[Music] foreign [Music] [Music] foreign [Music] and what we were discussing we were discussing about the different properties of the enzyme in the course enzyme sense and technology and in the couple of modules we are discussing about the enzyme Productions and if you recall what we have what we what we have discussed that the uh you can actually be able to utilize the different approaches to isolate the gene of your interest which is going to code the enzyme and you can have the option of using the genomic library or cdna library to screen out the gene of your interest or you can actually be able to use the site specific primers to amplify the gene of your interest and that you can be able to put it into the cloning vectors with the help of the resection enzyme and the ligates enzymes so uh once you've got a clone then you can actually be able to deliver that clone into a suitable host uh it can be a bacterial host or the eukaryotic host so in the bacterial host we have discussed about the different approaches what you can use for protein production and then we have also discussed about the protein production in the yeast bacteriophage based systems each sexual lines and as well as the mammalian system so once you have generated the product then the next task is that you are actually going to utilize the different techniques to purify the protein and in this current module we are discussing about how you can be able to isolate the uh the protein or the enzyme from the cell and how you can be able to utilize the different techniques to purify the enzyme so uh if you recall in the previous lecture we have discussed about the cell disruption methods and then very briefly we have discussed about the basic principle of chromatography and we have also discussed about the uh purification system and how you what are the different components are present in the purification system and how they are actually going to be helpful for the protein purifications now in today's lecture we are going to discuss more about the chromatography so let's start today's discussion now what we want to do is we want to purify a protein okay so remember that when we were discussing about the protein folding so protein is going to be reduced from the ribosome and uh it is actually going to be produced as a chain right a chain of amino acids right so all these chain of amino acid as soon as they comes out from the ribosome they start folding because of the intramolecular interactions and as a result they will actually going to folds around a central okay and as a as a result of this folding it is actually going to arrange all the amino acid in such a way that if you are actually going to see the cross section of a protein what you will see is that it is actually going to have the uh hydrophobic core at the center so this is actually the hydrophobic core and in the center and on the periphery it is actually going to have the polar residues after that it's also going to have the multiple patches these patches can be recognized by the different system so if you talk about the enzyme they are actually going to give you the different properties that can be exploited in a typical chromatography so the end the amino acids are going to have the different amounts different types of amino acids and that's why they can be able to have the positively charged amino acids or the negatively charged amino acid and that can be exploited in a chromatography technique similarly it can also have the hydrophobic residues so that also can be exploited in a chromatography technique apart from that proteins are globular in nature so they are actually acquiring a surface area and that surface area could vary between the different proteins because the different proteins are going to be of different diameters and that's how they are actually going to be of the different surface area so that also is a very very crucial property that can be also be exploited apart from that it can also have the exclusive region which will have the exclusive affinity for a particular molecule and that also can be exploited in the Affinity uh also so based on these four criterias you can have the different types of chromatography techniques so if you are going to exploit the charge whether it is the positive charge or the negative charge you are actually going to exploit that in a chromatography which is called as ion exchange chromatography similarly if you are going to exploit the presence of the hydrophobic amino acids then this chromatography is called as the hydrophobic interaction chromatography or the HIC similarly if you since the proteins are actually going to have the balls of the different sizes uh you can actually have you can expect that the proteins are going to be of different surface area that can be also be exploited in it in acrobatoreography technique which is called as the gel filtration chromatography and as we said you know proteins are also going to have the region which are going to be having the exclusive affinity for a particular molecule and that also can be exploited in a technique which is called as the Affinity chromatography so in this particular module we are going to discuss about these different chromatography techniques and how you can be able to utilize them to purify the enzyme of your interest so let's start our discussion with the first technique and that is the ion exchange chromatography so as a name says the I OS 10 chromatography is the Democracy where you are actually going to play a role of the different types of ions okay ions could be positively charged ions or ions could be negatively charged as so what is the principle of this technique so this