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Gas Chromatography

Gas chromatography (GC) is a technique used to separate and analyze mixtures of substances. It works by vaporizing the sample and carrying it by a carrier gas through a column coated with a stationary phase. Components interact differently with the stationary phase and emerge from the column at different retention times, allowing for separation. Key aspects of GC include the carrier gas, column parameters like temperature and phase, and detectors used for analysis. GC is useful for characterizing compounds and quantifying components in mixtures.

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1 Nabin Bist Gas Chromatography
2 Principles ■ A gas mobile phase flows under pressure through a heated tube stationary phase ■ The analyte is loaded onto the head of the column via a heated injection port (evaporates) ■ It condenses at the head of the column, which is at a lower temperature ■ The oven temperature is then either held constant or programmed to raise gradually. ■ Separation of mixture occurs according to the relative lengths of time spent by compounds in stationary phase ■ Monitoring of the column effluent can be carried out with detectors 2
3 Applications ■ Characterization of some unformulated drugs with regard to detection of process impurities ■ Limit tests for solvent residues and other volatile impurities in drug substances ■ Sometimes used for quantification of drugs in formulations, particularly if the drugs lacks a chromophore ■ Characterization of some raw materials used in synthesis of drug molecules ■ Characterization of volatile oils (excipients) ■ Cough mixtures, tonics, fatty acids in fixed oils ■ Measurement of drugs and their metabolites in biological fluids 3
4 Strengths ■ Capable of quantitative accuracy ■ Greater separation power than HPLC when used with capillary columns ■ Used to determine compounds which lack chromophores. ■ The mobile phase does not vary and does not disposal –cheap than HPLC solvents 4
5 Limitations ■ Only thermally stable and volatile compounds can be analysed ■ Sample may require derivatisation to convert it to a volatile form ■ Quantitative sample introduction is more difficult because of the small volume of sample injected ■ Aqueous solutions and salts cannot be injected into the instrument 5
6 ■ A simple GC system consists of: ■ 1. Gas source (with pressure and flow regulators) ■ 2. Injector or sample application system (sample inlet) ■ 3. Chromatographic column (with oven for temperature control) ■ 4. Detector & computer or recorder 6
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8 ■ Carrier gas: He (common), N2, H2 Pinlet 10-50 psig Flow = 25-150 mL/min packed column Flow = 1-25 mL/min open tubular column Column: 2-100 m coiled stainless steel/glass/Teflon/fused silica, packed vs. unpacked ■ Oven: 0-400 °C ~ average boiling point of sample Accurate to <1 °C ■ Detectors: FID, TCD, ECD, NPD, MSD. (SINGLE OR TANDEM) 8
Carrier Gas ▪ The purpose of carrier gas is to transport the sample through the column to the detector. ▪ The selection of proper carrier gas is very important because it effects both column and detector performance. ▪ Carrier gas usually used in GC are : ➢Helium ➢Hydrogen ➢Nitrogen ➢mixture of 95% Argon and 5% methane. ▪ The detector that is employed usually dictates the carrier to be used 9
10 ➢ TCD : Hydrogen and Helium most suitable carrier gases ➢ FID : Nitrogen and Helium ➢ ECD : mixture of 95% argon and 5% methane. ➢ Cylinders holding carrier gases are painted with different colours to identify carrier gas. ➢ Hydrogen is painted red ➢ oxygen painted black ➢ argon painted green. ➢ High purity carrier gases are used for quantitative analysis. 10
11 Injection port ■ Injection port : Sample is introduced into Gas Chromatograph ■ Sample introduced in the form of solution by a microliter syringe ■ A high temperature resistance rubber septum is kept in the injection port. ■ Injection port temperature = Max. 450o C. ■ Sample is vaporized as soon as it is introduced into the injection port. ■ The sample vapour along with the carrier gas enter the column. 11
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13 Column ■ Column is the heart of the chromatographic system ■ Sample mixture is separated into individual compounds depending on the principle of partition or adsorption. ■ Column : stainless steel – 2 to 18 feet length, 1/8II dia. ■ Stationary phase: Adsorbent (or) liquid phase coated on solid support (GSC/GLC) ■ Glass wool inserted into the tubing ends. ■ Common stationary phases ➢ Methyl silicone , SE-30 (non polar) ➢ Phenyl silicones (Medium polar) ➢ Diethylene glycol succinate (DEGS) (polar) 13
14 Capillary Columns ■ Goley introduced capillary columns ■ Capillary columns are made from fused silica, usually coated on the outside with polyamide to give the column flexibility. ■ The wall of the column is coated with the liquid stationary phase (0.1 -5 µm) ■ Most common coating is based on organo silicone polymers. ■ Length = 25 to 100m , 0.15 to 0.5 mm dia. ■ They are two kinds ➢ Wall coated open tubular columns (WCOT) ➢ Support coated open tubular columns (SCOT) 14
