1. prof. aza Basic gas chromatography prof. aza Department of Pharmacy, Andalas University STIFI Perintis, Padang STIFAR, Pekan Baru STIFI Bhakti Pertiwi, Palembang

2. prof. aza Definition The basis for gas chromatographic separation is the distribution of a sample between two phases. One of these phases is stationary bed of large surface area, and the other phase is gas which percolates through the stationary bed. If the stationary phase is a solid, we speak of Gas- Solid Chromatography. This depends up on the adsorptive properties of the column packing to separate samples, primarily gases. If the stationary phase is a liquid, we speak of Gas liquid Chromatography (GLC). The liquid is spread as a thin film over an inert solid and the basis for separation is the partitioning of the sample in and out of this liquid film.

3. prof. aza Schematic drawing of Gas Chromatographic System

4. prof. aza GC/MS Gas Chromatography/Mass Spectrometry (GC/MS) is a combination of two instrumental techniques, gas chromatography and mass spectrometry. The gas chromatograph is used to separate a mixture into component parts and deliver them to the mass spectrometer. The mass spectrometer breaks the molecules into ions and records the resulting spectrum. This spectrum conclusively identifies the compound.

5. prof. aza

7. Chromatogram, A Typical gas- liquid chromatography displays each component as peak

8. prof. aza Resolution The resolution of chromatographic peaks is related to two factors: column efficiency, and solvent efficiency. Column efficiency is concerned with the peak broadening of initially compact band as it passes through the column. The broadening results from the column design and operating conditions, and can be quantitatively described by the height equivalent to a theoretical plate. The HETP is that length of column necessary for the attainment of solute equilibrium between the moving gas phase and stationary liquid phase.

9. prof. aza Hypothetical representation of Chromatographic separation

10. prof. aza Advantages of Gas Chromatography Speed, the entire analysis is completed within 23 minutes. High Resolution. By using selective solvents, GC can provide resolution impossible by distillation or other techniques. Qualitative analysis. The retention time is that time from injection to the peak maxima. This property is characteristic of the sample and the liquid phase at a given temperature. Each compound has only one retention time. This retention time is not influenced by the presence of other component.

11. prof. aza Advantages of Gas Chromatography, continued Quantitative analysis. The area produced for each peak is proportional to that peak’s concentration. This can be used to determine the exact concentration for each component. Sensitivity. The simplest forms of thermal conductivity cells can determine down 0.01 % (100 ppm). The flame detector easily sees parts per million, and the specific electron capture detector can measure parts per billion or picograms (10 -12 g). Simplicity. GC are simple to operate and understand. Interpretation of the data obtained is usually rapid and straight forward.

12. prof. aza Temperature Injection-port temperature should be hot enough to vaporize the sample so rapidly that no loss in efficiency results from the injection technique and must be low enough so that thermal decomposition or rearrangement is avoided. Column temperature should be high enough so that the analysis is accomplished in a reasonable length of time. The retention time doubles for every 30 0 C decrease in column temperature. For the most samples the lower the column operating temperature, the higher the ratio of partition coefficient in the stationary phase and the better the resultant separation. Detector temperature should be high enough so that condensation of the sample and or liquid phase does not occur.

13. prof. aza DetectorSensitivity TCDAll compound 10 ppm FIDOrganic substances 0.1 ppm ECD0.1 ppb

14. prof. aza Thermal conductivity Detector (TCD)

15. prof. aza Flame ionization Detector (FID)

16. prof. aza Electron capture detector (ECD)

17. prof. aza Carrier gas Commonly used gases are hydrogen, helium, und nitrogen. The carrier gas should be : Inert to avoid interaction with sample or solvent. Able to minimize gaseous diffusion. Inexpensive. Suitable for detector used.

18. prof. aza Solid support The purpose of the solid support is to provide a large uniform, inert surface area for distributing the liquid phase. Some desirable support properties are : Inert (avoid adsorption). High crushing strength. Large surface area. Regulator shape, uniform size.

19. prof. aza Stationary phase Ideally the solvent should have the following characteristic: Samples must exhibit different distribution coefficients. Sample should have a reasonable solubility in the solvent. Solvent should have a negligible vapor pressure at operating temperature.

20. prof. aza Resolution The resolution of chromatographic peaks is related to two factors: column efficiency, and solvent efficiency. Column efficiency is concerned with the peak broadening of initially compact band as it passes through the column. The broadening results from the column design and operating conditions, and can be quantitatively described by the height equivalent to a theoretical plate. The HETP is that length of column necessary for the attainment of solute equilibrium between the moving gas phase and stationary liquid phase.

