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Gas Chromatography. Gas Chromatography (GC) This method depends upon the solubility and boiling points of organic liquids in order to separate them from.

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Presentation on theme: "Gas Chromatography. Gas Chromatography (GC) This method depends upon the solubility and boiling points of organic liquids in order to separate them from."— Presentation transcript:

1 Gas Chromatography

2 Gas Chromatography (GC) This method depends upon the solubility and boiling points of organic liquids in order to separate them from a mixture. It is both a qualitative (identity) and quantitative (how much of each) tool.

3 Gas Chromatography (GC) Introduction – Overview of Topics Applications – Most common for volatile compounds – More common for non-polar to moderately polar compounds Columns (packed vs. open tubular) Sample Injection Detectors

4 Process Flow Schematic Carrier gas (nitrogen or helium) Sample injection Long Column (30 m) Detector (flame ionization detector or FID) Hydrogen Air

5 Gas Chromatograph Components Flame Ionization Detector Column Oven Injection Port top view front view

6 GC Theory An inert gas such as helium is passed through the column as a carrier gas and is the moving phase. A sample is injected into a port which is much hotter than the column and is vaporized. The gaseous sample mixes with the helium gas and begins to travel with the carrier gas through the column. As the different compounds in the sample have varying solubility in the column liquid and as these compounds cool a bit, they are deposited on the column support. However, the column is still hot enough to vaporize the compounds and they will do so but at different rates since they have different boiling points. The process is repeated many, many times along the column. Eventually the components of the injected sample are separated and come off of the column at different times (called "retention times"). There is a detector at the end of the column which signals the change in the nature of the gas flowing out of the column. Recall that helium is the carrier gas and will have a specific thermal conductivity, for example. Other compounds have their own thermal conductivities. The elution of a compound other than helium will cause a change in conductivity and that change is converted to an electrical signal. The detector, in turn, sends a signal to a strip chart recorder or to a computer. Detectors come in several varieties, for example, thermal detectors, flame-ionization and electron capture detectors.

7 GC Theory To detector Analyte Column + packing Time

8 Separations To detector Time 1Time 2Time 3

9 GC Process

10 GC General The chromatogram shows the order of elution (order of components coming off the column), the time of elution (retention time), and the relative amounts of the components in the mixture. The order of elution is related to the boiling points and polarities of the substances in the mixture. In general, they elute in order of increasing boiling point but occasionally the relative polarity of a compound will cause it to elute "out of order". This is analyzing your sample.

11 Elution Order Compound Boiling Point ( 0 C) pentane 36 hexane 69 cyclohexane 80 isooctane 99 toluene methyl-2-pentanone 117 octane 126

12 Example Chromatogram The observed elution pattern appears below. Notice the reversed elution of toluene and 4- methyl-2-pentanone.

13 GC Chromatogram

14 Components of GC: Column, oven, injector, and detector. These parameters (HETP, etc) are affected by the various components of the instrumentation. Perhaps the column is the most important component of the GC. With it, different separations can be accomplished.

15 GC Instrumentation Carrier gas The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities. Sample injection port For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution. The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample. For packed columns, sample size ranges from tenths of a microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample, typically around 10-3 mL. For capillary GC, split/splitless injection is used. Have a look at this diagram of a split/splitless injector;

16 Sample Injection and Pretreatment Gas Sample: if present at moderate to high concentration, may be directly put into column via gas- tight syringe. Another technique is to use a gas-tight valve: For trace levels like volatile organic compounds (VOC), the sample may need to be preconcentrated by passing it through a solid-absorbing cartridge or using a cryogenic trap.

17 Sample Injection and Pretreatment Liquid Samples: direct injection of a relatively large sample (6-10 mL). A calibrated microsyringe may be injected through a gas-tight septum into a heated chamber that volatilizes the sample. Note that there are direct, split, and splitless column styles. Sometimes pretreatment requires dervitatization or moving the sample into a different solvent (remove from H 2 O). Static or Dynamic Headspace analysis of vapor phase above a sample avoids water in the sample.

18 Sample Injection and Pretreatment Solid Samples: First extract compounds of interest from a solid matrix by liquid-liquid extraction or supercritical fluid. Samples are placed in solvent, then treated as liquid samples. Thermal desorption may be used on some solids as analytes may be collected during heating of a solid. Pyrolysis GC is employed for substances that are not volatile and cannot be easily derivatized to volatile forms. The solid is heated in a controlled way to break it into smaller, more volatile pieces separated by the column to form a pyrogram, which can be matched to known standards.