15 ■ SCOT- very high temperature work ■ WCOT- 325 - 370oC ■ Non-silicone based polymers can not be bonded onto the wall of the column-less stable. Eg: carbowax (240oC) 15
16 - many bonded phases exist, but most separations can be formed with the following phases: Dimethylpolysiloxane Methyl(phenyl)polysiloxane Polyethylene glycol (Carbowax 20M) advantages: - more stable than coated liquid phases - can be placed on support with thinner and more uniform thickness than liquid phases Bonded-Phase 16
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18 Classification of stationary Phase ■ Kovats Retention Indices ( I ): ■ Determination of carbon number of any volatile organic compound by using the net retention time of normal paraffin's and the volatile organic compound under investigation. ■ I = 100n + 100 log Rx – log Rn log Rn+1- log Rn ■ Where R= specific retention volume ■ n = carbon number of the n-alkane eluting before substance X. ■ n+1 = carbon number of the n-alkane elutes after substance X. ■ For reference purpose, Retention Index of pentane (C5) =500, hexane (C6) = 600, Heptane (C7) = 700 .. ■ If the retention index of the compound is nearer to the retention index of hexane (C6). = Compound contains 6 carbon atoms. 18
19 McReynold’s constants (σI) ■ Measure of the polarity of the stationary phase is given by McReynold’s constant. ■ Based on the retention time of Benzene, n-butanol, pantane-2-one, nitropropane and pyridine on a stationary phase. ■ Squalane as non polar reference. ■ The sum of the (σI) obtained may be considered as the average polarity of the stationary phase. ■ The higher the (σI), the more polar the phase. ■ McReynold classified the stationary phases available in GC as non-polar, medium polar and polar depending on the value of (σI). 19
20 Phase Chemical type McReynold’s constants Squalane Hydrocarbon 0 Silicone OV-1 Methyl silicone 222 Silicone SE-54 94% methyl, 5% phenyl, 1% vinyl 337 Silicone OV-17 50% methyl, 50% Phenyl 886 Silicone OV-225 50% methyl, 25% cynopropyl, 25% phenyl 1813 Carbowax Polyethylene glycol 2318. 20
21 GC Oven ■ GC oven incorporate a fan-ensures uniform heat distribution throughout the oven. ■ Temperature conditions : ➢ isothermal ➢ Gradual increase in temperature ■ Oven programming rates can range from 1o C/min to 40oC/min. ■ Eg:60oC (1 min)/5oC/min to 100oC (5 min)/10oC/min to 200oC (5 min). ■ Materials of different volatilities can be separated in a reasonable time and also injection of the sample can be carried out at low temperature, where it will be trapped at the head of the column and then the temperature can be raised until it elutes. 21
Column Oven Isothermal Analysis Temperature Programming Analysis
23 Column Temperature Eg.1 􀂄Separations/Resolutions are good, but the analysis time is too long.
24 Column Temperature Eg.2 ❑ Short analysis time, but separations/resolutions are not good.
25 Column Temperature Eg.3
26 ■ Start from low temp and then raise the temp to a higher one in a certain rate of increase. ■ e.g.: 50 C (2 min) →20 C/min C/ → 200C (5 min) ■ 80 C (5 min) → 10C/min → 150C (3 min) → 5C/min → 180C (15 min) ■ The lower the rate of temp increase, the better the resolution is.
27 Column Support ■ Inert materials used ■ Acid washing- to reduce peak tailing ■ Deactivation of support surface ■ 10-15% (w/w) of stationary phase is coated on the support ■ Common supports : diatomaceous earth materials ■ Commercially known as chromosorb : different grading – P,W, G etc., 27
28 Theory of Gas Chromatography ■ Adsorption: the molecules of analyte reside on the stationary phase for sometime and then desorbed and eluted out along with the mobile phase. ■ Solid adsorbent is used as a stationary phase. ■ Partition : the molecules of analyte get distributed between the mobile and stationary phases. ■ Stationary phase : high molecular weight liquids. ■ If the con. Of the analyte is more in stationary phase, the elution takes more time. ■ If concentration of analyte is less in stationary phase, the elution takes less time. 28
29 Partition coefficient (β) ■ GC separation = distillation separation. ■ β = weight of the solute per gram of the stationary phase Weight of the solute per cc of the carrier gas ■ β is characteristic of each one of the components ■ In addition to partition, other physical interactions like – hydrogen bonding, wander Waal’s forces ■ Nature of analyte, stationary and mobile phases. 29
30 Height Equivalent to a Theoretical Plate (HETP) ■ Efficiency of a chromatographic separation is expressed in terms of the number of theoretical plates (N) ■ Number of theoretical plates (N) = 16 (tR/Wb)2 ■ Where t = the net retention time of the sample ■ Wb = width of the peak at the base ■ HETP = L/N ■ L = Length of the column, N = number of theoretical plates ■ If the value of N is more the efficiency of the column is more. If the value of N is less the efficiency of the column is less. 30
