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Applications of infrared spectroscopy—V1
Talanta. 1961. Vol. 8, pp. 497 fo 504.
APPLICATIONS
Pergamon Press Ltd.
Printed in Northern Ireland
OF INFRARED
SPECTROSCOPY-V*
THE RETENTION OF WATER AND ORGANIC CARBOHYDRATE MATERIALS
SOLVENTS
BY
D. M. W. ANDERSON@ and N. J. KING Department
of Chemistry, The University, Edinburgh 9, Scotland
(Received 6 February 1961. Accepted 2 March 1961) Summary-Studies have been made of the extent to which some carbohydrate materials retain water and organic solvents. The water evolved was determined gravirnetrically, the solvents by an infrared method. “Drying to constant weight” in an oven at 103”, or in vacua at 80”, does not give true “moisture” contents. Both water and organic solvents are retained up to temperatures at which the carbohydrate materials begin to decompose: the polarity and boiling point of the solvent do not determine the extent to which it is retained. Freeze-dried samples can retainorganicsolvents,and have significant moisture contents. The possibility of inaccurate results and misleading artefacts arising from such retentions or from interaction with solvents is stressed.
ORGANICsolvents are generally employed at some stage in the extraction or purification of carbohydrate materials. Thus, water-soluble polysaccharides are isolated, and plant gums purified, by precipitation from aqueous solution; Soxhlet extraction or refluxing with solvents removes plant pigments and soluble sugars, and inactivates enzymes; starches are “defatted” with 80% methanol or methanol-benzene, and are subsequently fractionated by the use of butan-l-01 or other polar organic solvents. The purified material is often “dried” by successive immersion in acetone, ethanol and ether, with final oven drying at temperatures slightly above the boiling points of the solvents used. Since such treatment may irreversibly decrease the solubility of some materials, e.g. starches, these are often stored under methanol or toluene until required. The retention of moisture and organic solvents by pectins,l cellulose,2 starches,3*4 gum arabic5 and biological polymer@ has been reported : a mathematical treatment of moisture desorption isotherms has been given. ’ For cereals, “free water” (i.e. water lost at a stated drying temperature) has been distinguished8 from “bound water”, which is difficult to remove and is considered to be associated with the protein content. The effect of bound water has been considered in ultrasonic studies of hydration effects in sugars.g In this paper we report quantitative observations on some polysaccharide materials in the belief that the extent to which retention of moisture and solvents can occur is not widely appreciated. The experiments were based on the use of a micro-scale, vapourphase, quantitative infrared technique, developed recentlylO for the examination of fractions separated by GLC and usedli for a study of the Zeisel alkoxyl reaction. EXPERIMENTAL Drying methods Method I: Oven drying; temperatures and periods as stated. Method ZZ: Drying over phosphorus pentoxide in a conventional organic solvent and evacuated by a water-pump. * Part IV: D. M. W. Anderson and J. L. Duncan, 497
Talanta,
pistol-dryer,
1961, 8, 241.
heated by refluxing
D. M. W. ANDERWNand N. J. KING
498
Me&d 111: Vacuum drying (usually at 80”) in a small electrically heated, thermostatted, glass drying chamber attached to a suitable high vacuum line (< 0.02 mm mercury pressure). Method IV: Heating in a stream of dry nitrogen. The sample was weighed in a small 3-necked flask, which was then placed in a temperat~e~ontro~~ oil-bath. The central neck of the flask carried a mercury thermometer pocket which was in contact with the sample. One of the two outer necks served as an inlet for CO,-free nitrogen, dried by passage through Anhydrone; the third neck served as the outlet, to which was connected a tared Anhydrone absorption tube. This served in turn as the inlet to a cold trap immersed in Iiquid nitrogen. The outlet of the trap was fitted with an Anhydrone guard-tube (~6 ref. 11). The nitrogen flow-rate was 15 ml per min.
-u
60-
3
Solvent, FIG. l.-Infrared
t-6-q
calibration curves for determination 0 Ethanol x Ether q Acetone A Methanol
of solvents
(1070czx1) (1140 cm’) (1740 cm-l) (1040 cm-‘)
Determination of water released The Anhydrone absorption tube was weighed at intervals. not retain any of the solvents involved in these experiments,
Anhydrone
(B.D.H.. M.A.R.) does
Determination of organic solvents released These were quantitatively retained in the cold-trap, and were subsequently determined by a quantitative vapour-phase infrared technique. This has been described,‘O together with details of the design of trap, method of quantitative transfer from trap to gas-cell, and methods of constructing calibration curves for each solvent involved (cjI ref. 11). The absorption peaks given by acetone at 1740 cm-l, ethanol at 1070 cm-l, methanol at 1040 cm-l, and ether at 1140 cm-l were used for calibration; the calibration curves obtained are shown in Fig. 1. (Jt is, of course, fortuitous that the sensitivity of detection of acetone, methanol and ether were all identical, as shown by their sharing a common calibration curve). No overlap or interaction occurred for the absorption peaks chosen, so that all four solvents could be determined simultaneously. The results from some typical experiments are reported below.
