to get more and more finer and accurate separation and accurate results.

[00:11:01]So, the classification of HPLC. So, HPLC can be classified into four major categories, namely the normal phase HPLC, the reverse phase HPLC, the ion exchange HPLC, and the last one, the size exclusion HPLC. In normal S phase HPLC, the stationary phase the stationary phase is generally stationary phase is generally, you can say, polar in nature.

[00:11:30]It is polar in nature, and your mobile phase is actually nonpolar.

[00:11:38]It is nonpolar.

[00:11:40]Now, the common example of polar stationary phases is, for example, alumina. For example, silica. For example, polyvinyl alcohols.

[00:11:52]They are the examples of polar stationary phase. Similarly, they are example of of nonpolar mobile phase heptane, hexane, benzene. Now, here the different components get separated on the basis of the difference in their polarity.

[00:12:12]Now, this normal phase HPLC is particularly beneficial for the separation of nonpolar, actually, compounds. And why I'm saying that nonpolar? Because if your compound is nonpolar, then it is going to interact with your polar compound. And since it's a combination of a nonpolar polar, then the interaction between the solute molecules, which are nonpolar here, and the stationary phase, which is polar, so the interaction will be not too much.

[00:12:47]The The interaction will be weaker. And if the interaction is weaker, then they are going to elute at a good rate. They are not going to have They are not going to stay for longer times.

[00:13:00]But on the other hand, if you are using this normal phase HPLC for the separation of some polar compound, for example, for pharmaceutical drugs, which are actually most of the time polar, then those pharmaceutical components present in that particular drug is going to interact very strongly with the stationary phase. Then the elution will become a problem. It will get taking too much long time.

[00:13:24]So, this normal phase HPLC is very good for the separation of nonpolar compound, for example, for the separation of of essential oils.

[00:13:37]Okay? Essential oils.

[00:13:41]So, the normal phase HPLC is based on the difference in the polarity. Or here, polarity is actually the principle of separation of different components of a mixture. Similarly, in reverse phase HPLC, the stationary phase here, the stationary phase is generally nonpolar.

[00:14:09]The stationary phase is generally nonpolar.

[00:14:13]While the mobile phase, while mobile phase here mobile phase here is actually polar.

[00:14:25]So, hydrophobicity is the basis of separation of reverse phase HPLC. And since the stationary phase is nonpolar and the mobile phase is polar, that is why it is also this it is also called as the reverse phase HPLC. Opposite to the normal phase HPLC. Now, this type of HPLC is good for the separation of, you can say, polar compound. Because the underlying theme of separation is that if your stationary phase is nonpolar, then it is very well It is effectively separate the polar compounds. So, it is very good for the separation of pharmaceutical for the pharmaceutical analysis. This reverse phase HPLC is there. And most of the time, nowadays, we generally use reverse phase HPLC.

[00:15:21]Then comes the ion exchange HPLC. Here, the separation occurs on the basis of the difference in charge.

[00:15:29]Difference in charge.

[00:15:35]Okay? So, the different components are separated on the basis of the difference in the charge or difference in polarity.

[00:15:43]Then comes the size exclusion HPLC. Here, what happens is that the separation occurs on the basis of difference in the size of the analytes which are to be separated. Generally, the stationary phase taken in this size exclusion HPLC is the porous medium. So, what happens is that the solute molecules enters into the pores of the the stationary phase. And those solute molecules, which are small in size, they just enters into the pores, while the large size particles elute steadily. So, here the principle of separation is the difference in difference in size of particles, size of particles.

[00:16:45]So, this is how these four methods are based on different separation technique.

[00:16:50]Normal phase HPLC means that is here, they are separation is based on the polarity. In reverse phase HPLC, the separation is based on hydrophobicity.

[00:17:00]Here, in ion exchange one, the separation is based on the difference in charge, while in size exclusion, that is based on the difference in the size of solute particles.

[00:17:11]Another specialized technique is the affinity HPLC. This type of HPLC is most frequently applied in the separation of biomolecules. And here, your stationary phase actually acts as a perfect ligand, and it binds with the predetermined target molecule. And on the basis of the targeted binding, the separation occurs.

[00:17:38]That is in affinity HPLC. It is mostly used for separation of biomolecules and other biological samples.

[00:17:47]Then comes the important aspect that what are the components which are to be used in HPLC system?

[00:17:55]So, just take a quick review or quick overview what are the different systems. So, of course, we have a solvent reservoir.

[00:18:05]Then we have a pumping station.

[00:18:07]Then again, we have an injector.

[00:18:09]And from injector, the sample, along with the solvent, here solvent and the sample mixes, and that goes to the column, which is actually the heart of HPLC, just like that in the GC.

[00:18:27]In GC, the column were also regarded as the heart of GC. Similar to that, in HP also HPLC also, the columns may be regarded as the heart of HPLC system.

[00:18:41]Then comes the detector. That is the eyes of the HPLC.

[00:18:46]Detectors are very important because, you know, the separation has been done.

[00:18:51]The separation has been done by column.

[00:18:53]Now, how you actually identify?

[00:18:56]So, identification is done by this detector.

[00:19:00]And the signals of a detector in terms of readable units, in terms of readable graph, is given by this data system in the form of a chromatogram.

[00:19:13]So, these are the essential components of an typical HPLC system. So, what is the basic principle of HPLC? Of course, here the separation in HPLC is done by the difference in the distribution constant of different analyte of different components present in the analyte.

[00:19:34]So, the separation occurs on the basis of differential distribution of various components present in that given mixture.

