The High Resolution Mass Spectra of Aliphatic Esters J. H. BEYNON, R. A. SAUNDERS, and A. E. WILLIAMS Dyestuffs Division, Imperial Cbemicol Industries, Ifd., Blackley, Moncbesfer, hgland b The high resolution mass spectra of 2 7 aliphatic esiers have been obtained. It is possible to assign a formula for each fragment ion in the spectra, which makes it easier to identify a compound from ils spertrum and, in pariicular, to disiinguish an ester from compounds such as isobaric alcohols, ethers, and acids.
T
of 27 aliphatic esters have been recorded under conditions of high resolution. It is hoped that this will supplement the information given by Sharkey, Shultz, and Friedel ( I I ) , n-ho have obtained the low resolution spectra of many of these compounds, and also that given in the recorded spectra of a number of esters of long-chain carboxylic acids (1, 6, 10). Accurate mass measurements of the ions detected were made t o obtain their empirical formulas ( 2 ); thus, the various more important fragmentation proccsses were able to be deduced. Esters of dibasic acids have been studied by Kourey, Tuffly, and Yarborough (6) and an extensive correlative study of the spectra of aromatic esters has been made by Emery (4) and McLafferty and Gohlke (8). The correlations of the spectra with the molrcular structure proposed by Sharkey et al. have been largely confirmed. Many of the peaks in the low resolution spectra have proved t o be doublets, as expected. I n all cases, however, the larger component has been of the expected formula. Because the parent peaks produced by most esters are either very weak or absent altogether, the identification of an unknown ester is often very difficult. It was hoped, therefore, that this investigation would produce useful information whereby the detection and identification of e s t m in unknown samples would be made easier. This is also a very good instance of the advantages to be gained by the use of a double focusing mass spectrometer even if only a modest resolving power of about 2000 is available. Apart from the cases of isotope peaks i t is only necessary to resolve the CH4-O doublet, for which at mass 100, M / A M = 2748, or the CzHs-02 doublet (such as might occur a t mass 44 between the ions C02& and C,H,+), which requires only half tliis resolving power. HE MASS SPECTRA
Table 1.
Mass 12 13 14 15 15.5 16 17 18 19 25 26 27 28 29
Mass Spectra of Aliphatic Esters of Formic Acid.
RCethj 1 3 7 27 5
0 75 0 75 2 5 0 22
Ethyl 0.19 0.58 2.1 5.5 0.58 0.19 0.19 1.1 3.9 0.66 7.3 29.1
5 9
62 3
30
0 702
31 32 33 39 40 41 42
100 0 -
33 6 0 7%
81.9 23.7 27.8 1.6 0.62 100.0 1 3i 0.06
0 19 0 28
0 40 0.67
43
0 23
6.7
44
1 6
1.5
45
31,l 1 2
46 47 48 53 54 55
1.49 9.8 0.12
56
4.2
57
0.14i
58
0.29
59
0 92
1.23
60
38 5p
0.21
n-Propyl
0.02i 1.3 6.5 1.1 0.03 1.6 13.9 1.1 3.5 7.3 7.4 1.7 0.16 100.0 1.6i 0.0% 0.2 0.9 8.9
0.2 0.2 0.1 1.1 4.3 0.4 0.90
1.3
p = parent ion; i are underlined.
