Lecture 28

Added: 2026-05-09

[00:00:20]Hello students, welcome to this massive open online course on advanced analytical technique.

[00:00:28]My name is Shahab Alazar Nami and presently I'm serving as associate professor of chemistry in department of industrial chemistry Aligar Muslim University Aligar. So this is week seven of our course and in this series of lecture we will be discussing the chromography. We will be discussing important basic terms which we are using in our coming slides in our coming lectures and we also study the various chronographic separations.

[00:00:59]So chronography is actually a branch of science that deals with the separation of constituents of a mixture. Have you ever wondered that whenever you write using a uh pen on a moist paper then you can see that the it actually ink separates into a colorful rainbow.

[00:01:20]Similarly, have you ever wondered that how the scientists are able to detect uh very small quantities of a particular constituent in a given sample that is sometimes up to a level of nanog sometimes up to a level of phentogram.

[00:01:38]So the answer to these two intriguing questions is the same that is chromography is the answer. Chromography may be defined as the art of separation of different constituents of a given mixture. Actually the separation science is regarded as the heart of chemistry.

[00:02:00]From time im memorial humans are using different methods to separate the analytes of a given mixture. So initially the humans have used filtration, decantation and a number of methods. So it was the genius of a Russian botonist whose name was Mikail Sweat who actually invented this technique this technique of separation that that actually called as chromography. The word chromography in fact is made up of two Greek words that is chroma and graphy.

[00:02:44]This chroma which means colors and graphi means studies.

[00:02:51]So actually Mikail Su in 96 invented this beautiful versatile separation technique to separate different plant pigments particularly chlorophyll chlorophylls and zenthophils. So chronography may be defined as a analytical technique for identification for separation for identification for qualitative estimation as well as for the quantitative estimation of different components of a given mixture. Now there are certain terms which we shall be using throughout uh this unit and in the coming unit also. So let us you know go through those basic terms. The first and the foremost term which we are using which we'll be using here is the stationary phase. A stationary phase may be defined as a fixed medium. It may be solid or it may be liquid immobilized on some solid support and it is the stationary phase on which the separation occurs. Similarly we will be using mobile phase. Mobile phase will be defined as a solute. A solvent may be defined as a solvent. It this solvent may be in liquid state or sometimes it may be gaseous by which the you actually mix your uh compound which is to be separated and by using the mobile phase you actually uh separates on the stationary phase that is the uh partition or you can say the separation of different constituents occurs on the stationary phase. So here we have a quick view that how actually this separation occurs.

[00:04:45]It's a typical chromogram. You can see that here the bands the three different colors are moving. So these this space is called as the stationary space and you keep on adding mobile phase fresh mobile phase each each time and because of the addition of mobile phase your different components gets uh separated in the form of bands. So you can see that these bands are going down. This is the column and these bands are going down. The movement of your mobile phase is by the action of gravity and the passing away of uh the mobile phase is actually called as illusion.

[00:05:36]Illusion that is washing away. Illusion may be simply defined as the washing away or simply passing away. So the terms which we are using is the stationary phase a fixed medium it may be solid or it may be a liquid immobilized on a solid surface.

[00:05:53]Similarly the mobile phase which actually helps the uh solute particles to separate on the stationary phase.

[00:06:02]Then we have we are using the word analyte. Analyte will be defined as the component or a mixture of component which has to be separated. we have illusion that is the passing away of the mobile phase along with the different constituents through the column and this illusion occurs by the action of gravity. So here what happens is that the why the different uh components elute from the uh uh from the column at different times is because that each component interacts with the stationary phase in a different manner.

[00:06:40]And this is because of the differential migration of the different components this illusion take place. That is what in simple sense you can say that the different components interact with the stationary phase differently and each component has a different interaction in terms of strength.

[00:07:06]So greater the strength of interaction, greater will be the time it will spend with the stationary phase and greater will be its you can say retention time. Retention time means that is the time taken by a particular uh you can say component with the stationary phase. So what happens is that here the basic principle of separation is the differential you can say migration rate and this differential migration rate occurs because of the difference in the interaction of the different constituents with the stationary phase. So the separation occurs because of the differential interaction that leads to differential migration.

[00:07:56]So here the solutes get partitioned between the stationary phase and the mobile phase.

[00:08:03]Now [clears throat] the fundamental principle or the basic principle of the chronography is the differential affinities.

