Running Your Chromatograph

While the sample is cooling, prepare a chromatographic column. Take a 25-mL buret. Add a small piece of glass wool. With the aid of a glass rod, push it down near the stopcock. Add 15-16 mL of petroleum ether to the buret. Open the stopcock slowly, and allow the solvent to fill the tip of the buret. Close the stopcock. You should have 12-13 mL of petroleum ether above the glass wool. Weigh about 20 g of alkaline aluminum oxide (alumina) in a 100-mL beaker. Place a small funnel on top of your buret. Pour the alumina slowly, in small increments, into the buret. Allow it to settle to form a 20-cm column. Drain the solvent but do not allow the column to run dry. 

The crude carotenoid is to be chromatographed on a 12-cm column of acid-washed alumina (Merck), prepared with petroleum ether (37-53°C) as solvent. Run out excess solvent, or remove it from the top of the chromatography column with a suction tube, dissolve the crude carotenoid in afew milliliters of toluene, and then transfer the solution onto the chromatographic column with a Pasteur pipette. Elute the column with petroleum ether, discard the initial colorless eluate, and collect all yellow or orange eluates together in a 50-mL Erlenmeyer flask. Place a drop of solution on a microscope slide and evaporate the rest to dryness (rotary evaporator or aspirator tube). Examination of the material spotted on the slide may reveal crystallinity. Then put a drop of concentrated sulfuric acid beside the spot and mix with a stirring rod. Compare the color of your test with that of a test on the other carotenoid. 

Make up thin layer plates as described above. Chromatograph the standards and the unknown(s) in toluene and locate the spots by iodine staining. Calculate the R( values and hence determine which of the five standard compounds the unknown(s) contain. Confirm your conclusions by running a further chromatogram using the unknown mixture as one spot and the suspected mixture as a second spot. (The Rf values may vary with the quality of the TLC plate and hence comparisons between the unknown and the standard must finally be made on the same plate.)... 

When you think of installing a gas chromatograph, you need to consider the entire system in order to run the new chromatograph(s) to peak efficiency. This means thinking of all aspects of the installation. The information in this chapter will help you design your gas management system wisely and obtain efficient performance from one or many gas chromatographs in a laboratory. 

To determine how many hydrogen generators you will need, calculate expected flow needs according to the number of gas chromatographs and the types of detectors and other equipment you will be using (Tables 10.1 and 10.2) or specific flow information from your instrument manuals). Once you know how much hydrogen you will need, you can determine which model or models will meet that need from Table 10.3. It is always best not to run at full capacity and to purchase a unit with excess capacity. 

---