Hello everyone, this is Doctor Vishal Trivedi
from department of biosciences and bioengineering IIT Guwahati. And in the course experimental biotechnology
we are dealing with the different techniques so that you can be able to utilize these techniques
for tackling the different scientific problem through designing the better experiments and
so on. So in this series today we are going to start
the discussion about the chromatography. So chromatography is technique which actually
allows you to separate the molecules and when you have a crude mixture you can able to purify
the molecule of your interest. So let us discuss about the chromatography. So chromatography was first discovered by
a Russian Italian botanist Mikhail Tsvet and the chromatography literally means that you
are writing in the color. So as you can see what the Mikhail has performed
that he has applied a small plant extract on to a column, and when he has dissolve this
sample what he could found that the sample is being dissolve into the different bands
of different color. And that is how he has given the name as the
chromatography or the color writing. The chromatography is a technique which is
being used to separate the molecules. But before getting into the details of discussing
the different aspects of chromatography let us discuss what is the basic; principles of
separation? So what is mean by the separation or the isolation,
so you can imagine that you have a mixture of the molecules, you can have the molecules
or the different colors some are yellow in color, some are red, some are blue, some are
pink. And what you have to achieve is the isolation
of these pink color compounds. So that is what is called as the separation
or the isolation of the compound. So how you can achieve that suppose we give
you the balls of the different colors and we ask you to separate the balls of the pink
color then what you have to do is? You have to use some ways so that you can
be able to recognize or you can be able to distinguish the pink color from the rest of
the color and that is how you can able to separate these pink color spots. That is what exactly is this separation techniques. The separation techniques always utilize a
exclusive parameter which can be utilized or which can be exploited to isolated the
compounds. Now you can take an example of these 3 molecules. So these 3 chemical molecules are being present
and we have taken these compounds only to discuss the basic principle of separation. So suppose you have the mixture of any of
these 3 compounds then you can be able to separate them exploiting the different physical
as well as the chemical properties. What are the chemical properties you can exploit? You can exploit the molecular weight. You can exploit the boiling points in case
these molecules are of liquids. Then you can use the freezing points, you
can use the crystallization, you can use you can do something that the one compound is
going to be crystallized. Whereas the other compound is going to be
present in the solution and that is how the crystallized compound can be filtered out
and can be separated from the rest of the molecules. Similarly you can actually play very nicely
with the solubility as well as the density parameters. So all these falls under the physical properties
exactly the same as you can actually also play with the chemical properties, for example
you can have the different types of functional groups. For example for this case the phenol as a
functional group whereas the aniline has NH2 as a functional group. So these 2 functional groups are very different
from the benzene so you can actually exploit the presence of functional group. And once the functional groups are different
their reactivity of one or other reagent is also going to be different. So when you react them some of the molecules
will react the other molecule will not react. So the molecule which will react to form the
complex this complex can either change the physical properties in such a way so that
the complex is going to be more soluble or the less soluble. And because of this you can be able to purify
your desire compounds. Let see how you can exploit these physical
or chemical properties for these three set of molecules. So these 3 set of molecules benzene, phenol
and aniline now you can see that the molecule weight, the molecule weight of the benzene
is 78:1. Whereas phenol is 94.1 and the aniline is
93. So if I have to purify the benzene and the
phenol I can actually exploit the differences between the molecular weight then you can
also see the boiling point, the boiling point of the benzene and phenol is different. So that also can be utilized. Let see show the boiling point can be exploit
to separate the 2 molecules using the technique called fraction distillations. So initially what we have? We have a mixture of benzene and the aniline. So when you have a mixture of benzene and
aniline what you have to do is you have to keep this sample in a flask and then you connect
this flask to a condenser. If you all do not know about the condenser,
the condenser is a instrument which actually allows you to distill the compound. So what happened is that the water vapors
or the vapor of the compound goes into this central tubing and then this central tubing
is surrounded by the water jacket. So when the water comes goes out and comes
out it this this tubing becomes cooled. So when the vapor goes in to this central
tube it at actually get cooled and because it get cooled it turns in to the liquid face
and that is how this water vapor can be collected in the second in the separate flask. So this distillation pretest can be used for
distillation of different liquids or the mixture of a different liquid. So what you have to do is you have a mixture
