Gas Chromatography

SND COLLEGE OF PHARMACY,BABHULGAON
FINAL YEAR B.PHARMACY.

What is Gas Chromatography?
• It is also known as…
– Gas-Liquid Chromatography (GLC)

Sample to be separated is
converted into vapour And
mixed with gaseous M.PComponent more soluble in the S.P → travels slower
Component less soluble in the S.P → travels faster
Components are separated according to their
Partition Co-efficient
Criteria for compounds to be analyzed by G.C
1.VOLATILITY:
2.THERMOSTABILITY:

What is Gas Chromatography?
• The father of
modern gas
chromatography is
Nobel Prize winner
John Porter Martin,
who also developed
the first liquid-gas
chromatograph.
(1950)

How a Gas Chromatography Machine
Works
– First, a vaporized sample is injected onto the
chromatographic column.
– Second, the sample moves through the
column through the flow of inert gas.
– Third, the components are recorded as a
sequence of peaks as they leave the column.

Chromatographic Separation
– Deals with both the stationary phase and
the mobile phase.
• Mobile – inert gas used as carrier.
• Stationary – liquid coated on a solid or a solid
within a column.

• Chromatographic Separation
– In the mobile phase, components of the sample are
uniquely drawn to the stationary phase and thus,
enter this phase at different times.
– The parts of the sample are separated within the
column.
– Compounds used at the stationary phase reach the
detector at unique times and produce a series of
peaks along a time sequence.

(continued)
– The peaks can then be read and analyzed by a
forensic scientist to determine the exact
components of the mixture.
– Retention time is determined by each component
reaching the detector at a characteristic time.

Chromatographic Analysis
–The number of components in a
sample is determined by the
number of peaks.
–The amount of a given component
in a sample is determined by the
area under the peaks.
–The identity of components can be
determined by the given retention
times.

PRACTICAL REQUIREMENTS
• Carrier gas
• Flow regulators & Flow meters
• Injection devices
• Columns
• Temperature control devices
• Detectors
• Recorders & Integrators

CARRIER GAS
» Hydrogen
better thermal conductivity
disadvantage: it reacts with unsaturated
compounds & inflammable
» Helium
excellent thermal conductivity
it is expensive
» Nitrogen
reduced sensitivity
it is inexpensive

Requirements of a carrier gas
 Inertness
 Suitable for the detector
 High purity
 Easily available
 Cheap
 Should not cause the risk of fire
 Should give best column performance

Flow regulators & Flow meters
 deliver the gas with uniform pressure/flow
rate
 flow meters:- Rota meter & Soap bubble
flow meter
Rota meter
placed before column inlet
it has a glass tube with a float held on to a
spring.
the level of the float is determined by the
flow rate of carrier gas

Soap Bubble Meter
◊ Similar to Rota meter & instead of a float,
soap bubble formed indicates the flow rate

Injection Devices
 Gases can be introduced into the column by
valve devices
 liquids can be injected through loop or
septum devices

COLUMNS
• Important part of GC
• Made up of glass or stainless steel
• Glass column- inert , highly fragile
COLUMNS can be classified
 Depending on its use
1. Analytical column
1-1.5 meters length & 3-6 mm d.m
2. Preparative column
3-6 meters length, 6-9mm d.m

Depending on its nature
1.Packed column: columns are available in
a packed manner
S.P for GLC: polyethylene glycol, esters,
amides, hydrocarbons, polysiloxanes…
2.Open tubular or Capillary column or
Golay column
 Long capillary tubing 30-90 M in length
 Uniform & narrow d.m of 0.025 - 0.075 cm
 Made up of stainless steel & form of a coil
 Disadvantage: more sample cannot loaded

3.SCOT columns (Support coated open
tubular column
 Improved version of Golay / Capillary
columns, have small sample capacity
 Made by depositing a micron size
porous layer of supporting material on
the inner wall of the capillary column
 Then coated with a thin film of liquid
phase

Columns
• Packed
• Capillary

Equilibration of the column
 Before introduction of the sample
 Column is attached to instrument &
desired flow rate by flow regulators
 Set desired temp.
Conditioning is achieved by passing
carrier gas for 24 hours

Temperature Control Devices
Preheaters: convert sample into its vapour
form, present along with injecting devices
Thermostatically controlled oven:
temperature maintenance in a column is
highly essential for efficient separation.
Two types of operations
Isothermal programming:-
Linear programming:- this method is
efficient for separation of complex mixtures

