Low-pressure liquid chromatography (LPLC) and high performance liquid chromatography (HPLC) Video Lecture - Biotechnology Engineering (BT)

in previous lectures we have dealt with
the basics in chromatography that is we
have discussed the basic concepts in
chromatography and we've also discussed
about the various performance parameters
of chromatography now in this lecture we
are going to extend our discussion on
column chromatography that is the most
widely used chromatographic method what
are the basic components of column
chromatography now column chromatography
is consists of a column a column could
be made up of a glass it could be a
glass column or a metal column or it
could be a plastic column then another
component is a stationary phase which is
packed in the column now it could be a
simple stationary phase or a stationary
phase supported on a matrix or support
material then a mobile phase which
passes through the stationary phase and
due to distribution coefficient the
partitioning or the separation of
analytes takes place there is a pump or
we can say delivery system for mobile
phase particularly in high pressure or
high-performance liquid chromatography
techniques you certainly require a pump
and even in low pressure chromatography
column chromatography
you can do it by gravity feed but it is
better to have a pump like peristaltic
pump for a constant flow rate then you
need a simple loading system for low
pressure chromatographic systems you can
load the samples manually also but for
advanced systems there has to be an
injector system attached to the to the
apparatus then finally a detector and
charter talk record of detector
different types of detectors are used as
we will see and chart recorder gives the
readout samples could be collected in a
fraction collector if they are required
for like say you want to purify or
analyze them afterwards
so frakkin fractional collector
different Peaks can be collected and
then it could be used for further
analysis
now column chromatography system can be
classified according to the back
pressure generated within the column
during separation process so low
pressure liquid chromatography systems
generate pressure which is less than 5
bar or 0.1 mega Pascal medium pressure
liquid chromatography generates pressure
between 6 and 50 bar and high pressure
liquid chromatography generates pressure
above 50 bar nowadays like both medium
pressure and high pressure gives
excellent resolution and this particular
demarkation has been kind of gone and
now they are called both of them are
called high performance liquid
chromatography system so HPLC or high
performance is preferred rather than
high pressure or medium pressure all
right so if you consider this is a very
simple schematic of a chromatograph or
equipment part if you can see here there
is a column and it is a marriott flask
above this where the solvent or the
mobile phase is there now this will be
run on gravity and so although this kind
of maintains the pressure but still the
flow rates are may vary as per the level
of the mobile phase finally this will go
to detected and fragile as eluate comes
out it will go to fractional collector
and through detector the improve systems
even for low pressure low pressure
chromatography here you can see one
there is
eluent reservoir and there is a pump
it's usually peristaltic pump as you'll
see later on the pump kind of drives the
mobile phase into the column there are
two plungers here and this is nylon
discs or nylon filters are attached to
it so stationary phase is supported on
to here as the mobile phase passes it
comes out here and goes to detector and
finally it could be collected in a
fraction collector so that's a very
simple arrangement and we'll discuss
each of these things individually so
let's start with the columns now these
columns mostly these are glass columns
are used and should have these columns
should have a means of supporting the
stationary phase near the base and that
is important that first thing is you
have to support so that could be staying
there and another is you have to
minimize the dead space below the column
support so that post column mixing of
separated analytes
will not occur now commercially
commercial columns purses either a
porous glass plate fused on the base of
the column or a suitable device for
supporting a replaceable nylon nylon
filter or net is there so which will
support the stationary phase so a column
like I said even syringe columns could
be used like syringes could be used as
columns and glass pool could be used to
support the stationary phase for simple
chromatography matrix materials which
are used which support the stationary
phase or stationary phases attached to
them matrix materials should have I will
discuss about what kind of matrix
materials are used but they should have
certain properties it has needs to have
say high mechanical stability to
maintain good flow rates you have to
have the particles or the stationary
phase with good mechanical stability so
they are not compressed or flow rates
are not altered actually it should have
good chemical stability as you are going
to use different kinds of buffer
solvents other things so chemical
stability is also required then there
should be functional groups on
stationery on matrix to facilitate the
attachment of stationary phase because
most of the time like say for attaching
ionic groups or say affinity ligands you
need to have certain functional groups
where these could be conveniently a test
then it should have high capacity that
is you can support large stationary
phase so that amount of sample to be