19 Sample Injections Next, the sample injection system. Here it is important that the sample be injected onto the column as a plug and of a suitable size. Also, the injector should provide consistent and reproducible injections. See Figure 27-3, Pg 704. The micro- syringe is used to load the sample onto the column. The syringe should be clean and accurate and gas tight. The syringe is injected through a rubber septum. The septum should be replaced after many injections to insure gas tightness onto the column. An auto sampler can be used to inject the samples. Typical volumes range from 0.2 to 20 Ls. With capillary columns it is necessary to use a splitter. (See Figure.) A suitable solvent is also necessary for the proper separations and injections.

20 GC Injector

21 GC Injection Gas Samples – various methods including injectors used for liquid samples (below), fixed loop injectors (described later with HPLC), and solid phase microextraction (SPME) Liquid Samples – on-column injection (needle goes into column) – direct injection – above two methods mainly for packed columns – split/splitless injector (next page)

22 GC Sample Injection – Liquid Samples Split/Splitless Injectors – Injectors capable of running in two modes: split and splitless – Split injections used to avoid overloading columns Injection Process – Syringe pierces septum and depressing plunger deposits liquid – Analyte volatilizes – Part injected (usually smaller fraction) – Part passed to vent – Fraction vented depends on split valve outside Inside oven Septum Syringe port He in To Column Split vent Split valve liner

23 GC Sample Injection – Liquid Samples Split injection is used for: – Higher concentrations – Smaller diameter (OT) columns – Greater need for high resolution than high accuracy In split injection, solvent overload is less problematic Splitless injection is used for trace analysis (~50% of injected sample put on column)

24 Carrier Gas This is the mobile phase and should be pure gas so as not to react with the column or analyte. Gas is usually He, Ar, N 2, or H 2. Choice will depend on the type of detector used. He and H2 give better resolution (smaller plate height) than N 2. Pressure is also important and as expected the system comes with regulators. Can you find where in GC equations that are dependent on pressure?

25 Columns The column is the most important component of GC. Here is where the separations take place. All the various equations we discussed above are dependent on properties of the column. There are four types of columns: wall-coated open tubular (WCOT), support coated open tubular (SCOT), micropacked, fused silica open tubular (FSOT), and packed column. The FSOT column is the most flexible. Open tubular is also capillary. Particle size is important because the efficiency of GC column increases rapidly with decreasing particle size of the packing material.

26 Column The column sits in a temperature controlled environment that is Temperature is very important in GC. Can you remember what equations are affected by temperature? See page 706 Fig for temperature effects on separations. Normally, one does a temperature program to get the various analytes off the column for better separations (resolutions).

27 Columns There are two general types of column, packed and capillary (also known as open tubular). Packed columns contain a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are m in length and have an internal diameter of 2 - 4mm. Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns. In 1979, a new type of WCOT column was devised - the Fused Silica Open Tubular (FSOT) column;

28 FSOT column

29 Capillary column

30 GC Columns Two Common Formats – Packed columns (most common with bonded liquid coating) – Open tubular (typically long columns with small diameters) Advantages of packed columns – Greater capacity (can handle larger masses injected on the column) – Lower cost/somewhat more variety of stationary phases (common as specialty column) Bonded liquid in green Packed Columns Open Tubular (end on, cross section view) Column Wall (fused silica) Mobile phase Stationary phase (wall coating)

31 GC Columns Advantages of Open Tubular Columns – Best resolution (negligible A term, small C term in van Deemter Equation) – More robust – Better sensitivity with many detectors (due to less band broadening vs. lower mass through column) Column Selection – High resolution (thin film, 0.25 mm diameter, 60 m) vs. higher capacity (thick film, 0.53 mm diameter) – Stationary phase based on polarity

32 GC Stationary Phase Selection of stationary phase affects k and α values Main concerns of stationary phase are: polarity, functional groups, maximum operating temperature, and column bleed (loss of stationary phase) More polar columns suffer from lower maximum temperatures and greater column bleed TypeFunctional GroupsPolarity OV-1methylNon-polar OV-1750% methyl/50% phenyl Somewhat polar OV-225Cyanopropyl, methyl, and phenyl More polar carbowaxEther groupspolar

33 GC Adjustments k is adjusted by changing temperature (higher T means smaller k) the values are adjusted by changing column (will work if there is a difference in solute polarity) – example: separation of saturated and unsaturated fatty acid methyl esters (FAMEs). – Retention of C18:0 and C18:1 FAMEs on OV-5 columns is very similar (due to similar boiling points) – Retention on more polar columns (e.g. OV-17) is greater for the more polar unsaturated FAMEs Changing carrier gas has no effect on retention C18:0 FAME C18:1 FAME bp = 369°C bp = 352°C

34 GC Gradients In GC, a gradient elution can be performed by increasing the oven temperature during the run This is useful when sample solutes have a wide variety of volatilities (or at least retention times) This results in a continuation of narrow peaks and less time between peaks, less total time and better late peak S/N ratios Note: Data from HPLC, but looks very similar to GC chromatograms for a series of compounds with extra –CH 2 - groups Isocratic Run Gradient Run

35 GC Detectors Performance criteria – limit of detection (low is better) – Linear range (or useful range for non-linear detectors) – Destructive vs. non-destructive – Other (cost, speed, size, precision, etc.) Most GC detectors perform well for cost vs. HPLC

36 Instrumentation Detectors There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound. Detectors can also be grouped into concentration dependant detectors and mass flow dependant detectors. The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas.

