Thin-layer Chromatography of Steroid Hormones

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liver. An increased secretion of the glucocorticoids is found in Cushing’s syndrome (adrenal hyperplasia), pregnancy, and stress due to disease, surgery, and burns. carry others. If the process is halted before all the steroids have been washed off the top of the plate, some will have migrated farther from the origin than others. THIN-LAYER CHROMATOGRAPHY In this exercise, an attempt will be made to identify two unknown steroids that are present in the same solution. To do this, you must (1) separate and (2) identify these steroids by comparing their behavior with that of known steroids. Since each steroid has a different structure, each will have a different solubility (ability to be dissolved) in a given solvent. These differences will be used to separate and identify the steroids on a thin-layer plate. The thin-layer plate consists of a thin layer of porous material (in this procedure, silica gel) that is coated on one side of a plastic glass, or aluminum plate. The solutions of steroids are applied on different spots of the plate (a procedure called “spotting”), and the plate is placed in a solvent bath with the spots above the solvent. If this chromatography were repeated using the same steroids and the same solvent, the final pattern (chromatogram) would be the same as obtained previously. In other words, the distance that a given steroid migrates in a given solvent, relative to the solvent front, can be used as an identifying characteristic of that steroid. We can give this identity a numerical value by calculating the distance the steroid traveled relative to the front (the R value) follows. f We can identify the unknown steroid by comparing its R f value in a given solvent with the Rf values of known steroids in the same solvent. As the solvent creeps up the plate by capillary action, it will wash the steroids off their original spots (the origin) and carry them upward toward the other end of the plate. Since the solubility of each steroid is different, it takes longer for the solvent to wash and carry some than to wash and 4
CLINICAL SIGNIFICANCE The chromatographic separation and identification of steroid hormones has revealed much about endocrine physiology that is clinically useful. It was learned, for example, that the placenta secretes estrogens that are more polar (water soluble) than the predominant ovarian estrogen, estradiol. These polar placental estrogens—estriol and estetrol—are now measured clinically during pregnancy to assess the health of the placenta. Chromatography of androgens recovered from their target tissues (such as the prostate) has revealed that these tissues convert testosterone into other products. Further, these products appear to be more biologically active (more androgenic) than testosterone itself. Testosterone secreted by the testes is thus a prehormone, which is enzymatically converted in the target tissue into more active products — dihydrotestosterone (DHT), in many tissues. Males who have a congenital deficiency in 5-reductase, the enzyme responsible for this conversion, therefore, show many symptoms of androgen deficiency even though their testes secrete large amounts of testosterone. Procedure 1. Using a pencil, make a tiny notch on the left margin of the thin-layer plate, approximately 1½ inches from the bottom. The origin of all the spots will lie on an imaginary line extending across the plate from this notch. 2. Using a capillary pipette, carefully spot steroid solution 1 (estradiol) about ½ inch in from the lefthand margin of the plate, along the imaginary line. Repeat this procedure, using the same steroid at the same spot, two more times. Allow the spot to dry between applications. 3. Repeat step 2 with each of the remaining steroid solutions (2, testosterone; 3, hydrocortisone; 4, cortisone; 5, corticosterone; 6, deoxycorticosterone; 7, unknown), spotting each steroid approximately ½ inch to the right of the previous steroid, along the imaginary line. 4. Observe the steroid spots at the origin under an ultraviolet lamp. (Note: Do not look directly at the UV light.) 5
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Page 3: The glucocorticoids, secreted by th

Thin-layer Chromatography of Steroid Hormones

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