Water and organic solvents in carbohydrate materials
499
RESULTS
A sample of the gum from Combretum leonend was precipitated with acidified acetone (O*lNwith respect to HCI) and dried with 5 changes of acetone(eachincontact for 2 days, the powder being progressively ground as it dried). The powder was stored in a desiccator (continuously evacuated) for 2 weeks at room temperature, and was then dried for 1 hr at 60” by method II. A weighed sample (554 mg) was then treated by method IV at various temperatures for lengths of time as shown in Table I. The total 1.
Time, FIG.
hr
2.-Release of water and acetone from a sample of C. Ieonensegum. Curve OA-water Curve O&acetone
amounts of water and acetone evolved were found gravimetrically and spectrometrically respectively. Several measurements were made within each of the periods quoted in Table I; the results are shown graphically in Fig. 2. After heating for 525 hr, weight of gum recovered = 469 mg, i.e., loss in weight = 85 mg. The weights of water TABLEI
IPeriod of heating, hr
l-20 20-264 264-288 288-406 406-454 454-525
Temp., “C
55 75 95 104 123 137
T
Total weight released from 554-mg sample. water,
acetone,
mg
mg
7.9 29.2 33.0 38.5 46.3 60.0
2.2 9.8 10.0 12.3 13.1 14.3
+ acetone found total only 74.3 mg. Very slight decomposition of the gum began at 123” and was significant at 1370; in the period 430-525 hr (Fig. 2) 6 mg of CO, were evolved. This evolution of CO, would be accompanied by water produced in the decomposition, and this explains the sharp rise in the amount of total water released between 430 hr and 525 hr in Fig. 2.
500
D. M. W. ANDERSON and N. J. KING
2. Crude C. leonense gum was precipitated with acidified ethanol, dehydrated with 5 changes of acetone (as in 1 above) and finally washed with ether. After preliminary drying by method II for 6 hr at 60°, a weighed sample (474 mg) was dried by method IV at 70” for the period l-5 hr, at 90” for 5-68 hr, at 105” for 68-116 hr, at 130” for 116 164 hr, at 140” for 164-188 hr, at 150” for 188-236 hr, and at 155” for 236-260 hr. The total weights of water, ether, ethanol and acetone liberated are shown graphically in Fig. 3. Decomposition of the gum was slight at 130” and pronounced at 140”: the graphs show that evolution of organic solvents continued steadily after the onset of
0
40
60
I20 Time,
FIG. 3.-Release
of water, ether, ethanol, Curve Curve Curve Curve
I60
200
240
hr
and acetone from a sample of C. Ieonense gum. OA-water OB--ether OC--ethanol OD-acetone
decomposition. It is noteworthy that, of the three solvents involved, ether was the most strongly retained: this is surprising, since (a) its boiling point is the lowest, (6) it is the least polar, (c) it was used in smallest amount for the shortest contact time and only for superficial washing. Apparently the ethanol (used in the precipitation process) and the acetone (used in the dehydration procedure) are accessible to and extracted by the ether used in the final washing stage. This effect was verified in the next experiment. 3. A further sample of crude C. leonensegum was precipitated withacidifiedethanol, dehydrated with absolute ethanol (5 changes, each in contact with the gum for 2 days), progressively ground to a powder, and finally washed with ether. After air drying, a weighed sample (845 mg) was dried by method II at 60” for 24 hr, when the total loss in weight (i.e., water + solvents) was 3.5 %, as represented by the curve OA given in Fig. 4. The sample was then quickly transferred and dried by method IV at 78” for a further 138 hr, i.e., for the composite drying period of 24-162 hr. This gave part AB of the dehydration curve. Further drying at 108” for the period 162-234 hr gave curve BC; drying at 120” for period 234-258 hr gave curve CD. The dotted
Water and organic solvents in carbohydrate
materials
501
AE, AF and AG give the weights evolved of water, ether and ethanol respectively; curve ABCD gives the sum of AE + AF + AG. The curves clearly show that solvents are still being liberated after drying for 280 hr, and confirm that the ether used as a final wash is more strongly retained than the ethanol used for both precipitation and dehydration.
