1438
P. R. SENTIAND L. P. WITNAUER [CONTRIBUTION PROM THE
Vol. 70
EASTERN REGIONAL RESEARCH LABORATORY']
Structure of Alkali Amylose BY F. R. SENTIAND L. P. WITNAUER X-Ray diffraction studies of the structure of amylose and amylose complexes have been limited for the most part to preparations which give powder patterns. Less ambiguous interpretations of structure can be given fiber patterns, but oriented preparations are required, which are diEcult to prepare by conventional methods owing to the low wet strength and hydrophilic character of amylose films. These dificulties can be circumvented by orienting a non-hydrophilic derivative of amylose and then converting this derivative to amylose under such conditions that orientation is retained. Amylose triacetate serves this purpose well, for it is easily oriented by stretching in glycerol a t 170'. Oriented alkali amylose is produced directly on deacetylation of clamped filaments in alcoholic alkali solution. Alkali amylose can be converted to the A, BN2and V structural modifications by methods previously described.8 All structural modifications give well-defined fiber patterns. The alkali amyloses give patterns especially rich in reflections and, because they constitute an isomorphous series, their patterns can be more readily interpreted than those of the other modifications.
a t 25' in 0.01 to 0.30 N potassium hydroxide (carbonate-free) in 75% ethanol. Completion of deacetylation has been checked by analysis of the filaments and is also evidenced by the disappearance of the amylose triacetate reflections from the diffraction pattern That ethanol is not an integral part of the structure is demonstrated by the identical patterns produced by filaments deacetylated in 0.25 N potassium hydroxide in 75% ethanol, 75% methanol, saturated butanol or in water. Deesterification proceeds slowly in the last two media and is accompanied by breakage of many filaments in the aqueous alkali solutions. Filaments from which the alkali has been removed by extraction with absolute methanol give amorphous diffraction diagrams. Extraction with 75% methanol or ethanol results in fiber patterns characteristic of the "V structure. Reconstitution, by soaking in 0.2 N potassium hydroxide in 7570 ethanol, restores the original diffraction pattern. Amylose triacetate filaments deacetylated in 75% ethanol, 0.01 to 0.30 N in lithium or cesium hydroxide, give the characteristic diffraction patterns of lithium hydroxide amylose and cesium hydroxide amylose. At the higher concentration Experimental of alkali (above 0.3 N potassium hydroxide, for Amylose was prepared by twice fractionating example) the characteristic fiber pattern diminautoclaved potato starch with nitroben~ene.~ishes in intensity and is gradually replaced by a Acetylation was carried out by the method of diffuse pattern of the V-type. Sodium hydroxide and guanidine in 7574 ethaWhistler, Jeanes and Hilbert,6 and films about 0.25 mm. thick were cast from chloroform solu- nol ljkewise deacetylate amylose triacetate and tion. Strips 2 to 4 mm. wide were oriented by produce the corresponding alkali amyloses with stretching in glycerola a t 170'. Films stretched characteristic X-ray diagrams. No systematic 500% are highly oriented, as evidenced by their study of the composition of these filaments or diffraction pattern, and are suitable for deacetyla- those of ammonium hydroxide amylose, prepared as described below, has been made. The similartion. In the deacetylation experiments, 3- to 10-cen- ity of their diffraction patterns to those of lithium, timeter filaments of oriented amylose triacetate potassium, and cesium hydroxide amylose, howwere held taut in stainless steel clamps and sus- ever, indicates that they have the same structure pended in the various deacetylating solutions. and composition as the latter. Preparation of an alkali amylose is not limited Unless clamped, the filaments retract considerably, especially a t higher alkali concentrations, to the deacetylation of amylose triacetate by the and give disoriented diffraction diagrams. The corresponding hydroxide, but is also accomplished characteristic diffraction pattern of potassium hy- by the exchange of one alkali for another. For droxide amylose is given by filaments deacetylated example, cesium hydroxide amylose results when potassium hydroxide amylose is immersed in al(1) One of tha Laboratories of the Bureau of Agricultural and Incoholic cesium hydroxide. Thallium hydroxide dustrial Chemistry, Agricultural Research Administration, United States Department of Agriculture. Article not copyrighted. amylose has been obtained in similar manner, (2) Fiber patterns of the B structural modification have been Barium hydroxide can be exchanged for potasdescribed by R. E. Rundle, L. Daasch and D. French, THIS JOURNAL, sium hydroxide, but the resulting fiber gives a 66, 130 (1944). (3) P. R . Senti and L. P. Witnauer, ;bid., 68, 2407 (1946). poorly defined diffraction pattern. Attempts to (4) R. L. Whistler and G. E. Hilbert, ibld., 67, 1161 (1945). prepare ammonium hydroxide amylose by deacet(5) R. L. Whistler, H. Jeanes and G. E. Hilbert, {bid., in press ylation have failed, whereas exchange with alco(1948). holic ammonia solutions has been successful. (6) Method of N. C. Schieltz, private communication.