31 Derivatization Techniques ■ i) To permit analysis of compounds not directly amenable to analysis due to inadequate volatility or stability ■ ii) To improve the analysis by better chromatographic behavior or detectability. ■ Replacement of N-H, O-H and S-H groups by alkylation, acylation, silylation increases the volatility. ■ Closely related compounds, optical isomers can be separated after derivatization. 31
32 Silylation Typical reagents ■ i) Hex methyl disilazane (HMDS) ■ (CH3)3SiNHSi(CH3)3 ■ ii) Trimethyl chlorosilane (TMCS) ■ (CH3)3SiCl ■ Iii) Dimethyl chlorosilane (DMCS) ■ (CH3)2SiCl ■ iv) Tetramethyl disilazane (DMCS) ■ (CH3)2HSiNHSi(CH3)2H ■ The reagent is used to replace an active protons to improve thermal stability or to reduce tendency to form hydrogen bonds. ■ Ex: sugars, thiols, steroids, amino acids 32
33 Acetylation ■ To replace active hydrogen ■ Alcohols, amino acids, 1 or 2 amines ■ Amino acids are usually esterified to methyl or butyl esters prior to acetylation. ■ Typical reagents : acetic anhydride & Trifluoroacetic anhydride dissolved in pyridine & DMF. ■ Trifluoroacetyl derivatives. 33
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36 Esterification ■ Carboxylic acid analogs, long chain fatty acids derivatized by using HCl in methanol or diazomethane. ■ Aminoacids can be esterified by reacting with butanol in 3N HCl at 150ο C for 10 minutes. ■ Diazomethane – more specific, minimal side reactions. ■ Disadvantage : toxic and potentially explosive 36
37 Analysis of fatty acid composition BP Monographs- GC analysis to confirm the content of fatty acid composing the triglycerides Almond oil states the composition of the fatty acids making up the triglyceride : Palmitic acid (16:0) 4.0-9.0% Palmitoleic acid (16:1) < 0.6% Margaric acid (17: 0) < 2.0 % Stearic acid (18:0) 0.9-2.0% Oleioc acid (18:2) 62.0-86.0% Linoleic acid (18:2) 7.0-30.0% Linolenic acid (18:3) < 0.2% Arachidic acid (20:0) < 0.1% Behenic acid (22:0) <0.1% 37
38 Methanolysis of triglycerides prior to GC analysis 38
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40 GC performance ■ Carrier Gas: Hydrogen or helium are used in GC Typical flow rate = 30-50 cm/s ■ Nitrogen has its optimum flow rate = 10-20 cm/s ■ Gas flow rate decrease with increase with column temperature (column efficiency) ■ Modern instruments have flow programming so that the flow can be set to remain constant as the temperature raises. 40
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42 Column Temperature ■ As column temperature increases, the degree of resolution between two components decreases. ■ Degree of interaction with stationary phase is reduced as the vapour pressure of the analytes increases. ■ Lower temperatures produce better resolution 42
43 Column length ■ Separating power of column varies as the square root of its length. ■ If two fold increase in resolution is required, a four fold increase in column length. ■ So, four fold increase in analysis time ■ Decrease in temperature, ensuring more interaction with stationary phase- better resolution. ■ 43
44 Film thickness phase loading ■ The greater the volume of stationary phase, the more a solute will partition into it. ■ If the film thickness or loading of stationary phase doubles then, the retention of an analyte should double. ■ Thicker films are used for very volatile materials to increase their RT. ■ To increase resolution between analytes without increasing the column length. 44
45 Internal diameter ■ The smaller the internal diameter of a capillary column, more efficient. ■ Mass transfer characteristics of the column are improved ■ Analyte being able to diffuse in and out of the mobile phase more frequently because of the shorter distance for transverse diffusion. 45
46 Question ? ■ A fixed temperature is used and the head pressure is adjusted so that the linear velocity of a helium carrier gas through the following capillary column is 20 cm/s: OV-1 film ■ (i) 30 m × 0.25 mm i.d × 0.25 µm ■ (ii) 15 m × 0.15 mm i.d × 0.2 µm ■ (iii) 12 m × 0.5 mm i.d × 1.0 µm ■ A) List the columns in the order in which they would increasingly retain a n-hexadecane standard ■ B) list the columns in order of increasing efficiency. 46
47 Detector ■ 1. Detector is to indicate the presence and measure the amount of component eluted out from the column. ■ 2. High detector temperature should be set to prevent condensation of sample
48 Type of Detectors 1. Flame Ionization Detector (FID) 2. Thermal Conductivity Detector (TCD) 3. Flame Thermionic Detector (FTD ) 4. Flame Photometric Detector (FPD) 5. Electron Capture Detector (ECD) 6. Mass Spectrometer Detector (MSD) TCD, FID, MSD are general detectors.
49 Type of Analysis ■ FID ::-Detects any compounds that can be oxdised in hydrogen/air flame ■ ECD:-Selective to electronegative moieties e.g.halogens ■ FTD:-Selective to organic N or P compounds ■ FPD:-Selective to P or S containing compounds ■ TCD:-Detects any component including N2 and O2 except the gas used for the carrier gas.
