Lecture 29

Added: 2026-05-12

[00:00:03][music] [music] Hello students, my name is Shahab Aliyazar Nami and I am associate professor of chemist. chemistry in the department of industrial chemistry Aliger Muslim University. So today we are going to extend our concept of chronography which we have already discussed in our previous slides about the introduction of chronography about the basic principle of chronography and we have also covered the classification of chromography on the basis of the principle on the basis of the format and of course the most important one on the basis of the mobile phase. So today we are going to explore the illusion chronography with regard to columns.

[00:01:11]Illusion chronography is defined as it is the basic chronography or you can say simplest chronography among all the various chronographies which we have described in our previous slides. So today we are going to explore the basic concepts involving illusion chronography, the migration rates, the retention parameters and also we are going to explore the selectivity of different chromographic columns with regard to mobile phase and also the somewhat about the detectors. So illusion chromography as the name suggest. So we will be you know this is these are our learning objectives which we have already you know said to you. So now quickly move towards the illusion chronography.

[00:02:07]As the name suggest, illusion may be in simple words, it may be defined as the washing away of an [clears throat] light by the continuous application of the mobile phase.

[00:02:22]For example, if you consider a simple column, for example, if you consider a simple column and here it is column, simple column is there.

[00:02:34]Now this column is actually suppose it is packed with some stationary phase.

[00:02:40]Let me show you with this cross lines that this column is pegged.

[00:02:45]And now you have to separate your analyte which is like this.

[00:02:54]You have to separate this analyte and for say let us suppose that it contains two constituents A and B.

[00:03:03]So initially what happens is that it shows you a very very narrow band. This mixture actually existed as a very narrow band. And the moment and the moment you apply your mobile phase what happens is this that this band actually moves a little downward.

[00:03:35]This band moves downward little bit and it broadens.

[00:03:43]This band broadens and this broadening of band this broadening of band is actually called as the this broadening is called as analyte dilution.

[00:03:58]This is very interesting fact. this analyte dilution and this column.

[00:04:04]This is a column. Now this band move for a little downward and this is called as analyte dilution. Now you can compare that here this band is narrow and the moment you start adding the mobile phase you continuously add mobile phase. So this band broadens and this band broadening actually tells you the story that now the migration or the distribution of the components has started and this column is actually connected to the detector. This column is also connected to the detector. And as you keep on adding the mobile phase, you keep on adding the mobile phase. I will show you here. You keep on adding the mobile phase. What happens is the band actually it starts separating into its individual components. For example, initially we have a two component system that is A and B. Now after a certain period of time after the continuous addition of the mobile phase these two bands separated for example let us say this one is A and this one is B and this I've already told you that this is connected to the detector.

[00:05:32]Okay. Now as you keep on adding the mobile phase after a certain period of time you will see that it the component that is the B component actually that this B component is on the verge of illusion.

[00:06:01]This actually this is how the two components start separating.

[00:06:08]And these two components start separating because A has different affinity to bind with the stationary phase while the component B has different affinity to bind with the stationary phase. And because of the difference in affinities, they are actually have a weaker and stronger interaction with the stationary phase.

[00:06:28]And stronger the interaction the more time they are going to stay with the stationary phase that is they are going to use at a later time. Now if you draw a detector response like this, if you draw detector response obtained from the column and you put plot for example on the basis of time then what you observe you observe initially you observe is this peak then you observe two peaks.

[00:07:07]Now as you see that initially you add a and b and finally the first component to lute was b. So in the chromog you will get first peak b and then you will get a. This pictorial representation of the migration of the two solutes or of the component with respect to detector response is called as this figure is called as chromatto.

[00:07:43]This called as chronog that is in simple words it can be regarded as the migration fingerprint of different analytes of the substance which is to be separated. So what we actually see that those solute actually interacted with the stationary phase and because of the difference in interaction they have traveled at different speeds that is they have different rate of migration different migration rates different migration rates and because of the difference of migration rate they have eluted at a different times. As you can see here that here this if you draw a perpendicular then this is the time taken by B you can say TB while this is the time taken by TA.

