Lecture 30

Added: 2026-05-09

[00:00:03][music] [music] Now let us explore the dead time.

[00:00:26]Now before giving you an idea of dead time let us draw a chromatoggram.

[00:00:34]This time I will drawing a little big one so that we can define certain terms.

[00:00:38]Now again on the y-axis we have the same thing that is the detector response and on the x-axis we have time.

[00:00:48]Now what what can be done is that you can also plot x-axis with regard to the mobile phase mobile phase which you are adding on a continuous basis. So it can also be plotted with regard to the mobile phase but let us for simplicity let us take in terms of time. Now for a two component or let us suppose for a simplicity let us suppose a one component system. Now if you draw the detector response of the illusion of a single component you will see an initial first peak like this.

[00:01:26]Then you will see a peak like this.

[00:01:33]Now on the x-axis it's time let us draw a perpendicular.

[00:01:39]Let us draw a perpendicular here.

[00:01:42]Now this time this time is called as tn.

[00:01:49]Okay. And this time is called as TR.

[00:01:58]And this time in between it is called as TRdash and sometimes you can find it as written as TS.

[00:02:11]Now we have three different times that is the TM that is the TR and the TR dash or TS. Now this TM gives you the time when you pass only the solute.

[00:02:30]Simply the need solute is passed that gives you the peak that gives you the detector response in terms of peak. That first peak actually tells you that this your solute is not having any sort of interaction with the stationary phase and that is why it is called as dead time or sometime it is also called as void time that is unwitten solute and it is actually you can say it's a standard like in for example In NMR we call it TMS. So in chronography the TMS can be equated with the TM. Now you actually record all your responses with regard to TM because TM is the time taken by the mobile phase which does not contain any of your analyte that is for your need mobile phase need solute which you have initially passed through the column.

[00:03:38]Now there are certain markers there are certain compounds which does not re uh gets retained which does not interact with the stationary phase and the examples are the methane in gas chronography. Similarly uracil in high performance liquid chronography these are the substances which does not react or which has zero interaction with the stationary fluids. So TM actually provides you a baseline for relative retention calculations.

[00:04:08]So it's a sort of reference point. It's sort of starting point that is on the basis of this starting point you measure the retention time of the other solutes or other components present in your separating mixture.

[00:04:27]So advantage is that it acts as a reference point and the limitation is that ideally speaking it requires non-interacting compounds but practically speaking there's certain interaction but that can be ignored because we are starting from this. So the relationship can be written as TR R is equal to T M plus T R dash or it may be written as TS.

[00:05:00]Now the point which is to be elaborated at this juncture is this that this TM is because of the non-interaction of your mobile phase and this TR retention time is because of the interaction of your components with both a stationary phase and mobile phase. Mind that it's not alone with the stationary phase because you have added TM also because your TR contains TM part.

[00:05:37]Your TR contains GM part. So it is already accounted the interaction with the mobile phase.

[00:05:45]So TS actually is simply the plane interaction of your components with the stationary phase alone.

[00:05:56]Are you getting my point my students? So this is actually very interesting that is on the basis of time you have quantified it in three terms that is the TM that is the TR and of course the TR dash or TS. So you have accounted for the interaction of the mobile phase. You have also accounted for the interaction of a stationary phase. And all these three quantities you can measure directly from the chromatoggram.

[00:06:28]So what is the difference between KDA and TM and TR and TS is that the KD gives you a very good idea about the column performance. It gives you about the interaction but it cannot be measured in. But on the other hand, you can measure all the TRS that is you can measure TR, TM and TR dash directly from the chromoggram. So now it's time to actually you know to actually establish a relation between these two because one is measurable while the other is not measurable but it is you know giving you very important information and similar to KD TR is also very specific very characteristic for any chronographic separation we will going to see this in our coming slides. So TR is actually the time taken by the solute from the injection point from the injection point from the injection point. Now suppose this is your packed column packing starts from this point. So, tr actually the time taken from this to this up to the illusion.

[00:07:48]So, we have three things. Number one is the TR that tells you about the retention time taken by the solute in the mobile phase to reach up to the column to reach up to the detector sorry. And then the second time is the void time and third one is the time taken by the interaction for the for the stationary phase interaction of the solute particles with the stationary phase. So we have three things we have we have TR we have TM and we have TR dash. So it gives you interaction of the stationary phase stationary and mobile.

