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Thin-layer chromatography consists of a stationary phase immobilized on a glass or plastic plate, and an organic solvent. The sample, either liquid or dissolved in a volatile solvent, is deposited as a spot on the stationary phase (TLC plate). The constituents of a sample can be identified by simultaneously running standards with the unknown. The bottom edge of the plate is placed in a solvent reservoir, and the solvent moves up the plate by capillary action. When the solvent front reaches the other edge of the stationary phase, the plate is removed from the solvent reservoir. The separated spots are visualized with ultraviolet light or by placing the plate in iodine vapor. The different components in the mixture move up the plate at different rates due to differences in their behavior between the mobile liquid phase and the stationary phase.

The purpose of this lab was to use this thin-layer chromatography (TLC) technique to try and identify the active ingredients of several oral contraceptives. The four fused carbon ring structure is the trademark of steroids and forms the steroid nucleus on the molecular level.

Procedure
We first spotted a TLC plate with known steroids. These will be the standards, which will run along side steroids extracted from contraceptive pills. We Applied 10 µl of each steroid standard and 20 µl of the extracts from the contraceptive pills. The steroids were applied using a micropipeter. A small amount was applied then we allowed 30 sec for the solvent to evaporate then applied some more of the steroid to the plate keeping the spot as small as possible. The smaller the starting sample zone, the sharper the separation.

The plates were then developed in a TLC tank containing as solvent of 3% methanol in chloroform. It takes about 1.5 hours for the solvent to move 2/3 of the way up the plate by capillary action. The principle of separation here has to do with the relative polarity of the steroids. The more polar steroids (i.e., those with more polar functional groups - hydroxyl, carbonyl, double bonds) absorb more tightly to the silica gel and move slower up the plate than the more nonpolar steroids.

After the plate is taken from the TLC tank, the steroids, being colorless compounds, have to be detected with a spray reagent. That reagent will be 50 % SULFURIC ACID (CAUTION). Dr. Allen applied the spray for us, under the fume hood.

We then heated the plate in an oven, at 140 °C. If the plate is left in the oven too long, all of the steroids will be carbonized and appear as black spots on a white background. The plate should be taken out of the oven a couple of minutes after closing the oven door. The steroids will be colored in specific fashions. Standard joke is that the "little boy" steroids will turn blue and the "little girl" steroids pink. We had to identify the steroids in the various contraceptive pills by comparing the colors and Rf values to the standards. An Rf value is the distance a steroid moves up the plate divided by the distance the solvent front moves up the plate.

Results

Rf = d1      d2 = solvent front = 9cm
        d2

       Rf Values
 s) 7.4/9cm = .82cm

1) 7.5/9cm = .83cm
2) 3.5/9cm = .39cm
3) 5.5/9cm = .61cm
4) .4/9cm = .044cm
5) 2.6/9cm = .29cm
6) 5.7/9cm = .63cm
7) 8.9/9cm = .99cm
8) 8.4/9cm = .93cm
9) 6.5/9cm = .72cm
10) 7.5/9cm = .83cm
11) .5/9cm = .056cm

Anabolic steroids are synthetic substances related to the male sex hormones (androgens). They promote the growth of skeletal muscle (anabolic effects) and the development of male sexual characteristics (androgenic effects), and also have some other effects. Anabolic steroids were developed in the late 1930s primarily to treat hypogonadism, a condition in which the testes do not produce sufficient testosterone for normal growth, development, and sexual functioning. The primary medical uses of these compounds are to treat delayed puberty, some types of impotence, and wasting of the body caused by HIV infection or other diseases. During the 1930s, scientists discovered that anabolic steroids could facilitate the growth of skeletal muscle in laboratory animals, which led to use of the compounds first by bodybuilders and weightlifters and then by athletes in other sports. Steroid abuse has become so widespread in athletics that it affects the outcome of sports contests.

Not all steroids however, are anabolic steroids. Other types of steroids include anti-inflammatory drugs such as cortisone and prednisone, which are used to treat severe inflammation and asthma. Estrogens and progestagens are naturally occurring female sex steroid hormones, which are also found in oral contraceptive pills.

ZOOLOGY 310 GUIDE TO STEROID HORMONES