purified could be enhanced then this
would be available stationary this
matrix should be available in a range of
particle size and also in porous form
for certain kinds of chromatographic
methods the surface of matrix needs to
be inert to minimize nonspecific
adsorption of analytes otherwise the
chromatic chromatographic separation
will suffer as peak trailing or other
problems might occur due to
non-selective at your adsorption on the
matrix now there are different types of
matrices which are being used and some
commonly used matrices are example
agarose now this agarose is a
polysaccharide made up of d-galactose
and 3/6 and Hydra one galactose units it
is the unbranched polysaccharide chains
are cross linked with agents such as the
2-3 die bro bromo propanol and to give
gels that are stable in pH range from 3
to 40 and examples are like sepharose
and bio gel a which are frequently used
second material could be poly acrylamide
it's a polymer of acrylamide
cross-linked with miss acrylamide you
will see this is quite used both agarose
and polyacrylamide are used in
electrophoresis in the next section poly
acrylamide is also pretty stable in pH
range of 2 to level and example is bio
gel P that's a commercially available
then cellulose could be used as matrix
material it's a polysaccharide of beta
one full-length glucose units and cross
linked with epichlorohydrin it has good
pH stability and very good flow
properties and is hydrophilic in nature
dextran that could be another polymer
it's a polysaccharide containing alpha
one six linked glucose units and this
will also be cross linked with
epichlorohydrin it's less stable to acid
hydrolysis and it's stable up to pH say
12 its hydrophilic and one example is
sephadex which is commercially available
in different forms then polystyrene that
is synthetic sort of material could be
utilized it's a polymer of styrene
cross-linked with dye vinyl Bengie it
has a good stability overall pH and
mostly used for exclusion and ion
exchange chromatography then there is
another one called silica it's a
polymeric material produced from
orthosilicic acid and it is stable in pH
range of three to eight so these are
some of the examples of the matrices
which are being used now on these
matrices attached is a stationary phase
which in which is involved in the
purification process so the chemical
nature of the stationary phase depends
upon the particular form of
chromatography to be carried out say if
it's ion exchange you will be
immobilizing ionic groups of affinity
and so on
now most stationary phases are available
attached to the matrices in a range of
size and shape commercially so both
properties of size and shape are
important because they influence the
flow rate and the resolution
characteristics now column packing once
you have selected the matrix with
stationary phase the next step is to
pack the column with the stationary
phase and that's very important part of
chromatographic separation
now packing a column is normally carried
out by gently pouring a slurry of the
stationary phase in the mobile phase
into the column and column is gently
tabbed to ensure that no air bubbles are
trapped and the packing settles evenly
now pore column packing will give rise
to uneven flow and reduced resolution
and once the column has been prepared it
is very important that it does not to
make sure that it does not run dry so it
should be all the time kept wet actually
or mobile phase should be there if it
gets dries dried then it will break or
crack and that again you have to pack it
again so let me show you how column is
packed and how a sample is applied onto
the column on your screen so please see
on our scheme so what is done if you
have a glass column or any other column
and you can see there is a support at
the base which will stabilize the
stationary phase now what happens is
your stationary phase is filled in
through a glass rod either you have to
have a glass rod here and you will pour
your stationary phase onto the through
this glass rod so that it will pass and
it will settle down it will go to the
base through the walls of the column now
advantage is that you will and it's
refilled from the base that if you
suddenly add this to the column the air
bubbles will be trapped but with this
method air bubbles will not be trapped
at the base of the column other way
could be that you can tilt the column
rather than if you don't want to use the
glass rod then column can be twisted and
then material could be could be poured
in through the sides of the wall so this
way what you can do is you can pour and
see that you when you are pouring this
material you do it evenly like
continuously without if you without
creating gaps
or demarcations like for example there
could be a column which could be pegged
in this may that there is a some
unfilled areas could be there and this
will again give uneven flows and will
hamper the chromatographic procedure a
separation so these things has to be
looked into now how simple is applied on
to the column let's see that so when you
have to apply the sample onto the column
what is done is again you have a packed
column once you have packed and you have
made sure you have equilibrium the
column with the particular buffer in
which chromatographic separation needs
to be done then you have equilibria at
the column the or stationary phase with
that now your so what we'll have is you
have a stationary phase filled in here
and you have your mobile phase which is
here and you will have either you have
closed it with plunger or with a certain
cork with tubing in that so they'll be
tubing which is attached to your mobile