50 FID ■ Most organic compounds can be detected. These compounds are oxidized in hydrogen / air flame
51 Flame Ionization Detector (FID) ■ For organic compounds analysis ■ Hydrogen and air are needed to create the flame ■ Sample is brought to hydrogen flame and converted into ions. Current will be generated. ■ The current is proportional to the amount of the organic compound present ■ Advantages like
52 ■ High Sensitivity to organic compounds ■ Little or no response to water, CO2, the common carrier gas impurities hence zero signal to when no sample is present ■ Stable baseline which is not mostly affected by fluctuations in carrier gas temp, flow rate and pressure. ■ Good linearity over a wide range of sample conc. Range ■ Presence of halogens in the compound decreases sensitivity
53 TCD ■ Any components including N2 and O2 except the gas used for carrier gas can be detected by TCD ■ Wheatstone bridge is used as principle of detection.
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55 Thermal Conductivity Detector (TCD) ■ Detects almost all compounds except the carrier gas. ■ The TCD filament is heated by applying a current ■ When carrier gas + sample gas passes over the filament, the temperature ■ Of the filament increases, because the thermal conductivity of the sample compounds is less than that of the carrier gas alone. ■ The changes in filament temperature affect its resistance ■ The resistance change is measured and produces the signal
56 ■ – Advantages over FID ■ Responds to all organic as well as inorganic compounds ■ Non destructive type of detector hence can be used for trapping separated components and preparative analysis ■ Low cost and versatile.
57 Electron Capture Detector (ECD) 􀂄Very selective and sensitive to electrophilic compounds (e.g. halogens)
58 ECD 􀂄Detects electrophilic compounds e.g.: halogenated compounds 􀂄Good for low concentration of these kind of compounds 􀂄Contains 63Ni radioactive 􀂄Examples of analyses: ■ analysis of PCBs (polychlorinated biphenyls) ■ analysis of PBBs(polybrominated biphenyls) ■ analysis of PBDEs (polybrominated diphenyl ethers) ■ analysis of organochlorine pesticides (DDT, BHC, etc.)
59 ECD Application
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63 I = 100n + 100 log Rx – log Rn log Rn+1- log Rn
64 Quantitation Methods ■ Area Percent Method : ■ Area percent is the simplest quantitation method. ■ This method assumes that thedetector responds identically to all compounds. ■ This assumption, however, is not valid. ■ This method provides a rough estimate of the amounts of analytes present. 64
65 ■ To calculate area percent take the area of an analyte and divide it by the sum of areas for all peaks. ■ This value represents the percentage of an analyte in the sample. 65
66 Single Point External Standard ■ method requires the analysis of more than just the sample of interest. ■ Analyze a sample containing a known amount of analyte or analytes and record the peak area. ■ Then calculate a response factor using Equation 1. ■ 66
6767 ■ Inject a sample with the unknown analyte concentration and record the peak area. ■ Then calculate the amount of analyte using Equation 2. ■ Calculate an individual response factor for each compound of interest.
68 SINGLE PT. EXT. STD. EXAMPLE ■ An injection containing benzene at a concentration of 2,000 μg/ ml is made and results in a peak area of 100,000. ■ Calculate the response factor for benzene ■ An injection of the sample with the unknown concentration of benzene has a peak area of 60,000. ■ Calculate the amount of benzene present
69 Single Point Internal Standard ■ The analyte chosen for the internal standard has a predictable retention time and area, allowing it to be used to determine if abnormalities have occurred. ■ The Single Point Internal Standard method requires at least two analyses. ■ The first analysis contains a known amount of internal standard and the compounds of interest.
70 ■ Then add a known amount of the internal standard to the sample containing analytes of unknown concentrations. ■ Calculate the amount of the unknown analyte using Equation 4.
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72 ■ In this method an internal standard is chosen which is similar in structure to the components of the mixture under investigation. ■ The internal standard chosen should be such that ■ 1. it should separate from all the components of the sample mixture under investigation ■ 2. it should not react with the components of the sample ■ 3. it should be stable at the operating temperature of the column ■ 4. it should have a fairly good detector response
73 SINGLE POINT INTERNAL STANDARD EXAMPLE ■ Prepare a sample containing 2,000 μg/mL of toluene (the internal standard) and 1,000 μg/mL benzene (the analyte). ■ Then inject the sample. The resulting peak areas are 120,000 for toluene and 67,000 for benzene. ■ Using Equation 3 the response factor for benzene is:
74 ■ Inject the sample containing 2,000 μg/mL of toluene and an unknown amount of benzene using the same chromatography conditions. ■ The resulting areas are 122,000 for toluene and 43,000 for benzene. ■ Calculate the amount of benzene present ?
Headspace (HS) Extraction/Injection

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Gas Chromatography

  • 1.
  • 2.
    2 Principles ■ A gasmobile phase flows under pressure through a heated tube stationary phase ■ The analyte is loaded onto the head of the column via a heated injection port (evaporates) ■ It condenses at the head of the column, which is at a lower temperature ■ The oven temperature is then either held constant or programmed to raise gradually. ■ Separation of mixture occurs according to the relative lengths of time spent by compounds in stationary phase ■ Monitoring of the column effluent can be carried out with detectors 2
  • 3.
