Gas chromatography

GC and its application

1. Prepared by Sneha A.Chavan Department of Pharmaceutics M pharmacy 1 st year IInd semester GAS CHROMATOGRAPHY AND ITS APPLICATION 1

2. What is gas chromatography  It is a technique where by the components of a mixture in the gaseous state are separated as the sample passes over a stationary liquid or solid phase and a gaseous mobile phase. 2

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4. Basic principle 4

5. Gas solid chromatography (GSC) Mobile phase – gas Stationary phase – solid Limited application as compared to GLC Interaction of solute with stationary phase is in the form of their adsorption on it. And adsorption is non - linear. K = Cs / Cm this ratio does not remain constant during separation. K = distribution coefficient of the solute Cs = concentration of drug in stationary phase Cm = concentration of drug in mobile phase K (GSC) >>> K (GLC) hence, solute in the gaseous form are not eluted out completely from solid stationary phase. 5

6. Cont…. This leads to semi permanent retention of solute on the stationary phase. longer time require for elution of solute. I t result in problem of tailing or broadening of elution peak. Tailing brings about remixing of separated compound. Stationary phase is molecular sieves or porous polymer 6

7. Cont… Molecular sieve Molecular sieves are resin like zeolite which are manufactured carefully so as to control the pore size in them. Pore or holes are of molecular dimension. The molecule that are smaller than pore, penetrate into sieve of molecule, where they can be adsorbed. Bigger molecule cannot enter the interstices of the sieves. For smaller molecule, larger surface area is available for the adsorption as compared to that for larger molecule. Hence, sieve can used to separate solute based on their size and adsorption coefficient. 7

8. Cont…. The molecules having smaller size, e.g., O 2, CO, H 2 , N 2 and methanol, are able to enter interstices of molecular sieve but can be separated from each other because of the difference in their adsorption coefficient. Problem the molecular sieve as the stationary phase is that the polar compound like CO 2 are permanently adsorbed in the pores of the sieves and thus block the surface to other species. 8

9. Cont…. Porous polymer Another stationary phase is in the form of porous polymeric beads of uniform size. A polymer can be in the form of styrene cross linked with divinyl benzene. Marketed trade name Porapak Pore size of polymer is controlled by controlling degree of polymerization. Being hydrophobic, this polymer find its main use in the separation of gases, which are polar in nature ( oxides of nitrogen, methanol, vinyl chloride, CO 2, H 2 O ) 9

10. Application Technique is useful for separation of species such as nitrogen oxide, H 2 S, CS 2 CO, CO 2 and rare gas that are not retained on liquid column. 10

11. Gas liquid chromatography Mobile phase – Gas Stationary Phase – liquid which is immobilized by supporting on some solid matrix Mobile phase (gas) does not interact much with the solute to be separated. Role of mobile phase is to push the solute, which are desorbed from stationary phase, out of the column. Hence, mobile phase is referred to as carrier gas Sample should be in the form of vapor. This vapor is introduced at head of column. The solute having greater solubility in the stationary liquid phase remain in smaller concentration in it. 11

12. Cont… Thus solute distribute themselves between the phases according to their distribution coefficient. Solute having lesser interaction with stationary phase are easily desorbed from it and eluted out faster. The elution of desorbed compound is achieved by forcing an inert carrier gas such as nitrogen or helium through the column. Chromatogram obtained in GLC, also record elution curves for the eluted compounds, which are bell shaped. 12

13. Application Peak height or peak area of an elute from GC column ha been widely used for quantitative and semi quantitative analysis GLC is commonly used for analysis of volatile compound or compound which can be vaporized at temperature of the sample port of the gas chromatograph. Applied for separation of complex mixture, organometallic compound and biochemical system. Powerful fractionating tool in conjunction with superior analytical instruments such as mass, IR, UV and NMR spectrometer. 13

14. Quantitative Analysis 14 Quantitative analysis involves the following steps :- The generation of signal for each compound in the mixture by the detector. The measurement of some parameter for the peak obtained in a chromatogram. Calculation of separated compound from measured parameter.

15. Measurement of peak parameters in chromatography 15 Analysis based on Peak Height

16. Cont… 16 Advantage Measurement of the peak height gives accurate result, provided the width during the period required to obtain chromatogram for a sample and a standard, the column temperature, the flow rate of the mobile phase and rate of sample injection are controlled properly . Suitable for narrow peak (height measured properly and accurately)

17. Cont… 17 Disadvantage Variation in peak width observed which result in change in peak height leading to inaccuracy in measurement.

18. 18 B. Analysis Based On Peak Area Advantage Preferred parameter for asymmetric or skewed peak . Measurement of peak area is independent of band broadening and offer advantage of reproducibility. This is because band broadening is inversely proportional to height. Disadvantage Measurement of peak area is not easy as measurement of peak height

19. Measurement of peak area 19 Peak measurement by triangulation technique A = ½ × w × h w - width of peak h – height of peak Advantage Simple formula for calculation of area under the curve Disadvantage Useful only for symmetrical peak Tedious job for putting tangent on side of triangle. Require operator skill Slight error in drawing tangent can have profound effect on height measurement.

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21. 21 b) Peak measurement by width at half the height method A = h × ( ½ w) Advantage Method reduces error due to tailing, in the measurement of area. More accurate, rapid, simpler compared to triangulation technique. Disadvantage Useful only for symmetrical peak

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23. 23 c) Area measurement by planimetry Advantage Does not provide precise measurement of peak area. More accurate than triangulation technique. Useful for unsymmetrical and skewed peak . Disadvantage Precision and accuracy depend on skill of operator Time consuming and tedious method. Same peak is traced several times and average of all value is calculated.

