HPLC (High Performance Liquid Chromatography), Downstream Processing (Lec -33 ) - Biotechnology Video Lecture - Biotechnology Engineering (BT)

today we are going to talk about the
HPLC HPLC is an analytical tool which is
used for identifying a unknown
metabolite or a protein or a peptide as
well as it can be used for
quantification so HPLC has been the
short form of high-performance liquid
chromatography or high pressure liquid
chromatography so HPLC also uses the
techniques of chromatography so you have
different types of HPLC s which operate
using different principles as we
discussed the various principles of
chromatography so the separation is
achieved either through hydrophobic
interactions or polar interactions or
ionic forces and so on actually so we
are going to look at the HPLC today
which is a very important tool which can
be used for both small molecules as well
as large proteins as well so it's a very
powerful analytical tools for
identifying and quantifying small
molecules peptides proteins metabolize
drugs natural chemicals natural products
phytochemicals and so on actually it's
extremely powerful and you achieve very
good separations by changing the
compositions of the mobile phase or
changing the stationary phase and so on
actually so what happens in an HPLC you
have a set of solutes and the solute
partitions between the stationary phase
and the mobile phase so it is basically
related to solubility between two phases
actually okay so the more the solvent
which is you are a continuous phase or
the mobile phase and you have the bonded
phase or the stationary phase so you are
a compound gets equilibria at between
these two phases and the separation
happens because of the four
that act on the solute and hence you
have a equilibrium process taking place
okay so we have two types of HPLC
the normal phase and the reverse phase
so what's a normal phase normal phase
has a polar stationary phase whereas
your reverse phase has a non polar
stationary phase so in a normal phase
the stationary phase is a polar or a
hydrophilic pond and the solvent is a
non polar whereas in a reverse phase you
have a nonpolar or hydrophobic compound
on the stationary phase and you use a
polar solvent so nowadays the reverse
phase chromatography has become very
popular
whereas originally we had the normal
phase chromatography being the problem
that means in a normal phase you have a
hydrophilic stationary phase so
hydrophilic compounds get adsorbed on to
the hydrophilic stationary phase whereas
in a reverse phase hydrophobic compounds
get adsorbed or get more partitioned and
hydrophilic compounds travel faster so
in a reverse phase what are the solvents
used as I said in in Reverse phase the
stationary phase is a hydrophobic
compound so these the continuous phase
is a hydrophilic or a polar so you use
methanol you use acetone nitrile you can
use tetrahydrofuran tetrahydrofuran is
also called THF or you use water or even
combinations of these two sometimes we
use a certain light rail water
combination if you want to play around
with the certain dielectric constant or
other parameters so if you look at this
solvent in a reverse phase predominantly
you will be using water and water is a
very very cheap that is why a reverse
phase chromatography has become very
popular whereas if you use a normal
phase chromatography the stationary
phase is a polar like a silica for
example
then you have to use a hydrophobic
solvent like hydrocarbon so that's very
very expensive and normally in this type
of HPLC systems the amount of solvent
which we use is quite a lot so the
solvent cost is extremely high and then
generally we try to recover the solvent
in a reverse phase chromatography use a
certain nitrile water and so you can use
predominantly water and small amount of
a certain nitrate so you will lose only
small amount of a certain level so
operating cost in a reverse phase
chromatography is much less when
compared to the operating cost in a and
normal phase chromatography and
sometimes we also use three solvents as
I said if you want to play around with
the dielectric constant and separations
we sometimes use say methanol a certain
nitrile water and so on actually if you
look at some of those solvents let's we
proceed in this course so what are the
columns used suppose you have solid
support usually about a 10-5 or three
micron silica or polymeric particles
okay so on top of that you have bonded
phases functional groups linked those
functional groups are covalently linked
to the solid support you also have a god
column this is a small column which is
placed before the original large column
so this god column acts as a filter so
it captures particles it captures
polymeric materials and other unwanted
materials thereby it is prolonging the
life of the analytical column after some
time after about 10,000 hours of use or
something you can throw away the god
column and replace it with another got
caught up so what are the bonded phases
we use in a reverse phase chromatography
we use a Ethel's silyl so the et al is a