1.1
sec-Bu tyl
1.12
2.7
0.O l i 0.16 0.84 1.4
0.031
8.4 2.5 27.6
1 .o
53.2 7.7 31.4 0.47 1.4 1.1 100.0
0.12
0.15 0.46 2.7
1.2 17.4 0.04 6.2 4.6 15.1 0.63 0.34 30.3
1.8 14.4 1.0 4.4 6.2 18.6 0.37 0.42 14.4
1.35 6.5 1.6 56.3 0.73 32.3 1.3 21.0 0.69 1.6 0.69 4.7
6.3 1.1 25 8 0.77 1.21 7.7 0.53 0.32 3.6 100.0 -
0.161 3.3 0.04 0.70 0.72 0.17 10.8
2.31 0.68 0.93 0.48 0.24 4.1
0.05 0.39 0.29 4.8 0.29 0.16i
35.9 3.6 12.4 0.47 0.53
100.0 1.o
7.91
0.02 0.31 0.69 0.26 2.4
17.4 0.5%
0.47 0.14 24.3
0.30 0.33
0.17 1.7 0.24 17.5 1.2
5.4
0.06i
0,86i
0.54
0.10 0.40 0.20p 0.20
0.14i 0.12 0.79~ 0.34
0.15 0.20 0.05 0.09 0.06 0.12p 0.12
0.20 =
4.5 0.41
2.3i 9.1 0.12
1.4
75 87 88 89 101 102 103 117
1.1 0.80 8.3 0.45 0.30
6.6 0.08
73
15.7~
0,05i 0.05 0.14 4.6 0.06 1.2 20.7
-
0.05 0.37
74
4.7
73.8 0.9 6.3 1.6 0.2 0.21 4.7 0.08
71 72
0 891
butyl
0.12
0.3 1.7
1.4 5.8 0.50 1.3 0.30 0.03 0.02 0.02
61
Isopropyl
0.571 0.19 3.3 0.201 0.45~ 0.031
ions in which isotopic contribution forms major part; base peaks ~
~
VOL. 33, NO. 2, FEBRUARY 1961
~
~
_
221
_
_
_
Table II.
Mass 12 13 14 15 16 17 18 19 25 26 27 28 29
30
Ion
C CH CH, CH,
Rlethyl 0.11 0.49 2.2 18.1 0.19i 0.09 0.12 0.03 0.26 0.3T 0.67 0.75 0.37 5.1 0.19 0.56
3.0 0.41 0.21
31 :3 2 33 38 39 40 41
2 2 0.37i 0.08
0.57 12.1 0.29i
0.08
0.56 8.2 100.0
44
45 46 53 54 55
Ethyl 0.06 0.30 1.9 9.6 0.14i 0.27 0.03 0.43 0.10 1.8 9.4 0.43 3.0 3.1 14.0 0.29 0.29i 1.3 0.14 0.09 0.07 0.29 0.29 5.4 0.29 99.9 0.10 0.43 2.2
42
43
Mass Spectra of Aliphatic Esters of Acetic Acidu
-
n-Prop yl
Isopropyl
57
0.14
58
0.05
59
0.21 5.3 0.06i
0 31 3.3 0 07i 0.22 1.2 0.20
0 02 2.8 0.04i
0.35 4.7 0.25 0.38 0.79 0.34
3.9 0.11
1.1
0.18 1.9 0,54 5.1 2.3 7.3 87.8 12.2
0.29 2.7 0.69 0.09 8.2 1.5 5.2 86.2 13.8
14.6 3.2 1.4 97.8 2.2
2.5 0.41
1.9 0.46
2 7 0.07
2 5
2.4 0 26
1.4 0.09
2.1 0.06
0.79
0 75
0.39 0.79
,3 0 0 0
7.3
2 1
39.6
19 7 0 83 5 8 0 11 0 22
1 11
1 6 3 8
2 li 1 8 0.14 2.4
0.20 0.30
0 98 0 10
1 0 0 13
0 25 0 81
13.6 0.39
4 -l
0.57 5.2 0.68 0.68 1.3 0.99 0.31
17.3 0.10
n-Amyl
0.06 0.70 4.2 0.13 0.06 0.25 0.10
0.08 0.14
sec-Butyl
0.60 5.4 0.09i 1.9 10.9
0.14
56
n-Butyl
0 0 0 0 0
ANALYTICAL CHEMISTRY
55 18 95 25 8 07 4 12
34 31 13 13 90
0.39 0.13 0.62 0.26
p = parent ion; i = ions i n which isotopic contribution forms major part: major contributions to base peaks are underlined.
