separation, but with tailing. Nickel filaments were tried and gave approximately 50% of the sensitivity of the tungsten along with some stability problems. Teflon-clad filaments were also tried and found to give only about 10% of the sensitivity of the tungster.. Examination of the tungsten filaments after a number of determinations showed no evidence of corrosion. It was necessary to saturate the system with HCI prior to injecting the sample. This was done routinely by injecting 5 ml of HCI gas prior to each determination. Low HC1 results were obtained if this step was omitted. The lower limit of detectabi iity of all three components is believed to be about 0.1 %, however no samples were run containing less than 10 H2Sor H"O and conditioning might be necessary for them also at the lower levels. Air in the samples was there only as an impurity and, indeed, most samples showed much less than is shown in Figure 1. The glass stopcock
sampling valve was found to be less than ideal, and the Teflon modification as shown by Graven and Harmon (7) would surely be an improvement. EDWARDL. OBERMILLER GEORGE 0.CHARLIER Research Division Consolidation Coal Co. Library, Pa. 15129 RECEIVED for review October 10, 1966. Accepted January 3, 1967. (7) W. M. Graven and H. R. Harmon, ANAL.CHEM., 37, 1626 (1965).
Degradation of Glutamine at Elevated Temperatures in Ion Exchange Chromatography Mixtures of 1.0 pc of I4C-labeled glutamine (Schwarz Bioresearch, Inc., Orangeburg, N. Y.; specific activity 6.58 mcjmm) and 0.25 pmole of unlabeled glutamine (L-glutamine, CP, Mann Research Lab., New York, N. Y . ) were chromatographed at two temperatures. In the first experiment, the column temperature was maintained at 30" C for 41i2 hours and then raised to 60" C. In the second experiment, a uniform column temperature of 60" C was maintained. Amino acid solutions were freshly prepared in distilled water just prior to use. Eluting buffer was pumped through the column at the rate of 0.50 mljminute. It was determined that 0.1075 ml/minute of column effluent was normally discarded via the h overflow tube just before the proportioning pump manifold. In these experiments, this overflow was collected in 10-minute fractions (1.075 ml). Portions were subsequently assayed for I4Cradioactivity with a liquid scintillation counter. Recovery of total added radioactivity was 100 in both experiments.
SIR: In a previous >itudy( I ) of amino acid separation by ion exchange chromatclgraphy, it was found that glutamine could not be quantitatively recovered at elevated column temperatures. At temperahres of 25" C, glutamine recovery was 100%; in contrast, at 60" C, only 24% of added glutamine could be accounted for. Loss of glutamine was not paralleled by formation or increase of glutamic acid. This finding indicated that hydrolytic dzamidation of glutamine to glutamic acid was not the cause of glutamine loss during chromatography a t elevated temperatures. To clarify this question, chromatographic experiments employing carbon-14-labeled glutamine were performed. Chromatography was carried out with the Technicon Co. AutoAnalyzer system of arnirio acid analysis as previously described
(0. (1) I. Oreskes, F. Cantor, and S. Kupfer, ANAL.CHEM., 37, 1720 (1965).
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Figure 1. Chroniatography of glutamine at column temperature of 30" C from 0 to 270 minutes, then 60" C thereafter VOL. 39, NO. 3, MARCH 1967
397
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Figure 2. Chromatography of glutamine at column temperature of 60" C
Absorbance us. time: upper dotted plot 570 mr, lower dotted plot 440 mr. Counts per minute lo-minute eluate fraction from column
The results of the experiment carried out at an initial temperature of 30" C are shown in Figure 1 . The first ninhydrin peak obtained a t 287 minutes was that for glutamine. (Elapsed time was determined between the start of experiment and the appearance of the ninhydrin peak maximum on the chart recorder, Time required for a particular amino acid t o pass through the column only was 20 minutes less.) This peak was associated with 90.7% of the added radioactivity. Later, a t 374 minutes, there was a trace of another ninhydrin positive substance, apparently glutamic acid. This was associated with about 1.0% of the added radioactivity. The glutamic acid appeared to be a n impurity present in the 14Clabeled glutamine judging from the paper chromatographic analysis supplied by the manufacturer. Approximately 8.3 % of the added radioactivity was found prior to the glutamine peak in a diffuse region between 102 and 272 minutes. This activity was not associated with any observable ninhydrin peak. The results of the 60" experiment are shown in Figure 2. At this elevated temperature, the elution time for glutamine was 212 minutes, and only 29.6% of the added radioactivity was associated with this peak. The chromatogram exhibited a trace of glutamic acid a t 266 minutes which accounted for 2.2% of the added 14C activity. The major portion of the added radioactivity was found in the preglutamine region; 68.3% of the total activity was found between 45 and 195 minutes. Most of it was spread diffusely through this region of the chromatogram except for a peak of activity at 70 minutes. As in the previous experiment, none of the preglutamine radioactivity was associated with any observable ninhydrin peak. The two experiments differed in one respect, namely, that in the first case glutamine was entirely eluted from the column a t 30" C whereas in the second case, glutamine was subjected to a 60" C column temperature during elution. As a consequence, there was extensive degradation of this amino acid. Recovery of glutamine at 60" C as compared to 30" C was
398
ANALYTICAL CHEMISTRY
us.
time: solid bar plot, radio-activity per
33% determined by radioactivity or 30% determined by the ninhydrin reaction. Extensive loss of glutamine observed was not primarily due to its hydrolysis to glutamic acid and ammonia as shown by the fact that the relative concentration of glutamic acid at 60 " C was only 2.2 %. The fact that a t a column temperature of 60" C, the major part of the radioactivity emerged diffusely before glutamine suggested that glutamine was continually being degraded on the warm column to a nonninhydrin reactive substance characterized by an elution time less than that for glutamine. While this process was most pronounced at 60" C, some degradation occurred a t 30" C as well. The product of glutamine degradation has not been characterized in this study. Judging from its formation at elevated temperatures (2), and its more rapid elution than glutamine (3) in ion exchange chromatography, it is likely that this material is pyrollidone carboxylic acid.
ACKNOWLEDGMENT The technical assistance of F. Cantor is gratefully acknowledged.
IRWIN ORESKES SHERMAN KUPFER Clinical Research Center and Department of Medicine Mount Sinai Hospital New York, N. Y. 10029
RECEIVED for review November 25,1966. Accepted December 30, 1966. Work supported by U. S. Public Health Service Grant FR-71. (2) P. B. Hamilton, J. Bid. Chem., 158, 375 (1945). (3) R. Rosenblum, Proc. SOC.Exptl. Biol. Me& 119,763 (1965).
