Chapter 11 Conventional Gas Chromatography

Transcription

1 Chapter 11 Conventional Gas Chromatography

2 Gas Chromatography GC is the first instrumental chromatographic method developed commercially It is relatively easy to introduce a stable flow and pressure for the mobile phase, that is the carrier gas The equipment is simple. All that is really needed is cylinder of compressed air, pressure regulator and a valve

3 Introduction Principles Partition of molecules between mobile phase and stationary phase Separation technique Gas is the mobile phase and liquid (GLC) or solid (GSC) is the stationary phase GLC: liquid is coated on an inert solid; separation is the result of variable solubilities in the liquid phase GSC: Particulate solid like molecular sieve is the stationary phase; separation is the result of variable adsorption on the solid surface

4 Abundanc e Gas Chromatography E A D Gas Chromatograph C Sample: mixture of volatile liquids (~1 L) B Gas Chromatogram A B C E D Time (minutes)

5 Uses of GC Separation and analysis of organic compounds Testing purity of compounds Determine relative amounts of components in mixtures Compound identification Isolation of pure compounds (micro scale work)

6 Advantages of the GC Speed: minutes or seconds Resolution: complex samples Sensitivity: 10-9 or even g/s Versatility: Gases, liquids or solids (qualitative & quantitative) GLC is more common than GSC in terms of flexibility and resolution Salts and other ionic compounds + high molecular weight may not be determined by the GC. They can be determined by liquid chromatography or pyrolysis chromatography

7 GC Process 1. Column is selected, packed with liquid phase, and installed 2. Sample injected with microliter syringe into the injection port where it is vaporized and mixed into the carrier gas stream (helium, nitrogen, argon). 3. Sample becomes partitioned between the moving gas phase and the stationary liquid phase. 4. The time spent by different compounds of the sample in vapor phase is a function of their vapor pressure 5. The more volatile compounds arrive at the end of the column first and pass into the detector

8 Factors Affecting Separation Boiling Points of Components in Sample Low boiling compounds have higher vapor pressures. Boiling point increases with increasing molecular weight Flow Rate of Carrier Gas Choice of Liquid Phase (Solubility in the liquid stationary phase determines the retention time in the stationary phase) Molecular weights, functional groups, and polarities of component molecules are factors in selecting liquid phase. Length of Column Similar compounds require longer columns than dissimilar compounds. Isomeric mixtures often require quite long columns

9 Mechanism of Separation of Components of a Mixture by Chromatography Stationary Phase Start Mobile Phase Affinity of molecule For the stationary Phase determines Residence time In this phase. high affinity low affinity

10 GC Instrumentation Carrier gas Sample Injector Column Detector

11 Schematic Diagram of a Gas Chromatograph

12 RESET Gas Chromatography System Filters/Traps Data system H Regulators Syringe/Sampler Inlets Air Hydrogen Gas Carrier Column Detectors gas system Inlet (Injector) column detector Readout system

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14 Gas Flow

15 Carrier Gas Carrier gases, must be chemically inert, Include helium, argon, nitrogen, carbon dioxide, and hydrogen. The choice of gases is often dictated by the detector used. Associated with the gas supply are pressure regulators, gauges, and flow meters. The carrier gas system often contains a molecular sieve to remove water or other impurities.

16 Purity of Carrier Gases Impurities (particularly O 2 & H 2 O) can chemically change the liquid phase and thus the t R and reduce the lifetime of the column. False peaks may appear Liquid stationary phases (polyesters, polysiloxanes, polyamides) degrade by O 2 & H 2 O Contaminant from column may desorb in H 2 O causing a high detector background (baseline drift and noise) Traces of hydrocarbons cause a high background in the FID Purity should be >99.995%

17 Carrier gas: mainly He and N 2 Two stage pressure regulator 1. It indicates the pressure left in the cylinder (min. 40 psi) 2. It indicates the increasing pressure delivered to the GC (min. = 20 psi)

18 Function Introduce sample Vaporize sample Split sample Injection systems Main Components It is a metal block containing: Heaters Temperature sensors Septum holder on the front Connection for the column on the rear