[00:08:50]We are going to see this in terms of the retention times in our coming slides. So what happens is that your solute actually gets partitioned between the stationary phase and the mobile phase and because of the partition because of the different migration rates they elute at different times and that is why this chromography in simple terms is called as illusion chromography.

[00:09:18]So what we have done we have achieved separation on the basis of the partitioning and we get a fingerprint that is called as the chronoggram.

[00:09:30]So this is how you know the chron uh the illusion chronography works. Okay. Now moving forward to it that how we are going to build up the setup. So initially what are needed is initially we need a stationary phase that is a stable phase a stable phase generally we'll use silica we use aluminina sometimes we use some resins like doex etc etc so these stationary phase are chosen on the basis of the type of compound which is to be separated into its constituents.

[00:10:14]Similarly, we have to choose mobile phase. Now, mobile phase is very important in the sense that again we have to choose the mobile phase with respect to the compound which is to be separated. For example, we have to start from non-polar. So, we have to start from initially we have to start from hexane.

[00:10:36]Then we have to move towards after hexane we move to benzene.

[00:10:43]Okay. Then we move to polars like like acetone.

[00:10:49]Then we move to more polar like methanol.

[00:10:53]And finally we end up on water which has the highest polarity among the common solvents. So we have to develop a polar non-polar versus polar gradient and in the mobile phase. So the stationary phase and mobile phase are chosen with regard to their interaction with the with the solid molecules. Now the important part is the detection. Detector we have to use detector because it is the detector which records the signal with regard to the separation of peaks.

[00:11:32]Okay. So detector is the instrument which records the illusion pattern and moreover it is as I've told you earlier that it is the you know it is versatile technique which not only tells you about the nature it also tells you about the nature of constituents as well as and their quantity that is very important and this quantity can be calculated from the chromogram by calculating the area area under the peel. If you remember in our previous slide we have we have a b eluted and then a eluted and this illusion pattern actually tells us about the that how much strong interaction the different components have with the stationary phase. So we are going to explore this when we took some real life example for example the high pressure liquid chronography system. So we will see this these real examples in our coming slides. So this is how we have to carry out our setup that is first of all we have to choose our stationary phase.

[00:12:46]Then we have to assertain our mobile phase and these two are chosen keeping in mind that which type of you know which type of uh uh the mixture we are going to separate. Then we have to choose the detector accordingly. That is we can have a number of detectors. For example, u visible detector, fluoresence detector, we can have conductivity detector, we can have thermal detector, we can have a marit of detectors depending upon the type of illusion. So now comes the migration rate. Now this migration rate is very important in the sense that the effectiveness of any column with regard to the separation actually partially depends upon the migration weight rates of the different constituents and this migration rate is actually measured in terms of their equilibrium constant. Let us suppose that we have a solute. Let us suppose we have a solute A. Let us suppose then this solute A is being transferred when we you know when we apply the solute to any chromatographic chromatographic column.

[00:14:05]Then what happens that the solute actually gets transferred between the solute get transferred between the mobile phase and stationary phase that is an equilibrium is achieved in the stationary phase as well as the mobile phase.

[00:14:29]Now the equilibrium constant for the distribution of this analyte in the stationary phase it may be defined as the KD.

[00:14:40]So this KD actually may be defined as like this that is activity of solute A in the stationary phase upon activity of solute A in the mobile phase. So this is how actually we define the equilibrium constant and this KD is actually the dissociation constant which we are going to discuss in our next coming slide. So at this point of time this migration of solute A is being done in a stationary versus the mobile phase. Now let us suppose a case that for example let us suppose let us suppose that that concentration of A is small or small or and A is some nonionic species nonionic species let us suppose let us suppose an interesting case that that A is nonionic actually then in that case what What we can do? We can define this activity in terms of its molar concentration. Then KD can be defined as CS upon CN.

[00:16:08]Okay. Now let us discuss the migration rate.

[00:16:13]The effectiveness of any chronographic column actually depends upon the migration of different solutes present in a given mixture and this migration rate can be determined in terms of the equilibrium constant. Let us suppose a solute A for example. Let us suppose a solute A. Now the solute A when added in the column it gets transferred it gets transferred it gets transferred between the mobile phase between mobile phase and stationary phase and stationary phase. Now what happens is that we can write in terms of its equilibrium constant that is a in the mobile phase equilibriated with the A in the stationary phase.

