Table
IV.
Rate Data o f Reaction Between Sarin and Enzyme Preparation" k? x 10-4 k o b s d . = 22 liters Sarin. [Sarin 1, 1Iole-' 1\IoleII Liter A h . --I Nin. -* 1 37 X 1 W 6 1 . 9 3 X lo-' 1.31 1 40 x lo-' 2.02 x 10-2 1.58 2 !12 x lo-' 3.44 x 10-2 1.38 2.80 X lo-" 2.85 X 1.39 4 28 x 5 96 x IO-' 1.22 .Iv. 1.38 'I
pI-1 7.4 to 7.8.
S o attempt ivas made to inhibit further reaction between the inliihit'or and the enzyine during the time the enzyme was hydrolyzing the acetate substrate. The error which might be introduced clue to t'liis reaction 11-odd be very sinal1 :inti could he ignored. Results are slioir-n in Table IT. The rate of reaction of Sarin with tlie esterase is. as in the case of the sub, independent of p H o i w 1%\vide range. Thus. the use of a strong1)- buffered solution is not necessary. I n fnct. it is preferable to use a low concentration of buffering materials to niinimize the nonenzymatic hydrolysis of Sarin. A final concentration of lniffer of 4 t o 5 X 10-41f (0.1 1111.
0.025Jl in about 6 nil.) is adequate to maintain a reasonably constant pH and presents no interference in the reaction between Sarin and the esterase. Miscellaneous. The sensitivity of the test can be increased by simultaneous or individual manipulation of several of t h e controlled features in the proccdurr-viz., t h e concentration of the acetate substrate, the incubation time of the inhibitor nith tlie enzyme preparation, or the temperature. As a n example, if it is desired that the standard curve be most usable between the range of 0 and 0.2 p.p.ni. of Sarin, calculations may be made for the proper incubation time for the inhibitor and enzyme preparation (using the same temperature and same concentration of acetate as in the above procedure), A colorimetric reading of approuimately 60 units will represent the loner limit of intensity (Figure 1). The activity of enzyme necessary to produce this intensity of color for a reaction time of 10 minutes can be shown to be approximately 207, of the original (Table 111). The problem is to calculate the time required for 0.2 p.p.m. of Sarin solution (1.4 X 10-6Jf) to reduce the actii-itjof tlie standard esterase solution to 20% of its original concentration. Because J22 is equal to the observed first-order velocity constant divided by the molar concentration of Sarin. the pseudo first-
order constant can be shown to be 1.93 X minute.-l The time is then calculated from the equation t = 1.303,'k o h s d . log 5 = 83.3 minutes. I n actual experiment a solution containing 0.2 p.p.ni. of Sarin solution incubated with the esterase reagent for 80 minutes, followed by addition of tlie substrate solution, gave a colorinietric~ reading of 65 after 10-minute reaction time; the same experiment. except for a n increase of incubation time to 90 niinUtes.. gal-e a colorimetric rending of 56. LITERATURE CITED
Aldridge, K. S., Riochem. J . 53, 110 (1853). .immon, R., PJuger's A r c h . ges. Physiol. 233, 486 (1934).
Barnett, R. J., Seligman, A. LI.,
Science 114, 579 (1951). Cook, J. IT.,J . Assoc. O j i c . Agr. Chemists 37, 561 (1954). Giang, P. A,, Hall, S. il., -4s.~~. CHEM. 23. 1830 11 ' Y51). ( 6 ) Glick, D., B k h e m . ' J . 31; 521 (1937); J . Gen. Physzol. 21, 289-97 (1938). (7) Hestrin, S., J . Bid. Chem. 180, 249-
61 (1949).
( 8 ) IIetcalf, R. I , , J . Econ. Entomol. 44,
883-90 11951). ( 9 ) llichel, H 0.. J . Lab C l ~ nJ l e d . 34,
1564-8 (1919). (10) Xachlas, >I. M.,Seligman, A. 11.. J . S a t l Cancer Inst. 9, 415 (1949).
RECEIVED for revieiy Augiist 15, 1956. Accepted February 6, 1957.