[00:08:40]It give interaction only with the only mobile and it gives you only station and fortunately you can measure all the three you can measure all these three quantities from the protogram. So let us you know so this is DM is called the hold up time or the dead time or the void time and this is the additional retention time due to interaction with the stationary phase. So you can see TI or sometimes you can see TS also. So don't worry this both are the same.

[00:09:17]Okay. So a classical example is the benzene. Benzene actually has a TR of 5.2 2 minutes and choline has a TR of 7.4 minutes. Why the difference is there? The difference is because of the difference in polarity. Simple. It's non-polar. So, it is eluting very fast.

[00:09:38]It is a little bit polar tolene.

[00:09:42]So, it is eluting at a low rate or it is taking more time as compared to benzene.

[00:09:49]This data is from the GC that is gas chronography. So what we infer is that retention time of any compound is specific under given conditions. Now what are the given conditions? Given conditions are of course it is the temperature again temperature and effect and the pressure.

[00:10:12]Now what we can see we can say that KD and these are thermodynamic in nature.

[00:10:19]They are thermodynamic parameters.

[00:10:21]They're not simple parameters. They are thermodynamic. So what are the factors which affect this TR? Number one is the column length. Now again I'm correcting it simply column length or packed column length or packed column length. Ped column length this is more specific.

[00:10:42]Then of course flow rate that is V.

[00:10:46]Okay. and specifically again average flow rate because we always talk about the average quantities similarly parameter and the station so these are the factors which affect the retention time TR okay so now it's time to relate the two that is the retention time and the distribution constant or distribution coefficient. So what we have seen we have seen that that higher K value means that longer retention. Now if you remember from our previous slide we have this relation TR R is equal to M plus TS or TRdash. Now let us drive in terms of the KD value.

[00:11:34]So what we were having is this.

[00:11:38]Let us now relate the two quantities. One is the measurable quantities and the other other is the non-measurable quantities.

[00:11:46]We have seen our previous slides that KD is giving you information but it cannot be measured directly and what you can measure directly is the TR is the TM and of course it is the TR dash. So on the basis of this an approximate relation can be drawn like TR that is the detention time can be defined as TM into 1 + K upon VS upon VM. VS is the volume of solute in the stationary phase. VM is the volume in the mobile phase. Now TR is the retention time and TM is the dead time. So we have already seen that if any substance has higher K value higher KD value then it means that it is having a stronger inter interaction and the stronger retention actually gives you larger or longer retention time. So this actually retention reflects a partition equilibrium that is where the partitioning is going to happen more. We can also drive another relation. For example, we have described that the average velocity for example the average velocity we have defined in our previous slides of solute in the mobile phase in the mobile phase we have defined by new. While the average velocity average velocity of mobile phase only mobile phase only was given by U. It is new and it is U. Now let us draw a relation.

[00:13:35]Now you can define like this that new may be defined as or let me draw it more clearly. New may be defined as U into the fraction fraction of solute in the mobile phase. This we can have this we can have the relationship that is the average velocity of solute in the mobile phase can be equated with the velocity of the pure mobile phase into the number of onto the fraction of solute in the mobile phase. Okay, it's a clear calculation. So this can also be written as U into now the fraction that is this fraction can be defined as CS then the mobile phase we can say that it is actually the CM into VM that is the solute or number of moles in the mobile phase upon number of moles in the mobile phase and Number of moles in the stationary phase we can write like this.

[00:14:58]That is what we have done. We have done in terms of number of moles of solute present in the mobile phase in terms of CMV. Because if you remember we have described it as C M upon V S C C C C C C C C C C C C C C C C C C C C Cs upon VS we have described like this like this. So it is the same. So what happens is now if you take this and solve this you can get divided by cm vm upon cm vm divided by cm vm upon cm vm plus cs vm upon cm vn.

[00:15:50]Okay, this is the relationship. What we have done, we have divided with the similar quantities. So this gets cancelled with this giving you one.

[00:16:02]This gets cancelled with this because numerator and denominator are the same.

[00:16:06]Here also it gives you one. And here if you solve it you will get like this.

[00:16:14]If you solve it further, you will get like this. U into 1 upon 1 + CS V upon it's actually upon CM VN here also it is CS it is also CS.

[00:16:41]So what we get u + u into 1 upon 1 + it is nothing but it is cs it is actually kd upon vs into vm.

[00:16:56]So you can drive in terms of your dead time. You can also drive in terms of your mobile phase velocity only. So you can drive in both ways. Whatever actually suits you. what type of data is given to you. Okay. So you can have two relations that is you can drive it in terms of V and you can drive in terms of U and the beauty is that these are can be measured.

