2 Brief request for next week’s lecture FT-IR. OutlineGC TheoryWhat are the separations?InstrumentationApplicationsConclusionsBrief request for next week’s lecture FT-IR.
3 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.
4 GC TheoryAn 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.
6 TheoryIn order to understand GC, need to focus on the general principles of separations which has its roots in solvent extraction. The theory of solvent extraction are used to explain all forms of chromatography i.e. HPLC, LC, GC, EP, and TLC.Consider two solvents S1 and S2 and solute X that is in S1. The partition between the two phases.X(S1) X(S2);
7 GC TheoryPartition coefficient K is an equilibrium constant is K = [X]S1/[X]S2. Suppose that solute X in V1 (water) is extracted with V2 (CCl4). Let m be the moles of X in the system and let q be the fraction of X remaining in phase 1 at equilibrium. The molarity in phase 1 (water) is therefore qm/V1.
8 GC TheoryThe fraction of total solute transferred to phase 2 (CCl4) is (1-q) and the molarity in phase 2 is(1-q) m / V2. ThenK = ((1-q) m / V2)/ q m V1.For GC:K = Cs/Cm where Cs is the concentration in the stationary phase (column) and Cm is the concentration in the mobile phase (gas).
9 Suppose I2 in H2O is 1 and I2 in CCl4 is 0. After shaking and letting settle, I2 in H2O is and I2 in CCl4 is ThereforeK = 7 x Fraction (q) remaining after the 1st extraction is:q = V1/(V1+KV2). Fraction remaining after n extractions isqn = (V1/(V1+KV2))n. It is more efficient to do several small extractions than one large one. This extraction can be used to describe GC where the liquid is the mobile phase and the column is the stationary phase. Each extraction is a theoretical plate.
10 Theory of separation Water + I2 Water + I2 Water + I2 CCl4 + I2 CCl4 Shake 1StartShake 2
11 Theory GCAs the gas moves the solute (analyte) through and over the stationary phase, the solute will be in equilibrium with the gas and the solid phase. Since there is a mobile phase, the separation will appear as a chromatogram showing the separation of the analytes.
12 Diffusion in and out of column pores Solute in and out of packing by diffusion.
15 Capacity Factor (k’)• While inside the column, a retained component spends part of its time on the stationary phase and part time in the mobile phase• When in the mobile phase, solutes move at the same speed as the mobile phase• this means that all solutes spend the same amount of time in the mobile phase (to)• the amount of time the solute spends on the stationary phase is equal to tR- to(adjusted retention time, t’R)•the ratio t’R/ to is the capacity of the column to retain the solute (k’)tRtt’oRk’ = (tr - t0) / t0k’ = (t’r ) / t0.InjectUnretainedSolute
17 Column Efficiency (N) to t1 t2 Solutes are placed on an GC column in a narrow band• Each solute band spreads as it moves through the column due to diffusion and mass transfer affects• The later eluting bands will spread more• Peak shape follow a Gaussian distributiontot1t2Band spreading eventually causes peaks to merge into the baseline. We want to minimize band spreading as much as possible.
22 GC GeneralThe 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.
23 Elution Order Compound Boiling Point (0C) pentane 36 hexane 69 cyclohexane 80 isooctane toluene 4-methyl-2-pentanone 117 octane
24 Example ChromatogramThe observed elution pattern appears below. Notice the reversed elution of toluene and 4-methyl-2-pentanone.
26 Column performancePlate height H = L / N where L is the length of the packed column and N is the number of theoretical plates.For example: A solute with a retention time of 407 s has a width at the base of 13 s on a 12.2 m long column.N = 16 * 4072/ 132 = 1.57 X 104.H = 12.2 / 1.57 X 104 = 0.78 mm;Van Deemter equation:H = A + B / x + C x. All three terms A, B, and C contribute to band broadening.A is for multiple paths, B / x is due to longitudinal diffusion,C x is due to equilibration time.
27 Peak performanceLongitudinal diffusion is important because diffusion takes place along the axis of the column and contributes to peak broadening. The faster the linear flow, the less time spent in the column and the less diffusional broadening occurs.
28 Separation efficiency Efficiency of separations will depend on:1. Elution times of the peaks,2. Broadness of the peaks.2 = 2Dm t = 2DmL /x.Plate height due to long diff:HD = 2Dm /x = B / x where Dm is the diffusion coefficient of the solute in the mobile phase and t is time and L is column length.
29 Column efficiencyC x comes from the finite time required for a solute to equilibrate between the mobile phase and the stationary phase. Although some solute is stuck in the mobile phase, most of it will move on and elute. The plate height for finite equilibration time i.e. mass transfer is:
30 More column stuffDs = is the diffusion coefficient of the solute in the stationary phase, d is thickness of the stationary phase, and k’ is the capacity factor. Effected by temperature and thickness of stationary phase.
32 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 portFor 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;
33 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.
34 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.See Figure 27-1 Pg 703 of text for instrumentation.
36 Column temperatureFor precise work, column temperature must be controlled to within tenths of a degree. The optimum column temperature is dependant 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.
41 Sample InjectionsNext, 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 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.
43 Carrier GasThis is the mobile phase and should be pure gas so as not to react with the column or analyte. Gas is usually He, Ar, N2, or H2. Choice will depend on the type of detector used. He and H2 give better resolution (smaller plate height) than N2. Pressure is also important and as expected the system comes with regulators. Can you find where in GC equations that are dependent on pressure?
44 ColumnsThe 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.
45 ColumnThe column sits in a temperature controlled environment that is 0.50. 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).
46 ColumnsThere 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;
49 DetectorsHow is the analyte detected? Several detectors are available for GC.FID (flame ionization detector) is the most widely used detector. See figure 27-6, Pg Based on the production of ions when compounds are burned then detecting the current produced from the ionization. What compounds can not be detected with this detector?TCD (thermal conductivity detector). Operates on the changes in the thermal conductivity of the gas stream brought about by the presence of analyte molecules. See Figure 27-7 on page He is the carrier gas most often used with this detector because it has a high thermal conductivity.
50 DetectionThe 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.
52 Detectors continuedECD (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. See Figure 27-8 page 709.AED (atomic emission detector). It is a AA unit using MIP that accepts the output from the GC. See Figure 27-9, Page 709.Mass spectrometer
54 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.)
57 PesticidesAnalysis 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
59 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.
61 HSGCExamples 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.
62 Food analysisAnalysis 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.
63 GC FoodGC 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.
64 Food and CancerChemicals 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.
65 DrugsThere 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.
66 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.
67 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.
68 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.
69 GC applicationEvery 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.
70 Various configurations: Column: capillaryAutosamplerComputer 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.