9, 477-483 (1965)
Microdetermination of Primary Amino Groups' ROBERT
Brooklyn College 0/ the 'City University 0/ New York Received May 10, 1965
Many methods have been developed for the determination of the amino group as a functional group. The Van Slyke (7) technique, with several important improvements, has remained a standard method for the determination of the primary amino group. In this method nitrogen is generated by the following reaction:
Oxides of nitrogen (being formed by the action of acids on sodium nitrite) are used to remove the air from the apparatus and subsequently sweep the nitrogen into a modified Hempel pipet. The oxides of nitrogen are then absorbed by an alkaline permanganate solution and the purified nitrogen gas is measured in a special gas buret. Errors are introduced by the incomplete absorption of nitric oxide, incomplete transfer of the nitrogen to the measuring buret, and clogging of the latter by precipitated manganese dioxide. Hussey and Maurer (3) developed a modified micro method by using carbon dioxide as a sweep gas and chromic acid and an alkaline solution of potassium permanganate as scrubbers to remove the oxides of nitrogen. The nitrogen and carbon dioxide are passed into a micronitrometer where the nitrogen is colleted over 50% potassium hydroxide solution. While working with this method, Hoffmann and Lysyj (2) found that the chromic acid reacted slowly and tended to lose its activity rather quickly, and the precipitated manganese dioxide eventually plugged the apparatus. Kainz (4) used bromic acid to absorb the nitric oxide and a saturated solution of sodium thiosulfate to remove the bromine which was 1
Paper presented at the International Symposium on Microchemical Techniques
-1965, held at The Pennsylvania State University, University Park, Pennsylvania, U.S.A., August 22-27, 1965.
ROBERT K. MAURMEYER
liberated. The latter had to be removed to prevent the formation of mercuric bromide, which would clog the nitrometer. Ma, Maurrneyer, and Monaco (6) designed an improved apparatus which was less rigid. However, the bromic acid was found to be rather unstable and had to be prepared freshly after every third analysis. The saturated sodium thiosulfate solution also tended to precipitate some colloidal sulfur. Since the solubility of the nitric oxide is less than 5% by volume at room temperature, long absorption tubes and a very slow flow of the gas stream is required to remove the nitric oxide satisfactorily. For reasons mentioned above, the use of a solid scrubber was deemed to be advantageous and therefore investigated. The following substances which react with nitric oxide were tried: (1) chromic trioxide, CrD a ; (2) silver oxide, Ag20; (3) manganese dioxide l\ln02; (4) silver permanganate, AgMn04' Although chromic trioxide reacts with nitric oxide at room temperature, it was found to be very hygroscopic and would readily cake up in an absorption tube. Auden and Fowler (1) found that silver oxide reacts with dry nitric oxide at room temperature to form silver nitrite and metallic silver. The basic silver oxide, however, reacts with the carbon dioxide forming silver carbonate. Manganese dioxide combines with nitric oxide very slowly at room temperature and has to be heated to 216°C to give any appreciable reaction, as stated by Auden and Fowler (1). They also investigated the reaction of silver permanganate with nitric oxide and found that the reaction proceeds at room temperature and is instantaneous at 80°C, leaving metallic silver, silver oxide, silver nitrate, and manganese dioxide as the final products. Silver permanganate has the additional advantage that it is crystalline, nonhygroscopic, and only slightly soluble in water. Although its decomposition (at 120°C) is spontaneous and has an explosive character, it is quite safe to handle at room temperature. MATERIALS AND METHODS
Reagents Silver permanganate. This substance was prepared following the directions given by Lysyj and Zarembo (5): 46.0 g of potassium permanganate are dissolved in 11. of hot distilled water (9O-95°C) and then 51.0 g of silver nitrate dissolved in a minimum volume of hot water are added slowly with constant stirring. After cooling to O-lOoC in a refrigerator for several hours the crystalline precipitate of Agl\ln04 is collected on a medium porosity fritted glass funnel by using suction and washed with
MICRODETERMINATION OF PRIMARY AMINO GROUPS
500 ml of cold water. The precipitate is finally dried in a vacuum desiccator at room temperature. Sodium nitrite, 1070. Dissolve 50 g of sodium nitrite in 450 ml of water. All solutions were made with freshly boiled distilled water. Acetic acid-sodium acetate mixture. Dissolve 10.0 g of sodium acetate trihydrate in 250 ml of water. Add 250 ml of 50% (v/v) acetic acid. Potassium bromide solution 1070. Dissolve 50 g of potassium bromide (reagent grade) in 450 ml of water. Cupric chloride solution, 4 1\1. Dissolve 268 g of cupric chloride in 500 ml of water. Potassium hydroxide 5070. Apparatus
The apparatus (Fig. 1) consists of a reaction vessel, A, of 10-ml capacity; a gas delivery tube, B; a funnel, C, for the addition of the sodium nitrite solution; a two-way stopcock, D, which connects the delivery tube to the funnel and the carbon dioxide generator. The absorption tube, E (Fig. 2), is made from Pyrex tubing of 12-13 mm a.D., 8 mm I.D., and 200 mm in length. It is attached to a standard taper joint 14/35 Pyrex
FIG. 1. Amino apparatus. A, reaction vessel; B, gas delivery tube; C, funnel; D, two-way stopcock; E, absorption tube; F, nitrometer.
ROBERT K. MAURMEYER Asbestos
Ag Mn 0 4
~'Dmlllltl.laIIIIIIII~~ ~mm=~-~---~------200mm------- _.
FIG. 2. Absorption tube.
No. 6560 and connected to the reaction vessel and the nitrometer, F, by means of ball joints. The absorption tube, E, is filled with alternate layers of 3-4 mm thickness of loosely packed Gooch filter asbestos fibers and crystalline silver permanganate, for a total of about 24 layers of silver perrnanganate containing 15 g of the latter substance.
