two peaks taken together can be seen to confirm the molecular weight of the compound. Other unusual peaks in the spectra are formed in intermolecular reactions. Thus, in several of the spec- t ra is seen a peak corresponding to ions ( p + H ) + where p represents the parent molecule. Such a peak can be recognized as due to intermolecular action, in which relative abundance in- creases relative to the parent ion as either the repeller voltage is reduced or the sample pressure is increased. It is particularly useful in confirming the molecular weight of the ester, and is accompanied by a smaller peak cor- responding to the ion ( p + RlCO)+, confirming the formula of the radical R1. As an example, n-propyl formate iniolecular wt., 88) s h o m pressure- dependent peaks at masses 89 and 117. Often these peaks are not large enough to be shown in the tables, but if a n ester is suspected such peaks can be augmented by merely increasing the sample pressure. Llclafferty ( 7 ) has made use of this same phenomenon in determining the molecular weight of aliphatic ethers. The peak a t ( p + H1’ is large enough t o be recorded in several of the spectra-e.g., ethyl formate and iFopropy1 acetate-and is too large to be accounted for by the isotopic contributions of the parent ions (Tables I to IV).

The series of rearrangement ions observed by Sharkey, Shultz, and Friedel at masses 60, 74, 88, and 102 are not particularly prominent in most of these lower esters. Mass 60 [(C2H402)+, 21.5%] and mass 88 [(CIH80p) +, 46.8%] in ethyl n-butyr- a te and mass 74 [(C3H&&)+, 76.0701 in methyl n-butyrate are the most noticeable examples. The occurrence of the peaks corresponding to ions such as (CH50)+, (C2H?O)+, and similar species, more heavily hydrogenated than neutral molecules, suggests tha t in these cases the positive charge is located on a n oxygen atom, and Of, being trivalent, can accommodate a n extra hydrogen atom.

The main advantage of high resolu- tion in the examination of an ester lies in the fact that any such compound submitted for qualitative identifica- tion can immediately be recognized as containing two oxygen atoms. Frag- ment ions in the mass series 59, 73, 87, 101, etc., can contain two osygens, as can the rearrangement ions a t masses 47, 61, 75, 89, etc. Determination of the composition of these ions gives definite proof that one is not dealing with a n alcohol or ether; these groups of compounds have the same nominal molecular v-eights as esters. The isomeric carbosylic acids cannot, of course, be distinguished on this count,

but the very prominent rearrange- ment peak in acids at mass 60 is very much less pronounced in the case of esters, and the rearrangement ions at ( p + H ) + and ( p + RICO)+ also render identification of the ester pos- sible.

LITERATURE CITED

(1) Aseelineau, J., Ryhage, R., Sten- hagen, E., Acta Chem. Scand. 11 , 196 (1957).

(2) Beynon, J. H., Nature 174, 735 (1954).

(3) Craig, R. D., Errock, G. h., “Ad- vances in Mass Spectrometry,” J. D. Waldron, ed., pp. 66-85, Pergamon Press. London. 1959.

\ - - - - ,

(7) McLafferty, F. W., Ibid., 29, 1782 (1957).

(8) McLafferty, F. W., Gohlke, R. S., Ibid., 31, 2076 (1989).

(9) Kier, A. O., Roberts, T. R., Phys. Rev. 81, 507 (1951).

(10) Ryhage, R., Stenhagen, E., i l r k i v Kemi 13,523 (1959).

(11) Sharkey, A. G., Shultz, J. L., Friedel, R. .4., ANAL. CHEM. 3 1, 87 (1959).

RECEIVED for reviev June 27, 1960. -4ccepted October 21, 1960.

Absence of an Isotope Effect in the FractionaI Rec rysta I I i z a t i o n of a I p ha -D- G I u cose -7 - t HORACE S. ISBELL, HARRIET L. FRUSH, and NANCY B. HOLT Nctional Bureau of Standords, Washingfon, 0. C.

b An isotope effect in the fractional recrystallization of a-D-glucose- 7 -f has recently been reported. Because of the significance of this result in the use of tritium-labeled carbohydrates as tracers, the fractional recrystalliza- tion of both a-D-glucose-7-f and a-D- glucose-6-t has been investigated. Under the conditions of crystallization used in this laboratory, no isotope effect was found for either substance.

