An Introduction to Gas Chromatography: Evaluating Experimental Parameters That Influence Gas Chromatographic Performance

5.16.1 Background and Summary of Method

Gas chromatography (GC) is the most widely used instrumental technique for the determination of trace concentrations of volatiles (VOCs) and semi-volatiles (SVOCs) organic pollutants found in environmental samples today! Its origins stem from the pioneering work of Martin and Synge in 1941 to the development of open tubular gas chromatographic columns advanced by Golay to the fabrication by Dandeneau and Zerenner [7 High Resol. Chromed. Chromatgr. Commun 2 351(1979)] at Hewlett-Packard of the fused-silica wall-coated open tubular (WCOT) gas chromatographic column which revolutionized the practice of gas chromatography. It must be recognized, however, that approximately 20% of all of the organic compounds that exist and could possibly make their way into the environment are amenable to GC techniques without prior chemical modification! Despite this limitation, over 60 organic compounds classified as VOCs have been found in drinking water, groundwater, surface water, and wastewater and are routinely monitored. Over 100 SVOCs have also been found, which include phenols, polycyclic aromatic hydrocarbons, mono-, di-, and trichloro aromatics and aliphatics, nitro aromatics, polychlorinated biphenyls, organochlorine pesticides, organophosphorus pesticides, triazine herbicides, and phthalate esters, among others.

The theoretical principles that underlie GC are found in numerous texts and monographs. Specific methods that incorporate GC as the determinative instrumental technique are to be found in a plethora of analytical methods published by the Environmental Protection Agency (EPA), the American Public Health Association / American Water Works Association / Water Pollution Control Federation (Standard Methods for the Examination of Water and Wastewater), and the American Society for Testing Materials (ASTM, Part 31, Water).

This exercise introduces the student to those experimental GC parameters that exert a major influence on GC performance. These include (1) detector selectivity, (2) injection volume vs. chromatographic peak shape, (3) the effect of changing the carrier gas flow rate on column efficiency, and (4) the effect of column temperature on chromatographic resolution and analysis time. This exercise affords the student the opportunity to vary these parameters and assess the outcomes. This is a qualitative analysis exercise only and involves making and recording observations and doing some calculations from information found in the chromatograms.

5.16.2 Brief Description of Gas Chromatographs Located in the Hazardous Waste Analysis Lab at Michigan State University

The Autosystem® PerkinElmer gas chromatograph (GC) consists of a dual-injector port, dual- capillary-column configuration, and dual detectors including the flame ionization detector (FID) and the electron-capture (ECD)) connected via an analog-to-digital (A/D) interface to a personal computer (PC) workstation. The PC is driven by the Turbochrom® (PE Nelson) chromatography data processing software. You will encounter two types of A/D interfaces in the laboratory. The 600 LINK interface provides for both data acquisition and instrument control. The 900 interface provides for data acquisition only.

The front injector consists of a split/splitless capillary column type and is connected to a 0.25 mm (i.d.) x 30 m (length) wall-coated open tubular (WCOT) column (referred to as a narrow-bore WCOT column). The column is coated with a DB-5 liquid phase (5% phenyl dimethyl siloxane) that is chemically bonded to the inner tubing wall. This type of liquid phase is appropriate for the separation of SVOCs whose boiling points are much greater than 100°C. The optimum volumetric flow rate (i.e., the flow rate that gives a minimum in the van Deemter curve) is between 1 and 3 cm' per minute. To obtain such a low flow rate, a split vent is required to remove most of the gas. Refer to the instruction manual for setting the split ratio. Typical split ratios are 1:25, 1:50, or 1:100, and this ratio refers to the ratio of gas flow through the column to that through the vent. The outlet end of this column is connected to the inlet to the ECD. This detector requires an additional source of inert gas, commonly called makeup gas. The flow rate for the makeup should be approximately 30 cm'/min. Using the digital flow check meter (refer to the instruction manual), measure the initial flow rate, then adjust to the optimum for operation of a narrow-bore WCOT column.