Procedure In order to test the efficacy of the silver permanganate absorption tube several blank determinations were made with 2 ml of the acetic acid mixture alone and subsequent addition of 1 ml of the 10j~ sodium nitrite solution, with and without the use of the absorption tube. The results are shown in Fig. 3, where the time of the blank run is plotted against the nitrometer 1601 150 .:
A· Without absorption tube B· With absorption tube
~ 0.70z 060--
o 10 _ o t,
HOAc + NaN0 2
L .1 __ J __ .1
5 10 15 20 25 30 35 4045 50 55 60 min
Results of blank determinations with and without the absorption tube.
reading. However, before the blank determinations are made, a small amount of air is introduced into the nitrometer and the starting volume is accurately determined. This is necessary in order to read the very small
MICRODETERMINATION OF PRIMARY AMINO GROUPS
volume of the blank by difference. For a determination, an accurately weighed sample containing about 0.1 meq of amino group is introduced into the reaction vessel A, and 2 ml of the acetic acid-sodium acetate mixture is added . The tube is swirled until the sample dissolves , and then 1 ml of potassium bromide and 1 ml of cupric chloride are added. The latter reagents act as inhibitors to minimize the decomposition of the nitrous acid . Finally 1 ml of the 1070 sodium nitrite solution is placed in funnel C. After having purged the apparatus free of air, the stopcock to the nitrometer is turned 90° and the two way stopcock D turned to allow the flow of the sodium nitrite into the reaction vessel. Both stop cocks are then turned to their original positions . When no more nitrogen gas is collected in the nitrometer, 0.2 to 0.3 ml of the sodium nitrite is introduced into the reaction vessel to check if there was sufficient nitrite to react with all the amino group. Calculations The percentage of NH 2 is obtained as follows: (mmoles N:!l X 16.02 X 100
% NH 2 = ----------wt. of sample in mg
The number of mmoles of nitrogen produced is found by the following expression. (Vs-O.Ol Vs-Vb) X F , mmoles N 2 = 28.016 where Vs equals observed volume, 0.01 Vs equals correction due to vapor pressure of KOH and drainage, F equals wt in mg per milliliter of nitrogen at the particular temperature and pressure, and Vb equals blank correction. RESULTS AND DISCUSSION
A number of assayed amino acids from the Mann Research Laboratories, N.Y.C. were analyzed by the above procedure and their results are listed in Table 1. The use of a solid scrubber to remove the nitric oxide, which is always produced to some extent the decomposition of nitrous acid, is advantageous. An absorption tube filled with alternate layers of asbestos fibers and crystalline silver perrnanganate was found to be most propitious. The number of ball joint connections and the resistance of liquid absorbents to the gas flow, which may give rise to leaks, have been reduced. The
ROBERT K . MAURM EY ER
TABLE 1 DETERMINATION OF PRIMARY AMINO GROUPS IN AMINO ACIDS NH2 <% )
Lysin e HCI C4HH02N 2H CI mol. wt. 182.65
7.765 8.287 5.547
17.54 17.54 17.54
17.4 17.6 17.5
Glutamic acid C5 H g 0 4 N mol. wt. 147.13
9.591 9.155 5.655
10.96 10.96 10.96
10.8 10.9 11.1
Ty rosine C9HIIO:IN mol. wt . 181.19
8.104 9.964 8.135
12.21 12.21 12.21
12.4 12.2 12.1
C OH r:P 2N mol. wt . 131.17 Alan ine C aH 702N m ol. wt. 89.09
5.949 8.040 7.828
17.98 17.98 17.98
18.1 17.9 18.2
NH 2 range <%)
capacity for the absorption of the nitric oxide has been increased many fold and the removal of the nitric oxide was found to be complete even at high concentrations. SUMMARY In th e gasornet ric determination of primary am ino groups, crystalline silver permanganate is recom mended as a solid ab sorbent for the complete removal of nitric oxide, th e forma tio n of which al wa ys occurs to some extent from th e decomposition of nitrous acid . E limination of th e wash solutions reduces both the number of connections in the app a ratus and it s internal resista nce, which may give rise to leaks . The time required for the complicated proced ure of purging the apparatu s fr ee of a ir is appreciabl y minimized. REFERENCES AUDEN, H . A., AND FOWLER, G . J., Th e action of nitric oxide on certain salts. Chern. News 72, 163 (1895). 2. HOFFMANN, E. R., AND LYSYJ, 1., Determination of primary amino groups by gas chromatography. Microchern. J . 4, 45-49 (1962). 3. H USSEY, A. S., AND MAURER, J. E ., Modified Van Slyke method for microquantitative determination of aliphatic am in o grou ps. Anal. Chern. 24, 1642-1645 ( 1952) . 4 . KAINZ, G., Mikrobestimmung Primarer Aminogruppen in Alipha tischen und Aro matischen Verbindungen. M ikrochim . Acta 1953, 347. 1.
MICRODETERMINATION OF PRIMARY AMINO GROUPS
5. Lvsvj , I ., AND ZAREMBO, J. E., A rapid semimicro method using silver permanganate , Mlcrochem, J. 2, 245-252 (1958) . 6. MA, T . S., MAURMEVER, R. K., AND MON"CO, M. A., Improved amino apparatus In "Organic Functional Group Analysis by Micro and Semimicro Methods" (N . D. Cheronis, and T . S. Ma, eds.), p. 235. Wiley (Interscience), New York, 1964. 7. VAN SLYKE, D. D., Eine Methode zur Quantitativen Bestimmung der Aliphatischen Amino Gruppen, 43, 3170 (I 910) .