of the benzene and aniline. You start heating these with the help of split
lamp or the Bunsen burner. And as the temperature will go up the benzene
which is actually having the lower molecule boiling point will have the tendency to go
and remain in the vapor phase. Whereas the aniline which; is having the higher
boiling point will remain with the liquid phase. Which means; that the benzene will go and
go with the vapor phase whereas the aniline will remain with its own phase. So as a result what will happen is when you
go with the first round of distillation you are going to have more and more amount of
benzene in the upper flask, whereas you are going to have the aniline in the lower flask. So if you repeat this whole exercise multiple
times or if you use the fractional distillation operators you could be able to separate these
2 molecules. Because they are having a very huge separation
in terms of their boiling point the benzene has a lower boiling point whereas aniline
has a higher boiling point. And because of the difference in the boiling
point the benzene will evaporate faster and evaporate sooner. So that benzene will remain the vapor phase
whereas the aniline will tend to remain in the liquid phase. Another result you will collect the aniline
in the lower chamber and lower flask. And whereas the benzene you will collect in
the upper flask. So at the end of these distillations you will
be able to separate the aniline from the benzene. If you can be able to distribute the molecules
into the 2 different phases whereas for example in this case you have the 2 phases one is
the vapor phase and the other one is the liquid phase. This whole this whole phenomenon can be monitored
or can be used to separate the molecules and it is. So the distribution coefficient which actually
explains this distribution is to describe the distribution of compound one between the
2 phases A and B, which means in this case the you have the distribution coefficient
of benzene for vapor phase and a liquid phase. Whereas you are; also going to have the distribution
for aniline which is different. So in this case the phase A is going to be
the vapor phase, phase B is going to be the liquid phase. So the Kd is going to be the concentration
of compound in phase A verses the compound concentration of the compound in phase B.
So as the names as the distribution coefficient is a ratio of the concentration of compound
in phase A verses the phase B it actually does not have any unit and it is actually
also a virtual number because as soon as you change the pairs of the compounds or as soon
as you change the condition the distribution of the molecules are also going to be different
between the different phases. And as a result, the distribution coefficient
always depends on the number of counter ions or number of compounds which are present in
the mixture. And it is also a not a fixed number it is
a dynamic number which actually depends on what a way the compound is going to be distributed
into the different phase. Now let us come to the chromatography. So chromatography the purpose of the chromatography
is to separate a complex mixture into the individual component exploiting the partitioning
effect. So partitioning effect means you are distributing
the molecule between the 2 phases which distribute the molecules into the different phases and
because you can do the partitioning with the help of during the chromatography. The partitioning can be done in 2 different
ways and that is how the chromatography can be divided into the 2 different types. One is called as the partition chromatography,
the other one is called as the absorption chromatography. So in the partition chromatography the analyte
distribute themselves into 2 phases. One is liquid stationary phase and the other
one is the mobile phase. So in the partition chromatography always
occurs in a places where you have the 2 liquids which are not immiscible or the 2 liquids
which are not mixing with each other, and you can put a compound and then the compound
is going to distribute between the these 2 liquids. Which means if I have a, 2 liquids and if
I put a compound X what will happen is the X will have the lower solubility in this one
or it can have the higher solubility in to the solvent 2. In that case what will happen is the X is
going to partition between the 2 compounds. So the partition chromatography, always exploit
the partitioning of the molecule between the 2 phases. In these 2 phases the 2 phases are always
been liquid. The major advantage of this chromatography
is that it is simple low cost and has a broader specificity. It is further divided into the liquid liquid
chromatography or the bonded phase liquid chromatography. So the in the liquid-liquid chromatography
you are actually utilizing the 2 different liquid of the different solubility and the
compounds are going to be partitioned between the 2 liquids. For example, if I can take the mixture of
hexane and water. So suppose I my compound is hydrophobic and
it is not soluble in water then what I can do is I can just simply add the hexane mix
it into the very vigorously. So in that process what will happen is the
compound will going to partition between the aqueous phase as well as the hexane phase. And the sum of my compound which is actually
more soluble in hexane phase will transfer from the aqueous phase into the hexane phase. On the other hand the compound which, are
more of polar in nature probably will remain the aqueous phase and that is how you can
actually be able to separate the molecules utilizing the different partitioning. And that you can do in the liquid-liquid chromatography. Whereas in the bonded phase liquid chromatography