[00:17:23]So retention factor now retention factor is very important. It gives you an idea a quantified idea that how much the solutes is going to retain in the both phases that is in the mobile phase and in the stationary phase and this is actually denoted by K dash and Kdash can simply be defined as TR minus TM upon TM that is the retention time minus the dead time upon dead time. In fact, you can also quantify it like you can also quantify K dash is equal to the K A or V S upon B. You can also define like this.

[00:18:10]Okay. If you are dealing with solute A or you can also write like this that KD into VS upon VN the solute in the stationary phase divide by solute in the mobile phase. So what does actually K tells? K tells you that if the value of K is zero then your solute remains unin unretained.

[00:18:36]That is there is no interaction. your solute does not interact with the stationary phase and as the value of K increases it implies that it is going to retain in the column for longer times and longer retention means stronger interaction. So for an optimum analytical separation the K value must lie between 1 to 5 1 to 5 at the most up to 10. But if it is greater than 10 then we can get a broad peak like this.

[00:19:09]And broad peak means that your column is actually inefficient not working not working ideally.

[00:19:21]So the K value actually decides the the utility or the importance of your separation. Now what are the significance? We have seen that in terms of value that if the K value is 1 to 5 it is you know good. So it is actually a dimensional parameter and it is independent of the flow rate of the mobile phase and it is very ideal for comparison of more than one solute or more than one uh you can say that uh components and it gives you a balance between the resolution and time efficiency.

[00:20:04]So now comes the selectivity factor. The selectivity factor is very important because it gives you an idea about the relative uh interaction of more than one solute.

[00:20:16]For example, for two solutes, you can define this selectivity factor as K2 upon K1 that is component two and component one. And this K dash is actually the which we have discussed is the retention factor. So now you have another parameter which gives you an idea about the interaction of your solutes with the stationary phase and mobile. So for example that K2 value K2 value dash upon K1 value is giving you that this K2 or component two actually component two is having more retention time having more it is actually giving you an idea that is having more retention And if alpha that this alpha is equal to one then you you have no separation that is your peaks are actually you know overlapping on each other.

[00:21:24]This is actually the story with it.

[00:21:26]So always remember that that two means that it its peak is actually if you have a two peak system like this then you can have this is component one and this is component two.

[00:21:41]So the upper part is actually telling you that this is going to retain for a longer time and this is actually eluting at a faster time.

[00:21:50]So always always for practical separation the the limit is alpha should lie between 1.05 to two and again it's a dimensionless quantity it does not have any unit.

[00:22:06]So for for example we have given an example of leucine and phenylen in reverse with H sphere see its value lies the value of alpha lies 1.8 Okay, it means it's a good separation and moreover if its value is too high then the peaks are you know far more to a part again this is not a good separation because it is telling the story that it is taking too much time so always you have that you know that range that is 1.05 05 to 2 that is the ideal range. If it is one then you have an overlapping peak. Okay. And if it is greater than one then it's fine but it not must be greater than two. So that is actually gives you about the relative separation. So for an ideal separation you have two well definfined peaks whose bandwidth is to be kept as small as possible. So this is an ideal chromatoggram and here you have the detector response detector response.

[00:23:14]So this is how your alpha actually dictates the whole process of separation. So what are the factors which affect the selectivity? Number one is the mobile phase composition. This is very important. Again solvent polarity dictates all these factors. that is how much polar solvent you are taking and you are taking solvent on the basis of the nature of the compound which is to be separated. Similarly the stationary phase chemistry that is which type of a stationary phase you have chosen that is you have chosen a polar stationary phase or slightly polar or exactly non-polar and again temperature comes into play pressure comes into play. We are going to see the effect of temperature uh in a very detail in our uh you know next slides where when we are going to discuss the gasograph.

[00:24:09]So what what are the basically advantages of this of this the first advantage is it is one of the most powerful factor in improving the separation.

[00:24:20]So on the basis of the value you actually decides that whether you have achieved a good separation or not.

[00:24:28]And the limitation is that it is difficult to optimize predictively. You cannot you cannot initially you cannot say like this that it is going to have a good separation or not. So you have to run you have to run chronogram each time otherwise you cannot say anything about it. So the interpretation of selectivity that how selectivity is significant. Selectivity is actually a measure that how different solutes in a column are interacting with each other.

[00:25:01]Okay. So for a good separation they should they should have a good separation between among them. Okay. So their values must lie between 1.05 to2 and similarly we can change we can you know modulate the selectivity factor by changing the stationary phase.