chromatography distributes the analyte molecule as per their charge and their Affinity towards the opportunity charged Matrix the analyte bound to The Matrix are exchanged with a competitive counter ion to elute the interaction between the Matrix and the analyte is determined by the net charge ionic strength and the pH of the buffer so what we are going to say is that in a ion exchange chromatography it is actually a chromatography where you are actually going to exploit the different types of ions so if you are going to talk about that the positively charged proteins so it is actually going to have an affinity with negatively charged Matrix okay so in this case you can actually having a competition with the positively charged ions okay and that's how uh sorry negatively charged ions and that's how you are actually going to have the competition so it is actually a competitive analogy essay where you are actually first going to bind the protein to the matrix by the which is going to be a positively charged and then you are going to elute it with the help of the competition the interaction between the Matrix and the analyte is determined by the net charge onto the protein called the enzyme any strength of the buffer and the pH of the buffer okay and this all we are going to discuss in detail then you will be able to understand these processes so imagine that we have uh analyzed a complex mixture of these protein molecules okay so you have a protein molecule which has no charge you have a protein molecule which has a negatively charged this is the positively charged and then you also have a protein which has the uh one negative charge so you have a protein which is one negative charge you have the protein which is two negative charge one positive charge and the protein which has no charge so when a mixture of positively charged analytes such as M M plus M minus 1 and M2 minus are loaded onto a positively charged Matrix the neutral or the positively charged minorite will not actually going to bind The Matrix where as the negatively charged analyte will bind as per their relative charge and in needed the higher concentration of the counter ions to elute from The Matrix so what we have is we have this mixture which we have loaded onto a matrix which is positively charged so you know that the positive is actually going to attract negative but positive is actually going to Ripple the positive right whereas the neutral is anyway not going to bind right so in this particular column when you are going to load this particular mixture where you have the m0 M minus 1 m 2 minus 1 and M plus M plus and m0 is actually going to not binding the column because the m0 will not have a charge so it will not going to have any affinity and M positive is actually going to get rippled from the column similarly but the M minus 1 and M2 minus is actually going to have the affinity for this column but the strength of this Affinity is going to be proportional to the amount of charge what is present on this particular molecule so the M minus 1 is actually having the lower strength and M2 minus is going to have the higher strength and that's why the M2 is actually going to bind to the column in the beginning of the column and M2 M minus 1 is actually going to bind in the lower portion of the column and when you are going to elute the small amount of the counter ions right so for example in this case the negatively charged ions are actually going to elute the m minus 1 first because the strength of the M minus to the to the Matrix is lower right so it will come out first right so it is going to come out first and M2 minus is going to have higher strength so it is going to come out second that means you have started with the four molecules you started with M 0 m plus n minus 1 and M2 minus 1 and at the end you are going to get all the molecules separately this means you have achieved the purification with the help of the ion exchange chromatography and as I said you know ion exchange chromatography is going to deal with the positively charged ions or the negatively charged and and that's how the based on these ions the an exchange chromatography can be of different types so the Matrix used in the ion exchange chromatography is present in the ionized form the reversibly bond ion onto the Matrix the ion present on The Matrix participate in the reversible exchange process with the analyze hence there are two types of ion exchange chromatography you can have the cation exchange chromatography or the anion exchange chromatography in a cation exchange chromatography in cation exchange chromatography The Matrix has negatively charged functional group with affinity towards positively charged molecules the positively charged analyte replaces the reversibly bound cation and binds to The Matrix in the presence of a strong cation such as sodium in the mobile phase The Matrix bound positively charged analyzed is replaced with the illusion of an analyte similarly you can have the anion exchange chromatography in anion exchange chromatography Matrix has a positively charged functional group with a Affinity towards the negative charged molecules the negatively charged analyte replaces the reversibly bound anion and bound to The Matrix in the presence of a strong anion such as chloride in the mobile phase The Matrix bound negatively charged analyte is replaced with the illusion of the analyte so you can have the cation exchange chromatography or the anion exchange chromatography and you can use the different types of Matrix for example you can have the strong cation exchangers or the weak weak cation exchangers strong Alliance and weakness so these are the different examples of