Colorimetric Determ inuti on of Molybdenum in Scheelite Ores and Concentrates R. P. HOPE King Island Scheelite ( 7 947),Itd., King Island, Ausfralia
b The need for a r a p i d and sensitive method, free from tungsten interference, for the determination o f molybdenum in scheelite ores and concentrates has resulted in the development o f a procedure satisfying these conditions. Small amounts of molybdenum can b e accurately estimated in the presence o f a large excess of tungsten, using potassium iodide as a reducing agent, prior t o color formation with ammonium thiocyanate. Sodium sulfite i s used to decolorize liberated iodine and hydrochloric acid concentration should be maintained a t 2M. The sample i s taken into solution b y fusion with sodium hydroxide, followed by water extraction, filtration, and washing with hot 0.570sodium hydroxi d e solution.
E
methods for the thiocyanate determination of molybdenum in the presence of tungsten, using stannous chloride ns a reducing agent, suffer, in general, from lon- sensitivity with an attendant loss of precision, and are susceptible to interference by tungsten. The reduction of molyhdenum(T'I) by stannous chloride results in only partial conversion to molyl)denuni(S7), the desired form for complexition. Ilon-ever, if reducing agents with a single available electron, such 3 s cuprous cliloride in aqueous 4 J I hydrochloric acid medium ( I ) or potassium iodide in aqueous 2 X hydrochloric acid medium (3, 4)>are employed, complete or near complete formation of molvhdenum(V) is obtained, resulting in grestlv inXISTIKG
creased color intensity and freedom from tungsten interference. EXPERIMENTAL
Rate of Color Development. I n this laboratory, 231 rather t h a n the 3 t o 431 hydiochloiic acid of Ginzberg and Lur'e (4) is the optimum value for color development, maximum intensity being reached after 20 minutes and color remaining Etable for 1 hour before fading commences. K i t h . 331 hydrochloric acid, full color is obtained in about 2 minutes hut begins to fade immediately and n ith 1-ll hydrochloric acid, color develops more slon ly, reaching its peak value after 40 minutes, but maximum intensiti- is not obtained. VOL. 29, NO. 7, JULY 1957
1053
The increased color intensity realized in the presence of traces of iron and copper, when reducing with stannous chloride, has been mentioned by various investigators (1, 2 ) . The addition of 1 drop of a 0.1X solution of cuprous or cupric chloride, or ferrous or ferric sulfate in concentrated hydrochloric acid to an acidified solution of molybdenum (prior to reduction Tvith potassium iodide and complexation with ammonium thiocyanate) will in each case increase the rate of color development without altering the final intensity. The effect of copper is more pronounced than that of iron. The order of addition of potassium iodide and ammonium thiocyanate is without importance in color developnient . Effect of Temperature on Color Development. Several runs conducted a t 17' and 30" C. were compared t o ascertain t h e necessity for maintaining a constant temperature during color development. A 5% decrease in color intensity was noted a t t h e higher temperature, a change t h a t was not reflected in t h e reagent blank, indicating t h e desirability of carrying out determinations within a relatively nnirow temperature range. Neutralization of Sample Solution. Seutralization with hydrochloric acid prior to color development of the alkaline solution obtained from t h e sample givcs rise to low results, t h e discrepancy becoming larger with a n increase in standing time between ncutraliaation and complexation Table I shows the results when color intensities were read 30 minutes after t h e addition of color-developing reagents when stable mauimum values had been obtained.
Table I. Effect of Lapsed Time on Color Development after Neutralization
(Total MoOI present, 0.300 n x . ) Lapsed Time from Xeutralization to Reagent Addition, Min. 45
30 I5 12
9 6 3 1
11001 Found, hlg.
0,273 0.290 0.293 0.302 0.311 0,320 0.328 0 . 330
This does not occur when all of the acid required for color development is added and the acidified sample solution
1054
ANALYTICAL CHEMISTRY
is allowed to stand before the addition of the remaining reagents. Table I1 shows the results when color intensities reach a stable maximum and are read 30 minutes after the addition of the remaining color-developing rengents.