37 GC Universal Detectors Thermal Conductivity Detector (TCD) – Principle: heat conduction by gas (dependent on gas molecular weight) – All gases detected, but variable sensitivity. – Not very sensitive Flame Ionization Detector (FID) – Principle: GC effluent burned in hydrogen flame producing ions measured by electrode – Good sensitivity – Hydrocarbons are detected with reduced response for other functional groups – Quantitation is good and possible without standards

38 GC Detectors - Classes Universal Detectors – Should detect wide range of compounds – Response should be based on mass or moles of analyte (so can quantitate with surrogate standards) – Used for comprehensive understanding of samples Selective Detectors – Should detect only specific types of compounds – Best for selected analytes in complex samples Multi-dimensional Detectors (selective and universal – MS is most common)

39 GC Selective Detectors Based on Gas Ionization – Electron Capture Detector (ECD) Uses β emitter to produce electrons that cause current Very sensitive detector Selective for compounds with electronegative groups (e.g. halogens, nitrogroups, etc.) – Photoionization Detector (PID) Uses UV light to photoionize compounds (M + h ν M + + e - ) Sensitive to unsaturated compounds Element Specific Detectors (common for S, P, and N)

40 GC Instrumentation

41 Column temperature For precise work, column temperature must be controlled to within tenths of a degree. The optimum column temperature is dependent upon the boiling point of the sample. As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution time of minutes. Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as separation proceeds.

42 Mobile Phase (gas)

43 GC Under the hood

44 GC Column and Oven

45 Typical GC (dual column)

46 Detection 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 compounds is measured. FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector's response. The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the sample.

47 Detector types are: 1) FID ( flame ionization detector ) 2) TCD ( thermal conductivity detector ) 3) ECD ( electron capture detector ) 4) FPD ( flame photometric detector ) 5) HID ( helium ionization detector ) 6) NPD ( nitrogen-phosphorus detector ) 7) PID ( photo ionization detector ) 8) TID ( thermionic ionization detector ) 9) CCD ( catalytic combustion detector ) 10) NPD/DELCD ( combination NPD and dry electrolytic conductivity detector ) 11) FID/DELCD ( combination FID and dry electrolytic conductivity detector ) 12) FID/FPD ( combination FID and FPD ) 13) Dual FPD ( dual wavelength FPD for both sulfur and phosphorus ) 14) FID dual FPD ( dual FPD plus FID combination )

48

49 Detectors continued ECD (electron capture detector). Uses Ni-63 as a radiation source to cause ionization of the substance using N as the carrier gas. Detector is sensitive to functional groups containing electronegative species such as halogens, quinones, peroxides, and nitro groups. Hence, very good detector for environmental analysis where pesticides need to be measured. AED (atomic emission detector). It is a AA unit using MIP that accepts the output from the GC. Mass spectrometer

50 Different GC detectors

51 Detectors: Selective Detectors Specific for specific types of chemicals (i.e., nitrogen- phosphorus detector, NPD) Measures ions produced from eluting N or P compounds, but generates e – from a heated surface (not flame) that combine with electronegative elements to form negative ions Good selectivity, good LOD, but must periodically change heated material

52 Detectors: Electron Capture Detects electronegative atoms or groups (Cl, -NO 2 ) and also polynuclear aromatics and conjugated carbonyl compounds These groups capture e – that are produced from nuclear radiation from 3 H Carrier gas hit by these e – can release secondary e –, which will be absorbed by analyte Has good LOD, but a narrow linear range and requires radioactive source

53 Detectors: Mass Spectrometry Detects and measures by converting eluting analytes into gas phase ions (forming a molecular ion or fragmenting analyte [and ionizing]. Compare patterns of ions and fragments to known values. Intensity relates to amounts. A portion of analytes is converted to anions via electron impact or chemical impact (softer, less fragments). Gas phase ions are separated by mass/charge ratio using a quadrupole mass analyzer. (Uses four parallel rods with well-defined potentials so that only certain mass/charge species may pass [sorted])

54 Detectors: Mass Spectrometry A Mass spectrum plots intensity of mass/charge vs. timecould be set a certain retention time in a GC. Mass chromatogram

55 Thermal Conductivity Detector (TCD) Introduction A TCD detector consists of an electrically-heated wire or thermistor. The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it. Changes in thermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance. The TCD is not as sensitive as other detectors but it is non-specific and non-destructive. Instrumentation 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, and 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 bridge circuit allows amplification of resistance changes due to analytes passing over the sample thermoconductors and does not amplify changes in resistance that both sets of detectors produce due to flow rate fluctuations, etc.