curves
I 40
I 80
I I20 Time,
FIG. 4.-Release
I I60
I 200
I 240
I 2fN
hr
of water, ether, and ethanol from a sample of C. leonense gum. Curve OABCD-total % loss in weight Curve AE-water contributing to total AD Curve AF-ether contributing to total AD Curve AG-ethanol contributing to total AD
4. To test the effect of particle size on the retention, a sample of crude C. leonense was precipitated and dehydrated with methanol (6 changes, each in contact for 2 days). After air-drying, the sample was ground finely and sieved. Two fractions (a) passing 200 mesh and (b) passing 100 mesh were retained, and dried by method II at 60” for 9 hr. Weighed samples were then dried by method IV at 65” for the period O-365 hr, at 95” for 365-437 hr, at 98” for 437-509 hr, and at 101” for 509-629 hr. For the 200-mesh sample, the weights of water and methanol evolved are given in Fig. 5 by curves A and B respectively, and for the lOO-mesh sample by curves C and D respectively. These curves again emphasise that the drying temperature is more effective than the period of drying; methanol was still being released at the end of the experiment. 5. Parallel experiments to those described for C. leonense gum were made on samples of gum ghatti; l3 the retention of water and solvents by the two gums was very similar. 6. Further experiments were made with gum ghatti to test whether (a) solvents less polar than ethanol, methanol, acetone and ether e.g., carbon disulphide and dioxan, or (b) solvents containing bulkier functional groups e.g., isopropanol would be less strongly retained. For all of these solvents, however, retentions similar to those for acetone, ethanol, methanol and ether were given. 7. The retention of methanol by some starches was investigated. (a) A sample of potato starch (500 mg) was refluxed with methanol for 4 hr. After heating by method gum
4
502
D. M. W. ANDERBDNand N.
J.
KING
III at 80’ for 24 hr, it was then dried by method IV at 98’ for O-18 hr, at 102” for 18-40 hr, at 108” for 40-64 hr, at 112” for 64-84 hr and at 134” for 84-88 hr. As much methanol was evolved in the fhral4-hr period of heating at 134”, after steady evolution for 84 hr, as was evolved in the 18,hr initial period at 98”. {b) Rye starch, which had been stored under methanol at 0” for 2 years was treated by method II at 61’ for 24 hr.
Time,
FIG. 5.-Effect
hr
of particle size on release of water and methanol from a sample of C. leonen.segum. Curve A-water Curve C-water Curve D-methanol
I from ‘oo-mesh
A sample (500 mg) was then dried by method IV, the evolution of methanol being reported in Table II (+ and + + indicate O*1 - O-5% and 0.5 - 1% w/w respectively). Similar results were obtained with other starch samples. 8. A sample of C. I~UWPWgum which had been purified by ethanol precipitation, dialysed, and freeze-dried for 4 days was found to lose 1% by weight after heating at TABLE IX
Temperature,
66-90
90-106
106-154
154-l-78
178-202
Methanol evolved
103” for 5 hr, and a further I % on raising the temperature until d~rn~sitio~ began. Similar experiments with a freeze-dried sample of a peetie acid isolated from N&&z transiucenP gave a loss in weight of 6.4 % (duplicate runs) when the sample was taken direct from the freeze-drier; samples stored in a desiccator overnight after freezedrying gave 8.5 % loss in weight when treated by method III.
Water and organic solvents in carbohydrate
materials
503
DISCUSSION
The results show that both water and organic solvents are retained tenaciously, even on prolonged drying under vacuum at temperatures much higher than the boiling point of the solvents concerned. Indeed, retention continues up to the temperature at which the polysaccharide material begins to decompose, as revealed by yellowing in colour and the starting of the evolution of CO,. Furthermore, although apparent “constant weight” is reached after drying at a certain temperature for a determinable number of hours, continued drying at a slightly increased temperature gives further liberation of water and solvents, until eventually “constant weight” is again reached. This cycle can be repeated many times, by raising the temperature in small increments, until eventually the decomposition temperature of the carbohydrate material is reached. Freeze-dried samples also retain moisture tenaciously, although in smaller amount; this agrees with Robson, l6 who found that a solution containing 16 % glucose + 3 % gelatin required freeze-drying for 500 hr at room temperature for all moisture to be removed. In practice, freeze-drying is not normally continued for longer than about 100 hr, and it is often assumed that samples so treated have a negligible moisture content: many quantitative inaccuracies must arise in this way. Freeze-dried samples may also retain organic solvents ; cellulose retains 1.5 % (w/w) of benzene on freezedrying.16 Water and vapours can be occludedl’ in crystalline sugars; indeed, it