Production of
linical-Ty
PARTIAL HYDROLYTIC DEPOLYMERIZATION AND FRACTIONATION OF THE DEXTRAN PROM Leuconostoc rnesenteroides STRAIN KRKL B-512 IV-AiX A. TOEFF, C. L. JIEHLTRETTER, R. L. JIELLIES, P. R. Wa4TSON. B. T. HOFREITER, P. L. PATRICK, 4XD C. E. KIST ,Vorthern Regionwl Research Laboratory, Bureau of Agricultural and Industrial ChemisLry, LT.S . Depurtment of Agriculture, Peoria, I l l .
I X T R A K has bern partiall) depolymerized by acid (8, 16), alkaline ( $ 6 ) )or enzymatic ( 0 ) hydrolysis, ultrasonics (14, 16, 23), or heat (84,$0) for the piepalation of polysaccharide fractions having value as blood-volume extenders for use in the treatment of shock and burns. At present, acid-hydrolysis procedures appear to be favored because the reaction can be simply carried out in conventional equipment, can be readily controlled, and does not require isolation of the native dextran in dry form. The industrial production of clinical dextran has recently been described (1). The partial acid hydrolysis of dextran followed by precipitation of the depolymerized product with alcohol was described in 1940 ( 2 ) . More recently Swedish and English investigators have reported, chiefly in the patent literature (8, 16),the fractionation of partial dextran hydrolyzates with such watermiscible nonsolvents ~ t 9the lower molecular weight alcohols, acetone, and dioxane. There is not available in the scientific literature detailed information on the production of a dextran fraction which meets the current chemical and physicochemical requirements ( $ 6 ) for making clinical-injection solutions. Publication here of the results of systematic studies on the preparation and properties of such fractions, termed “clinical dextran” or “clinical fraction” in this paper, fulfills the need for such data, A large number of polysaccharides classifiable as “de\traii” because their constituent units are linked predominantly by a-l,6’-glucosidic bonds have been isolated at the Northern Regional Research Laboratory (IO)from cultures of a nide variety of bacterial strains. These dextrans differ in the amount and type of non-l,6’- linkages which they contain. The authors have investigated the partial acid hydrolysis of several of these dextran types to obtain fractions with charactei istics now required for the production of clinical-injection solutions. This report is concerned only with the partial hydrolytic depolymerization and fractionation of dextran from Leuconostoc rnesenteroides NRRL
and variation in conditions of fractionation, on the course of dextran degradation and on the type of product obtained. The ltinctics of the hydrolysis is also discuqsecl. MATERIALS AXD METHODS
DEXTRAN.The samples of KRRL B-512 dextran used m r e
produced in whole culture and were purified by several reprecipitations (12). Typical analyses on the purified dextran studied m r e nitrogen, 0.03%; ash, 0.1 to 0.3%; apparent fructose, 0.03 to 0.2% [modification of Gray’s ( 7 ) method]; and [7] = 1.1 t o 1.3 dl. per gram in water at, 25” C. HYDROLYSIS PROCEDURE. Hydrolyses c re re carried out by the following procedure, except where deviations were made for the study of a given variable, a6 indicated in succeeding sections.
A slightly greater than 5% (w./v.) aqueous solution of the &xtran was heated to the temperature of hydrolysis in a constant-temperature bath. The amount of 6 S acid required to give the desired pH was then added as quickly as possible with rapid stirring. This quantity of acid vas determined in advance by titration of a known volume of the dextran solution. The hydrolysis was timed from the moment, all of the acid had been introduced. The relative viscosity of the solution, measured at 25“ C., was the criterion for ,judging the extent of hydrolysis, m-hich allowed selection of the time for ptopping t,he reaction a t a desired degree of depolpmerization. The relat,ive flow time of the hydrolysis solution (heSeaftrr for c o n v e n 1 e n o e r eferred t o as rclative viscosity) x a s measured by us(’ of a jacketed capillary tube viscometer (Figure 1 ) . A similar t e c hili q u e Water, has been used for Jacket folloiTing t h e enzymatic hydrolysis of starch (13).
B-512. Dexti an KRRL B-512 has a weight-average molecular weight of 30,000,000 to 50,000,000 as determined by light-scattering methods (20). Periodate oxidation ( I f ) has shown that the ratio of 1,6’- to non-1,6’- glucosidically linked units in its structure is approximately 16 to 20 to 1 (94 to 96% apparent 116’linkages). Recent military specifications (26) for clinical dextran requiie a product having a weightewerage molecular weight of 75,000 & 25,000, determined by the light-scattering method. The distribution of molecular sizes is further specified by stating that the 5 t o 10% fraction of the sample having the loTvest molecular weight shall not have an average molecular weight below 25,000 and the 5 to 10% fraction having the highest molecular weight shall not have an average molecular weight above 200,000. The authors’ hydrolysis experiments were cariied out with the objectives of obtaining maximum yields of material meeting these specifications and of demonstrating the effects of different variables in the various processing steps. They report here studies on the effect of pH-temperature-time interrelationships during hydrolysis, kind of acid, dextran concentration, extent of hydrolysis,
4 k16rnm. O.D. Figure 1. Viscometer Used for Following Course of Dextran Hydrolysis
370
The relative viscosity d e c r e a s e d rapidly at first. but under most of the selected conditions changed rather slowlybetxeen relative viscosities of 3.5 and 2. Several typical curves illustrating the change in viscosity during the course of hydrolysis are shown in Figure 2. The hydrolysis was readily st)opped xithin kO.05 rela-
February 1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
tive viscosity unit from a preselected value by rapid cooling of the hydrolyzate and simultaneous neutralization with slightly less than the required quantity of 12N sodium hydroxide solution. Final neutralization of the solution a t room temperature (with more dilute alkali) was to pH 6.5 to 7.0. The relative viscosity of the neutralized hydrolyzate was usually very close to the value
[;IL\ IO
SO', pH 1.03, H,SO,
371
determined by drying to constant weight a t 100' C. in vacuo over phosphorus pentoxide. The percentage fie]& of dextran fractions given here were based upon the total weight of hydrolyzed dextran used for fractionation rather than upon the initial starting weight, since in these small scale laboratory runs 4 to 5% of the dextran was used in multiple sampling during hydrolysis. An overall recovery of 98.3% of the original dextran, in the form of the various fractions, was obtained in a hydrolysis conducted on a pilot plant scale. VISCOSITY.Viscosity measurements on frac* tions out inofconventional partially hydrolyzed fashion bydextran the usewere of Ostwaldcarried
99: pH 2.1, 75:HCI pH 105,HCI