phase it may be through pump or it may
be without pumps so you can do it either
by gravity or may be without gravity now
how do you load your sample one is if
you have lot of sample and it's like
sayin exchange chromatography things it
could be directly loaded through
peristaltic pump but other methods could
be that you have to remove this top
plunger or or core whichever is there
and then what you have to do is then
once you have you have this material and
so you have to remove this mobile face
from here very slowly without disturbing
the stationary phase bed once you have
removed then very nicely with a micro
syringe or a pipette you can you can
inject the sample or you can load the
sample in
here in a smaller volume and then very
slowly let this sample get into the
column and once it gets into the
stationary phase then you can put the
mobile face again and then close put the
plunger on and start the chromatographic
procedure another method could be you
could have high density solutions like
you can use sucrose density sucrose or
glycerol in your sample which gives them
high density and then with you don't
have to really remove this mobile phase
because you can put in when your mobile
face is there you can put in the
material and since it has high density
it will settle in here without any
problem and so you'll be able to do it
without removing the mobile phase so
these are a few ways you can do it like
say for high concentration of protein
solutions are quite dense and they could
be we done like that alright let's come
back to our discussion and so this was
about the column packing and remember
per column packing will certainly rise
to uneven flow and will certainly reduce
the resolution so it is very important
that no air bubbles no gaps no uneven
settling of the stationary phase and all
these things has to be taken care of
then application of sample as we have
discussed is a very important part that
you can do manually or through injector
system mostly low pressure
chromatography you can do it manually
like I have shown you like you can
remove the mobile phase or you can use a
high density like sucrose or glycerol or
you can use peristaltic pumps so once
the sample is loaded then you have to
develop the column and Aleut the analyte
so the components of the applied samples
are separated by continuous passes of
the mobile phase through the column now
separated analytes
will be removed from the column by
illusion and
this illusion processes essentially that
the flow of mobile face is maintained at
a constant rate and in certain systems
mobile the column certain systems the
analyte s per retention time will stay
in the column and then slowly will be
moved and since they have different
distribution coefficient they will be
separated and they will retained
separately or different very differently
they will be removed so it's a isocratic
elution there could be also gradient
elution where you are utilizing an
element containing other substances like
say for example salt Ellucian you might
use sodium chloride and that replaces
the the analyte and then analyte is
eluted so there you can have gradient
elution or that could be step gradient
or that could be continuous gradient
elution could be done and there are
gradient makers which are available to
make the illusion to make the gradient
to produce a suitable gradient two or
more elements have to be mixed like for
example you can have zero molar nacl in
a buffer and you can have say 1 molar
NaCl in that buffer and though two can
be combined in correct proportion prior
to entering the column this could be
achieved by using two different
peristaltic pumps to deliver the
separate elements and they will be mixed
in a chamber before entering the column
gradient could be a linear gradient or
it could be a convex or concave gradient
convex gradients better resolution
initially whereas concave will give
better resolution at the end this is a
peristaltic pump a very simple schematic
of a peristaltic pump it is a roller low
roller type peristaltic pump and you can
see these rollers here what they do is
they compress the tubing and as they
revolve it's the this revolution occurs
these rollers move compressing the
tubing and due to the compression the
mobile phase is sucked in from the
and then we'll be at fixed flow rate it
will be supplied to the column now this
has to be standardized in a sense that a
different tubing will give you different
flow rates as per the revolution of this
desk actually so that has to be
standardized but that's a very simple
peristaltic pumps there is planetary
type also are available and both of them
can be used for providing even flow or a
constant flow
this figure shows this is a gradient
maker you have one solution here that is
donor chamber one is recipient chamber
and then as it could be mixed with the
magnetic stirrer and a gradient could be
made in a different proportion this is
like a linear gradient or you can have
convex or concave gradient as per your
requirement so this is a very simple
method of illusion where you get your
analyte out and you can collect it in a
fraction collector you can detect it
through a different kinds of detectors
so method of detection and collection
we'll be discussing later on with HPLC
section as many things are very common
only differences detectors are for HPLC
are more sensitive and they are more
quick to detect certain things rather
than a low pressure chromatography
system where a simple UV monitor could
be utilized all right so this was about
the low pressure chromatography system
now let's shift to high-performance
liquid chromatography and how you will