[00:25:21]Okay. By changing the actually changing stationary phase mean changing the polarity of stationary phase. Similarly, we can change the polarity of the solvent system also. That is whether we are using some non-polar or polar.

[00:25:38]Now again the thumb rule comes into play. For example, if our stationary phase is non-polar and we are using a polar solvent, then it will you know it will elute very slow.

[00:25:50]it it will take too much time. But again, for a non-polar non-polar combination, it is a good combination.

[00:25:56]It will elute in an optimal way.

[00:25:58]Similarly, we can also uh change the selectivity factor by adjusting the pH in case of exchange photography where the separation occur on the basis of charge.

[00:26:09]So charge comes and the pH comes into play because then acid base character also comes into play. So by adjusting pH in ion exchange photography. So what is the relation? Let us you know briefly see that what is the relation between retention time and retention factor. The retention factor is K dash. We have described it with K dash. It is TR minus TM upon TM. So if we have we have taken a very small example very simple example. If TR for example if you draw a chronoggram then what is happening is that you have TM coming at 2 minute then you have the TR which is coming at 12 minute now this is coming at 2 minute 1 and this is coming at 12 minute for example okay now you have four you have 6 you have 8 you have 10 and you have 12 so k dash can be calculated you have to simply put the value 12 - 2 12 is the tr and 2 is the tn that the dead time which is this peak dead time t and this is the tr this is the tr which is actually 12 minutes and this is tn this is 2 minute so 12 - 2 is equal to 10 upon 2 which is 5 so solute This implies that solute spends 5% more time in the stationary phase than the mobile phase. This is how you relate your numeric with the interaction.

[00:27:52]So it is interacting five times more in the stationary phase than the mobile phase. Now resolution another important feature on which this is actually the basic feature resolution resolution with regard to peaks. So greater the resolution better will be the separation between the peaks. So we have to have as much resolution between the peaks as possible. Again it is for a two component system it is given as RS may be defined as TR2 minus TR1 upon W1 + W2. Now where what are W1 and W2? Let us see. Now we have a chronoggram. We have a TM.

[00:28:33]Then we have a this.

[00:28:38]Okay.

[00:28:39]Now these are two well definfined well resolved peaks. Now let us draw a perpendicular.

[00:28:45]Let us draw a perpendicular here.

[00:28:49]Now for the example this is peak number one and this is peak number two. Then this actually this is called as W1 up to this point and if you draw a perpendicular now from where you have to draw a perpendicular that is important. So you have to take the fronting part here and you have to take the tailing part here. Similarly for dropping the perpendicular you have to take the fronting and you have to take the tailing part. So you have to take that the the medium you know where maximum uh illusion is taking place. So you have to take the maximum part this is w1 and this is w2.

[00:29:35]This is tm half of this.

[00:29:39]This is TM and this is you can say TR R 1 up to this point and this is TR R2.

[00:29:55]So your resolution is defined as TW of TR2 minus TR R1 upon W1 + W2. So W1 and W2 are the baseline peak widths. For a good resolution, RS must be greater than 1.5.

[00:30:16]That is this distance must be greater than 1.5.

[00:30:20]Okay. So for an effective chromographic separation, RS value RS value must always be always be greater than 1.5.

[00:30:30]for effective separation.

[00:30:38]Now let us see in terms of migration rate. Migration rate we have already told you is given by the velocity of migration or migration rate is given by in terms of uh the solute movement in given by v is equal to l upon dr that is the length of the packed column. TR is the detention time. So if you know if you have a large value of V large value of V means that you have a small value of TR that is it is weakly retained it is going to elute very fast. Similarly if you have strongly ret you have a small V values.

[00:31:19]Now a migration rate with with regard to retention factor we are going to see. So what are the factors which affect the retention parameter? Of course it's the column length and the packing in it that is the greater the column length greater will be the separation or resolution. We have to adjust the flow rate we have seen the previous slide temperature again important we will be going to discuss it mobile phase polarity and pH and the nature of a stationary phase whether it is you know polar or non-polar that is hydrophilic or hydrophobic or ionic. So these are the factors which directly affect the retention parameters that column length and packing that longer the length greater will be the resolution flow rate flow rate must be optimized. Similarly temperature it has to be maintained for isotherm. We will see that we have to have isothermal ovens and the mobile phase polarity. It must match with the polarity of the stationary phase because again we have a strong strong interaction for polar polar and we have a weak interaction for polar and non-polar.