Cannon-Fenske capillary tube viscometers. Inherent viscosities [designated { q ) in this paper] (S), determined a t relative viscosities between 1.1 and 1.2, were found to approximate closely the intrinsic 0: 4 4 L99*, pH 1.6, HCI viscosities of the degraded dextran samples (see 2 also 27) and were therefore used in all routine deSO', pH 03, HCI I I I I I 1 I I I I I terminations. When intrinsic viscosities were 0 I lo I' le measured, the concentrations were chosen such TIME, HOURS that the relative viscosities fell between 1.1 and 1.5; the intercept at Zero concentration of the Figure 2. Variation of Relative Viscosity of Dextran Hydrolyzates (5Yo Dextran Concentration) with Time under Different Conditions of qSpl vs. c curve was determined mathematically Temperature and Acidity from the lea& squares straight line through the points. The viscosity measurements were made a t 25' C. obtained before neutralization. The initial concentration of the NUMBER-AVERAGE hIOLECCLAR \.~TEIGHTS. Number-average molecular weights were deduced by measurement of the reducing dextran solution was chosen so that the neutralized hydrolyzate had a concentration of 5 =!= 0.05%. power of the dextran fractions, assuming one reducing end group FR.4CTIONATION PROCEDURE. The neutralized dextran parper molecule. Such assumption is justified from the agreement tial hydrolyxates were fractionated by the graded addition of found between number-average molecular weights determined in either methanol or 95% ethanol. The standard fractionation this way and those measured by the osmotic pressure method in scheme, illustrated diagrammatically in Figure 3, involved a this laboratory. primary separation of material of the desired molecular-weight distribution (Bor "crude clinical" fraction), followed by two purification stages, or refractionations of this middle cut, further to narrow the molecular dispersity. The correct solvent limits for I'reci itates Keutral Hydrolyzate Supernatants obtaining the maximum yield and desired type of fraction for (5% Dextran Concentration) J. clinical use were ascertained experimentally. to X % Precipitant Concentrations of precipitants in the ensuing discussions are expressed in per cent by volume of methanol or of 95% (specific gravity 25"/25" 0.8091) ethanol, calculated without regard for volume contraction on mixing. Fractionations were carried to Y % Precipitant out a t 25' & 1" C. The precipitant was added slowly to the stirred dextran solution, and the mixture was allowed to stand I for at least 1.5 hours for aettling of the precipitated liquid phase. I1 R Fraction ("('rude" ('liniral Dextran) CJ Fraction In the laboratory, centrifugation of the mixture preceded separa-
5
'
P
tion of the phases, but this step was unnecessary if a sufficient length of time was allowed for settling. If the mixture containing precipitated dextran was allowed to stand for several days, the precipitated phase often solidified and became less readily watersoluble. However, complete solution in water was always achieved a t 80" C. The composition by weight of the precipitate was 35 to 4Q% dextran, approximately 15% alcohol, and the rest water. Calculation of the amount of precipitant to add in the refractionation stages was based on the total volume of 5% dextran solution without regard for the alcohol content resulting from carry-over with the precipitate. Fractions were recovered in solid form, when desired, either by removal of alcohol by concentration in vacuo, followed by lyophilization, or by dehydration of the dextran by additon of its solution to 10 t o 15 volumes of alcohol, with vigorous stirring. The granular, dehydrated products from the latter process were separated by filtration, washed with additional alcohol, and dried in vacuo over anhydrous calcium chloride. Equilibration a t 60 to 65% relative humidity for several days removed any residual traces of alcohol and produced a product having a moisture content of 14 t o 16%. Moisture in solid dextran samples was
to 5% Concentration in Water to S% Precipitant
1
II
B-1 Fraction
t.0 Y % I'recipitant
I
I1
B-2 Fraction
I
B-3 Fraction
to 5% Concentration in Water to Xy0 Precipitant
"
to YyOPrecipitant
13-2-a Fraction
/I
B-2-b Fraction (Final Clinical Fraction) Figure 3.
I
B-2-c Fraction
Fractionation Scheme Used on Dextran Hydrolyzates
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
372
Reducing power measurements were carried out by the procedure of Soinogyi ( R I ) , using 6- [a-r~-glucopyra~iosyl]-u-giucose (isomaltose) as a reference standard. Isomaltose and isomaltotriose, containing a-1,6'- linkages, show good agreement in molar reducing powers, providing further justification for the use of the former as a standard substance for evaluating dextran fractions of higher degree of polymerization. The number--average degree of polymerization was calculated from the reducing power, calculated as C1,R?,OII,H?O, by means of the equation nig. of saniple X 2 Dpz. = ___-__-
mg. of isomaltose monohydrate X 0.9
The co11ce11DETERMISATIOK OF DEXTRAX COKCESTRATIOK. tration of dextran in solution \vas most conveniently determined by measurement of the optical rotation of the solution, assuming a specific rotation (sodium D line) of +200°, which is within 1% of t,he average value, for the dextran. The concentrations of solutions too turbid for polarimetric analysis werr determined colorimetrically by only minor niodificatioii of a puhlished anthrone procedure (fa). SEP.4RATIOX OF' HIGH ASL) h V 5 TO 1079 FROM (:LIXIl!.%T, FRACTIOKS. Two procedures were used for separation of tlie high and low 10% fractions of clinical-type dextran. I n the first procedure, applied to those samples of Tables 1. and V I foi, which light-scattering molecular weight3 are reported, the s e p rations were carried out in the presence of O.Oyosodium cahloritlc by a slight modification of the method proposed in the inilituy specifications ( 2 6 ) , directions ivhich m r e kindly supplied tiy 8.G. Keissberg, National Burc:iu of Standards. In earlier runs, reprewiting the rest of the Iiydrolyees listed in Tables V and Trl, the separations Jvew cwried out as follow.
Vol. 46, No. 2
srw
STUDY OF VARIABLES IT THE HYDROLYSIS
EFFECT O F D E X T R A N C O . TIOX. Considerable leeway was possible in selectioii of ran concentration to i)e for hydrolysis, without any alteration of the conditions of time, temperature, or acidity required. I n Table I it, can IJf, seen that under a selected set of hydrolysis conditions, the dist'ribution of products rvas not greatly changed, notwithstanding the wide variation in de LII concentration. Pro[iuct,s rollectt:d between the same methanol limits also had similar viscosities. This may be an important factor in commercial practice, whercl maximum output from the hydrolysis lrettle is required. Solutions of B-512 dextran containing up to approximatdj- 42 gram!a per 100 nil. of solution could be adequately stirred at 99": higher concentrations gave rise to solutions whic:h were too viscous for effective agitation.
