Gas-liquid Chromatography - Biotechnology Engineering Video Lecture - Biotechnology Engineering (BT)

till now we have discussed three
chromatographic techniques that is ion
exchange chromatography gel filtration
chromatography and affinity
chromatography all these three
techniques the mobile phase is liquid or
a solvent today in this lecture we are
going to discuss a technique which is
where mobile phases gas rather than a
liquid so this particular technique is
gas chromatography so we are going to
discuss about in particular gas liquid
chromatography gas chromatography is the
technique used for separation of
thermally stable and volatile organic
and inorganic compounds so gas
chromatography like if a particular
analyte has two separate or has to
partition between a liquid phase and
mobile gaseous phase then it has to be
in vaporized state and so only those
analytes which are thermally stable and
volatile could be utilized or could be
separated in here now there could be two
kinds of gas chromatography one is gas
liquid chromatography which accomplishes
the separation by partitioning the
components of a chemical mixture between
a mobile gas phase and a stationary
liquid phase held on a solid support
then there could be another
chromatography that is gas solid
chromatography which uses a solid
adsorbent as the stationary phase so in
this lecture we are going to focus on
gas liquid chromatography technique now
if we compare gas chromatography with
normal or other liquid chromatography
techniques which is in terms of like say
either low pressure
or high-performance liquid
chromatography techniques and all the
general principles and the performance
parameter applies here also so here like
say for example retention volume or
retention time is a characteristic of an
analyte and that will characterize its
particular it's a characteristic of a
particular analyte so if you have to
find if you have to know about or
identify a particular analyte then if
you know the retention time or a tension
or volume or elution volume you can
identify that particular component from
a mixture so then other parameters like
we have talked about resolution capacity
ratios and all those things we'll be
applying in here also so it's a similar
kind but the difference is that here
only volatile and thermally stable
components of a mixture could be
separated now guess chromatographs
or the gas it could gas chromatography
equipment is a little different from the
normal chromatography equipment the
major components of a gas chromatograph
are a constant flow of a carrier gas
that's the mobile phase as we have
discussed and you have to control the
flow flow rates of that carrier gas
through different valves and flow meters
this would be a simple system of
introducing the sample into the gas flow
and then into the column so there is a
sample Inlet point which is very
important then after that a column has
to be housed in an oven that is because
you are doing all the experiment or
separation procedure at a very high
temperature and ovince should be
temperature programmed so column your
column in gas chromatograph is based in
an oven which is temperature control and
then finally when analyte comes out of
the column
then a detector amplifier and data
recorder system needs to be there to
analyze the chromatography of a
particular analyte so these are the
important components of a gas
chromatograph and we are going to
discuss all different components in
detail now here in this diagram if you
can see on your screen
it's a estimating of a schematic of a
gas chromatograph and it very nicely
depicts what are the different
components of the gas chromatograph now
if you see from starting here this is
the carrier gas supplied in a particular
tank or cylinder this carrier gas is
supplied from here from this tank and as
it goes through the tubing there are
pressure regulators pressure meters and
there are flow meters which can regulate
the flow then there is a sample Inlet in
here which lets the sample in a
vaporized form - so it will be around
somewhere here vaporization will take
place so there's a sample inlet from
your sample will be injected now this
could be a direct injection as we'll see
later on or it could be a automatic
sampler could also be used in here then
there is a column here this column is
it's a very long column and since it's a
long column it is coiled actually here
which is not seen so in this oven
there's a coiled long column but very
thin now diameter is very very less
there are very narrow bore columns and
then this column length is required for
proper separations and then this column
is comes out here and the analyte from
the column will pass through detector
and then certain electronics and
microprocessor amplifiers and finally
data recorder and you will see the peaks
in here as you see in other
chromatography techniques
so the system or the principles are same
as per their partitioning between the
guests and the liquid phase
these analytes will elute at different
times as per their retention times or
their distribution coefficient here and
so the separation will take place and
like I said and every analyte on a
particular standard condition will have
a characteristic retention time or
elution volume and through which you can
identify the particular analyte even for
an unknown sample you can make kind of a
standard curve or you can run standard
standard compounds and then you can plot
it like we've seen in other
chromatographic techniques and then
unknown could be a unknown sample