hydrophobic or you can use octal silyl
or we can use octo the Kyle silyl that's
just see a teen or we can even use a
sino profile silence so what we have we
we tell a rock tile or after deck I'll
so as we go down the series we are
making it more hydrophobic and
hydrophobic okay so that's the advantage
of it and the c18 type of columns have
become extremely popular and it has also
become ubiquitous that means it's used
in quite a lot of applications
practically separation of many
phytochemicals or even organic
metabolites so a typical setup of an
HPLC so we have the column here column
will be in the order of several meters
okay then we have a pump here and we
have several tanks having various mobile
phases either you can run one single
mobile phase that's called an isocratic
system or we can have two mobile phases
we can shift from one mobile phase to
another over a period of time so that we
get some sort of a gradient which can
improve the separation efficiency so
there might be one pump if you're in
isocratic or you may have two pumps
pumping two different solvents so you
have the pump and then there is the
injector
that's where you are injecting your so
youth mixture okay and then finally you
have the detector there are different
types of detectors that are used in HPLC
depending upon the type of components
you're separating we can have refractive
index director you have UV detectors and
we have diode array detectors light
scattering detectors even mass spec acts
as a detector so you can have a HPLC
mass spectrometer connected to each
other
that's called an LC ms so mass spec will
detect the mass of the analyte that's
coming out and the NC or the HPLC does
the real separation of the various
components so this is a typical setup
the pump is the most important in the
sense it has to deliver very high
pressure me in HPLC the pressures are
extremely high 30 40 50 bars because you
are using very fine particles in micron
sized and so the back pressure developed
is also very large so we are going to
look at different types of detectors
there are advantages and disadvantages
during the course of this lecture so you
may have a UV detector that means a
single wavelength UV detector exactly
matching one particular wavelength if
you know the components come at this
particular wavelength we just have a
single UV detector it's very cheap or we
can have a variable wavelength detector
using a monochromator or a you having a
multiple wavelength detector okay so
these are more expensive than a single
wavelength you can have a fluorescence
detector if you know that compounds are
fluorescing we can have an
electrochemical detector we can have a
mass spectrometric detector so lot of
detectors depending upon the type of
compounds and how they behave
typically a column specification looks
like this you know the C 18 column this
is the column dimension it's a four
point six meter long column 255 it's a
particle size is five micron and we have
30 and strong pore size that means the
pore size and the particles which are
supported hard 300 angstrom so this is a
typical specification for a column now
the output from HPLC will be a
chromatogram okay it will be like a
Gaussian distribution or normal
distribution and so on but if you have
multiple components and if your
separation is not very very good you may
have Peaks coming out like this this is
a peak these another peak but both are
overlapping so much we cannot
differentiate the area of this peak or
area of this big or you may have a
reasonable separation but still this is
not a good separation again you have
some overlap in this region and this is
the best separation one can hope for you
get a very good baseline separation
these two components are and well
separated so we can measure the area
under this curve we can measure the area
under this curve independently without
any
so we can determine what's the
concentration of the species what's the
concentration of this species so ideally
one would like to have this type of
separation but one may end up like this
and this is extremely poor and imagine
if you have many components you are
going to have many Peaks overlapping
some of them overlapping some of the
male spread out so you the challenge is
to separate each one of the component
and clearly at the baseline so that you
can measure the area under the curve and
hence the composition of that particular
component of interest so the x-axis you
have the time and the y-axis will be a
detection signal so this is called the
retention time that's the time at which
you get the maximum of this particular
peak so the retention time is very
characteristic of a particular component
for a given HPLC hardware as well as
given solvent and solvent flow rate if
you change solvent flow rate you are
going to change your retention time or
if I use different column I am going to
get a different retention time so the
retention time is very very
characteristic in a HPLC system so if I
want to detect a compound and if I know
what that compound probably is what do I