    3 Applications ■ Characterization ofsome unformulated drugs with regard to detection of process impurities ■ Limit tests for solvent residues and other volatile impurities in drug substances ■ Sometimes used for quantification of drugs in formulations, particularly if the drugs lacks a chromophore ■ Characterization of some raw materials used in synthesis of drug molecules ■ Characterization of volatile oils (excipients) ■ Cough mixtures, tonics, fatty acids in fixed oils ■ Measurement of drugs and their metabolites in biological fluids 3
  • 4.
    4 Strengths ■ Capable ofquantitative accuracy ■ Greater separation power than HPLC when used with capillary columns ■ Used to determine compounds which lack chromophores. ■ The mobile phase does not vary and does not disposal –cheap than HPLC solvents 4
  • 5.
    5 Limitations ■ Only thermallystable and volatile compounds can be analysed ■ Sample may require derivatisation to convert it to a volatile form ■ Quantitative sample introduction is more difficult because of the small volume of sample injected ■ Aqueous solutions and salts cannot be injected into the instrument 5
  • 6.
    6 ■ A simpleGC system consists of: ■ 1. Gas source (with pressure and flow regulators) ■ 2. Injector or sample application system (sample inlet) ■ 3. Chromatographic column (with oven for temperature control) ■ 4. Detector & computer or recorder 6
  • 7.
  • 8.
    8 ■ Carrier gas:He (common), N2, H2 Pinlet 10-50 psig Flow = 25-150 mL/min packed column Flow = 1-25 mL/min open tubular column Column: 2-100 m coiled stainless steel/glass/Teflon/fused silica, packed vs. unpacked ■ Oven: 0-400 °C ~ average boiling point of sample Accurate to <1 °C ■ Detectors: FID, TCD, ECD, NPD, MSD. (SINGLE OR TANDEM) 8
  • 9.
    Carrier Gas ▪ Thepurpose of carrier gas is to transport the sample through the column to the detector. ▪ The selection of proper carrier gas is very important because it effects both column and detector performance. ▪ Carrier gas usually used in GC are : ➢Helium ➢Hydrogen ➢Nitrogen ➢mixture of 95% Argon and 5% methane. ▪ The detector that is employed usually dictates the carrier to be used 9
  • 10.
    10 ➢ TCD :Hydrogen and Helium most suitable carrier gases ➢ FID : Nitrogen and Helium ➢ ECD : mixture of 95% argon and 5% methane. ➢ Cylinders holding carrier gases are painted with different colours to identify carrier gas. ➢ Hydrogen is painted red ➢ oxygen painted black ➢ argon painted green. ➢ High purity carrier gases are used for quantitative analysis. 10
  • 11.
    11 Injection port ■ Injectionport : Sample is introduced into Gas Chromatograph ■ Sample introduced in the form of solution by a microliter syringe ■ A high temperature resistance rubber septum is kept in the injection port. ■ Injection port temperature = Max. 450o C. ■ Sample is vaporized as soon as it is introduced into the injection port. ■ The sample vapour along with the carrier gas enter the column. 11
  • 12.
  • 13.
    13 Column ■ Column isthe heart of the chromatographic system ■ Sample mixture is separated into individual compounds depending on the principle of partition or adsorption. ■ Column : stainless steel – 2 to 18 feet length, 1/8II dia. ■ Stationary phase: Adsorbent (or) liquid phase coated on solid support (GSC/GLC) ■ Glass wool inserted into the tubing ends. ■ Common stationary phases ➢ Methyl silicone , SE-30 (non polar) ➢ Phenyl silicones (Medium polar) ➢ Diethylene glycol succinate (DEGS) (polar) 13
  • 14.
    14 Capillary Columns ■ Goleyintroduced capillary columns ■ Capillary columns are made from fused silica, usually coated on the outside with polyamide to give the column flexibility. ■ The wall of the column is coated with the liquid stationary phase (0.1 -5 µm) ■ Most common coating is based on organo silicone polymers. ■ Length = 25 to 100m , 0.15 to 0.5 mm dia. ■ They are two kinds ➢ Wall coated open tubular columns (WCOT) ➢ Support coated open tubular columns (SCOT) 14
  • 15.
    15 ■ SCOT- veryhigh temperature work ■ WCOT- 325 - 370oC ■ Non-silicone based polymers can not be bonded onto the wall of the column-less stable. Eg: carbowax (240oC) 15
  • 16.
    16 - many bondedphases exist, but most separations can be formed with the following phases: Dimethylpolysiloxane Methyl(phenyl)polysiloxane Polyethylene glycol (Carbowax 20M) advantages: - more stable than coated liquid phases - can be placed on support with thinner and more uniform thickness than liquid phases Bonded-Phase 16
  • 17.
  • 18.