was found recently18 that when phenylboronate ester derivatives of methyl glycopyranosides were recrystallised from benzene, significant amounts of solvent were retained unless special care was taken to ensure its complete removal. Other authorslg have recently reported similar solvent occlusion effects. To obtain accurate quantitative results with carbohydrate materials, it is clear that great attention must be paid to drying procedures. Furthermore, any previous treatment of a sample with organic solvents must be considered if inaccurate and misleading functional group analyses e.g., methoxyl, acetyl are to be avoided (c$ ref. 20). Application of the infrared alkoxyl methodl’ has shown that mere reflux with ethanol can create artefacts e.g., ethoxylation of the fructose from lucernezl and of certain plant gum components:22 we are grateful to Dr. R. J. Ferrier for the informatiorP that a dry, powdery xylan of normal appearance prepared by him contained 70 % of ethanol. These findings serve to support the timely warning given by Belles regarding the possibility of formation of non-reducing ethyl glycosides when plant materials are treated with hot 80-95 % ethanol. It now appears likely that the occurrence of an ethyl riboside,* so far unconfirmed,25 also arose in this way. Acknowledgements-We thank Professor E. L. Hirst, C.B.E., F.R.S., for his interest in these studies, and the Department of Scientific and Industrial Research for a maintenance grant (to N. J. K.). We are grateful to Dr. R. S. Fanshawe, Dr. R. J. Ferrier and Dr. E. E. Percival for supplying materials for examination, and for providing experimental details. Zusammenfassung-Es wurde untersucht, bis zu welchem Ausmasse einige Kohlenwasserstoffe Wasser und organische Liisemittel festhalten. Das Wasser wurde gravimetrisch und die Solventien mittels einer Infrarotmethode bestimmt. “Trocknen zu konstantem Gewicht” bei 103°C im Ofen oder bei 80°C im Vakuum gibt nicht den “wahren Feuchtigkeitsgehalt”. Wasser sowohl als such organische Losemittel werden bis hinauf zu Temperaturen festgehalten, wo bereits Zersetzung der Kohlenwasserstoffe stattfindet. Polaritiit und Siedepunkt sind fur das Ausmass der Retention nicht bestimmend. Frostgetrocknete Proben kiinnen organische Lijsemittel zurtickhalten und zeigen
504
D. M. W. ANDERSONand N. J. KING
signifikante Gehalte und Feuchtigkeit. Die Moglichkeit ungenauer Ergebnisse und irrefuhrenden Verhaltens, die durch diese Art von Retention hervorgerufen werden konnen, werden betont. RBsmn&-Les auteurs ont Ctudie l’importance avec laquelle les hydrates de carbone retiennent l’eau et les solvants organiques. L’eau fide etait dosee par gravimetrie, les solvants par une methode infra-rouge. Le “sechage a poids constant” dans un four a 103” ou sous vide a 80” ne donne pas des “teneurs en humidit.? reelles. L’eau et les solvants organiques sont retenus tous les deux jusqu’a des temperatures auxquelles les hydrates de carbone commencent a se decomposer; la polarite et le point d’ebullition du solvant ne determinent pas l’importance de sa liaison. Des echantillons La seches a froid peuvent retenir des solvants organiques et ont des teneurs en eau importantes. possibilite de resultats inexacts et de produits aberrants provenant de fixation ou dint&action avec les solvants est soulignee. REFERENCES 1 E. F. Jansen, S. W. Waisbrot and E. Rietz, Analyt. Chem., 1944, 16, 523. 2 H. Staudinger, Z. angew. Gem., 1952, 64, 149. 3 M. Ulmann and F. Schierbaum, KolloidZ., 1958, 156, 156. 1 Idem, Die Stiirke, 1959, 11, 203. j L. K. H. van Beek, J. Polymer Sci., 1958, 33,463. 6 G. Champetier and J. Neel, Bull. Sot. Chim. BZoZ.,1958, 40, 1773. ’ H. A. Becker, Canad. J. Chem., 1958,36, 1416. 8 D. W. Kent-Jones and A. J. Amos, Modern Cereal Chemistry., The Northern Publishing Co. Ltd., 5th Edn. 1957. g H. Shiio, J. Amer. Chem. Sot., 1958, 80, 70. lo D. M. W. Anderson, Analyst, 1959, 84, 50. I1 D. M. W. Anderson and J. L. Duncan, Talanta, 1960, 7, 70; 1961, 8, 1. I2 D. M. W. Anderson, E. L. Hirst and N. J. King, Talanta, 1959, 3, 118. I3 G. 0. Aspinall, B. J. Auret and E. L. Hirst, J. Chem. Sot., 1958, 4408. I4 D. M. W. Anderson and N. J. King, Biochem. Biophys. Acta, 1961, in the press. I5 E. M. Robson, Vacuum, 1956,4, 60. I6 M. Kouris, H. Ruck and S. G. Mason, Canad. J. Chem., 1958, 36,931. I7 H. E. C. Powers, Nature, 1958, 182, 715. I8 R. J. Ferrier, personal communication. In C. E. Childs and E. B. Henner, Chemist-Analyst, 1960, 49, 26. 2oD. M. W. Anderson and J. L. Duncan, Talanta, 1961, 8, 241. 21R. S. Fanshawe, personal communication. 22D. W. Drummond and E. E. Percival, personal communication. 23D. J. Bell, in Modern Methods of Plant Analysis, Vol. II, Edited K. Paech and M. V. Tracey. SpringerVerlag, Berlin, 1955. 24L. B. Winter, Biochem. J., 1927, 21, 467. z5 R. W. Jeanloz and H. G. Fletcher, in Advances in Carbohydrate Chemistry. Academic Press, New York, 1951, Vol. VI, p. 159.