[00:08:12]So each component interacts with the stationary phase in a different manner and since the interactions are different so each component is going to stay for a different time with the stationary phase and on the basis of the difference in the staying there is a difference in the illusion or you can say in the going away of that component from the from the column that is Each molecule actually [clears throat] has a different interaction with the stationary phase and because of that each component has a different retention time. Retention time is the time taken by the particular component with the stationary phase. So stronger the interaction greater time it will have with the stationary phase and greater the time that is you can say greater the retention time it is going to stay for more time or you can say then it will elude last. Okay. So similarly on the contrary if the interaction is less then the illusion will be fast. Okay. So as a whole you can define as the the components gets partitioned between the stationary phase and the mobile phase and you can say if you have a single component system then that component will going to equilibrate between the stationary phase and the mobile phase. That is there is an equilibrium. And if you want to uh give this equilibrium in terms of K then that may be defined as equilibrium of K of stationary phase upon concentration of A in the mobile phase.

[00:10:07]Similarly this is also called as the distribution constant. And this distribution constant may be simply defined by capital K that is CS upon CN concentration of the solute in the stationary phase divided by the concentration of the solute in the mobile phase. You can also define this K as the number of moles of solute in the stationary phase divided by the volume in the stationary phase upon number of moles of solute solute in the mobile phase upon volume of the mobile phase.

[00:10:45]You can also define like this.

[00:10:47]So this distribution constant which is actually different for each component is actually the measure of the interaction with the stationary phase and the mobile phase. Now the problem with that distribution constant that you cannot measure it directly. So in the coming slides we will see that how we are going to quantify this important characteristic feature that is the distribution loss.

[00:11:17]Let us look into the historical evolution that how this technique gots evolved and how different you know stages are there. So in 96 it was the genius of Mikail Sweat the Russian botonist who invented this technique to separate different plant pigments like chlorophyll zanthophils on a glass column filled with calcium carbonate. So he used actually calcium carbonate to separate chlorophyll and zentoils.

[00:11:47]Then comes another advancement that is called as the paper chronography.

[00:11:53]This technique was exclusively used by the organic chemist. And here in paper chronography they actually use a strip of paper and on that paper they take the mixture which is to be separated and they also take some authentic sample so that they can be compared and then they put this paper in some cylinder or in some beaker with certain solvent which acts as a mobile phase. They dip it little bit and then what happens is the solvent system rises and this also rises and after a certain time they show a different colors and on the basis of color you actually identify that if there are three spots then you can say that it's a three component system and each component is have a separate RF value that is called as the retention factor and this RF value is correct characteristic for each component.

[00:12:56]So that was the advent of paper chronography and by using the prop paper chronography you actually identifies a particular component. You actually check the purity of the sample. So already chemistry people use this technique to monitor their reactions also. Similarly in 1950s there comes a revolution and this technique of initially the technique of illusion chronography got advanced and it evolved as gas chronography and the gas chronography is the versatile technique of separation of volatile compounds.

[00:13:36]So it was the best technique to separate volatile compounds in just a matter of few hours. You can separate a multicomponent system, a multicomponent mixture. But the issue was that this technique was good for separation of only the volatile components. Then comes the high performance liquid chromography or HLC.

[00:13:59]Sometime it is also called as high pressure lipid chronography because here the pressure you need is very much sometimes you need a pressure of suppose 5,000 PSI this technique was versatile in the nature that you can use HLC to separate nonvolatile compounds also you can separate the thermally label compounds by using this HLC so HLC has changed the whole scenario of separation science Then comes the most advanced technique that is called as UHPLC or ultra high performance liquid chromography.

[00:14:38]So this was more advanced than HLC. And now you can separate even ftograms of your compound. Okay. Ftograms.

[00:14:48]This is highly sensitive. This is highly specific technique of separation. Then comes the LCMS. Then the microchip, microchip based chronography that is now you can have instead of having using a very big big instruments now you can separate your mixture in just by using a just a chip and you only need nanol of sample.

[00:15:17]So this has actually changed the whole scenario and again the sensitivity is very high specificity is very much and these techniques are fully automated in nature. So this has changed the whole aspects of separation.

[00:15:33]Now this chronography is not only important for the chemistry persons to identify the unknown compounds or to check the purity of samples but they are also good for the biology people because this chronography can be exclusively used to separate biomolelecules to separate proteins to separate nucleic acid to separate peptides and there's a specific chronography which we are going to see that is called as affinity chronography. in the coming slides where you can exclusively se separate your biomolelecules. Then comes the pharmaceuticals.