    18 Classification of stationaryPhase ■ Kovats Retention Indices ( I ): ■ Determination of carbon number of any volatile organic compound by using the net retention time of normal paraffin's and the volatile organic compound under investigation. ■ I = 100n + 100 log Rx – log Rn log Rn+1- log Rn ■ Where R= specific retention volume ■ n = carbon number of the n-alkane eluting before substance X. ■ n+1 = carbon number of the n-alkane elutes after substance X. ■ For reference purpose, Retention Index of pentane (C5) =500, hexane (C6) = 600, Heptane (C7) = 700 .. ■ If the retention index of the compound is nearer to the retention index of hexane (C6). = Compound contains 6 carbon atoms. 18
  • 19.
    19 McReynold’s constants (σI) ■Measure of the polarity of the stationary phase is given by McReynold’s constant. ■ Based on the retention time of Benzene, n-butanol, pantane-2-one, nitropropane and pyridine on a stationary phase. ■ Squalane as non polar reference. ■ The sum of the (σI) obtained may be considered as the average polarity of the stationary phase. ■ The higher the (σI), the more polar the phase. ■ McReynold classified the stationary phases available in GC as non-polar, medium polar and polar depending on the value of (σI). 19
  • 20.
    20 Phase Chemical typeMcReynold’s constants Squalane Hydrocarbon 0 Silicone OV-1 Methyl silicone 222 Silicone SE-54 94% methyl, 5% phenyl, 1% vinyl 337 Silicone OV-17 50% methyl, 50% Phenyl 886 Silicone OV-225 50% methyl, 25% cynopropyl, 25% phenyl 1813 Carbowax Polyethylene glycol 2318. 20
  • 21.
    21 GC Oven ■ GCoven incorporate a fan-ensures uniform heat distribution throughout the oven. ■ Temperature conditions : ➢ isothermal ➢ Gradual increase in temperature ■ Oven programming rates can range from 1o C/min to 40oC/min. ■ Eg:60oC (1 min)/5oC/min to 100oC (5 min)/10oC/min to 200oC (5 min). ■ Materials of different volatilities can be separated in a reasonable time and also injection of the sample can be carried out at low temperature, where it will be trapped at the head of the column and then the temperature can be raised until it elutes. 21
  • 22.
  • 23.
    23 Column Temperature Eg.1 􀂄Separations/Resolutionsare good, but the analysis time is too long.
  • 24.
    24 Column Temperature Eg.2 ❑Short analysis time, but separations/resolutions are not good.
  • 25.
  • 26.
    26 ■ Start fromlow temp and then raise the temp to a higher one in a certain rate of increase. ■ e.g.: 50 C (2 min) →20 C/min C/ → 200C (5 min) ■ 80 C (5 min) → 10C/min → 150C (3 min) → 5C/min → 180C (15 min) ■ The lower the rate of temp increase, the better the resolution is.
  • 27.
    27 Column Support ■ Inertmaterials used ■ Acid washing- to reduce peak tailing ■ Deactivation of support surface ■ 10-15% (w/w) of stationary phase is coated on the support ■ Common supports : diatomaceous earth materials ■ Commercially known as chromosorb : different grading – P,W, G etc., 27
  • 28.
    28 Theory of GasChromatography ■ Adsorption: the molecules of analyte reside on the stationary phase for sometime and then desorbed and eluted out along with the mobile phase. ■ Solid adsorbent is used as a stationary phase. ■ Partition : the molecules of analyte get distributed between the mobile and stationary phases. ■ Stationary phase : high molecular weight liquids. ■ If the con. Of the analyte is more in stationary phase, the elution takes more time. ■ If concentration of analyte is less in stationary phase, the elution takes less time. 28
  • 29.
    29 Partition coefficient (β) ■GC separation = distillation separation. ■ β = weight of the solute per gram of the stationary phase Weight of the solute per cc of the carrier gas ■ β is characteristic of each one of the components ■ In addition to partition, other physical interactions like – hydrogen bonding, wander Waal’s forces ■ Nature of analyte, stationary and mobile phases. 29
  • 30.
    30 Height Equivalent toa Theoretical Plate (HETP) ■ Efficiency of a chromatographic separation is expressed in terms of the number of theoretical plates (N) ■ Number of theoretical plates (N) = 16 (tR/Wb)2 ■ Where t = the net retention time of the sample ■ Wb = width of the peak at the base ■ HETP = L/N ■ L = Length of the column, N = number of theoretical plates ■ If the value of N is more the efficiency of the column is more. If the value of N is less the efficiency of the column is less. 30
  • 31.
    31 Derivatization Techniques ■ i)To permit analysis of compounds not directly amenable to analysis due to inadequate volatility or stability ■ ii) To improve the analysis by better chromatographic behavior or detectability. ■ Replacement of N-H, O-H and S-H groups by alkylation, acylation, silylation increases the volatility. ■ Closely related compounds, optical isomers can be separated after derivatization. 31
  • 32.