the Matrix what is available for the ion exchange chromatography so you can have these strong anions in which you can have the sulfonic acid or the SP sephorus so you can have and the PH range in which you are going to this these matrixes can be worked is a 4 to 13 you can have the multiple examples like bsp Chef rows SP shepherdx textual CM Shuffle cellulose and CM sephorus similarly you can have the V cation so your V cation you can have the functional group carboxylic acid which is going to be attached to the Matrix and you can use this column in the range of 6 to 10 and the examples are CM sepharos CM sulfur Dex CM surface cl6b and TSK gel CMS 5B okay uh in apart from that you can also have the strong anion or the vegan and so strong anion where you can have the quaternary amine so quaternary amine can be used in a range of 2 to 12 the examples are two sephorus Dao X and all that then we can have the weak anions so in the weak anion you are going to have the primary amines secondary amines and tertiary amines such as Dae and the range in which you are going to use this is two to nine uh examples are dhaff rose capto and the D A Cell rows now how the ion exchange chromatography is going to use is it is actually going to be dependent on the competition between the counter ions so for example in the cation exchange chromatography you have the Beats which is negatively which is attached to a negatively charged groups and on this negatively charged group you have the counter ion that is the sodium which is immobilized so in the presence of a positively charged protein what it's going to do is it is going to do a competition with the immobilized uh sold cation onto the Matrix and in this process what will happen is that immobilized cation is actually going to be replaced and the protein will actually going to bind and as you remember when we were discussing about the different types of analyte when you have loaded onto the positively charged Matrix the charge the amount of charge onto this protein is going to be different so if suppose you have the plus one charge plus two charge plus three charge so this guy is actually going to have the maximum strength this guy is going to have a middle range sense and this guy is going to have the least strength whereas the negative charge molecule will not bind or the neutral molecule will not bind so since the proteins are going to have the different amount of the ionizable groups these charges are going to be different and that's why the strength of the interaction of the protein to The Matrix is also going to be different and then what you are going to do if you are going to again doing a competition with the counter ion so in this case we have supplied the sodium so once you supply the sodium sodium is going to replace the protein which is bound to the uh to The Matrix and this protein is going to replace right but the amount of sodium what you are going to use to replace the plus three protein or plus two protein or plus one protein is going to be different and that's why these proteins are going to be eluted at the different concentration of the sodium and ultimately you are going to regain the Matrix at the end same is true for the anion exchange chromatography the only difference is that the bead is going to be positively charred and it is going to have the negatively charged and and as the counter ion and in this case the protein is going to be negatively charged and therefore the negatively charged protein is going to bind The Matrix and then when you supply the anions it is actually going to replace the protein and that's how you're you are going to get the Matrix bags so whether you you want to use the cation has to change chromatography it depends what kind of charge is actually going to be present onto the protein and based on this charge you can be able to use either the cation exchange chromatography or the anion exchange chromatography now how you are going to run the chromatography ion exchange chromatography so when you want to start the running of the ins change chromatography you have to follow the three steps number one you are going to select the Matrix right so we have discussed many types of Matrix like the uh weak cations and strong cations and weak uh anions and all that so you have to choose the Matrix then you have to prepare the Matrix bead for the chromatography so these beaks are always been supplied as a powder right so this powder has to swell into a buffer where you are actually planning to perform the chromatography and that's how you are going to prepare the beads and then you are actually going to do the ion exchange chromatography but I Has Changed chromatography operation of the ns10 chromatography is also a multiple step process because how you are going to select the Matrix how you are going to prepare the Matrix and how you're going to operate the ion exchange chromatography is depends on the multiple factors so let's see uh how you can be able to select the Matrix when you're going to select the Matrix what are the different points what you have to always keep it in mind first is you are going to be considered the pi values and the net charge onto the protein so you know that the pi charge pi value is the pH at which at which the protein is going to be protein is going to be neutral so keeping this pH and you are actually going to calculate the charge so you can actually be able to vary the pH of the buffer and that's how the net charge on the protein is also going to be vary when you do the pH variation it is actually going to disrupt not only the it is also imparting the charge onto the protein molecule but it also going to disrupt many of