Example

This section provides an example elucidating the handling and operation of the chromatograph. The fundamentals of sample introduction are explained first; followed by an example of analysing a hydrocarbon mixture and evaluating the resulting measurement. In this example, the benzene content in supergrade petrol is determined.

General note using this software

The examples can be comprehended with the CGA21-Software not only with the gas chromatograph itself, but also by means of simulation software on every Windows workstation in your lab. Of course you can’t make real measurements with a PC, but evaluation of measurements as well as the single steps that lead to the actual measurement can be demonstrated in the lecture hall. The USB pen drive, that’s included in delivery contains the system software of your CGA21 in portable form running on every Windows PC using Windows XP up to Windows 7 without any installation.

Here’s how to use the pen drive with the CGA21 software:

· Plug the flash drive into an USB connector of your Windows PC.

· Open the Explorer (e.g. right click Start button, than select Explorer) If autorun comes in your way, cancel it.

· Search for the pen drive, its name is CGA21, navigate into folder _CGA21 and double click gc.exe

· Depending on your operating system you have to agree to security warnings

Starting the software on a workstation

After the program has been started a GC measurement is loaded automatically so you can immediately start using the program. The software may be handed over to employees, students or pupils of your institution.

No software is perfect. Please help improving this program (or the manual)! Send bug reports or suggestions to frank.beer@s-lg.de

Notes on injection

Sample introduction is the only step in the operation of a chromatograph which requires a certain amount of skill and practice in order to achieve reproducible results.

To fill the syringe, push the plunger in to the limit, immerse the needle in the liquid, and then draw the plunger slowly to a point far beyond the required volume. Observe the column of liquid in the syringe. If it is largely free of bubbles - very small air bubbles with a volume of approximately 0.01µl cannot be avoided in practice - then push the plunger forward until the required volume is reached. Any air bubbles still remaining can be compensated.
Example: The column of liquid contains an air bubble approx. 0.1µl in size and the required volume is 1µl; consequently, the plunger is set to the 1.1µl mark. If the column of liquid contains too much air, then "pump" the plunger briskly - but not too briskly - down to its limit, draw in liquid again slowly, and observe the liquid column. If the amount of air appears sufficiently small, then proceed as before and adjust the sample volume, allowing for the air quantity.

During injection, a compromise needs to be struck between two conflicting requirements. On the one hand, the injection must be rapid as the thin needle of the syringe heats up very quickly in the hot injector; if the sample in the needle starts boiling, part of the substance will be ejected prematurely from the syringe. This will result in widened, deformed or even duplicated peaks. On the other hand, the relatively thin and sensitive needle needs to penetrate the septum of the injector, an act which requires a certain amount of effort as well as caution. If you only hold the glass cylinder of the syringe, the needle will bend sideways and, in the worst case, become unusable. For this reason, you should use one hand to hold the glass cylinder and guide the needle with the thumb and index finger of your other hand. Once the needle has been fully inserted, press the plunger down quickly and then withdraw the syringe immediately. This procedure should be coordinated in such a way that the plunger is pushed down as soon as the countdown of the chromatograph has elapsed to zero.

Determining the benzene content in supergrade petrol

In some countries it is not allowed to carry out experiments with Benzene in school labs. To comply with applicable regulations use other substances eg. Toluene instead of benzene.

Identifying a substance in a chromatogram

The investigation of mineral-oil products is an important domain in high-resolution gas chromatography, as these complex mixtures cannot be analysed so elegantly using any other method. The content of benzene, present in varying quantities of up to a few percent in carburetor fuel, is to be measured in this example. The measurements are in directory _CGA21\_GC_Mes

One of the peaks in the chromatogram above is benzene. Which one is it?

To identify a particular peak, add a certain amount of the substance you are searching for, i.e. benzene in this case, to the sample of the test mixture. The quantity of the added substance should be roughly equal to that already present in the mixture. In this example, 10% by volume of benzene has been added to the supergrade petrol. The sample is then analyzed with the same parameters as used for the original sample, and the two chromatograms are subsequently compared.

For comparison, click “Compare chromatogram” in the main menu and observe both the chromatograms on a suitable scale. If the test substance contains benzene, then the second chromatogram should contain a peak also present in the original one, but considerably smaller there. Fig. 13 shows the comparison identifying the benzene peak, which is much larger in the upper chromatogram than in the lower one.