[00:19:45]Now, we have a stationary phase.

[00:19:48]Just like the other chromatographic process techniques, we have a stationary phase.

[00:19:52]Now here the stationary phase is actually a packed column.

[00:19:57]And the stationary phase is actually composed of finely divided finely divided uniform size particles.

[00:20:06]Okay.

[00:20:08]So here the column is uniformly packed by the smallest particle possible smallest particle.

[00:20:15]Then we have a mobile phase that is called as a solvent system. And again, you see a typical chromatogram.

[00:20:23]Now from just from seeing this chromatogram, we can infer that this is actually a chromatogram for four component system for four component system.

[00:20:39]Four component system.

[00:20:43]Now you can clearly see that if you call this component one, you call this component two, you call this component three, you call this component four.

[00:20:57]Then >> [clears throat] >> from the chromatogram, it is quite clearly visible that initially component one is coming at at at TR equal to 2 minute.

[00:21:13]Component two is coming at TR 5 minute.

[00:21:17]Component three is coming at TR 9 minute and the last component is coming at TR is equal to 12 minute.

[00:21:26]So these different components are coming at different times and this time is called as the retention time.

[00:21:32]This retention time may be regarded as a time elapsed during the interaction of component one with the stationary phase.

[00:21:42]TR2 represents the time elapsed time taken for the interaction of the stationary phase with the component two. Similarly for three, for four. Now these different times actually telling you that the component four is having the strongest interaction with the stationary phase.

[00:22:06]That component two is eluting at last and that implies component component four is having strongest interaction.

[00:22:24]strongest interaction with the stationary phase with stationary phase.

[00:22:37]Similarly, component one which is eluting which which is eluting the most quick in the most quick manner can be regarded as component one has strongest interaction strongest interaction with mobile phase.

[00:23:06]With mobile phase. So you can see that how actually chromatogram tells you about the various about the possibility of various interactions with the stationary phase or the mobile phase. So the component which is eluting last is having the strongest interaction with the stationary phase and the component which is eluting first has the strongest interaction with the mobile phase. And since component one has the strongest interaction with the mobile phase, that is why it is eluting most quickly then two, then three, then four.

[00:23:38]So as you progresses from here to here, as you go from here to here, the interaction with the stationary phase increases.

[00:23:47]Or with the mobile phase vice versa.

[00:23:51]So the point which is to be remembered is that if elution for first elution means that strong interaction with the mobile phase and last eluting species means the strongest interaction with the stationary phase. Okay? Clear now? Look, okay. Let's move further. Now theoretically, how we can define partition coefficient?

[00:24:16]Because this separation is based on the principle of partition coefficient.

[00:24:21]Different components of analyte separate on the basis of the difference in their partition coefficient. Which may simply be defined as the concentration of the solute in the stationary phase upon the concentration of the solute in the mobile phase.

[00:24:41]You can also define in terms of number of moles like this. Number of moles of solute in the stationary phase divided by volume of solute in the stationary phase upon number of moles of solute in the mobile phase divided by volume volume of mobile phase. So you can define in terms of like this.

[00:25:07]NS upon VS divided by NM upon VM.

[00:25:10]Okay? Where NS is the number of solute in stationary phase, NM number of moles of solute in the mobile phase, VS and VM are the volume in stationary and mobile phases.

[00:25:22]So the important thing which is to be remembered is that okay, all the components are separating on the basis of the difference in their partition coefficient. Or similarly, we can also decide this in terms of distribution constant which is the same as that of partition coefficient which is described here.

[00:25:44]But the problem is we cannot measure these partition coefficient or the distribution constant of an analyte directly. So we need certain quantity.

[00:25:57]We need certain measurable quantity which must be proportional to this partition coefficient or parti- or you can say distribution constant. So the answer is we have the retention time which is directly related to K. And we can measure this retention time that is the TR. And this TR is specific for every compound. And this TR is specific and it is unique and it actually helps in understanding the quantitative and qualitative idea about the presence of any analyte present in the sample which is being analyzed by HPLC.

[00:26:45]So there are certain modes of HPLC separation based on the different principles. For example, we have adsorption chromatography.

[00:26:56]Adsorption chromatography is actually based on the adsorption of various components on the stationary phase.

[00:27:05]And that because of the difference in the adsorption rates, we actually all the analyte of all the components of the analyte comes with different migration rates and elute at different times giving you a good separation. We have partition chromatography which is also called as the liquid-liquid partition.

[00:27:24]Here the analyte gets partitioned between the liquid liquid which is actually immobilized on the solid surface and the station and the liquid phase which is also liquid. So the analyte is get partitioned between those two liquid and that is why it is called also called as liquid-liquid partition. So we have adsorption chromatography and the then the third one is the ion exchange chromatography.

[00:27:51]This type of chromatography is based on the charge separation.

[00:27:56]Okay? Similarly, the we have size exclusion chromatography. This size exclusion chromatography is based on the difference in size of the solute. So that if the here the stationary phase is generally porous in nature and those solute molecules whose size is less than the pores of the stationary phase, they just go inside and they get trapped, they gets retained for larger times. While the particles of the solute which are bigger than the cavity of that pores, they just go out with quickly. That is the elution uh actually happens very quickly. In affinity chromatography, it is very specific type of chromatography. Here the stationary phase actually acts as a ligand and it binds the specific target of the solute. So this type of chromatography is generally used in analyzing the biological biological samples for the separation of proteins, for the separation of enzymes, DNAs, etc. etc. >> [music]