Use of Capillary Trap in Microdetermination of Carbon BEN D. HOLT Chemistry Division, Argonne N a t i o n a l Laboratory, Lemont,
111.
A rapid and precise manometric method is described
EXTENDED RAYGE
for the measurement of carbon dioxide produced by combustion in microdetermination of carbon. In the very low range 1 y of carbon correspondsto a manometer reading of 4 mm. Excluding the effects of sample loading, the blank is negligible. With the sacrifice of some precision the measuring unit may be revised to possess a dual range, accommodating both micro and macro amounts of carbon.
The range of measurement was extended to accommodate macro as well as micro quantities of carbon by constructing a unit with two additional modifications. A hollow stopcock reservoir (Figure 2) was sealed on between the manometer tube and the U-bend; and the lower portion of the V-bend was made of a 5-em. section of 7-mm. tubing containing a plug of glass wool. This arrangement offered a choice of two calibration curves. For low carbons the reservoir was not used, whereas for higher percentages it was opened to increase the volume of the unit, so that the scale divisions represented correspondingly larger amounts of carbon. The unit used had a low scale range of 0 to 400 y and a 7.5-ml. reservoir which afforded a high scale range of 0 t o 2500 y. The carbon in a 0.1-gram sample running as high as 2.5% could be measured on the high scale, while that in a 1-gram sample of a material containing as low as 0.001% could be measured on the low scale. The low scale of such a revised unit is not as highly sensitive as the unit described above because of the increased volume of the glass-wool section. The ratio of scale reading to weight of carbon was about 1 mm. per y.
A
SINGLE unit for measuring carbon dioxide has been developed by Smiley ( 4 ) which he has used in methods for determining oxygen and carbon. I n view of considerable interest shown recently in methods for determining low carbon in metals (1-3,6), the following brief description is given of a very satisfactory adaptation of Smileg’s capillary trap t o the standard combustion method. The result of the adaptation is a simple, rapid method, giving good precision in the microgram range. Figure 1 shows a schematic diagram of the all-glass apparatus. The indicated components were made large enough to accornniodate an oxygen flow rate of 150 ml. per minute, and were connected together by semiball joints. The manometer used was constructed with dimensions approximating those described by Smiley ( 4 ) ,except that the manometer tube was made of 1.0-mm. precision-bore capillary and was made longer because of lower altitude. Another minor change was the substitution of 1-mm. oblique-bore stopcocks for the brass valves. The stopcocks were sealed on t o become permanently incorporated in the calibrated unit. The outer stems were equipped with 18/9 semiball joints t o facilitate easy dismantling from the line for cleaning. The inlet stopcock had a grooved plug t o give precise control of gas flow. For this manometer 1 y of carbon (or 0.0001% for a 1-gram sample) was equivalent t o about 4 mm. a t the upper portion of the scale and t o about 2 mm. a t the lower. T h e maximum amount of carbon measurable with this manometer was about 200 y. The various purification tubes indicated in the diagram adequately protected the cold trap of the measuring unit from contaminants such as moisture and oxides of sulfur and selenium. A liquid nitrogen trap, not shown, was used between the measuring unit and the Duo-Seal pump to protect the unit from pump oils. PREHEITER TUBE CUD AT O O O o c INDlCbTlNG SlLlCb @EL SlLICb
OXYOEN FROM
ISCARITE
A
ANHIDRONE
BUBBLER
AMbLQAMbTED Cu TURNINOS
I I
bNHVbRONE-+I S.
r
.
COMBUSTION
TUBE
.
I UMEbSUREMENT NIT FOR
I mm.CAPILLARY
EINFORCEMENT
I mm. CAPILLARY-
-7 GLASS
Figure 2.
Hollow stopcock reservoir (left) and glass wool U-tube ( r i g h t )
The glass-wool section was found to be necessary to condense out completely and to retain all the carbon dioxide coming from a sample exceeding about 200 y of carbon. The plain capillary U-bend adequately retained all the carbon dioxide below this amount; but as additional amounts entered the cold zone of the trap carbon dioxide crystals apparently grew t o the extent that some of them broke loose from the walls of the capillary into the oxygen stream, which carried them out of the measuring unit, thus giving slightly low results. This explanation of the low results was verified by operating two measuring units in series. The combined amounts in the two units gave complete recoveries. After the glass-wool section was added to the primary unit it
SB R E M O V I L TUBE
Table I.
0
Figure 1.
mm. TUBING
WOOL
Schematic diagram of carbon train
Analyses of National Bureau of Standards Standard Samples 166 and 59 Carbon Recommended,
The time required for measurement of the carbon dioxide after completion of sample combustion was 2 minutes or less. This constituted a considerable saving of time over the previous method .of weighing Ascarite tubes on a microbalance. Another outstanding advantage over the gravimetric method was the absence of a blank attributable t o the carbon dioxide measurement. When only the sample loading operation was omitted from the procedure the manometer gave zero readings. Results obtained on two Xational Bureau of Standards samples containing low carbon are shown ic Table I.
Carbon
Found,
%
%
Sample SBS steel 166
0.0274
NBS ferrosilicon 69
0.015
Average
0.0275 0.0275 0.0273 0.0281 0.0281 0,0272 0.0276 i= 0 . 0 0 0 3 0.0158 0.0148 0.0155
0.0151
Average
1500
0.0153
* 0.0004
1501
V O L U M E 2 7 , NO. 9, S E P T E M B E R 1 9 5 5 Table 11.
dioxide were involved, but all permitted varying degrees of loss. The glass wool not only served as a filter to retain the crystals but it also substantially increased the cold surface on which the condensation could occur. Table I1 shows the results obtained on materials containing carbon in the macro range, using the modified measuring unit.
