Gas Chromatography

Gas Chromatography - 8. GAS CHROMATOGRAPHY Gas...

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Unformatted text preview: 8. GAS CHROMATOGRAPHY Gas chromatography (GC). also known as gas-liquid partition chromatography (GLPC) or vapor- phase chromatography (VPC), is a useful technique for separating and analyzing the components of a volatile mixture. This method of separation uses an instrument called a gas chromatograph. Gas chromatography shares with all chromatographic techniques the partitioning of the components of the sample between a mobile phase and a stationary phase. The theory behind the separation of components by partitioning in gas chromatography is the same as that discussed in the TLC chapter (see Chapter 7 in Techniques). In gas chromatography the components of the sample distribute themselves between a mobile gas phase and a stationary liquid phase. The mobile phase is called the carrier 9 as. The most commonly used carrier gas in gas chromatography is helium, but nitrogen or any other inert gas can also be used. The carrier gas carries the components of the mixture along the stationary phase (just as the mobile liquid phase carried the components of a sample up the solid Figure 1 . Gas chromatograph stationary phase in TLC). In gas chromatography a small diameter column holds the stationary phase, a finely divided porous solid substance whose surface is coated with a high boiling liquid. The non-volatile liquid on the surface of the solid support serves as the stationary phase in the separation. The components of a volatile mixture partition between the stationary liquid phase and the mobile gas phase, causing the various components to be carried through the column at different rates and giving the separation. A schematic diagram of a typical gas chromatograph is shown in Figure 2. To separate a mixture on a gas chromatograph, a small volume (0.5 — 5 uL) of a liquid sample is injected through a rubber septum into the heated injection port using a microliter syringe. The sample immediately vaporizes in the heated injection port and is swept onto the column by the carrier gas. The column which contains the stationary adsorbent is located in an oven to maintain a constant temperature for the system during the separation. As the sample passes through the column, its components partition between the stationary T8-1 . Recorder ‘—‘- Carrier gas (He) Injection Syringe or! (heated) Figure 2. Schematic diagram of a gas chromatograph liquid and mobile gaseous phases. As each component of the mixture elutes from the column it passes through the detector- The detector produces an electronic signal which is sent to a chart recorder which records the chromatogram. After passing through the detector the vapors are either collected or released into the atmosphere. A. Retention time The time that elapses from the injection of the sample into the injection port until a component elutes from the column is called the compounds retention time. Since strip chart recorders move the chart paper at a constant rate, the distance on the chromatogram from the point of injection to the maximum height of a peak gives the corresponding compound’s retention time (Figure 3). Compounds with short retention times move through the column taster than those compounds with larger retention times. For a given set of conditions (column temperature, column length, choice of liquid phase, carrier gas flow rate, injection port temperature), the retention time is characteristic of the compound in Detector response / Injection Time—> Figure 3. Measuring retention times on a chromatogram T8-2 question (just as the R, value was characteristic of a compound in TLC) and can be used as a means to help identify the compound. Unfortunately, gas chromatography gives no direct information about the identities of the compounds being separated. While an unknown compound and a known compound with the same retention time might be identical, a definitive identification of the unknown compound can be made only by using some type of spectroscopy. in general, four factors affect the separation obtained on a gas chromatograph: a) the type of stationary liquid phase used, b) the column’s length, c) the column temperature, and d) the flow rate of the carrier gas. B. Stationary phase The heart of a gas chromatograph is the packed column where the separation occurs. GC- columns are usually made of 1/4 or 1/8 inch diameter copper, stainless steel, or glass tubing, packed with the stationary phase, a finely divided, inert support material coated with a non-volatile liquid. Columns are identified by the type of packing that they contain. Many different types of non- volatile liquid phases are available for use in gas chromatography: hydrocarbon waxes and greases, silicone oils, polyesters, polyethylene glycols, etc. (Table 1). While packing a GC-column is not difficult, most columns used today are purchased pre-packed from various manufacturers. The choice of which type of column to use for a separation depends on the types of compounds in the mixture. To understand the partitioning process behind gas chromatography, the solubility of organic compounds in liquids needs to be considered. In general, the more volatile a compound is (i.e., the lower its boiling point), the faster it travels through a GC-column. A more volatile compound vaporizes more readily than a less volatile one, so it spends less time adsorbed