Temperature Control
• Isothermal • Gradient
240
200
160
120
80
40
0
0 10 20 50 6030 40
Time (min)
Temp(degC)
Instrumentation - Oven

DETECTORS
Heart of the apparatus
The requirements of an ideal detector are-
 Applicability to wide range of samples
 Rapidity
 High sensitivity
 Linearity
 Response should be unaffected by
temperature, flow rate…
 Non destructive
 Simple & inexpensive

Measures the changes of thermal conductivity due
to the sample (g). Sample can be recovered.
1.Thermal Conductivity Detector
(Katharometer, Hot Wire
Detector)

Thermal Conductivity Basics
When the carrier gas is contaminated
by sample , the cooling effect of
the gas changes. The difference in
cooling is used to generate the
detector signal.
The TCD is a nondestructive,
concentration sensing detector. A
heated filament is cooled by the flow
of carrier gas.
Flow
Flow

When a separated compound elutes from the
column , the thermal conductivity of the
mixture of carrier gas and compound gas is
lowered. The filament in the sample column
becomes hotter than the control column.
The imbalance between control and sample
filament temperature is measured by a simple
gadget and a signal is recorded
Thermal Conductivity Detector

□ Measures heat loss from a hot
filament–
e filament heated to const T
• when only carrier gas flows heat loss to
metal block is constant, filament T remains
constant.
• when an analyte species flows past the
filament generally thermal conductivity goes
down, T of filament will rise. (resistance of
the filament will rise).

Relative Thermal Conductivity
Compound
Relative Thermal
Conductivity
Carbon Tetrachloride 0.05
Benzene 0.11
Hexane 0.12
Argon 0.12
Methanol 0.13
Nitrogen 0.17
Helium 1.00
Hydrogen 1.28

Advantages of Katharometer
Linearity is good
Applicable to most compounds
Non destructive
Simple & inexpensive
Disadvantages
 Low sensitivity
 Affected by fluctuations in temperature and
flow rate
 Biological samples cannot be analyzed

Flame Ionization Detector
 Destructive detector
 The effluent from the column is mixed with H
& air, and ignited.
 Organic compounds burning in the flame
produce ions and electrons, which can
conduct electricity through the flame.
 A large electrical potential is applied at the
burner tip
 The ions collected on collector or electrode
and were recorded on recorder due to
electric current.

FIDs are mass sensitive rather than
conc. sensitive
ADVANTAGES:
• µg quantities of the solute can be
detected
• Stable
• Responds to most of the organic
compounds
• Linearity is excellent
• DA: destroy the sample

Argon ionization detector
 Depends on the excitation of argon atoms to a
metastable state, by using radioactive energy.
Argon→ irradiation Argon + e- →collision Metastable
Argon→ collision of sub. → Ionization →↑Current
ADVANTAGES
1.Responds to organic compounds
2.High sensitivity
DISADVANTAGES
1.Response is not absolute
2.Linearity is poor
3. Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR
The detector consists of a cavity that
contains two electrodes and a radiation
source that emits  - radiation (e.g.63Ni,
3H)
The collision between electrons and the
carrier gas (methane plus an inert gas)
produces a plasma containing electrons
and positive ions.

• If a compound is present that contains
electronegative atoms, those electrons are
captured and negative ions are formed, and
rate of electron collection decreases
• The detector selective for compounds with
atoms of high electron affinity.
• This detector is frequently used in the
analysis of chlorinated compounds
• e.g. – pesticides, polychlorinated biphenyls

ADVANTAGE
Highly sensitive
DISADVANTAGE
 Used only for compounds with electron
affinity

RECORDERS & INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORS
Record the individual peaks with Rt, height….

Derivatisation of sample
Treat sample to improve the process of
separation by column or detection by
detector.
They are 2 types
Precolumn derivatisation
Components are converted to volatile &
thermo stable derivative.
Conditions - Pre column derivatisation
Component ↓ volatile
Compounds are thermo labile
↓ tailing & improve separation

Post column derivatisation
 Improve response shown by detector
 Components ionization / affinity towards
electrons is increased
Pretreatment of solid support
To overcome tailing
Generally doing separation of non polar
components like esters, ethers…
Techniques: 1. use more polar liquid S.P
2. Increasing amt. of liquid phase
3.Pretreatment of solid support to remove
active sites.