you have the liquid layer which is actually been absorbed on to the solid support and
then the molecules are been portioned between the liquid which is bonded on to a support
verses the liquid as a mobile phase. The classical example of the liquid-liquid
or the partition chromatography is the gas liquid chromatography, thin layer chromatography
and the paper chromatography. In all these 3 cases some places you are using
the liquid-liquid partition chromatography and some places you are using the bonded phase
liquid chromatography. Now the second way in which you can actually
the partition the molecule is called as the adsorption chromatography. In this form of chromatography, the matrix
molecule has the ability to hold the analyte on their surface through a mutual interaction
due to the different types of the forces such as the hydrogen binding, electrostatic interactions,
vander waal etc., So in this adsorption chromatography what
you are doing is? You have the small beads on which you have
the functional groups these functions group may facilitate the different types of the
forces. For example you they can have the hydrogen
bonding they can do the vander wall interaction or they can have the electrostatic interaction
or they can have the hydrophobic interaction with the groups present on to the protein. And as a result the molecules are going to
be absorbed on to these groups. So molecules are present in free flowing into
the mobile phase whereas the molecules are also being absorbed onto these beads. And then ultimately these molecules are going
to be removed when you do the washing and the subsequent steps. Whereas beads absorb molecule can be removed
from the column using the different types of elution techniques. And that is how the absorption chromatography
is very much different from the partition chromatography So the partition chromatography is simple
low cost and it is broad specificity the partition chromatography is not very much towards the
specific molecules. Whereas the absorption chromatography can
be modified or can be adjusted in such a way that it should be it is going to be even for
the specific molecule. So it actually provides the specificity if
becomes the user friendly and it is more convenient to perform compared to the partition chromatography. The classical example of the absorption chromatography
is ion exchange chromatography, hydrophobic interaction chromatography and the affinity
chromatography. Irrespective of whether you do the partition
chromatography or the absorption chromatography the basic principle of the chromatography
remains the same that you are actually distributing the molecule between the 2 different phases. So the distribution in the case of chromatography
is also been defined by the same formula that is the concentration of the molecule in phase
A verses the concentration of the molecule in the phase B. In the case of column chromatography
it is well understood that the phase A is always been the stationary phase or the matrix
whereas the phase B is the mobile phase or the buffer. So in the case of other cases the distribution
coefficient can have the multiple phases can have any phase in the phase A or phase B. But in the case of column chromatography for
the for purpose of convenience, we always keep the stationary phase as the phase A and
we keep the mobile phase as the phase B to calculate the Kd values. Now let us see how the molecules are being
separated when they are being subjected to a chromatography. So we have taken a mixture of the compound
and we would like to understand how the Kd is being used in a column chromatography to
separate the 2 molecules. So we had taken a example of a molecule which
is called X and Y. And the Kd value of the X is 1. Whereas the Kd value of Y is 9 ok. If the Kd value is 1 which means if every
ml of the mobile phase the molecule is going to be distributed evenly between the stationary
phase as well as the mobile phase. Which means if I have the X and Y if I loaded
the X and Y mixture on to the column, as the molecule will run to the column it will actually
going to distribute between the matrix as the on the mobile phase. And because the X has the Kd value of 1 it
is actually going to distribute always in terms of 1 is to 1, which mean if I pass through
the 1 ml of the buffer the X is going to distribute 50-50 between the matrix as well as the stationary
phase. Whereas the Y is going to be distributed in
terms of 1 is to 9 which means the Y is going to be remain with the stationary phase whereas
the X will also is going to remain with the mobile phase. So as the sample will run through the column
with the water as a mobile phase as they will travel the X and Y will partition between
the stationary phase and the mobile phase. As there is a huge difference in Kd values
the Y is always been associated with the matrix or the stationary phase and remain on top
of the column. Whereas the X will move along the water phase
because as you can see that the X has a lower Kd value so it will always going to be partitioned. And that partition will going to be keep distributing
it towards the mobile phase. At the end of the chromatography X will come
out first whereas Y will come out at the end. So this is the X molecule and this is the
Y molecule and because they have the very wide separation or wide difference in terms
of the Kd values the Y is going to be separated from the X. And that is how you an actually exploit the
distribution of the 2 molecules based on the distribution coefficient. Now once you monitor the elution of these
molecules from the column you can use the any parameter to monitor. For example, you can monitor it simply by
