INSTRUMENTAL ANALYSIS AND SEPARATIONS GAS CHROMATOGRAPHY - LECTURE 20 Mobile and Stationary Phases Columns for Gas Chromatography

Capillary columns are commonly made from high purity silica which contains no significant metallic oxides that may react with the sample. The surface however does contain many active silanol groups. Generally prior to the application of the stationary phase coating, the inner surface is deactivated. One method of deactivation is to react the free silanol groups with a reagent such as trimethylsilyl chloride. The stationary phase is then applied, usually by a method known as static coating. The column is filled with the stationary phase dissolved in a volatile solvent. Then one end of the column is sealed, a vacuum is applied and the solvent is evaporated, leaving an even coat of the stationary phase on the column wall. The stationary phase is held in place only by surface tension forces and it can be disturbed by solvents in the sample or excessive temperature. Increased stationary phase stability can be achieved either by polymerization of the coating or by covalently linking it to the column surface. In addition to increasing durability, immobilization of the stationary phase by these processes allows a much thicker film to be applied which is important especially for separating highly volatile compounds. It is also possible to regenerate immobilized stationary phases by washing the column with solvents such as methylene chloride or tetrahydrofuran to remove soluble contaminants that accumulate in the column(this can extent column life). The outer surface of the column is coated with a polymer called polyimide which makes the column strong and flexible. Any flaw in the coating, and the column can break very easily. There are three major types of gas chromatography columns, packed columns, porous layer open tubular (PLOT) columns, and wall coated open tubular (WCOT) columns. The geometry of WCOT capillary columns is fairly simple, consisting of length, internal diameter, and stationary phase thickness. Nevertheless, there are endless possible combinations of these three factors that could be used for optimizing chromatography. Doubling the column length effectively doubles the number of theoretical plates but the resolution between any two compounds is proportional to the square root of the plate number so doubling the column length only increases resolution by about 40%. Doubling the column length also will result in longer analysis times and small peak heights, so longer columns are not the answer to poor resolution. However, longer columns do spread out the peaks and as a general rule, the more complex the sample, the longer the column should be.

Column Diameter

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The diameter of a column and the thickness of the stationary phase should always be considered together because they interact with regards to column performance. However, the general effect of decreasing column diameter is to increase the speed of analysis. This is because the optimal carrier gas velocity increases as the diameter decreases (assuming that the retention factor and plate number are held constant). Smaller diameter columns usually have lower plate height (higher N) because of improved mass transfer. Increasing carrier gas velocity results directly in faster analyses. On the other hand, larger diameter columns have increased sample capacity which can provide higher detectability with mass sensitive detectors such as an FID. Another consideration of column diameter is how it affects the amount of sample that can be injected. Larger samples require a larger amount of stationary phase to interact with, otherwise the column will be overloaded, resulting in poor chromatography. Larger diameter columns have higher capacity and less problems with overloading. Overloading results when a band of a compound in the sample totally saturates the stationary phase. Once the stationary phase is saturated, no more of the compound can interact with the stationary phase so it will move through the column as if it were an unretained compound. Since some of the compound is moving faster than it should, it will elute from the column a little earlier which will show up on the chromatogram as a fronting peak. If a compound overloads the mobile phase it will condense on the inner surface of the column (without partitioning into the stationary phase). This effect is most often seen as later eluting and tailing peaks. It is important to note that overloading is related to specific compounds in the sample. Generally if complex samples are injected, some compounds will be overloaded, while some compounds may not show up on the detector because their concentration is below the detection limit. Therefore, the amount of sample injected is based on a balance between these effects. Thickness of Coating The thickness of the stationary phase coating is very important. Thinner coatings usually provide higher efficiency (higher N or lower HETP) because mass transfer is quick between the mobile and stationary phase. However, the column capacity is low and the column is easily overloaded. Very volatile compounds should be analyzed using thick films because there is not sufficient interaction with thin films and the result is poor separation. Very nonvolatile compounds should be analyzed using thin films, otherwise the retention in the stationary phase is too long. Polar compounds have a tendency to have excess tailing of peaks. This peak tailing can be reduced with thicker stationary phase coatings