222
0 0 0 0 6 0 1 0
0.31 0.15
EXPERIMENTAL
The spectra were obtained using a l\letropolitan-Vickc.rs Type 1188 mass 3pectrometc.r (S), Ivhich follows the design of Nier and Roherts ( g ) . The ions are accelerated through a potential of 4 hv. and deflected through a n electrostatic analyzer and then a magnetic aiialywr, the radius of this latter qec-tion being 6 inchcs. With beanidcfining slits of 0.001 inch. the resolution n as approximately 2500. The samples were ionized with 50-volt elcctrons, with a source temperature of 250' C.; the spectra nere scanned hy n r y i n g the magnetic field. The compountis T W I supplied ~ inaiiily by the Fine Chemicals Service of these laboratories. The n-propyl n-butyrate arid n-butyl and isobutyl propaiioates w r e as supplied by British Drug Houses. Ltd., Eiiglancl. Each conipound was first analyzed by gas-liquid chromatography and found to give n qingle peak in the chromatogram.
Table 111. hInss 14
15 16 17 18 19 26 27 28 29
XIethvl
Ethyl
n -Pr opyI
hopropy1
n-Butyl
Ieobutyl
0 17 0 0 0
0.24 1.1 0.07 0.21 1.3 0.49 1.6
0.38 0.56 0.17 0.24 1.5 0.07 0.77 8.1 1.1 3.2
0.19 1.7 0.01i 0.27 I .4 0.03 0.95 10.1 0.54 2.8 0.68 28.1
0 08 0 41
0.08
03
0.02 0.09 0.08 0 52 6.7 0 ZJ 1.0
48
5 302 06 30
2 1
12 3 0 9; 7 3 2 5 67 4
30
0 39 1.5
31
4 2 0 16 0 71
32 33 38 39
40 41 4"
DISCUSSION A N D RESULTS
43
TI-riting the formula of these esters a-
Ion
Mass Spectra of Aliphatic Esters of Propionic Acid.
44
'I
nlicrr R1 and R2 are alhyl radicals, i t io scen, in Tables I to IV, that peaks d ~ c , to Rl+, (RICO) -> (COOR2) +, (OR2)+,and R2+nere detected in most r a s ~and this agrees n i t h the pre~ i r m results ( I O , 2 1 ) . The ions given in these tables are all singly charged n c c p t where othern-ise indicated, and ~ s o t o p arc c ~ only noted where they contribute the major portion to the peaks. Tlic spectra have been normalized by tnhing the sum of the components of thc largest peak in earh spectrum as I00 units. The peaks due to (RICO)" ions are the most prominent in the spectra of th(, acetates and the propaiioates. I n the case of tlie butjrates they are ;till fairly strong, falling only to 34.57, 1~1thisopropyl isobutyrate. This trend :ilw pwsists for some of the esters of thc higher acids. Kithout the aid of accwrate mass nicasurtment and high I cwlution, these ions can easily be confused w t h those of hydrocarbons, and the ~~rosc~ncc of the o\-gm atom cannot hc dctwted directly. The peak a t i n a s 29 is not as characteristic of format(,.: as the oncs a t masses 31 and G, uliich are more prominent with niothyl, c>thyl, and n-propyl formates :iiitl isopropyl and sec-butyl formates, rwpectivcly. These are all rearrangemr~iitions (,wept in thc case of m r t h j 1 fo Ininte. .\ pclak characteristic. of many of th(w rstcrs a t mass 19 is due to the H 3 0 + ion, which is usually small but not so easily overlooked. Vnfortunately this ion does not occur solely in esters; it is also i n the spectra of many