Comparison of gasoline and gasoline with benzene added

For the sake of convenience, the peaks in Fig. 14 already have been marked. Fig. 15 shows an enlarged section of the region where the benzene is expected. If, however, we assume there is a substance different from benzene with a retention time very close to that of benzene we would be misguided. Our assumption about the identity of peak 1 in the diagram only has a certain probability and is far away from certainty. To come closer to this, additional analysis would be necessary, e. g. a mass spectrum of the peak would give clear statement about the nature of the substance behind it.

Section from above

Quantitative analysis:

Now that we know the peak that represents the benzene, the next step is the determination of the content of the sample. This can be done in two different ways:

• Comparison of total peak area and single peak area: Estimation of the content by comparing the area of all peaks with the area of the desired peak.
• Determine the absolute mass through a comparison with the peak of the reference substance

Comparison total area / single peak area

Quick and dirty method for a rough estimate. Step by step procedure:

· Mark the benzene peak.

· Mark entire chromatogram before the benzene peak and after the benzene peak. See Fig. 15. The benzene peak is marked for clarity.

· The peak list now shows three peaks. One of the peaks is benzene, the other peaks represent all hydrocarbons present in the probe. The percentage of benzene can now be read directly from the peak table. Although we obtain a rasonable value of 2,7%, it must be stressed that this can be regarded only as a rough estimate. The reasons are manifold. One of the most important is the fact, that the sensitivity of the flame ionisation detector is dependent on the nature of the analysed substances.

Comparison total/single peak area

Comparing two peaks

The classical procedure to determine the quantity of a substance in a sample mixture is to make two measurements and compare the peak areas of interest:

1. Perform a measurement using the substance containing the benzene in an unknown concentration, termed main measurement here. The benzene peak must be identified as shown in Chapter 3.3.2. Mark the benzene peak.
2. Perform a measurement using a standard solution containing benzene in a known concentration. This measurement is termed reference measurement here. Mark the benzene peak. It is recommendable to use a concentration roughly equal to the expected value, i.e. 4% in this case. For comprehension use file Heptane_benzene_mark_quant.gcx The two measurements should be performed under identical conditions (T-program, gas flow etc). For best results you should make two consecutive measurements.
3. Assign the mass of the benzene contained in the injected reference substance to the benzene peak in the reference measurement.
4. Compare the areas of the reference benzene peak with the identified benzene peak in the main measuremet.

For clarity steps 3 and 4 from above are explained in full details:

Step 3:

• Click “open measurement” and load the reference measurement into the main display.
• Mark the benzene peak by double clicking start and end position of peak. The peak now appears as a line in the peak list.
• Double click the name field and enter “benzene”, then double click the mass field and enter 17.5 The mass of the injected portion of benzene is calculated from the volume of the injected mixture, its content, and the density of the benzene:

m(Benzene) = V(Injection) × w(Benzene) × r(Benzene)
m(Benzene) = 0.5 µl × 4% × 0.874 g/ml » 17.5 µg

• The benzene line in the peak table is now updated and the color of the benzene peak turns to green, meaning that this peak is calibrated with a known mass.

The chromatogram of the reference measurement is shown in Fig. 17. The benzene has already been marked and assigned peak mass and name.

Refererence measurement ‘benzene in heptane’, entering peak mass

Step 4:

Now we can finally compare the gasoline measurement and the benzene reference measurement:

• Open the main measurement by clicking “open measurement” in the tool bar on top
• Click “compare measurement” in the tool bar, now two data displays are visible.
• Open the reference measurement by clicking the “open reference” button on the left hand side of the reference display.
• Adjust the display excerpt to suit your requirements. The two selected peaks should now be roughly at the same position, as shown in Fig. 18. If this is not the case, use the pan and zoom buttons to move both maxima into about the same position.
• Click the “compare” button left to the peak list, move the cursor into the main measurement display where it turns into a line cursor. If the line cursor is inside the benzene peak, click the left mouse button.
• This triggers the following actions: From the known mass of the reference peak and its area a factor is determined that allows for conversion of peak area to absolute mass. This factor is immediately used to compute the mass of the benzene peak in the gasoline probe. You should give this peak a name by double clicking the name cell and entering the name in the pop up window. Now don’t forget to save your measurement!

Comparing refererence measurement and main measurement

You have determined the mass of benzene present in our sample of supergrade petrol. By balancing a simple equation, you can now calculate the volume percentage of benzene in the supergrade petrol:

m(Benzene) = 8.342µg

v(Benzene) = 8.342µg / 0.874g/ml = 9.54nl

w(Benzene) = 9.54nl/0.5µl » 2.0 Vol%