VOL. 18, No. 4, 1964 107

(8) A. O. Nler in Nuclear Masses and Thetr Delermina- hon, H. Hmtenberger, Ed., Pergamon Press, Lon- don, 1957, p. 185

(9) J. H. Beynon and A. E. Williams, Mass and Abun- dance Tables for Use in Mass Spectrometry, Elsevier

Pubhshing Co., Amsterdam, 1963 (10) E. G. Carlson, G. T. Pauhssen, R. H. H u n t and

M. J. O'Neal , Jr., AI, CAL. CHEM. 32, 1489 (1960) (11) E. Kendrick, IBID. 35, 2146 (1963) Submltted June 4, 1963

T

Moisture-Induced Transformations of a - and B-D-Glucoses As Revealed by Infrared Spectroscopy

Leon Segal

Plant Fibers Pioneering Research Laboratory*

New Orleans, Louisiana

Abstract Spectral data are presented showing that the fl form of glucose

can be changed to the a form whale m the sohd phase, but th~s ~s not reversible. The existence of B-D-glucose hydrate xs postulated. Anomer transformatmn occurs only w~th the hydrated sugar R~gorous drying of mfrared-quahty KBr was found unnecessary for use with the sugars

Introduction D-glucose is available in three forms, anhydrous a-D-

glucose, a-D-glucose monohydrate, and anhydrous B-D- glucose. Differentiation between the a and fi forms by means of infrared spectra has been established by several workers (1, 2, 8, I0, 11). The distinguishing features are very prominent, characteristic bands located in the 1000- 700 cm -x region. The three forms of glucose given above are considered to be reasonably stable forms of the com- pound. Barker and coworkers (3), however, have presented hmited infrared data suggesting that a- and B-D-glucose (as well as other sugars) change during the preparation of KBr specimen discs. Band shifts observed in the 930- 750 cm -1 region were at tr ibuted to the high pressure ap- plied in making the discs, but no conclusions were ar- rived at concerning what changes in molecular structure could give rise to these shifts. Later, Barker and others (4) published evidence showing that B-D-glucose while in the KBr disc, changed to the a form. Here the spectral changes, again in the 1000 - 700 cm -1 region, are ascribed not to pressure but to traces of moisture in the KBr. Farm- er (5) later found that spectral shifts took place when KBr containing between 0.01 and 0.17% NaBr was used and that these arose because of the formation of a com- plex between the sugar and NaBr. Farmer indicates that the spectra of the complex and of a-D-glucose monohy- drate are very similar and questions some of Barker's conclusions.

I t is well known that a- and B-D-glucoses dissolved in water immediately mutarotate, ending in an equilibrium mixture of the two forms. Mutarotat ion has not yet been reported to give only the pure form of the other anomer. Indeed, there is little in the literature to indicate that any such complete transformation is accomplished by other than the tra&tional phase diagram route involving solution concentrations and temperatures. Yet this change seems to

"One of the laboratories of the Southern Utdlzatlon Research and Development Dlwsmn, Agricultural Research Service, U S Depart- ment of Agriculture.

be suggested not only by Barker's (4) findings with glu- cose but also by observations made by Norris and Green- street (7) with lactose. In the present paper further spec- tral data are presented, which confirm these suggestions. A rather subtle effect of moisture on the anomers of glu- cose is discussed as well as several anomalies that have been observed in connection with it.

Experimental Two lots of anhydrous a-D-glucose were used (East-

man t white label grade and Malhnckrodt t Analytical Reagent grade). The a-D-glucose monohydrate was Baker t Analyzed Reagent grade material, while the B-D-glucose was Calbiochem t Grade C. Melting points of these sub- stances and optical rotatory constants agreed very closely with the published values. Harshawt powdered infrared- quality potassium bromide without any special predrying was used for preparing the discs and was stored in closed screw-cap bottles in a desiccator over P205. The discs were prepared, and the infrared spectra were obtained with the same equipment described by O'Connor, DuPr~, and Mc- Call (9).

Portions of the sugars were stored in a closed desiccator over '~syrupy P205" (P20.5 covered with a layer of syrupy liquid). Other portions were exposed to atmospheric moisture. The KBr discs containing the sugars were stored after use in individual paper envelopes in a laboratory atmosphere of 35% relative humidity. Drying of the KBr discs was done by storing them over PeO5 in vacuo for one week at room temperature.