retention time can be calculated for
that so this is how you can there's a
very simple way to depict how across gas
chromatograph will look like or what are
the different components of gas
chromatography all right so let's start
with the first thing as we've seen in
the gas chromatograph
it's a carrier gas supply that's the
most important at the beginning so you
have to choose a mobile phase and that
circuit a gas phase so a constant supply
of a carrier gas with proper controls is
so very important necessary aspect of
gas chromatograph norm normally helium
nitrogen or carbon dioxide or hydrogen
can be used these are the most commonly
used carrier gas or mobile phase
obviously hydrogen has the disadvantage
of explosion length danger there are
suitable flow regulator walls and means
of reducing the flow rate I think all of
you understand that flow rate has to be
reproduced to reproduce a
chromatographic separation or to to
maintain a particular retention time or
elution volume a flow rates change the
retention times and
and illusion volumes also change and
then you cannot compare the two
experiments a typical meter like soap
bubble meter can be used for this
particular purpose
so carrier gas is one part where you
have to choose particular carrier
carrier gas as a mobile phase now as
carrier gas goes on the next thing is
the sample injection now like I showed
you sample injection like sample has to
be many times a certain chemistry has is
involved to prepare the sample and maybe
derivatized ition or other things might
be required to vaporize the sample
because not all the compounds could be
vaporized
so this is a limitation of gas
chromatograph so sample injection when
we have to do what it has to be done is
that the sample has to be vaporized so
that it can partition between the liquid
mobile a liquid phase that is stationary
and mobile gas phase so there are two
ways you can do it one is direct
injection the sample could be directly
injected and it is the standard mode
suitable for 95 percent of packed
columns like we will discuss what is
packed columns then sample is injected
by a hypodermic syringe through a
self-sealing silicon rubber septum onto
a glass inner glass liner with in a
metal block where it is vaporized and
swept into the column so what is done
there is a heat block or metal block
which is heated to a certain temperature
higher than oval temperature maybe and
the instantly as the sample is put on
there it is it vaporizes and it is swept
onto the column now the block is heated
at a fixed temperature which is
sufficiently high to convert liquid
sample into a plug of vapour so what you
are doing is you are injecting a liquid
sample which could be vaporized and it
is done very fast samples can be
measured with
a calibrated loop then introduced into
the flowing gas stream by means of wall
for capillary columns we'll see what our
capillary columns as we go along a
reduction in sample volume is necessary
because capillary columns capacity is
very low and this will be accomplished
by an injector splitter where one micro
liter sample can be injected but only
say point zero one microliter enters the
capillary so what is done is as you are
injecting the 1 micro litre sample there
will be a injector splitter actually
which will split the sample where only
point zero 1 microliter goes to the
capillary column and rest of it is
vented to the waste spiritless injection
is required for very small amounts of
sample the entire sample is injected
onto the open tubular column through a
modified heated flesh vaporizer so there
are could be many ways you can do it
now this is a very typical
representation of typical vaporizer
where you can inject the sample in here
as you can see on your sample on your
screen there is a syringe which which
injects the sample in here through a
rubber septum as I was talking to you
and this is there is a heat block here
if you can see this is your heat block
and this is heated to a very high
temperature so that they could liquid
could be converted to vapor instantly
and then once it is done this will be
flushed into the column where it can
then be separated so this is a typical
vaporizer you can see a flash vaporizer
then you can have you can do injection
by or you can load your sample by
automatic sampler also so sample vials
are a glass throwaway type whipper like
septum caps vapor tight septum caps so
these vapor tight septum caps could be
taken now the procedure of sample
injection
if you see what is done is the sample
flushes the syringe with a new sample to
remove traces of the previous sample
that's very important that you do not
kind of get contamination from the
previous run now in the sample injector
the pumps then what is done is new
sample will be pumped into the series to
eliminate any bubbles and then we were
taken in a precisely measured amount of
sample taken then it will be injected
into the gas chromatograph so you have
to very careful when you are utilizing
automatic sampler and the automatic
samplers provide like highly
reproducible results and they are much
more precise so you you can very nicely
load your sample through automatic
samplers alright so this was about how
sample injection could be done on the
gas chromatographic column now let's
come on to the next part that is gas
chromatograph columns actually the two
basic types of columns which are
generally used in gas chromatography
these are called packed columns as we
mentioned earlier another one will be
open tubular or called capillary columns