do I get a pure authentic sample from a
manufacturer inject it in HPLC and see
the retention time and then when I
inject the mixture and I see a compound
coming out at the same retention time
then I can tell probably this particular
compound is present in that mixture so
that is how we do identification of
components so suppose we are looking at
three or four and what we know and what
we expect it will be there in my mixture
I'll buy those three or four authentic
samples from a manufacturer inject each
one of them independently and check its
retention time and when I inject the
mixture and if I get those three or four
peaks and those retention times then I
can tell that my mixture contains these
components so that is the identification
part in
B&C how about quantification in a
quantification what do we do and the
inject authentic sample at certain
concentration and we identify the area
under the curve so using that
relationship between area under the
curve and concentration I can tell how
much concentration of a another
component is present in the mixture by
measuring the area under the curve so
typically the output from an HPLC will
look like this you will have your nice
looking Gaussian distribution and we
talked quite a lot about gaussian
distribution and the type of equation
your normal distribution will have and
so on so we can apply those knowledge
and those equations in HPLC as well okay
so normally you will get a nice-looking
Gaussian distribution and this is called
the retention time okay and this is
called the height of the peak and this
is called a half the ID and this is
called the width at half height so here
this is called the bit of the pealed
which is done by drawing two parallel
lines okay and you get the width at the
base and the width at half the height
that means you check what is the half
the height of this and then measure the
width
that's called with that half mile now
there is a parameter called theoretical
plates just like we study the
theoretical plates with the
chromatography here also you have the
concept of theoretical plates and the
three theoretical plates could be
estimated by measuring the retention
time as well as either the width at the
base or width at the half maximum okay
theoretical plates is a measure of the
efficiency of the HPLC column so if a
column has large number of theoretical
plates as against another column which
has a smaller number of theoretical
plates we can say the one which has
large number of theoretical plates
to be more efficient in separating
components
so these are some formulae which can be
used to measure the theoretical plates n
is the theoretical plates TR is the
retention time W is the width at the
base so you have n equal to 16 into TR
by W whole square or we can use this
equation if you are measuring W per half
that means if you are measuring the
width at half maximum then we use
instead of 16 here you will get five
point five four okay or there is another
equation is called n equal to 25 into TR
by dub W Phi s whole square where W Phi
s is the peak with that four point four
percent of the peak height okay this is
the peak height four point four will
come somewhere there okay so this width
will be like this no the bigger width so
if you have that then you have to
multiply by 25 so W Phi s will be with
somewhere here whereas W is the width
when you draw two tangents to this
Gaussian distribution so we can use
different equations to calculate the
number of theoretical plates and and
there will be small differences
depending upon the equation which we use
because although we said this is a very
Gaussian looking distribution it's never
so there will be small differences in
its normal distribution shape so the N
which we calculate from this equation or
this equation or this equation will be
slightly different but we can use the
same equation if you want to compare
different columns so that we don't get
confused with the value of n which we
get so all you have to do is inject the
sample determines its retention time
determine its width at half maximum use
this equation and calculate your number
of theoretical plates and then if you
take another column do the same job get
the number of theoretical
so we can use this equation to compare
quantitatively between two different
columns and say which column is more
efficient in separating a mixture and
and which column is less efficient okay
now there are many issues if you are
performing a HPLC it's not so easy this
is this example is taken from a book
school and Leary principles of
instrumental analysis you see we are
having a six components so we are
getting two nice Peaks and after some
time we get a slightly broader peak and
after a very very long time we get
extremely shallow Peaks peak number five
and six so these Peaks may be coming at
a very long time number one number two
they are so broad area under the curve
when you measure there will be a lot of
errors okay so to overcome the problem I
may change the solvent conditions that
means I may change type of solvents when