Results and Discussion The infrared spectra of the three forms of D-glucose

taken over the rock salt region 5000 - 650 cm -1 are given in Figure 1. The spectra are in good agreement with those obtained in KC1 for a- and B-D-glucose (6, 10) and a -D- glucose monohydrate (I0). Other published spectra do not permit accurate identification of the compounds because the labeling of either the spectra or the chemicals used to obtain the spectra give insufficient information. Thus, Sadtler Standard Spectra t No. 1040-B and 5455 are labeled D-glucose and glucose, respectively, but actually these are both the spectrum of the monohydrate (Figure 1B). Coblentz t Spectrum No. 95 labeled D-glucose, a n -

t i t ~s not the pohcy of the Department to recommend the products of one company over those o£ any others engaged m the same business.

] 08 APPLIED SPECTROSCOPY

WAVENUMBER (CM "1)

o -

zd- . gz I z o e -

_ 14oo -

$ -

~ . ~ -

(1 -

tY tv,_l ,,io "° ; I I i , I i r i I ~

WAVELENGTH (MICRONS)

FIG. 1. INFRARED SPECTRA OF VARIOUS ISOMERS OF GLUCOSE

A--Alpha-D-glucose anhydrous, B--alpha-D-glucose monohydrate, C--beta-D-glucose anhydrous

hydrous, agrees very closely with Figure 1A (a-D-glucose anhydrous). Some of these &screpancies have been dxs- cussed by Tlpson and Isbell (10).

The effect of moisture on B-D-glucose is shown m Figure 2. Spectrum A of Figure 2 was taken after a seven- day exposure of the anhydrous sugar alone to the air of the laboratory. Within this length of time room humidi ty did not affect the sugar. Then, after seven days more of drying of the KBr disc, spectrum B was obtained. As it is identical with spectrum A, no adverse effects were intro- duced by the KBr or by the storage con&tions under which the &sc was kept. Spectrum C, however, showing marked changes, was obtained after the anhydrous sugar had been stored 21 days in a desiccator over "syrupy PeOs" and then a disc prepared. The 1618-cm -1 hydrate band greatly increased in intensity, while resolution of the rest of the bands became very poor. The sugar at this point was still dry and crystalline m appearance. The spectrum has some resemblance to that of B-D-glucose. Spectrum D, which appears to be quite different, was obtained 43 days later and after drying the KBr disc containing the hydrated B-D-glucose (spectrum C) . Comparison of spectrum D with spectrum E, that of a-D-glucose mono- hydrate, indicates that the sugar is now mostly in the a form. It appears that the hydrated B-D-glucose was trans- formed to a-D-glucose monohydrate while being dried.

The spectrum taken after anhydrous B-D-glucose had been exposed to moisture (Figure 2 C) bears a most star- t l ing resemblance to spectra published by Higgins, et al. (6) and Tipson and Isbell (10) for equilibrium mixtures prepared by mutarotat ion of glucose in aqueous solution and freeze-drying. There is also a very marked similarity of Higgins ' spectrum for the freeze-dried B-D-glucose (6) to the present spectrum and to the spectra of the freeze- dried equilibrium mixtures. These similarities introduce complications m interpretation at this point. Where the sugars were dissolved in water and the solutions freeze- dried, presumably only amorphous material remained. The entire system is now different. That loss of crystal struc- ture markedly affects the infrared spectrum was recognized by Higgins and coworkers but was not mentioned by Tipson and Isbell. In the present study the anhydrous crystalline sugar never contacted liquid water, nor lost its crystalline appearance. I t was stored in an environment such that the hydrated sugar could be expected to be

WAVENUMBER (CM" )

ua

{

i 2 3 4 5 6 7 8 9 I0 II 12 13 14 15

WAVELENGTH (MICRONS)

FzG. 2. INFRARED SPECTRAL CHANGES IN BETA-D- GLUCOSE CAUSED BY MOISTURE

A--Beta-D-glucose, spectrum obtained after 7-day exposure of sample to laboratory air, B--dried KBr disc of A, spectrum obtained 7 days after that of A, C--portton of A after 21 days m closed desiccator over "syrupy P.,Os," D--dried KBr disc of C, spectrum obtained 43 days after that of C, E--alpha-D-glucose monohydrate.