APPLICATIONS
Pergamon Press Ltd.
Printed in Northern Ireland
OF INFRARED
SPECTROSCOPY-V*
THE RETENTION OF WATER AND ORGANIC CARBOHYDRATE MATERIALS
SOLVENTS
BY
D. M. W. ANDERSON@ and N. J. KING Department
of Chemistry, The University, Edinburgh 9, Scotland
(Received 6 February 1961. Accepted 2 March 1961) Summary-Studies have been made of the extent to which some carbohydrate materials retain water and organic solvents. The water evolved was determined gravirnetrically, the solvents by an infrared method. “Drying to constant weight” in an oven at 103”, or in vacua at 80”, does not give true “moisture” contents. Both water and organic solvents are retained up to temperatures at which the carbohydrate materials begin to decompose: the polarity and boiling point of the solvent do not determine the extent to which it is retained. Freeze-dried samples can retainorganicsolvents,and have significant moisture contents. The possibility of inaccurate results and misleading artefacts arising from such retentions or from interaction with solvents is stressed.
ORGANICsolvents are generally employed at some stage in the extraction or purification of carbohydrate materials. Thus, water-soluble polysaccharides are isolated, and plant gums purified, by precipitation from aqueous solution; Soxhlet extraction or refluxing with solvents removes plant pigments and soluble sugars, and inactivates enzymes; starches are “defatted” with 80% methanol or methanol-benzene, and are subsequently fractionated by the use of butan-l-01 or other polar organic solvents. The purified material is often “dried” by successive immersion in acetone, ethanol and ether, with final oven drying at temperatures slightly above the boiling points of the solvents used. Since such treatment may irreversibly decrease the solubility of some materials, e.g. starches, these are often stored under methanol or toluene until required. The retention of moisture and organic solvents by pectins,l cellulose,2 starches,3*4 gum arabic5 and biological polymer@ has been reported : a mathematical treatment of moisture desorption isotherms has been given. ’ For cereals, “free water” (i.e. water lost at a stated drying temperature) has been distinguished8 from “bound water”, which is difficult to remove and is considered to be associated with the protein content. The effect of bound water has been considered in ultrasonic studies of hydration effects in sugars.g In this paper we report quantitative observations on some polysaccharide materials in the belief that the extent to which retention of moisture and solvents can occur is not widely appreciated. The experiments were based on the use of a micro-scale, vapourphase, quantitative infrared technique, developed recentlylO for the examination of fractions separated by GLC and usedli for a study of the Zeisel alkoxyl reaction. EXPERIMENTAL Drying methods Method I: Oven drying; temperatures and periods as stated. Method ZZ: Drying over phosphorus pentoxide in a conventional organic solvent and evacuated by a water-pump. * Part IV: D. M. W. Anderson and J. L. Duncan, 497
Talanta,
pistol-dryer,
1961, 8, 241.
heated by refluxing
D. M. W. ANDERWNand N. J. KING
498
Me&d 111: Vacuum drying (usually at 80”) in a small electrically heated, thermostatted, glass drying chamber attached to a suitable high vacuum line (< 0.02 mm mercury pressure). Method IV: Heating in a stream of dry nitrogen. The sample was weighed in a small 3-necked flask, which was then placed in a temperat~e~ontro~~ oil-bath. The central neck of the flask carried a mercury thermometer pocket which was in contact with the sample. One of the two outer necks served as an inlet for CO,-free nitrogen, dried by passage through Anhydrone; the third neck served as the outlet, to which was connected a tared Anhydrone absorption tube. This served in turn as the inlet to a cold trap immersed in Iiquid nitrogen. The outlet of the trap was fitted with an Anhydrone guard-tube (~6 ref. 11). The nitrogen flow-rate was 15 ml per min.
-u
60-
3
Solvent, FIG. l.-Infrared
t-6-q
calibration curves for determination 0 Ethanol x Ether q Acetone A Methanol
of solvents
(1070czx1) (1140 cm’) (1740 cm-l) (1040 cm-‘)
Determination of water released The Anhydrone absorption tube was weighed at intervals. not retain any of the solvents involved in these experiments,
Anhydrone
(B.D.H.. M.A.R.) does
Determination of organic solvents released These were quantitatively retained in the cold-trap, and were subsequently determined by a quantitative vapour-phase infrared technique. This has been described,‘O together with details of the design of trap, method of quantitative transfer from trap to gas-cell, and methods of constructing calibration curves for each solvent involved (cjI ref. 11). The absorption peaks given by acetone at 1740 cm-l, ethanol at 1070 cm-l, methanol at 1040 cm-l, and ether at 1140 cm-l were used for calibration; the calibration curves obtained are shown in Fig. 1. (Jt is, of course, fortuitous that the sensitivity of detection of acetone, methanol and ether were all identical, as shown by their sharing a common calibration curve). No overlap or interaction occurred for the absorption peaks chosen, so that all four solvents could be determined simultaneously. The results from some typical experiments are reported below.