56 Schematic of a bridge circuit for TCD detection Two filament in one cell ( reference side ) --- carrier gas only The other cell ( sample side ) --- carrier plus sample flowing 1. Universal 2. Used primarily for gas analysis 3. Sensitive few ppm

57 Detectors: Thermal Conductivity TCD can be used for organic and inorganic analytes. The key aspect is the ability of the carrier gas and the analytes to change the conductivity of a wire filament, which will vary with different analytes. The carrier gas should have different thermal conductivity of analytes. TCD is a non-destructive type of detection that uses a Wheatstone bridge style. Downsides are the response to impurities, leakage in air, and poor response to LOD.

58 Wheatstone Bridge TCD Most common carrier

59 Detectors: Flame Ionization FID uses fuel mixed with carrier and organic analyte. Analyte forms ions in the flame. Cations from the flame are gathered by the negative electrodeproduces a current. Advantage:inorganics do not respond (He carrier gas), so the low background signal allows for LOD 100- to 1000-fold lower then TCD. Disadvantage: destructive

60 Flame Ionization Detector Introduction The flame ionization detector (FID) is the most sensitive gas chromatographic detector for hydrocarbons such as butane or hexane. With a linear range for 6 or 7 orders of magnitude (10 6 to 10 7 ) and limits of detection in the low picogram or femtogram range, the FID is the gas chromatographic detector for volatile hydrocarbons and many carbon containing compounds. FID Responds to all organic compounds except for formic acid. Response greatest with hydrocarbons and decreases with substitution. Except for vapor of elements in Groups I and II, does not respond to inorganic compounds. Sensitivity high due to low noise level. Insensitivity to water, the permanent gases, and inorganic compounds simplifies the resolution of components in analysis of aqueous extracts and in air pollution studies.

61 Flame Ionization Detector Consists of a stainless steel burner assembly installed in the detector compartment and a electrometer system in a separate unit adjacent to the gas chromatograph Often it is installed in the tandem with the thermal conductivity cell Effluent form the column enters burner base through millipore filters which remove contaminates Hydrogen mixed with gas stream at bottom of jet and air or oxygen is supplied axially around the jet. Hydrogen flame burns at the tip, which also functions as the cathode and is insulated form the body by a ceramic seal Collector electrode is above the burner tip and is made of platinum

62 An FID consists of a hydrogen/air flame and a collector plate. The effluent from the GC column passes through the flame, which breaks down organic molecules and produces ions. The ions are collected on a biased electrode and produce an electrical signal. The FID is extremely sensitive with a large dynamic range, its only disadvantage is that it destroys the sample. FIDs are normally heated independently of the chromatographic oven. Heating is necessary in order to prevent condensation of water generated by the flame and also to prevent any hold-up of solutes as they pass from the column to the flame. With the flame extinguished, the column end should be passed up through the jet and then lightly held in position by slightly tightening the coupling. Gradually draw the column end back into detector jet until it is approximately mm below the jet tip. Then tighten the coupling to retain it in position. Do not over tighten couplings on capillary columns.

63 Mechanism The effluent from the column is mixed with hydrogen and air and then ignited electrically at a small metal jet. Most organic compounds produce ions and electrons that can conduct electricity through the flame. There is an electrode above the flame to collect the ions formed at a hydrogen/air flame. The number of ions hitting the collector is measured and a signal is generated. In series with flame gases is a selection of resistors 10 7 to ohms. Vibrating reed electrometer used to provide sensitivities up to 5 × Amps. Carbon counting device that produces a current proportional to number of ions or electrons formed in the flamed gases. The organic molecules undergo a series of reactions including thermal fragmentation, chemi-ionization, ion molecule and free radical reactions to produce charged-species. The amount of ions produced is roughly proportional to the number of reduced carbon atoms present in the flame and hence the number of molecules. Because the flame ionization detector responds to the number of carbon atoms entering the detector per unit of time, it is a mass-sensitive, rather than a concentration-sensitive device. As a consequence, this detector has the advantage that changes in flow rate of the mobile phase has little effects on detector response.