formed. In the preceding transformation (Figure 2) , there is only litt le indication that B-D-glucose was stdl present after the drying step. Better evidence for both forms was found, however, when the times of exposure were changed. This is shown in Figure 3 where spectrum A is again that of B-D-glucose anhydrous but from a newly opened bottle. Spectrum C was taken after the sugar had been held 15 days in the desiccator over "syrupy PeOn." The spectrum of this sample, now, is less like those of the freeze-dried sugars and more like that of the anhydrous sugar, except for the water-of-hydrat ,on band. Spectrum D was obtained seven days later after drying the KBr d i sc - - the B anomer band is still present at 900 cm -1 although most of the sugar is now in the a form. Again spectrum B, which was obtained from the dried KBr disc containing the anhydrous sugar, ,s identical with spectrum A. In this case, however, the spectrum from the dried d,sc was recorded 17 days after spectrum A was recorded.

The significance of these findings is three-fold. First, here is evidence of complete anomer transformation taking place in the solid phase-- the P form has been transformed to the a form. This confirms the glucose band shifts re- ported by Barker (4) and substantiates the claims of Nor- ris and Greenstreet (7) regarding a similar shift with lactose.

Second, the transformation requires that the B-D-glu- cose be hydrated. This is apparently the first recognition of hydrated B-D- glucose.

Third, the claim of Barker and coworkers (4) that rigorously dried KBr (650 ° for four hr) is necessary to prevent such shifts do not seem to be completely just,- fled. Moisture causes the shift, and the presence of a cer- tain level of water content, whether in the sugar itself as in the present instance or in the KBr as Barker advo- cates, will do it. But the use here of KBr not specially dried did not cause spectral changes even after 17 days,

VOL. 18, No. 4, 1964

WAVENUMBER (CM "~1

WAVELENGTH (MICRONS)

FIG. 3. CHANGES OBSERVED IN THE iNFRARED SPECTRA OF BETA-D-GLucOSE AFTER DIFi~ERENT EXPOSURE CON-

DITIONS

A--Beta-D-glucose, freshly opened bottle, B--dried KBr disc of A, spectrum obtained 17 days after that of A, C--portion of A after 15 days in closed desiccator over "syrupy P205," D--dried KBr disc of C, spectrum obtained 7 days after that of C, E--alpha-D-glucose monohydrate

whereas Barker and coworkers (4) state that KBr not thoroughly dried caused immediate changes which neared completion in about seven days. A similar observation was made with anhydrous a-D-glucose. This is Illustrated by spectra A and B in Figure 4. The time lapse between spectrum A and spectrum B is five years and five months. The KBr used five years ago had not been specially dried nor had the disc been stored under anhydrous conditions. These findings point to some factor other than moisture as being responsible for the shifts reported by Barker and co- workers. Farmer (5) found no in&cation of glucose hy- drate formation or any other spectral change when pressed KBr discs were stored 14 days in laboratory air at 50% relative humidity. Shifts did occur, however, when traces of NaBr were present in the KBr. The fact that Norris and Greenstreet (7) reported spectral changes with lactose on storage in spite of following Barker's recommendations suggests that this impuri ty may have been present in their KBr. I t is regrettable that m many publications where the KBr technique is used, no mention is made of the puri ty or source of the KBr used.

Long exposure of anhydrous a-D-glucose to the hu- midi ty of the laboratory atmosphere caused a partial hydra- tlon of the sugar as can be seen in spectrum C of Figure 4. Band changes occurred in the 1430 - 1000 cm q region, but the more marked and probably more significant shifts are those which took place in the 870- 715 cm -1 region. Here the bands of the anhydrous ~ form shifted to the locations in which are found the bands characteristic of the monohydrate (see Figure 4 F) . The 1618 cm -1 band characteristic of hydrates is not evident in this spectrum. This band does appear in spectrum D which was obtained after the partially hydrated sugar had been held over "syrupy P20.~" for two months.

