Introduction and Basic concepts in chromatography 1 Video Lecture - Biotechnology Engineering (BT)

in coming lectures we are going to start
our new topic chromatography now
chromatography in chromatography we are
going to discuss about basic concepts of
chromatography as well as some
techniques particularly the techniques
of column chromatography now as we all
know that there are a lot of biochemical
processes which which are going on
inside the cell now to understand the
biochemical processes it is very
important to understand the function and
physical and chemical properties of the
constituents or the active participants
of these biochemical processes and to
understand their function or their
physical or chemical nature it is
important to get these constituents in
pure form so that we can understand the
particular function of an analyte or a
particular molecule inside a cell so if
if we analyze our understanding of
biochemical processes have gone in
parallel with our ability to isolate and
purify biological molecules which
includes macromolecules such as proteins
and nucleic acids and also other lot of
other biological molecules
now most biochemical investigation
requires the they require that material
may be purified so that that is free of
contaminants and this biological
material then can be utilized for
characterization in terms of its
physical and chemical nature typical
cell contains thousands of different
kinds of molecules and many of them may
be closely resembling in physical and
chemical properties so it becomes a
challenging task to obtain of
homogeneous solution of a particular
substance so if you see many times these
molecules of interest which needs to be
investigated they exist in very small
amount that is they might be like 0.1 or
maybe less than of the total cell
content and another problem which comes
in is that they may be unstable actually
so when a substance is present in a very
small amount and this has to be brought
to a purity level of almost above or
around 98% and you have to have
substantial quantity for analysis or
characterization so the task of
purifying biological molecules is quite
challenging and in modern times it
relies heavily on chromatographic
methods
now these chromatographic methods
started with paper chromatography so
these include paper chromatography then
thin layer chromatography came and in
now adays column chromatography is
widely used now column chromatography in
particularly is of the most prevalent
method in biochemistry for purification
and also in certain like clique clinical
applications and other areas of
biotechnology it is a continuous
separation process where separated
components can be collected into
separate fractions for analysis now high
performance liquid chromatography has
been has revolutionized the technique
where a very high degree of purity could
be achieved so column chromatography is
one where we can use it for getting a
good amount of pure compound to be
analyzed now let's introduce little bit
chromatography and let us get into a bit
of history of chromatography now
chromatography it comes from Greek where
chroma means color and graphene means to
writes it's like color writing so it's
usually
you introduced as a technique for
separating and identifying or we can say
or identifying the components of a
mixture now chromatography literally
meaning color writing was first employed
by a Russian scientist
mikail sweat in 1900 and he continued to
work with chromatography in the first
decade of 20th century and primarily for
the separation of plant pigments such as
chlorophyll carotenes gentle fields and
so on so that's how the chromatography
started chromatographic techniques then
developed substantially as a result of
the work of two scientists that is
Martin and since during the 1940s and
1950s they established the principles
and with basic techniques of partition
chromatography and their work encouraged
the rapid development of several
chromatography methods some of them we
are going to discuss in this section now
the basic principle of chromatography is
that the components in a mixture have
different tendencies to adsorb onto a
surface or to dissolve in a solvent now
in all forms of chromatography methods
there will be a stationary phase now
this stationary phase could be solid gel
or liquid and this is immobilized then
there will be a mobile phase that is it
could be liquid or it could be gaseous
and it will this mobile phase flows over
or through the stationary phase so what
you have essentially is an immobilized
stationary phase and a mobile are moving
mobile phase which moves through the
stationary phase now separation of
various components of a mixture is based
on differential partitioning or we can
say distribution behavior between these
two phases that is a stationary phase
and mobile phase so the
on how a particular component divides or
distributes itself into the two phases
so chromatography may be prepara tip or
analytical now the purpose of propriety
of chromatography is certainly to
separate the component and get in a
substantial amount so it's a form of
purification and in analytical
chromatography is done normally with
smaller amounts of material and is for
measuring the relative proportions of
analytes in a mixture and two are both
both are used extensively and they are
not mutually exclusive now let's get
into little bit the general principle of
the chromatography
the basis of all forms of chromatography
is partition or distribution coefficient
as we were talking about and it is
represented by KD that is dissociation
or distribution sorry that is
distribution coefficient now this
distribution coefficient describes the
way in which a compound distributes or
partition itself in between two
immiscible phases now the various
components of a mixture will get
separated due to varying affinities or
we can say distribution behavior of
those components for the stationary
phase and the mobile phase so if we
consider two immiscible phases a and B
that could be stationary or mobile phase
now the value for distribution
coefficient will be a constant at a