you do that I may get five and six
closer these are nice and sharp peaks
but then you see one and two has
overlapped and three and four also has
overlapped we cannot differentiate one
and two but we can slightly
differentiate three and four so okay
again we play around with the with the
solvent mixtures and so on five and six
look reasonably good three and four very
good separations but still one and two
is not very very good separation because
there is a large overlap so if you are
measuring area under the curve for one
and two we are going to have large error
so again this is also not very good so
we need to again work on the solvent
optimization until we get reasonably a
good baseline separation for one and two
so these are some issues we call it
Aleutian issue issues so you need to
play around with the various solvents in
the continuous phase that's very very
important okay
there is another chromatography which
should we use quite a lot in
synthetic organic chemistry and I also
talked about it for the past ten
lectures
that's called thin layer chromatography
so we can use it for qualitatively
looking at a mixture of biological or
small-molecule chemical components the
advantages are its low cost you need
very little sample for that we can use
different mobile faces to perform it's
very flexible very easy sample detection
we can even go for high sample loading
ease of handling is all good so if I am
doing your synthetic organic chemistry
where I am performing a reaction a plus
B and I want to know whether C is formed
or D is formed all I have to do is run a
thin layer chromatography and see
whether I get new spots so thin layer
chromatography is based on spots which I
find on the plate chromatographic plate
okay so that's the main advantage we can
monitor a reaction suppose I have a raw
material and I'm slowly getting a
product initially the raw material will
give one spot as the product is formed I
will get two spots one for the raw
material as well as one for the product
after some time when the raw material
completely gets converted and the raw
material spot will disappear and I will
have only the product spot so
beautifully we can monitor reactions so
organic chemists use this TLC technique
and very extensively practically every
day for all their synthetic organic
chemistry and synthesis work so how does
it work so you may have a glass plate I
may coated with silica on the glass
plate okay then I put a initial mixtures
spot and then take a solvent which is
the element okay so now it's a silica so
it's a normal phase chromatography
silica is a hydrophilic compound or a
polar component so I have another
solvent which moves as it travels upward
due to capillary effect
it carries components with it
component which are tightly bound to
silica remain at the bottom component
which are loosely bone will start start
traveling so very long period of time
I'm going to get various products in the
mixture as various spots ideally I would
like to get clean sharp separated spots
but sometimes what happens is you may
get overlap of spots so it's a very
simple system all I need is a glass
plate and I quote just silica and I put
it in a solid mixture so I can play
around with different types of solid
mixture so that I change the polarity
and hence the movement of the solute
from bottom to top this is called a thin
layer chromatography and it's as I said
widely used in a synthetic organic
chemistry it's also used in downstream
processing if I am looking at
separations of small peptides or
biomolecules okay so how does it happen
you have a capillary action the so the
solvent and the solution mixture so
you'd mixture flows upwards so based on
the partition coefficient the solid will
adsorb fraction of each component of the
mixture remainder will be in the
solution will be traveling up so if it
is tightly bound on silica it tri
becomes very hard for it to travel up
first
whereas if it is loosely bound and
silica it becomes much easy for it to
move upwards so substance that is
strongly adsorbed by the solid matrix
will have a greater fraction of it
absorbed so it will spend more time on
this support and less time in the
solution whereas a weakly adsorb stands
will have a smaller fraction of it
adsorbed hence it will move fast so
weakly adsorbed substance will move up
the plate and strongly adsorbed
substance will stay near the bottom of
the plate so and from the location of
the various parts we can tell components
which are strongly adsorbed on silica
component which are weakly adsorbed on
silica so alumina is the strongest and
job and followed by charcoal and
Florizel
silica and hydras and so on and silica
gel is the least absorbing in this
particular group of components actually
with alumina that zorb and we can use
different types of solvents depending
upon whether I want least eluting power
or whether I want strong eluting power
okay so with the alumina has that jorb
and solvent with the least electric
power our petroleum ether like excellent
pentane and then goes cyclohexane then