109

An anomaly observed now is that the partially hy- drated sugar still had the melting point and specific ro- tation of anhydrous a-D-glucose. The same anomaly was found with a second lot of a-D-glucose which gave spec- t rum E of Figure 4. In this spectrum the principal bands of anhydrous a-D-glucose are shown in the 750 - 930 cm -1 region, but the 853 cm -1 band of the monohydrate form appears also. The spectrum in this region confirms that given by Barker and coworkers (5) for a-D-glucose after three days in a "moist KBr disc." The spectra of the present two part ial ly hydrated a-D-glucoses indicate struc- tural changes in the crystal, conformational changes in the molecule of the original anhydrous compound, or the existence of a hemihydrate. Yet these changes apparently are not great enough to cause changes that can be de- tected in melting point and specific rotation.

No a t tempt was made to at tain complete hydration of the anhydrous a form. I t might be noted that the same atmosphere which so strongly hydrated the fl form (spec- tra C, Figures 2 and 3) failed to do the same to the a form. Furthermore, whereas the fl anomer changed over completely to the a, no evidence was found for a changing to ft. From these observations one could specu- late as to whether the a anomer is the more stable form and if so why, as well as to delve into why merely changing the configuration at the C1 atom has such an influence as to make considerable changes in the infrared spectra.

Acknowledgments The author is indebted to Mr. G. J. Boudreaux and

WAVENUMBER (CM ~,

WAVELENGTH (MICRONS)

FIG. 4. INFRARED SPECTRA OF ALPHA-GLucOSE SHOW- ING CHANGES CAUSED BY MOISTURE

A--~nhydrous alpha-glucose, spectrum obtained in 1958, B--spectrum of A obtained again from same KBr disc in 1962, 5.4 years later than A, C--portion of A after long exposure to laboratory atmosphere, D--pornon of C after 2 months in closed desiccator over "syrupy P..O~," E--sample of supposedly anhydrous alpha-glucose, F--alpha-D- glucose monohydrate.

110 APPLIED SPECTROSCOPY

Mrs. Sylvia Miles for the infrared spectra and to Dr. D. J. Stanonis for the melting point and optical rotation data.

Literature Cited (1) S. A. Barker, E. J. Bourne, M. Stacey, and D. H.

Whiffen, J. CHEM. Sac. 1954, 171 (2) S. A. Barker, E. J. Bourne, R. Stephens, and D. H.

Whiffen, IBID. 1954, 3468 (3) S. A. Barker, E. J. Bourne, W. B. Neely, and D. H.

Whiffen, CHEM. AND IND. ( L O N D O N ) 1954, 1418 (4) S. A. Barker, E. J. Bourne, H. Weigel, and D. H.

Whiffen, IBID. 1956, 318

T

(5) V.C. Farmer, IBID. 1959, 1306 (6) H. G. Higgins, C. M. Stewart, and K. J. Harring-

ton, J. POLYMER SCI. 51, 59 (1961) (7) K. P. Norris and J. E. S. Greenstreet, NATURE 181,

265 (1958) (8) L. P. Kuhn, ANAL. CHEM. 22, 276 (1950) (9) R. T. O'Connor, E. F. DuPr6, and E. R. McCall,

IBID. 29, 998 (1957) (10) R. S. Tipson and H. S. Isbell, J. RESEARCH NATL

BUR. STA~NTDARDS 66A, 31 (1962) (11) R. L. Whistler and L. R. House, ANAL. CHEM. 25,

1463 (1953) Submi t t ed Apr i l 19, 1963

Notes

X-Ray Diffraction Powder Data for Some $~-Pregnanes

Jean M. Ohrt and Dorita A. Norton Department of Biophysics, Roswell Park Memorial Institute

Buffalo, New York

X-ray diffraction data for seventeen 5c~-pregnanes were collected using a General Electric XRD-5 diffractometer with a nickel-filtered CA-7 copper target tube operated at 40 kv and 20 ma in conjunction with a scintillation counter and a No. 2 SPG preamplifier. The linear scale of a Leeds and Nor thrup Speedomax G recorder was used to record the X-ray powder spectra. Samples were run at a speed of 0 .2° /min using a take-off angle of 2.0 °, a 3.0 ° beam slit, a medium resolution Soller slit, and a 0.1 ° de- tector slit. The number of cps was recorded by a digital printer that was synchronized with the 20 scan. The digital tape readings and Speedomax charts were compared to determine maximum peak heights and locations in accord- ance with the accepted methods outlined by Klug and Alexander (2) and by Clark (1).