[00:17:17]Now in terms of equilibrium constant how can we define it? We can define this transfer of analyte between the two phases as like this. K of activity of analyte A in the stationary phase upon activity of analyte A in the mobile phase. Since this analyte A gets distributed between the stationary phase and the mobile phase.

[00:17:51]So we can fairly write like this KD.

[00:17:55]Now since it's a distribution so we call it distribution constant. We call it distribution.

[00:18:06]We call it distribution constant. And this is this KD is actually very unique for each chromographic separation. This is something you can say a characteristic for any chronographic separation that is KD and how the value of KD actually affect the migration rates the column performance. This this these are very interesting and we are going to dwell in these aspects in our coming slides. So at this point of time we have defined KD in terms of the activity of analyte A in the stationary phase as well as the mobile phase. Now let us suppose that if your analyte is nonionic for example let us suppose it is nonionic and has low concentration and low concentration suppose then what we can do we can replace this activity by the molar concentration by the molar concentration that is we can write like this that is molar concentration like This we can write KD is equal to CM CS upon CN that is the molar concentration of analyte A in the stationary phase upon molar concentration of analyte A in the mobile phase. Now it's the molar concentration we can surely convert it in terms of the number of moles. So how we can define it? number of moles of solute okay in the stationary phase that SS subscript is there upon the volume in the of the stationary phase. Similarly we can write n m upon v mm and this is actually this kd can now be defined as the partition coefficient.

[00:20:10]This can be defined as in terms of partition coefficient. So KD can also be called as the partition coefficient.

[00:20:24]Now for example suppose that in certain cases that CS gets proportional to CN then we can have KD value equal to unity. This implies that the concentration remains constant for a very long range.

[00:20:49]So the two solutes actually gets migrated on the basis of their interaction with the stationary phase.

[00:20:59]So this migration actually depends upon two factors. Number one is the strength of the interaction with the mobile phase and number two is the flow rate of mobile phase. Now this is the point which we have to discuss the flow rate of mobile phase. Now actually there are two flow rates and specifically we have to say in terms of average flow rate.

[00:21:25]Average flow rate in chromographic systems we always talk about the average parameters.

[00:21:32]Okay. So now suppose you have a column.

[00:21:37]You have a column and this flow rate actually this flow rate may be defined as V may be defined as L upon TR R. Now this L is actually the packing height of the column. It is not the length of the column. It is the packing height. This is L.

[00:22:05]This is not L.

[00:22:08]That is L is the packing height of the column. And this TR is called as the retention time. Now what is the retention time? At this point of time, we can say that retention time is the time taken by the solute to elude from its injection point to the final point.

[00:22:28]At this point, we know it is connected to the detector.

[00:22:32]So this is the TR. Now we are going to discuss this TR in our coming slides.

[00:22:39]There is another point which is called as the flow rate of this is actually the flow rate of the solute. Now there's another flow rate which is called defined by U and this is defined by L upon TM.

[00:22:57]This TM is actually the dead time. It is called as a dead time and sometimes you can find it written like void time.

[00:23:06]This is actually defined as the time where the interaction with the stationary phase is minimum or nil that is only passing of the mobile phase alone. So what we'll have what we actually are going to do we are going to quantify the migration rate.

[00:23:32]So TR is the retention time here. It was the retention time.

[00:23:37]It was the retention time.

[00:23:41]Okay. And this this was the dead time.

[00:23:44]TM was a dead time. So the two solutes actually are distinguished on the basis of their migration rates on the basis of their TR and the equation is V is equal to L upon TR. For example, if you are going to define the average flow rate of uh the mobile phase alone, mobile phase alone it becomes U and it becomes L upon T M.

[00:24:19]While for the solute it becomes V upon T R. Okay. So we have to you know keep in mind the difference between the average velocity of the mobile phase and the average velocity of the mobile phase carrying the salute with it. Okay, we have to have very you know very clearcut distinction between the two where L is the length of column that is length of the column packed.