now let us discuss each of them one by
one now packed columns they are
constructed from tubing of stainless
steel or nickel or maybe glass now
remember all of these things has to be
heat-resistant because they will be kept
in an oven at high temperature so the so
these are they can withstand that heat
the inner diameter of these columns
range somewhere around two to four
millimeter and they could length they
could go like I said they are very long
up to one to three meters length could
be there now these columns are packed
with stationary phase which is coated
onto an inert silica support
the peg means the completely packed
they're fully like pegged and the
support should not take part in the
separation like as we have discussed in
other tomato graphic techniques also so
it should be inert it must be thoroughly
in activated when polar compounds are to
be analyzed so it's a liquid phase or a
stationary phase is on support and
support has to be nert as we have
discussed earlier also now there are
some commonly used stationary phase
which are could be polyethylene glycols
methyl phenyl and methyl vinyl silicon
gums esters of a depicts and such scenic
and telecare sets likewise which are
available actually there could be beta
cyclodextrin based faces for chiral
separations the most commonly used
support material is so light or
diatomaceous silica or earth we call it
which is treated so that hydroxyl group
are modify modify to avoid support
sample interaction and this could be
achieved by salinization of the support
of it compounds like hexa methyl die
solid gene so you can you can use this
this is most commonly used support
columns are then dry packed under a
slight positive gas pressure and after
packing must be conditioned for 24 to 48
hours by heating to near upper
temperature limit where the carrier gas
at normal flow rates is passed through
the column now during this conditioning
the column should be disconnected from
the detector because you don't want to
follow the detector to pramana and you
don't want to contaminate the detector
now what happens is when you are
conditioning dis columns there might be
a situation where column bleeding is
taking place where you're losing
stationary phase or support material due
to heating and in that case it will
certainly contaminate the detector so
that has to be and so before you
a column is commercially provided or you
make it for use it has to be conditioned
so that there is no bleeding or it is
minimized
now special packing materials may be
needed for a particular applications for
example very tightly loaded glass bits
are used for very rapid analysis well
below the boiling point of the simple
components they might be sieved teflon
supports used where a corrosive
substances substances are handled and
then in gas solid chromatography the
packing material is an adsorbent like
silica gel or a bonded phase support or
a molecular sieve so you can have
different types of support for different
types of applications the peg columns
are very commonly used columns but then
there are capillary columns and our
capillary columns are different from
packed columns they have an internal
diameter of say one millimeter or less
so they are very narrow bore as compared
to back columns they are constructed of
fused silica having higher degree of
cross-linking within the silicon oxygen
matrix there is a high tensile strength
of silica tubing and permits the
construction of thin walled flexible
columns for protection of thin wall
against scratches coating of polyamide
is applied to the outer wall now there
are two types of capillary columns and
these are could be packed columns or
open tubular columns
now when you say packed columns this is
having solid particles over the whole
diameter of the column like we have seen
packed columns but open tubular columns
they are different in in a sense you
have what you have is you have an open
and unrestricted flow path through the
middle of the column divided into like
for example let me show you this on your
screen so what you have is
so when you say packed column and if I
have a very thin column of or very small
diameter column when you say it is a
packed column then it is filled in it is
all over like so you have beats which
are a small stationary support which is
filling of itches all over completely
over the diameter so it is like filling
the whole area in here and there is no
open space in middle action but when you
are saying open tubular column then an
open tubular column you will have beads
on the walls inner walls of the so you
will have beads here but and you will
have weeds here but you have an open
area in middle so this open area lets
the gas flow without any problem so
there is a clear difference between a
packed column and a capillary column so
hope you have able to understand this
all right so let's return to our
discussion with an open and unrestricted
flow and open tubular columns capillary
columns there is of like there is a flow
path which is quite unrestricted here
and the gas can flow very easily here
now there are different kinds of open
tubular columns one is called wall
coated open tubular column in short form
you can say WC OT wall coated open
tubular column now then here the
stationary phases thinly coated directly
onto the walls of the capillary then
there could be another one called
support coated open tubular column now
here in support coated like SCO T
support matrix is bonded to the walls of
the capillary column so here the