45
-
0 0 0 0 0 0
36 75 10 4c)
23 68
2 1 1 0
46
47
0.70 31.3 0.21 0.77 4.2
0.51 0.19 1.1 2.4 0.60 0.26 0.09 0.55 11.9 0.12 0.33 0.11 0.06
6.9 0.35 5.2 0.18 11.6 2.0 0.39i
0.18 0.11
9 0 1 0 0 0
20.7 41.6
0 0 0 0
0.51i 1 li
0.27 3.1 0.13 0 ,07 0.04 0,09
54
0 13
0.11
55
4 1
1. 3
56
3 1
3.5
-57
100 0
05.1
58
3 3
3.1
59
30 5 0 2ii 1 li
1.2
0.81 0.41 1.1 3.8 100.0 3 , :3
83 7 047, 28
4 24 0 59 37
20 28 15 13 57
0 03 0 28 0 04 0 15 0 65
7 9 0 33 A- .7
,
91.4 -8.6 :3 . 3
0 39
0.60
4.4
0.14
61 70
4
1 5 0 :33
10.0 0.68 4 .3
0 19
60
0 3 0 0
0.15 2.4 0.54
53
0.81 0.07 1.9 0.25 99.7 0.30 3.6
16 11 72 6 54 1 61
0 13 0.64 1 . .5
0.0% 1.3 0.39
0 23 0 13 0 10
0 RI-C-O-RQ
13.1 0.05 2.1 2.0 98.0 0.26 2.3 2.5 0.02i
0 0 0 0 7 0 1 0 37
0 I9
10.0
0 242
0.43 0.02
0.47 30.3 0.12 0.68 3 .0 0.031 0.15 0.10 2.4 0.50 10.4
0.29 2.1 0 87 5 6 0.15 0.08 13.11
0.12 I .8 IO,01
0.02 0.18 0.03 0.08 0 ,-14 1.9 1.1 25.2 95.7 ____ 4 , :3 :1 2 0.26 0,0.5 0.43
0.40 0.!)5
0.08 I3 1-5 "8 I),22 0.35 1.9 0,90 0 54 6 i 0,23 ,
71 72
73
i,4
0.70
74
12.8
2.5
3 .
13.6 0.52 0.14 0.31 1.6
32.4 1.1 0.14i
10
-76 i I
83 84 86
87 88
89
a
1 9 23 l p 1 42
1.4 0.49
0"
6.3p
03 14 15 16 28 29 30
0.76
0.49 6.2
0.28i
0.17~
0.01i
0.95 0.95 5.1
Y3,5 1.2
1(1 0 22 0 17 0 01 1 3
12 8 0 35
0.18i
:3 , 5
0.1% 0.08
5 4 0 302 0 22
0,28 0.6i 0 . 5:3p 0 . 04i
0.07 m i
l
0 .59i 0.11 ,
0 ,08
0.02 0.02 0 . lop
Pee footnote in Table 11.
VOL. 33, NO. 2, FEBRUARY 1961
223
alcohols in addition to aanrarinn in the spectrum of mater if the pressure admitted to the ion chamber is sufficiently high. iis it was possible that the peaks a t masses 17 and IS, due to HO+ and H*O+, were formrd to some extent from fragmentation of the ester molecules as well as bcing part of the background spectrum, they have been included in the spectra. For the same reasons the peaks due to C 0 2 +and COions at masses 44 and 28 have also been included. It is usually very difficult to distinguish the ion R1+ formed from an ester from the corresponding ion in hydrocarbon spectra, but the alkyl group a t the other end of the molecule often gives an olefinic rearrangement peak due to (R, - H ) + when R 2 > methyl. This is particularly noticeable in the case of the formates and the other esters when Rz> propyl, as seen in Table V. There is also another rearrangement peak, formed from the remaining part of the molecule when the above olefinic fragment is removed from the molecule. This corresponds to the 0 L
Table IV.