Water and organic solvents in carbohydrate materials
499
RESULTS
A sample of the gum from Combretum leonend was precipitated with acidified acetone (O*lNwith respect to HCI) and dried with 5 changes of acetone(eachincontact for 2 days, the powder being progressively ground as it dried). The powder was stored in a desiccator (continuously evacuated) for 2 weeks at room temperature, and was then dried for 1 hr at 60” by method II. A weighed sample (554 mg) was then treated by method IV at various temperatures for lengths of time as shown in Table I. The total 1.
Time, FIG.
hr
2.-Release of water and acetone from a sample of C. Ieonensegum. Curve OA-water Curve O&acetone
amounts of water and acetone evolved were found gravimetrically and spectrometrically respectively. Several measurements were made within each of the periods quoted in Table I; the results are shown graphically in Fig. 2. After heating for 525 hr, weight of gum recovered = 469 mg, i.e., loss in weight = 85 mg. The weights of water TABLEI
IPeriod of heating, hr
l-20 20-264 264-288 288-406 406-454 454-525
Temp., “C
55 75 95 104 123 137
T
Total weight released from 554-mg sample. water,
acetone,
mg
mg
7.9 29.2 33.0 38.5 46.3 60.0
2.2 9.8 10.0 12.3 13.1 14.3
+ acetone found total only 74.3 mg. Very slight decomposition of the gum began at 123” and was significant at 1370; in the period 430-525 hr (Fig. 2) 6 mg of CO, were evolved. This evolution of CO, would be accompanied by water produced in the decomposition, and this explains the sharp rise in the amount of total water released between 430 hr and 525 hr in Fig. 2.
500
D. M. W. ANDERSON and N. J. KING
2. Crude C. leonense gum was precipitated with acidified ethanol, dehydrated with 5 changes of acetone (as in 1 above) and finally washed with ether. After preliminary drying by method II for 6 hr at 60°, a weighed sample (474 mg) was dried by method IV at 70” for the period l-5 hr, at 90” for 5-68 hr, at 105” for 68-116 hr, at 130” for 116 164 hr, at 140” for 164-188 hr, at 150” for 188-236 hr, and at 155” for 236-260 hr. The total weights of water, ether, ethanol and acetone liberated are shown graphically in Fig. 3. Decomposition of the gum was slight at 130” and pronounced at 140”: the graphs show that evolution of organic solvents continued steadily after the onset of
0
40
60
I20 Time,
FIG. 3.-Release
of water, ether, ethanol, Curve Curve Curve Curve
I60
200
240
hr
and acetone from a sample of C. Ieonense gum. OA-water OB--ether OC--ethanol OD-acetone
decomposition. It is noteworthy that, of the three solvents involved, ether was the most strongly retained: this is surprising, since (a) its boiling point is the lowest, (6) it is the least polar, (c) it was used in smallest amount for the shortest contact time and only for superficial washing. Apparently the ethanol (used in the precipitation process) and the acetone (used in the dehydration procedure) are accessible to and extracted by the ether used in the final washing stage. This effect was verified in the next experiment. 3. A further sample of crude C. leonensegum was precipitated withacidifiedethanol, dehydrated with absolute ethanol (5 changes, each in contact with the gum for 2 days), progressively ground to a powder, and finally washed with ether. After air drying, a weighed sample (845 mg) was dried by method II at 60” for 24 hr, when the total loss in weight (i.e., water + solvents) was 3.5 %, as represented by the curve OA given in Fig. 4. The sample was then quickly transferred and dried by method IV at 78” for a further 138 hr, i.e., for the composite drying period of 24-162 hr. This gave part AB of the dehydration curve. Further drying at 108” for the period 162-234 hr gave curve BC; drying at 120” for period 234-258 hr gave curve CD. The dotted
Water and organic solvents in carbohydrate
materials
501
AE, AF and AG give the weights evolved of water, ether and ethanol respectively; curve ABCD gives the sum of AE + AF + AG. The curves clearly show that solvents are still being liberated after drying for 280 hr, and confirm that the ether used as a final wash is more strongly retained than the ethanol used for both precipitation and dehydration.