[00:24:48]Okay, it is not the length of the full column. Mind that L is the packed length of the column up to which the stationary phase is packed. Okay. Now we have also given an example that methane for example if you pass methane then it is not it it does not have any interaction with the stationary phase and that is why it has zero retention and it moves or eludes very fast. Why? If we have some aromatic hydrocarbon which is polar in nature, it is going to retain and the retain means that is it is going to have some interaction and that interaction gives you TR.

[00:25:24]Okay. So any sort of interaction will give you TR and any sort of non-interaction or zero interaction gives you TM. So migration rate actually reveals the balance of interaction forces between the components which are two separated along in and with the stationary phase. Okay. So migration rate actually gives you the idea about the interaction of different components with the stationary phase.

[00:25:56]So here comes the distribution cost. You know this is something very unique something you can say uh this is a unique feature of every chronographic system that is KD the dissociation or the distribution constant is defined as CS that is the molar concentration of an ally in the stationary phase upon the concentration of an ally in the mobile phase and we have also resolved it in terms of number of moles of solute upon volume of solute into number of moles of mobile phase upon volume of mobile phase. So we have also discussed it in the previous slide. Now what are the actually what are the importance of this? Why we are so concerned about KD?

[00:26:41]Actually KD is very diagnostic. A large value of KD means this is quantity which is large.

[00:26:49]This quantity is large means that we have a very strong interaction that is with the mobile phase that is solute prefer the stationary phase. Solute are going to spend more time and if they are going to spend more time they have slower migration.

[00:27:08]Similarly on the basis of similar KD values we can infer the opposite that is smaller KD value means this quantity is high cm is high and cm is high that is your solute is preferring the mobile phase so if it is preferring the mobile face it's going to very fast because you know it is happy with the mobile phase okay I'm with the mobile face so I'm going out this is like this so it is actually getting faster migration that is If we have KD value high for two components for two components we have A and B and for B if the KD value is high then we get in the chromograph first peak of B then we got peak of A B and A that is here your actually B component is loving the mobile phase and because of its love it is eluting very fast and if it is eluting very fast it means that it has less retention time.

[00:28:08]So this is how KD is diagnostic with regard to the illusion of different components. Now moving forward.

[00:28:18]So polar solute polar solute in silica has high KD values. Now the polar solute solute actually binds effectively with the stationary phase with the silica column and because of this strong binding with the stationary phase the CS is high and since CS high the KD value is high this is like this simple the opposite also true that the non-polar solute in the reverse phase HBZ has gives low KD value the analog is the And a very beautiful example is the sugars.

[00:28:58]Sugars are polar and it is reverse phase HLC they gives low KD values because they elute very rapidly. They go go out of the column very rapidly. They loves you know they loves the polar solvent because you know the basic theme is like dissolves like that is called as the thumb rule.

[00:29:23]Thumb rule that is called as the thumb rule. Thumb rule of chemistry that is thumb rule. So non-polar prefers non-polar and polar prefers polar.

[00:29:35]So the moment you change the combination the things will change.

[00:29:40]So this is about the KD value. Now KD is very beautiful, very interesting.

[00:29:47]But the irony with KD is that it cannot be measured.

[00:29:51]This is the actually problem. The problem is that we cannot measure KD directly from a chronoggram.

[00:29:58]So we have to have certain factor certain factor on which KT depends actually because we cannot measure KD.

[00:30:05]So we have to find some factor which actually depends on KD and that factor can relate to KD and that is measurable in nature. So we have the answer and before the answer let us you know briefly uh explore what are the factors which affect KD. Number one we have as we already discussed it is the polarity of the stationary phase the chemistry of the stationary phase the what are the different functional groups that are present and that actually decides the retention time that decides the interaction similarly we have solvent polarity is there we have solvent polarity which type of solvent we are using whether we are using the non-polar solvent like hexane or benzene these are non-polar or we We are using less polar like chloroform or dicchloromthane or more polar like alcohol or a very polar like water.

[00:31:08]Which type of solvent we are using and this solvent polarity is important with regard to the nature nature of the component which are to be separated.