Dextran Concn. Graruii/lOo MI.' Dilute FICl 6h
Yield of Fraction between Indicatwl __ Afethanol _ _ _ _ Limits, % 40-16 45-.511 50--5:, B5-fiC ~~
0-40
0 I8 30 1C 0 2 24 30 18 0 6 21 37 13 tlydrolyzed a t Hilo C. arid p I I 1 .6 f o r 1 hour with !I(;] Itelatire viscosity of final hydrolyzate was 2 . 5 . 21 nii.l 35 pl~a:nsilOOml. of solritioii, respectively. 95' 30
" "
~
.-
>BO
H I (J
21 18
II
I6
In each of the experiments listed in Table I fractionation ~vpls (tarried out a t 570 dextran roncentration. This is a h the most desirable concentration for. hydrolysis if the course of renction is to be followd by viscosity change as described aiiove, and waq therefore adopted as the Ptandard. It would undoubtcdlp I)I> The same precipitant, either niethanol or (35% ethanol, XIF possible to follow the hydro1 of the more concentrated dextraii used for evaluat,ing the molecular distrihution of the final clini fraction as had been used in tlie original fractionation. T solutions by viscosity change if one chose a tcmperaturc highel, portions, each containing a minimum of 5 grams of dextran, of than 25" for viscosity measurement, or a viscometer deeigned f o r 5% aqueous clinical dextran solution were placed in 250-mi. use with more viscous solutions than was that sho\r-n in Figure I , centrifuge bottles suspended in a constant teinperaturc bath maintained a t 25" To one portion V:LS added slorvly with or if a sample portion viere diluted to 5% dextran concentration stirring thc amount of precipitant required to take the mixture for viscosity nicasurement. Experience is required at this stage just beyond tlie cloud point. USE o r H Y D R o C H L o R I C 1's. ~ I - L K R I C ,ICIU. In TtLble 11 cn1iifor judging the degree of turhidity which \vi11 represent betv-een parison is made betneen hydrochloric and sulfuric acids in tiextrit 11 5 and 10% precipitation; the volume per cent of precipitant hydrolysis. At the same pH value, measured a t room temperarequired is about 2% beyond the lower alcohol limit ( X % in Figure 3) employed in the original precipitation of the 73 fraction. ture, hydrochloric acid was the more a c t i n Homver, whtw After equilibration for an additional 15 minutes (stirring durhydrolysis was carried to t,he Pame extent with the two a,cids, thr ing first 5 minutes) the mixture T T ~ Qcentrifuged arid the amount molecular distribution of the products, as indicatcd by fractionaprecipitated determined by difference, based on the optical rotation of the supernatant liquid. If the amount precipitated ~ n s tion with methanol, \\-vas generally similar. The differences that less than 5y0, further adjustment with alcohol was made; if were found may be attributable to the differing amount>sand kind more than IO%, sufficient water was added to redissolve the of salts present which can shift the precipitation range of thr. precipitate and another trial was made. For separation of the dextran, and could be compensated by slight shiEting of alcohol lowest 3 to log0,precipitant \vas added to the ot,her portion until limits if desired. The crude clinical fractions from a sulfuric t,he concentration ivas the same tis (for methanol) or 1.5Y0 higher than (for 95% ethanol) that used in the primary precipitation of acid hydrolyzate commonly contained 2 to 4% of sodium sulfate, the fraction. As before, the mixture wvas centrifuged, the amount while those from hydrochloric acid hydrolyzatcs rctrtined only precipitated determined, and further adjustment of precipitant about 0.5% sodium chloride. Jn either case the clinicrtl maconcentration made as needed until the amount of dextran left terial obtained after refractionation had an extremely low w h in the supernatant liquid ims between 5 and 10% of the starting amount, The high and low 5 t o l O 7 fractions ~ were next freed content,, well within allon-able limits. from alcohol by concentration in vacuo and made up to known The higher activity shown by the hydrochloric acid can be :it volume in water for viscosity determination, and the solid was least in part explained by the fact that its acidity WB,S not lowered recovered by freeze-drying and used for 3f.y determination b.i. as much with rise in temperature as vas t,hat of solutions ('on-. reducing power.
49 15 ( 0 . 2 8 8 ) b 9 34 (0.278) 17 49 ( 0 . 2 6 8 ) 23 61 0 263) 31 61 {0:249) 34 57 (0.246) 40
___ 0-42 76 50 28 7 5 2
Hydrolysis of 5 % dextran solution with HzS04 at 80" and pH 1 . Values in parentheses are inherent viscosities of fraction.
373
ume % of methanol. The variation in amount of this crude dextran fraction a t different extents of hydrolysis is shown in Table IV. As expected, there was a progressive increase in the amount of lower molecular weight fraction and concomitant decrease of higher molecular weight material during the course of the hydrolysis. As the quantity of the desired fraction was a t a maximum when the relative viscosity of the 5% dextran solution a t the end of the hydrolysis period fell between 2.4 and 2.85, hydrolyses were stopped within this range. The intrinsic viscosity of a whole neutralized hydrolyzate with qrei = 2.45 was 0.193. It is also noteworthy in Table IV that the inherent viscosity of the crude clinical fraction approximated that of the final, twice refractionated product from a similar run (Table VI), and that the viscosity values of the 42 to 49% methanol fraction were somewhat dependent on the molecular size distribution of the hydrolyzate fractionated, becoming lower as the average molecular size of the entire hydrolyzate decreased. It is, thuR, not adequate to specify solvent limits for the isolation of clinical dextran unless these data are accompanied by information on the extent to which hydrolysis was carried, if close agreement between experiments is desired. STUDY OF FR4CTIONATION CONDITIONS
taining sulfuric acid. Thus, of two 5 % dextran solutions made to pH 1.05 a t room temperature, the one containing hydrochloric acid had a pH of 1.14 at 80" while that containing sulfuric acid was 1.21. The choice of acid in dextran hydrolysis should be governed by whether or not the nonvolatility and lesser corrosiveness of the sulfuric acid are considered important or whether the lesser solubility of sodium sulfate in aqueous alcohol and its tendency to be precipitated with the dextran fraction outweigh the a,dvantages of the sulfuric acid. TIME-TEMPEHATCRE-pH RELATIONSHIPS.Hydrolyses of 5 % dcxtran solutions to the same final relative viscosity were carried out for time periods varying from 10 minutes to 46 houri and a t temperatures varying from 70" to 120" C., by suitable adjustment of the acidity. At high acid concentrations-IN hydrochloric acid, for example-sufficient salt was present to make deionization desirable for comparison with other runs. Fractionation of the several runs with methanol indicated a similarity in the distribution of molecular sizes in these hydrolyzatcs (Table 111). In the absence of any marked difference in the product, choice of hydrolysis conditions becomes a matter of convenience, the only essential factor being careful control of the extent to which the reaction is carried. EXTENT OF HYDROLYSIS.As is shown below, the crude cliniea1 dextran fractions could be precipitated between 42 and 49 vol-
TABLE V.