[00:16:12]The this chronography is exclusively used in pharmaceutical uh research or pharmaceutical analysis because it can uh give you an idea about the purity of your drug about an idea about you can do some bioequivalence studies by using this. You can actually standardize your drug by using different chronographic techniques. Foreign6 nowadays this GC or this HLC has also gained very much uh importance in the area of foreignics. In fact, now on this is chronography by which a traffic police person can check uh that whether a driver has drunk or not by just checking the alcohol that whether he has uh taken some alcohol or not. Similarly, you can also check narcotics even nowadays uh in various accidental cases and in various uh suicides. This is checked by using the various chromographic techniques that is the whether a person has consumed poison or not. Similarly in environment it has made a big throw that is now you can check the trace uh uh level pollution by using different chromographic techniques. Similarly in food industry it becomes mandatory to have a proper you can say specification of the different constituents present in a given food item. So this chronography is actually become a multifaceted technique and it is it has found significance in almost all the branches of science.

[00:17:53]Now why chronography is gaining so much importance? It is because of the high sensitivity. Now you can detect fto level concentration that is up to a level of 10 to 15 g. You can understand the sensitivity such high sensitivity.

[00:18:10]Similarly, versality you you can you can uh you can identify gases, you can identify solids, you can identify liquids, you can identify polymer material, you can identify biomolelecules, you know just name the compound and the answer is with the cryptography. So versality is there as I have already discussed that it is a technique which can give you identification which can give you a qualitative idea as well as the quantification of different constituents present in a given sample. So it is it serves all the purpose of separation.

[00:18:49]Similarly the size you know you can only need you only just need nano levels of or nano liters of sample and in just such a small quantity you can check all the components. Okay. So scalability you can you have seen the nano you have seen the lower limit and now you can see the higher limit you can use this technique for industrial purpose also for large scale industries also.

[00:19:19]Okay. So these features make this chronography a versatile technique. Now how this chronography actually works?

[00:19:27]What are the different you know uh what are the different you can say steps involved in the chronographic separation. So initially you have to have your sample. So you must you must have a sample which is to be separated into its constituent uh into its constituents. Then you have to have a certain mobile phase. It may be a it may be a liquid. It may be a gas depending upon the nature of analyte which is to be separated depending upon your requirement of separation.

[00:20:01]Similarly, now after choosing the mobile phase, you have to have a stationary phase also. Then what happens is that partition occurs between the different constituents and because of the difference in partition they migrate differently and because of the difference in migration they elute at different times. So you have a visualization and because of the illusion at different times they appears as in the form of bands they appears in the form of zones peaks because you get a chromogram which is like this on the x-axis you have time and this time is generally called as retention time.

[00:20:43]Retention time and on the y-axis you have detector response.

[00:20:50]detector response.

[00:20:53]So you need you actually get this type of this is for a two component system. For example, you have two component A and B.

[00:21:08]So each component actually comes in the form of a peak or in the form of zone which we have seen in the previous slide. And this peak actually gives you an idea about the mobile phase illusion of mobile phase alone. We will discuss this point. So this time up to half of this peak is called as TR of A. And this time half of this peak is called as TR of B. That is retention time of component A. T R A retention time of component B that is the time taken by the component to pass from the column. Okay. So detection you can get a quantitative idea as well as a qualitative idea that is you can have information that how many different components are there and if you know the number of component it also gives you that how much each component is there in the given mixture. So you can classify the chronographic techniques into a number of ways. You can classify on the basis of the principle of separation on the you can classify chronographic techniques on the type of or on the state of mobile phase used. You can also qualif you can also classify the chronographic techniques on the basis of the format on the basis of shape. So on the basis of principle of separation chromographic techniques may be classified as absorption chronography, partition chromography, ion exchange chronography, size exclusion chronography and the specific and specialized affinity chromography which is exclusively used for the separation of biomolelecules. So we are going to see each type of chromography in detail in the coming slide. Similarly on the basis of the state of mobile phase we can classify the chronography as gas chronography where the mobile phase is gas. We can also classify it as liquid chronography where the mobile phase is liquid.

[00:23:19]Similarly on the basis of format or shape we can classify them as column chromography as well as the planer chromography. We are going to study each of these in the coming slides in detail.