the crucial interactions so in those cases you also have to see that particular pH uh whether the there will be a structural stability or not and when you are trying to you know purify the enzyme for example it's very important that the enzyme should have been active at that particular pH it should not be the case that you are purifying an enzyme which is not active at that particular pH because if that happens the enzymes are you know going to be dead before you actually going to purify so there is no point in verifying the enzyme which is dead actually now how you are going to to exploit the pi value and you are going to calculate the net charge so Pi is the is the information of the pi will allow you to calculate the net charge at a particular pH on a protein A cation exchange chromatography can be used below the pi whereas the anions chain chromatography can be used above the pi so if you have the pi at this pH it is actually going to be neutral which means the amount of positive and the negative charge are going to be said but if you go lower to this pH it is going to be positively charged so you can be able to use the cation exchange chromatography if you go to the above to this particular pH it is actually going to be an ion exchange chromatography because the protein is going to be negatively charged now the question comes how you can be able to determine the pi of a protein right because that is very crucial and important uh information what is required to perform the ion exchange chromatography calculating the pi of a protein you have multiple options one is theoretical calculations so that you can manually you can will do right so individual amino acids and their PKA will be used to calculate the pH at which the net charge will be zero so you can actually have the forearm University if you take the four amino acid and histidine so if you calculate the PK values of the number of amino acids like number of lysines number of arginine number of glutamic acid number of aspartic acid and number of histidines if you calculate all these and if you use the PK value of these amino acids it's the average molecule is actually going to tell you what will be the charge and at what PH it's actually going to have the zero charge or net charge as zero charge the second option is the web sources so what you can do is you can actually be able to go to the xpac website and you can given you the link also so if you go to that expressive website and you say the compute the pi values so what you have to do is you have to just put the amino acid sequence in this particular column and it is actually going to do exactly the same way it is actually going to put the theoretical values of the API of the the EK values and of the individual amino acids and that's how it's actually going to tell you the calculation at which pH the pi is actually going to be at which the charge is going to be 0 net charge is going to be 0. and that is going to be your Pi values the third is uh the experimental method so experimental methods where you are actually going to use uh in those cases so the first two options only possible when you are actually going to know the protein sequence okay if you don't know the protein sequence you only have a protein but you don't know the protein sequence then you actually going to have no option but to do experimental calculations how you are going to do the experimental calculations so experimental calculation is based on the basic principle that the proteins have the minimum solubility in a solution with the pH corresponding to their PF and often precipitated out of the solution which means at Pi the net charge is actually going to be 0 right so net charge is zero and because the net charge is zero it is not going to interact with the solvent molecule and as result it is actually going to have the minimum solubility and how you are going to monitor the solubility because if it is having the lower solubility it is actually going to form the precipitate and these precipitates actually are going to scatter a light and that can be measured with the help of the spectrophotometer okay so what you see here is I have knew you a curve between the solubility versus the pH so what I have done is I have taken the protein I have incubated that into the different reactions of PH2 ph3 four five six seven eight nine and ten so all the pH I have prepared right so all these pH are actually going to change the total charge on the protein so it possible that in some charges some places the protein is going to have acquire or reach to a pi values and in that that value the solubility is going to be very minimum so what I have done is I have taken the protein same amount of protein incubated in ph3 four five six seven eight nine and ten and then I have incubated it for some time and then I have measured this scattering at 660 nanometer in a spectrophotometer so scattering is going to tell me that how much the protein is actually going to form the particulate matter so at that I can use to calculate the solubility so initially it is actually going to have the high solubility because it is actually going to have the uh charges so if it's having a charge it's going to interact with the solvent system and that's how it is actually going to this remain solution a as the protein will reach to its pi value its solubility is actually going to go down and that's how you see here is this is the point at which the pi is that the protein is actually having the minimum solubility this means at this particular point the main protein has the minimum uh charge right it could be neutral or it could be minimum charge so at this point this is the point at