Analyses for Carbon in Macro Range' Using Revised Measuring Unit Carbon Recommended
Carbon Found,
%
Sample CaCOa
%
12.00
h-BS steel 82. 8 samples
2.78
NBS steel 132, 6 samples
0.803
Average
11.97 12.08 11.99, 12.04 12.02 12.05 12.14 11.87 11.98 12.00 1 2 . 0 1 =t0 . 0 5
-4verape
2.79 i0.09
Average
0 . 7 9 3 i 0.005
ACKNOWLEDGMENT
The author wishes to acknowledge the valuable assistance supplied by W.G. Smiley of the Los Alamos Scientific Laboratory in this application of the capillary trap. Private correspondence and a visit to his laboratory yielded many helpful suggestions. LITERATURE CITED
gave complete recoveries and no carbon dioxide was found to escape into the secondary unit. Other designs tried for the cold trap before adopting the U-bend containing glass wool were a capillary helix coil, a multiple loop coil, and a U-bend containing a fritted-glass filter. These were all improvements over the plain capillary U-bend when large amounts (over 200 y) of carbon
(1) Bennet, E. L., Harley, J. H., and Fowler, R. AI., ANAL. C H E M . , 22, 445 (1950). ( 2 ) Fowler, R. hl., Guldner, W. G.. Bryson, T. C., Hague, J. L., and Schmitt, H. J., Ibid.. 22, 486 (1950). (3) Pepkowitz, L. P., and Rloak, W.D., Ibid., 26, 1022 (1954). (4) Smiley. 1%'. G., Ibid., in pres*. (5) Wooten, L. A., and Guldner, W.G.. IND. EXG.CHEX, - h . t r , . ED., 14, 835 (1942).
RECEIVED for review
November 2 , 1954. Accepted April 1, 1955. Based o n urork performed under t h e a u p i c e s of U. S. Atomic Energy Commission.
Detection of Amino Acids on Paper Chromatograms J. A. ClFONELLl and FRED SMITH Department
of
Agricultural Biochemistry, University
of Minnesota, St. Pad, M i n n .
Amino acids and other amino compounds after being oxidized by periodate may be detected on paper chromatograms by benzidine and by starch-iodide reagents. These methods have enabled certain of the amino acids to be distinguished. Mixtures of tert-amyl alcoholI-propanol-water have been found useful as irrigating solvents for separating amino acids.
N
INHYDRIN is a satisfactory reagent for detecting amino acids on paper chromatograms (5) and the more recent method involving chlorination followed by a starch-iodide spray makes it possible to detect proteins as well as amino acids ( 2 , 8). However, the identification of the amino acids in a mixture still presents considerable difficulty. Since no single solvent mixture permits the separation and identification of all the amino acids on a single one-dimensional chromatogram, recourse has been had to the use of two dimensional chromatography ( S ) , and of specific tests. Thus diazo reagents have been employed for detecting tyrosine and histidine ( 4 , 5, 9); p-aminobenzaldehyde for glucosamine ( 7 ) ; and sodium azide ( I ) , iodoplatinate ( I I ) , and nitroprusside (10) for locating sulfur-contnining amino acids. In an attempt to simplify the identification of amino acids on paper chromatograms the authors have found that certain periodate sprays with or without a subsequent benzidine spray are useful for detecting certain amino acids and groups of amino acids. These new reagents, which have already proved valuable in the paper chromatographic analysis of carbohydrates ( 2 ) are also valuable for the identification of the difficultly separable amino acids such as glycine and serine, threonine and alanine, and valine and methionine on one-dimensional chromatograms. EXPERIMENTAL
Solvents for Irrigating the Chromatograms. >fixtures containing tert-amyl alcohol and 1-propanol were found to be useful
as a second solvent following phenol in two-dimensional chromatographic separation of amino acids. Such solvent mixtures can also be used for one-dimensional chromatograms (eee Table
11). REAGENTS
Benzidine Reagent A. Mix equal volumes of 0.1M benzidine in ethyl alcohol and 0.8N hydrochloric acid. Benzidine Reagent B. Mix 10 volumes of 0.1V benzidine in 50% ethyl alcohol with 1 volume of 0.2N hydrochloric acid and add 2 to 3 volumes of acetone t o dissolve the benzidine hydrochloride. Neutral Periodate. Saturated aqueous solution of potassium metaperiodate. hlkaline Sodium Iodide. A 4% aqueous sodium iodide solution saturated with sodium bicarbonate. Starch, 0.1% aqueous solution. Sodium Iodide, 5 % aqueous solution. Glycine, 5% aqueous solution. PROCEDURE AND R E S U L T S
Treatment of Chromatograms with Periodate. The chromatograms were freed from volatile solvents by allowing them t o stand in air. When phenol was used it was removed by extraction with either ether or acetone-ether. The papers were then sprayed with the saturated aqueous potassium metaperiodate solution and allowed to stand for 1 to 2 minutes. Procedure 1. Detection of Amino Acids with Benzidine Reagent A. When the periodate treated paper is sprayed with Reagent A it gives within 1 minute a blue color with glycine and a yellow color with methionine on an almost colorless background. With insufficient hydrochloric acid in the reagent, the background will turn blue, while too much acid reduces the sensitivity of the reagent. Maximum sensitivity for all reactive amino acids is obtained by spraying the chromatogram lightly with Reagent A and, after the blue spots appear, respraying with Reagent A; this inteneifies the spots due to the sulfur-containing amino acids and tryptophan. The blue spots generally fade under this treatment, but