[00:23:30]So on the basis of principle we can classify it into five categories namely the absorption the partition ion exchange size exclusion and the affinity. In absorption chromography the principle of separation of different constituent is the differential absorption of components on the stationary phase.

[00:23:55]That is here the principle is the surface binding because absorption is a surface technique.

[00:24:03]Similarly the partition chromography here the different constituents of a sample separate on the basis of the differential partitioning of components between the two immissible liquids.

[00:24:20]Okay, we are going to see it in more detail in the coming slide. Similarly, we have ion exchange chronography where the principle of separation is the difference in charge. Okay. So, different constituents separate on the basis of difference in charge with regard to the stationary phase.

[00:24:36]Similarly, we have size exclusion chronography where the principle of separation is the difference in the size of the different constituents of the mixture. Similarly, we have the affinity chromography which is very specific one where your actually stationary phase acts as a liant and it captures a specific targeted molecule uh in the which is coming uh as a analyte.

[00:25:09]Now absorption let us dwell into the mechanism here. The separation occurs on the basis of the absorption of the different constituent of the analyte and the different components get absorbed on the surface by weak interactions that may be wonderall interaction that may be hydrogen bonding or there may be some weak electrostatic forces. Here generally you take stationary phase as a silica gel you take aluminina sometime you also take other stationary phases like polyvinyl benzi polyvinile alcohols etc etc depending upon the nature of the analyte here you take actually the mobile phase mobile phase is generally you can say organic compound organic liquids you start from non-polar you start from benzene hexane chloroform methanol and you keep on changing and this type of chronography where you keep on changing the polarity is sometimes also called as the gradient illusion chronography. Okay. And on the other hand if you are taking the solvent or if you are have a fixed solvent system then that type of chronography is called as isocratic illusion chronography and the common examples are the TLC that is the thin layer chronography or column chronography. This type of chronography is very good for the separation of natural products, for the separation of common organic compounds and also for checking the purity of a given sample.

[00:26:45]Then comes the partition chromography.

[00:26:48]Here the basic principle of separation of different components is the differential partitioning of the components between the two immissible liquid. Here what happens is the stationary phase is actually gets quoted by a mobile phase.

[00:27:09]Okay. And now the solid is coated with a mobile phase or you can say immobilized on that solid support and your liquid which is a mobile phase. And now the partitioning of different constituent occur between this stationary phase and the mobile phase and these two liquids must always be immissible.

[00:27:33]Okay. So here the basic principle is the partitioning of component between two immissible liquid. So this type of uh chronography is used for the separation of amino acids for the separation of pharmaceutical drugs. Similarly this is also suited for the separation of metabolomics. Okay. So this is about partition napy. Then comes the ion exchange roy. Here the separation occurs because of the difference in the electrostatic charges which are present in the solute or in the or in the mixture. Here the sep stationary phase contains certain charge group and because the eludes or the you can say your components also bear certain charge. So they opposite charge get attracted and they on the basis of the attraction certain ions pass quickly while certain retain and after some time they also pass. So this type of chromography is very useful for separation of amino acids for the separation of enzymes and also for water dalination. Here we use aquis electrolytes as the mobile phase.

[00:28:58]Then comes the size exclusion chronography. This is also called as gel filtration chronography. Here the stationary phase contains certain pores components. According to their size, the sum of the components which have comparable size with the pores that are present in the stationary phase, they get trapped. While the particles which are greater than the size of that pores, they just pass through. And the particles which are bigger in size than the pores of the stationary phase takes shorter path or you can say elute quickly. Here illusion.

[00:29:36]Illusion is quick.

[00:29:40]And the molecules which gets entered or get trapped they elute elute later. Okay. On this basis of the difference in the elusion pattern the different components get separated and this type of chronography is very good for determining the molecular weight of polymers as well as for the protein fractionation. Okay. So now comes the apheric chronography. This type of chronography is very good for the separation of biomolelecules. What happens is that for example if you have a column if you have a column and this column is made up of you can say is made up of a specific type of a specific type of ligance. Please.

[00:30:53]Now let us suppose they have a specific type of ligant which have this type of you know stationary phase and you have mixture which contains this type of particle triangular circular and some squares.

[00:31:13]Then when you pour this mixture along with mobile phase, mixture plus mobile phase, then what happens that those which have which are square in shape which are square in shape they get trapped.