Parameters used in GC
Retention time (Rt)
It is the difference in time b/w the point of
injection & appearance of peak maxima. Rt
measured in minutes or seconds
(or) It is the time required for 50% of a
component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is
required to elute 50% of the component from
the column.
Retention volume = Retention time ˣ Flow rate

Separation
factor (S)Ratio of partition co-efficient of the two
components to be separated.
If more difference in partition co-efficient b/w two
compounds, the peaks are far apart & S
Is more. If partition co-efficient of two compounds
are similar, then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on
a chromatogram is measured by resolution
It is the measure of both column & solvent
efficiencies
R= 2d
W1+W2

THEORETICAL PLATE
 An imaginary unit of the column where
equilibrium has been established between
S.P & M.P
 It can also be called as a functional unit of
the column
HETP – Height Equivalent to a Theoretical
Plate
 Efficiency of a column is expressed by the
number of theoretical plates in the column or
HETP
 If HETP is less, the column is ↑ efficient.
 If HETP is more, the column is ↓ efficient

HETP= L
N
A + B +Cu
u
(length of the column)
(no of theoretical plates)
HETP is given by Van Deemter equation
HETP=
A = Eddy diffusion term or multiple path diffusion
which arises due to packing of the column
B = Molecular diffusion, depends on flow rate
C = Effect of mass transfer,depends on flow rate
u = Flow rate

Efficiency ( No. of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no. of theoretical plates
Rt = retention time
W = peak width at base
The no. of theoretical plates is high, the
column is highly efficient
For G.C the value of 600/ meter

Asymmetry Factor
 Chromatographic peak should be
symmetrical about its centre
 If peak is not symmetrical- shows Fronting or
Tailing
 FRONTING
Due to saturation of S.P & can be avoided by
using less quantity of sample
 TAILING
Due to more active adsorption sites & can be
eliminated by support pretreatment,

Asymmetry factor (0.95-1.05)
can be calculated by using the
formula AF=b/ab & a calculated at 5% or 10% of the peak
height

ADVANTAGES OF G.C
Very high resolution power, complex
mixtures can be resolved into its
components by this method.
Very high sensitivity with TCD, detect down
to 100 ppm
It is a micro method, small sample size is
required
Fast analysis is possible, gas as moving
phase- rapid equilibrium
Relatively good precision & accuracy
Qualitative & quantitative analysis is possible

Applications of G.C
• G.C is capable of separating, detecting &
partially characterizing the organic
compounds , particularly when present in
small quantities.
1, Qualitative analysis
Rt & RV are used for the identification &
separation
2, Checking the purity of a compound
Compare the chromatogram of the std. & that
of the sample

3, Quantitative analysis
It is necessary to measure the peak area or
peak height of each component
4, used for analysis of drugs & their
metabolites.

Semi-Quantitative
Analysis of Fatty Acids
DetectorResponse
C14
C16
C18
PeakArea
Sample Concentration (mg/ml)
2
4
6
8
10
1.50.5 1.0 2.0 2.5 3.0
C
C + C + C

= t h e c o n t e n t % o f C14 f atty a c i d s
Retention Time
T h e c o n t e n t % o f C1 4 fatty a c i d s =

Tentative Identification of Unknown
Compounds
Response
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Octane Decane1.6 min = RT
Hexane
Response
Unknown compound may be Hexane
1.6 min = RT
Retention Time on Carbowax-20 (min)

Response
GC Retention Time on SE-30
RT= 4 min on SE-30
Unknown compound
Response
GC Retention Time on SE-30
RT= 4.0 min on SE-30
Hexane
Retention Times

Advantages of Gas Chromatography
• Very good separation
• Time (analysis is short)
• Small sample is needed - l
• Good detection system
• Quantitatively analyzed

How a Gas Chromatography
Machine Works
– First, a vaporized sample is injected onto the
chromatographic column.
– Second, the sample moves through the
column through the flow of inert gas.
– Third, the components are recorded as a
sequence of peaks as they leave the column.

contents
 Derivatisation techniques:
 Applications of gas
chromatography
GC DERIVATIZATION

• What is GCDerivatization?
• Derivatization is the process of chemically modifying
a compound to produce a new compound which has
properties that are suitable for analysis using aGC.
WHAT IS DERIVATIZATION?