goes carbon tetrachloride
then goes benzene and dichloromethane
chloroform ether ethyl acetyl acetone
ethanol methanol water and pyridine so
with alumina as adsorbent the solvent
with greatest eluting power will be
organic acids so if I am using alumina I
need to play around with these solvents
depending upon whether I want our least
eluting or I want a greatest eluting
part so most strongly adsorbed or acids
and bases and least strongly adsorbed
are saturated hydrocarbons because
you're using a stationary phase like
silica or alumina which are polar or
hydrophilic so least strongly adsorbed
or saturated hydrocarbons alkyl halides
unsaturated hydrocarbons Hulk canal
highlights aromatic hydrocarbons here'll
halides poly halogenated then comes
hydrocarbons ethers esters aldehydes
ketones and alcohols so alcohols because
they are hydrophilic will get strongly
adsorbed
and SM hydrocarbons which are extremely
hydrophobic or leastly adsorbed so the
relationship between the distance the
solutes travel because of the solvent
after they have reached equilibrium is
given by something called an RF value so
RF value is a characteristic of the
compound for a given stationary phase so
RF value is the distance travelled by
the solute divided
made distance travel very solvent friend
okay so it's the distance travel by the
solute even have a distressed I'll be
the solid friend so in the hour of value
is very very large
obviously the compound travels fast or
very far after it has achieved
equilibrium the RF value is very small
then you can say that compound has not
traveled very far in the system so for
example I have TLC okay then I go to
your reversed-phase hplc how do i
connect the spots I get on TLC with the
peaks that I get in the HPLC so that
depends upon the type of HPLC whether I
am using a normal phase hplc or whether
I am using a reverse phase hplc now in a
TLC we use generally silica so it is
almost like a polar stationary phase now
if I take your normal phase
chromatography I will again use a polar
stationary phase so the order of the
components in a TLC which uses silica as
against your normal phase chromatography
will be the same so if I take a TLC and
I see your spot see which is right at
the bottom and then I see B and then I
see a okay that means here C is very
very high polar and he is the least
polar so in a normal phase what will
happen a will come out fast B will come
out second C will come out third that
means a will have the least
Ellucian time or retention time we will
have the text retention time and C will
have the highest retention time so in
normal phase hplc we are using silica so
that's also a polar so they will behave
in the same fashion as a nor
my face TLC which also use a silicon
whereas if I use a reverse phase hplc
where I am using a hydrocarbon like a
c18 as the stationary phase okay so now
the stationary phase is hydrophobic or
lipophilic then what happens so in a
hydrophobic reverse phase chromatography
I use a polar as the mobile phase so
what will happen see which is more polar
will come out first B which is next will
come and finally a which is hydrophobic
or nonpolar or a lipophilic will come
out last so you see the order of the way
these three components ABC come in a
normal phase visa is reverse phase are
very very different so you should not
get confused you should know if I get
you know TLC three components a
traveling the farthest be in the middle
C traveling the least if you are using a
normal phase you will have a a coming
out first
that means a having the lowest retention
time followed by B and C will have the
highest retention time whereas if I use
a reverse phase chromatography C will
have the least
retention time and Yi will have the
highest retention time so you see that
there is a completely reverse happening
in the normal phase in the reverse phase
chromatography
okay so you need to understand this
concept very very well so that if I do a
TLC during initial downstream processing
and then later on I am going to HPLC I
should be able to tell depending upon
the type of HPLC I am using then each of
the components will come with respect to
the spots which I observe in my TLC okay
now let us go forward so you have a
normal phase chromatography I have a low
polarity mobile phase okay it's a normal
phase chromatography so I mean the
stationary phase is polar don't forget
that so I have components like this see
be a so if I am increasing the polarity
what happens I am having a polar
stationary phase and I am increasing the
polarity so obviously these three will
come together okay so that means if I am
using a normal phase chromatography and
if the peaks are very close I want to
move the peaks further apart I reduce
the polarity that means I relatively
make it more hydrophilic sorry
hydrophobic so in a normal phase
chromatography if the peaks are very
close and I want to move the peaks
further apart I use a slightly more
hydrophobic system that means I reduce
the polarity of these mixture now let us
go to reverse phase suppose I have a