given temperature and is given by the
expression if you see on your screen the
that is concentration in phase a that is
and divided by a concentration in phase
B equals the distribution coefficient or
KD value so this will give you a sense
of how a particular analyte distributes
itself between the two immiscible phases
now there is another term which is known
as effective distribution curve
efficient and it is defined as the total
amount rather than concentrations of a
compound present in one phase divided by
the present in a phase now this will be
like so here it's a concentration of a
compound presenting one phase divided by
the total amount present in the other
phase so it is the distribution
coefficient multiplied by the ratio of
the volumes of the two phases present so
what does that mean rather you are not
talking about concentration as such in
effective distribution coefficient the
total amount is being taken so if the
distribution coefficient of a compound
say for example between the two phases
is one that is it is equally distributed
then but if you have volumes between say
5 ml and 1 ml then the concentration in
two phases will be the same but the
total amount present in the two phases
will be different that is one phase will
contain five times more than the other
phase so these two terms kind of one
signifies the distribution in terms of
concentration and another signifies
distribution in terms of amount of
substance present now there could be
different combinations of stationary and
mobile phases they could be selected on
the basis of a particular compound to be
separated and it should be selected so
that if you are taking and unlike it
could be separated and it has a varying
distribution coefficients from other
analyte so different combinations could
be and we will be discussing these in
detail as we go along and also we are
going to discuss certain techniques here
particular techniques in column
chromatography based on these
combinations so one combination could be
based on equipped partition equilibrium
or partition equilibrium here in
partition equilibrium occurs between us
Neri liquid phase and a mobile liquid
phase or a gas phase in this case like
for example there could be a partition
chromatography there could be reversed
phase chromatography there could be
gas-liquid chromatography these are the
examples of partition chromatography
there could be an adsorption equilibrium
so an adsorption equilibrium occurs
between a stationary solid and a liquid
mobile phase and example of this
particular type of chromatography
adsorption chromatography then there
could be an exchange equilibrium in an
ion exchange equilibrium it occurs
between a stationary ion exchanger and a
mobile electrolyte phase so the typical
example of this is an exchange
chromatography then an equilibrium could
occur between a liquid phase trapped
inside the pores of a stationary porous
structure and a mobile liquid phase and
a typical example is exclusion
chromatography or gel chromatography
another combination could be equilibrium
between a stationary immobilized ligand
and a mobile liquid phase so where what
what has been done is a ligand has been
immobilized onto the stationary phase or
it acts as a stationary phase and a
mobile liquid phase passes on through it
and typical examples of this type of
cryptographic techniques include
affinity chromatography techniques
different types of affinity
chromatography technique techniques so
you see there could be lot of different
combinations of stationary and mobile
phase and these will be discussed in
detail now there are three modes or
rather we can say essentially two modes
of chromatographic separations now here
three modes where paper chromatography
could be one mode but it has been kind
of replaced
a thin layer chromatography paper
chromatography and thin layer
chromatography comes under planar
chromatography as the bed or you can say
the chromatographic material or bed
where this separation is taking place is
present on a planar surface so paper
chromatography and thin layer
chromatography bed is present on a
planar surface as far as column
chromatography is concerned the in
column chromatography the stationary
phase is packed in a tube or in a column
so that's why it's called a column
chromatography
so let's little bit discuss about paper
chromatography and thin layer
chromatography and then we'll move on to
column chromatography in particular
alright paper chromatography is an
analytical technique essentially and it
is utilized for separation and
identification of mixtures that may or
may not be colored like earlier started
with the color like we were discussing
about say pigments from the plants and
other colored substances but it you can
also use substances which are not
colored but could be stained but or
could be analyzed by different methods
in this technique stationary phase is
supported by the cellulose fibers of a
paper sheet and hydrated cellulose
fibers or we can say absorb water in
fibers acts as a stationary phase the
mobile phase relatively less polar
solvent system will pass along the paper
sheet either by gravity field or by
capillary action so if you see here that
stationary phase is a paper which is
cellulose fiber off a paper sheet and
the this cellulose fibers or a paper
could be what meant paper
there could be modifications in this
paper where you can change its property
in terms of say ionic
imparting ionic properties or other
properties to the paper could be an
option likewise the water in fibers acts
as a particularly acts as a stationary
phase here so you can say that hydrated
cellulose fibers act as a stationary
phase now mobile phase which is
relatively less polar as compared to
water will pass along the paper sheet
and will carry the mixture of substances
which are present as a on the paper so
what is done is the mixture of
substances are distributed between
stationary and mobile phase as per their
affinities for two phases so substance
having higher affinity for mobile phase
will certainly move rapidly as compared
to those having higher affinity for