64 Normal Combustion: i.e. burn methane in air and get carbon dioxide and water vapor... CH 4 + O 2 CO 2 + H 2 O or: CH 4 + 3O 2 CO 2 + 2H 2 O Flame Ionization: during combustion, a uniform proportion (about %) of the molecules in this reaction do this instead: (simplified for clarity) CH 4 + O 2 C + + H 2 O + e - CO 2 + H 2 O or: CH 4 + 3O 2 C + + O 2 + 2H 2 O + e - CO 2 + 2H 2 O These oppositely-charged, intermediate products can then be detected by the FID:

65 Limitations Molecules that contained only carbon and hydrogen respond best in this detector but the presence of "heteroatoms" in a molecule, such as oxygen, decreases the detector's response. For instance, the FID's methane response (CH 4 ) is fabulous but formaldehyde's (CH 2 O) is quite poor. Therefore, highly oxygenated molecules or sulfides might best be detected using another detector instead of the FID. Sulfides determination by the flame photometric detector and aldehydes and ketones analyzed with the photoionization detector are alternatives to the use of the FID for those molecules.

66 Functional group, such as carbonyl, alcohol, halogen, and amine, yield fewer ions or none at all in a flame. In addition, the detector is insensitive toward noncombustible gases such as H 2 O, CO 2, SO 2 and NO x. Selectivity: Compounds with C-H bonds. A poor response for some non- hydrogen containing organics (e.g., hexachlorobenzene). Sensitivity: 0.1 ~ 10 ng Linear range: 10 5 ~ 10 7 Gases: Combustion - hydrogen and air; Makeup - helium or nitrogen Temperature: °C; °C for high temperature analyses

67 Detector Construction FID is constructed of a small volume chamber into which the gas chromatograph's capillary column in directly plumbed. Usually the small diameter capillary is fitted directly into the bottom of the detector's flame jet. The gaseous eluents from the column are mixed with separately plumbed in hydrogen and air and all are burned on the jet's tip. After the fuel (H 2 ) and oxidant (O 2 in air) are begun, the flame is lit using a electronic ignite, actually an electrically heated filament that is turned on only to light the flame. The charged particles created in that combustion process create a current between the detector's electrodes. One electrode is actually the metallic jet itself, another is close by and above the jet. The gaseous products leave the detector chamber via the exhaust. The detector housing is heated so that gases produced by the combustion (mainly water) do not condense in the detector before leaving the detector chimney.

68 Flame Ionization Detector FIDTCD View of TCD and FID of HP5890 GC

69 Flame Ionization Detector

70 Makeup Gases The total volume of gas in the FID that yields the most sensitive and widest linear response is not the same volume of gas when the column effluent flow (~ 1 mL/min) and hydrogen and air flows are flowing; these gases' total flow into the detector is too small. Another way to say this is that the optimum column flow to maintain the best chromatography and the best fuel and oxidant flows for the best flame conditions--all added together-- don't create the best gas flow for the FID detector's design. This means that to maintain the best analytical conditions, additional gas must be constantly flowed into the detector. This gas makes up the additional needed gas flow and so is termed makeup gas. Since the gas needs to be inert so that its addition doesn't upset the fuel and oxidant balance and since it needs to be added in relatively large amounts (~30+ ml/min in some detector designs) nitrogen is usually the gas of choice. Helium would work also but is a nonrenewable resource and more expensive. All gas flows are controlled by adjustable gas regulators.

71 Electron Capture Detector (ECD) The ECD uses a radioactive emitter (electrons) to ionize some of the carrier gas and produce a current between a biased pair of electrodes. When organic molecules that contain electronegative functional groups, such as halogens, phosphorous, and nitro groups pass by the detector, they capture some of the electrons and reduce the current measured between the electrodes. The ECD is as sensitive as the FID but has a limited dynamic range and finds its greatest application in analysis of halogenated compounds. Schematic of an ECD

72 ECD Selective in its response and highly sensitive Hewlett Packard makes one with a detection limit of less than 8 fg/sec for lindane Sensitive toward molecules with electronegative functional groups (halogens, peroxides, quinones, nitro groups) Insensitive towards amines, alcohols and hydrocarbons A leak test of an ECD containing nickel-63 ( 63 Ni) must be performed at intervals not to exceed six months. The test must be performed in accordance with the manufacturer's instructions, or by wiping the gas intake and outlet surfaces. NOTE: Never attempt to directly wipe the inner surface of the component containing the radioactive material. This might cause the ECD to fail and will contaminate the ECD, the gas chromatograph and the surrounding area. Never open the detector cell for any reason.