    32 Silylation Typical reagents ■ i)Hex methyl disilazane (HMDS) ■ (CH3)3SiNHSi(CH3)3 ■ ii) Trimethyl chlorosilane (TMCS) ■ (CH3)3SiCl ■ Iii) Dimethyl chlorosilane (DMCS) ■ (CH3)2SiCl ■ iv) Tetramethyl disilazane (DMCS) ■ (CH3)2HSiNHSi(CH3)2H ■ The reagent is used to replace an active protons to improve thermal stability or to reduce tendency to form hydrogen bonds. ■ Ex: sugars, thiols, steroids, amino acids 32
  • 33.
    33 Acetylation ■ To replaceactive hydrogen ■ Alcohols, amino acids, 1 or 2 amines ■ Amino acids are usually esterified to methyl or butyl esters prior to acetylation. ■ Typical reagents : acetic anhydride & Trifluoroacetic anhydride dissolved in pyridine & DMF. ■ Trifluoroacetyl derivatives. 33
  • 34.
  • 35.
  • 36.
    36 Esterification ■ Carboxylic acidanalogs, long chain fatty acids derivatized by using HCl in methanol or diazomethane. ■ Aminoacids can be esterified by reacting with butanol in 3N HCl at 150ο C for 10 minutes. ■ Diazomethane – more specific, minimal side reactions. ■ Disadvantage : toxic and potentially explosive 36
  • 37.
    37 Analysis of fattyacid composition BP Monographs- GC analysis to confirm the content of fatty acid composing the triglycerides Almond oil states the composition of the fatty acids making up the triglyceride : Palmitic acid (16:0) 4.0-9.0% Palmitoleic acid (16:1) < 0.6% Margaric acid (17: 0) < 2.0 % Stearic acid (18:0) 0.9-2.0% Oleioc acid (18:2) 62.0-86.0% Linoleic acid (18:2) 7.0-30.0% Linolenic acid (18:3) < 0.2% Arachidic acid (20:0) < 0.1% Behenic acid (22:0) <0.1% 37
  • 38.
    38 Methanolysis of triglyceridesprior to GC analysis 38
  • 39.
  • 40.
    40 GC performance ■ CarrierGas: Hydrogen or helium are used in GC Typical flow rate = 30-50 cm/s ■ Nitrogen has its optimum flow rate = 10-20 cm/s ■ Gas flow rate decrease with increase with column temperature (column efficiency) ■ Modern instruments have flow programming so that the flow can be set to remain constant as the temperature raises. 40
  • 41.
  • 42.
    42 Column Temperature ■ Ascolumn temperature increases, the degree of resolution between two components decreases. ■ Degree of interaction with stationary phase is reduced as the vapour pressure of the analytes increases. ■ Lower temperatures produce better resolution 42
  • 43.
    43 Column length ■ Separatingpower of column varies as the square root of its length. ■ If two fold increase in resolution is required, a four fold increase in column length. ■ So, four fold increase in analysis time ■ Decrease in temperature, ensuring more interaction with stationary phase- better resolution. ■ 43
  • 44.
    44 Film thickness phaseloading ■ The greater the volume of stationary phase, the more a solute will partition into it. ■ If the film thickness or loading of stationary phase doubles then, the retention of an analyte should double. ■ Thicker films are used for very volatile materials to increase their RT. ■ To increase resolution between analytes without increasing the column length. 44
  • 45.
    45 Internal diameter ■ Thesmaller the internal diameter of a capillary column, more efficient. ■ Mass transfer characteristics of the column are improved ■ Analyte being able to diffuse in and out of the mobile phase more frequently because of the shorter distance for transverse diffusion. 45
  • 46.
    46 Question ? ■ Afixed temperature is used and the head pressure is adjusted so that the linear velocity of a helium carrier gas through the following capillary column is 20 cm/s: OV-1 film ■ (i) 30 m × 0.25 mm i.d × 0.25 µm ■ (ii) 15 m × 0.15 mm i.d × 0.2 µm ■ (iii) 12 m × 0.5 mm i.d × 1.0 µm ■ A) List the columns in the order in which they would increasingly retain a n-hexadecane standard ■ B) list the columns in order of increasing efficiency. 46
  • 47.
    47 Detector ■ 1. Detectoris to indicate the presence and measure the amount of component eluted out from the column. ■ 2. High detector temperature should be set to prevent condensation of sample
  • 48.
    48 Type of Detectors 1.Flame Ionization Detector (FID) 2. Thermal Conductivity Detector (TCD) 3. Flame Thermionic Detector (FTD ) 4. Flame Photometric Detector (FPD) 5. Electron Capture Detector (ECD) 6. Mass Spectrometer Detector (MSD) TCD, FID, MSD are general detectors.
  • 49.
    49 Type of Analysis ■FID ::-Detects any compounds that can be oxdised in hydrogen/air flame ■ ECD:-Selective to electronegative moieties e.g.halogens ■ FTD:-Selective to organic N or P compounds ■ FPD:-Selective to P or S containing compounds ■ TCD:-Detects any component including N2 and O2 except the gas used for the carrier gas.
  • 50.
    50 FID ■ Most organiccompounds can be detected. These compounds are oxidized in hydrogen / air flame
  • 51.