[00:31:39]they get trapped while the other one they go as such. So here you can see that illusion occurs very in a very specific manner. Now this may also be regarded as a lock key model. So here the liant actually immobilized on a resin and it has a specific you know a specific uh opening where a particular biomolelecule gets trapped. So it is very specific for a specific type of separation. And here the final illusion occurs by the salt gradient or by competitive binding. And this type of affinity chromography is very good for the separation of biomolelecules for the DNA protein binding etc etc. So the separation the chronographic classification on the basis of the mobile phase on the basis of mobile phase you can classify for example if your mobile phase is exclusively for example is if it is exclusively a gas then you can call it the gas chronography and generally in gas chronography the mobile phase is taken an inert gas generally the helium argan and sometimes times nitrogen also. So in gas chronography the mobile phase is always a gas while in liquid chronography the mobile phase is always a liquid solvent. Okay. Similarly we have another type of chronography which is called as supercritical fluid chromography or sfc. Here the mobile phase actually a super critical fluid generally super critical carbon dioxide.

[00:33:31]So we are going to study the gas chronography in unit four after this unit.

[00:33:37]Now the gas chronography it is one of the versatile technique of separation.

[00:33:42]This technique has actually made a breakthrough in the separation science.

[00:33:46]Here the mobile phase is always an inert gas and the use of this the main objective of the mobile phase is to take your sample to the column where actual separation occurs. So mobile phase only act as a carrier and it must be inerted because if it is not inert then it then there are chances that it will react with the different constituents of the mixture and that actually leads to improper separation.

[00:34:19]Similarly in gas chronography you actually coat your stationary phase with certain uh liquid and then the separation occurs. So GC is very useful that is grass chronography is very useful for the detection of environmental pollutants for the detection of uh uh for petrochemicals for checking the purity of drugs etc etc. Now the biggest advantage of using GC is that it gives you very good well- resolved peaks and it's a very rapid techniques but at the same time you can only separate compound separate mixtures of limited size of limited volume. Okay, this is one of the drawback and this technique is only good for volatile volatile compounds.

[00:35:13]Okay, you cannot separate non-volatile compound by using GC because your sample is to be in the volatile state so that it can mix with the carrier gas. So if your sample is a liquid then it is to be initially it is to be volatized before passing it to the main column where separation occurs in the gas chronograph. Then comes the liquid chronography. Here the mobile phases always are liquid or you can also use aquis systems aquis buffers you can take. Here the stationary phase may be a solid or it may be a solid which is coated by some liquid. So paper chronography is the best example. In paper chronography the it generally the paper is a cellulosic paper. So the holes of that paper contains the water and that trapped water actually acts as a stationary phase and the liquid the solvent in which you dip your paper acts as a mobile phase. So the common example is TLC. Another that is the thin electronography. This is HLC that is the high performance liquid chromography.

[00:36:29]We will be you we will be studying this chronography in detail after GC in unit 4. So this is very good for the bioctive profiling for the pharmaceutical uh quality checks and for the metabolic profiling also.

[00:36:46]Now the superc critical fluid chromography this is very specific technique not most not commonly used but again it has very specific applications here actually we take super critical we take carbon dioxide as the superc critical substance and above its superc critical point the beauty is that it acts as a gas-like and it also have some liquidlike properties. So it combines with the speed of gas chronography and it has the selectivity of the liquid chronography. So you can actually monitor you can separate the thermally labile compounds by using the SFC which you cannot separate by using simple GC.

[00:37:28]Similarly you can separate kyal compounds using this sfc that is super critical fluid chronography. Now comes the format based. You can also classify the chronography on the basis of the format that is whether your stationary phase is packed in a vertical column then you call it a column chronography because it is packed in a column. Now if the stationary phase is spread in a 2D surface for example the paper which we have given to you this paper contains pores and in these pores the water is trapped and this type of chronography is called as the planer chromography.

[00:38:03]Similarly, we have a capillary chromography where the separation occurs in the micro capillaries.