which you can say that it is actually a pi if you want you can make it more precise you can what you can do is for example in this case this is a value which is closer which is in between the five and six so what I can do is I can just prepare another pH okay of 5.1 5.2 and 6 okay so if I prepare some more pH I will actually going to get very precisely I will know the number that okay the API of this particular protein is 1.16 5.16 so that's a way you have to do right initially you have to start with the broader range when you see that okay the precipitate is maximum between 5 and 6 X then you can actually be able to prepare a buffer of 5.126 and you will know that at 5.16 the precipitate is maximum this means this is actually going to be the pi value of this particular protein now once you know the pi values you can be able to use that for calc for selecting The Matrix so the pi value and net charge so the information of the pi value will allow you to calculate the net charge at a particular pH A cation exchange photography can be used to below the pi value and enhance what it can be used above once you know this you can also be talk about the structural stability so the 3D structure of a protein is maintained by the electrostatic and wonderball interaction between the charge amino acid Piper interactions and so as a result the protein structure is stable in a narrow range around its pi and a large deviation from it may affects its three dimensional structure so that is very very important then it's enzymatic activity similar to the structural stability enzymes are active in a narrow range of pH and this range should be considered for choosing a ion exchange comparator of a because you always want to purify the enzyme which is very very active so as I said you know if you go if you want to use the cation exchange chromatography you have to go below the pi values and if you want to use the line exchange chromatography you have to use the above to DNA stage chromatography now the second step is preparation of the Matrix for the chromatography so the first step is swelling of the Matrix or swelling of the media which is called recycling so swelling makes the functional group to be exposed for a ion exchange for photography the swelling of the anion chromatography is usually been creating by treating it first first with an acid and then with the base so all this is been done so that the Matrix functional groups are going to be you know denatured and therefore they are actually going to be exposed exactly the reverse is the case with the cation exchange The Matrix can be treated with EDTA for the impurities eliminations then you also have to remove these small particles so these fine will decrease the flow rate and unsatisfactory reactions to remove the fines the exchanger is repeatedly suspended in a large volume of water and after the large polymer has settled down the slowly sedimentation decanted which means you are going to wash the Matrix multiple times so that the fine particles can be removed because they are actually going to interfere in terms of reducing the flow rate and this fine particle can also interact with the proteins then you are going to recuperate with the counter and so for example uh this is accomplished by washing that stranger with the different reagents depending upon the desired counter ion to be introduced for example you can wash it with the Noh if you want the NaOH and sodium plus to bind so that will be you going to do for the cation exchange chromatography you can use the HCL if you want to introduce the h plus and so on so once you are going to prepare the Matrix it is actually going to be like this you are going to have the positively charged ions and it is actually going to have the counter ion in terms of the negative ions now when you at this stage you can be able to have the different choices of the buffers what you are going to use for performing the ion exchange chromatography so these are the some of the buffers what you can use and you can choose the buffers based on the working consultation so what is mean by the working computation is that it is always going to be plus minus 1 from the PK values for example in this case it's a 3.1 so it is actually going to be from 2.6 to 3.6 which means 0.5 on this side and 0.5 on this side okay similarly this side also this one also so depending on and what buffer range you are working you can be able to choose these buffers and you can actually be able to work so these are the buffers for the anion exchange chromatography now the second point is that how you are going to perform the ion exchange chromatography so ion exchange performance of the operation of the ion exchange chromatography is a three-step process in the step one you are going to prepare the column Matrix and the stationary phase so column material should be chemically inert to avoid the destruction of the biological sample it should allow the free flow of liquid with the minimum clogging it should be capable to understand the back pressure and it should not compress or expand during the operation so this is going to be the Matrix when you are going to prepare it's going to have the positively charged groups and that are going to have the counter ions then the step two you are actually going to incubate the column with the mobile phase this mobile any strength and the pH are the crucial parameter to influence the property of the mobile phase and what will happen is that once you are going to supply the mobile phase it is actually going to have the counter ions what are going to be present in the buffer and that's how this bead or this Matrix is now ready to bind The Columns or bind