AS FRACTIONPreparation of Clinical Dextran. 95% ETHAVOL
AGENT. Experiments on the use of 95% ethanol for the fractionation of dextran hydrolyzates are summarized in Table V. Yields of up to 47y0 of clinical dextran within specification limits (26) were obtained by the process described. It can be seen that, further improvement of yield is possible as the molecular weight of the upper 10% fraction is still considerably below the permitted top limit of 200,000. Variation in the lower alcohol limit ( X % in Figure 3) appears to have a greater influence than the upper limit in governing the molecular size of the clinical dextran fraction. The correct lower limit for obtaining dextrans of the proper molecular weight, under experimentd conditions, was found to be about 397, of 95% ethanol. ) r l m H A w o r d AS FRACTIONATION ~ E N T . D a t a obtained on the fractionation of dextran hydrolyzates with methanol are summarived in Table VI. By suitable adjustment of precipitant ranges clinieal dextran fractions of similar composition could be obtained in comparable yields to those realized with 95% ethanol. Choice between these two solvents becomes then a matter to be based on factors of economics or convenience to the producer. For a given spread of concentration limits methanol precipitated a lesser amount of clinical dextran from the total hydrolyzate than did 95% ethanol. For example, a 6% ethanol range yielded 26 to 41% of twice refractionated clinical sample, while ATION
YIELDSAND PROPERTIES OF HYDROLYZED~ DEXTRAN FRACTIONS OBTAINED WITH ETHANOL AS THE FRACTIONATING AGENT qrel
at End Range of of 95% HydrolEtOH ys~s 39-46 2.50 39-45 2.54 39.5-45.5 2 . 4 8 40-47 2.51 40-46 2.54 40-45d 2.43 40-444 2.39 40.5-45.5d 2 . 4 3 41-47d 2.33 41-45d 2.39
Amount of Fraction,b
~
-4 0 1 2 8 11 3 2 12 16 14
% of Hydrolyzed Dextran B- n- B-
B 68 64 64 69 59 56 50 52 55 46
C B-1 B-3 31 5 11 35 4 12 33 12 11 23 6 11 30 13 9 40 11 11 47 18 11 36 11 11 30 10 10 41 19 9
2-a 2-e 2-b 1 6 47 2 6 41 1 4 36 5 6 40 2 4 30 5 5 25 4 3 13 6 4 20 4 4 26 4 3 11
Properties of B-2-ti Fraction lnl Mw AWN 0.247 7 4 , 6 0 0 4 6 , 2 0 0 0 . 2 5 7 8 1 , 2 0 0 50,900 0.231 58,200 38,900 0 . 2 1 9 50,700 34,900 0 . 2 2 4 5 2 , 8 0 0 38,600 0.236 . , . 38,900 0.238 . . . 39,900 0.218 ... 32,900 0.205 8,500 0.210 .. .. .. 232,400
High Molecular Weight Fraction of B-2-b
blc01101
limit 43.6' 43.4: 44.3 44.7: 44.6 42.6 42.6 42.4 42.6 43.0
Low Molec@ar Weight Fraction of B-2-b Alco-
% 7 7 7 10 7 10 10 10 5 10
lql
Mw
0.325 129 000 0 . 3 3 3 12O:OOO 0.284 87,000 0.271 78,100 0.268 74,100 0.277 ... 0.291 ... 0.266 ... 0:235
.. .. ..
MN 70 500 741500 57,300 52,500 52,700 50,700 47,000 43,400 36,800 37,100
1101
limit %. 51.5' 6 50.0: 9 50.7 9 52.0: 8 50.7 9 4 7 . 1 10 47.1 8 48.5 7 49.0 7 47.6 9
l.t.l .l
0.162 0.180 0 163 O:l50 0.164 0.180 0.179 0.163 0.149 0.169
Nw 26,200 30 200 25'800 21:200 26,200
.,
.
... ...
.. .. ..
Mv 20,700 23 500 20'700 17:300 20,400 21,900 22,400 18,000 19,800 20,100
All hydrolyses carried out at 5% dextran qoncentration, using HzSO4 at pII 1 and 80' C . ; fractionation at 5 % dextran concentration. Fractions designated according to scheme In Figure 3. Separation carried out using MeOH at 6% dextran concentration in the presence of 0.9% NaC1. -4 different preparation of the N R R L B-512 dextran was used for these five runs than for the first five; weight-average molecular weights may he estimated from the inherent viscosities by use of the relation given in the text. a
Vol. 46, No. 2
INDUSTRIAL A N D ENGINEERING CHEMISTRY
374
TABLE k-1.
YIELDS A N D PROPERTIE'
0 1 I%kDROLYZhD
D E X T R ~FRkCTIO\S \"
rjrd
R~~~~
of h&hanolb
at Amount of Fracrion 5 End of as % of Hydrolyzed D&tran HydrolB- B- Bysis A B C B-1 8-3 2-a 9-c 2-b 2.59
0
66
34
2 . 6~3 1 ~ 63 37 ~ 42-30 2.61 0 68 31 42.5-49.je 2 . 4 0 2 56 41 42.5-48.5e 2 . 4 0 2 51 47 43-48e 2.40 3 44 52 45-508 2 . 4 2 26 35 38 2 . 6 8 0 60 4U (
~
Properties of B-2-b Fraction
[VI
Mw
.Ila
6
8
4
4
43
0.243
73,000
43,600
12 )
8
0
0 3 2 2 2 1
37 25 19 16 9 5 39/
0.253 0.221 0.224 0.223
8 2
22 21 23 23 18 t I1 10
i
d
6
.
.
.