hydrophilic stationary phase so those
compounds which are relatively nonpolar
will move in the mobile phase but those
which are not will be sticking to the
stationary phase so this is how the
separation in paper chromatography will
take place now here the sample or more
than one sample could be placed onto the
paper as a small spot or line of sample
solution on a chromatography paper I'll
show you in a little while the whole the
procedure here now once these are spots
are placed and spot one has to be
careful that how much sample is being
put you should not put to less or to a
high amount of sample and it should be
on a clear spot should be placed onto
the paper in a if more than one sample
is there it's they are in a in a single
line
once you have spotted and write the
paper then it will be placed in jar
containing a shallow layer of mobile
phase
remember spots should not be dipping in
the mobile phase they should be above
the mobile phase and the jar is sealed
actually as the mobile phase rises above
the stationary phase and encounters
mixture it carries sample with it now
the different components of the sample
will travel different distances
according to how strongly they adsorb
onto the stationary phase as compared to
dissolve in the mobile phase as we have
discussed earlier
now the compounds within the mixture
will travel further if they are nonpolar
as compared to pure components which
will interact with hydrophobic
stationary phase or surface so this is
how a chromatogram will be run as far as
paper chromatography is concerned once
the it has been run you can take it out
and then detection of the result
compounds has to be performed so the
detection of result compounds may be
performed either visually if they are
colored or by physical and chemical
detection for colorless compounds so the
detection methods may include that could
be staining with certain dyes or other
reagents it could be autoradiography if
you are utilizing radioisotopes it could
be fluorescence or other physical
methods could be utilized
now after detection the result compounds
may be identified based on their
retardation or retention factor that is
our F value our F value is defined as
the ratio of the distance traveled by
the analyte to the distance traveled by
the solvent front and from a definite
point actually our F value will be
affected by type of mobile phase that is
solvent like say if you are utilizing
different types of solvents certainly
our F value will change the temperature
in different temperatures you will have
different RF values nature of the sample
and people will also affect RF value
now let's little bit get into like let
me show you one at your screen the whole
procedure here
all right so now in paper chromatography
what is done is like we were discussing
first thing is you have to take the
proper paper that is cellulose paper and
it could be what main paper now this
paper
now this paper like I said first thing
is you have to spot your samples on this
paper this paper could be simple paper
or it could be derivatized for other
applications so what you can do is a
very thin line could be drawn or you can
do it visually and here you have to spot
your samples and these spots has to be
appropriate or accurate now sports more
than one spot could be placed on a line
here on one side of the paper all right
now once you have spotted this then this
could be dried and then paper could be
put in a jar like I said a jar
containing a shallow jar containing a
shallow mobile face let's see what does
that mean so what you have is that you
have a particular kind of say container
or a jar
there's a very simple way to make a
container so you have a container here
and in this container what you have to
have a paper here if I can have a rod
too to support the paper this is a rod
to support the people now what is done
is you have a people which is hanging
sort of or it is supported paper which
can remain straight in here the main aim
to put a rod and this jar has to be
sealed completely so me name to have a
rod or a support system is to keep the
paper straight now this paper here and
you can see there will be spots in here
as we have spotted there and it be a
mobile face here now remember the spots
are not submerged here in this one so
what you have is you have this is
chromatographic chamber this is
chromatographic chamber then this is
chromatographic people which is you are
utilizing here this is mobile phase
which could be your now mobile phase
mostly is like I said it's a relatively
non-polar as compared to water now so
this one contains what you call it could
be say organic solvent water combination
and other additives could be utilized
like say it could be phenol water it
could be it could contain methanol or
lot of other different types of
combinations are available so what you
have is that once you have placed this
then automatically they will be and this
is your spots here which are many spots
are being put in here so once you have
placed this and this
remember spots are not dipping in here
the water will move or the mobile phase
will move up so this is this is called
ascending order or you can say it is
being run in ascending fashion there
could be another way that is it could be
the paper could be upside
down and that would be called the
standing manner where you can run that
particular chromatogram also paper
chromatogram also so once these is as
they run these sports or the samples
will be carried up in here as per the
affinities for the mobile or stationary
phase and what you are going to have
finally you will be developing a
chromatogram so finally what you will
have you will have a paper which could
be taken out from the jar and what you
will see is you will see various spots
in here as per how many spots you have
loaded and these a' spots could be done
either if they are colored you can see
them visually or if they are not colored
you can see them by staining them with
different agents or like say for example
in hydrogen is utilized to see amino
acids there could be a lot of other
different reagents or you can use