Samples were ground to the reouired fineness by hand in an agate mortar. An amount sufficient to cover an area 1.5 cm x 1.5 cm with a thickness of approximately 0.2 mm was placed on a ~lass slldo thinly coated with vaseline and then gently flattened with a spatula. Care was exer- cised to assure uniform sample preparation by flattening each sample u~in~ a vertical compression rather than a shearing type of horizontal motion.

T A B L E I . X - R A Y P O W D E R D A T A O F S U B S T I T U T E D 5 ~ -

P R E G N A N E

3~8, 20a-dtol dtacetate (m.p. = 161-163°C)

d, A I/I~ d, A I/I~ d, A 1/I1 d, A I/I~

13 96 100 4 58 8 2 9 3 4 1 2.252 2 9 7 1 4 4 4 8 8 2 854 2 2 239 2 8 17 3 4 30 12 2 822 1 2 219 2 6 98 3 4 16 9 2 794 3 2 168 5 5 99 39 3 88 4 2 746 1 2 126 2 5 73 25 3 79 5 2 637 1 2 092 5 5.54 26 3 65 2 2.563 1 2 080 6 5 34 6 3 56 5 2 502 1 2 042 2 5 16 7 3 42 9 2 404 13 2 018 1 5 01 7 3 25 3 2 386 2 1.999 1 4 81 20 3.12 3 2.365 2 1 969 I 4 7 3 38 3 03 2 2 300 3 1.932 2

1.855 1

T A B L E I . X - R A Y P O W D E R D A T A O F S U B S T I T U T E D Jc~-

P R E G N A N E - - (Cont.)

3fl, I I~ , 21-triol-20-one-t l-acetate ( m p . = 188-190°C)

d , A I/I~ d , A l/I~ d , A I / Iz d , A I / I ,

9 04 38 4.52 59 3 36 18 2.708 11 6 0 1 100 4 2 4 61 3 28 3 2.614 12 5 87 46 4 06 41 3.19 5 2.564 3 5 69 29 3 77 49 3.07 6 2.377 14 5 28 63 3.73 48 3.00 3 2 319 3 4 94 66 3.56 13 2 897 3 2 207 16 4 61 46 3.44 2 2.774 13

3 l~, 17 a-dtol-2 0-one (decamp. at 264"C)

d, A 1/11 d, A I/I~ d, A 1/11 d, A I / I ,

11 42 14 3 97 8 2.687 2 2 169 1 6 50 3 3 72 1 2.570 4 2.109 9 5 90 100 3.56 10 2 491 6 2.042 8 5 64 29 3.36 1 2 429 2 1.986 2 5.12 36 3.13 7 2 374 3 1.810 1 4 94 4 3 04 14 2 318 2 1 767 1 4 54 43 2 840 8 2.287 1 1.742 1 4 14 1 2 791 4 2 194 1 1.568 1

3~, l l B , 201~, 21-telrol (decamp. at 268-274°C)

d, A I/I~ d, A I/I~ d, A I/I~ d, A I/I~

10 97 24 4.90 21 3 01 2 2.427 10 10 15 14 4.52 3 2.896 3 2.328 13

8 27 3 4.11 31 2.850 3 2.269 1 6 98 46 3 92 9 2 794 4 2.212 3 6.32 54 3 69 14 2.707 4 2 161 3 5 83 100 3 54 4 2 598 3 2.119 1 5 68 63 3.34 7 2 534 8 2.087 4 5 37 69 3 19 9 2.475 1

3, 6, 20-trlone (m.p. = 234-237°C)

d , A I/I~ d , A I/I~ d , A I/I~ d , A I/I~

11.76 8 4.94 90 3 23 16 2 192 1 7 52 4 4.58 17 2.914 16 2,152 2 6 77 19 4.24 4 2 785 10 2,134 3 6.44 7 3.73 19 2.463 5 2,080 4 5 83 100 3 60 13 2 4 2 5 6 2,055 4 5 63 37 3 45 9 2.402 9 2,002 1 5 12 14 3 27 15 2.324 1 1.965 1