Topermit analysis of compounds not directly amenable to
analysis due to, for example, inadequate volatility orstability.
Improve chromatographic behavior or detectability.
Many compounds do not produce a usable chromatograph
(i.e.multiple peaks, or one big blob), or the sample of interest
goes undetected. As a result it may be necessary to derivatize
the compound before GCanalysis isdone.
Derivatization is a useful tool allowing the use of GC and
GC/MS to be done on samples that would otherwise not be
possible in various areas of chemistry such as medical,
forensic, and environmental.
WHY DERIVATIZATION

Increasesvolatility (i.e. sugars):
 Eliminates the presence of polar OH,NH,& SHgroups
Derivatization targets O,S,Nand Pfunctional groups
(with
hydrogens available.
 Increases detectability, I.e. steroids/ cholesterol
Increases stability.
Enhancessensitivity for ECD(Electron Capture
Detection). Theintroduction of ECDdetectable groups,such
ashalogenated acyl groups, allows detection of previously
undetectable compounds.
WHAT DOES DERIVATIZATION
ACCOMPLISHED

TypesofDerivatization
 pre-column derivatization
 post-column derivatization
 Precolumn derivatisation:
Components are converted to volatile &thermo stable
derivative
Conditions - Precolumn derivatisation
Component ↓ volatile
Compounds are thermo labile
↓ tailing & improve separation

Post columnderivatisation
 Improve response shown by detector
 Components ionization / affinity towards electrons is
increased
Pretreatment of solidsupport
Toovercometailing
Generally doing separation of non polarcomponents
like esters, ethers…

TECHNIQUES OF DERIVATISATION
 SILYLATION
 ACYLATION
 PERFLOURO-
ACYLATION
 ALKYLATION
 ESTERIFICATION
 CONDENSATION
 CYCLISATION

Acylation
• Acylation reduces the polarity of amino, hydroxyl, andthiol
groups and adds halogenated functionalities for ECD.In
comparison to silylating reagents, the acylating reagents
target highly polar, multifunctional compounds, such as
carbohydrates and amino acids.
• Acyl derivatives are formed with acyl anhydrides,acyl
halides, and activated acyl amide reagents.
• Theanhydrides and acyl halides form acid by-products
which must be removed before GCanalysis.

CONT…..
• Activated amide reagents, such asMBTFA,have the
advantage of not yielding acidby-products.
• Fluorinated acyl groups, going from trifluoracetyl to
heptafluorobutyryl , canbe used to increaseretention
times.

AcylatingReagents
• 1.Fluorinated Anhydrides:-
• TFAA-Trifluoroacetoic Anhydride
• PFPA-PentafluoropropionicAnhydride
· Most commonly used reagents, asderivatives are suitable
for both FIDandECD.
• · Reactswith alcohols, amines, and phenols to
produce stable and highly volatile derivatives
• · Theacid by-product should be removed, via astream of
nitrogen, before injection onto column. Bases,such as
triethylamine, canbe added asan acid receptor and
promote reactivity
• ·Ability to adjust retention times for ECD

• 2. Fluoracylimidazoles
• TFAI-Trifluoroacetylimidazole
• PFPI-Pentafluoropropanylimidazole
• HFBI- Heptafluorobutyrylimidazole
• · Usually abetter choice for making ECDderivatives
• · Reactunder mild conditions and their by-products,
the imidazole, is not acidic so it will not harm column.
• · Reagentsare extremely sentive towater- will react
violently to it.
• · Works best with amines and hydroxycompounds

Cont..
• 3.MBTFA{N-methyl-bis(trifluoroacetamide)}
• · Reactswith primary and secondary amines, slowly
with hydroxyl groups and thiols.
• · Conditions are mild and the by-products are
relatively inert and are nonacidic
• 4.PFBCI-PentafluorobenzoylChloride
• · Phenols most receptivesite
• · Usedfor making derivatives of alcohols andsecondary
• amines. Secondaryamines will react with this
compound

Ex:
1. Esterification with n-propanol, acidic catalyst and
benzene for remove water azeotropically, the
ester were acylated with acetic anhydride and
finally derivatives extracted and diluted for GC.

Esterificationwith n-propanol, acid catalystand benzene removes
water azeotropically.
Later,Esterwas acetylatedwith aceticanhydride toyieldthe
acetylatedderivative.

Advantages and
Disadvantages of Acylation
• Advantages: Addition of halogenated carbonsincreased
detectability byECD.
• Derivatives are hydrolytically stable.
• Increased sensitivity by adding molecular weight
• Acylation canbe used asafirst step to activate carboxylic
acids prior to esterfication(alkylation).

Disadvantages
• Acylation derivatives canbedifficult toprepare.
• Reactionproducts (acid by-products) often needto
be removed beforeanalysis.
• Acylation reagentsaremoisture sensitive.
• Reagentsarehazardousand odorous.