21. prof. aza Resolution, continued Solvent efficiency result from the solute- solvent interaction and determines the relative position of solute bands on a chromatogram. Solvent efficiency is expressed as the ratio of peak maxima (adjusted retention times). Column efficiency is measured by the number of the theoretical plates.

22. prof. aza Resolution, continued The true separation of two consecutive peaks is measured by the resolution. Resolution is a measure of both the column and solvent efficiency. If R = 1, the resolution of two equal-area peaks is approximately 98% complex. If R = 1,5, base line separation (99,7% resolution) ia achieved.

23. prof. aza Separation of two bands as a function of resolution (Rs) and relative band size (I/1, ¼, 1/16).

24. prof. aza Solvent efficiency and Separation Factor

25. prof. aza Normal Increased Column Efficiency (more theoretical plate) Increased Solvent Efficiency (greater ratio of retention times)

26. prof. aza Effects of a change in k’, N, or a on the resolution of two bands

27. prof. aza Calculation of Theoretical Plate HETP = L/N

28. prof. aza Band asymmetry, (a) Definition of asymmetry factor, As; (b-e) examples of band asymmetry

29. prof. aza Rate theory, van Deemter The three principal contributions to the broadening of a band are : Multipath effect or eddy diffusion (A term) Molecular diffusion (B term). Resistance to mass transfer (gas and liquid, C term).

30. prof. aza Plot of HETP against gas velocity

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34. van Deemter equation HETP = A + B/µ + C.µ µ = length of column (cm)/ retention time (seconds) HETP = 2 dp + 2  Dgas + 8 k’ d f 2 x µ µ  (1+k’) 2 D liq

35. prof. aza van Deemter equation, continued 2 dp = a constant which is a measures of packing irregularities. dp = average particle diameter of the solid support. 2  Dgas µ Dgas = diffusivity of the solute in the gas phase. µ = the linear gas velocity

36. prof. aza van Deemter equation, continued 8 k’ df 2 Fliq = fraction of cross section occupied by the liquid phase Fgas = fraction of cross section occupied by the gas phase df = effective thickness of the liquid film which is coated on the particles of the support. Dliq = diffusivity of solute in the liquid phase  (1+k’) 2 Dliq k’ = capacity factor = k ( F liq / F gas ) k = partition coefficient of the solute, expressed as the amount of solute per unit volume of liquid phase divided by the amount of solute per unit volume of gas phase

37. prof. aza Illustration of Multipaths Multiple Path (Eddy diffusion) term, A = 2 dp In any packed column solute molecules and carrier gas molecules travel along many paths. These path have different lengths, therefore the solute molecules have different residence time. This adds to peak broadening. This broadening depends upon the size of the particle constituting the packing, the shape, and the manner in which they are packed and the column diameter.

38. prof. aza To improve column efficiency Particle diameter: Column efficiency is improved by the use of small, uniform particle size. Diatomaceous earth type with 100 – 120 mesh range. Flow rate: For maximum efficiency the column must be operated at the optimum flow rate. In practice, operating at flow rates slightly higher than optimum will decrease the analysis time and not materially effect the HETP. Carrier gas: The detector employed usually dictates the choice of carrier gas. For highest efficiency a high molecular weight gas should be choice. Where rapid analysis time is required and highest efficiency is not necessary a low molecular weight carrier gas such as helium or hydrogen would be preferred.

39. prof. aza To improve column efficiency, continued Type of liquid phase: A low viscosity, low vapor pressures solvent with good absolute solubility for the sample should be used. To obtain a separation, it must exhibit a differential solubility. Amount of liquid phase: Low liquid loadings (thin film) 1% - 10% have the advantage of fast analysis and lower temperature operation.

40. prof. aza To improve column efficiency, continued Temperature: Resolution can usually be improved by lowering the column temperature. Lowering the temperature also decreases decomposition of the compounds but may increase adsorption. Pressure: The majority of practical gas chromatographers work at an outlet pressure of one atmosphere so that operation at the optimum flow rate fixes the inlet pressure. Best efficiency is obtained at low inlet-to-outlet pressure ratios. Column diameter: Capillary and preparative column experiments indicate that efficiency is improved with decreasing internal diameter. Thus 1/8 – 1/16 inch OD are used for highest resolution.

41. prof. aza Basic measurement

42. prof. aza Science for a better future 1955 – 2005 FMIPA UNAND