HE USE of radioactive compounds T as tracers depends on the feasi- bility of isolating and purifying prod- ucts by recrystallization or other means, without altering the specific activity. Recently, an isotope effect nas reported (4) in a fractional re- crystallization of a-D-glucose-I -t equi- librated by the addition of dilute aqueous ammonium hydroxide. The

specific activities of five successive crops varied progressively from 104.2 to 98.2YG of the specific activity of the original sugar. This variation was con- sidered to be due to a secondary iso- tope effect associated with the kinetics of the opening and closing of the sugar ring during isomerization in solution. It was concluded (4) tha t such an effect can occur when the carbon atom bound to tritium undergoes isomerization, and the subsequent isolation step is in- complete.

Because of the far-reaching implica- tions of this result, a study was made of the recrystallization of e-D-glucose-I-t and a-D-glucose-6-t under the conditions ordinarily used for preparing position- labeled, radioactive D-glucoses. The use of dilute, aqueous ammonium hy- droxide to equilibrate the solution be- fore concentration was omitted, be-

cause i t is not a customary step in the crystallization procedures employed in this laboratory. I n a-D-glucose-6-t the carbon atom joined to tritium is not involved in the isomerization. It would therefore be expected that, if a n isotope effect exists in the recrystalliza- tion of a-D-ghcOSe-I-t, there rrould be a detectable difference between the behavior of a-D-glucose-I-t arid CY-

D-glucose-6-t on fractional recrystalliza- tion.

In the experiments described belolv, no such effegt has been found. Care- ful assays of successive fractions, in the recrystallization both of a-D-glucose- I-t and of a-D-glucose-6-t have shown no significant variation in specific activity. Hence, it must be concluded that , under the conditions for recrystallizing a- n-glucose-I-t used in this work (con- ditions generally employed for recrystal-

VOL. 33, NO. 2, FEBRUARY 1961 225

Table I. Radioassay of Fractions Obtained in the Recrystallization of a-D- Glucose- I -f and a-~-Glucose-6-f

Material Original a-D-glucose-

Fraction 1

Fraction 2

Fraction 3

Fraction 4

Fraction 5 Original a-D-glucose-

Fraction 1

Fraction 2

Fraction 3

Fraction 4

Fraction 5

I - t

6-t

T t . , Gram 1.000

0,483

0.220

0 128

0.098

0,041 1,000

0.511

0.184

0.131

0.081

0.044

x 100. a d t o t a l counts total counts

Sample, Mg.

25 247 25 350 25 514 25 060 25 363 25 452 25 247 25 103 25 200 25 184 25 370 25 046 25 300 25 055 25 240 25 439 25 041 25 035 25 260 25 152 25 163 25 160

Probable Counts Statistical in 500 Srariazon,o

Seconds 15,503 15,398 15,647 14,997 15,446 15,500 15,500 15,297 15,356 15,248 15,500 13,893 14,103 13,800 14,000 14,051 13,948 13,681 13,952 13,873 11,247 13,700

YO

1 0 . 8 0 0 .80 0.80 0.81 0.80 0.80 0.80 0.81 0.81 0.81 0.80

1 0 . 8 5 0 .85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0 .95 0.85

Specific kctivity, pc./Mg. 0.1598 0.1581 0.1596 0.1588 0.1585 0.1585 0.1598 0.1586 0.1586 0.1576 0.1590 0.1444 0.1451 0.1434 0,1444 0.1438 0,1450 0,1423 0,1438 0,1436 0.1454 0.1418

Relative Radio-

activity, %

100.00

99.21

99.72

100.16

99.47

100,03 100.00

99.41

99.76

98,83

99.48

97.96

lizing sugars), an isotope effect does not exist.

The work does not disprove the exist- ence of a n isotope effect in the rate of anomerization reactions, or in side reactions that may accompany crystal- lization processes. Either of these may account for the striking isotope effects observed by the previous authors in the recrystallization of D-mannose-I-t phenylhydrazone.