73 Nitrogen Phosphorous Detector Specific: sample must contain nitrogen or phosphorous Destructive LOD : 0.4 pg N / sec 0.2 pg P / sec Linear range : ~ 10 4 Mode of operation: essentially a modified FID Active element acts to block undesired species

74 Flame Photometric Detector The determination of sulfur or phosphorus containing compounds is the job of the flame photometric detector (FPD). This device uses the chemiluminescent reactions of these compounds in a hydrogen/air flame as a source of analytical information that is relatively specific for substances containing these two kinds of atoms. The emitting species for sulfur compounds is excited S 2. The lambda max for emission of excited S 2 is approximately 394 nm. The emitter for phosphorus compounds in the flame is excited HPO (lambda max = doublet nm). In order to selectively detect one or the other family of compounds as it elutes from the GC column, an interference filter is used between the flame and the photomultiplier tube (PMT) to isolate the appropriate emission band. The drawback here being that the filter must be exchanged between chromatographic runs if the other family of compounds is to be detected. Instrumentation In addition to the instrumental requirements for 1) a combustion chamber to house the flame, 2) gas lines for hydrogen (fuel) and air (oxidant), and 3) an exhaust chimney to remove combustion products, the final component necessary for this instrument is a thermal (bandpass) filter to isolate only the visible and UV radiation emitted by the flame. Without this the large amounts of infrared radiation emitted by the flame's combustion reaction would heat up the PMT and increase its background signal. The PMT is also physically insulated from the combustion chamber by using poorly (thermally) conducting metals to attach the PMT housing, filters, etc. The physical arrangement of these components is as follows: flame (combustion) chamber with exhaust, permanent thermal filter (two IR filters in some commercial designs), a removable phosphorus or sulfur selective filter, and finally the PMT.

75 Schematic of a gas chromatographic flame photometric detector Specific: phosphorous or sulfur Destructive LOD: 20 pg S /sec, 0.9 pg P / sec Linbear range: ~10 4 P, ~10 3 S

76 Photoionization Detector Introduction The reason to use more than one kind of detector for gas chromatography is to achieve selective and/or highly sensitive detection of specific compounds encountered in particular chromatographic analyses. The selective determination of aromatic hydrocarbons or organo-heteroatom species is the job of the photoionization detector (PID). This device uses ultraviolet light as a means of ionizing an analyte exiting from a GC column. The ions produced by this process are collected by electrodes. The current generated is therefore a measure of the analyte concentration. Theory If the energy of an incoming photon is high enough (and the molecule is quantum mechanically "allowed" to absorb the photon) photo-excitation can occur to such an extent that an electron is completely removed from its molecular orbital, i.e. ionization. A Photoionization Reaction

77 If the amount of ionization is reproducible for a given compound, pressure, and light source then the current collected at the PID's reaction cell electrodes is reproducibly proportional to the amount of that compound entering the cell. The reason why the compounds that are routinely analyzed are either aromatic hydrocarbons or heteroatom containing compounds (like organosulfur or organophosphorus species) is because these species have ionization potentials (IP) that are within reach of commercially available UV lamps. The available lamp energies range from 8.3 to 11.7 ev, that is, lambda max ranging from 150 nm to 106 nm. Although most PIDs have only one lamp, lamps in the PID are exchanged depending on the compound selectivity required in the analysis.

78 Selective detection using a PID Here is an example of selective PID detection: Benzene's boiling point is 80.1 degrees C and its IP is 9.24 ev. (Check the CRC Handbook 56th ed. page E-74 for IPs of common molecules.) This compound would respond in a PID with a UV lamp of 9.5 ev (commercially available) because this energy is higher than benzene's IP (9.24). Isopropyl alcohol has a similar boiling point (82.5 degrees C) and these two compounds might elute relatively close together in normal temperature programmed gas chromatography, especially if a fast temperature ramp were used. However, since isopropyl alcohol's IP is ev this compound would be invisible or show very poor response in that PID, and therefore the detector would respond to one compound but not the other.

79 Instrumentation Since only a small (but basically unknown) fraction of the analyte molecules are actually ionized in the PID chamber, this is considered to be a nondestructive GC detector. Therefore, the exhaust port of the PID can be connected to another detector in series with the PID. In this way data from two different detectors can be taken simultaneously, and selective detection of PID responsive compounds augmented by response from, say, an FID or ECD. The major challenge here is to make the design of the ionization chamber and the downstream connections to the second detector as low volume as possible (read small diameter) so that peaks that have been separated by the GC column do not broaden out before detection. Specific: compounds ionized by UV LOD: ~ 2 pg Carbon / sec Linear range : 10 7