see how it is different from low
pressure chromatography now the
resolving power of chromatographic
column increases with column length as
we were discussing earlier and it
depends on number of theoretical plates
per unit length but there is a limit to
or there is a practical limit to the
length of the column because of peak
broadening and other problems so what
you have is that you cannot really
increase the length to after certain
certain
particular length of the column so you
cannot increase it to like there is a
limit to increase of the column length
so what has to be done to increase the
theoretical plates what could be done is
that in a smaller or in a particular
length column you can have a stationary
phase with support particles of course
with a smaller particle size so if you
have a smaller particle size of a
stationary phase the better resolution
could be achieved because you will have
higher number of theoretical plates as
you can compare there's a course
material or higher size particles which
will pack in the column and will
certainly have low number of theoretical
plates but if you have a very small
particle size they'll pack much better
and they will have higher number of
theoretical plates but problem is that
as the pack very nicely there will be
lot of resistance due to smaller
particle size and which will create back
pressure in the column and simple pumps
are not really sufficient to drive the
flow and many times this matrix
collapses actually because of high
pressure now this problem has been
solved by a lot of advancement where
development of small particle size
stationary phase which can withstand
these pressures very high pressures
could be withstand by these particles
and so now this development has resulted
in faster and much better resolution and
the HPLC or high-performance liquid
chromatography has emerged as the most
popular very powerful and versatile form
of chromatography and it is also very
quick it doesn't take much time as the
low pressure chromatography because it
is done at very high flow rates or very
high pressure limits a very simple
schematic of different components of
isocratic HPLC system if you see what
you have is a reservoir or xi reservoir
now solve
razor wire then it there is a
high-pressure pump so we will discuss
these are different from simple
peristaltic pump and then you have from
pump it is the the solvent will be
directed to the column many times there
is a guard column here which kind of
cleans up the system and remember all
mobile phase has to be de guest and or
and filter so that it does not like
precipitate things in there and leads to
generation of high pressure because of
that and because of the choking of the
column column which is a small column we
will see how big these columns are but
length is certainly less than normal
columns because you can pack more number
of theoretical plates in here it goes to
the detector and then to fraction
collector and you can collect it you can
detect and readout could be seen so this
is a very simple schematic of isocratic
HPL system if it is a gradient then you
have to have two pressure pumps and a
gradient mixture which will allow you to
supply gradient and Aleut the sample
alright so let's discuss different
things different parts of this HPLC
first thing is and most important is
column now conventional column used for
SPLC are generally made up of stainless
steel and they can be withstand pressure
very high pressure up to say 5 point 5
into 10 raised to power 7 Pascal's
columns are generally 3 to 50 centimeter
long and 4 millimeter
an internal diameter with flow rates
which could reach up to 1 to 3 ml per
minute
there are also micro bore or open
tubular columns and they have internal
diameter of say 1 to 2 millimeter and
they are generally up to 50 centimeter
long and can sustain flow rates of 5 to
20 milli micro liter per minute so there
are advantages also of microbe or
columns like the elude consumption is
reduced as due to slower flow rates I
l for interfacing with mass spectrometer
due to reduced flow rate increased
sensitivity and higher concentration of
analyte could be used here now matrices
and stationary phase if you see the
three forms of stationary phase material
are available these are micro porous
support where micro pores ramified
through the particles which are
generally five to ten millimeter in
diameter then there is a película
support which is porous particles coated
on to the inert solid core such as glass
bead of about 40 millimeter in diameter
and then there is a bonded phase where
the stationary phase is chemically
bonded on to the inert support say
silica so these are some of the
stationary phases available for
adsorption chromatography adsorbents
such as silica or alumina are available
as micro porous or película forms with a
range of particle size and for partition
chromatography system the stationary
phase may be coated onto the inert micro
porous or película support for ion
exchange chromatography system
cross-linked micro porous polystyrene
resins are widely used película resins
forms are also available and a bonded
phase exchanger covalently bonded to a
cross-linked a silicone network are also
used
now application of sample is very
important in this HPLC if you could
recall and low pressure you can do it
manually but in HPLC which is a advanced
and enclosed system this should be a
very like very accurate or automated
system of application of sample so
application of sample on to s peak LC
columns is done through like for
successful separation it is done through
a loop injector sectioning and not