81,300 48,500 56,500 3 6 , 5 0 0 ... 36,100 . . . 313,400 0.228 . . , 38,100 ... . . . 0.186 0.262 76,400 44,100
FRWTIOVATI\C .'GEYT
OBT4IiYED M ITH h f h l ' H 4 V O L A S
High Molecular Weight Fraction Low 3Iolecrilar Weight Fraction of B-2-b - --__ of B-2-b Alcohol hol limit 7; (7) JIw .Vs limit % [v! Xiv MN
=-42.gd
6
0.301
13.2d 10 0.316 43,ld 8 0.254 44.4 8 0.257 44.2 7 0.262 ,
..
,
,,,
. . . . . . . .
...
10
0.337
110,000
-
60,600
50.Zd
7
0.164 26,700 20,100
128.000 71,300 14.0d 10 0.183 35,100 26,400 66,400 49,200 51.0d 4 0 . 1 6 3 28,200 20,400 . . . 44,700 3 0 . 3 8 0.173 . . . 20,900 ... 47,000 4 9 . 0 9 0.173 ... 21,100
. . . . . . . . . . . . . .
. . I
. . . . . . . . . . . . . .
...
132,000 74,800
...
9
0.159
... ...
26,300 16,500
All hvdrolyses carried out a t 5% dexrriin noiicentration, rising HzSOI a t pH 1 and 80' C.: fractionation a t 5% dextran concentration. Valiit% given in parentheses a r e methanol liniih used in the two refractionation stapes. Fractions designated according to achenie iu Figure 3. Separation carried o u t a t 6% dextran concentration in the presence of 0.9% PiaC1. e A different preparation of the K R R L B-512 dextran was used for these five run3 t h a n used for the first three; w e i y h - a v e r a g e inol~cularweights may b e estimated from the inherent viscosity by use of the relation given in the text. I B-2 fraction. Fractionation carried through only t,wo stages.
the highly purified samples uEed in the work reported here, have also been found to give the same results in the hydrolysis and fractionation procedures described. A single refractionation (Table VI) using appropriate precipitant limits gave a fraction sufficiently homogeneous as regards molecular weight distribution to meet the specifications, but the distribution of molecular sizes \vas broader (for comparable yields of clinical fraction) than a twice refractionated mateiial. The ash content was high (0.4 to 0.5%) in the once refractionated as contrasted with the tnice refractionated dextran (0.1% or lese; sodium sulfate ash). EFFECTOF TEMPERATURE. Fractions of degraded dextran were found to have a fairly high temperature coefficient of solubility in aqueous alcoholic solutions. For this reason temperature control during fractionation was required for good reproducibility between runs, The influence of temperature variation on the yield and properties of clinical fractions, other factors being held constant, is illustrated in Table VI1 and Figure 4.
60r
-~
'E-2-bn Fraction
0 401
10 20
I
22 24 26 28 TEMPERATURE OF FRACTIONATION,
30 O C
.Figure 4. Effect of T e m p e r a t u r e o n Fractionation of Dextran Hydrolyzates with M e t h a n o l
TABLE
Fractionation Temp., O
c.
20 23
a 6% methanol range gave only 16%. E'o? a 5% spread the respective yields with 95% ethanol and with methanol were 20 to 25% and 5 to 9%. The lower precipitation limit for obtaining clinical-size dextran was about 3 % higher (42%) for methanol than for 95% ethanol. Another outstanding different property of methanol ab coinpared with ethanol was the unusually large amount of B-1 fraction produced by tpe former solvent whenever the same solvent limits were used for refractionation as were originally employed. This effect was eliminated, with consequent improvement in yield, by changing the methanol limits in the refractionation stages over what they were originally, as shown in Table VI. Recommended for use to obtain clinical material would be either fractionation at 42 to 50%, with refractionation a t 41 to 50%, or fractionation a t 42 to 49% with refractionation at 41 to 49%. These recommended fractionation limits supersede those (45 to 50%) proposed earlier (1'7) on the basis of preliminary studies, which are inadequate (Table VI) in the present procedure. Several different preparations of dextran synthesized by the NRRL B-512 organism either in whole culture or by a culture filtrate (65), and used a t different stages of purification varying from a crude gum containing 6 to 9% fructose and 1.6 to 1.9% ash to
ON I-IELD AUD YII, E F F E C T O F TEMPCRATURE PROPERTIES OF THE B-2-b FRSCTION~
25
27 30
Yield,
%
3Iethanol (Initial 42 to 1 9 % : Recuts 41 to 49%) 0.217 34,000 21 0.249 40,300 27 0.259 44,000 36 46,200 35 0 259 48,400 29 0 267
98% Ethanol (40 t o 46%) 32,400 0.206 20 40,000 0.234 33 47 600 0,258 33 30 a Hydrolyjis of 5% dextran solution with HzSOr a t pII 1 and 80' t o relative viscosity of 2.4-2.6.
20 25
~
The data show that the yield and molecular size of the clinical fraction are less affected by slight temperature increase than by lowering of the temperature a corresponding amount, under the indicated conditions. Lowering of the fractionation temperature 2" to 5" below the usual 25" caused a profound change in amount and properties of the B-2-b fraction. As the fractionations were carried out at successively higher temperaturesj the solubility of the dextran increased (Figure 4), so that there mas a progressively greater quantity of dextran left in the various supernatant liquids and lesser amounts in the fractions less soluble than the clinical cute. This naturally resulted in a progressively higher average molecular weight for the clinical fraction. Similar trends were noted for both methanol and 95% ethanol.
February 1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
375
weight of the hydrocarbon radical of the alcohol used, and (c) were correlated with electrical properties of the precipitantnamely, its molar polarization (6) and the quantity p2/s, where M is the dipole moment and E is the dielectric constant of the precipitant. These results on the relative precipitating power of the alcohols are generally similar to those of Erbring and Wenstop ( 8 ) on the precipitation of the polar polymer cellulose acetate from acetone solution.
‘ 90 O0L
T.4BLE VIII. RELATIONS BETWEES DEXTRAYPRECIPITATED AND PRECIPITANT USED IS DEXTRAN-WATER-ALCOHOL SYSTEM
Dextran Pptd.,
%
0 (Incipient precipitation) 50 60 70 80 90 95
Moles Alc. Required/100 M1. 5 % Dextran Soln IsoNeOH EtOH PrOH 1 92 k 13 0.71 2.09 1.16 2.24 2.35 2.60 2.96
1.21 1.24 1 .27 1.32 1.42 P .53
Molar Ratio of MeOH to IsoE t O H a PrOH” 1.70 2.70
0.75 0.76 0.78 0.82 0.88 0.96
1.73 1.74 1.76 1.78 1.83 1 .9a
2.79 2.84 2.88 2.86 2.95 3.08
Molar polarization (5) b 37.1 50 9 63.2 (Dipole moment) 2 X 1037 0.8.52 1.31 1 80 Dielectric constant a C2Hs/CHs = 1.93. CsH1/CHa = 2 87 Values of density And dielectric constant a l e for 20’ C.