19 Packed column injection Injector is a simple portion of the GC system when packed columns are used There are two basic approaches for these injectors Injection ports Sampling loops/ valves

20 Various Types of Injection ports Split - only a small portion (10:1 and 100:1) of the injected sample goes on column Splitless - all material injected goes on column Usually split/splitless modes are provided to the same instrument. Cool on-column injection (Direct on column cold injection. It is used for sensitive materials) Programmed temperature - sensitive materials (more durable method than OC)

21 Rubber septum serves for about 30 injections in ordinary care 5-10 injections in case of large syringes

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23 Injection Port/ Temperature Effects The purpose of the port is to flash vaporize the sample and introduce it into the column T injector should be > 50 o C above T column Septum of the injector must be: Stable at the T inj Replaced regularly to maintain seal

24 Syringe Injection Methods Major source of precision error results from poor injection technique Both automatic and manual injection methods are available If automatic equipment is present, use it. If not, several approaches can be tried to help reduce injection errors

25 Injection of liquid sample 10 µl syringe is the most popular device Load the desired volume of liquid, then draw the plunger back to pull the liquid out of the needle Insert the needle quickly through the septum as far as it will go Depress the plunger and immediately remove the needle from the injection port

26 Gas Sampling Loops (Sampling Valves) Introducing a constant amount of a gas can be difficult with a syringe Gas sampling loops and valves offer a high precision (+/- 1%) means of introducing gases Equipment is relatively inexpensive and only requires a constant temperature for easy use

27 Gas Sampling Loops/Valves Valves give better reproducibility Require less skill Can be easily automated

28 Injection valve

29 Injection Port Temperature The temp. should be o C hotter than the boiling point of the least volatile component But low enough to prevent sample decomposition and septum bleed Temp. may be checked by raising it and watching : Position, or area, or shape of the peaks. Drastic changes mean the temp. setting is high It should be 10% above that of the column to ensure rapid volatilization of the sample. The efficiency of the column is almost constant under this condition Components may be vaporized at a temp. ~100 o C below its atmospheric boiling point

30 Very high boiling point or temp. sensitive material can be handled by dilution with volatile solvent that permits lowering the injection temp. This will lower the sensitivity! Try various temperatures until peak broadening becomes apparent With temp. programming techniques low injection temps become very practical. No rush to vaporize the high boiling components

31 Effect of injection port temperature on resolution a: Methanol; b: Ethanol; c: ispropanol Boling points: o C

32 Gas Chromatographic Columns 1. Packed Columns for GLC 2. Packed Columns for GSC 3. Capillary Columns

33 Types of columns Packed columns 1/8 1/4 OD, stainless steel or glass tube 6-20 feet in length Capillary (open tubular) columns mm ID meters in length

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36 Capillary (Open tubular columns)

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39 Packed Columns 1. Column material 2. packing material (solid or inert support) 3. Stationary phase

40 Column Material Stainless Steel : most common. adsorbs some compounds particularly polar ones & especially water. Copper tubing. It is good for trace water analysis. Plastics: are limited due to permeability & temperature limit (used for reactive or highly corrosive chemicals H 2 S, HF Teflon, polypropylene and nylon tubing are available. Glass : If glass were not difficult to form into columns & relatively fragile it would be the very best choice for tubing ( used for pesticides & steroid).

41 Solid Support (Stationary Phase Support) Thermally stable Diatomaceous earth is the most common (nearly, all silica) solid support. Diatomaceous earth: Skeletons of microscopic unicellular algae (diatoms) consisting of microamorphous hydrous silica marketed under Chromosorb Polar analyte species such as, Alcohols or Aromatic hydrocarbons are adsorbed physically on the silicate surfaces of diatomaceous earth. Adsorption results in distorted peaks broadened with tail This catalytic activity may lead to sample decomposition

42 Reasons for adsorption activity Silicates + Water Silanol groups on the silicate surface OH OH OH OH Si O Si O Si O Si Si-OH groups have strong affinity for polar organic molecules