Mass Spectra of Aliphatic Esters of Butyric Acid.
n-Butyrates
Isobutyrates
Mass
Pvlethyl
Ethyl
14 15 16 17 18 19 26 27 28
0 38 15 2 0 312
0.42 2.8 0.11 0.25 1.4 1.3 2.1 29.2 0.88 6.1 2.8 70.5 0.56 1.6 0.77 0.15
0 36 13 18 5 0 46 23 12 35 0.30 0 11 31 0 26 1 1 0 66 77 14 24 5 62 65 29 4 70 6 0 34 19 24 15 16
29 30 31 32 33 38 39 40 41 42 43
-
44 45 46 47 53
0.09 0.17 0.13 8.0 * 0.60 0.3%
54 55
56
n-
57 58
59
5.4 1 .o 23.6 6.6 8.9 6.9 88.8 1.3 1.1 3.0 0.18 4.9 1.7 0.68
1.3 9.3 0.05 0.lli 0.05 0.24 0.12 0.78 0.85 17.9 14.4 0.42 2.1 1.5 0.76 0.76 5.3 0.84 0.20 0 21 0.16 14 3.9 0.08 1.1 0.20 0.35 5.0 5.5 1.0 1.3 21.3 27.1 3.2 15.9 12.5 1.4 4.1 77.0 98.6 0.17 0.31 0.30 0.06 2.6 3.3 0.31 1 . 1 0.72 1.5 0.03 0.24
3.5 0.25 0.32
0.29 0.13 52 0 07 0 35
22
0.87 0.07 0.20 24 1
n-Propyl Methyl Ethyl
3.4 0.35 0.28 0.31
60 61 62 68 69
0.37 2.5 0.12 0.09 0.48 1.2 2.0 56 0.84 4.7 1.6 58 2 0.44 1.2 80 0 09i
Iso-
propyl
1.2 0 .Oli 0.22 0.12 0.55 9.3 0.33 1.5 0.56 0.42 0.86
0.19 0.25 4.7 4.2 1.0 0.94 21.9 14.9
0 85 0 17 61 2 27
71
72 73
- -
0.17 1.7 0.17 0.44 1.3 0.70 1.3
0 48
0 08 27 0 5.4 26 13.9 1 li 45 7.9 0.22 0 10
0.13 0.07 14 0 14 0 52 0 09 56 0.90 0 271. 0 41 1.5 0 24 0 13
4.7 0.22 0.11
0.68 0.25 0.06 0.30 0.10 1.8
0.08 0.55
--I S
2.7i 0.43
76 85 86 87 88 89 90 101 102 103 104 114 115 116 117 129 130 144
18 4 0 821 0 1Oi 097 1 3p 0 71
0.33 0.15
0 74
0 1.3 0 27
0.14
0.81
10
13
0.35
100.0 4.5 8.5 18 0 0.27i 0 27i 0,32i 1.6 0.98 0.49 0.07
0 14 74 46 8 13 0 0 63 64 0 56i
0.12 0.07 0.81 24 2 1 12 4.0 49.3 0 132 2.2 0 74 10 6p 1 li 0 06 0.14 0.34
ANALYTICAL CHEMISTRY
0 08 0 77 84 49 0 24 15 0 61 0 07
43.8 4.1 30.6 1.4i
Table
V.
34.5 1.5 1.9
100.0 4.5 3.2 2.0
0.11
0.36 2.2 15.7 0.69
0.33 0.20 0.34 0.19 0.08 0.71 1.9 19.9 0.91 4.9 0.30i 0.08
4.3 0.33i 0.55 0.llp
(R2-Wf,% Ethyl n-Propyl Isopropyl n-Butyl sec-Butyl
(Ri.CO.0
Ethyl n-Propyl Isopropyl n-Butyl sec-B;tgl n-Amy1
Isoamyl
Formates Mass 47 81.9 (CzH4)' 98 73.8 (C&)+ 66 53 2 (C3Hs)' 9.1 100 0 (CdH8)+ 3.3 (C4H8)+ 0.68 35 9
3.0 7.3 5.2 39.6 19.7 16.3 39.7
Mass 61
(GHa)+
2.1 5.2 4.3 6.7 25.2
10.5
29.0 (C3H6)+ 21.5 (C,Ha)+ 13.6 (CcH,)' 4.5 (CGHIO)+ 23.4 (CsHlo)+ 12.3 (C3H6)'
Propionates Ethyl n-Propyl Isopropyl n-Butyl Isobutyl
+
m+, %
Acetates
n-Propyl Ethyl
0.11 0.06 o.08P
Rearrangement Ions in Esters
Olefin Fragments,
0.26 7 3p 0 672
0.17~
a
39 5 18 60 0 31 0 29 5 5 0 82 0 57 0 19i 0 77
42 6 19
+
2H)+ ion and occurs at masses 47, 61, 75, and 89. A list of such ions is also given in Table V. The information gained from these
0 36
0.30
2.5p 0.77
See footnote in Table 11.