The rear injector consists of a packed column adapted for connection to a 0.53 mm (i.d.) x 30 m (length) capillary column (referred to as a megabore column). The column is coated with a cyanopropyl dimethyl polysiloxane liquid phase that is chemically bonded to the inner tubing wall. This type of liquid phase is appropriate for the separation of VOCs. The optimum volumetric flow rate is between 5 and 15 cm'/min. The column outlet is connected to the inlet to the FID. This detector does not require makeup gas. The FID, however, requires a 10:1 ratio of airflow to hydrogen flow. Conventional flow rates are 300 cm'/min for air and 30 cm'/min for H,. Once the air/fuel ratio has been established, the FID can be ignited. Sometimes, a slightly fuel- rich ratio is necessary to ignite the FID.

5.16.3 Principle of Separation in GC

When two compounds migrate at the same rate through a chromatographic column, no separation is possible. Two compounds that differ in retention times, /R or capacity factor, k', and appear to separate, do so because of differences in their equilibrium distribution constants, denoted by Kp. If KD is independent of sample size, Gaussian elution bands (i.e., symmetrical peaks) are observed. This is the case of linear elution chromatography. In other words, a plot of the concentration of analyte in the stationary phase to the concentration in the mobile phase yields a straight line whose slope equals Kp. If the amount of analyte increases either by injecting equal volumes of solutions whose concentrations are increasing or by injecting increasing volumes of a solution whose concentration is fixed, nonsymmetrical chromatographic peaks result. Kp is now dependent on the amount of solute, and either peak tailing or peak fronting results. This is the case of nonlinear elution chromatography. Gaussian or symmetrical peak shape is a chief objective when GC is used to perform trace quantitative analysis. The following equations relate the parameters discussed above:

where p is the volume (mobile phase)/volume (stationary phase), tR is the retention time for a retained peak, and t0 is the retention time for an unretained peak.

5.16.4 Experimental

Gas chromatograph interfaced to a PC that is loaded with chromatographic software. In our lab, an Autosystem® (PerkinElmer) is interfaced to a PC workstation that utilizes Turbochrom® (PE Nelson) for data acquisition, processing, and readout.

5.16.4.1 Preparation of Chemical Reagents

Note: All reagents used in this analytical method contain hazardous chemicals. Wear appropriate eye protection, gloves, and protective attire. Use of concentrated acids and bases should be done in the fume hood.

  • 5.16.4.2 Accessories to Be Used with the GC per Group
  • 1 Digital flow check meter.
  • 1 GC syringe with a beveled end that includes a Chaney adapter. Do not confuse with the blunt-end syringe used for HPLC.
  • 1 GC test mix for each of the studies discussed below.
  • 5.16.4.2.1 Summary of Turbochrom Methods to Be Used in This Experiment

5.16.4.3.1 Measurement and Adjustment of Carrier Gas Flow Rate and Split Ratio

Order

Turbo Method

Remarks

1

FLOWRATE

2

DETSENS

Neat acetone—FID

Neat methylene chloride—ECD

3

INJECD

Inject increasing amounts of 10 ppm 1, 2, 4-trichlorobenzene

INJFID

Inject increasing amounts of hexadecane at 225°C

4

PLATES

Temperature program: 265°C (0.1) to 285°C (10.0) at 6°C/min; multi- component organochlorine test mixture

5

TMAX

Isothermal at 285°C

TMIN

Isothermal at 200°C

5.16.4.3 Procedure

Refer to Summary of Turbochrom Methods (above) for a definition of each of the Turbochrom methods created in support of this experiment. These previously created methods are illustrative of how chromatography-based software can be used to teach fundamental principles of GC.

5.16.4.3.1 Measurement and Adjustment of Carrier Gas Flow Rate and Split Ratio As you approach the gas chromatograph, you will find it in an operational mode, with carrier gas flowing through both capillary columns. If not already set up, retrieve the Turbochrom file titled “FLOWRATE” and download this method. Your first task will be to measure the flow rate of the carrier gas through both capillary columns with the makeup gas off. After turning the makeup gas on, measure the split ratio through the capillary injector using the digital flow check meter. Record flow rate data in your lab notebook.

Once the optimum carrier flow rates have been established, the dual detector method titled “DETSENS” can be retrieved from the Turbochrom software, then transferred to the instrument via the interface (a process known as download) and the comparison of detector sensitivity can be undertaken. Ignite the FID (refer to the operator’s manual for the Autosystem GC from Perkin Elmer for the specific procedure).

