Microdiffusion methods. Ammonia and urea using buffered absorbents (revised methods for ranges greater than 10μg. N)

Abstract

Microdiffusion methods for determinations of minute amounts of ammonia, urea, carbon dioxide, the halogens etc., are now established in many laboratories. A general account of apparatus and several applications have been given elsewhere [Conway, 1939]. Recent extensions include submicro-determinations of total N by Needham & Boell [1939], as well as a further very accurate submicro-total N method by Tompkins & Kirk [1942], also amide-and nitrate-N etc. by Borsook & IDubnoff [1939], quantitative acetone determination by Werch [1940] and an accurate acetone method using bisulphite absorption by Winnick [1941; 1942], who has also extended the technique to lactic acid and threonine estimations. As shown elsewhere micro-NH3 determinations by the microdiffusion technique can be brought to any desirable level of accuracy. Recoveries are better than with aeration methods, the apparatus required,is simpler and large numbers of determinations can be carried out in one series. Using the standard unit and 0-2 ml. of the NH3-containing fluid the full absorption time at room temperature is 45 min., but with rocking this can be reduced to 10 min. Such times arise out of the principles discussed and the experimental results described. (Borsook & Dubnoff [1939] have erred in claiming an absorption time of 90 min. as an advance on the microdiffusion technique.) The present study is directed especially to the removal of certain objections to the clinical use of the microdiffusion method for urea, but has also a more general application for concentrations of NH3 beyond a certain level. The blood urea method as originally described [Conway, 1933] had the disadvantage for clinical use, as commented on by Lee & Widdowson [1937], that the upper limit was 100 mg. urea/100 ml. Occasional blood ureas exceed this figure, though as pointed out [Conway, 1939], the use of 0.1 ml. as well'as 0-2 ml. blood in a duplicate estimation raised the limit without any extra trouble to 200 mg./100 ml. However, this disadvantage is fully removed in the method described below, in which there is no upper limit. With this revised method also the amount ofcleaning required need not exceed that given to ordinary analytical glassware, nor is any standardization of solutions required, apart from the original making up of N/50 1ICI, which remains indefinitely stable. This removes the objection raised by Dukes [1939] who considered the microdiffusion method unsuitable for occasional blood urea determinations as distinct from large series. In the new method for urine and blood urea, as also urine NH3 or NH3 in quantities greater than about 10 jg. NH3-N, there is used the principle introduced into the micro-diffusion technique by Abelin [1938], and further studied by Kawerau [1941], of absorbing the NH3 into a solution such as dilute boric acid and titrating back with standard HCI. Improvements are included arising out of a further study of the conditions of the micro-diffusion procedure, an account of which is given at the outset. The boric acid procedure of Abelin [1938] was criticized by one of us [Conway, 1939] as giving a poor end point with the Tashiro indicator compared with the barium hydroxide titration, but it was noted that the principle was certainly a good one and that a change Biochem. 1942, 36 (655) 43