224
0 32 0.17 0.75 2.9
;I
(R1.C. 0
1.1
100 0 45
0 2% 76.0
74
0
10 2
0.06 0.64 0.25 0.26 1.5 0.08 0.49 10.8 0.71 2.4 0.56 16.5 0.11 0.37 3.0 0.45 2.3 0.19 5.0 1.2 30.2
12.9 8.6 7.8 2.8 1.7 1.2 97.2 98.3 - 84.1 0.59 0.26 0.31 0.08 0.39 0.13 3.3 2.8 3.3 12.5 0.42 0.46 6.5 0.42 0.77 0.17i 0.42
0.16
70
Iso-
butyl
_
Mass 75
(CzH4)+ 13.6 (C3H6)*
(CaH,) (C4Ha) (CaH,)'
32 4 33.5 12.8 6.7
n-Butyrates Mass 89 6.1 (CpHI)" 13.0 15.9 (C,H,)+ 49 3
Isobutyrates Mass 89 Ethyl 4.7 (C,H4)+ 4 9 Isopropyl 8.6 (CaH,); 15,7 Isobutyl 43.8 (C,H,) 19.9
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 spectra is seen a peak corresponding to H ) + where p represents the ions ( p parent molecule. Such a peak can be recognized as due to intermolecular action, in which relative abundance increases 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 corresponding 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 pressuredependent 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-butyrate 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 t h a 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 resolution in the examination of an ester lies in the fact that any such compound submitted for qualitative identification can immediately be recognized as containing two oxygen atoms. Fragment 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 rearrangement 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 possible.
+
+
LITERATURE CITED
(1) Aseelineau, J., Ryhage, R., Stenhagen, E., Acta Chem. Scand. 1 1 , 196 (1957). (2) Beynon, J. H., Nature 174, 735 (1954). (3) Craig, R. D., Errock, G. h.,“Advances 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., ilrkiv 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 Recrysta IIiz a t io n of a Ipha-D- GIucose-7 - t HORACE S. ISBELL, HARRIET L. FRUSH, and NANCY B. HOLT Nctional Bureau o f 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 recrystallization of both a-D-glucose-7-f and a-Dglucose-6-t has been investigated. Under the conditions of crystallization used in this laboratory, no isotope effect was found for either substance.
T
HE USE of radioactive compounds as tracers depends on the feasibility of isolating and purifying products by recrystallization or other means, without altering the specific activity. Recently, an isotope effect nas reported (4) in a fractional recrystallization of a-D-glucose-I -t equilibrated by the addition of dilute aqueous ammonium hydroxide. The
specific activities of five successive crops varied progressively from 104.2 to 98.2YGof the specific activity of the original sugar. This variation was considered to be due to a secondary isotope effect associated with the kinetics of the opening and closing of the sugar ring during isomerization in solution. It was concluded (4) t h a t such an effect can occur when the carbon atom bound to tritium undergoes isomerization, and the subsequent isolation step is incomplete. Because of the far-reaching implications 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 positionlabeled, radioactive D-glucoses. The use of dilute, aqueous ammonium hydroxide to equilibrate the solution before 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 recrystallization of a-D-ghcOSe-I-t, there rrould be a detectable difference between the behavior of a-D-glucose-I-t arid CYD-glucose-6-t on fractional recrystallization. I n the experiments described belolv, no such effegt has been found. Careful assays of successive fractions, in the recrystallization both of a-D-glucoseI-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 an-glucose-I-t used in this work (conditions generally employed for recrystalVOL. 33, NO. 2, FEBRUARY 1961
225