[00:32:26]So let us briefly go through some of the practical examples that is caffeine. It is actually here the water methanol it is a mixture and the stationary phase is C8 silica gel we observe a caffeine peak and retention time of 6 minute it's at at about 6 minute okay 6 minute here time and time is in minute it is the detector response so caffeine it is eluting at after 6 and sugar elute earlier because it has a polar and it has a weak interaction. So the weak interaction will come first.

[00:33:10]The sugar will come first then caffeine comes then the caffeine caffeine comes because it is going to retain and this is actually how the K dash and alpha gives you a good idea about the separation. Another example is a gas chromographic separation of hydrocarbons where we have used an hexane versus octane. Octane has a very strong interaction and a strong interaction means higher K value that is large TR value that is it is going to retain it is coming very slowly. So it will come at the end of the chronog large value.

[00:33:55]Similarly, selectivity always it is should be greater than when so that we can have a visible peak separation. That is the peak should not like this.

[00:34:06]It should be clearly resolved.

[00:34:10]Okay, it should not superimpose on each other. So it's not good. It's an ideal system. Okay, and this can be achieved if we have alpha that is selectivity factor greater than one. Now another example is the protein ions in chrography. Here the proteins have different charges and because of different charges they have different retention times because they migrate at different rates. Now by altering the pH or the salt gradient it can be tuned because now you induce uh certain parameters and because of that certain factors of positive and negative charge comes into play that positive will attract the negative and because of that the selectivity factor can be changed or it can be fine-tuned.

[00:34:54]Now let us summarize let us summarize the various equations which we have derived. So initially first of all we have dived the distribution constant KD we have written it like this KD. So do not get confused where is the small TS it is all the way same that is small KD or KD. So it we have defined in terms of CS upon CM that is the molar concentration of analyte in the stationary phase upon the mobile phase.

[00:35:22]Similarly, it depends upon the affinity and here it is the it is because of the migration equivalent. So an example is the polar analyte in the silica gel.

[00:35:34]Similarly we have defined TR we have if you remember it's again it is TR like this. So we have defined in terms of TM that is the dead time and the extra retention time or it is because of the interaction with the stationary phase.

[00:35:50]Now it depends on the flow rate as well as the KD value. The KD value significance is time of detection. So we have seen example is the caffeine peak at 6 minute while the sugar soluted very fast. We have seen it again it's the K dash that is the you can say retention factor. It was defined in terms of TR upon minus TM upon TM. Again it depends on TR and TN.

[00:36:20]Okay. So the significance it tells you about the relative retention rates. For example, it's a two component system or three component system. It can be decided on the basis of its value that is K dash value. Similarly we have the selectivity factor which actually relates the separation of the components. we have seen in terms of octane and hexane and the last one is the resolution which was given in terms of an equation that is trus tr1 upon the width of the peaks that is w1 and w2 so these are the actually factors on which it depends and it it gives you about the significance of the peak resolution the significance is the peak resolution we have a very good resolved peaks and a common example is the amino this separation on the basis of their positive and negative charge. So what are the practical advantages of illusion photography? Illusion photography gives you a very good resolution and of course it is reproducible. You can do five, 10 or 20 separations and you will get the same values each time. So this is actually the reproducibility. So it has very good reproducibility. Now it can separate complex mixtures. Okay.

[00:37:37]Similarly again it is quantitative and qualitative information. Qualitative it gives you the nature and quantitative you can calculate under the area of the peak. Okay. So versality is it can be used in GC HLC ion exchange as well as size exclusion chromograph.

[00:37:57]So what are the there are certain limitations also. Now the first or the foremost limitation is the time.

[00:38:04]Sometime it becomes very time consuming because sometimes they different uh components which are to be separated it get it's it gets very strong bond with the stationary phase and once they get strong interaction they are going to retain for longer times and this longer retention actually gives you a longer you know uh application or or longer time for separation. Peak tailing occurs due to second interaction that is the peak is not sharp. It actually you go you can see that it goes on tailing.

[00:38:39]Similarly sometimes fronting also it takes very long time to reach the ideally your peak must be gshian like this. Ideal peak is this gshian peaks.

[00:38:50]No fronting and no tailing. This is tailing and this is fronting. So an ideal peak must be gshian.

[00:39:02]Ideally goshian peaks are there. Goshian peaks are to be obtained.

[00:39:16]Sensitivity to temperature that is on increasing the temperature you can change it. Okay. Sometime it it is beneficial while sometime it is not.