Perflouro-Acylation
•Thisgroup increases the mol.wt of the sample relative tothe
analogous hydrocarbon.
•Best method to increase the retentiontime.
•Eg.
• N-Triflouro acetic anhydride
•Direct acylation with Triflouro acetic anhydride in triflouro
acetic acid followed by methylation with diazomethane in
methanol.

Alkylation
• Alkylation reduces molecular polaritybyreplacing active
hydrogens with an alkyl group. These reagents are used to
modify compounds with acidic hydrogens, such as
carboxylic acids and phenols. These reagents make esters,
ethers, alkyl amines and alkylamides.
• Reagents containing fluorinated benzoyl groups canbe used
for ECD.
• Theprincipal reaction employed for preparation ofthese
derivatives is nucleophilic displacement.
• Alkylation is used to modify compounds with acidic
hydrogens, suchascarboxylic acids and phenols.

•Alkylation canbe used alone to form esters, ethers and
amides- or they canbeused in conjunction with acylation
or silylation.
•It is generally used to convert organic acids into esters.As
the acidity of the active hydrogen decreases, thestrength of
the alkylatingreagent
must be increased. Theharsher the reaction conditions or
reagents, the more limited the selectivity and applicabilityof
this method.

1.DMF (dialkylacetals)
·These reagents work quickly, derivatizing upon dissolution.
Suitable for flash alkylation, where derivatization takes place
in the injectionport.
·Thedifferent alkyl homolgues allow formation of avarietyof
esters. polarity and volatility of the samples canbe adjusted,
thereby changing retention time.
·They will react with water to give the corresponding alcohol.
Tracesof water will not affect the reaction aslong asyou
have an excessof acid.
ALKYLATING AGENT

·Froms butyl ester, which will allow longer retentiontimes
·Usedmost commonly for low molecular weightacids
3.BF3in methanol orbutanol
·Convenient and inexpensive method for forming esters
4.PFBBr (Pentafluorobenzylbromide)
Esterifies phenols, thiols, and carboxylic acids
2.TBH (tetrabutylammonium hydroxide)

anticonvulsants and barbiturates.
Themost common derivative is
methyl imide, which canbe formed
on column by using trimethyl
ammonium hydroxide.[TMAH]

Cont…
•Alkyl esters have excellentstability and canbe
isolated and stored for long periods oftime.
•A two step approach is commonly used in
derivatization of aminoacids, where multiplefunctional
groups on these compoundsmay
necessitate protection duringderivatization.

Advantages
Wide range of alkylation reagents available
Reaction conditions canvary from strongly acidicand
strongly basic.
Somereactions can be done in aqueoussolutions.
Alkylation derivatives are generally stable.
Disadvantages
Limited to amines and acidichydroxyls.
Reaction conditions are frequently severe.
Reagentsare often toxic.

esterification
Esterification:
Esterification is used to prepare derivatives of carboxyl group.
The conversion of the carboxyl group to ester increases volatality by
decreasing hydrogen bonding.
Ex:- Analgesics, prostaglandins, aminoacids, & anti-inflammatory
agents.
Derivatization by esterification can be carried out by using Fischer
esterification procedure in which strongly acidic conditions are present.
H+R` - COOH + R -OH R`- COOR + H2O
BF3

Amino acids : E.x. Alanine, α-amino butyric acid, valine,leucine,
isoleucine.
1. α-chloromethyl esters:
prepared by treating the amino acid with a mixture of concentrated
nitric acid and Hydrochloric acid.
Aminoacid Chloro methyl ester
R – CH-NH2 – COOH Hcl/ HNO3 R – CH – COOCH3
Cl
2. Methyl ester salts:
Esterification of 1-leucine, 1-methionine with methanol & thionyl
chloride.

Silylation•Silylation produces silyl derivatives which are more volatile,
lessstable, and more thermally stable.
•Replacesactive hydrogens with aTMS(trimethylsilylgroup).
•Silylation occurs through nucleophilic attack (SN2).The
better the leaving group, the better the siliylation.
•Silylation reagents will react with water and alcohols first.
Caremust be taken to ensure that both sample andsolvents
are dry.
•Solvents should be aspure aspossible. Thiswill eliminate
excessivepeaks.Tryusing aslittle solvent aspossible asthis
will prevent alarge solventpeak.