43 Treatment of Solid Supports Non-acid washed (NAW) an untreated form Acid washed (AW) use HCl Removes metals, impurities, Reduces surface activity and absorption Acid washed Dimethyldichlorosilane treated (AW-DMCS) Cl OH Si + (CH 3 ) 2 SiCl 2 HCl CH 3 O Si CH 3 Si CH 3 CH 3 O Si CH 3 Si HCl CH 3 OH

44 Stationary Liquid Phase

45 Characteristics of Stationary Liquid Phase The stationary phase should provide separation of the sample with a reasonable column life Suitable phase is chosen on the basis of : Experience or Experiment. It is desirable to have maximum information about the sample composition : bp.range, components expected & their structure Stationary phases should have similar chemical structure to the sample components

46 ( is the separation factor)

47 Commonly Used Liquid Phases PHASE TEM. LIMITS Good for 1. SQUALANE 0/125oC Nonpolar 2. OV-1, SE /350 oc 3. DEXSIL /350 Oc (Most thermally stable) 4. OV-17; SP Moderately polar 5. QF-1; OV-210; SP CARBOWAX-20M 60/225 Strongle polar 7. DEGS 20/ OV /250

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49 Squalane Saturated, highly branches, C-30 hydrocarbon Non-polar Limited temperature range: oc Separate hydrocarbons Standard reference for Rohrschneider and McReynolds constants

50 Non-polar Temperature range: oC Most widely used liquid phase Separate all sample types OV-1 ( SE-30 )AND SP-2100

51 OV-17 (SP-2250) 50% methyl, 50% phenylpolysiloxane Semi-polar Temperature range : 0-350oC Widely used to separate drugs, steroids, carbohydrates

52 Carbowax 20-M HO ( - CH 2 CH 2 O-) n H Polymeric polyethylene glycol Polar Temperature range : oC Widely used for polar samples

53 Column Temperature 1. As the Column temp. increases a sample component spends more time in the mobile phase. * This will cause a decrease in the t R (Faster separation) * t R doubles for every 30 o C decrease. 2. Increasing the temp. decreases the band broadening since it leads to a decrease in the available time for diffusion in the column. 3. The lower the temperature the better is the separation * The column temp. should not be less than about 10 o C below the bp of the highest-boiling sample component (other wise distorted peaks may result). * Roughly, a temp. equal or slightly above the average b.p of a sample results in a reasonable elution time (20 to 30 min.). 4. Optimum column temp. depends upon: b.p. of the sample Degree of separation required.

54 Isothermal and temperature programming analysis The column is placed in the oven If the temperature is held constant during the entire analysis then it is called analysis under an isothermal mode. If the temperature is varied during the analysis, then the process is called temperature programming

55 Column Temperature Effect (Isothermal Analysis) Isothermal chromatographic analysis is one which is performed at a constant column temperature.

56 Conclusion Higher temp. enables rapid analysis but loss in resolution. Lower temp. achieves better resolution but longer analysis time

57 Narrow Boiling Range Samples Isothermal column temperature should be used. Select temperature o C lower than boiling range of sample when thin films are handled. Use highest temperature that still allows adequate resolution and stability to shorten analysis time.

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59 PERCENT LOADING OF STATIONARY PHASE How much liquid phase should be coated on the support? For analytical columns: 5 10% Higher loading ( up to ~ 30% ) can be applied for light gases (C 1 to C 4 hydrocarbons) Low Loading (1-5% ) with very high boiling compounds The liquid phase should cover the support almost completely. If the support is inert, this condition is not a must

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61 LIQUID PHASE AND TEMPERATURE LIMIT To avoid decomposition, column bleeding and high retention Practical Temperature Limits depend upon the following: * Nature of liquid phase * Percent loading * Sensitivity of detector Temp. limits reported in most cases for TCD Limits; for FID may be about 100 o C less than that for the TCD.