[00:39:25]Again the high cost for advanced column for example if you are using uh sophisticated columns made of a glass or made of a steel sometime clogging occurs and because of this it limits its use.

[00:39:38]Okay.

[00:39:40]So what are the real life applications where we can use the illusion photography? Of course we are exclusively used in pharmaceuticals to for the purification of drugs for profiling for checking the impurities.

[00:39:54]Similarly, foreign we have already discussed that it is used to assertain the poisoning to assertain the drug abuse or sometimes to check the rash driving where people drink alcohol and they go on rash driving. So that alcohol content can be checked by simple test.

[00:40:13]Similarly in food safety it is very important because now it is mandatory to give you a clearcut profile about the different ingredients present in it.

[00:40:22]Similarly with regard to environment it gives you a very good idea about the certain pollutants present even at trace and nano levels. So drug control studies paracetamol tablet retention time actually confirms the drug identity.

[00:40:39]Similarly resolution gives you an idea about the purity that how much pure your sample is and the selectivity confirms that it does not contain any exipients.

[00:40:52]So it is actually very good with regard to drug control. Similarly advanced insights what are the you know what are the things which are going under the background. So retention behavior is actually influenced by certain weak interactions. Number one is the pi pi interaction which generally happens in case of aromatic compounds. Similarly hydrogen bonding when uh when when electro when an atom actually atom gets in between two electrogative centers two electrogative atoms then hydrogen bonding comes into play.

[00:41:38]So similarly we have ionic interactions because of the charged solute.

[00:41:43]Now we have seen a lot of advantages of KD but KD alone cannot explain all the things. this KD can be write this okay KD so it's not something different it's the same distribution concept so what are the hidden retention concepts so practically TR must range from 2 to 15 minutes for example in HLC case the shape of the uh peak must be gshian in nature and there must be no tailing that is there must be no secondary binding with the stationary phase so what is the optimization strategy. How we can have good results from the column or how can we have effective column separation? Number one is we have to ensure the retention factor that it should range between 2 to 10. We have to adjust the selectivity factor according to column and according to solvent with regard to the polarity.

[00:42:38]Similarly, we have to monitor RS that is the peaks must be well resolved. It's not overlapping or it's not superimposing on each other. it must be separate. Okay. And the peak must be have as small width as possible.

[00:42:55]So it is very important to optimize the column. And these are the three important steps to optimize your column.

[00:43:03]So in the end let us summarize what we have you know uh what we have studied initially. We have to inject our sample.

[00:43:13]Then the there's a dead time or void time that is the time taken by the mobile phase alone to elute from the column it is given by dead time. Then the retention time that is the time taken by the solute along with mobile phase that is called the TR. Similarly capacity factor the retention factor you can say like select uh retention factor and capacitive factor are those same. Then we have the selectivity and finally we have the resolution. On the basis of resolution you can say whether you have done a good separation or not. If not then you have to optimize your separation. And how we have optimized we have seen that the optimization. So initially you carry out the injection of your compound which is to separated then you get the TM value dead time. Then you get DR on the basis of DR you you calculate K dash because KD is not measurable directly. So you uh run all the data in terms of TR then you calculate K dash then alpha then the resolution and then after certain optimization if needed you get the optimized separation.

[00:44:22]Okay. So this is how the elution pornography works. Okay. So what are the future perspectives? Now nowadays you know we are more concerned with the environment. So we have some eco-friendly solvents. We do not have to use every time very toxic solvents. So most of the time we have to rely on the eco-friendly and the most eco-friendly solvent is of course you know our delicious water but the water is actually making problem because it is very polar. So we have to optimize in terms of green solvent. Similarly, we have we have to have small we can separate uh we can be able to separate very small samples that is we can have micrfluidity columns because these traditionally we use very long columns because sometimes this long column is needed to have a better efficiency because new is equal to L upon TR.

[00:45:23]So we cannot compromise with the length of the call.

[00:45:28]Okay. So nowadays you know AI is of every way. AI means we have to use the artificial intelligence in a very intelligent manner along with the machine learning that is AML. So we have to use AI and ML hand in hand to have a very good retention prediction before you know running in a in a lab. we can get an idea that which type of solvent is better for which type of stationary phase. So if we get an idea then it will be definitely it will save our time it will save our resources then we can use the more hyphenated technique like liquid chromography mass spectrometry or the gas chromography mass spectrometry. So we are have to use all these in our future. So future for chromography is very bright. So with this, thank you very much. Thank you all. [music]