stationary phase is not directly linked
to the wall of the column rather it is
link to link through a support
supporting matrix so the stationary
phase is coated onto the support and
support is
coated onto the wall then there could be
another one called porous layer open
tubular columns which is kind of support
coated used for adsorption work so the
two mean our wall coated and support
coated open tubular column now in terms
of capacity certainly the amount of
material which support coated columns
can open tubular columns can carry will
be higher than wall coated columns so
commonly used stationary phases which
include like we discussed earlier like
polyethylene glycol on Italian soil poly
salon chains and other materials are
utilized they are caught coated onto the
supporting matrix to give 1 to 25
percent loading depending upon the
analysis so the capacity of the support
coated columns is very high considerably
high and the wall coated columns there
are certainly advantages of capillary
columns the capillary columns with
particularly open tubular columns they
are having shorter retention time they
have greater inertness the longer life
long lower bleed which is higher in
packed columns they have higher
efficiencies and greater reproducibility
so these are certain advantages of
capillary columns remember but the
capacity of these columns is very low so
many times they could be only for
analytical purpose rather than the
propriety purposes all right so we have
discussed about the column now column is
contained in an oven which is
temperature controlled so
chromatographic columns they are quiet
and they are held in a basket that is
mounted inside an oven now column must
be able to be rapidly heated and cooled
in this oven so this requires the
well-designed and very adequate system
of air flow ovince are usually
constructed of low mass stainless steel
and the working temperature ranges
chosen to give a balance between
peak retention time and resolution now
column temperature is controlled to say
plus minus 0.1 degree Celsius and
analyte partition coefficients are
particularly sensitive to temperature so
that's very important analysis times may
be regulated by the adjustment of the
Coulomb oven which can be operated in
different modes like say it could be
operated in either isothermal analysis
where a constant temperature is applied
and should be maintained here there
could be temperature programming as well
so you can have isothermal as well as
you can have temperature programming
where the temperature is gradually
increased to facilitate the separation
of compounds of widely different
polarity or molecular weight where
temperature should be maintained within
plus minus 2 degree Celsius you can have
both ways you can you operate the oven
that is isothermally or by programming
the temperature now liquid phase
stationary phase or coated on inside the
column or on the support material as we
have discussed it is like here
partitioning takes place and it
separates the sample compounds
components into discrete peaks it should
be chemically and thermally very stable
because otherwise like we were talking
about column bleed and other problems
could occur which will certainly damage
or will result into not so good
chromatographic separation on gas
chromatography operations are always
kept 10 to 15 degree Celsius below the
upper temperature limit of the face
because again column bleeding which will
be much higher if you go at the upper
limit then the amount of column bleed
must be minimized and it will certainly
prolong the column life prevent any
fouling of the detector and maintain
baseline stability on the chromatogram
so that's a very important part that you
column bleed could be mean
to enhance the quality of the separation
now bonding the stationary partitioning
liquid to the inner surface of capillary
columns or to the column packing
permanently anchors the stationary phase
to the surface commonly used phases
which we have already discussed includes
a polyethylene glycol methyl phenyl and
methyl vinyl silicon gums esters of
adipic Saxena canned telecasts episode l
and so on so there could be different
kinds of depending on what temperatures
what analytes you are going to use you
can use different kinds of liquid phases
once the analyte has passed through the
column it has partitioned and it has the
mixture has different analytes in a
mixture has separated they will come out
of the oven and will be passing through
the detector like they will be eluted
from the column and passing through the
detector so that they could be
identified or those chromatogram could
be obtained now detectors there are many
different types of detectors as we'll
see now detector is located at the exit
of the separation column and there are
like what it does is it will sense the
presence of an individual component as
they leave the column and there could be
different ways methods to sense that
particular analyte in a vaporized phase
now volume must be small to prevent the
remix ting of the component separated so
that should reach the detector fast
their electrical analog output of the
detector is amplified and then sent to
the strip chart recorder or converted to
a digital signal and sent to a computer
system then computer system can process
the data store them and display the
chromatograph chromatogram with
electrical results on your screen or a
recorder and there are several types
like I said on detectors let's see let
us discuss some of these detectors here
they're very simple type of detector are