fluorescence or autoradiography to see
these so this is about a little bit
about the paper chromatography now let's
move continue our discussion here so
hope you have been able to understand
the simple methodology of paper
chromatography here the next one that
planar chromatography system is thin
layer chromatography now thin layer
chromatography this technique has almost
replaced the paper chromatography with
the advantages of speed that is it could
be run fast better separations due to
high resolving power so you have much
higher resolution here it's a much
faster and you can have you have a
choice between different kinds of
stationary phases which could be used
now this could be used for both
analytical as well as propriety purpose
so thin layer chromatography could be
both analytical as well as propriety now
as we have discussed in pupae
but chromotography lot of things are
same so we are not going to repeat them
but a few things about a thin layer
chromatography now the stationary phase
or the adsorbent material is mostly like
silica gel or aluminum oxide or
cellulose paper and other agents are
also other adsorbent materials are also
utilized so they'll be coated thinly on
to a glass or a plastic or a metal foil
plate and the mobile liquid phase will
pass across the thin layer plate held
either horizontally or vertically by
capillary action now the process is
similar to paper chromatography now the
these plates or thin layer plates as we
see how these are prepared so for
preparation of these plates layers of
powdered solids in form of slurry can be
applied to a clean glass plate with a
binder and then they could be dried and
activated by heating now TLC plates or
thin layer chromatography plates are
commercially available with standard
particle size ranges for reproducibility
you have to have standard particle size
so that you can reproduce your results
now they are prepared by mixing
adsorbent say silica gel or alumina with
a small amount of inert binding agent
for example calcium sulfate or gypsum
could be used with water the mixture is
spread as a thick slurry on an inert
carrier sheet and then like I said it
could be glass alumina or plastic then
plate is tried and activated by heating
in an oven at say 100 220 degree Celsius
particularly mostly 110 degree Celsius
for 30 minutes now plates of uniform
thickness are obtained now here for
analytical purpose the plates of 0.12
dura point five millimeter and for
propriety purpose 0.5 to 2 millimeter
plates could be prepared so
like like I said it could be
commercially you can obtain them without
any problem as per your requirement and
you can use them now TLC can be used for
a lot of different applications like
paper chromatography and TLC almost have
similar applications like in paper
chromatography it could be used for
analysis of lot of different samples
right from say nucleotides amino acids
peptides drugs pesticides and in
cosmetic industry food industry a whole
lot of things a whole lot of areas it
could be utilized
likewise TLC can be also utilized for a
lot of different applications now before
we go into the applications I will just
like to show you how it is done like I
said it is almost similar to what you
call it is almost similar to the paper
chromatography now here rather than a
people what is there is a glass or say
certain plate which could be plastic or
tin foil now on this tin foil you will
have a say slurry which is prepared in
here and this will be poured on this
glass plate and we spread evenly which
will be put in whole place and it is
like this slurry here access it will
have certain thickness and this slurry
will be utilized or will be acting as
the stationary phase which could be like
it says silica gel or alumina gel or
others so all other things are as as the
paper chromatography except for the
stationary material but it has lot of
like I said lot of advantages over paper
chromatography all right so now TLC
could be used for monitoring the
progress of reactions for example it
could be used for monitoring of organic
chemistry reactions say at different
stages
the compounds which are made could be
identified by their RF values then
identifying the compounds in a mixture
or to assess the purity of the compound
that's also a very important application
of both paper and thin layer
chromatography where you have a pure
compound and you can assess the purity
of that compound you can use it for
separating a lot of different kinds of
substances or compounds for example you
can use it to separate lipids steroids
alkaloids sugars phenols nucleotides
amino acids and peptides and many others
actually it could be used for detection
of pesticides and insecticides in food
and water it could be utilized for
identification of constituents of a
medicinal plant it could be utilized in
forensic science and it could be
utilized to assay the ready
radiochemical property of radio
pharmaceuticals and so on so there are a
whole lot of different applications of
thin layer chromatography now practical
advantage of thin layer chromatography
or paper chromatography over column
chromatography is that large number of
samples can be studied simultaneously
here rather than in column
chromatography you can use at a time one
mixture sample mixtures could be could
be analyzed all right so we have
discussed about two planar
chromatography techniques one paper
another is thin layer chromatography
now let's discuss about the column
chromatography and which which is the
main topic here now in column
chromatography this is a separation
technique in which stationary phase or
stationary phase on a suitable supported
matrix is packed in a column or a tube
made of it could be made of glass metal
or polypropylene that is plastic now the
mobile phase passes through the column
either of a gravity feed or by use of
pumping system as we'll discuss in
coming lecture and the method has been
has become the most commonly used mode
of chromatography so let me show you the
how column looks like actually now if
you see the column the column is a
simple tube like or column structure