Table 11. Effect of Lapsed Time on Color Development after Acidification with 1 to 2.4M (or 3.2M) Hydrochloric Acid
(Total 1100,present, 0.390 mg.)
Lapsed Time from Acidification to Remaining Reagent Addition. Min.
11003
Found, l l g . 0 38s 0.386 0.394 0.390 0.388
45
30
15 10 5
It is, therefore, most important t h a t the sample solution should not be neutralized before color is deveIoped . Interferences. Interference due to tungsten has been studied in some detail. Over 50 mg. of tungsten per 5 ml. can be tolerated, provided 5 ml. of 30y0 by weight per volume of ammonium citrate solution is added to prevent precipitation of tungstic acid during color development. In general, this is necessary when the tungsten level exceeds 7 mg. per 5 ml of sample solution. Antiniony(II1) forms a weak vellow complev with potassium iodide in hvdrochloric acid solution; home\-er, up to 5 mg. may be present during color development, without interference, and larger amounts map be tolerated, provided a blank determination is performed in the absence of thiocyanate. Antimony(V) which is reduced to antimony(II1) by the procedure with the liberation of iodine, may be present at about the same concentration level as antimony(III), provided sufficient potassium iodide and sodium sulfite are present. Although Ginzberg and Lur'e (4) have reported interference due to other elements, these were not examined in this study as none are present in significant quantities in King Island ores. PROCEDURE
Solutions Required. Sodium hydroxide wash, 5 grams in 1 liter of hot water. Hydrochloric acid, 1 to 2.4, 280 ml. of concentrated hvdrochloric acid d u s 680 ml. of water.
.41ii:iion1uiii thiocyanate, 23 grama of best reagent grade in 100 ml. of water Potassium iodide, 50 grams in 100 nil of water. Sodium sulfite, 1 gram in 100 ml. of n-ater. Ammonium citrate, 30 grams di+ solved in miter and diluted to 100 nil. Standard molybdenum trioxidr, 0.1000 gram of best reagent grade plus 1 grain of sodium hydroxide dissolved in water and diluted to 1 liter; 1 ml = 0.1 mg. of molybdenum trioxide. Sample Preparation. Fuse 0.2 t o 2.0 grams of finely ground sampl? with about 15 grams of sodium hydroxide in a 1.5 X 1.5 inch iron crucible at dull red heat. (Crucibles should be prepared by adding sodiiim hydroxide, fusing t o dehydrate, allon ing t o solidify, and storing in a desiccator or a n oven a t 110' C. until required for use.) Allow the melt to solidify and cool to such a temperature that, when it iq placed in a covered 250-ml. beaker containing about 80 ml. of nearly boiling n-ater, a vigorous reaction ensues. Where 1- or 2-gram charges are employed, solution of the melt may bc facilitated by cautiously cooling thc outside of the crucible in cold watm and/or tapping the crucible on a solid object after solidification of the melt. When solution is complete, remove the crucible and rinse with hot 0.5% sodium hydrovide solution. Filter through a 12.5-cm. Green's No. 795 or similar paper, wash well with hot 0.5% sodium hydroxide solution, and cool. Make up to the required volume so that the aliquot for color development contains 0.05 to 0.5 mg. of molybdenum trioxide. Color Development. If tungsten is present in the sample solution in excess of 7 mg. per 5 ml., use a 5-ml. aliquot in 5 ml. of 30% ammonium citrate solution. Otherwise a suitable portion u p t o 10 ml. may be used. diluting t o 10 nil. with water. Pipet t h e aliquot into a 50- or 100ml. beaker provided with a glass rod. Stirring after each addition, add 32 nil. of 1 t o 2.4 hydrochloric acid, 3 nil. of 25% ammonium thiocyanate solution, 3 ml. of 50% potassium iodide solution, and 2 ml. of 1% sodium sulfite solution. Allow t o stand for 30 minutes and read a t 460 mp. Calibrations. This work was carried out using a Spekker Model Hi60 absorbtiometer with a Kodak S o . 2 filter, 1-em. cells, and a water setting of 1.0.