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25. 25 d) Area measurement by cut and weight method Advantage Superior than triangulation method Useful for asymmetric or skewed peak Disadvantage Accuracy depend on accuracy of cutting chart paper. Time consuming Very high quality chart paper is essential in order to get reproducibility in the measurement.

26. 26 e) Area measurement using ball and disc integrator Advantage Used for skewed peak also. Provide high degree of automation and higher accuracy as compared to other technique.

27. Method of quantitative analysis 27 Area normalization technique Internal standard method External standard method Preparation of calibration curve

28. Area Normalization Method 28

29. Cont… 29 For this method to be accurate, the following criteria must met All analyte must be eluted All analyte must be detected All analyte must have same sensitivity (response/mass) Advantage Simple technique to calculate % of compound when limited number of compound present in mixture Does not require reference standard for finding concentration of compound

30. Cont… 30 Disadvantage Elution of all compound in the sample is required even when % of only one compound is to be determined. Method is not used when number of compound is more.

31. Internal standard method 31 Amount of each compound in mixture is calculated by using a reference standard. The reference compound is mixed along with mixture to be separated and this mixture is injected in column. Hence reference compound is called is called as internal standard . Choice of internal standard is made based on the following criteria It must have completely different retention time than for the compound to be separated. Must be elute out near the peak of interest. Must be in similar in concentration to compound of interest. Must be chemically inert. Must be absent in sample.

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33. Cont… 33 Internal standard and compounds in mixture separate depending on their retention time value. From height or area of peak for reference standard and eluted compound, the concentration of each compound in mixture can be found out as follows : h (std) × c (std) = h (sample) × c (sample) Advantage Higher accuracy. Error associated with apparatus and procedure can also be eliminated by use of an internal standard. Disadvantage Sometime difficult to get suitable internal standard. And it may not elute out near compound of interest. It may not soluble in the solvent in the concentration range corresponding to compound to be separated. It may not be chemically inert.

34. External standard method 34 Standard is not mixed along with the sample to be separated. Separate solution of standard and sample are prepared. External standard should be 100% pure (AR grade) or it should be of known purity. Both solution are injected separated in the column which result in produces two separate chromatogram. The height of sample is compared with height of standard and their concentration are determined. Advantage No need to find suitable chemical as standard.

35. Cont… 35 Disadvantage Error may occur during weighing and dilution, which make the process less accurate as compared to internal standard. Require precise control on analytical technique, particularly while injecting the solution in the column. Two chromatogram are required to be recorded. More time consuming.

36. Preparation of calibration curve 36 Standard solution of compound of interest are prepared. Equal volume of solution injected in chromatograph and recorded. Area under the curve is measured and concentration plotted against the area. Unknown concentration of sample can be obtained by measuring area under the peak and then dropping a perpendicular on concentration axis.

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38. Qualitative analysis by GLC Useful tool for identifying separated compounds. Retention time Retention time (RT) is a measure of the time taken for a solute to pass through a chromatography column. Characteristics parameter of solute which help in identification. Change with change in temperature of column and flow rate and pressure of carrier gas. Precise control needed. Chromatogram recorded number of time hence, process become tedious and expensive . 38

39. Cont… 39 Relative retention time ( α ) It describes the retention time of solute in relation with the retention time of standard.

40. Cont… 40 Retention volume (Vr) It is defined as the total volume of carrier gas required to elute out the compound completely from the column. If column temperature is increased, the volume of gas is increased because of which flow rate of carrier gas is also increased, but retention time is reduced.

41. 41 Specific retention volume (Vg) Volume of gas per unit weight of the stationary phase, required to elute the compound completely is called specific retention volume .

42. Gas Chromatography Environmental Analysis 42 used in quantification of pollutants in drinking and waste water. identification of unknown organic compound in hazardous waste. used in analysis of industrial products as well identification of reaction products. Environmental toxins can be identified with gas chromatography. Air samples can be analyzed by gas chromatography for quality control. Pesticides , fertilizers can by analyzed by GC.

43. Gas Chromatography Clinical Analysis 43 Blood , Saliva and other secretions which contains large amount of organic volatiles can be easily analyzed by gas chromatography. Isolation of volatile proteins , lipids, carbohydrates  by gas chromatography. used to analyze the chemicals and drugs present in the body. Various biological volatile organic compounds can be analyze by gas chromatography. Urine sample is also analyzed by gas chromatography.

44. Gas Chromatography Food Analysis 44 Food products can be analyzed by gas chromatography. used in analysis of alcohol beverages. Analysis of various solvents in food preparation. analysis of dairy products such as milk, butter, cheese etc. Quantification of  volatile organic food products 

45. Gas Chromatography Forensics 45 determination of blood alcohol content in the body. Certain drugs are prohibited in certain states, so in order to check whether person has taken drugs in the body used to test samples found at crime scene.

46. Reference 46 1. Instrumental method of analysis, Supriya Mahajan, page no. 283 - 293 2. Principles of Instrumental Analysis, Douglas A. Skoog, F. James Holler, S tanley B. Crouch, page no.865 – 892 3. Basic Gas Chromatography, Harold Mcnair, James Miller, 2 nd edition, page no. 138- 143 3. http://frndzzz.com/Applications-of-Gas-Chromatography/GC-Pharmaceutical-Analysis

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