This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1. Presenter Dr Anurag Yadav Morderator Dr Avinash S S Column
Chromatography 1 Dr Anurag Yadav
2. Capsule: Chromatography is a technique for separating
mixtures into their components in order to analyze, identify,
purify, and/or quantify the mixture or components. Supporting
medium planar column MECHANISM ION EXCHANGE PARTITION ADSORPTION
AFFINITY SIZE EXCLUSION 2 Dr Anurag Yadav
3. Contents Gas Chromatography: Definition Types of Gas
chromatography Basic principle Instrumentation Practical
consideration Applications 3 Dr Anurag Yadav
4. The father of modern gas chromatography is Nobel Prize
winner John Porter Martin, who also developed the first liquid- gas
chromatograph. (1950) First separated compound was fatty acid.
History 4 Dr Anurag Yadav
5. 5 Dr Anurag Yadav
6. Column chromatography GC LC 6 Dr Anurag Yadav
7. Gas chromatography Gas chromatography is a type of column
chromatographic technique that can be used to separate volatile
organic compounds. Mobile phase: inert gas: nitrogen, helium,
hydrogen carrier gas. Stationary phase: liquid/solid. 7 Dr Anurag
Yadav
8. Types of Gas chromatography GSC : Packed columns filled with
solid sorbent (stationary phase) support particles. GLC : Support
particles coated with thin liquid layer 8 Dr Anurag Yadav
10. 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 the10 Dr Anurag Yadav
11. 11 Dr Anurag Yadav
12. 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. 12
Dr Anurag Yadav
13. Sample to be separated is converted into vapour And mixed
with gaseous M.P Component 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: 13
Dr Anurag Yadav
14. Chromatographic Separation 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. 14 Dr Anurag
Yadav
15. Separation 15 Dr Anurag Yadav
16. Chromatographic Separation (continued) The peaks can then
be read and analyzed to determine the exact components of the
mixture. Retention time is determined by each component reaching
the detector at a characteristic time. 16 Dr Anurag Yadav
17. 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. 17 Dr Anurag Yadav
18. Peaks and Data 18 Dr Anurag Yadav
19. Instrumentation Carrier gas (mobile phase) supply: N2, He,
H2 Flow control Injector Column Detector Computer/recorder 19 Dr
Anurag Yadav
20. Carrier gas supply Function: to provide carrier gas to
chromatographic column Carrier gas carries sample to column. Tank,
needle valve, flow meter, pressure gauge Type of carrier gases
depends on column & detector Capillary columns: H2, He. Packed
columns: N2 TCD, ECD: N2 FID: He 20 Dr Anurag Yadav
21. Carrier gas supply Ideal carrier gases: pure & dry
Impure & moisture: harm the column, performance of detectors,
adversely affect quantification of trace analysis. Measures: Tubing
(gas sourceGC)uncontaminated. Molecular sieve beds moisture,
hydrocarbon, oxygen content. 21 Dr Anurag Yadav
22. 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 22 Dr Anurag
Yadav
23. Flow control Regulates the carrier gas flow in GC Constant
flow of carrier gas column efficiency & reproducible elution
time. Magnitude of carrier gas flow rate depends type of column
Packed column 10-60ml/min Capillary column 1-2ml/min 23 Dr Anurag
Yadav
24. Injection port The injection port consists of a septum
through which a syringe needle is inserted to inject the sample.
The sample is injected into a stream of inert gas usually at an
elevated temperature by a microsyringe. The vapourized sample is
carried into a column packed with the stationary phase. To ensure
rapid & complete solute volatilization temp of injector 30-50
degree celsius>column temp 24 Dr Anurag Yadav
25. Injection techniques Split Splitless 25 Dr Anurag
Yadav
26. 26 Dr Anurag Yadav
27. Split Injection Advantages & Disadvantages Advantages
1.Simple to Use 2.Rugged Design 3.Narrow analyte band on column
4.Protects column from involatile sample components 5.Easy to
Automate Disadvantages 1.Not suitable for ultra- trace analysis
2.Suffers from Discrimination 3.Liner geometry dictates injector
settings 4.Analytes susceptible to thermal degradation 27 Dr Anurag
Yadav
28. Splitless Injection Advantages & Disadvantages
Advantages 1.Simple to Use 2.Rugged Design 3.Excellent for trace
analysis 4.Less Risk of Analyte Discrimination than Split Mode
5.Easy to Automate Disadvantages 1.Need to carefully optimise
conditions 2.Risk of backflash 3.Analytes susceptible to thermal
degradation 28 Dr Anurag Yadav
29. 29 Dr Anurag Yadav
30. 30 Dr Anurag Yadav
31. Common problems of injection port Backflash Septum leak
Adsorption of components from sample onto septum Septum
heatedDecomposition products leak into column ghost peak
(chromatogram) Measures Back flash: Septum purge, small injector
volume, larger vol injector liners. Teflon coated low leak septum
is used Inner surface is purged continuously with carrier gas
Septum should be replaced 100 injection31 Dr Anurag Yadav
32. Columns & its types Packed column Capillary (open
tubular) column 1 - 4mm ID; 1 - 5 m length Glass/stainless steel
coil Packed solid particles either porous/non-porous coated with
thin (1 m) film of liquid 0.1 - 0.5 mm I.D. (ID); 10 - 150 m length
Thin fused-silica. Inner wall coated with thin (0.1-5 m) film of
liquid (stationary phase) 32 Dr Anurag Yadav
33. Capillary (open tubular) column 3 layers 1. Polyamide
coating strong water proof barrier 2. Thin fused-silica- minimize
chemical reactivity, uniform surface for stationary phase 3.
Stationary phase 33 Dr Anurag Yadav
34. Stationary phase Polymer inner surface of fused silica
layer Thickness, uniformity, chemical nature influences the
separation of components in sample. Mc stationary phase silicon
polymer used polysiloxane. 34 Dr Anurag Yadav
35. GC Columns : Composition of stationary phase 100% dimethyl
polysiloxane: non polar; for drugs and amino acid derivatives
Polyethylene glycol : Polar ; for acids, ketones and alcohols.
Disadvantages: high susceptibilty of structural damage by oxygen at
high temperatures. GSC: PLOT: Polystyrene, aluminium oxide,
molecular sieve Separation: partition/adsorption 35 Dr Anurag
Yadav
36. Selection criteria for capillary column Stationary phase
close to polarity of solutes. Column diameter : small
diamter(0.25mm) when sample overloading is not a problem. Film
thickness: thin high boiling point solutes (TG, steriods) thick low
boiling point solutes Column length: 30mts most application 15mts
simple samples (