.
April, 1948
STRUCTURE OF ALKALI AMYLOSE
Many salts in aqueous alcohol solution can be exchanged for the alkali in alkali amylose. By this method we have obtained compounds of amylose with the iodide, bromide, acetate, formate and propionate of potassium. All give excellent fiber diffraction patterns, which will be described in another publication. Alkali and Water Content.-On removal from the deacetylating solution, the clamped filaments were wiped dry 6 t h absorbent tissue to remove adhering alkali solution. Adsorbed alcohol, which is di5cult to remove by vacuum drying, was removed by humidification of the filaments over water in a vacuum desiccator for sixteen to twenty-four hours. The vacuum-dried filaments were weighed, excess acid added, and titrated with sodium hydroxide. Figure l presents data on the alkali content of the lilaments as a function of the normality of the deacetylation solution. It is to be noted that the alkali content of the filaments increases rapidly with concentration of base in the deacetylation solution up to a normality of 0.02. Above 0.02 N , the alkali content of the fibers increases a t a much lower rate, and the composition curve is nearly horizontal over a range of normalities, particularly in the case of cesium and potassium hydroxide. At higher normalities, the slope of the composition curve again increases. These observations suggest stoichiometric compound formation between amylose and alkali a t a composition correspondingto the horizontal portion of the curve. For cesium hydroxide amylose, this composition corresponds to an alkali content of 26%, whereas for potassium and lithium hydroxide amyloses, the alkali contents are 11 and 5%, respectively. The observed values are in satisfactory agreement with those calculated from the formula lMOH.3C6H1006, which requires 23.7% cesium hydroxide, 10.3% potassium hydroxide and 4.7% lithium hydroxide. At both extremes of the composition curve, however, the alkali content of the filaments deviates considerably from that corresponding to the stoichiometric ratio lMOH :3C6H10O6, and the problem of the distribution of the alkali arises. One possibility is that the alkali is distributed a t random among a number of crystallographically related sites, which are progressively filled as the alkali content increases. On this hypothesis we should expect the relative intensities of the diffraction maxima to vary with the alkali content. Since the lithium, potassium and cesium amyloses appear to be isomorphous, the magnitude of the shift in relative intensities is indicated by a comparison of the patterns of cesium and potassium amylose having the same mole per cent. alkali. On the cesium amylose (1CsOH :3C6HloOs)pattern, I(101)> I(zoo,. If the amount of potassium is increased and the cesium is decreased until the two isomorphous structures con-
. 30
\
0
1439
QOH
0
0
0.100 0.200 0.300 0.400 Normality of deacetylating solution. Fig. 1.-Alkali content of lithium, potassium and cesium hydroxide amylose as a function of the normality of alkali in the deacetylating solution.