[00:38:11]Get few information simple information about chromograph about column chronography. Here the mixture is applied on a packed column and it is the column chronography which was the first type of chronography used by the Mikail SWE. Actually he taken a glass column and in that glass column he filled calcium carbonate. So calcium carbonate acted as a stationary phase and on that stationary phase he separated the plant pigments that is the chlorophyll zentos etc etc. So here the solvent flows you actually here you add you you have a column and this column contains certain stationary phase and what happens is on this You take your mobile phase plus your mixture which is to be separated and you just pour it. So it percolates and the sample travels by the action of gravity. Sometimes you can also put pressure also. In GC you actually use pressure. In HBCLC you'll use pressure because that column is very tightly pegged in HBLC and you use extra pressure sometimes up to 500 PSI you need pressure so that you can actually pump your solute along with the analyte along with the the uh you can say mixture where actually main separation occurs. So here the components elute at different times and because of the difference in illusion times you actually separate each component. So this is very useful for the separation of natural products for the separation of inorganic compounds also. Now the paper chronography it is one of the most used type of chronography in the laboratories to monitor the reactions also. Here the stationary phase is again water molecules bound or trapped in the in the in the pores of the paper. So here the mobile phase you can take any solvent and on the basis of change of polarity you actually get a good separation. So you keep on changing the polarity you keep on getting a good separation. So you can visualize these colored spots sometimes by a naked eye and sometimes you have to develop these in terms of in by putting it in the chambers of iodine where uh the components make reversible bond with iodine and they they actually comes in more bright colors. You can check uh the purity of food comp of food colors or amino acids and various pigments by using this paper chronography. Then the TLC here the stationary phase is silica. Generally this is also a laboratory technique where you use glass plates and on glass plates you coat silica or aluminina and then you uh put your sample by using a capillary.

[00:41:21]Again you have a glass slab and on that you actually coat it with silica silica gel and on that you then you add your mixture as a pores by using capillary fine capillary fine capillary and then you put it put it in certain beaker and in beaker for example is is a beaker and in beaker You use certain certain mobile phase and what you can do you change the polarity change the polarity of the mobile phase polarity of mobile phase and the spots rise and after that you take out and you sometimes the spot spots are visible in the naked eye otherwise you can put in in iodine chamber and after a certain time the spots uh they gets developed and you can they are more visible. So you can check the reaction that whether reaction is going on in the in the right direction whether a mixture of compound is forming or there is a single product formation so you can check it. So you can use u lamp you can use i addin chamber sometime you can also use fluosense tagging by adding some certain fluorosence agent so that you can get a good fluorescent uh peak. Now the hlc this is one of the most versatile technique. So here you use high pressure pumps instead of gravity alone you actually have need high pressure because the columns here are tightly packed by the stationary phase and in order to pump your mixture along with the mobile phase you need high pressure pumps. So this is very highly efficient technique. It gives sharp peaks. Very good quantitative idea. Very reproducible result you get.

[00:43:26]Now here in HLC you can have a number of different detector. You can use UV detector that is ultraviolet visible detector. You can use fluoresence detector. You can use diode array detector. You can use a number of detectors depending upon the nature of sample given to you for separation.

[00:43:46]Similarly, sometimes you can also use mass spectrometer detector and by using this MS detector that is a mass spectrometer detector, you can also get an idea in you can also get the structural information of the different components. So you can use a number of detectors and also you can use more than one detector at once that is you can use hyphenated detector.

[00:44:09]hyphenated detector that is more than one detector. For example, you can use UV plus MS. This is very good for detecting the drugs for checking the uh purity of drugs in clinical diagnosis as well as in the biomolelecule profiling. So this is one of the most advanced separation technique.

[00:44:34]Now there are other special technique also for special purposes. For example, we have capillary electrophorosis. Here the principle of separation is the charge and the size and the separation occurs in very narrow capillaries.

[00:44:48]Similarly, we can have liquid chromography mass spectrometry. here that we have already discussed. You couple H you couple your HLC equipment that is high performance liquid chromography equipment with mass spectrometer so that you can also get identification you get a qualitative idea you get a quantitative idea and by using the MS you get the structural information of the different analytes of the different or you can say or the different components present in the supplied mixture. Similarly nowadays you have a very small miniaturaturized systems where you can check the purity of a sample in just few minutes and you only need nanole of sample. You can analyze in a very small sample and you can also check the purity of that particular sample. Now let us get a comparative idea about the various techniques and the applications which each technique gives you. For example, absorption chronography. Here the basis was the surface interaction. In this case, the different uh components of your mixture gets a surface uh interaction, they get absorbed on the stationary surface and because of the difference in the absorption because of the difference in absorption strength they gets retained for different times and they elute at different times. So a common example is TLC and you can separate natural products by using absorption chromography. Similarly you have partition chromatography where the different components gets partitioned and the basis of separation was the solubility and the best example is the high pressure liquid chronography and you can separate different drugs different metabolites by using HLC.

