GAS CHROMATOGRAPHY

1. GAS CHROMATOGRAPHY LECTURE 8

2. PRINCIPLES In Gas Chromatography, the components of a vaporized sample are separated as a result of being partitioned between a mobile gaseous phase and a liquid or a solid stationary phase held in the column.

3. PRINCIPLES • Asample is being injected at the inlet/injector and vaporizedinto the chromatographic column. • The sample is transported through the column by the flow of inert gaseous mobile phase. • As the sample passes through the column, they are separated and detected electronically by detector.

4. Gas Chromatography • Gas is called “carrier gas”. • Typical carrier gas: helium or nitrogen. • Pressure from a compressed gas cylinder containing the carrier gas is sufficient to create the flow through the column.

5. Gas Chromatography There are two types. • Gas-liquid chromatography (GLC) mobile phase – gas stationary phase - liquid • Gas-solid chromatography (GSC) mobile phase – gas stationary phase - solid Shortened to Gas Chromatography

6. INSTRUMENTATION Carrier gas Flow regulator Injector Column Detector Integrator Display system -printer/monitor Thermostated oven Integrator

7. INSTRUMENTATION

8. INSTRUMENTATION Injection port and detector must be kept warmer than the column, • To promote rapid vaporization of the injected sample. • To prevent sample condensation in the detector.

9. Samples must be….. • Volatile • Thermally stable. • When injected onto the head of a chromatographic column and vaporized.

10. Mobile phase • Mobile phase transports the analytes (sample) through column. • Mobile phase can not interact with the molecules of the analyte. • Referred as carrier gas.

11. A. Carrier gas • Must be chemically inert. • Most common carrier gas is Helium(He) • Some specific detectors are using Nitrogen gas(N2), Hydrogen gas(H2), Carbon dioxide gas(CO2) and Argon. • The carrier gas should not contain traces of water or oxygen. Both are harmful to the stationary phase.

12. B. Flow regulator • The function of flow regulator is to control the flow rate of the carrier gas using the pressure regulators, gauges and flow meters. • The pressure at the head of the column is stabilized • mechanically OR • through the use of an electronic device.

13. C. Injectors • Functions • An inlet for the sample. • To vaporize and mix the sample with the carrier gas before the sample enters the head of the column. • Temperature is set about 50°C higher than boiling point of the least volatile component of the sample. • Modes of injection and characteristics of injectors vary depending on type of column used whether split/splitless.

14. Mode of Injections

15. D. Sample Injection System • Column efficiency requires sample to be…… • Of a suitable size • Introduced as a “plug” of vapor • Band broadening and poor resolution are caused by……. • Slow injection. • Oversized sample.

16. D. Sample Injection System • Sample introduction usually…… • In the form of neat liquid or solution. • Introduced in a small volumes. • 1 μL - 20 μL for packed column. • 1 x 10-3μL for capillary column.

17. D. Sample Injection System Examples • Direct injection using microsyringe • Loop injectors • Auto samplers • Headspace

18. D. Sample Injection System

19. D. Sample Injection System LOOP INJECTORS DIRECT INJECTION USING MICROSYRINGE

20. D. Sample Injection System Headspace • A headspace sample is normally prepared in a vial containing the sample, the dilution solvent, a matrix modifier and the headspace. • Volatile components from complex sample mixtures can be extracted from non-volatile sample components and isolated in the headspace or gas portion of a sample vial. • A sample of the gas in the headspace is injected into a GC system for separation of all of the volatile components.

21. G = the gas phase (headspace)The gas phase is commonly referred to as the headspace and lies above the condensed sample phase. • S = the sample phaseThe sample phase contains the compound(s) of interest. It is usually in the form of a liquid or solid in combination with a dilution solvent or a matrix modifier. • Once the sample phase is introduced into the vial and the vial is sealed, volatile components diffuse into the gas phase until the headspace has reached a state of equilibrium as depicted by the arrows. The sample is then taken from the headspace.

22. E. Oven • Must have sufficient space to hold the column. • Can be heated to the desired temperature for analysis. • Atmosphere inside the oven is constantly agitated by forced ventilation which has small thermal inertia. • Reproducible of retention time,tR which require control of the column temperature within a few tenths of a degree.

23. E. Oven • Optimum temperature depends on the boiling points of the sample components. • A temperature that is roughly ≥ the average boiling point of the sample results in a reasonable elution period. • Samples with broad boiling range, necessary to employ temperature programming.

24. Temperature Programming • Definition: A technique in which the column temperature is increased either continuously or in steps as the separation proceeds. • In general, optimum resolution is associated with minimal temperature. • Low temperature, result in longer elution times hence slower analysis.

25. Temperature Programming • Using Temperature programming, low boiling point constituents are separated initially at temperatures that provide resolution. • As separation proceeds, column temperature is increased so that the higher boiling point constituents come off the column with good resolution and at reasonable lengths of time.