tain equivalent alkali on the basis of scattering power, the ratio 1 ( 1 0 1 ) / 1 ( 2 ~ 0 )should be the same for the two patterns. Since the scattering factor of cesium is three times that for potassium, patterns of cesium amylose of composition approximately O.5CsOH :3C6HI0O6were compared with those of potassium amylose of composition approximately 1.5KOH:3C&@6. For each pattern, the ratio I(101)/1(200) was unchanged within the error of visual estimation, and it is certain that the inequalities indicated above were not reversed. Random substitution in a set of sites related by symmetry is thus not consistent with our observations. It appears more likely that the crystalline por'tions of the fibers are essentially constant in composition with respect to the symmetry-related alkali. Alkali present in other sites would not affect the relative intensities of the discrete diffraction maxima but would contribute to the amorphous background. Any large excess would be expected to distort the structure, causing a change in lattice constants and resulting ultimately in a new or amorphous structure. The possibility that the amorphous regions of the filaments increase in extent and contain an excess or deficiency of alkali a t the extremes of composition is not excluded. Experimentally, it is observed that the background scattering increases and the discrete maxima diminish in intensity when the filaments have either a low or high alkali content. The water content of lithium, potassium and cesium hydroxide amyloses was determined a t several humidities after preliminary humidification a t 85% R.H. to remove adsorbed ethanol. Humidities were maintained by saturated salt solutions in vacuum desiccators. Water contents on desorption are given in Table I. Diffraction patterns were taken of filaments enclosed in glass capillaries after equilibration a t the humidities listed in Table I. Below R.H., corresponding to a water content of about lo%, the patterns diminished in intensity, and there was a slight decrease in lattice dimensions. Precise values of the lattice dimensions of the dry fibers were not determined because accurate measurement of the
iMo
P. k. SBNWAND L. P. WITNAUER
v01. i 0
sumption that the lattice is orthorhombic, sin 0 values were computed and compared with the observed values. The results for potassium hydroxide amylose, based on the orthorhombic unit having a. = 12.7 kX, bo = 22.6 kX,and to = 9.0 kX, are presented in Table 11. A comparison of the TABLE I dimensions of the orthorhombic unit cells found MOISTURE CONTENT OF ALKALIAMYLOSE AT 25' IN PER for the alkali amyloses is given in Table 111. All CENT. OF DRYWEIGHT ON DESORPTION reflections were indexed on the patterns of lithium Relative ,Water, %, inand cesium hydroxide, whereas the unit cell dihumidity, % ' LiOH-Amylose KOH-Amylose CsOH-Amylose mensions of sodium, ammonium and guanidinium 12.9 11.4 50 12.6 hydroxide amylose were derived from measure9.9 9.0 30 9.7 ment of equatorial reflections and layer line sepa6.3 6.0 12.5 6.3 rations alone. The lateral dimensions increase It should be noted that lithium hydroxide am- regularly with increasing size of the cation exylose also occurs in a more highly solvated struc- cept for cesium hydroxide amylose, which has a ture. Filaments deacetylated in 0.2 N lithium smaller a0 than expected. Since the fiber repeat hydroxide in 75y0 ethanol and X-rayed while period remains the same, the decrease in a. is probmoist with deacetylating solution give an X-ray ably due to a small rotation of the glucose residues pattern showing the same fiber repeat period about the b-axis, and i t is unlikely that this will (22.6 kX), but a t least one lateral identity period affect the validity of the isomorphous relations larger than that of the normal hydrate to which which we apply. Potassium hydroxide amylose containing 10% the structure reverts upon drying in air. Under potassium hydroxide and 13% water has a density the conditions of deacetylation we have employed, neither cesium nor potassium hydroxide amylose (determined by flotation in carbon tetrachloridehas given any indication of a more highly solv- petroleum ether mixtures) of 1.53. If it is assumed that the water and alkali are uniformly disated structure. X-Ray Diffraction Patterns.