Gas chromatography is a technique used to separate mixtures by exploiting differences in how compounds interact with a mobile phase (carrier gas) and a stationary phase. Samples are injected into a column containing a stationary phase and separated components emerge from the column over time, detected, and recorded as peaks. Common applications include analyzing volatile organic compounds, flavors, fragrances, and petrochemicals. Limitations include only being able to analyze volatile compounds.

1. 

2.  “color writing”
 Isatechniqueused to separateand identify the
componentsof amixture.
 Ettre& Zlatkis, 1967 introduced “ThePracticeof
GasChromatography.
 Gaschromatography isatechniqueused for
separation of volatilesubstanceswherethemobile
phaseisgasand stationary phasemay besolid/liquid.

4.  A samplecontaining thematerialsto beseparated isinjected
into thegaschromatograph. A mobilephase(carrier gas)
movesthrough acolumn that containsawall coated or
granular solid coated stationary phase. Asthecarrier gasflows
through thecolumn, thecomponentsof thesamplecomein
contact with thestationary phase. Thedifferent componentsof
thesamplehavedifferent affinitiesfor thestationary phase,
which resultsin differential migration of solutes, thusleading
to separation

5. Syringe
Injector
Detector
Carrier Gas
Cylinder
Column
To Waste or Flow
Meter
Flow Controller
Two-Stage
Regulator

6. Hydrogen
Air
Capillary tube (column)
Platinum jet
Collector
Sintered disk
Teflon insulating ring
Flame
Gas outlet
Coaxial cable to
Analog to Digital
converter
Ions
Why do we need
hydrogen?

7.  Respondsto compoundsthat produceionswhen burned in
an H2-air flame
◦ all organic compounds
 Littleor no responseto (useaThermal Conductivity
Detector for thesegases)
◦ CO, CO2, CS2, O2, H2O, NH3, inert gasses
 Linear from theminimum detectablelimit through
concentrations timestheminimum detectablelimit107

8.  Thermal Conductivity Detector
◦ Differencein thermal conductivity between the
carrier gasand samplegascausesavoltageoutput
◦ Ideal carrier gashasavery low thermal
conductivity (He)
 Electron Capture Detector
◦ Specific for halogenated organics

9.  Requiresonly very small sampleswith little
preparation
 Good at separating complex mixturesinto
components
 Resultsarerapidly obtained (1 to 100 minutes)
 Very high precision
 Only instrument with thesensitivity to detect volatile
organic mixturesof low concentrations
 Equipment isnot very complex (sophisticated oven)

10.  Only volatilesamplesor thesamplewhich can bemade
volatileareseparated by thismethod.
 During injection of thegaseoussample proper attention is
required.
 Thesampleof gaswhich isabout to inject must bethermally
stableso that it doesnot get degraded when heated.

11. gas Compound must exist asa____ at atemperaturethat
can beproduced by theGC and withstood by the
column (up to 450°C)
 Alcoholsin blood
 Aromatics(benzene, toluene, ethylbenzene, xylene)
 Flavorsand Fragrances
 Permanent gases(H2, N2, O2, Ar, CO2, CO, CH4)
 Hydrocarbons
 Pesticides, Herbicides, PCBs, and Dioxins
 Solvents

13.  Originally referred to asHigh-Pressure Liquid
Chromatography
 Now morecommonly called High
Performance Liquid Chromatography
 HPLC isaform of liquid chromatography used to
separatecompoundsthat aredissolved in solution.
 Mobilephase– liquid
 Stationary phase- solid

14. 1. Solvent Reservoir: To carry sampleinto thecolumn
2. Pumps: To producean appropriatepressureto push solvent into the
sample.
3.Sample Injection System: Syringe/injector
4.Columns:straight, 15 to 150 cm in length; 2 to 3 mm id and packing -
silicagel, alumina.
5.Detectors:UV/Vis,Refractiveindex,Fluorescence,Evaporativelight
scattering (ELSD),MS,DiodeArray Detector (DAD).
6.Data Processing: Receivetheinformation from HPLC machineand
present it asagraph using softwares.
 Thegraph describesabout qualitativedata(Retention time) and
quantitativedata(areaunder curve)
7.Waste

16. HPLC COLUMN

18.  Thesolvent ispumped from thesolvent reservoir
through thepump passing through thefiltersand the
sampleisinjected through thesampleport and gets
mixed with thesolvent and ispassed into theHPLC
column and thesamplerunson thesilicagel likepaper
chromatography and theeluted sampleat theend of the
column isdetected by thedetector and thesignal is
passed to acomputer wherethesignal isconverted to a
chromatogram and displayed.

19. Column Parameters
 Column Material
 Stationary Phase
 Coating Material
Instrument Parameters
 Temperature
 Flow
 Signal
 SampleSensitivity
 Detector
Sample Parameters
• Concentration
• Matrix
• Solvent Effect
• SampleEffect

21.  Speed in minutes
 High resolution
 High accuracy and reproducibility.
 Automated

22.  Need askill to run theinstruments
 High cost
 Complexity
 Low sensitivity for few compounds.

23. 1. Pharmaceuticalsindustry
 To control thedrug stability
 Quantity of drug determination from pharmaceutical dosage
forms, ex. Paracetamol determination in panadol tablet
 Quantity of drug determination from biological fluids, ex:
blood glucoselevel
2. Analysisof natural contamination
- Phenol & Mercury from seawater
3. Forensic test
- Determination of steroid in blood, urine& sweat.
- Detection of psychotropic drug in plasma

24. 4. Clinical test
- Monitoring of hepatic chirosispatient through
aquaporin 2 in theurine.
5. Food and essencemanufacture
- sweetener analysisin thefruit juice
- preservativeanalysisin sausage.

26.  1930s,first developed by A.WILHELM TISELUIS-a
Scottish biochemist won theNobel prizein 1948.
 Used to study enzymesand other proteins.
 Relieson theaffinity of variousbiochemical
compoundswith specific properties.

27.  Antigen antibody
 Antibody antigen
 Substrate enzyme
 DNA HISTON
 Hormone receptor

28. Specificity isbased on threeaspectsof affinity :
1. Matrix-for ligand attachment
2. Spacer arm-used to bind ligand to matrix
3. Ligand-moleculethat bindsreversibly to aspecific
target molecule

29.  Thesampleisinjected into theequilibrated affinity
chromatography column.
 Only thesubstancewith affinity for theligand are
retained on thecolumn.
 Thesubstancewith no affinity to theligand will elute
off.
 Thesubstanceretained in thecolumn can beeluted off
by changing thepH of salt or organic solvent
concentration of theeluent.

32.  Used in Genetic Engineering for nucleic acid
purification
 Vaccineproduction-antibody purification from blood
 Basic metabolism research-protein or enzyme
purification from cell freeextracts.

33. 1. Extremely high specificity
2. High degreesof purity can beobtained
3. Theprocessisreproducible

34.  Expensiveligands
 Leakageof ligand
 Degradation of thesolid support
 Limited lifetme
 Non specific adsorption
 Relatively low productivity