the proteins in the third step you are going to prepare the sample so the sample is prepared in the mobile phase and it should be the free of suspended particle to avoid the clogging of the column the most recommended method to apply this sample is to inject the sample with a syringe so this is the protein purification system and you can actually be able to use a syringe to inject the sample onto the column then in the step 4 you are going to do the illusion so there are many ways to dilute the analyte from the ion exchange column you can do the stepwise gradient or you can actually be able to do the continuous gradient by the salt or the pH so illusion can be done by increasing the ionic strength using a gradient displacing the bound protein as iron in the buffer competing with the binding sides so you can imagine that if I add Supply more amount of negatively charged ions it is actually going to compete for the protein which has negative charge and that's how it is binding to The Matrix and as a result it is actually going to have the competition so these negative ions are actually common bind to The Matrix and the protein which is bound is actually going to be replaced and therefore it is actually going to come out into the solution if you further increased in the ionic strength the displaced proteins that are more highly charged so it's actually going to be in proportion to the amount of protein so initially you see a very I when I supplied the low amount of ions only the two charged protein are going to be eluted but when I am supplying the more amount of charge the three and four uh charge containing proteins are also going to be eluted once you are done with the chromatography the last step is actually the column regenerations so after the illusion of the analyte and exchange chromatography columns requires a regeneration step to use for the next time column is washed with a salt solution with a ionic strength of two molar to remove the all non-steptically bound analyte and also to make all the functional group in a ionized form to bind the fresh air light so this is what going to happen when you when you regenerate you wash it with the very high ionic salt it is actually going to remove all the proteins and it's also going to bring the counter ion back because this counter ion is actually going to replace when you are going to load the protein with the lignition in charges so this is all about the ion exchange chromatography now let's move on to the next technique and next technique is called is going to utilize the hydrophobic groups what is present onto the protein structures and the technique is called as the hydrophobic interaction chromatography so hydrophobic interaction from micrography as the name suggests is going to exploit the ability of a strong interaction between the hydrophobic groups attached to the Matrix and the hydrophobic patches present on the analyte such as protein so what happen is that when the protein is going to be produced from the ribosome it is going to be produced as a string of the amino acids which are attached through the peptide bond but when it rotates it actually try to hide the hydrophobic groups from the polar residues or from The Polar environment right and that's why it actually keeps these hydrophobic groups in the center and all the protein actually rolls over this so that these hydrophobic groups should not be able to see the polar environment because you are outside it is polar because you have the water molecule outside right and that's how it is actually going to give you the functional fully developed or folded protein where all the hydrophobic groups are going to be present in the center and all the polar groups are present outside because in outside you are going to have the water molecules now the question is because you want to utilize this for the hydrophobic interaction chromatography these hydrophobic patches should be available for the protein to interact with the hydrophobic groups what are present onto the Matrix and that you are going to achieve by a process which is called as the salting a so what has happened is that if you are actually going to add the small amount of salt what will happen is that it is actually going to remove the bound protein molecule so it's actually going to remove the some of the water molecules and as a result it is actually going to bring more and more space into the solution and that's why it is actually going to increase the solubility of the protein molecule and this effect is called as the salting in so addition of a low amount of salt to the protein solution results in the displacement of the bound water molecule with the increase in protein solubility and this effect is called as salting in in the presence of more amount of salt water molecule shielding water protein side chains are displaced completely with an exposure of hydrophobic patches on the protein surface to induce the protein precipitate or decrease in solubility this effect is called as salting so what happen is if you allow if you add the small amount of salt it is actually going to remove the you know the water molecules and that's how it's actually going to bring more space and that's how it's actually going to increase the solubility but if you increase more amount of salt right then it is actually going to remove the uh even the it is going to expose the hydrophobic patches and as a result the protein protein molecules are actually going to interact with each other and they are actually going to form the precipitate and this effect is called as the salting out the phenomena of this salting out is modulated so that the addition of a salt induces exposure of