reverse phase chromatography and my
Peaks are like this C B and hey now my
solvent mixture or mobile phase is a
high polarity if we reduce the polarity
to medium then they will come closer so
in a reverse phase chromatography if the
peaks are very very close all I have to
do is increase the polarity of the
mobile phase then the peaks will get
separated out so in a normal phase
chromatography if the peaks are closer I
reduce the polarity for the peaks to
separate out whereas in reverse phase
chromatography if the peaks of closer I
increase the polarity so that the
components get separated out so you see
the strategy which you adapt and in a
normal or a reverse phase changes
dramatically so if you want to move from
a close to a further separated system
so what are the various factors that
affect the column efficiency factors
which affect the column efficiency or
the linear velocity of the mobile phase
so if the velocity is high then
obviously my retention time goes down
relatively a diffusion coefficient in
the mobile phase diffusion coefficient
in the stationary phase that means how
the components diffuse inside the pores
of the stationary phase then we have the
retention factor that means how much of
the solute is taken up by the stationary
phase diameter of the packing material
thickness of the liquid coating on the
stationary phase so if you have thick
material you are going to have mass
transfer resistances if you have very
thin coating you are going to have less
must answer if assistance so all these
factors affect the column efficiency so
you can play around with many of the
factors but once I select my column when
some of the factors get fixed whereas I
can still play around with the the
solvent flow rate or I can play around
with the mixtures of solvent so that I
affect my retention factor as well as I
effect my linear olicity of the mobile
phase so imagine I have our three
components and I am using a UV detector
that means the components are detected
using a UE
so if I set the detector to 240
nanometers and suppose I have three
components like this aspirin as terminal
fin and caffeine I may get the area
under the curve for aspirin 19.5 the
acetaminophen has 50 caffeine has 20 now
if I change the detector setting to 254
nanometers then this becomes 7.3 area
under the curve goes down dramatically
and this becomes very very large eighty
one point nine caffeine becomes ten
point eight now we have set the detector
to 80 nanometer
aspirin becomes 24 I still my fin
becomes 39.3 caffeine becomes 35.9 now
how much is the real amount we don't
know because they take depending upon
the detective setting I am getting
different person area under the curve
percentage area under the curve why is
it so that's because of the lambda max
if the lambda max for each of the
components are very different depending
upon where I said the lambda max that
particular component will show a very
high area so that is another big
challenge when you are doing HPLC so I
may get mistaken whether the component s
terminal fin is very very large or it is
equal to caffeine so you see that I am
able to get different area under the
curves for aspirin depending upon the
lambda max of my UV detector so I need
to have some idea about the lambda max
for the components which I am measuring
in my in my solution or in my solute
mixture if I do not know then I may end
up with this type of misunderstanding of
the whole system
so you see UV max for aspirin is 225 and
296 okay as soon my finish 248 so at 254
I get a very very large number so when I
go away from 248 you see the extreme
enough in keeps going down now for
caffeine it is 272 so as I keep
increasing see the caffeine percentage
goes up so the main problem here is the
lambda max
aspirin has lower lambda max caffeine
has the next one
sorry is terminal finish the next one
and caffeine has the third-largest so
the numbers keep dramatically changing
so you see the aspirin it goes down and
again increases because aspirin has two
different lambda maxes so there is one
lambda max here that is why you get some
number
as we move out from that particular
number the value goes down and again it
goes up it picks up because aspirin has
two lambda max so the first thing is to
you we use variable system just to find
out the lambda max of various components
present in my mixture and then you
decide which lambda max to use if I want
to use a constant UV detector and then
do all your standardization measurements
and so on actually
so understanding the lambda max for each
one on the component is very very
important that's what this particular
example tells you otherwise you can
totally get mistaken okay
now there are different types of liquid
chromatography we can use depending upon
the type of molecule which you are
trying to detect this is taken from a
book by Lindsey and bonds from join
Miley publication it's called high
performance liquid chromatography now if
it is a small molecule like molecular
mass less than two thousand it could be
soluble in water that means its