called thermal conductivity detector or
tcd we call it a thermal conductivity
detector are one of the earliest
detectors actually which will develop
for use with gas chromatography now what
it what it does is it utilizes a heated
filament placed in the emerging gas
stream so the amount of heat lost from
the filament by the conduction to the
detector walls will depend on the
thermal conductivity of the gas phase
I'll show you that in a little while now
within a cavity in the metal block their
extents are tightly coiled filament like
I said this is heated filament filament
is heated to a constant temperature but
less than a dull red condition by a
regulated current supply now heat loss
from the filament to the metal block is
a constant is constant when only carrier
gas is flowing through the detector so
when only like certain carrier gas and
there is no analyte in it then the heat
loss will be a particular amount to the
walls of the detector now the thermal
conductivities of say hydrogen and
helium are roughly 6 to 10 times greater
than those of the most organic compounds
- what will happen if there is an
analyte or organic compounds present the
presence of even small amounts of this
material organic material will cause a
relatively large decrease in thermal
conductivity of the column effluent so
what will happen that the amount of heat
lost or which is conducted to the walls
of the detector will be less so the
filament Hritik will retain more heat
and its temperature will rise so that
particular its electric electrical
resistance will go up so the advantage
of and you can measure that the
advantage of thermal conductivity
detector is its simplicity its large
linear dynamic range which is around 10
is 205 its general response to both
organic and inorganic species it doesn't
differentiate between them and it is
it's a non destructive character like it
is not destroying the sample so you can
collect also the sample after detection
limitation one limitation could be it is
low it has low sensitivity so that could
be one limitation now this is a very
simple schematic of this thermal
conductivity detector as you can see
there is a filament here this filament
is heated now when it is heated and
there is a gas flow here which is only
carrier gas the the heat generated from
here we'll go to the walls of the
detector now when there is an analyte
organic compound mixed with the carrier
gas then the conduction of heat will be
lower and that could be measured
actually because resistance will go up
here and that's how you will get the
signal like we said it's non-destructive
so flow out here you can collect your
sample after it has been detected so
thermal conductivity detector is one
type of detector but it has a low
sensitivity as we were talking about now
second most commonly used detector is
flame ionization detector or we call it
fi D now if I D are the most what you
call generally applicable and it's the
most widely used detector like I said a
mixture of hydrogen and air is
introduced onto the detector to give a
flame so it's a flame ionization to
flame is an important part of this
detector so a flame forms one electrode
so what is done is a mixture of hydrogen
and air is introduced into detector to
give a flame and the jet of which forms
one electrode the other other electrode
will be a brass a platinum wire mounted
near tip of the flame so this detector
will have one electrode in form of flame
another in form of a wire so what
happens is when sample components will
emerge from a column they will be
ionized in the flame
and when they are ionized they will
result in increased signal being passed
to the recorder and that will be
measured upper temperature limit here is
400 degree Celsius and minimum detection
quantity is the order of like say 5 into
10 raise to power minus 12 gram per
second if you compare with TCG it was 10
is 2 or minus 8 actually which is lower
than this so this has much higher
sensitivity here one thing has to be
remembered this flame ionization
detector they responds proportionately
to the number of ch2 groups so if you
have 2 ch2 groups then signal will be a
particular amount and if you have 4 ch2
group it will double up that likewise so
it is like directly proportional to the
number of ch2 groups but there is a
problem that no response from the fully
oxidized carbons will be obtained so the
response from carbons attached to say
certain groups is like say hydroxyl
groups and I mean groups is lower and
oxidized carbons will not give any
signal so that's a disadvantage now it
is a advantageous to use fi d that is
flame ionization detector because in
Traktor is unaffected by the flow rate
it is non combustible gases and it is
not affected by flow rate or non
combustible gases or water now the
properties allow fi D high sensitivity
and low noise the unit is both it is
very reliable and very easy to use but
you require a flame here flammable gas
here and also the sample is destroyed so
unlike a thermal conductivity detector
where it was not destructible this is
destroys the sample if you see the very
simple schematic here of flame
ionization detector then you can see
here there is a flame here and there's
this collector you can say collector
assembly the wire so what happens is as
the exit or material is put in this
flame
Dionysius and then a current is passed
and the signal is recorded and like I
said it's a destructible there are like
grounded Chet there are a lot of
hydrogen air flame is here hydrogen is
supplied from here