the hydrophobic patches on the protein but does not cause the precipitation or aggregation the exposure of hydrophobic patches facilitate The Binding of protein to the non-polar ligand present onto the Matrix when the concentration of the solids decrease the exposure it's 4 both hydrophobic patches onto the protein reduces the Affinity towards the Matrix and as a result it is actually going to elute so what we are doing is we are actually adding the high salt so that it is actually going to expose the hydrophobic patches right and hydrophobic groups on the protein right and as a result it is going to bind The Matrix now what you are going to do is you're going to reduce the salt so if you reduce the salt if you reduce the salt the hydrophobic patches are going to be covered with the pore with the water molecules and as a result it is actually going to destroy the interaction between the hydrophobic patch and the functional group what is present onto the Matrix there are different types of choice the choice of the Matrix what you can use you can use the butyl Chef rules you can use of Rose when is approach a high sub and low sub you can use the Capital Finance approach and you can also use the octails of course so choosing a suitable Matrix is essential to achieve the best results the strength of The Binding of analyte on the HIC column is governed by the length of the aliphatic linear ligands right Matrix with the aromatic Rings containing ligand waves additional pipeline correction and they will bind analyte more strongly than the same number of carbon aliphatic ligands in addition the presence of Piper interaction gives the selectivity as well as at least At Last the ligand density play a vital role in the strength of binding of an analyte to The Matrix hence this point should be considered to choose a suitable Matrix for the uh for the purifications for example you have the two choices you can use the finance of course low substituted and phenolative process High substituted low substituted means low concentration of the phenyl group what is present onto the Matrix whereas here you are going to have the high concentration so depending on your Affinity of the depending on the Affinity of a protein for a matrix you can actually be able to choose whether you want the low substituted or the high substituted now how you are going to perform the hydrophobic interaction chromatography so what you are going to do is first you are going to do the equilibration so in the step one you are going to do the equilibration so HIC column Matrix packed in a column and included with a buffer containing 0.5 to 1.5 molar ammonium sulphate right so when you are actually going to put the high side into the equilibration buffer the start must be below the concentration where it has a salting own effect so when you put the high concentration it is actually going to remove dehydration shell from the protein and it is actually going to expose the hydrophobic patches and as a result the the functional group what is present onto the protein of on The Matrix is actually going to bind these hydrophobic patches now in the Second Step you are going to do the you're going to do the washing right so your and then the third step you're going to do the illusion so there are many ways to elute and analyte from the hydrophobic interaction chromatography you can decrease the salt concentration so you if you decrease the salt concentration what will happen is that it is actually going to bring the hydration shell back and once it actually brings the hydration shell back all these hydrophobic patches are actually going to be covered by the water molecule and as a result it will not be able to make an interaction with the HIC Matrix and that's all it's actually going to come out into the solution then you can also change the polarity of the mobile phase such as alcohol right so if you actually change the polarity of the mobile phase then also you are actually going to do do the same because then there will be a competition of the uh molecules what is present in the uh in the mobile phase so for example if you are going to use the alcohol right uh alcohol molecules then are going to compete for the hydrophobic patches with the Matrix and as a result the alcohol is actually going to bind these hydrophobic patches and it is actually going to come out uh buy a detergent to replace the bound protein so you can also use a detergent to dislodge or destroy this interaction between the uh match IC Matrix and the protein and therefore you can be able to use that for illusions now in the step four you have to do a column regeneration so after the illusion of the analyte HIC column requires a regeneration step to use it for the next time column is washed with a six molar ulia or bolognium hydrochloride to remove all non-specifically bound protein the column is then incubated with a mobile phase to regenerate the column the column can be stored at 4 degree Celsius in the presence of the 20 alcohol containing point zero five percent sodium aside so this is all about the chromatography techniques where we have actually discussed about the exploitation of the charge so we have discussed about the different steps what we have to do for the ion exchange chromatography and then we also discuss about the hydrophobic interaction chromatography so we have discussed about the uh what are the different steps how you are going to perform the hydrophobic interaction chromatography so with this I would like to conclude my lecture here in our subsequent lecture we are going to discuss more about the gel filtration chromatography and as well as the Affinity chromatography thank you [Music]