VOL. VI, PAGES 45-49 (1962)
Determination of Primary Amino Groups by Gas Chromatography ERIK R HOFFMANN and IHOll LYSY.J, Eihicon Inc., Someroille, New Jersey
Introduction The Van Slyke l technique, together with its many modifications, has remained a standard method for the determination of primary amino groups. The method is based on reaction between primary amino groups and nitrous acid under proper conditions. As the result of reaction, nitrogen is generated. It originates half from the sample and half from the nitrous acid. The evolved nitrogen is chemically separated from the nitrogen oxides, resulting from decomposition of nitrous acid, and determined either manometrically or volumetrically. The air in the system prior to analysis is swept out either by the nitric oxide, or, in a later modification, by carbon dioxide. 2 In our work with these methods, we have been troubled by the alkaline permanganate 01' chromic acid wash solutions. The manganese dioxide which precipitates, eventually plugs the apparatus. The chromic acid in the Hussey and Maurer method 2 tends to lose activity rather quickly. Blank values tend to increase at times, and leaks are sometimes hard to detect in the involved glassware with its many connections. We decided to rid ourselves of these sources of error by eliminating the wash solutions, the complicated glassware, and the need for chemical separation of the nitrogen from the other gases. In the method described, the sample is introduced into the reaction flask. After sweeping out the system with helium, nitrous acid is introduced. The evolved nitrogen-nitrogen oxide mixture is passed through a suitable chromatographic column, The nitrogen peak is then read off the recorder strip chart and calculated. 45
E. R. HOFFM ANN AN D I. LYSYJ
The Perkin-Elmer model 154 gas chromatograph was modified by inserting a gas sampling fixture into the sample line (Fig. 1). This allows a glass reaction chamber either to be inserted into the system or to be bypassed by manipulation of only three brass stopcocks. The reaction cell (Fig. 2) (1.5 ml. volume) was fashioned from a T joint. Two of its tubes were connected to stopcocks 1 and 2 by means of pressure tub ing. The third was closed with a rubber sept um for
Fig. 1. Schematic outli ne of gas chromatograph modification ; 8 1, 8 2, 8 3, stopcocks; R, reaction cell; C, column.
Fig. 2. Reaction cell. MICROCHE}UCA L JOURNA L, VOL. VI , ISSUE 1
DETERMINATIO N OF PRIMARY AMINO GROUPS
Fig. 3. Arrangement for sample introduction.
th e introduction of sample and reagents by means of a hypod ermic syringe. Th e complete set up is shown in Figure 3.
Reagents and Procedure Saturated aqueous sodium nit rite (de-aired) and glacial acetic acid were used. The following operating conditions were found to be satisfactory for the separation of nitrogen: column, 3 ft . long, 1/ 4 in. o.d. copper tubing; pa cking, 80-100 mesh molecular sieve, 5A.; gas, helium, inlet pressure 10 lb., rate of flow, 60 ml.ymin .; temperat ure, 40°C.; sensitivity, 16, 32, 64. The procedure used is as follows. Introduce 1 to 5 mg. of sample and 0.3 ml. of sat urated solut ion of sodium nitrite into reaction cha mber. Connect the chamber to st opcock 1 and 2 by means of pressure t ubing (Fig. 3). Close st opcock 3 and open 1 and 2. Sweep out with helium for 30 sec. Close st opcocks 1 and 2 and open 3. Introduce 0.15 ml. de-aired glacial acetic acid through the septum by mean s of It hypodermic syringe. ~h!lke the chamber for 1 minute with a
E. R. HOFFMAN N AND 1. LYSYJ
Fig. 4. Gas chromatogram of reaction gases ; first peak, nitrogen ; second peak, nitrous oxides. 7
Fig. 5. Calibration curve for nitrogen.
rubber-covered electric vibrator. After waiting 10 min., close stopcock 3 and open 1 and 2 simultaneously for 30 sec. Then close 1 and 2 and open stopcock 3. The nitrogen appears as a symmetrical peak and is determined by the peak height method. Run a calibrat ion curve on aliquots of a 5% glycine solution. A typi cal chromatogram is shown in Figure 4, and a calibration curve in Figure 5.
Results and Discussion A number of acidic, basic, and neutral I-amino adds (chromatographically pure, Mann Research Laboratories, N.Y. C., N.Y.) were analyzed by the above procedure. Five percent aqueous solutions of basic and neutr al amino acids were prepared, and 50 to 100 MICROCHEMICAL JOURNAL, VOL. VI, ISSUE 1
DETERMINATION OF PRIMARY AMINO GROUPS
ul-aliquote were used. Acidic amino acids were dissolved in O.5N sodium hydroxide, in order to prevent immediate reaction upon contact with the sodium nitrite. The results are presented in Table I. TABLE I Compound
Percentage of Nitrogen Found Theory
Glycine (aminoacetic acid) C2H 502N
2 5 2 .5 2.5 5.0 5.0 5.0 5.0 5.0 5.0 5.0
]S tif> ]S.65 IS.6.5 IS.(i5 18.65 ]8 65 ]865 IS.65 IS.65 ]S(i5
IS40 ]S.3.5 1900 ]8.70 18.60 18.40 1920 ]8.72 ]8.60 IS.40
-.25 -.30 -.35 +.05 -.05 - .25 +.55 +.07 -.05 -.2.5
I-Glutamic acid C5H 904N
5.0 5.0 5.0
\l.52 \l.52 9 ..52
9.50 \l.30 9.60
-.02 -.22 +OS
l-Tyrosine C 9H n0 3N
5.0 5.0 5.0
7.n 7.n 7.n
8.10 8.00 7.S6
+.37 +.27 +.13
5.0 5.0 5.0
15.62 15.45 15.10
+.30 +.]3 +.23
5.0 5.0 .5.0
11. so lUJ6 1l.!16
+.24 +.32 +.42
I-Valine C5H n0 2N
The microsamples were run at sensitivities of 16, ;~2, and 64. Ultramicrosamples could be run by employing higher sensitivity. Other gas evolving reactions may be studied with this apparatus without interference with normal gas chromatographic work.
References 1. Van Slyke, D. D., Ber., 43, 3170 (1910). 2. Hussey, A. S., and J. E. Maurer, Anal. Chem. 24, 1642 (1952).
Received July 7, 1961 Revised October 10, 1961