62 Conditioning the column Why? Remove: impurities in liquid phase ; residual solvent from coating How? Install the column but do not connect to the detector Set the carrier flow 30 ml/min for 1/8 inch Heat for 1 hr at 100 o C Raise the temperature to slightly below the b.p. of liqid phase (about 30 o C higher than expected operating column operation temp.(. Continue heating over night Cap the column when it is removed from the instrument

63 Deatectors Features of Detectors A device that measures physical properties (preferred), not chemical properties The detector generates an electrical signal proportional to the sample concentration Detector and connections must be hot enough (20 to 30 o C above the column temp. or the boiling point of the highest boiling component) so that condensation of the sample or liquid phase does not occur

64 Peak broadening or disappearance is characteristic for condensation in the connections Ionization type detectors must be maintained at temp. high enough to avoid not only condensation of sample but also the water or by-products formed in thr ionization process

65 Most Common GC Detectors Most common detectors roughly in order from most common Thermal Conductivity Detector (TCD or hot wire detector), Flame Ionization Detector (FID), Electron Capture Detector (ECD), Photo Ionization Detector (PID), Flame Photometric Detector (FPD), Thermionic Detector VERY expensive choices: Atomic Emission Detector (AED) Ozone- or Fluorine-Induced Chemiluminescence Detectors. All of these (except the AED) produce an electrical signal that varies with the amount of analyte exiting the chromatographic column. Fourier Transform Infrared Detector (FTIR) Mass Spectrometer (MS) Other: UV, FT-NMR

66 Schematic of a thermal conductivity detector, TCD

67 Two pairs of TCDs are used in gas chromatographs. One pair is placed in the column effluent to detect the separated components as they leave the column. Another pair is placed before the injector or in a separate reference column. The resistances of the two sets of pairs are then arranged in a bridge circuit. The heated element may be a fine platinum, gold, or tungsten wire or, alternatively, a semi conducting thermistor. The resistance of the wire or thermistor gives a measure of the thermal conductivity of the gas. Elution heat loss increased resistance needed to balance bridge = recorded

68 Flame Ionization Detector Basic Principle The effluent from the column is mixed with hydrogen and air, and ignited. Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame. A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame. The current resulting from the pyrolysis of any organic compound is measured which is proportional to the carbon content of the molecule entering.

69 Ring electrode: stainless Steel gauze (+ve electrode) Flame jet serves As Ve Electrode (FID) sample burned in H 2 /air flame sample must be combustible must use electrometer ppm sensitivity destructive

70 Qualitative analysis by Chromatographic methods Qualitative analysis is based on retention data Retention time t R is characteristic of a substance, compared to a standard. Reproducibility of retention depends upon several experimental conditions: column length and diameter, stationary and mobile phases, column packing, column temperature, mobile phase flow rate and others

71 Retention time, t R t R : It is the time elapsed from the point of injection to the peak maximum Adjusted t R : It is the time from the maximum of unretained peak (the peak of the mobile phase or the air peak) to the peak maximum of a certain component t M (hold up time): is the time required for the mobile phase to be eluted completely from the column

72 Same column under Same conditions Has been used Unknown alcohol components Standard sample

73 Component 1 is used as the reference; it should be present or added to the sample and compatible with the sample Peak of component 1 must be close (but resolved) to the sample peak

74 When component 3 is suspected, add more of this component to the sample and watch any change in its peak

75 Basis for Quantitative Analysis The peaks in the chromatogram are the basis for quantitative analysis Peaks of interest should fulfill the following requirements: must be undistorted must be well separated Must have a large S/N ratio must have a flat baseline Peak shape: The ideal chromatographic peak is symmetric and narrow Peak integration The peak height or, better, the area must be determined and this is done by the computer Calculation External standard method Internal standard method Internal normalization

76 External standard method This method is common to most quantitative analysis techniques. It allows the measurement of the concentration of one or more components that elute in a chromatogram containing, perhaps, many peaks. This method, employing the absolute response factor, K, is used in the following way (Single point calibration method)

77 Multilevel calibration In a multilevel calibration several different amounts of the standard are prepared and analyzed. A regression method is used (e.g. linear least-square) and this leads to a more precise value for C unk. This quantitative method is the only one adapted to gaseous samples. This simple method is used in industry for repetitive analyses. For such analyses, chromatograph must be equipped with an autosampler,

78 Multilevel External Calibration of Fatty Acids Detector Response C 18 Peak Area (cm 2 ) 10 C C Retention Time The content % of C fatty acids = 14 C C + C + C Sample Concentration (mg/ml) = the content % of C fatty acids 14