Before you read this supplement, you should have an understanding of liquid chromatography and UV absorbance detection. See your lecture notes, your textbook and the SRIF backgound paper on HPLC.
As you noted in the introductory lab sesssion, the HPLC system gives out two types of data images. One is called a chromatogram, and the other is called a spectrum. The chromatogram is a graph that monitors the signal in the detector over time. As chemicals are detected by the instrument, the signal increases, and the chromatogram displays a "peak." Each peak in the chromatogram indicates the presence of a chemical in the sample. The chromatogram from last week is shown in Figure 1 below.
Each peak is labeled with retention time. Retention time indicates how long it takes for a compound to come out of the HPLC column. The first few small peaks in the chromatogram (around 2-3 minutes) are "noise" from the injection we made. We ignore those. The larger peaks represent chemicals in the sample. Notice there are 5 peaks. The peak labeled 9.994 minutes is actually methylnaphthalene and the peak at 13.468 minutes is phenanthrene.
Figure 1: HPLC-UV Chromatograph of PAHs
If we use the exact same HPLC method, both methylnaphthalene and phenanthrene will come out at nearly identical retention times every time we run them. We can use retention time as a way to determine the presence of these chemicals in other samples.
Qualitative analysis involves running a standard that contains the target analytes. We note the retention times. This technique is called "calibrating" the instrument. Standards that are used for calibrating an instrument are called "calibration standards," for obvious reasons. In our LC Chemstation software, we can build a "calibration table" containing retention times for the analytes we are interested in.
Now we can run the sample(s) of interest. If we see peaks present in the sample that correspond to peaks in our calibration standard, we have a probablility of a match. For example, if we ran a calibration standard containing heroin and found it's retention time was X minutes and then ran a blood sample from Subject A and saw a peak at X minutes, we might say that Subject A was on heroin! Of course, we would want to confirm that before we made any accusations. The next graphic tells us of a way to do that.
The other graphic produced by our HPLC-UV system is called a UV spectrum. UV spectra are signatures produced by each chemical in the detector. Our HPLC system scans for these spectra every second during the sample analysis. If we click on the peak in the chromatogram while we are in the Sepctral Task mode of LC Chemstation, we can obtain the spectra from any point in time. Of course, the spectra of greatest interest to us are the ones that are scanned when the analytes are present in the detector. Therefore we select the sepctra at the apex of the peaks. Figure 2 is the spectrum from the 9.994 minute peak (methylnapthalene). Notice that the spectrum has UV absorbance wavelengths along the x axis. This spectrum is relatively unique for methylnaphthalene, but other chemicals may have very similar spectra.
Figure 2: UV Spectrum of methylnaphthalene.
Figure 3 is a UV spectrum of phenanthrene (the 13.468 minute peak). Notice is is quite different from the spectrum in figure 2. This difference allows us to confirm the identity of analytes in a sample. However, some spectra have small differences and cannot be absolute confirmations by themselves. Analytical chemists use both retention time and spectra to determine a probability of identifying a chemical in a sample.
Figure 3: UV Spectrum of phenanthrene.
Our HPLC system is used to determine the presence of specific chemicals (analytes) in a sample. We accomplish this by first running a standard* containing our target analytes using a specific method. Then we note the retention times and their UV spectra.
HPLC is big business! The manufacturers of HPLC systems and supplies offer an abundant supply of information. They want you to use their products, and they will educate you so that you do! Try browsing their sites and their online catalogs. You may find a project title!
Here is a partial list of some of the suppliers of HPLC products.
Search the Waters Corporation site for further information about our experiment. Use their search engine in the Applications link.
More information is available from the Hewlett Packard web site. Look for their chemical analysis pages. Then search their literature.