PRECIPITANT CONCENTRATION, PERCENT
Figure 5. Comparison of Methanol, Ethanol, and 2-Propanol as Dextran Precipitants
The results emphasize the importance of using designated solvent concentrations for fractionations only a t the temperature for which they were evolved. Maintenance of as constant a temperature as possible will assure uniformity of product produced from different runs. No extensive study of the fracEFFECTOF CONCENTRATION. tionation of dextran hydrolyzates a t various polysaccharide concentrations was made. As the dextran concentration was raised, less precipitant was required t o insolubilize a given proportion of the dissolved dextran. For example, precipitation of 50% of the dextran from a hydrolyzate a t 2, 5, 10, and 15% dextran concentrations required 52, 51, 46, and 44 volume % methanol, respectively. As the efficiency of fractionation of high polymers has usually been found to be improved a t lower concentrations (4),one might expect that less homogeneous fractions would be obtained from solutions more concentrated than those described in this work. PHASE RELATIORS IN DEXTRAN FRACTIONATION
RELATIVEPRECIPITATING POWEROF ALCOHOLS.Although the theoretical basis for comparing the precipitating action of various alcohols in a system such as dextran-water is not well defined ( 4 ) , certain empirical relationships are of interest because of their similarity to those obtained with other polymers. I n Figure 5 comparison is made among methanol, ethanol and 2-propanol as precipitants of a typical B-2-b dextran fraction. Qualitatively it may be seen by inspection that the precipitating efficiency of the solvents is in the order MeOH < EtOH < isoPrOH. The sensitivity of the precipitation is in the reverse order, as precipitation of 95% of the dextran from solution required a 10.8% range of methanol, and 7.4 and 7.1% ranges of absolute ethanol and 2-propanol, respectively. These relationships are expressed quantitatively in Table VIII, in which it can be seen that the relative (to methanol) precipitating powers of the alcohols used ( a ) did not vary greatly over the entire range of precipitation, ( b ) were related to the molecular
NATUREor THE P R C C I P I T ~ TPHASE. ED A typical B fraction was precipitated from the same hydrolyzate with methanol (42 to 500/0) and with 95% ethanol (40 to 46%). These sirupy fractions had the following properties: % of % of Total hlolee original Solution % b r Weight in Moles ROH/ PreDextran Volume Fraction , M e O H / Base cipitant in B in B DexMole Mole Used Fraction Fraction ROH tran Hz0 Sp. Gr. R O H a Dextran .MeOH 62.5 3.5 17 41 42 1.14 1 2.1 EtOH 63.5 4.3 13 35 52 1.14 1.66 1.3 a Calculated for equal weights of dextran.
The data of this section are indicative of the amount of carryover of precipitant from one fractionation stage to the next and enable calculation of the actual amount of precipitant present in refractionation. Thus a t nominal methanol and 95% ethanol concentrations (as previously defined) of 42 and 40%) respectively, for precipitation of a B-1 fraction, the actual amounts of precipitant present would be 43.7 and 41.5%. PROPERTIES O F FRACTIONS O F PARTIALLY HYDROLYZED DEXTRAN
VISCOSITY-MOLECULAR WEIGHT RELATIONSHIP. The inherent viscosities and average molecular weight (MN and Mw) values of the fractions listed in Tables V and VI were plotted on log-log graph paper. The points were well represented by straight lines. The equations of the regression lines, calculated from 48 points on the M N curve and 27 points on the M w curve, were { q ] = 8.85 X l O - 4 i M ~ o . 5 2 8and { q } = 2.03 X 10-3 Mw0.431. The average percentage deviations of the experimental values of .?IN and M w from the values derived from the regression lines by use of the viscosities of the fractions were found to be 5.2 and 5.5(ro9rei spectively. Wales et al. (87) and Senti and Hellman ( 2 0 ) also propose relationships for estimation of molecular weights of acidhydrolyzed B-512 dextran fractions. The equations given above are intended particularly for application to dextran fractions obtained by procedures like those described in this paper. RATIOOF 1,6’- TO No~-1,6’-LINKAGES.Formic acid produced by periodate oxidation (11) was measured on a number of degraded B-512 fractions having differing molecular sizes [ { q ) from 0.12 to 0.371. In the usual periodate analysis procedure (11)cal-
376
I N D U S T R I A L AND E N G I N E E R I N G CHEMISTRY
culations of the ratio of 1,6'- to non-l,6'- linkages are made, using the msumption that one mole of formic acid is produced from each anhydroglucose unit linked through the 1- or the 1- and 6- positions only, and that there is a negligible contribution from reducing end groups. As each &linked reducing end group can produce 4 moles of formic acid on periodate oxidation, and the number of aurh terminal group8 is no longer negligible in fractions of hydrolyzed dextran, the formic acid values were corrected by means of the formula ('orrected HCOOH (nioles/mole anhydroglucose) =
HCJOOH found (moles/mole anhydroglucsose)
-
3 I)P.\
There was no syatematicn change in linkage ratio between the more and less highly degraded fractions. The mean ratio of 1,6'- t o non-1,6'- linkages (corrected by the above equation) in 20 tiamples was 22 (standard deviation, 3.5) as conipared with a value of 19 for the original material. This change in ratio i d considered to be too close to the limits of error of the analytical determination to vcarrant any conclusion as t o whether or not a real inweave in the percentage of 1,6'- linkages has occurred. The OpwcAL R O T A T I O ~ . specific optical rotations of typical €3-2-b fractions, meamred at 5% dextran concentration (25'; sndium L) line) varied from $198' t o +200", averaging 199". OTHER PROPERTIES.Tlie amounts of vaiious trace constituents and the buffering capacity of the clinical dextran fraction prepared according t o the proceis dewibed wwe alwv:tyi w a l l within allowable limits ( 2 6 ) .