hydrophilic or it could be soluble in
organic solvent that means its
hydrophobic now in soluble in water it
could be ionizable or it could be non
ionic or ion pad type of system so if
it's an ionic then I can use a ion
exchange or ion chromatography it's a
non ionic or ion pad okay then I can use
a bonded normal phase chromatography
okay so it's a normal phase that means
the stationary phase will be hydrophilic
now soluble in water now let's go to the
system the system is soluble in organic
solvents that means its hydrophobic now
here we can have two different things
one is soluble in nonpolar to be clip
polar solvents soluble in moderately
polar to polar solvent okay
so if it is nonpolar tweekly then we go
to reverse phase chromatography here we
can use CIT
CHC 4c2 or phenol or sign on this is
completely hydrophobic material so we go
for reverse phase chromatography whereas
it's moderately polar to polar solvents
so it may be soluble in chloroform or a
methanol so if it is soluble in
chloroform we go to adsorption type of
chromatography that means the stationary
phase forces are just based on and softy
forces or it's soluble in alcohol
acetone nitrile or ethyl acetate so here
we use bonded or normal phase
chromatography okay again we see we use
a normal phase chromatography bid sino
or amino or dowel as the stationary
phase so depending upon this is for
small molecules depending upon whether
molecule is soluble in water or it is it
is soluble in organic solvent if it is
soluble in water is it ina easily
ionized or it's non ionic or I inferred
we may have two different types of
chromatography either ion exchange or
normal phase now on the other side if
it's soluble in organic solvents then
one could be soluble only in weak polar
or completely nonpolar or the other one
could be moderately polar solvents
completely nonpolar we may be using a
reverse phase chromatography whereas in
a moderately polar we can have two
systems one is the chloroform soluble
other one is the alcohol soluble normal
suta nitrile soluble so in a chloroform
soluble memory using a silica based
chromatography whereas in the other one
we may be using a normal phase
chromatography like cyano amino Dowell
and so on actually now let's look at
molecules which are larger than 2000
that means there's much larger molecules
whereas so far we looked at molecules
which are smaller than 2000 okay so
again this is taken from the same book
high-performance liquid chromatography
second edition Lindsey and Mars okay
again you have soluble in water soluble
in organic solvents
so soluble in water again it can be
ionic it can be non ionic around paired
if it is ionic we can use a ion exchange
chromatography if it's non ionic it
could be small molecular sizes or large
molecular size so if it's a small
molecular size we can use a reverse
phase or hydrophobic interaction
chromatography like different types of
stationary phases CA T in Ch C for if
it's a large molecular size 32 400
nanometers then we can use a molecular
exclusion chromatography okay it's that
size exclusion chromatography we talked
about it long time back it's called a
size exclusion or gel permeation so it
is based on size or molecular weight
alone so larger sized polymers to
smaller size polymers I just say is a
metabolize to smaller size availablee or
larger molecular weight metabolized to
smaller molecule right now that is to do
with the solubility in water what
happens if they are not soluble in water
but they are soluble in organic solvents
again you can have small molecules and
large molecules small means less than 30
nanometers large means 30 to 400
nanometers so large molecules we can
again go for the exclusion
chromatography like size exclusion or
gel filtration chromatography whereas
small molecule we can go for reverse
phase chromatography c 18 c HC 4 so
again large systems we again have a
water-soluble large systems or solvent
organic solvent soluble large systems if
the size of the molecules or molecular
weights of the molecules are very large
we can go for gel permeation
chromatography of course if it's ionic
we cannot use a gel permeation
chromatography because as you can see
here you can use earlier and exchange
chromatography gel permeation
chromatography is good for non ionic or
and paired systems actually which is
based on just play size separations so
smaller molecules will get entrapped in
the
pores of your stationary phase whereas
larger molecules do not get entrapped so
they travel faster so larger molecules
will come out of the column faster
whereas smaller molecules will take much
longer time because they interact so
this table gives you what type of
chromatography to use depending upon
whether it's a small molecule or whether
it is a large molecule so the molecules
are large we can go for an exchange
chromatography or we can go for gel
permeation chromatography or we can go
for hydrophobic interaction or reverse
phase chromatography if the molecular
sizes are very small again we go for an
exchange chromatography normal phase
chromatography adsorption chromatography