Pyridine is the most commonly used solvent. Although
pyridine may produce peak tailing, it is an acid scavangerand
will drive the reactionforward.
In many cases,the need for asolvent is eliminated with
silylating reagents. (If asample readily dissolves in the
reagent, it usually asign that the derivatization iscomplete).
.

 Easeof reactivity of functional groups towardssilylation.
Many reagents require heating (not in excessof 60 degreesC
for about 10-15 minutes, to preventbreakdown).
Hindered products may require long term heating

Theeaseof reactivity of the
functional group
towardsilylation
follows the order:
Alcohol >Phenol >Carboxyl >Amine >Amide
General
Reaction
R-OH + (CH3)3 – Si - Cl
Mechanism
R – O – Si - (CH3)3 + HCl
TrimethylsilyletherTrimethylchlorosilane

Silylating Reagents
1.HMDS (Hexamethyldisilane).
·Weakdonor, asit hassymmetry
·If used will attack only easily silylated hydroxylgroups
·Sometimes found in combination with TMCS
2.TMCS (Trimethylchlorosilane).
·Weakdonor, again not commonly used
·Often found asacatalyst to increase TMSdonorpotential
·Badby-product, HCL
3.TMSI (Trimethylsilylimidazole).
·Not aweak donor, but it is selective (will not target N
compounds)
·Reactsreadily with hydroxyls but not withamines
·Since it is selective, it will target the hydroxyls in wet sugars.
It will derivatize the acid sites of amino acids, and will leave
the amino group free forfluorinated derivatization (ECD)

4.BSA (Bistrimethylsilylacetamide).
·First widely used silylating reagent
·Strong silylating reagent- acetamide is agood leaving group.
Reactsunder mild conditions and produces relatively stable
by-products
·Drawbacks: by-product, TMS-acetamide, will sometimes
produce peaksthat overlap those of other volatilederivatives.
BSAmixtures also oxidize to form silicon dioxide, which can
foul FIDdetectors
TMS-DEA(Trimethylsilyldiethylamine).
·Reagent is used for derivatizing amino acids and carboxylic
acids
·Targetshindered compounds

5.BSTFA
(Bistrimethylsilyltrifluoroacetamide)·Developed by Gerhke in 1968
·Reactssimiliarly to BSAbut the leaving group is
trifluoroacetamide, soit acts faster and more completelythan
BSA
·BSTFAis highly volatile, and produces by-products that are
more volatile than BSAby-products, thus there is little
interference
with early elutingpeaks
·It canact asits own solvent
·Combustion product silicon trifluoride, does not foul
detectors

Advantages and Disadvantages ofSilylation
Advantages
•Ability to silylate awide variety ofcompounds
• Largenumber of silylating reagents available.
• Easily prepared.
Disadvantages
•Silylation Reagentsare moisture sensitive
•Must useaprotic (no protons available) organic
solvents

Condens
ation:If ketone or aldehyde is present in a sample, it is frequently
derivatized to prevent hydrogen bonding due to enolization & helps in
resolution from an interfering substance.
The most commonly used reagent is methoxylamine to protect
enolizable ketogroups in steroids by formation of methoximes.
Cyclization:
Cyclization is performed on compounds containing two functional
groups in close proximity so that 5 or 6 membered Heterocyclic rings
can be formed.

• Heterocycles formed are ketals, boronates, triazines &
phosphites.
E.g: Cyclization of α – OH ketones (present in corticosteroids)
with formaldehyde forms bismethylene dioxy derivatives
which are thermally stable & permit resolution of
corticosterone from a mixture of steroids.

applications
Qualitativeanalysis:
Retention time data should be usefulfor
identification of mixtures.
Comparing the retention time of the sample as
well asthe standard.
Checking the purity of acompound: comparthe
standard and sample.
Additional peaksare obtained…..impurities are
present….compoundis not pure.

Elementalanalysis
Determination of C,H ,O ,S and N .
Determination of mixture of drugs
Isolation and identification of drugs
Isolation and identification of mixture of components(amino
acids ,plant extracts ,volatile oils)
GS-MSis one of the most powerful tool in siologicaland
chemical studies.
Other app…like
Analysis of dairy prod.., aldehydes, ketones etc.. Whichare
present in pharm..,Rancidity in fattyacids.
Assayof drugs, purity of compounds, determinationof
foreign or relatedcompounds.

