How to Identify Unknown Peaks in Chromatography

2013-07-05 09:35:20 liting

Laboratories commonly use the process of chromatography to analyze samples to determine their chemical composition. In gas chromatography, a diluted sample is vaporized and flushed through a column packed with material that separates the components. In liquid chromatography, a solution of the sample in solvent is pumped through a column and separated. In both cases, a detector at the end of the column responds to chemicals as they exit. The amount of time it takes a given chemical to travel through the column is generally constant and you can use that time to identify unknowns.
1. Examine the detector output --- the "chromatogram" --- produced by the chromatograph instrumentation in response to testing the unknown sample. The chromatogram is a plot of the detector output with output intensity on the vertical axis and elapsed time since the sample was introduced into the instrument on the horizontal axis. The chromatogram shows peaks (sharp increases in detector output) at the times where various chemicals exited the column.
2. Note the exit time --- often also called the "retention time" --- of each peak in the chromatogram. There will be one peak and therefore one retention time for each unknown chemical in the mixture that was tested. For example, you might see peaks at 7.8 and 10.2 minutes.
3. Obtain standard pure samples of the chemicals believed to possibly be present in the sample that was tested. This require some guesswork, but a laboratory will often have a good idea, based on the type and source of the sample. For instance, a soil sample from an abandoned gas station will likely have traces of gas components such as benzene and toluene.
4. Prepare dilute solutions of the chemical standards in a suitable solvent; one solution for each chemical. Generally you use the same solvent in which you dissolved your sample when preparing it for analysis. For liquids, use a pipette to transfer a known quantity into a volumetric flask. For solids, weigh out a known amount and transfer. In each case, you then fill the flask with solvent and mix. You will have to estimate the concentration to use for each standard solution. In general, for gas chromatography you can produce samples in the low parts per million (ppm) range and for liquid chromatography in the high ppm range.
5. Inject each of the standard chemical solutions onto the chromatographic instrumentation and examine the resulting chromatograms.
6. Compare the retention times obtained for each chemical standard to the retention times of the unknown peaks in the sample chromatogram. If the retention time of a standard matches that of a peak in the sample, you can identify that unknown peak as being due to that sample. In the case of the example with peaks at 7.8 and 10.2 minutes, if your benzene standard peak was at 7.8 minutes, you would conclude the first unknown peak in the sample was due to benzene.