taking the absorbance. So what you will see is that you will see
a pattern in terms of the multiple peaks ok. And what you can see in this and this pattern
where you are going to see the pattern of elution with multiple peak is called as the
chromatogram. So the plot of elution column along with the
absorbance or any other parameter is known as the chromatogram. So on the Y axis you will put the detector
signal which means it could be a absorbance, it could be fluorescence, it could be a refractive
index and so on. And on the other hand on this side you are
actually going to put the elution volume or the elution time and this pattern is known
as the chromatogram. So in the chromatogram what you can see is
that you have the 4 peaks A peak, B peak, C peak and the D peak. So A and B are the 2 separate peaks which
means the column is good enough to separate the A from the B. Whereas the C and D are
actually are the fused peaks and how the C and D are fused peak because the C is eluting
like this. Whereas the D is eluting like this which means
they are actually sharing a broad base and because their base is broad they are interacting
with each other and as a result they are actually being eluted as the fused peaks. And why it is happening so? It is happening because these column is good
enough to separate the A and B but it is not good enough to separate the C and D which
means the column has the lesser ability to separate the peaks C and D and the ability
of the column to separate the 2 peaks and give you the 2 molecules into 2 separate peak
is known as the resolution. So what is mean by the resolution? The ability of a chromatography column to
separate the 2 analyte peak from one another is known as the resolution, which means in
the earlier figure the A and B the column is separating the peak A and Peak B, and that
is called as a resolution. And it is defined as the ratio of difference
in the retention time between the 2 peaks and the average of the base of the peak width. Which means the resolution is directly proportional
to the difference of the retention time between the 2 peaks and it is the inversely proportional
to the average of the base of the peak width. So when you have the resolution of 1 which
means the retention time and the average width of the peak is going to be equal the separation
of the 2 peak is going to be performed with an efficiency of 97.7%. But if you have the resolution which is more
that, 1.5 it will actually going to give you the separation of the 2 molecules by more
that, 99%. The number; of distribution event govern the
ability of a column to separate the 2 analytes. In another words resolution is directly proportional
to the number of distribution events. In column chromatography each thin layer of
the column matrix participates in distribution of the molecules. So you can assume that the height of the distribution
plain is H which means the beads what you are using has a diameter of the H. And the
length of the column what you are taking is L. If that is the case the number of distribution
plain is going to be equivalent to the length divided by the diameter of the individual
beads. Which means those many number of beads are
going to be present in the column. And the N is the directly proportional to
the 16 tR / W whole square or N is directly proportional to the 5.54 tR / W average the
whole square. Let us see how the distribution plain, are
going to improve the resolution of a column. So number of distribution plain in a column
is controlling 2 parameters. Number of distribution as the number of distribution
plain will go up it will allow the analyte to travel for longer period of time consequently
it will increase the distance between the 2 peak. Which means as the distribution plain will
go up or as the number of distribution plain will go up that the length or the distance
between the 2 peak is going to be more and more which means the delta t is going to be
on a larger scale. And if you remember the r is directly proportional
to delta t. On the other hand, as the number of distribution
plain will go up it will reduce the width of the base of the peak as it results into
the peak of the more sharp peaks. You can take the example imagine that I have
packed a column with the varying number of distributions plain. So if have the distribution plain of n = 10
I am going to see a peak width with the base of this much. Whereas if I increase the number by n by = 100
I am going to reduce the base and it is going to give me the shrink base. And if I increase the distribution plains
further up which means if I increase the distribution plain by another 10 times, I am going to see
a sharp peak with a very, reduced base. So that actually proves that as you increase
the distribution plain you are actually going to reduce the average peak width of the base. And that actually is going to increase the
resolution because the R is inversely proportional to the W average. So as the number of numbers is of the tripling
plain is increasing the peak is increasing. Hence the number of distribution plain is
indirect way to measure the column efficiency. Higher the number is desirable for better
separation. Now if you would like to perform the chromatography
you can utilize a chromatography system. And in a typical chromatography system you
are going to have the multiple components. Let see what are these components? So you can have the reservoirs. The reservoirs you can have 1 or 2 reservoirs
where you are going to keep the mobile phase, the buffers. In some cases, even you can have the 4 reservoirs. So that you can be able to perform or you
can be able to prepare the gradients between the 2 buffers. Then next to the reservoir you are going to