which is closed on one side it will be
closed on both sides while running but
to support the matrix there is a filter
or disk suppose with a filter and the
material will be filled in this here
which is your matrix so stationary phase
or matrix is put in here the mobile
phase will be will be put in from top
now through gravity or pumping system as
I will show you in the coming two
lectures and then drops which are which
like comes out and as per the affinities
to the stationary mobile phase this
analyte will be separated and these
fractions can be collected separately so
this is a simple depiction of column
chromatography and we are going to see
it more as we go along alright so let's
move on on this lecture so what we have
done is in this lecture what we have
seen is you have like different
combinations of stationary phase and
mobile phase and there is a distribution
coefficient or term known as
distribution coefficient which defines
how a particular analyte will separate
between the two phases that is a
stationary phase and a mobile phase now
that depends on the affinity of that
particular analyte for two phases and
the separation is based on the of
different analytes in a mixture or in
that that they have different affinities
for the two phases
there are different modes of
chromatography systems and there could
be different combinations of a
stationary and mobile phase as we have
seen adsorption equilibrium or ionic an
exchange equilibria ssin or say
partitioning liberation and there a
whole lot of different combinations
could be there and the modes of
chromatography there are paper
chromatography and thin layer
chromatography which are planar
chromatography techniques and then there
is column chromatography where you have
a stationary phase being packed into a
column now before we end this lecture
let us let's see as we are going to
discuss more about column chromatography
a few terms and we'll continue to
discuss these terms in the next section
but few terms which we which are
important let's discuss them here one is
chromatography as we are listening this
term what is chromatography means here
now chromatography is a physical method
of separation where components to be
separated are distributed between the
two phases that is stationary and mobile
phase so that's what chromatography
means and second term is chromatograph
now here if you see chromatograph is an
equipment that enables the separation in
chromatography so chromatography is the
technique whereas chromatograph is the
equipment or the instrument that enables
the separation in chromatography and
that or it enables to do the
chromatography now there is another term
called chromatic graph now chromatogram
is the visual output of the
chromatograph in the form of different
Peaks or patterns like say when you are
separating different analytes then as
they come out of the column then they
will be coming out in a particular
pattern and these are represented as
Peaks as will be showing you that in a
little while in the next lecture so
chromatograph chromatograph Graham is
another important term here
then stationary and mobile phase that is
the substance which is fixed in place
for the chromatography method and it is
immobilized on a support or stationary
phase could also be separate and could
be immobilized on a support particle and
can be packed
it could be solid gel or liquid then
there could be mobile phase which is the
phase which moves on to the stationary
phase or through the stationary phase
and then it carries the sample as per
the affinities with it so that the
separation could take place so like here
you have different ways you can like we
said different combinations of
stationary and mobile phase now mobile
phase is also there are two more terms
one is called element element is the
carrier portion of the mobile phase and
it moves the analyte in the
chromatography or chromatograph that is
instrument so element is the solvent
that is part of the mobile phase but it
is the carrier portion and eluate
another term is eluate which is the
mobile phase leaving the column and it
consists of both carrier and the analyte
material so these two terms are
important one is alumina and another is
eluate eluate is which is coming out of
the column element is the carrier
portion of the mobile phase then analyte
analyte is the substance to be separated
during chromatography so analyte could
be if you are assessing the purity it
could be single analyte analyte could be
present in a mixture or a sample
containing lot of different analytes
which needs to be purified say one
analyte could be purified from mixture
or there will be more than one and a
light could be purified from the sample
so these are few terms which are related
to the to the chromatography where
we have discussed about chromatography
chromatograph chromatogram stationary
phase mobile phase and analyte so in the
in the next lecture we are going to
discuss certain terms which define the
performance of chromatography systems
like for example retention time
retention volume capacity factors
resolutions and all different kinds of
terms we are going to discuss to
summarize this lecture what we have
learnt today's in this lecture is that
there is one the the chromatography
separation occurs due to the varying
affinities of a particular analyte in
the two immiscible phases that is phase
a and phase B and their distribution is
measured by distribution coefficient
that is if say distribution coefficient
is 1 that means they have distributed
themselves equally then there is
effective distribution coefficient that
is the amount in the two phases rather
than two concentration in two phases
there could be lot of different kinds of
combinations and stationary phases
mostly solid or liquid whereas mobile
phases either liquid or gas there could
be different modes of chromatographic
systems like paper thin layer and column
chromatography and our discussion in the
next lecture is going to be on basic
concepts relating to performance of the
chromatographic system and various
techniques about the column
chromatography thank you