manually a now this consists of a metal
loop of fixed small volume and they
could be of varying volume from very
small to very large and
that can be filled with the sample and
then 11th from the pump is then
channeled through those loop by means of
wall switching system and the sample is
flushed on to the column why the loop
outlet without interrupting the flow of
the alumina to the column so that's a
very important part now let us see let
me show you on your screen this loop
injector so that you can appreciate how
does it look like and how this whole
thing is happening all right so loop
injectors like I said they are very
important part now a loop injector is
simply and there are different ports and
let me make this for you here
it's a disc sort of and you have
different ports here and we can number
them like say 1 2 3 4 5 6 then there
will be a loop which is connected if you
see there is a loop here which is off so
this is a loop here which is being
formed now at this this is a now there's
two positions one is loading position if
I could say this is loading position
then what will happen loading position
means you are loading your sample onto
the loop so what is the configuration
here configuration is that you have a
particular element or the solve solve
mobile phase from pump this is going to
the port number 1 and from the port
number 2 it is going to the column so
still you have not loaded so now you
this is the loading position where you
can load your sample if I say this is
your knob which wood has to be shifted
then this is the loading position and
this is your loop this is loop is of
fixed volume now this could be like I
said it could be like 200 microliter 2
say a few ml also now what will happen
so you have to load this mr. port where
you will be loading the sample actually
so this is Inlet port for loading the
for loading sample by micro syringe
alright now once you have loaded so once
you put in your micro syringe and you
are loading your sample through a micro
syringe in this you have to fix micro
syringe then what will happen this will
go here your sample will move from into
this loop and it will fill this loop
here alright so what will happen say if
this is 200 micro litre loop so if you
load 250 micro liter that 200 micro
Newton will be contained in this loop
and rest of it will go to through the
port number five like it is connected
six loop is connected to SiC and six is
connected to five here so this will go
to waste so these are both things will
go to waste so what will happen you have
loaded the loop extra sample will go to
the waste
alright now this is loading position now
let's see that you have to inject it on
to the column so what will happen what
is the difference here now what is these
are all things are same that you have
these ports here which is one two now
and this is your loop here and this is
connected here so what was the situation
here then here it is from Alou and from
pump is coming here and this is two
column
so what will happen now here if you see
this knob will shift to injecting
position now this knob which was here
this will be shifted to injecting
position and will be you can just rotate
it to this position and now the
configuration will change the
configuration will change in a sense
that they Lewin twitch was here going to
port number 2 directly rather as you
change this knob this will go to port
number 6 and now port number 6 and 5 are
not connected and so what is happening
the element is going to port number 6
then it goes to the loop and and from
the loop finally it goes through port
number 3 to port number 2 and then to
the column so you can see the change
here as you put it from loading to the
injecting position the element is
directed through the loop and so the the
Lu and pushes the sample onto the column
and then this run could go on when you
have to inject again what will do again
it will be brought to the loading
position and then again you have to do
the same thing
these will go to the waist actually both
of them so this is the very good system
or injector loop which could be used for
for loading the or for application of
the sample all right so let's return to
our discussion all right so we are
discussing about the application and as
I have shown you this is how the
application will be performed
application of sample will be performed
now mobile phase depends upon what
separation needs to be achieved if it is
a isocratic elution it could be made
with a single pump using a single
element or two or more events pre-mixed
in a mixed proportion
but if it is a gradient elution then you
have one needs to use two pumps and
there will be gradient mixture or
programmer has to be used all wealth
must be specially purified because
traces of impurities can affect the
column and interface with the detection
system it is also essential that all
elements be D guests and they should be
filtered for high resolution the next
component important component of HPLC is
the pumps actually the means feature of
good pumping systems and SPLC is that it
is capable of output of at least say 5
into 10 rates per 7 Pascal's and ideally
there must be low pulses as this may
affect the detector response so this
would be like your flow capabilities of
at least like say 10 centimeter cube per
minute or up to 100 centimeter cube per
minute for prepara t'v systems now the
pumps which are used in here are mostly
constant displacement pumps and
reciprocating pumps like now all pumps
which are used in here they incorporate
pulse dampeners to minimize the pulsing
effect and they have inbuilt safety
cutout mechanism to inactivate the pump
