Submitted by Gerald J. Cox and Harriette King.
Checked by H. T. Clarke and Jessica P. Leland.
1. Procedure
(
A)
Tryptophane.—In an
8-l. (2-gal.) bottle is placed 600 g. of commercial casein (coarse powder), which is then covered with about 3.2 l. of tap water at 37° (Note
1). The bottle is shaken until all the casein is moistened. A solution of
60 g. of anhydrous sodium carbonate (Note
2) and
6 g. of sodium fluoride (Note
3) in 1 l. of water at 37° is added. A thin paste of 20 g. of commercial pancreatin in 100 cc. of water (37°) is poured in. The mixture is covered with a layer of
toluene (80 cc.), diluted to 6 l., stoppered, shaken thoroughly, and placed in a warm room or bath at 37°.
After four or five days, with daily shakings, most of the casein is in solution and chalky masses of
tyrosine begin to separate. After five days, a second 20-g. portion of pancreatin in 100 cc. of water is added. After twelve days, the bottle is cooled in an
icebox overnight and the undissolved material is filtered (Note
4) and reserved for the preparation of
tyrosine.
The filtrate (6.9–7 l.) is measured into a
16-l. (4-gal.) stone jar, and for every liter there is added
163 cc. of dilute sulfuric acid (
one volume of 95 per cent sulfuric acid and one volume of water, cooled to room temperature). The first part of the acid must be added cautiously on account of the liberation of
carbon dioxide.
The
tryptophane is precipitated by adding a solution of
200 g. of mercuric sulfate (Note
5) in a mixture of
1860 cc. of water and 140 cc. of 95 per cent sulfuric acid. After standing for twenty-four to forty-eight hours, the clear liquid is siphoned out and the yellow precipitate is filtered and washed (Note
6) with a solution of
100 cc. of concentrated sulfuric acid in 1.9 l. of distilled water containing
20 g. of mercuric sulfate, until the filtrate is colorless and Millon's test is atypical (Note
7); about 1.5 l. is necessary. The precipitate is washed with three successive 500-cc. portions of distilled water to remove most of the
sulfuric acid.
The moist precipitate (120–130 g.) is suspended with mechanical stirring in 1.2–1.3 l. of distilled water, and a hot,
20 per cent aqueous solution of barium hydroxide is added until the mixture is permanently alkaline to
phenolphthalein (about 120 cc. is required). A rapid stream of
hydrogen sulfide is passed in with stirring until the
mercury is completely precipitated (Note
8). The precipitate is filtered and washed with water until a sample of the washings gives a negative test for
tryptophane with
bromine water (Note
9). The
barium is removed from the combined filtrate and washings by adding the exact amount of dilute
sulfuric acid (Note
10) and filtering. The filtrate is concentrated under reduced pressure to about 80 cc.
The
tryptophane is extracted from the aqueous solution by repeated shaking in a
separatory funnel with
25-cc. quantities of n-butyl alcohol; water is added from time to time to keep the volume approximately constant (Note
11). The
butyl alcohol extract is distilled under reduced pressure. After the water present has distilled, the
tryptophane precipitates in the distilling flask and may cause bumping. When all the water has been removed, as is indicated by non-formation of drops on the side of the condenser, the distillation is stopped and, after cooling, the
tryptophane is filtered and washed with a little fresh
butyl alcohol. Such extractions and distillations are continued until the quantities of
tryptophane obtained are negligibly small (Note
11).
The
tryptophane so produced (7–8 g.) varies somewhat in quality in different runs. It is purified by recrystallization from
60 cc. of dilute alcohol (two volumes of 95 per cent alcohol to one volume of water), filtering from the hot solution an appreciable quantity of insoluble matter, and subjecting this to a second extraction with an additional
10 cc. of aqueous alcohol. The solution is decolorized by the addition of
1 g. of Norite and allowed to stand in the icebox; the silvery leaflets of
tryptophane are filtered and washed successively with cold
70 per cent, 80 per cent, 95 per cent alcohol, and, finally, with a little
ether. Less than half the
tryptophane is obtained in each crystallization (Note
12). The yield of pure (Note
13)
tryptophane is
4.0–4.1 g., together with under 0.1 g. of less pure product.