and reverse phase chromatography so
selections based on the ionized ability
selection based on the size of the
molecule selections based on the
solubility of the molecule there is
something called isocratic elution that
means I use only one mobile phase it's
very simple very cheap I use only one
solvent I use only one pump the pump is
very very expensive in a HPLC so by
using only one pump I am able to bring
down the cost or if I want to use two
solvents I mix the solvents and make it
in a container and use that mixed
solvent for illusion okay but still I
used only one so it's simpler you don't
need a mixing chamber if I have two
solvents then obviously I need the
mixing chamber okay no but it has got
limited flexibility because I cannot
change the dielectric constant of the
operation it's good for routine
operation so if I am going to do the
same system like a QC lab where you are
going to do the same thing day in and
day out for very long time to come this
is the best system gradient elution
we're the mobile phase compositions are
changed as a function of time so
initially you may start with modern one
solvent as time proceeds I change the
solvent to something else okay
this is just like a gas chromatograph in
gas chromatograph and either you can do
the the chromatography in one single
temperature value that means the column
is placed inside the oven and the
temperature is maintained constant or as
a function of time I can change the
temperature of the column or if so I may
start with say 150 and in ten minutes I
may go to 250 and then maintain at the
temperature so exactly similar to
changing the temperature in a gas
chromatograph I am changing the solvent
dielectric constant in a HPLC system so
I may start with one solvent I may go
into another solvent or I may start with
one solvent mixture then I move to some
other solid mixture so that's called a
gradient elution which is opposite to UF
isocratic so you'll be using multiple
pumps so you can use two pumps or you
can use four pumps that means it can be
a binary system or it can be coronary
cistern and of course the cost is very
high because pump costs are very high so
more the pumps more is a cost so when I
change the mobile phase components I am
changing the polarity index so I can use
it to elute compounds that are not
coming out of my column things stick
inside they stay inside so that but the
problem is there could be some
additional wear on the stationary phase
after each run I have to again bring it
back to the original condition
so each run will take longer so I do a
run and then I will bring it back to the
original condition which requires again
several minutes so in a gradient elution
normally the time taken is much much
longer than yeah isocratic elution
because in high socratic you are not
changing the composition of the
solvent over a period of time so you do
not have to again recreate the system so
that is one of the disadvantages
gradient elution it takes much longer
time but we can get excellent
separations using two solvents or three
solvents or mixtures of solvents that's
the main advantage of this type of
gradient elution okay there are various
types of detectors that are possible in
HPLC depending upon the the properties
of the solutes top properties of the
amino acids or proteins or peptides
which are separating and we are going to
now look at some of the detectors look
at some the advantages disadvantages and
so on actually okay the most cheapest
one is the single beam UV visible with
the flow-through cell Kuwait that means
you take your sample inside equate and
there is a UV which measures the
absorbance of the solute mixture
generally it operates at at 254
nanometer default wavelength but we can
also set it for other wavelengths also
know some UV detectors you cannot set it
then it's very very cheap and there may
be UV do test where you can preset it to
some fixed value 3 for different
wavelengths or we can use even filters
we can also have adjustable wavelength
units which are cost-effective the main
advantage is the cost it's very very
cheap and then it's non-destructive ok
so the sample we can take it back and
use it for some other purposes so
especially if the sample amount is very
small you cannot have afford to have
your sample which is totally destroyed
after the analysis so that's the main
advantages but the disadvantage is not
all compounds will absorb the light
so you will not be able to detect a
compound which is not you be detectable
at all so that is a big problem or we
can have several cells with different
wavelengths fixed wavelengths so we can
look at various wavelengths in each one
of the cell so that is other approach by
which we can slightly improve the
flexibility of this type of UV detectors
now we will look at more detectors in
the next classes and as I said depending
upon the detector we can look at
compounds that are present in the
mixture - very very simple compounds to
very complex compounds so we shall
continue this particular topic in the
next class
you