curves
I 40
I 80
I I20 Time,
FIG. 4.-Release
I I60
I 200
I 240
I 2fN
hr
of water, ether, and ethanol from a sample of C. leonense gum. Curve OABCD-total % loss in weight Curve AE-water contributing to total AD Curve AF-ether contributing to total AD Curve AG-ethanol contributing to total AD
4. To test the effect of particle size on the retention, a sample of crude C. leonense was precipitated and dehydrated with methanol (6 changes, each in contact for 2 days). After air-drying, the sample was ground finely and sieved. Two fractions (a) passing 200 mesh and (b) passing 100 mesh were retained, and dried by method II at 60” for 9 hr. Weighed samples were then dried by method IV at 65” for the period O-365 hr, at 95” for 365-437 hr, at 98” for 437-509 hr, and at 101” for 509-629 hr. For the 200-mesh sample, the weights of water and methanol evolved are given in Fig. 5 by curves A and B respectively, and for the lOO-mesh sample by curves C and D respectively. These curves again emphasise that the drying temperature is more effective than the period of drying; methanol was still being released at the end of the experiment. 5. Parallel experiments to those described for C. leonense gum were made on samples of gum ghatti; l3 the retention of water and solvents by the two gums was very similar. 6. Further experiments were made with gum ghatti to test whether (a) solvents less polar than ethanol, methanol, acetone and ether e.g., carbon disulphide and dioxan, or (b) solvents containing bulkier functional groups e.g., isopropanol would be less strongly retained. For all of these solvents, however, retentions similar to those for acetone, ethanol, methanol and ether were given. 7. The retention of methanol by some starches was investigated. (a) A sample of potato starch (500 mg) was refluxed with methanol for 4 hr. After heating by method gum
4
502
D. M. W. ANDERBDNand N.
J.
KING
III at 80’ for 24 hr, it was then dried by method IV at 98’ for O-18 hr, at 102” for 18-40 hr, at 108” for 40-64 hr, at 112” for 64-84 hr and at 134” for 84-88 hr. As much methanol was evolved in the fhral4-hr period of heating at 134”, after steady evolution for 84 hr, as was evolved in the 18,hr initial period at 98”. {b) Rye starch, which had been stored under methanol at 0” for 2 years was treated by method II at 61’ for 24 hr.
Time,
FIG. 5.-Effect
hr
of particle size on release of water and methanol from a sample of C. leonen.segum. Curve A-water Curve C-water Curve D-methanol
I from ‘oo-mesh
A sample (500 mg) was then dried by method IV, the evolution of methanol being reported in Table II (+ and + + indicate O*1 - O-5% and 0.5 - 1% w/w respectively). Similar results were obtained with other starch samples. 8. A sample of C. I~UWPWgum which had been purified by ethanol precipitation, dialysed, and freeze-dried for 4 days was found to lose 1% by weight after heating at TABLE IX
Temperature,
66-90
90-106
106-154
154-l-78
178-202
Methanol evolved
103” for 5 hr, and a further I % on raising the temperature until d~rn~sitio~ began. Similar experiments with a freeze-dried sample of a peetie acid isolated from N&&z transiucenP gave a loss in weight of 6.4 % (duplicate runs) when the sample was taken direct from the freeze-drier; samples stored in a desiccator overnight after freezedrying gave 8.5 % loss in weight when treated by method III.
Water and organic solvents in carbohydrate
materials
503
DISCUSSION
The results show that both water and organic solvents are retained tenaciously, even on prolonged drying under vacuum at temperatures much higher than the boiling point of the solvents concerned. Indeed, retention continues up to the temperature at which the polysaccharide material begins to decompose, as revealed by yellowing in colour and the starting of the evolution of CO,. Furthermore, although apparent “constant weight” is reached after drying at a certain temperature for a determinable number of hours, continued drying at a slightly increased temperature gives further liberation of water and solvents, until eventually “constant weight” is again reached. This cycle can be repeated many times, by raising the temperature in small increments, until eventually the decomposition temperature of the carbohydrate material is reached. Freeze-dried samples also retain moisture tenaciously, although in smaller amount; this agrees with Robson, l6 who found that a solution containing 16 % glucose + 3 % gelatin required freeze-drying for 500 hr at room temperature for all moisture to be removed. In practice, freeze-drying is not normally continued for longer than about 100 hr, and it is often assumed that samples so treated have a negligible moisture content: many quantitative inaccuracies must arise in this way. Freeze-dried samples may also retain organic solvents ; cellulose retains 1.5 % (w/w) of benzene on freezedrying.16 Water and vapours can be occludedl’ in crystalline sugars; indeed, it