(
B)
Tyrosine.—The insoluble material (160–170 g.) obtained on filtering the digestion mixture
(p. 612) is suspended in 320 cc. of water and
80 cc. of 36 per cent hydrochloric acid, and the mixture is boiled gently for thirty minutes (Note
14). After straining through cheesecloth, decolorizing with
6 g. of Norite (Note
15), and filtering hot (Note
16), the warm (60–70°) solution is shaken with three
20-cc. portions of benzene (Note
17) and heated to boiling (Note
18). A slight excess
(120–150 cc.) of 28 per cent ammonia is cautiously added, and the mixture is allowed to stand overnight in the icebox. The crystalline product is then filtered and washed with three 40-cc. portions of ice-water. After drying, it weighs about 23 g. The mother liquor and washings are evaporated to about 200 cc., when a second crop is obtained, weighing slightly under 1 g.
The combined product is suspended in 400 cc. of water and dissolved by adding
8 g. of sodium hydroxide in 20–30 cc. of water (Note
19);
2 g. of Norite is added, and the solution filtered. The residue is washed on the funnel with 20–30 cc. of hot distilled water. The filtrate is heated to boiling (Note
18) and treated with
13 cc. of hydrochloric acid (Note
20), when crystallization usually begins. The mixture is then acidified to litmus with
acetic acid (Note
21) and allowed to stand overnight in the refrigerator. The resulting
tyrosine is filtered and washed with ice-cold distilled water (130–150 cc. is necessary) until the washings are free of chloride. The product is dried in air or in a vacuum oven. The yield is
17.0–18.2 g. of pure white, silky needles of
tyrosine. A second crop (about 0.5 g.) of a slightly less pure product may be obtained on concentrating the mother liquor to about 120 cc.
2. Notes
1. Tryptic action is more rapid if all water used is at 37°. Distilled water is not necessary at this stage.
2. This is a considerable excess of
sodium carbonate. Smaller quantities might be satisfactory.
4. This filtration may be slow.
Büchner funnels of 20-cm. diameter are best used; the material from a single filling is allowed to suck dry and the
filter paper then changed.
7. A persistent red color is always obtained in the filtrates, but the final color is distinctly different from that due to
tyrosine.
10. This amount is best determined in a 20-cc. aliquot sample, employing
2 per cent sulfuric acid in a
buret.
11. In checking, it was found satisfactory to extract in a continuous apparatus
.htm(Fig. 21). Extraction is continued until the liquid in the flask begins to bump on account of the separation of solid; a new charge of
butyl alcohol is then employed, about five charges being necessary. This process is repeated until the residue after a three-hour period of extraction fails to give the red color, characteristic of
tryptophane, with
bromine water. The time necessary, of course, depends upon the rate of boiling; in checking, it was found to be twenty-eight to thirty hours.
Fig. 21
12. The recrystallization of the crude
tryptophane is an extremely troublesome process. Not only must a less soluble by-product be removed, but the mother liquors contain a more soluble, gummy impurity in considerable proportion. After collecting each crop, the mother liquor must be evaporated to a small volume on the
steam bath and treated with a double volume of alcohol. This process is repeated until no further crystals are obtained, but only a gum.
13. The purity of the
tryptophane has been checked by the optical rotation (
[α]D = −28 to −33°) and by analysis for amino nitrogen (6.8–6.9 per cent) by Van Slyke's method.
14. The boiling acid solution hydrolyzes protein material that otherwise greatly retards filtration.
15. The
Norite used in this preparation is insufficient to decolorize the solutions completely but gives a white final product.
16. All the filtrations in the purification of
tyrosine, except possibly the last, are best done on a
20-cm. Büchner funnel. Whenever charcoal is used, kieselguhr may be employed to obtain a clear filtrate.
17.
Benzene extracts traces of substances, probably fatty acid, that retard filtration and greatly alter the quality of the final product.
18. The
tyrosine crystallizes in long, silky needles, easy to filter, if the solution is neutralized at the boiling point.
19. A small amount of flocculent impurity remains undissolved.
20. The
hydrochloric acid is added to provide chloride ion as an index of complete washing.
3. Discussion
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