26. Isothermal Elution A technique in which the column temperature is constantly maintained throughout the separation.

27. Isothermal at 1500C Temperature programmed: 500C to 2500C at 80C/min

28. F. Columns Two types of columns • Packed column • Capillary column Capillary Column: 10-100m in length, very small i.d Packed column: 1-5m in length, 2-4mm i.d

29. 1. Packed Column • Less commonly used • Made of glass or steel • Length: 1 to 5 m • Internal diameter: 2 to 4 mm • These column is densely packed with uniform, finely divided solid support, coated with thin layer (0.05 to1μm) of stationary liquid phase. • Accommodate larger samples. Cross-sectional view of packed column

30. 1. Packed Column • Carrier gas flow between 10 – 40 mL/min. • Not well adapted for trace analysis. • Contain an inert & stable porous support on which the stationary phase can be impregnated(coated) or bound. • Advantages: • Large sample size • Ease & convenience of use

31. 2. Capillary Column • Widely used in GC analysis • Also known as open tubular column • Length: 10 – 100 m • Coiled around a light weight of metallic support. • Types of capillary column • FSOT (Fused Silica Wall Coated) - i.d. 0.1 - 0.3 mm • WCOT (Wall Coated) - i.d. 0.25 – 0.75 mm • SCOT (Support Coated) -i.d. 0.5 mm

32. 2. Capillary Column • Advantages: • High resolution • Short analysis time • High sensitivity

33. Properties and characteristics of GC Column

34. G. Stationary Phase Desirable properties for the immobilized liquid stationary phase: • Low volatility (ideally the boiling point of the liquid at least 1000C higher than the maximum operating temperature for the column) • Thermal stability. • Chemical inertness. • Solvent characteristics such as k and α values for the solutes to be resolved fall within a suitable range.

35. G. Stationary Phase • Separation principles • Use the principle of “like dissolve like” where like refers to the polarity of the analyte and the immobilized liquid stationary phase. • Polarity of organic functional group in increasing order • Aliphatic hydrocarbons<olefins<aromatic hydrocarbons<halides<ethers< esters/ aldehydes/ketones<alcohols/amines< amides<carboxylic acids<water

36. G. Stationary Phase • Polarity of the stationary phase should match that of sample components. • When the match is good, the order of elution is determined by the boiling point of the eluents.

37. The choice of stationary phase should match that of sample components. Non polar Stationary phase Polar Stationary phase

38. Polarity of Stationary Phase Water Carboxylic acids Amides Alcohol/amines Esters/aldehydes/ketones Ethers Halides Aromatic hydrocarbons Olefins Aliphatic hydrocarbons Polar Non-polar

39. Aliphatic hydrocarbons < esters/aldehydes/ketones < alcohols/amines < water Non-polar Polar Pentane, Hexane Heptane, Octane Acetone, 3-pentanone Methyl ethyl ketone Propanol, Butanol Pentanol

40. G. Stationary Phase applications

42. G. Stationary Phase • Many liquid statationary phase are based on polysiloxanes or polyethylene glycol (PEG) Polyethylene glycol (PEG) Use for separating polar species Polydimethyl siloxane, the R groups are all CH3. (Non-polar)

43. H. Detectors • Some detectors are universal. • They are sensitive to almost every compound that elutes from the column. • Most detectors are selective. • They are sensitive to a particular type of compound. Give response that is dependent on the concentration of analyte in the carrier gas. • Yield(produce) simple chromatogram.

44. H. Detectors • Characteristics of ideal detector • High reliability & ease to use. • Similarity response toward all solutes or alternatively a high predictable & selective response toward one or more classes of solute. • Detector should be nondestructive.

45. H. Detectors • Characteristics of ideal detector • Adequate sensitivity. • Good stability and reproducibility. • Linear response to solutes that extends over several orders of magnitude. • Temperature range (from room temperature to at least 400 0C)

46. H. Detectors • Several types of detectors. • Flame Ionization Detector (FID) • Thermal Conductivity Detector (TCD) • Electron Captured Detector (ECD)

47. 1. Flame Ionization Detector (FID)

48. How does FID works? • Effluent from the column is passes through a small burner fed H2 and air. • Combustion of the organic compounds flowing through the flame creates charged particles (ionic intermediates are responsible for generating a small current between the two electrodes). • The burner, held at ground potential acts as one of the electrodes. • The second electrode called as a collector, is kept at a positive voltage & collects the current that is generated. • Signal amplified by electrometer that generate measurable voltage.

49. 1. FID • Advantages • Rugged • Sensitive (10-13 g/s) • Wide dynamic range (107) • Signal depends on number of C atoms in organic analyte - mass sensitive not concentration sensitive.

50. 1. FID • Disadvantages • Weakly sensitive to carbonyl, amine, alcohol & amine groups. • Not sensitive to non-combustibles analyte such as H2O, CO2, SO2, NOx. • Destructive method.