was found recently18 that when phenylboronate ester derivatives of methyl glycopyranosides were recrystallised from benzene, significant amounts of solvent were retained unless special care was taken to ensure its complete removal. Other authorslg have recently reported similar solvent occlusion effects. To obtain accurate quantitative results with carbohydrate materials, it is clear that great attention must be paid to drying procedures. Furthermore, any previous treatment of a sample with organic solvents must be considered if inaccurate and misleading functional group analyses e.g., methoxyl, acetyl are to be avoided (c$ ref. 20). Application of the infrared alkoxyl methodl’ has shown that mere reflux with ethanol can create artefacts e.g., ethoxylation of the fructose from lucernezl and of certain plant gum components:22 we are grateful to Dr. R. J. Ferrier for the informatiorP that a dry, powdery xylan of normal appearance prepared by him contained 70 % of ethanol. These findings serve to support the timely warning given by Belles regarding the possibility of formation of non-reducing ethyl glycosides when plant materials are treated with hot 80-95 % ethanol. It now appears likely that the occurrence of an ethyl riboside,* so far unconfirmed,25 also arose in this way. Acknowledgements-We thank Professor E. L. Hirst, C.B.E., F.R.S., for his interest in these studies, and the Department of Scientific and Industrial Research for a maintenance grant (to N. J. K.). We are grateful to Dr. R. S. Fanshawe, Dr. R. J. Ferrier and Dr. E. E. Percival for supplying materials for examination, and for providing experimental details. Zusammenfassung-Es wurde untersucht, bis zu welchem Ausmasse einige Kohlenwasserstoffe Wasser und organische Liisemittel festhalten. Das Wasser wurde gravimetrisch und die Solventien mittels einer Infrarotmethode bestimmt. “Trocknen zu konstantem Gewicht” bei 103°C im Ofen oder bei 80°C im Vakuum gibt nicht den “wahren Feuchtigkeitsgehalt”. Wasser sowohl als such organische Losemittel werden bis hinauf zu Temperaturen festgehalten, wo bereits Zersetzung der Kohlenwasserstoffe stattfindet. Polaritiit und Siedepunkt sind fur das Ausmass der Retention nicht bestimmend. Frostgetrocknete Proben kiinnen organische Lijsemittel zurtickhalten und zeigen
504
D. M. W. ANDERSONand N. J. KING
signifikante Gehalte und Feuchtigkeit. Die Moglichkeit ungenauer Ergebnisse und irrefuhrenden Verhaltens, die durch diese Art von Retention hervorgerufen werden konnen, werden betont. RBsmn&-Les auteurs ont Ctudie l’importance avec laquelle les hydrates de carbone retiennent l’eau et les solvants organiques. L’eau fide etait dosee par gravimetrie, les solvants par une methode infra-rouge. Le “sechage a poids constant” dans un four a 103” ou sous vide a 80” ne donne pas des “teneurs en humidit.? reelles. L’eau et les solvants organiques sont retenus tous les deux jusqu’a des temperatures auxquelles les hydrates de carbone commencent a se decomposer; la polarite et le point d’ebullition du solvant ne determinent pas l’importance de sa liaison. Des echantillons La seches a froid peuvent retenir des solvants organiques et ont des teneurs en eau importantes. possibilite de resultats inexacts et de produits aberrants provenant de fixation ou dint&action avec les solvants est soulignee. REFERENCES 1 E. F. Jansen, S. W. Waisbrot and E. Rietz, Analyt. Chem., 1944, 16, 523. 2 H. Staudinger, Z. angew. Gem., 1952, 64, 149. 3 M. Ulmann and F. Schierbaum, KolloidZ., 1958, 156, 156. 1 Idem, Die Stiirke, 1959, 11, 203. j L. K. H. van Beek, J. Polymer Sci., 1958, 33,463. 6 G. Champetier and J. Neel, Bull. Sot. Chim. BZoZ.,1958, 40, 1773. ’ H. A. Becker, Canad. J. Chem., 1958,36, 1416. 8 D. W. Kent-Jones and A. J. Amos, Modern Cereal Chemistry., The Northern Publishing Co. Ltd., 5th Edn. 1957. g H. Shiio, J. Amer. Chem. Sot., 1958, 80, 70. lo D. M. W. Anderson, Analyst, 1959, 84, 50. I1 D. M. W. Anderson and J. L. Duncan, Talanta, 1960, 7, 70; 1961, 8, 1. I2 D. M. W. Anderson, E. L. Hirst and N. J. King, Talanta, 1959, 3, 118. I3 G. 0. Aspinall, B. J. Auret and E. L. Hirst, J. Chem. Sot., 1958, 4408. I4 D. M. W. Anderson and N. J. King, Biochem. Biophys. Acta, 1961, in the press. I5 E. M. Robson, Vacuum, 1956,4, 60. I6 M. Kouris, H. Ruck and S. G. Mason, Canad. J. Chem., 1958, 36,931. I7 H. E. C. Powers, Nature, 1958, 182, 715. I8 R. J. Ferrier, personal communication. In C. E. Childs and E. B. Henner, Chemist-Analyst, 1960, 49, 26. 2oD. M. W. Anderson and J. L. Duncan, Talanta, 1961, 8, 241. 21R. S. Fanshawe, personal communication. 22D. W. Drummond and E. E. Percival, personal communication. 23D. J. Bell, in Modern Methods of Plant Analysis, Vol. II, Edited K. Paech and M. V. Tracey. SpringerVerlag, Berlin, 1955. 24L. B. Winter, Biochem. J., 1927, 21, 467. z5 R. W. Jeanloz and H. G. Fletcher, in Advances in Carbohydrate Chemistry. Academic Press, New York, 1951, Vol. VI, p. 159.