Qualitative Analysis of Gas Chromatography
By Harry G. Hajian and Robert L. Pecsok
Copyright 1990, New Jersey: Prentice Hall

In Experiment 2-1, Part A, you will determine the retention times of some standard pure substances (recall the use of standards of Experiment 1-1).  In Part B, you will obtain a chromatogram of a commercial product and by comparison of retention times to those of the standards, you will identify what components make up the mixture.  To be sure you understand the task ahead, try to identify the components of the unknown mixture used in the following hypothetical experiment.

A technician measures the following retention times for pure substances.
 

Use the retention times to identify the peaks in the chromatogram of the unknown mixture below.  (The chart speed was 1/2 inch per minute.)
 
 
 

SAFETY PRECAUTIONS
The vapors of many liquids used in this experiment are flammable.  Be sure that covers are replaced on their containers immediately after use; no open flames are permitted in the laboratory.  The injection port of a GC can be very hot - when injecting your sample don t touch the septum cover as this can result in serious burn.

Part A:  Preparing Standard Chromatograms

PROCEDURE
1.  Several labeled standard samples will be made available to you.  Each standard is a pure liquid.  You will inject each one into the column separately and measure its retention time.  Unless you are actually in the process of filling the syringe, the containers should be capped to prevent hazardous vapors from escaping.

2.  Before any of the following steps are attempted, check to make sure that your equipment is functioning properly.  (As the operation of all GCs varies from model to model, values for the instrument settings mentioned in this section will be given to you by your instructor.)

3.  Draw some acetone into the syringe; the plunger should operate freely.  Keep in mind that the syringe is an expensive and fragile piece of equipment.  Now rinse the syringe several times with acetone, drawing in fresh liquid and emptying it into a flask provided for waste liquids.  Acetone is often used to rinse liquid because it easily dissolves many impurities and evaporates quickly.

4.  Rinse the syringe several times with your first standard sample.  Empty it into the waste container after each rinse.  Finally, remove a 1.0 microliter µ sample and inject it, along with several microliters of air, through the septum and into the column as shown in Figure 2-15.

5.  Wait for the sample to completely emerge from the column.  This may require several minutes.  The process is complete when the pen has returned to the baseline after recording the sample peak.  If the sample peak is very small, or if it travels of the top of the chart, change the attenuation setting* on the instrument, or change the sample size, and run the standard again. (NOTE:  Most GC instruments feature an attenuation control which enables the user to increase or decrease the electronic signal sent from the detector to the recorder.  If peaks are so large that they run off the edge of the chromatogram, the attenuation control can be increased.  The signal intensity will be decreased ("attenuated"), and the peaks will consequently appear smaller on the chromatogram.)

6.  Tear the recorded chromatogram from the chart roll and label it with the following information:

8.  In your notebook prepare a table entitled "Retention Times of Known Substances."  There should be two columns, one headed "Substances" and the other "Retention Time (min)."

9.  Measure the retention times from the air peak of each of the standard samples.  Record your data in the table you have prepared.
   

Part B: Preparing Chromatograms of Unknowns

PROCEDURE

1.  Your laboratory instructor will assign one (or more) commercial liquids to you for analysis.

2.  If you have not been doing so regularly, recheck the column temperature and carrier gas flow rate before proceeding.  These should be the same as they were when the standards were injected.  It is important that all conditions remain constant when you are comparing retention times of standards and unknowns.

3.  Follow Steps 3 to 6 in Part A to obtain a chromatogram of your mixture.  In this case, you must be sure to let the recorder run until all the components have passed through the column.  You can assume in this experiment that some components of your mixture may have a longer retention time than the longest retention time of a standard.  Find this longest time from your table, and let the recorder run at least that length of time plus 3 minutes.

4.  Measure the retention times of all the peaks on your chromatogram.

5.  Now refer to the standard table of retention times prepared in Part A, and see if any correspond to those you have measured for your commercial products.  When you find a retention time which matches that standard, list the substance to which it corresponds in your notebook.

What happens if you can find no known retention time that matches a retention time in your unknown mixture?  First, carefully reexamine the listings in the table of retention times of known substances.  Check the flow rate of carrier gas and column temperature.  If you still cannot find a match, check your measurements.  (Possibly you have made a mistake.)

It is possible that your unknown contains a few substances which were not in your list of standards.  On your chromatogram simply label these as unidentified substances.

What the Experiment Shows

Once again it is time to stop and think about our results.  We have seen that if we are given a table of retention times, it is possible to analyze mixtures of unknown composition.  This can be done rather easily and rapidly.  Just a few years ago, before the gas chromatograph was developed, the analyses, which you have performed could have been done only by a highly skilled chemist or chemical technician and would have required a long period of time.  Of course, you were able to identify only substances listed in our table of retention times.  If you were working in an industrial laboratory you might have to prepare such a table yourself, and you would want to include all the substances that would likely appear in the mixtures you normally analyze.

It is important to remember that retention time for a given substance vary from column to column and that they are also affected by the flow rate of carrier gas and the temperature of the column.  That is why it was mandatory throughout the experiment to keep these values constant.  It is best to always determine new retention times for standards immediately before attempting any qualitative analysis with the gas chromatograph.

One further word of caution: It is possible that under certain circumstances, two different substances will have the same retention times.  Since this uncertainty always exists, an absolutely positive identification of a component in a mixture is only possible if several chromatograms of each standard and unknown are run under a variety of conditions.  Normally, one suspects only certain components to be present in a sample, and the GC as a quantitative analytical tool later in the text.

In fact, we often know before the analysis what is in the sample ? and are only interested in how much of each component is present.  By measuring the relative "sizes" of sample peaks of a chromatogram, the quantity of each substance in a mixture can be determined.  We will discuss the very important use of GC as a quantitative analytical tool later ion the text.

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