A plot of absorbance us. concentration using 0.05 to 0.5 mg. of molybdenum trioxide gave a linear relationship over the useful instrument range with almost twice the slope of a similarly prepared line for a frequently used stannous chloride reduction (6). If it is desired to calibrate in the presence of tungsten, care should be taken to see that the tungsten compound used is free of molybdenum. Should an appreciable quantity be present. as often occurs, this may be corrected for.
Reproducibility. Fifteen determinations conducted over a period of several days on a sample of scheelite concentrates containing 70y0tungsten trioxide gave a mean molybdenum content of 2.63% with a standard deviation of 0.03.
LITERATURE CITED
( l j Crouthamel, C. E., Johnson, C. E.,
ANAL.CHEM.26, 1284-91 (1954). ( 2 ) Dick, A. T., Bingley, J. B., Yuture
158. 516 11946). (3) Fishe; Scientific Co., Pittsburgh, Pa., Bull. hfo-301-E. ( 4 ) Ginzberg, L. B., Liir'e, Yii. Yu.,
Zmodskaya Lab. 14, 538-45 (1948 I Analyst 74, 281 (1949) (ahstrart) (5) Grimaldi, F. S., Wells, R. C., 1x0 ENG. CHEW,ANAL. ED. 15, 315
(1943). RECEIVED for review December 6, 1956. .4crepted February 23, 1957.
Use of Silica Gel and Alumina in Gas-Adsorption Chromatography S. A. GREENE
and H. PUST
Aeroief-General Corp., Azusa, Calif. ,The use of silica gel and alumina in gas-adsorption chromatography is compared b y analyzing a complex gas mixture. These adsorbents are generally similar, although alumina is superior for light hydrocarbon analysis. Carbon dioxide can b e eluted from silica gel a t a low temperature and can b e separated from near boiling hydrocarbons.
L
work has been done on the separation of gases using ndsorp-
ITTLE
F i g u r e 1. Separation of gases on silica gel column
IO
eral similarity of silica gel and alumina as far as retention times and order of elution of hydrocarbons are concerned. They report poor separation of hydrogen, oxygen, and methane, and somewhat better separation for light hydrocarbons with these adsorbents. A comparison has been made of the heparation of a mixture of some low hoiling gases and a spectrum of hydrocarbons on silica gel and alumina colunins. Silica gel is a versatile column
W
~
W
P
-3L
tion columns packed n i t h silica gel. Patton and coworkers ( 2 ) note the gen-
Y
packing generally similar to aluniiiiti except in the elution of carbon dioxide, which is irreversibly adsorbed in the latter, and in the position of acetylene. EXPERIMENTAL
The apparatus was described previously (1). Helium carrier gas flow rates were 70 cc. per minute. Columns contained 20 feet of 20- to 40-mesh adsorbents. The alumina used was Alcoa-F1, activated (Alcoa Chemical Products, Aluminum Co. of America) ; the silica gel was Davison Chemical Corp. silica gel-desiccant. The teclinique of programmed heating adsorption columns was used in both cases. RESULTS A N D DISCUSSION
v)
x W
8 0
a
0 0
Figure 2. Separation of gases on alumina column
Figure 1 shows the separation of a mixture of gases on the silica gel column. Column temperature, initially a t 5" C., was raised t o 155' C. in 65 minutes. Good resolution of lower boiling components was obtained, while higher boiling components were not satisfactorily separated. Figure 2 shows the separation of the same mixture in the alumina column under identical conditions. I n this case carbon dioxide is irreversibly adsorbed, but all other components are resolved. It seems likely that when the resolution of air into oxygen and nitrogen is not required, silica gel columns could be used for separations that are usually done on charcoal columns. LITERATURE CITED
Greene S. A., Moberg, M. L., Wilson, E. h.,ANAL. CHEM. 28, 1369 (1956).' ( 2 ) Patton, H. W.,Lewis, J. S., Kaye, W.I., Zbid., 27, 170 (1955). I 0
65 TIME
LMINJ
RECEIYED for review August 31, 1056. Accepted February 23, 1957. VOL. 29, NO. 7, JULY 1957
1055