-Patterns for in- tributed throughout the filaments, then the numdexing were taken in a cylindrical cassette of ber of glucose residues (mol. wt. 162) in the unit 5-cm. radius with filtered CuK, radiation. Fila- cell is 12.7 X 9.0 X 22.6 X 1.53 X 0.77 = 11.4 ments mounted with the fiber axis along the cylinN = 162 X 1.65 der axis show only the second-order reflection of the fiber repeat period. To observe the higher or- A similar computation for cesium hydroxide amders, filaments were oscillated with the fiber axis ylose (28.1% cesium hydroxide, 9.0% water, d = perpendicular to the cylinder axis. Superposition 1.86) gives a value of 11.3 for N. Consequently, of reflections from planes not perpendicular to the there are twelve glucose residues in the orthorhomfiber axis on the desired orders of the fiber repeat bic unit cell. period was prevented by restriction of the oscillaSpace Group.-If the alkali amylose structures tion range. By maintaining a common reflection are based on an orthorhombic lattice, as appears on succeeding photographs, it was possible to likely from the agreement of observed and compare the relative intensities of the various or- calculated sin 6 values, the space group must be ders. Relative intensities of the meridian (orders isomorphous with the point group D2. All other of the fiber identity period) reflections of lithium, point groups of the orthorhombic system contain potassium and cesium amyloses were placed on a planes of symmetry which are not permitted by common basis by the comparison of timed photo- the optically active amylose molecules. graphs of filaments of known thickness and alkali Of the reflections (OkO),only those with k even content. Intensities were estimated visually by were observed out to k = 16 in lithium, potassium comparison with a scale of known relative intensi- and cesium hydroxide amyloses. It is therefore ties. probable that the structures possess a twofold Unit Cell.-The fiber identity period of the screw axis parallel to the fiber axis. Observed realkali amyloses is readily determined from the flections of the form (h00) and (001) are consistent layer line separation on cylindrical cassette pat- in all cases with twofold screw axis in the directions terns or from the d-values of the various orders a and 6, although not sufficient orders were obof the fiber identity period obtained from the os- served to establish this beyond a possibility of cillation photographs. Determination of the doubt. The observed reflections thus allow space lateral identity periods is ambiguous. The as- groups P2121211 P22,2,, P ~ ~ 2 and ~ 2 ,P 2 ~ ~ 2 . sumption was made that a reflection corresponding Intensity considerations, however, permit only to a primitive lateral translation would appear on space group P21212,. This follows from the Pattera t least one of the eight layer lines observed. son projections P,(uw) of the lithium (Fig. 2) and Thus, the first and second nearest reflections to the potassium hydroxide (Fig. 3) amyloses. Since the meridian were indexed as OkZ and ZkO, respectively, amylose chains lie along b (fiber axis), the projecwhere k is the layer line index. On the further as- tion Pv(uw) should give lateral vector distances u, weak patterns was impossible. A water content of 10% corresponds to one molecule of water per glucose residue, and this is concluded to be the normal content in the crystalline regions of the filaments.
STRUCTURE OF ALKALI AMYLOSE
April, 1948
1441
TABLE I1 hkl
101 200 201 002 202 302 003 203
COMP.ARISON OF CALCULATED AND OBSERVEDSINti VALUES FOR POTASSIUM HYDROXIDE AMYLOSE Sin 13 Sin e Sin 0 Sin 8 (obs.)
(calcd .)
0.1049 .1207 .1488 .1708 .2107 .2495
0.1049 .1210 ,1483 .1714 .2098 .2496
.2563 ,2843 .3150
{ :;E}
[
.a1
.4005 .4653 ,4954
.3635 .4014 .4653 .4953
111 210 21 1 012
.lo97 ,1254 .1533 .1754
,1102 .I256 .1521 .1747
112 311 212 013 113 213
.I845 ,2034 .2118 .2585 ,2665 ,2862 ,3025
313 511 512
.3160 .3506 .3652
513 612 414 711 120 121 220 22 1 320 321 222 420
023 520 423 523
,4000
{ :E} .2035 .2125
{ :E} .2663 .2861 .2984
1 ;yz! 1 -izJ .3493
.3984
4213
,4209
I4337
{ ,:3”:i: )
,0905 ,1253 1391 1634 ,1945
2113 .2200 ,2512 .2672 ,3084 ,3574 .4023
{
WM \.li +
W
W+
600 204 602 305 703
,3637
I (est.)
S+ ’wW S-
W-
WWWS+
s+ W-
M+
W+ W W W W W-
mrW-
\v”-
:E; j
s
{ :E ] .3099 { :E} ,4027
.2071
132 33 1 133 233 432 531 532 234
.2246 .2831 .3009 .3148 .3303 .3613 .3780
140 041 141 240 241 042
.1483 ,1608 .1719 .1825 .2016 .2179 .2273
340 341
.2427
M M-
W Sf W+
,2205 .2513
(obs.)
.2905 244 640 642
353 551 160 161 26 1
WWWW
360 262 460 163 462
I
0.1183 .1461 .1580 .1797
{
{
1
I (est.)
WW-
11.1
w
:E)
M
.2249 .2&30 .3018 .3134 .3304 .3622 * 3774
M W+ WW diff. W diff. W diff. W diff.
,1484 .1603 .1714 ,1817 .2009 ,2185
S-
:z} ,2421 .2906
M LVIV =
M-
iv
+
M+ W
w
+
::E/
W-
.4230
.4236
W-
,1797 .1901 .1993 .2091 .2254
.1799 .1898 .1992 .2082 .2251
W- diff. M W-
.2486
{ :;E}
.3388
.150 051 151 250 251 152 351 153 253
(calcd.)