EXPERIMENTAL

a-D-Glucose-14. prepared as described previously (I), !vas recrystallized four times from methanol by the addition of isopropyl alcohol. It was then dried in a vacuum oven a t 60" C. for 4 hours and a t 70" C. for 1 hour, and

finally stored in a vacuum desiccator over anhydrous calcium chloride. CPD- Glucose-64, also prepared as dwcribed previously ( 2 ) , was similarly dried and stored.

In the fractional recrystallization, 1 gram of the labeled sugar (either a-D- &cow-1-t or a-D-glucose-6-f) was placed in a Tveighed test tube and dis- solved in 0.6 ml. of water. After a clear solution had been obtained by warming the mixture slightlr. a few drops of ethyl alcohol Tvere added. The solution was seeded with a trace of crystalline a-D-glucose, and the tube lvas stoppered and stored a t room tem- perature. After several hours, the mother liquor n-as separated from the crystals by a capillary pipet and trans- ferred to a second weighed tube. Ethyl alcohol vias added to thp mother liquor

Y

to the point of incipient turbidity, and a second crop of crystals was obtained. The mother liquor from this crop was concentrated and again treated with ethyl alcohol to yield additional crops of the sugar. The crystals in the tubes were drird in the same manner as the original sugar.

Radioactivity measurements were made a t 1800 volts with a commercial, 2 ~ , windowless, gas-flow, proportional counter (Uodel PC-3 of Suclear Meas- urements Corp., Indianapolis, Ind.) fitted with a water-cooled slide and a stainless steel sample cell (36 mm. in diameter) ( 3 ) . For radioassay, accu- rately \veighed samples of the fractions, in small test tubes, were dissolved in weighed amounts of formamide (about 1 ml. each). Each solution was counted for five successive periods of 100 seconds; there was no progressive change during these periods; hence, only the total count, corrected for back- ground, is recorded. The second series of determinations was made in a similar manner, after an interval of several days. The weights of the crops and their radioactivity measurements are given in Table I.

The radioactivity 15 as calculated from the empirical relationship,

A = amk, n-here A is the activity, in microcuries, corresponding to a, the observed counts per second, k is a calibration factor (0.112), and m is the combined weight of the solvent and solute. The specific activities in Table I are espressed in microcuries per milligram of the sugar.

LITERATURE CITED

(1) Isbell, H. S., Frush, H. L., Holt, S. B., Mover. J. D.. J. Research Natl. Bur. %andards WA', 177 (1960).

J. D., Ibid., MA, 359 (1960). (2) Isbell, H. S., Frush, H. L., Moyer,

(3) Schwebel, A., Isbell, H. S., Moyer, J. D., Ibid. , 53, 221 (1954).

Chem. Ber. 92, 1635 (1959). (4) Weygand, F., Simon, H., Keil, K. D.,

RECEIVED for review August 29, 1960. Accepted Xovember 2, 1960.

Resolution of Time-Dependent Gamma Spectra with a Digital Computer and Its Use in Activation Analysis OSWALD U. ANDERS and WILLIAM H. BEAMER

Radiochemistry Laboratory, The Dow Chemical Co., Midland, Mich.

b Recent developments in activation vation samples. Its use permits ob- analysis are emphasizing completely taining maximum information from instrumental methods employing V a Y mixed y-spectra and freedom from spectrometry- For such analyses data simultaneous irradiation of elemental handling is a problem. To make standards. Typical analyses using 5- better use of the obtainable data a minute irradiations are given and the small electronic digital computer has been programmed tocorrect,normalize, Peeling technique used described* and resolve time-dependent y-spectra Reference spectra have been collected from short-irradiation-time neutron-acti- and are available for 69 elements.

N INCREASING interest in short-ir- A radiation-time neutron-activation analysis can be noticed in .the recent literature (I, 3-7). A requirement of this type of activation analysis is rapid and efficient data taking, following either immediately after the activation or after rapid and efficient chemical group separations. In several cases much of the burden of the analysis is

226 ANALYTICAL CHEMISTRY