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Stationary Phases The different stationary phases available are described by the term polarity. This term is used in a loose sense as a measure of the interactions between the sample compounds and the stationary phase. These interactions are mostly based on dipoledipole attraction, induced dipole-dipole attraction and van der Waals forces. Because separations are so strongly influenced by temperature, there is no great need for many types of stationary phases. Common stationary phases are based either on polysiloxanes or polyglycols. The methylsiloxane group [Si(CH3)2O] can be polymerized to a wide range of thermally stable compounds ranging from low viscosity liquids gums to silastomer rubbers. These polymers can also be easily modified by substituting polar groups for methyl groups in the polysiloxane structure thereby producing a wide range of polarities. Table. A Few of the Available Stationary Phases For Gas Chromatography
Compound Poly(methyl siloxane) Polarity Low Max Temp oC 300-350 Column ID HP-1 AT-1 DB-1, SE-30 OV-1 ZB-1 RTx-1 BP-1 SPB-1 CP-Sil 5 CB HP-5 AT-5, EC-5 DB-5, SE-54 OV-5 ZB-5 RTx-5 BP-5 SPB-5,MDN-5 CP-Sil 8 CB HP-20M AT-Wax DB-Wax Carbowax 20M ZB-Wax Stabilwax BP20 Supelcowax 10 CP-Wax 52 CB HP-FFAP AT-1000 DB-FFAP OV-351 ZB-FFAP Stabilwax-DA BP-21 Nukol, SPB-1000 Manufacturer Agilent Alltech J&W Ohio Valley Phenomenex Restek SGE Supelco Varian Agilent Alltech J&W Ohio Valley Phenomenex Restek SGE Supelco Varian Agilent Alltech J&W Ohio Valley Phenomenex Restek SGE Supelco Varian Agilent Alltech J&W Ohio Valley Phenomenex Restek SGE Supelco Varian

95% Dimethyl, 5%phenyl Poly(methyl siloxane)

Low

300

Polyethylene glycol

Medium

250

Polyethyleneglycol Nitroterephthalic acid ester (free fatty acid phase)

Medium

250

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Mobile Phase Hydrogen, Helium and Nitrogen are the most common gases used as mobile phases. The lighter the carrier gas, the higher the speed of analysis. Therefore, hydrogen will give the most plates/sec. However, helium is often used because hydrogen is potentially explosive. High-speed analysis also results in narrower peaks and better detectibility. Longitudinal diffusion is lowest in the heavier gases such as nitrogen so nitrogen can give the highest N (lowest H) at low mobile phase velocities where longitudinal diffusion has the highest effect on efficiency.

Figure 1. Velocity vs Theoretical Plate Height for Common Carrier Gasses. Gases are forced through the column by an applied pressure at the head of the column. The outlet end of the column is usually at atmospheric pressure, or even vacuum (when attached to a mass spectrometer). Therefore there is a drop in pressure as the gas moves through the column. This drop in pressure causes the gas to expand which can result in peak broadening. It also causes the gas velocity to increase as it moves through the column (Figure 2). Therefore it becomes difficult to optimize gas velocity. Figure 2 shows the gas velocity profile along the column. The value pi/po is the ratio of the inlet velocity(pi) to the outlet velocity (po). Lecture 20 4

As the column oven is heated, the viscosity of the mobile phase increases. (the viscosity of gases generally increases with temperature, which is in contrast to liquids where the viscosity decreases with increasing temperature). Therefore as the column is heated during the temperature ramp, the flow rate goes down. In order to keep a constant flow (and reduce peak spreading of later eluting peaks) a process of pressure programming is used. The constant flow mode increases the pressure at the head end of the column, and keeps the mobile phase velocity constant. Pressure programming can also be used to increase mobile phase velocity as the temperature increases, further decreasing analysis time and increasing peak height.

Kovats Retention Index The retention time of an analyte provides at least some information on the chemistry of the compound. However, retention time is dependant on many operational factors such as temperature, column length, column diameter, coating thickness, etc. Therefore it is more satisfactory to use a relative retention value whereby many of these variations are compensated for. The Kovats retention index system is based on a scale defined by the elution of a series of n-alkanes. It is widely used, and information on the Kovats Index for many compounds can be found in the literature. In this system, normal alkanes (pentane, hexane, heptane, etc.) are given an index value 100 times their carbon number (ie. Pentane = 500). The elution time of an alkane gets longer with the addition of each additional CH2 group such that the a plot of the log of retention time vs carbon number is linear (there is some deviation, especially at lower carbon numbers). The

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calculation of the Kovats Index for a particular compound is obtained by interpretation of its position between the two nearest alkanes in the reference series. Kovats Retention Index = 100Cn + 100 [log tR(A) log tR(n)] [log tR(n+1) log tR(n) Where Cn is the carbon number of the alkane eluting just before the analyte tR(n) is the retention time of the alkane eluting just before the analyte tR(n+1) is the retention time of the alkane eluting just after the analyte tR(A) is the retention time of the analyte

The classical Kovts retention index is measured under isothermal conditions. However, in the case of temperature-programmed gas chromatography a similar value can be calculated utilizing direct numbers instead of their logarithm. In other words, an equation for the retention index can be developed using a polynomial regression of a series of alkanes vs. their retention times. This method of measuring reference retention is dependant only on the analyte, the temperature and the stationary phase composition. A Kovats index for an analyte should be the same on any column with the same stationary phase irregardless of column length, mobile phase, flowrate, etc. However it has been shown that in open tubular columns with thin films, adsorption effects can contribute to retention, affecting the accuracy of the Kovats index especially at low temperatures and with polar stationary phases. References. Grant, D. W. Capillary gas chromatography; John Wiley & Sons, Ltd: West Sussex, England, 1996; pp 295. Modern Practice of Gas Chromatography; 3rd. ed.; Grob, R. L., Ed.; John Wiley & Sons, Inc.: New York, 1995; pp 888.

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