automatically a pressure within the
chromatographic system changes from
preset limits now here is one example is
that is constant volume pump if you can
see here this constant volume pump
displacement pump it maintains a
constant flow rate through the column
irrespective of the changing conditions
within the column and this is which is
like constant volume pump it is a motor
driven pump we can say it's a series
type pump that delivers a fixed amount
of volume to the column by piston driven
by a motor so what happens is this pump
contains a screw jack it contains a
screw jack
driven by a stepper motor so on the
delivery stroke what has done it on the
delivery stroke the the solvent from
here goes and it is delivered to the
column at a constant rate so once it is
done then these valves will be then
accordingly they will be closed or open
like say for when it has to be the
element has to come from the reservoir
then this will be opened and then in the
next when the syringe pump strokes and
like when it has to deliver this
displace this element on to the column
this wall will be open and this will be
closed so this is like constant volume
pump and it is quite used and in the
HPLC systems there is another one which
is called reciprocating pump now in
reciprocating constant displacement pump
a motorized prank drives the piston and
check valves regulate and through your
filament to the column on the
compression stroke like here this one is
a driven drive motor and there's a chem
crank dry driver a motorized crank which
drives the piston so what happens is on
compression stroke the level Fuu is
forced from the pump chamber so element
will be forced from the chump pump from
the pump chamber to the outlet vault and
to the column and during the return
stroke the exit check wall closes and xi
is drawn in via the entry wall that is
the inlet wall so these two strokes that
is compression and return stroke one
will let the xi come into the system
another will forced element into the
column so these pumps produce small
pulses of flow and pulse dampeners as I
said are employed to minimize the effect
so these are the two kinds of pumps
which are utilized constant displacement
pumps
which is constant volume pump and Co and
reciprocating pump so the pump here are
important in a sense they will maintain
the constant flow rate irrespective of
the conditions in the in the column and
there is a cut-off limit that if the
pressure goes higher after certain set
limit then pumps will automatically will
switch off another important component
is detectors there are a lot of
different kinds of detectors which are
used now the quantity of material
applied to these columns is normally
small and it is very important that
sensitivity of the detector is
sufficiently high and stable to respond
to the low concentrations of each
analyte in the eluate now some of we are
taking some examples of detectors most
commonly used detector is the variable
wavelength detector it's a UV visible
detector based on UV visible
spectrometry it is capable of measuring
absorbance down to 190 nanometer and can
give full scale deflection for as little
as a point zero zero one absorbance
units sensitivity is almost like 5 into
10 is to about 10 gram per centimeter
cube and linear ranges is of 10 is to 5
so they allow continuous monitoring of
the column kill you it there could be
other detectors like a scanning
wavelength detector and it has a
facility to record the complete
absorption spectrum of each analyte and
display a plot on computer screen in
real time then there are fluorescence
detectors which are extremely valuable
Forex PLC because of their greater light
sensitivity like in order of 10 list or
minus 12 gram per centimeter cube then
UV detectors but they have reduced
linear range and they could be used but
limitation is that not many analytes
Floris so that's the limitation so they
are used for specific analytes
then there could be left row chemical
detectors which are selective for
electro active analyte analytes and are
potentially high sensitive highly
sensitive there are two types which is
amperometric and cola metric and they
have amperometric detectors have higher
sensitivity and then the kilometer ik
and these are also have greater linear
range then also you can attach to HPLC
mass spectrometry and HPS HPLC like here
you will enable the analyte to be
detected and its structure determined by
mass spectrometer it you can it's like
it's a mechanism for identification of
different kinds of analytes and also you
can see overlapping Peaks here then
there could be like certain times you
can use NMR spectrometer directly a test
to HPLC and it can again give structural
information about the analyte that is
complementary to the obtained by HPLC ms
that is mass spectrometry then they
could be refractive index detectors and
they depend on change in the refractive
index of the eluate as analyte emerges
from the column it will respond to any
analyte in any element and the change in
refractive index being either positive
or negative commonly used in for shape
analysis of carbohydrates and other
element another analytes then there
could be a operative light scattering
detectors which realized on the
vaporization of the eluate evaporation
of alumina and quantification of the
analyte by light scattering could be
done it can quantify analytes in flow
rates up to five centimeter cube per
minute very useful for the detection of
say fatty acid lipids and carbohydrates
so these were some of the detectors
which could be used but mainly it's a UV
vis detector which are the most common
for say carbohydrates refractive