[00:46:37]Similarly you have ion exchange and the separation basis was the difference in the charge.

[00:46:44]Now a good example is the protein purification where you actually get different positive and negative ends. So you can all the biotechnology laboratories actually use this ion exchange chronography for separating different components of protein or other biomolelecules. Similarly, you have size extography where the basis of separation was the difference in the the molecular size. And a good example is the gel filtration chromography where actually your stationary phase was a porous and it contains different pores of different sizes. And those molecules of the mixture which has comparable size to the pores, they get strapped, they get fit in. While the bigger molecules they just elute very quickly and after after a certain time the trapped molecules also eluted and a good separation was done.

[00:47:41]Similarly you can separate proteins and polymers by using size chronography.

[00:47:45]Then comes the last one which was very specific type of chromography called as the affinity chronography. It actually was based on a specific binding of the stationary phase with the analytes of the mixture and it is very good example is imuno affinity and you can separate monoconal antibodies by using affinity chronography. It was also good for separation of proteins also.

[00:48:11]Now what are the advantages of chronography? It gives you high selectivity and very good sensitivity.

[00:48:18]Selectivity was very high and sensitivity was very good. For example, sometimes of the range of 10 to the^ minus 15 depending upon the detector.

[00:48:32]In case of GC and HLC that which type of detector actually you are using so on that basis you can have selectivity as well as sensitivity. So you can separate complex mixture by using HLC and you need only microscopic amounts. You do not need samples in ml or in 10 ml sample you need or one liter sample you just need only nan of sample that is up to a level of 10 to the power - 9 liter.

[00:49:04]So you can see that in just such a small amount you can get an idea about the number of component present and you can also get an idea that how much each component is there. So by coupling different detectors you can also get structural information as you have seen that by using MS detector you al can also get a structural information of the components. Similarly, it has actually become a very important technique for almost all the branches of science wherever separation is involved and also in industry. So there are certain limitations also of this chronography that in certain cases you need very costly equipment. For example, HLC is very costly. Even GC is very costly. It comes in more a good GC with a very good sensitivity. It actually the equipment comes in 25 lakh rupees. So for such a costly equipment you also need very specialized or very technical type of person for doing the analysis for performing the analysis. Similarly certain chronographic techniques are consuming time consuming for large scale and there is actually nowadays we are using programmed GCS which has actually cut short the runtime that is skilled labor is required for example in GC and HLC and sample preparation is very important for example when we were when we will discuss the GC then we will see that sample injection is very important And there are different ways there are different techniques by which we can actually introduce our sample. We have splitless injection we have you know we have head-on injection. So depending upon the nature of the sample we have to decide the s the sample injector port also. So now there are certain real world example that where HPS where this chromography comes for our help. So food safety for example for checking the melamine content for checking the artificial dyes in food products. So HLC is the answer to detect all these. Similarly in foreign 6 for example if certain uh hanging is occurred or certain you know uh if certain poisoning is there. So autopsy in autopsy you need specialized uh GC for analyzing a particular body part. Similarly in pharmaceuticals to check the impur to assertain the the percent purity you need this DC to check this. Similarly in environment to trace the heavy metaline concentration to check the pesticide residue or other insecticide. So GC can give you a very good information. Similarly for the purification of uh hormones for example insulin or to a certain certain growth factor. So by using the affinity chronography you can have a very good idea you can detect to a very good sensitivity. So with this we can have the chronography. Now let us quickly recap and see that what we have learned today. So chronography is actually the heart of separation science.

[00:52:27]We can classify the different chronographic technique on the basis of principle on the basis of mobile phase and also on the basis of format. And we have seen that on the basis of principle we have classified as absorption partition size exclusion partition as well as affinity chronography. Similarly on the basis of mobile phase we have classified as in the form of gas chronography as well as liquid and also classified the format. So this chronography is vital for not only chemistry but for biology, medicine of course industry and environment.

[00:53:02]So nowadays the technique is so advanced that miniaturization has occurred and now we also have AI for our help. So by just coupling by combining these versile techniques with AI we can have a very realtime analysis of different constituents of a given mixture. So chromography is transforming the whole landscape of separation science.

[00:53:34]So with this quote we are finishing our uh lecture introductory lecture on chromography and in the coming lectures we are going to see illusion chromography. We are going to see the optimization and we are going to see more aspects of chromography. Thank you very much. Have a nice day. Thank you.

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