on the stationary liquid phase and is \ carried more quickly through the column by the carrier gas. A compound's volatility is not the only factor that affects its interactions with the stationary liquid phase; it is also necessary to consider the polarities of the compound and of the stationary liquid phase. The old adage that “like dissolves like” applies in gas chromatography just as it did in TLC. A polar stationary phase will interact strongly with polar compounds, retarding their movement through the column while non-polar compounds in the sample, which will not interact strongly with a polar stationary phase, move through the column more rapidly. These interactions allow partitioning to set up between the stationary liquid phase and the mobile gas phase, thus effecting separation. The reverse situation also T8-3 3:93 .mmEEoEm mEom >23 555m .228 8:99. 603583 @955 .228 .2285 muczanoo umficomofic 60:29. .mmEEmEm mucsanoo 3:38 26. can 2088063 209805? cousin—ow muczonEoo to mun—Pr = lwzoazo leolfoazo o 0 0° com .9328 a zomzofo +o£o£o *0: 0° omm 69% 9.92620; G 9+. o _ "1:35 ol_ m Io 5%:8 _ f 0 0° omm I mwm __o 2525 8x:__-mmob Sn .__o .392 0o mum 1 0mm 28:6 2 55:5 9.8% 3522 98.89%; 889m 0° com .. omm B 2352 2028931 2392.20... £3.5qu :oEmanoo can—E 2:53 2388—2 union."— cEEooéo coEEoo .F 035... 689 22603 _oo>_m 953505 88m - o8 3% 525880 oow-OD om-m_w 3m .2 .2 JV 53:? 0:52 5:300 T8-4 applies — a non-polar stationary liquid phase will interact strongly with and retard the movement of non- polar compounds through the column, while polar compounds which do not interact strongly with the non-polar stationary phase move through the column more quickly. C. Column length GC-columns are typically three to five feet in length but can be longer depending on the circumstances. Separation depends on the amount of partitioning of the components of the sample between the mobile and stationary phases. In general, the longer a column is the better the separation of the compounds in the sample. As the column length increases, there are more opportunities for interactions between the components of the sample and the stationary liquid phase. This increase in the amount of possible interactions between the components of the sample and the stationary liquid phase increases the partitioning of the components along the column, giving a better separation. D. Column temperature The temperature of the column also affects the separation in gas chromatography. The solubility of a gas in a liquid decreases with increasing temperature. The retention times of the components in a sample can be decreased by raising the temperature of the column. Raising the column temperature decreases the interactions with the stationary liquid phase, causing the component(s) to move through the column at a taster rate. it the column temperature is too high, however, then little or no partitioning of the components in the sample between the stationary liquid phase and the mobile gas phase occurs and the entire sample mixture travels through the column with the carrier gas, giving poor or no separation. Too low a column temperature will also result in a poor separation. Low column temperatures typically give long retention times for the components and broad peaks in the chromatogram; at too low a column temperature, the components of the sample remain adsorbed on the stationary liquid phase and never revaporize to get carried through the column by the carrier gas. Each type of column packing has a maximum temperature at which it can be used (see chart). This maximum column temperature should never be exceeded. Running a column above its temperature limit can result in the liquid phase becoming volatile to some extent, causing the adsorbent to bleed off of the column, interfering with the separation and making the column either less effective or useless for further separations. T8-5 E. Carrier gas flow rate The carrier gas moves the sample along the column, so increasing its flow will increase the rate that the compounds in a mixture move through the column. If the carrier gas is flowing through the column too rapidly, a poor separation will result because the components in the mixture have less time to partition with the stationary liquid phase. F. Detector Unlike TLC, where a visualization technique is used after the separation is finished to view the chromatogram, in GO an electrical device called the detector is used to continuously monitor the separation. As compounds elute from the column, they pass through the detector which sends an electrical signal to the recorder which draws the chromatogram on the chart paper. The most common type of detector used in gas chromatography is a thermal conductivity detector. This type of detector works as follows: a wire, heated by constant electrical voltage, is placed in the carrier gas stream at the column exit. As the carrier gas exiting the column passes over the heated wire, it cools the wire. When helium is the only gas passing through the detector, the temperature of the wire and its electrical resistance remain constant. Helium has a much higher thermal conductivity than most organic compounds, so when an organic compound elutes from the column and passes over the detector wire the exit gas stream is no longer as effective at cooling the wire. This causes the temperature and the electrical resistance of the wire to increase. The change in the resistance of the detector wire is measured