have the pumps depending on the type of the chromatography system you can have the 1 pump
or the 2 pumps. And they can individually be connected to
the individual reservoirs. So that you can be able to utilize, the different
pumps to produce the very precise gradients. Depending on the pressure level you can be
able to utilize the pumps which are made up of the glass or which are made up of steel. Then next to the pump you have the mixture. The mixture is the place where you are going
to mix the liquid which are coming from the individual reservoir. So that you can have the homogeneous mixed
buffer and that actually will go into the column. And column is made up of a glass or the steel
depending on the pressure limit. In most of the low pressure or the middle
pressure chromatography system you can be able to attach the glass columns. But when you go to the high-pressure columns
you have to use the column which is made up of steel. Next to the steel next to the column you are
going to have the detectors. Detector is something which actually detects
the analyte which are coming out from the column the detector could be or utilize the
multiple parameters. Detector could utilize the UV visible spectroscopy
UV visible absorbance. The detector could utilize the fluorescence,
detector could utilize the refractive index or detector can utilize many other parameters
like you can even attach the mass spectrometry to the, as the detector. And then in that case the chromatography system
will it will turn into the liquid LCMS or liquid chromatography mass spectrometry system. So the detector could be off depending on
what kind of molecule you are analyzing you can actually choose the detector. And that actually is going to do the online
monitoring system to test the presence of the particular analyze based on the different
type of properties. There are different types of detectors such
as UV visible detector fluorescence detector or RI detector and all that. Once you detect the molecule using the detector
because detector is going to give you the pattern or detector is actually going to give
you the chromatogram you can actually ask the machine to collect the peaks and that
you will do in the fraction collectors. And the fraction collectors will collect these
eluting molecules into the different fractions. And once and on the other hand detector will
also give the signal to the recorder and the recorder is going to show you the profile
of these eluting molecules in the form of a chromatogram. So this is all about the brief overview of
the chromatography system. Let see how a real chromatography system look
like. A real chromatography system for example in
this case we are showing the AKTA pure M. So the AKTA pure M has the different components
like you have the reservoir. So you can have the reservoirs then you have
the column. So next to the reservoir you have the pump
so in the AKTA pure M you have the 2 different pumps to and you can see the you have the
4 reservoirs. Then you have the mixer, so you have the mixer. Then you have the column so in this case you
will see it has a in the picture we are showing a high trap high resolution column. Then you have in this place you have a detector
and next to the detector whatever is coming out can be collected in the form a different
fractions, or you can actually be able to utilize a computer with the interface to the
system to visualize. What is visualize the profile of the eluting
molecule in the form of chromatogram. So this is all about the chromatography system. So when you would like to utilize the chromatography
to purify the molecule you have to decide a strategy and a strategy will let you to
purify or isolate the protein of your interest. And when you would like to design a strategy
you have to consider the many parameters. So when you want to do a protein purification
strategy. So suppose you are working with the mammalian
cells as soon as you break open the mammalian cells you are going to get the cell lysate. And once you are getting the cell lysate and
you would like to isolate the protein like actin. Now if I have to purify the actin I should
know the protein sequence. I should know the biophysical and biochemical
properties. I should know how the other people have purify
the actin in the previous research article. And then I should also know what are; the
affinity tag or the biological affinity of the actin towards a particular matrix so that
I can utilize that particular matrix in the absorption chromatography. And lastly, I should also know what kind of
projection level is our requirement. So if I; have a requirement of milligram level
or if I have a requirement of simply to isolate the actin for immunization purpose and so
on. Depending on what kind of what kind of the
production level you want you have to divide the strategy. Because if you are looking for the protein
in milligrams or gram level then you have to devise a strategy where you should preserve
each and every loses because when you perform the chromatography technique. You are going to lose the molecules or you
are going o lost the sample because some of the fraction you have to discard when they
do not have the essential or enough proteins of enough quality ok. So in this module we are going to discuss
about the different chromatography techniques. How these chromatography techniques can be
utilize to purify the proteins. And on the other hand we also be going to
discuss how you can utilize these chromatography techniques to answer your specific experimental
or the scientific questions. So with this I would like to conclude our
lecture here. In the subsequent lecture we are going to
discuss more about the different chromatography techniques. Thank you.