80 Atomic-Emission Detector (AED) This detector, while quite expensive compared to other commercially available GC detectors, is an extremely powerful alternative. For instance, Instead of measuring simple gas phase (carbon containing) ions created in a flame as with the flame ionization detector, or the change in background current because of electronegative element capture of thermal electrons as with the electron capture detector, the AED has a much wider applicability because it is based on the detection of atomic emissions. The strength of the AED lies in the detector's ability to simultaneously determine the atomic emissions of many of the elements in analytes that elute from a GC capillary column (called eluants or solutes in some books). As eluants come off the capillary column they are fed into a microwave powered plasma (or discharge) cavity where the compounds are destroyed and their atoms are excited by the energy of the plasma. The light that is emitted by the excited particles is separated into individual lines via a photodiode array. The associated computer then sorts out the individual emission lines and can produce chromatograms made up of peaks from eluants that contain only a specific element.

81 Instrumentation The components of the AED include 1) an interface for the incoming capillary GC column to the microwave induced plasma chamber, 2) the microwave chamber itself, 3) a cooling system for that chamber, 4) a diffraction grating and associated optics to focus then disperse the spectral atomic lines, and 5) a position adjustable photodiode array interfaced to a computer. The microwave cavity cooling is required because much of the energy focused into the cavity is converted to heat. Schematic of a gas chromatographic atomic emission detector

82 PDD (pulsed discharge detector) The VICI PDD (pulsed discharge detector) utilizes a stable, low powered, pulsed DC discharge in helium as an ionization source. Performance is equal to or better than detectors with conventional radioactive sources. In the electron capture mode, the PDD is a selective detector for monitoring high electron affinity compounds such as freons, chlorinated pesticides, and other halogen compounds. For this type of compound, the minimum detectable quantity (MDQ) is at the femtogram (10-15) or picogram (10-12) level. The PDD is similar in sensitivity and response characteristics to a conventional radioactive ECD, and can be operated at temperatures up to 400°C. For operation in this mode, He and CH4 are introduced just upstream from the column exit. In the helium photoionization mode, the PDD is a universal, non-destructive, high sensitivity detector. The response to both inorganic and organic compounds is linear over a wide range. Response to fixed gases is positive (increase in standing current), with an MDQ in the low ppb range. The PDD in helium photoionization mode is an excellent replacement for flame ionization detectors in petrochemical or refinery environments, where the flame and use of hydrogen can be problematic. In addition, when the helium discharge gas is doped with a suitable noble gas, such as argon, krypton, or xenon (depending on the desired cutoff point), the PDD can function as a specific photoionization detector for selective determination of aliphatics, aromatics, amines, as well as other species. (Click here for an ionization potential chart in.pdf format.) here

83 PDD detector

84 Various configurations: Column: capillary Autosampler Computer controlled for data acquisition and analysis of peaks. Some come with their own compressor for the air supply. Sensitivity is very important because of detection limits. More expensive system will use a MS for the detector to do GC-MS. Can do headspace analysis for volatiles in water etc.

85 GC Companies ThermoQuest Agilent (Hewlett Packard) Perkin-Elmer Buck Scientific Thermo Nicolet

86 Gas Chromatography Instrumentation 3.)Packed Columns The major advantage and use is for large-scale or preparative purification Industrial scale purification maybe in the kilogram or greater range 500 L chromatography column Oil refinery – separates fractions of oil for petroleum products

87 Qualitative analysis : t R Standard ---- methanol, MEK(t R ), toluene Unknown same t R (X) Conclude (X) = MEK X methano l MEK toluene tRtR Retention time limitations t R changes with flow rate, column temperature, liquid phase, column history, sample size *** WARNING identical retention times do not confirm peak identity

88 Spiking Step 1 Peak X --- toluene ? Step 2 Toluene added to sample Step 3 Peak X identified as toluene X methano l MEK toluene tRtR Toluene added to sample

89 Pesticides Analysis of pesticide residues in soil, water, and food is crucial for maintaining safe levels in the environment. The PDD in the ECD mode is highly selective for monitoring electron capturing compounds such as chlorinated pesticides and other halogens. This chromatogram illustrates the sensitivity of the non-radioactive PDECD for such compounds. Sample: Pesticide calibration mix Detector mode: Electron capture Detector temp: 330°C Column: 25 m x 0.32 mm x 25 µm, HP-5 Column temp: 150°C to 300°C at 10°C/min Sample volume: 1 µL, 10:1 split Discharge gas: Helium, 30 mL/min Dopant gas: 5% methane in helium, 2.4 mL/min Attenuation: 1

90 Pesticide separations Retention time (sec)

91 Headspace gas chromatography analysis Headspace GC (HSGC) analysis employs a specialized sampling and sample introduction technique, making use of the equilibrium established between the volatile components of a liquid or solid phase and the gaseous / vapor phase in a sealed sample container. Aliquots of the gaseous phase are sampled for analysis.