and air is supplied for the flame so
this is very useful and most widely used
detector and like I said it's a certain
limitation that oxidized carbons cannot
be sensed here then other groups like
hydroxyl will be have lower detection
limits and it is directly proportional
to the ch2 groups so it is very useful
for say environmental samples and a lot
of like saying pollution studies and a
lot of other studies it is utilized then
another version of flame ionization
detector is thermionic emission detector
or te D this is also called nitrogen
phosphorus detector and it is same as fi
d but it has you can say much cooler as
compared to the FI D and it has a
crystal of sodium salt fused onto the
electrode system or a burner tip
embedded in a ceramic tube containing a
sodium salt or rubidium chloride tip it
has an excellent selectivity towards
nitrogen and phosphorous it has been
modified for compounds containing
nitrogen and phosphorous containing
analytes upper limit or a range could go
up to 300 degree Celsius like I said it
is kind of cooler or flame is cooler
than the flame ionization tractor
detection limit is very high say up to
tellus 1-14 grams per second it is
widely used for organophosphorus
pesticide residue analysis and other
things so here there is a simple
schematic it's like a fighting only
there is detect here the flame which is
a it's a cooler flame like I said this
is a heat he took connect
the Whittemore caesium bead here anode
which is collector here and as the
Campania lights are ionized here then
mostly nitrogen and phosphorus compounds
then the signal could be or could be
recorded in here another detector which
has which is for a particular kind of
compounds it's called electron capture
detector electron capture detector only
responds to analytes that can capture
electrons as the name suggests a
particularly halogen containing
compounds it is widely used in analysis
of say poly chlorinated compounds like
pesticides DDT Aldrin dialed ring and
all those things it has a very high
sensitivity of around 10-ish / - 13
grams per second and the upper
temperature could go up to 300 degree
Celsius
now this detector works by means of a
radioactive source present on surface of
one electrode which will emit
high-energy electrons that is beta
particles if you could recall in
radioisotope technique we have discussed
about that radioactive substance will
emit certain particles or radiation now
these electrons bombard the carrier gas
as we have seen in gas ionization abased
detectors they're releasing an electron
and that gives the current across the
electrodes when a suitable voltage is
applied if you could recall GM counters
it's something like that now what
happens is when electron capturing
analyte emerges from so when you are
taking carrier gas the different current
is recorded but when you have an
electron capturing analyte in the
carrier gas the what will happen is the
ionized electrons will be captured by
these analytes and there will be a drop
in the current and this change could be
recorded so the carrier gas so
here that's the method and cobalt could
be utilized as a radioactive material or
other things could be all soot alized
the carrier guest most commonly used in
conjunction with a CD is nitrogen or an
argon plus 50
it's a nitrogen or an argon plus 5
percent methane mixture that could be
either nitrogen could be used or argon
mixed with methane and that is five
percent mission could be mixture could
be utilized so it's a very simple
schematic of electron capture detector
there is a radio active beta emitter and
there's a gas here and the electrodes
here are now radioactive material will
emit beta particles which will ionize
the gas and you will have a current as
the as you apply the suitable voltage so
when there is no analyte in the carrier
gas there will be a current and when
hello cell containing or electron
capturing analytes are there there will
be drop in current that would be
measured so electron capture detector
also has are very useful for analytes
which can capture electron and there are
other detectors also these were what we
have discussed were the most common
types of detectors but there are other
detectors also likes a flame photometric
detector which exploits the fact that
phosphorus and sulfur containing
analytes emit light when they are burned
in a fi D type detector and this light
is detected and quantified and a
detection limit can go up to 1 picogram
for phosphorous containing compound and
twenty peak around for sulfur containing
component then there could be like photo
ionization detectors which uses
ultraviolet radiation from lamps which
energy is ranging from nine point five
to level electron volt to produce
ionization of solute molecules the ions
are collected at a positively charged
electrode and the current is measured
compounds with ionization potential or
lower than the lamp ionizing energy will
give a response there could be another
like electrolytic conductivity detector
which operates
on electrolytic conductivity principle
so organic compounds eluting from the
column that is gas chromatography column
will be burnt in a say miniature furnace
to form simple molecular species and
that readily ionizes and contributes to
the conductivity of deionized water and
this change in electrolytic conductivity
will be monitored so so we have
discussed these are different types of
detectors which could be utilized and
they could be have applications for
different kinds of works or different
kinds of scientific techniques like say