Quantitation Introdution
• Sampling techniques for practical quantitative capillary GC have to meet
certain principal requirements. Both the absolute and the relative peak areas
(e.g. column loads) must be reproducible with high precision and at high
accuracy; discrimination of certain constituents according to their volatility
should not take place on sampling. On the basis of systematic studies, the
three most reliable sampling techniques used for GC analyses with the aim of
achieving precise and accurate quantitative data proved to be the following:
On‐column, injection, splitless PTV injection, and an optimized version of split
sampling called “cooled needle split” injection. The on‐column technique can
be optimized by using precolumns with wider internal diameters and without
stationary phase coatings to overcome the problems of large liquid sampling
volumes and for automation. The PTV technique should only be used in the
splitless mode because discrimination cannot be suppressed completely with
the split mode. All three of the techniques can be operated automatically,
either to avoid “human interference”, i.e. to improve precision or for
unattended operation to save man‐power.

What is Area Normalization method
• Normalization is a technique used for
quantitatively assessing a chromatogram to
provide a quantitative analysis of the mixture
being separated. ... The quantitative results
are obtained by expressing the area of a given
peak as a percentage of the sum of
the areas of all the peaks.
•

Internal Standard Method
• Description
– In this approach, an internal standard is added to the sample, and the
response from the analyte peak is compared to the internal standard. This
approach corrects for minor variations in the injection volume.
• Response Factor (RF)
– The response factor accounts for differences in the detector response
between the analyte and standard.
– Conc-sample = [( AreaIScalibrator) / ( AreaISsample)] x [Areasample /
Areacalibrator] * (Conccalibrator)
124

Sample Chromatogram and Integration Report
126
IS
X
AIS = 17.80
AX = 27.01
isx
isx
isx
cc
AA
R /

Calibration Curve with Internal Standard
127
Standards
• Each contains fixed mass of internal
standard, various masses of std analyte
• Calibration curve shows linear response.
Slope = response factor*
Unknown
• Add known amount of internal standard
• Inject and measure Ax/Ais
• Determine cx/cis for your unknown from
calibration curve. Since cis is known, cx for
your unknown is simply
cx = (cx/cis)cis
isx
isx
isx
cc
AA
R /
GC Calibration Curve for Cocaine with Internal Standard
Standard
Cocaine
mg/mL
Int. Std.
mg/mL cx/cis Ax Aix Ax/Ais
1 2.50 5.00 0.500 120 600 0.200
2 5.00 5.00 1.000 241 601 0.401
3 10.00 5.00 2.000 480 600 0.800
4 25.00 5.00 5.000 1198 600 1.997
Cocaine with Interal Standard
1.0 microliter injections
y = 0.3991x + 0.0013
0.000
0.500
1.000
1.500
2.000
2.500
0.000 1.000 2.000 3.000 4.000 5.000 6.000
cx/cis
Ax/Ais
*This expression for the response factor is not used directly in your calculations.
The following expression which accounts for the intercept is more rigorous (in
practice the intercept is very near zero). Calculations based on the calibration data
do take the intercept into account.
/
( intercept)x is
x is
x is
A A y
R
c c



Area Percent Method
• Area percent is the simplest quantitation method. This
method assumes that the detector responds identically
to all compounds. This assumption, however, is not
valid. This method provides a rough estimate of the
amounts of analytes present.
• Gas chromatography is a technique used to analyze
mixtures. The instrument allows mixtures to be
separated and the amount of each component to be
determined. ... Using the chromatogram, the identity
and the percent composition of each component in the
mixture can be determined.
•

Single Point Internal Standard
• The Single Point Internal Standard method requires at
least two analyses. The first analysis contains a known
amount of internal standard and the compounds of
interest. Calculate the response factor using
• Internal Response
• Factor = area IS x amount SC amount IS x areaSC IS =
Internal Standard SC = Specific Compound of Interest

Single Point External Standard
• Unlike the area percent method, the Single Point
External Standard method requires the analysis of
more than just the sample of interest. Analyze a
sample containing a known amount of analyte or
analytes and record the peak area. Then calculate a
response factor using
• response factor = peak area/ sample amount

Gas Chromatography
Quantitative Analysis
Chromatogram
The response must be linear
Concentration
Mass
The response factor of each
compound is different
for each compound
Parameters that can be used:
Peak Height
Peak Area

External standard
• For an external standard quantitation, known data
from a calibration standard and unknown data from
the sample are combined to generate a quantitative
report. It is called external standard because
the standard or known material is separate
orexternal to the unknown material.