    51 Flame Ionization Detector(FID) ■ For organic compounds analysis ■ Hydrogen and air are needed to create the flame ■ Sample is brought to hydrogen flame and converted into ions. Current will be generated. ■ The current is proportional to the amount of the organic compound present ■ Advantages like
  • 52.
    52 ■ High Sensitivityto organic compounds ■ Little or no response to water, CO2, the common carrier gas impurities hence zero signal to when no sample is present ■ Stable baseline which is not mostly affected by fluctuations in carrier gas temp, flow rate and pressure. ■ Good linearity over a wide range of sample conc. Range ■ Presence of halogens in the compound decreases sensitivity
  • 53.
    53 TCD ■ Any componentsincluding N2 and O2 except the gas used for carrier gas can be detected by TCD ■ Wheatstone bridge is used as principle of detection.
  • 54.
  • 55.
    55 Thermal Conductivity Detector(TCD) ■ Detects almost all compounds except the carrier gas. ■ The TCD filament is heated by applying a current ■ When carrier gas + sample gas passes over the filament, the temperature ■ Of the filament increases, because the thermal conductivity of the sample compounds is less than that of the carrier gas alone. ■ The changes in filament temperature affect its resistance ■ The resistance change is measured and produces the signal
  • 56.
    56 ■ – Advantagesover FID ■ Responds to all organic as well as inorganic compounds ■ Non destructive type of detector hence can be used for trapping separated components and preparative analysis ■ Low cost and versatile.
  • 57.
    57 Electron Capture Detector(ECD) 􀂄Very selective and sensitive to electrophilic compounds (e.g. halogens)
  • 58.
    58 ECD 􀂄Detects electrophilic compounds e.g.:halogenated compounds 􀂄Good for low concentration of these kind of compounds 􀂄Contains 63Ni radioactive 􀂄Examples of analyses: ■ analysis of PCBs (polychlorinated biphenyls) ■ analysis of PBBs(polybrominated biphenyls) ■ analysis of PBDEs (polybrominated diphenyl ethers) ■ analysis of organochlorine pesticides (DDT, BHC, etc.)
  • 59.
  • 60.
  • 61.
  • 62.
  • 63.
    63 I = 100n+ 100 log Rx – log Rn log Rn+1- log Rn
  • 64.
    64 Quantitation Methods ■ AreaPercent Method : ■ Area percent is the simplest quantitation method. ■ This method assumes that thedetector responds identically to all compounds. ■ This assumption, however, is not valid. ■ This method provides a rough estimate of the amounts of analytes present. 64
  • 65.
    65 ■ To calculatearea percent take the area of an analyte and divide it by the sum of areas for all peaks. ■ This value represents the percentage of an analyte in the sample. 65
  • 66.
    66 Single Point ExternalStandard ■ method requires the analysis of more than just the sample of interest. ■ Analyze a sample containing a known amount of analyte or analytes and record the peak area. ■ Then calculate a response factor using Equation 1. ■ 66
  • 67.
    6767 ■ Inject asample with the unknown analyte concentration and record the peak area. ■ Then calculate the amount of analyte using Equation 2. ■ Calculate an individual response factor for each compound of interest.
  • 68.
    68 SINGLE PT. EXT.STD. EXAMPLE ■ An injection containing benzene at a concentration of 2,000 μg/ ml is made and results in a peak area of 100,000. ■ Calculate the response factor for benzene ■ An injection of the sample with the unknown concentration of benzene has a peak area of 60,000. ■ Calculate the amount of benzene present
  • 69.
    69 Single Point InternalStandard ■ The analyte chosen for the internal standard has a predictable retention time and area, allowing it to be used to determine if abnormalities have occurred. ■ The Single Point Internal Standard method requires at least two analyses. ■ The first analysis contains a known amount of internal standard and the compounds of interest.
  • 70.
    70 ■ Then adda known amount of the internal standard to the sample containing analytes of unknown concentrations. ■ Calculate the amount of the unknown analyte using Equation 4.
  • 71.
  • 72.
    72 ■ In thismethod an internal standard is chosen which is similar in structure to the components of the mixture under investigation. ■ The internal standard chosen should be such that ■ 1. it should separate from all the components of the sample mixture under investigation ■ 2. it should not react with the components of the sample ■ 3. it should be stable at the operating temperature of the column ■ 4. it should have a fairly good detector response
  • 73.
    73 SINGLE POINT INTERNAL STANDARDEXAMPLE ■ Prepare a sample containing 2,000 μg/mL of toluene (the internal standard) and 1,000 μg/mL benzene (the analyte). ■ Then inject the sample. The resulting peak areas are 120,000 for toluene and 67,000 for benzene. ■ Using Equation 3 the response factor for benzene is:
  • 74.
    74 ■ Inject thesample containing 2,000 μg/mL of toluene and an unknown amount of benzene using the same chromatography conditions. ■ The resulting areas are 122,000 for toluene and 43,000 for benzene. ■ Calculate the amount of benzene present ?
  • 75.