92 Headspace sampling

93 HSGC Examples of HSGC are the forensic analysis of blood and urine alcohol levels, quality and production control of diesel fuel and beer constituents. Aromatic flavors and trace volatiles in foods and soft-drinks are also readily analyzed. and HSGC analysis of volatile free fatty acids produced by bacteria, particularly anaerobic bacteria, enables a fingerprint of the particular microorganisms to be obtained, which assists in the identification of the bacteria.

94 Food analysis Analysis of foods is concerned with the assay of lipids, proteins, carbohydrates, preservatives, flavours, colorants and texture modifiers, and also vitamins, steroids, drugs and pesticide residues and trace elements. Most of the components are non-volatile and although HPLC is now used routinely for much food analysis, GC is still frequently used. For examples, derivatization of lipids and fatty acid to their methyl esters(FAMEs), of proteins by acid hydrolysis followed by esterification (N-propyl esters) and of carbohydrates by silylation to produce volatile samples suitable for GC analysis.

95 GC Food GC quality control analysis of food products can confirm the presence and quantities of the analytes For example, fruits, fruit derived foodstuffs, vegetables and soft drinks, tea and coffee, were analyzed for their polybasic and hydroxy acid contents (citric, maleic acids) as TMS derivatives. All food and beverage products on sale today must be carefully assayed for contamination with pesticides, herbicides and many other materials that are considered a health risk. The analysis of food involves separating and identifying very complex mixtures, the components of which are present at very low concentrations. GC is the ideal technique for use in food and beverage assays and tests. Furthermore, the origin of many herbs and spices can often be identified from the peak pattern of the chromatograms from their head space analysis.

96 Food and Cancer Chemicals that can cause cancer have a wide variety of molecular structures and include hydrocarbons, amines, certain drugs, some metals and even some substances occurring naturally in plants and molds. In this way, many nitrosamines have carcinogenic properties and these are produced in a number of ways such as cigarette smoke. GC can be used to identify these nitro-compounds in trace quantities.

97 Drugs There are still numerous GC applications involving both quantitative and qualitative identification of the active components and possible contaminants, adulterants or characteristic features which may indicate the source of the particular sample. Forensic analysis frequently users GC to characterize drugs of abuse, in some cases the characteristic chromatographic fingerprint gives an indication of the source of manufacture of the sample or worldwide source of a vegetable material such as cannabis. Analytical procedures, chromatographic methods and retention data are published for over 600 drugs, poisons and metabolites. These data are extremely useful for forensic work and in hospital pathology laboratories to assist the identification of drugs.

98 Pyrolysis gas chromatography Pyrolysis GC (PGC) is used principally for the identification of non-volatile materials, such as plastics, natural and synthetic polymers, drugs and some microbiological materials. The thermal dissociation and fragmentation of the sample produces a chromatogram which is a fingerprint for that sample. The small molecules produced in the pyrolysis reaction are frequently identified using a GC-MS system and information on molecular structure for identification is also obtained.

99 Metal chelates and inorganic materials Although inorganic compounds are generally non- volatile, GC analysis can be achieved by converting the metal species into volatile derivatives. Only some metal hydrides and chlorides have sufficient volatility for GC. Organometallics other than chelates, which can be analyzed directly, include boranes, silanes, germanes, organotin and lead compounds.

100 Environmental analysis Environmental pollution is an age-old trademark of man and in recent years as technology has progressed, populations have increased and standards of living have improved. So the demands on the environment have increased, with all the attendant problems for the ecosystems. Combustion of fossil fuel, disposal of waste materials and products, treatment of crops with pesticides and herbicides have all contributed to the problems. Technological developments have enabled man to study these problems and realize that even trace quantities of pollutants can gave detrimental effects on health and on the stability of the environment. There is a vast amount of literature on the use of GC for studying a wide variety of these problems.

101 GC application Every year many new substances are synthesized that differ radically from the natural products that exist in biosystems. The Environmental Protection Agency is empowered to control water pollution and the production, use and disposal of toxic chemicals. It follows that detailed studies must be made of their effect on the environment and their method of movement through the ecosystem. Many of the compounds are not biodegradable and will thus progressively pollute the environment. There are a number of tragic examples of which DDT (dichlorodiphenyltrichloroethane) and the PCBs (polychlorinated biphenyls) are well known instances. The materials of interest are present in environmental samples at very low concentrations and are often to be found among a myriad of other compounds from which they must be separated and identified. It follows that GC, with its inherent high sensitivity and high separating power, is one of the more commonly used techniques in the analysis of environmental samples.


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