and sent electronically to the recorder which plots the change in resistance on the chart paper. Once the compound has completely eluted from the column, the exit gas stream returns to being just helium and the resistance in the detector wire drops back to the original level until the next component in the mixture elutes from the column and enters the detector. A flame ionization detector (FID) is a more sensitive way to monitor the separation on a gas chromatograph. In this type of detector, a portion of the gas stream from the column is directed into a hydrogen—air flame. When an organic compound elutes from the column and burns in the detector's flame, it produces ion fragments that increase the electrical conductivity of the hot gases above the flame. This change in conductivity is measured electronically and a signal is sent to the recorder which draws a peak on the chart paper. Although an FID is a more sensitive detector than a thermal conductivity detector, it has the drawback that the sample is destroyed during detection. T8-6 G. Peak resolution in any good chromatographic separation, the components of the sample are well resolved, i.e., they are completely separated from each other in the chromatogram. In gas chromatography, this means that after each component elutes from the column, the recorder pen returns to the baseline for a period of time before the next component passes through the detector. An incomplete separation of the components in a sample gives poor peak resolution and there is an overlap between adjacent peaks in the Figure 4. poor peak resolution good peak resolution chromatogram (Figure 4). Poor peak resolution can be caused by many factors, such as loading too much sample on the column, using too short a column, using too high a column temperature, using too high a carrier gas flow rate, etc. When the peaks in a chromatogram are poorly resolved, it is necessary to adjust one or more at the separation parameters until baseline resolution is obtained. H. Quantitative analysis GC-analysis gives no direct information about the identity of the individual compounds being separated. However, the mole percentage composition of a mixture can be obtained directly from the chromatogram by comparing the peak areas of the various components of the mixture. The area under each peak in the chromatogram is directly proportidnal to the number of moles of that compound present in the mixture injected onto the column. Many recorders'have integrators built into them that automatically calculate the areas under each peak in the chromatogram. Otherwise, the relative areas of the peaks in the chromatogram can be determined either by carefully cutting out the individual peaks and weighing them on an analytical balance (the area of a peak is proportional to the weight of the paper out out), or by using a geometrical approximation called triangulation. In this method, which works well when the peaks are symmetrical, the height of a peak (h) is multiplied by the width of the peak at half its height (w1 ,2) to give a good approximation of the area under the peak. T8-7 3—9 W l/2 area = h x w“2 Figure 5. Measuring peak area by triangulation Once the relative areas of allot the peaks in the chromatogram have been determined. the molar percentage of each component in the sample is obtained from the equation: mole percentage of _ area under individual peak x 100 Compound m m'Xture total area under all the peaks For example, assume that the GC analysis of a mixture of compounds gives the following chromatogram: Detector response Time ———> Figure 6. Three component chromatogram To determine the molar percentage of each component, first determine the area under each peak in the chromatogram. The area of each peak is given below. a 120 mm2 b 355 mm2 c 185 mm2 The sum of all of the peak areas is 660 mmz. To determine the mole percentages of the components in the sample, divide the area of each component's peak by the sum of all the peak areas and multiply by 100. T8-8 This gives the following molar ratios: a (120 mm2/660mm2) x 100 = 18.2% b (355 mm2/660mm5 x 100 = 53.8% c (185 mm2/660mm2) x 100 = 28.0% This gives the composition of the sample that was injected into the gas chromatograph. I. Questions 1. if the peaks obtained in a gas chromatogram are not well separated, what changes in conditions can you make to improve the resolution? 2. What would you predict as the order of elution of a mixture of p-xylene, o-xylene, toluene, and benzene from a carbowax GC-column? 3. A sample containing the isomers cyclohexylmethanol and cyclohexyl methyl ether is injected onto a carbowax column. Which of these two compounds would have the shorter retention time? Briefly explain why. 4. What happens to the retention time of a compound as you: a) decrease the column temperature? b) increase the length of the column? c) increase the flow rate of the carrier gas? 5. A liquid mixture has a boiling range of 100 - 132 °C. What initial column temperature might you use to separate this mixture by using gas chromatography? 6. What happens to a GC column if it is run above its maximum temperature limit? 7. A GC-traoe shows three peaks with the areas 29 mmz, 210 mmz, and 136 mmz. Calculate the mole percentages for the three components in the mixture. T8-9 ...
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Gas Chromatography - 8. GAS CHROMATOGRAPHY Gas...

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