Vol. 46, No. 2
considered as an approximation only, as the extremely low reducing power represented by the extrapolation is very ~ensitivet o small errors, and would alPo give misleading results if the nativcb dextran contained traces of adsorbed reducing materialx. First-order specific reaction rate constanti for the hytlrolysiv were 3.64 X lop3hour-' and 9.11 X 10 hour 1 :tt 80" itnd 70", respectively. The temperature coc42rient for a 10" r i v in trmperature is thus about 4. Thtx energy of wtivatioii ( t j ):IS calculated from these rate conbtants 33,300 c d . pc'r niolc, :i valuf' somewhat below the 36,000 figiirt~found by other worhcarq at thiu laboratory (28) but close to the 33,390 value reported 1)) llot>lwyn-Hughes (18) for the @-1.6'-linked disaccsharidt., grntiobio,ra If the latter value ic: independcnt of ariomeric configuration, ac is the case with the activation eiiergiec of maltow and ctallobio-ta (28), this figure would also reprebent the activation e w r g y of a i l a-l,6'- bond and would be in close agreement with the authoir' value for dextran. By use of the activation energy in conjunction with the. rea('tion rate constant at 50" or SO", the rate of reaction a t other ternpwatures may be determined. Since the inherent viscoritr (5% roncentration) of the hrdrolyzate bore a straight-linc. relationship to the DPN- (log-log plot oi data in Table IX), it is po'sible to determine from the data cited the time required to rradi :I desired relative viscosity at hydrolysis temperalwei othw Ih:tn those given for this particular dextran. 1)JSCIIRSJON 111 a continuing operation the yield of clinical dextran citn augmented by rehydrolysis of a number of combined A, B-1, niid R-2-a fractions. The hydrolysis of such a mixt.urc. (1'11 I, 80" C.) was found to bt conipiived with tha hytlrolysis of the native dextran (cf. 3.75 hours being required for thc viscosity of the 5% solution t'o decrease from 3.7 (origird) t o 2.6. This hydrolyzate WRE considerably less polydisperse t,hal-I a native dextran hydrolyzate, and gave 52% of twice refraction-. ated clinical size dextran (42 to 49% methanol; 41 to 4970 in wfractionat,ions\. Thus the yields of c1iriic:al dextran shown iii Tables V and V I may be augmented by several per cent. Other means for improving the yield of clinical dextran or for simplification of the prowssing have also been tried. For example, very slight hydrolysis t,o a relative viscosity of about 10, as shoxn in Table IV, followed by rehydrolysis of the 76% of higher molecular weight fract,iori to t,he usual extent, and processing of the combined B fractions froin t>hetwo stages resulted in lees (about 5%) convirsion to dextran fractions below clinical size. As a result, some improvement in yield of cliiiical material can be obtained. However. it is doubtful whether the gain is worth xhile in view of the e x ~ rhandling ~, and quantities of solvents required. The conditions ol fractiontition and hydrolysis cited in t,liis paper illustrate the yields to be expected with dextran produced by the SItItL B-512 organism, and cannot necessarily be t r a r i ferred to dextrans having other chemical structures. The authors have found definite differences between this arid other dexh n s ; these results will br the subject of a fut,ure publication. The possibility also remains that, niinor variations will be found between preparations of YRlLL E-512 dextran, which may necessitate some alteration of {he experimental condit,ions given. tf' care in the various manipulations as described here is maintained and rehydrolysis of combined high molecular weight fractioris (.%) B-1, and B-2-a) is carried out, yields approaching 50% of' clinical-type dextran should be obtainable in a plant., operating a t a steady state, under conditions selected from Table V or Table, TI. Should fut,ure clinical exl.":rimentatioii demonstrate :L di:graded dextran of different average molecular size and size ( 5 s tribution t)o be preferred over the currently tieaired product,, thtt procedures described hei hould be easily amenable to appropriate modification. b ( b
KINETIC STUDY OF B-512 DEXTRAN H Y n H O L Y S l i
of 5 % solutions of high molecwlar weight dextran were measured at 70" and 80" j= 0.2" C., using sulfuric, acid a t pH 1. Samples ivvpre withdrawn a t different time intervals, rapidly cooled to 25")neutralized, and used for measurement of relative viscosity and rrtlncing power. The data obt ainetl are shown in Table IX.
TAHLC Ix. RATEO F Time, Hours
~ c X l ' R A A 'HYDROLYSIS AT 70" A N I )
Relative Yiucosity of Hydrolyzate
DP\
80"
c.
"/o
Hydrolysis"
A t 70'
n R " . ._ 1 .o
2.0 4.0 6.0 8.0 12.0
7. R_
x.~ m
0.12 0.lj
390 230 160 125
0.2G
86
1.1G
610
46 22 10
8.8 5,2 3.7
0.44 0.62 0.78
A t 800
" Percentage of apparent dextrose as determined by reducing p o ~ e r , based on amount of dextrose theoretically formed after complete hydrolysis.
The extent of dextran hydrolysis at the stage at which the reaction had to be interrupted for production of good yields of clinical fraction was small, representing breakage of only about 2% of the bonds originally present in the polysaccharide. At, this small degree of degradation the hydrolysis could be represented as a zero or a first-order react,ion; either the quantity [lOOyo- % apparent dextrose (see note, Table I X ) ] or the logarithm of this figure gave a straight-line relationship when plotted against hydrolysis time. By extrapolation of t,hese straight lines t o zero time the reducing power, and hence the number average degree of' polymerization. of the original native dextran could be eatiiriatetl. The value obtained, DPs = 1430 ('WN= 230,000) c m be
~
INDUSTRIAL AND ENGINEERING CHEMISTRY
February 1954
ACKNOWLEDGMEYT
This research program on dextran has been carried out with the cooperation of several groups of workers a t the Xorthern Regional Research Laboratory. The authors wish particularly t o thank R. J. Dimler, H. M. Tsuchiya, J. Corman, V. Sohns, P. Rogovin, and H. Conway for their assistance in supplying the native dextran used as starting material; C. 5. Wise for carrying out analyses for fructose; J. C. Rankin for periodate analyses; R. L. Lohmar for performing the experiments on fractionation of dextran hydrolyzates at different polysaccharide concentrations; Allene Jeanes for supplying the isomaltose and isomaltotriose samples used as standards in reducing power determinations; and members of the Analytical, Physical-Chemical, and Physics Division for assistance in the physical and chemical characterizations and for discussions concerning fractionation procedurea. LITERATURE CITED
(1) Bixler, G. H., Hines, G. E., McGhee, R. M., and Shurter, R. A., IND.ENO.CHEM.,45, 692-705 (1953). (2) Colin, H., and Belval, H., Compt. rend., 210, 517-20 (1940). (3) Cragg, L. H., J . Colloid Sci., 1 , 261-9 (1946). (4) Cragg, L. H., and Hammerschlag, H., Chem. Revs., 39, 79-135 (1946). (5) Daniels, F., “Outlines of Physical Chemistry,” pp. 88, 365, Xew York, John Wiley & Sons, 1948. ( G ) Erbring, H., and Wenstop, K., Kolloid-Z., 85, 342-50 (1938). (7) Gray, D. J. S., Analyst, 75, 314-17 (1950). (8) Gronwall, A. T. J., and Ingelnian, B. G. A. ( t o Aktiebolaget Pharmacia), U. S. Patent 2,437,518 (March 9, 1948), 2,644,815 (July 7, 1953). (9) Ingelman, Bjorn, Acta Chem. Scand., 2, 803-12 (1948). (10) Jeanes, A,, Haynes, N‘. C., Wilham, C. A , Rankin, J. C.. and Rist, C. E., Division of Agricultural and Food Chemistry Symposium on Microbial Polysaccharides, 122nd Meeting, AMERICAN CHEMICAL SOCIETY, Atlantic City, N. J., 1952. (11) .Jeanes, A., and Wilham, C.A., .J. Am. Chcm. Soc., 72, 2655-7 (1950).
