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Lecture X. Gas Chromatography. Outline . GC Theory What are the separations? Instrumentation Applications Conclusions Brief request for next week’s lecture FT-IR. Gas Chromatography (GC).
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Lecture X Gas Chromatography
Outline • GC Theory • What are the separations? • Instrumentation • Applications • Conclusions • Brief request for next week’s lecture FT-IR.
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.
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.
Additional Information • From http://www.shu.ac.uk/schools/sci/chem/tutorials/chrom/gaschrm.htm
Theory • In 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);
GC Theory • Partition 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.
GC Theory • The fraction of total solute transferred to phase 2 (CCl4) is (1-q) and the molarity in phase 2 is • (1-q) m / V2. Then • K = ((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).
Suppose I2 in H2O is 1 and I2 in CCl4 is 0. • After shaking and letting settle, I2 in H2O is 0.0014 and I2 in CCl4 is 0.9986. Therefore • K = 7 x 102. Fraction (q) remaining after the 1st extraction is: • q = V1/(V1+KV2). Fraction remaining after n extractions is • qn = (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.
Theory of separation Water + I2 Water + I2 Water + I2 CCl4 + I2 CCl4 CCl4+ I2 Shake 1 Start Shake 2
Theory GC • As 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.
Solute in and out of packing by diffusion. Diffusion in and out of column pores
GC Theory Analyte To detector Column + packing Time
Time 1 Time 2 Time 3 Separations To detector
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(adjustedretention time, t’R) • •the ratio t’R/ to is the capacity of the column to retain the solute (k’) t R t ’ t o R k’ = (tr - t0) / t0 k’ = (t’r ) / t0 . Inject Unretained Solute
Column Efficiency (N) • 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 distribution to t1 t2 Band spreading eventually causes peaks to merge into the baseline. We want to minimize band spreading as much as possible.
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.
Elution Order • Compound Boiling Point (0C) • pentane 36 • hexane 69 • cyclohexane 80 • isooctane 99 • toluene 110 • 4-methyl-2-pentanone 117 • octane 126
Example Chromatogram • The observed elution pattern appears below. Notice the reversed elution of toluene and 4-methyl-2-pentanone.
Column performance • Plate 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.
Peak performance • Longitudinal 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.
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.
Column efficiency • C 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:
More column stuff • Ds = 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.
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;
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.
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.
Column temperature • For 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 2 - 30 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.
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.
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, 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?
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.
Column • The 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. 27-5 for temperature effects on separations. Normally, one does a temperature program to get the various analytes off the column for better separations (resolutions).
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 1.5 - 10m 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;
Detectors • How 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 707. 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 708. He is the carrier gas most often used with this detector because it has a high thermal conductivity.
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.