detectors could be based on refractive
index changed to be utilized then
fraction collector is utilized to
collect the fractions of the analyte
purified fractions now for study in
which the analyte in the eluate is to be
collected and studied further that could
be divided into different fractions and
automatic fraction collectors which are
programmed along with the whole system
and they could be designed either to
collect a selected volume of eluate or
to collect the eluate for a
predetermined period of time so both
ways it could be done so this was about
the high-performance liquid
chromatography and you could see that
there are a lot of changes in low
pressure and high-performance liquid
chromatography in terms of particle size
in terms of flow rates in terms of types
of pumps utilized in terms of high
sensitivity detectors utilized and it
terms of purity and high resolution so
there are a whole lot of differences and
HPLC is widely used for high resolution
work so both low pressure chromatography
or high performance compare graphi there
are a whole lot of different
chromatographic methods have been
developed for both of these techniques
that is low pressure and high
performance I'll get to just give you
certain examples here of certain
chromatographic methods which are widely
used and will be also discussing some of
them in the subsequent lectures one is
adsorption chromatography now adsorption
chromatography is probably one of the
oldest types of methods and it utilizes
a mobile liquid or gaseous phase that is
odd adsorbed onto the surface of a
stationary solid phase the equilibrium
between the mobile and the stationary
phase accounts for the separation of
different studies now you can see here
there is the adsorption the surface on
which this material a solute will adsorb
onto and this is the basis of
separation next is a partition
chromatography now this form of
chromatography is based on a thin film
you can see here thin film formed on the
surface of the solid support by a liquid
stationary phase and the solute
equilibria then between the mobile phase
and the stationary phase you can see
here solute is dissolved and in a liquid
phase coated on surface and mobile phase
passes on and this becomes basis like
for example reversed phase
chromatography and other systems then
there is an exchange chromatography here
what is happening you have immobilized
two ions actually like the use of this
sort of covalently attached anions or
cations on resin and the solute ions of
the opposite charge will be attracted
and will be binding to the stationary
phase and then you will have to have a
they will be binding to electrostatic
force and then you have to have say salt
or some other element in Ewald
containing those material to dilute the
analyte so this is another important
chromatography which we are going to
discuss then molecular exclusion
chromatography it is also known as gel
permeation or gel filtration
chromatography and it lacks an
interaction sort of thing between a
stationary phase and the solute but what
happens is here the pore like the
separation occurs through a porous gel
and the stationary phases the solvent or
the mobile phase trapped in the pores so
pores are normally small and exclude the
large solute molecules but allows water
smaller molecules to enter the gel
causing them to flow through a larger
volume and this causes the larger
molecules to pass through the column at
a faster rate than the smaller ones
because larger molecules will not be
able to enter the pores and will not be
retained for a longer period of prime as
compared to smaller
molecules you can see there are pores
and these molecules are kind of entering
and going out so they are they spend
more time in the column as compared to
the bigger particles which will not be
entering these pores then there is
affinity chromatography and it utilizes
the specific interaction between one
kind of solute molecule and second
molecule that is immobilized on a
stationary phase for example the
immobilized molecule may be an antibody
to a specific protein that is antigen
and when solute containing a mixture of
proteins are passed by this through this
chromatographic stationary phase only
the specific protein will bind and
others will not bind to it so you can
see like there is a ligand which is here
and this specific protein will only bind
to that ligand immobilized ligand so
that is another form of chromatography
which we are going to discuss now if you
see in this figure it is summarizes sort
of things where you have a certain
chromatographic like gel permeation
adsorption reversed-phase partition ion
exchange and here you can see increasing
polarity like a more polar ion exchange
chromatography could be utilized and
force a non-polar adsorption and other
cryptographic systems could be utilized
gel filtration of permission can be
realized for any kind of molecule
because it is less the particular
interactions of that sort so this
completes our section on introduction to
column chromatography where we have
discussed the low pressure column
chromatography or liquid chromatography
and high-performance liquid
chromatography we have gone through
different components of the both the
systems where we have discussed about
columns pumps stationary phase mobile
phase detectors and so on and in the
next section we are going to discuss
we'll start with a specific
chromatographic method which we are
going to discuss about ion exchange
chromatography gel filtration or
exclusion chromatography
affinity chromatography and also will be
discussing about gas liquid
chromatography systems thank you
you