[00:31:18]Similarly, temperature also comes into play and we will discuss the effect of temperature on a very large scale in GC when we discuss the gas chromograph where temperature controlling is one of the very vital things for the effective separation of different components of an analyte.

[00:31:40]Similarly, pH pH also becomes very important in case of ionizable compounds. For example, if we have some acid or bases, then pH also becomes very important. Similarly, the presence of modifiers that is the ion p reagent.

[00:31:55]These directly affect the value of KD.

[00:31:59]Now, what are the actually advantages of KD? The advantage of KD is that it's a direct measure of the partitioning. on the basis of the KD value on the basis of its magnitude we decide that it is partitioned where that is it is more towards the stationary phase or more towards the mobile phase. So this is the advantage that would give you a direct link a direct measure of the partitioning between the mobile phase and the stationary phase.

[00:32:31]It gives you a direct measure but at the same time it has a very strong limitation. So what are the limitations?

[00:32:38]There are certain limitations of KD also. Besides all these advantages there are certain limitations also. The first limitations of course uh it's KD KD cannot be directly measured. This is the foremost directly measured from the chromogen from the chromatoggram.

[00:33:05]That was the first limitation and the second limitation that it is strongly matrix dependent because you know the polarity and non-polarity comes into play. So if we have you know combination of polar polar then it's fine and sometimes it it interacts very much with the solute and the illusion becomes a problem. That is why it is its matrix actually sometimes creates problem and we cannot predict we cannot predict a sample before running because we never know what type of interaction it is going to hold. So this is the limitation. Okay. So now with regard to the distribution constant what is the role of KD actually KD actually gives an idea about the retention.

[00:33:54]Now this KD actually is directly linked with the migration rate. We have seen in the previous slide and at the same time this KD value influences the peak width and separation efficiency. Now this point is you know worth discussing that that influences the peak width and the separation efficiency. Now if you remember we have drawn a chromoggram where we have plotted detector response okay on the y-axis and we have plotted time.

[00:34:28]So for a two component system we have seen that initially we get a small peak then we get the peak of component one and then we get a peak of component two.

[00:34:42]So this time is running and these are the distracted responses. For example let us suppose let is let us call this B and this is called as component A. Now for an ideal illusion for an ideal illusion these two peaks must be as far as possible that is they must be as you know as far as possible within a given time. It's not that they are taking 2 hours or 3 hours. If they are taking more time then again it will compromise the efficiency of the column.

[00:35:17]So the they have a clearcut separation and the speed peak width that is that is this that is to be kept minimum that is the peak must be goshian type.

[00:35:32]It is not like this.

[00:35:34]This is a bad chronographic peak. This is not ideal. The ideal is the gshian.

[00:35:40]It is called as gshian peak. We will discuss this gshian peak and this gshian have a lower fronting.

[00:35:49]This is called as the fronting part of the fronting and this is called the trailing. The trailing is not too much. This is called tailing and this is actually the central part that is the part where most of the migration take place. So this KD value actually influences the peak width that is the peak width and it also influences the separation efficiency because a good separation means that is the peaks must be as far from each other as possible within a stupulated within given time that is you have to take time also into [clears throat] account. So solute with close K values for example if you get a separation like this then the values are too close and here you can say that the distance distance between the peaks is not well defined. defined defined or you can say peaks are not separated.

[00:37:15]Similarly, if you if you add some performance factor and you get this type of peak, although the peaks are separated but they are wide. So then ideal chronograph should like this.

[00:37:37]You can see this is an ideal chromatoggram where where you have good separation and the peak width is also to be minimized.

[00:37:52]So what we infer is that KD value actually dictates the distance and they also dictates the width of the peak. So width of the peak must be kept as small as possible and the separation of the two peaks must be kept as big as possible keeping in mind the time. Time is also important with regard to deciding the efficiency of any of any cryptographic.

[00:38:19]So solutes with K closed value of K does not give poor gives poor separation and we have optimal illusion when K values are significantly different. So K values can only be different in cases where we have the clearcut separation like this.

[00:38:39]Here the K value of for example of B and K value of A are different.

[00:38:51]in terms of their distribution constant.

[00:38:54]So this is how the role of KD. So with this thank you very much. [music]