it could be in environmental science or
medical or clinical research where gas
chromatography could be utilized or in
basic research like I said the thermal
conductive detector is very simple very
useful detector and the most widely used
is flame ionization detector there could
be many applications of gas liquid
chromatography there could be
qualitative or quantitative like for
qualitative analysis generally
chromatographic data is presented as a
graph of detector response against the
retention time like you can make a
standard curve or you can plot them it's
called a criminal guess chromatogram so
this provides a spectrum of Peaks for a
sample representing the analytes present
in a sample in looting from the column
at different times retention time can be
used to identify analytes if the method
contains conditions are constant by
comparing the retention time of the
sample as well as the standard and I
have shown you just when we have shown I
have shown you the chromatic guest
chromatograph and the schematic of the
gas chromatograph now this pattern of
the peaks obtained will be constant for
a sample under constant or standard
conditions and then it could be
identified from a very complex mixture
of analytes then you can also test the
purity of compound here by comparing the
standard
sample if additional Peaks are obtained
then impurities are present and
otherwise the compound is pure so if
there is any additional peak compound
it's not pure so the purity of a
purified compound could be tested in gas
chromatography in most modern
applications the gas chromatography is
connected to a mass spectrometer or a
similar kinds of kind of detector that
is capable of identifying the analytes
represented by the Peaks so the samples
could be passed to mass spectrometer and
where structural information through
molecular mass information you can
identify the samples and many more
things could be done on a mass
spectrometer where a lot of information
about the analyte could be obtained in
quantitative analysis
like for example area under a peak like
we've done for like you can do it for
other chromatographic techniques where
quantitative analysis could be done by
measuring the area under the peak so
here also area under a peak is
proportional to the amount of the
analyte present in the chromatogram and
by calculating the area of the peak
using the mathematical function of
integration the concentration of analyte
in the original sample can be calculated
concentration can be calculated using a
calibrated
calibration curve created by finding the
response for a series of concentrations
of analytes or by determining the
relative response factor of an analyte
so this is like you can do quantitative
analyst analysis relative response
factor is the expected ratio of an
analyte to an internal standard or
external standard and it's calculated by
finding the response of a known amount
of analyte and a constant amount of
internal standard added to the sample at
a constant concentration with the
distance and to have a distinct
retention time like you have a distinct
assent retention time or to Dynalite so
these are few methods for quantitative
analysis they're quite similar as for
other chromatographic techniques you can
use internal or
to standards and by various methods you
can do that
gas chromatography is utilized for a lot
of different applications like elemental
analysis and different fields of
biotechnology like you can determine say
elements like carbon hydrogen oxygen
sulfur and nitrogen you can use it for
analyzing mixture of drugs isolation and
identification of drugs could be done
you can have isolation and
identification of mixtures of components
for example you plant extracts volatile
oils amino acids and what others could
be could be tested and identified and
even isolated so there are a whole lot
of applications for gas chromatography
with lot of applications there are
limitations as we were talking here
samples which are analyzed are limited
because not all the compounds can be
volatile and thermally stable but many
times it can be made by a volatile by a
derivatized asian where a lot of
chemistry is in word sample must be
thermally stable to prevent degradation
when heated because if it is degraded
then it's no use to purify it or analyze
it the gas chromatography cannot be used
to prepare samples for further analysis
once separated because you don't get
whole lot of amount so there are certain
limitations also so gas chromatography
nowadays has been replaced by
high-performance liquid chromatography
for many applications but still gas
chromatography is used by many different
streams or biotechnology and like for
example clinical analysis environmental
science a lot of like testing for say
drugs the pharmaceutical industry in
like food industry a lot of analysis
cistern particularly of say certain
analytes or certain elements is done in
here with this lecture the week
to an end of chromatographic section so
in chromatography we have discussed lot
of different techniques we have
discussed basic concepts we have
discussed about liquid chromatography
where low pressure and high-performance
liquid chromatography
we have discussed and we have discussed
these four techniques so this completes
our chromatographic section in the next
lecture will start a new topic and that
is electrophoresis which is another very
important analytical method which is
routinely used in various laboratories
for both research as well as for
application purposes thank you