5.16.4.3.2 Comparison of the FID vs. the ECD Sensitivity

Allow time for the GC to equilibrate at the column temperature set in the method. Using the manual injection syringe (GC syringes are manufactured by the Hamilton Co. as well as by others), inject equal microliter (pL) aliquots of acetone into both injectors. Observe the appearance of a retained chromatographic peak found in both chromatograms. You cannot assume that tR will be identical on both columns! Compare the peak heights from both chromatograms. Inject equal pL aliquots into both injectors, as earlier, of the specific chlorinated hydrocarbon that is available. Record your observations and compare the peak heights as done previously. Each member of the group should have an opportunity to make these sample injections so as to gain some experience with manual syringe injection of organic solvents.

5.16.4.3.3 Injection Volume vs. GC Peak Shape

Retrieve the Turbo file titled “INJECD” and download. Inject a series of increasing pL aliquots of a reference solution labeled “10 ppm 1,2, 4-trichlorobenzene” into the front capillary injection port. Obsen’e and record the changes in chromatographic peak shape as the amount of analyte is increased. Retrieve the Turbo file titled “INJFID” and download. Repeat the series of injections as before using the reference solution labeled “hexadecane” and make these injections into the rear injector. Observe and record the changes in chromatographic peak shape as the amount of analyte is increased.

5.16.4.3.4 Flow Rate vs. Capillary Column Efficiency

A column’s efficiency is determined in a quantitative manner from the chromatogram by measuring the number of theoretical plates, N. The effect of carrier flow rate on capillary column efficiency is significant in GC and will be examined under isothermal conditions (i.e., at a fixed and unchanging column temperature). Retrieve the Turbo file “PLATES” and program this method for a high flow rate by increasing the head pressure. Save this change in the method and download the method. Turn off the makeup gas and adjust the actual pressure so as to nearly match that which is set in the method and measure the flow rate. Turn the makeup back on. Inject 1 pL of the test mix and observe the chromatogram. Retrieve the method a second time and reprogram the head pressure to a much lower value. Turn off the makeup, decrease the carrier head pressure, measure the new flow rate, turn the makeup gas back on, and then make a second injection using the same volume.

For the carrier gas flow rate that exhibited the highest efficiency, calculate the number of theoretical plates using equations from your text. In addition, for the optimum carrier flow rate, choose any pair of peaks and calculate the resolution for that pair.

5.16.4.3.5 Column Temperature vs. Capacity Factor

Retrieve the Turbo file titled “TMIN” and download the method. Inject approximately 1 pL of the multi- component organochlorine test mix at this column temperature of 200°C. Observe the degree of separation among organochlorine analytes and record your qualitative comments.

Retrieve the Turbo file titled “TMAX” and download the method. Inject the same volume of the multi-component organochlorine test mix and obseiye the chromatogram when the column temperature has been increased to 285°C.

5.16.5 For the Lab Notebook

Write a brief discussion on how your experimental observations connect to the theoretical relationships for GC introduced in various textbooks and journal articles.

Address the following:

  • 1. Explain why different GC detectors have different instrument detection limits.
  • 2. If you operated a GC at significantly reduced carrier gas flow rates, predict what you would observe in a gas chromatogram for the injection of organic compounds. What would be the principal cause for these observations?
  • 3. Explain why a symmetrical peak shape is important in gas chromatography.
  • 4. What happens to KD for a given organic compound when column temperature is varied?
  • 5. How efficient is your GC column? That is, what is the number of theoretical plates? How many plates per meter do you have?
  • 6. How is the phase ratio, (3, determined for capillary GC columns?
  • 5.16.6 Suggested Readings

To develop this experiment, the author consulted the following resources:

Sawyer D., W. Heineman, J. Beebe. Chemistry’ Experiments for Instrumental Methods, New York: John Wiley & Sons, 1984, pp. 321-343.

A thorough grounding in the principles and practice of the GC and GC-MS determinative techniques can be found among others in the resources shown below:

Perry J. Introduction to Analytical Gas Chromatography. New York: Marcel Dekker, 1981. Jennings W. Analytical Gas Chromatography. San Diego: Academic Press, 1987.

McNair H., J. Miller. Basic Gas Chromatography. New York: Wiley Interscience, 1998. Budde W. Mass Spectrometry: Strategies for Environmental and Related Applications, Washington, D.C.: American Chemical Society, Oxford University Press, 2001.

Grob R., E. Barry., Eds. Modern Practice of Gas Chromatography, 4th ed. Hoboken, NJ: Wiley-Interscience, 2004.

 
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