0.1188 .1457 .1571 .1806
ivf
.0908 .1249 ,1387 ,1630
.2118
hkl
130 131 230 231
.2629 .3136 .3314 .3573 .2129 .2287 .2535 .2718 .2918 .3162 .3335 .3593
.3874
.2626 ,3138 .3308
{ :;E } .2121 .2287 .2516
{ ,2921 :;E ) ,3160 .3333 .3595
w-
MWW W
w
+
WW‘ M M
M diff. M diff. W+
w
+
W
W W
M diff.
W+ diff.
w between the c:hains. Resolution of individual carbon and oxygen atoms cannot be expected,
since there will be much overlapping in the projection, and furthermore, early termination of the
F. R. SENTIAND L. P. WITNAUER
1442
TABLE I11 UNITCELLDIMENSIONS OF ALKALIAMYLOSES bo (fiber axis), Amylose
LiOH NaOII KOH ",OH CsOH C (NHdaOH
ao,
kX
12.1 12.3 12.7 12.7 12.4 13.1
kX
co,
22.6 22.6 22.6 22.6 22.6 22.6
Vol. 70
can be eliminated by comparison of their projected intermolecular vectors with the observed Patterson projection.
kX
8.8 8.9 9.0 9.0 8.9
-a
0
I-
-I
1/4
9.0
series required by the observed reflections results in low inherent resolution. We inter ret the maxima in Figs. 2 and 3 at (0, 0) and '/2) to indicate two amylose chains in the unit cell separated by the vector distance (I/2, '/2). Such an
(It,
0
1 +U
+
w
Fig. 2.--Patterson function Pl(uw) for lithium hydroxide amylose. 0
1 -U
Fig. 4.-Projection of the alkali amylose structure on (OlO), showing position of amylose chains and alkali ions.
Isomorphism of the Alkali Amyloses.-The near identity of the unit cell dimensions and the fact that all diffraction patterns are consistent with space group P21212,, indicate that the alkali amyloses are isomorphous. Further evidence is provided by a comparison of the IF\-values (Table IV) of the (OkO) reflections of lithium, potassium and cesium hydroxide amyloses. Filaments having the same content of alkali on a mole basis (1 mole alkali/3 glucose units) were used for this purpose. Appropriate corrections were made for the time of exposure, sample thickness, absorption, Lorentz and polarization factors. The assumption was made that all filaments are equally crystalline. This appears reasonable, since all filaments were prepared from amylose triacetate stretched and deacetylated under identical conditions except for the variation of the alkali. If the structures are isomorphous then
I F l o k o (KOH-Amylose) IFloko
(CsOH-Amylose)
- I F l m (LiOH-Amylose - IFloko (KOH-Amylose) fK+ fC.+
1
4 W
Fig. 3.-Patterson
function P,(uw) for potassium hydroxide amylose.
arrangement is consistent with space group P212121 as indicated in Fig. 4,where the chains lie on the screw axis along b. Since the amylose molecule does not possess twofold symmetry, it cannot lie on a twofold axis, and a structure based on P 2 2 , 2 would have four chains in the orthorhombic cell. Disposition of the four chains in the unit cell according to symmetry requirements and packing considerations demands a Patterson projection having maxima a t approximately (l/2, 0) and (0, '/e) in addition to the observed maximum a t l / ~ ) . Likewise, structures based on Pz,~,z or Pnlzl
- fLI+
- fK+
where thef's represent the ionic scattering factors. At low angles the value of the ratio i s '/z and decreases with increasing angles. Thus IF1 (OkoKOHAm lose) - IFloko(LiOH-Amylose) = '/a to l/,[~Ioro(CsOH-Amylose) - (Floko(KOH-Amylose)] for the orders considered. These differTABLEIV F-VALUESFOR THE (OkO) REFLECTIONS OF LITHIUM, CESIUM AND POTASSIUM HYDROXIDE AMYLOSES (1)
OkO 020 040
060 080 0.10.0
0.12.0 0.14.0 0.16.0
(2)
FLioa-
Amylose 1 (-)
7
0 (+I 17 0 (-)25