areas
%g 
PeakAreag
100
g
100g
(areai  fi )
PeakArea  f
%g 
Gas Chromatography
Quantitative Analysis
Chromatogram
Area Normalization
The sum of the areas of all the peaks
corresponds to 100% of the solutes separated.
Only true if:
All the compounds are eluted
Same sensitivity
As the compounds usually do not have the
same sensitivity a correction factor should be
applied
Calibration curve
area
fg

mass

Chromatogram Internal Standard
An internal standard is a compound, not present in the sample, that is added in a
constant amount to samples and calibration standards.
The peak of compound must not overlap with the peaks of the analytes.
SI Method
y=0.9978x
R2
= 0.9991
y=0.497x
R2
=0.999
0
1
2
3
4
5
6
0 2 10 124 6 8
masscompound/mass SI
Areacompound/area
SI
compoundA compoundB Lineal (compound A) Lineal (compoundB)
Advantages: manual injection
Disadvantages:
To analyse great number of
analytes
To find a good IS

Chromatogram External Standard
Advantages: simpler than IS.
Disadvantages: Sample injection reproducibility
Preferable Automatic injection or sample valve
ES Method
y = 1.9841x
R2
= 0.9991
y = 0.9981x
R2
= 0.9993
2
1
0
3
4
5
6
0 1 2 4 5 63
mass compound
Areacompound
compound A compound B Lineal (compound A) Lineal (compound B)

Derivatization is the process of chemically modifying a compound
to produce a new compound which has properties that are
suitable for analysis using a GC
WHY?
To permit analysis of compounds not directly amenable to
analysis due to, for example, inadequate volatility or stability
Improve chromatographic behavior or detectability.
Derivatization is a useful tool allowing the use of GC and GC/MS
to be done on samples that would otherwise not be possible in
various areas of chemistry such as medical, forensic, and environmental

•Increases volatility (i.e. sugars):
–Eliminates the presence of polar OH, NH, & SH groups
–Derivatization targets O,S, N and P functional groups (with hydrogens
available
Increases detectability, I.e. steroids/ cholesterol
•Increases stability
•Enhances sensitivity for ECD (Electron Capture Detection). The introduction of
ECD detectable groups, such as halogenated acyl groups, allows detection of
previously undetectable compounds
•in some cases: derivatization can also be used to decrease volatility to allow
analysis of very low molecular weight compounds, to minimize losses in
manipulation and to help separate sample peaks from solvent peak.

Gas Chromatography
DERIVATIZATION
Comments Advantages Disadvantages
Silylation Readily volitizes the sample
- Wide variety of compounds
-Large number of silylating reagents
available
-Easily prepared
-Moisture sensitive
-Organic solvents must be aprotic
(no protons available)
-WAX type columns cannot be
used
Acylation
-Used as the first step to further
derivatizations or as a method of
protection of certain active hydrogens.
-Reduces the polarity of amino,
hydroxyl, and thiol groups and adds
halogenated functionalities.
-Increased detectability by ECD
-Derivatives are hydrolytically stable
- Increased sensitivity by adding molecular
weight
-Acylation can be used as a first step to
activate carboxylic acids prior to
esterfication (alkylation)
-Difficult to prepare.
-Reaction products often need to
be removed before analysis
-Moisture sensitive
-Reagents are hazardous and
odorous
Alkylation
-Reduces molecular polarity by
replacing active hydrogens with an
alkyl group.
- modify compounds with acidic
hydrogens, such as carboxylic acids
and phenols.
-Reagents containing fluorinated
benzoyl groups can be used for ECD
-Wide range of alkylation reagents.
-Reaction conditions can vary from
strongly acidic to strongly basic
-Some reactions can be done in aqueous
solutions
-Alkylation derivatives are generally
stable
-Limited to amines and acidic
hydroxyls
-Reaction conditions are
frequently severe
- Reagents are often toxic
Formation of
perfluoro-
derivatives
Reagents containing fluorinated
benzoyl groups can be used for ECD
Wide range of application
Easy to prepare
Selectivity
GC-Quiral
Derivatiz.

Gas Chromatography
DERIVATIZATION
Derivatization Reaction Common Derivatizing Agent
Methylation of carboxylic acids Diazomethane,
methanol/sulfuric acid
Oxime formation of carbonyl functionality PFBHA
N-hexyl carbonate, carbamate, and ester formation
from hydroxylic, aminic, and carboxylic functionality
N-hexyl chloroformate
Heptafluorobutyramide formation from aromatic amines Heptafluorobutyramide
Some examples
And much more…

Gas Chromatography

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