- •Keizo Uenaka was the scientist who practically isolated adrenaline as crystal.
- •Adrenalin purification methods released in 1901 was based on Uenaka's hand-written document, “On adrenalin, Memorandum, July to December, 1900”.
- •Nagayoshi Nagai, Jokichi Takamine and Keizo Uenaka had crucial roles in chemical isolation of ephedrine and adrenaline.
|On adrenalin, Memorandum, July to December, 1900 (reference #3)||The American Journal of Pharmacy. 73: 523–535, 1901. (reference #5) Yakugakuzassi 260; 1044–1045, 1903 (in Japanese, reference #7)||Journal of Physiology (Proceedings of the Physiological Society) 1901; 27: XXIX – XXX. (reference #6)|
|Purification steps||(original date of description in the "memorandum" and summarized experimental note)||purification step||additional comments|
|1||July 20th: Extracting in water.|
July 30th: Steeping in 75–85° water for 8hours.
|Suprarenal capsules finely disintegrated by suitable means, are steeped in water or acidulated water for a period of about five hours at a temperature varying from 50–80° centigrade, with frequent agitation and with the addition of water as it evaporates.||Tips #1: As the active principle of the glands is prone to absorb the oxygen from the air, to form inactive substance, it is necessary to avoid exposure of the liquid to the air as much as possible.||Disintegrate the suprarenal glands of sheep and oxen, extracting them in water.|
|2||July 30th: Press and filter to separate lipid.|
September 10th: Keep at 60° water for 6hours, then heat to 95° to coagulate proteins.
September 30th: boil in 80° for 12hours and heat up to 90–95° at the end to coagulate proteins.
|The temperature of the mass is now raised from 90 to 95° centigrade for the period of one hour so as to coagulate as much albuminoid as possible. (with tips #1–3)||Tips #2: A layer of fat floating on the surface of the mass acts very conveniently for this purpose and at the same time it has an effect of retarding evaporation of water as well.||Rendered weakly acid by the addition of a few drops of acetic or hydrochloric acid, at 95 °C|
|3||September 11th: Press to separate lipid.|
September 30th: Press and separate solution.
|The mass is now pressed and separated from the liquid portion which contains the active principle. (with tips #4)||Tips #3: Other methods of preventing oxidation may be employed at this stage, such as conducting the steeping process in an atmosphere of carbonic acid gas.||The solid residue is pressed and reextracted.|
|4||September 30th: Concentrate in vacuum.|
October 20th: The squeezed solution is evaporated in vacuum.
|The clear extract is now evaporated in a vacuum pan to a suitable strength.||Tips #4: The mass is again steeped for hours in warm water slightly acidulated with acetic or hydrochloric acid, in order to extract the residual amount of active principle. The liquid separated from the mass is now added to the first extract and allowed to separate from the oil.||The liquid is filtered and then concentrated by evaporation.|
|5||July 30th: Add triple volume of 95% alcohol.|
September 11th: Add 95% alcohol to make 63% alcohol solution.
September 30th: Add alcohol to precipitate inorganic salts.
October 10th: Removal of inorganic salts with alcohol should not be skipped for successful purification.
|To this concentrated solution about two to three times its own volume of strong ethyl alcohol is added or more economically wood spirit, which will precipitate both inert organic and inorganic substances. The inert substances thus separated are washed with alcohol so as to free them from the active principle.||Alcohol is then added until no further precipitation occurs.|
|6||September 30th: The filtrate is evaporated in vacuum.||The alcohol solution is now evaporated preferably in vacuum still, whereby the alcohol used is duly recovered.||The filtrate is then evaporated in vacuo|
|7||July 20th: Alkalization with sodium hydrate.|
September 11th: Add sodium hydrate and ammonia to obtain crystal.
September 30th: Precipitate crystal with sodium hydrate and ammonia.
October 20th: Precipitation can be achieved solely with ammonia, without use of sodium hydrate and ammonium chloride.
November 6th: Precipitation method with ammonia is more effective than the method with sodium hydrate and ammonium chloride.
|To the residual liquid, ammonia is now added until the solution gets distinctly alkaline and left over for several hours. (Tips #5)||Tips #5: Instead of using ammonia, sodium hydrate may be used as a precipitant, but care must be taken not to use in excess, which redissolves adrenalin. In order to counteract caustic alkali, ammonium chloride or carbonic acid may be conveniently used.||Treat with ammonia or ammonium chloride or sodium hydrate until the solution is distinctly alkaline.|
|8||August 11th: Adrenalin crystal is easily combined with phosphate salts.|
September 30th: Wash and dry with alcohol in vacuum.
|A yellow brownish precipitate will be formed which is the crude adrenalin in a basic form. (Tips #6)||Tips #6: The impure adrenalin usually precipitates in a light yellow brownish tomato-shaped form, but not infrequently in needles. The former is an agglomeration of needle crystals and is more or less contaminated with coloring matters and some inorganic substance, chiefly phosphates.||Adrenalin crystallizes out in the course of a few hours.|
|9||July 21th: Adrenalin crystal can be dissolved in weak acetic acid, not in water, alcohol or ether.|
August 4th: Ether is added to remove lipid, and add sodium hydrate carefully for alkalization to facilitate crystallization.
August 4th: Adrenaline crystal is easily dissolved in weak sulfuric acid, not in water and alcohol.
August 5th: Adrenaline crystal is easily dissolved in weak acid and carbonated water.
August 13th: Crude crystal can be dissolved in acetic acid and precipitated with lead acetate, or crystalized by alkalizing with ammonia.
September 19th: For further purification, crude crystal is dissolved in acetic acid, and then 5–6 times volume 95% alcohol is added. Following precipitation of inorganic salts, the filtrated solution is alkalized with ammonia.
November 12: For further purification, crude precipitate is re-dissolved in weak acetic acid and precipitated with ample volume of alcohol, then half volume of ether is added. Adrenaline is precipitated by adding ammonia to the filtrate. The precipitate is again re-dissolved in acetic acid and re-precipitated with ammonia. The obtained crystal is washed and dried.
|The precipitate is now filtered, washed with water and dried.||Further purification: The crude adrenalin is dissolved in acid and alcohol and ether is added to a sufficient quantity. A brown colored precipitate is produced which chiefly consists of coloring matter and inorganic impurities. The precipitate is separated both by decantation and filtration. The filtrate is now treated by one of the above mentioned processes, when white crystalline precipitate of adrenalin will be obtained. It is quickly filtered, washed with water and then with alcohol and dried. The process of purification may be repeated, if desired, two or three times.||Adrenalin may be purified by dissolving in acid and re-precipitating.|
Personal history of Keizo Uenaka and adrenaline purification
Uenaka's supervisor, Nagayoshi Nagai, and ephedrine
Jokichi Takamine and isolation of adrenaline with Keizo Uenaka
Further researches on adrenaline after its purification
Declaration of Competing Interest
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