377
(12) Jeanes, A,, Wilham, C. A., and Miers, J. C., J. Riol. C h m . , 176, 603-15 (1948). (13) Landis, Q., and Redfern, S., CereaE Chem., 24, 157-66 (1947). (14) Lockwood, A. R., James, A. E., and Pautard, F. G., Research (London), 4, 46-8 (1951). (15) Lockwood, A. R., and Swift, G. (to Tell and Usher, Ltd.). U. S. Patent 2,565,507 (Aug. 28, 1951). (16) M f g . Chemist, 23 (2), 49-54 (1952). (17) Lfehltretter, C. L., in “Report of Working Conference OII Dextran,” National Research Council, Subcommittee on
Shock, and Northern Regional Research Laboratory, Peoria. Ill., October 29, 1951, p. 30. (18) Moelwyn-Hughes, E. A., Trans. Faraday Soc., 25, 503 20 (1929). (19)
(20) (21) (22) (23) (24)
Seifter, S., Dayton, S., Novio, B., and Muntwyler, E., Arch. Biochem., 25, 191-200 (1950). Senti, F. R., and Hellman, N. N., Abstracts of Papers, 121st Meeting, AM. CHEM.SOC.,Milwaukee, Wis., March 30 t o April 3, 1952, p. 8. Somogyi, M., J. Biol. Chem., 160, 61-8 (1945). Stacey, M., Abstracts of Papers, XIIth International (’oriqrrss of Pure and Applied Chemistry, New York, N. Y . , Sept. 10 to 13, 1951, p. 623. Starey, M., Research (London), 4, 48 (1951). Stacey, hf., and Pautard, F, G., Chemistry and frd,u&tri/, 1952,
1058-9. (25) Tsuchiya, H. M., Koepsell, H. J., Corman, J., Biysnt. G . , Rogard, M. o., Feger, V. H., and Jackson, R. J . Hncteriol., 64, 521-6 (1952). (26) U. S . Government, military medical purchase description for dextran injection, stock number 1-161-890, May 24, 1951. (27) Wales, hi., Marshall, P. A,, and Weissberg, S. G., J . Pollirn~r Sci., 10, 229-40 (1953). (28) Wilham, C. A,, and Jeanes, A., Northern Regional Laboratorv,
w.,
unpublished results.
(29) Wolff, I. A., Watson, P. R., Sloan, J. W., and Rist, C. E., ENQ.CHEX., 45, 755-9 (1953).
[VI).
RECEIVIOD for review June 20, 19% ACCJOPTED October 29, 1053. Presented before t h e Division of Carbohydrate Chemistry at the 124th Meeting of t h e A 4 x E R I C A N CHIMICAL SOCIETY, Chicago, Ill.
Enthalpy-Concentration Diagram for System Ferrous Sulf ate-Water J
KENNETH A. KOBE AND EARL J. COUCH, JR., Unicersity of Texas, Austin, Tex.
T
HE usefulnees of the enthalpy-concentration diagram in
making refrigeration, crystallization, and evaporation calculations for salt solutions has been pointed out by Bosnjakovic (6) and hlcCabe (26‘). I n view of the importance of the system ferrous sulfate-water in connection with processes for the recovery of waste pickle liquor, it is desirsble to have such a diagram for this system. PREVIOUS THERMAL DATA
.4 survey of the thermal data available in the literature is presented in Table I. Values selected for enthalpy calculations include the accurate heat capacity data for anhydrous ferrous sulfate determined by Moore and Kelley ( 2 8 ) ,for ferrous sulfate heptahydrate by Lyon and Giauque (26), and for ice b y Giauque and Stout (11); the extensive and accurate heat of solution data presented by Perreau (29) for ferrous sulfate heptahydrate; and the heat of formation data tabulated by Bichowsky and Rossini
(4) AFPARATUS AND PROCEDURE
The literature values for the heat capacity of ferrous sulfate solutions are considered to be inadequate as a basis for the calculation of enthalpies of ferrous sulfate solutions.
I n order t o measure the heat capaeitie.: of ferrous sulfate solutions, a calorimeter similar to that described by Kobe and Anderson (22) and Kobe and Sheehy (95’)was constructed as shown in Figure 1. Two 6-volt storage batteries served as a power source to the calorimeter heating element, which was constructed in a manner similar to that described by Randall and Taylor ( S I ) . The power input to the heating element was measured by the ammetervoltmeter method and the length of the heating period was nieasured by an electric stop clock to the nearest 0.1 second. The calorimeter temperature was measured by means of a calibrated thermometer graduated a t 0.1O C. intervals. Distilled water was used as the calorimeter fluid in determining the heat capacity of the calorimeter over the temperature range 5” to 95” C. A determination consisted of making time-temperature readings before and after a heating period during which the known weight of water was heated through a temperature rise of approximately 10’ C. ( 3 2 ) . The ferrous sulfate solutions used in this work were prepared from C.P. Baker’s analyzed ferrous sulfate heptahydrate. In order to minimize oxidation of the solutions, boiled distilled water, through which nitrogen was bubbled during cooling and storage, was used. A few drops of sulfuric acid were added to each solution as an oxidation inhibitor ( 7 ) . All containers for solutions were flushed with nitrogen and the solutions kept in a nitrogen atmosphere. A fresh solution was prepared immediately before each calorimetric dctcrmination.
