I-VDUSTRIAL AND ENGINEERING CHEMISTRY
240
with live steam and condensing the alcohol that is driven off. The refrigerating system, besides supplying cold brine to the condensers, chills a cold storage room and manufactures ice for numerous small refrigerators throughout the laboratory. Precipitations of the final product with alcohol are carried out in 12-liter balloon flasks. Such precipitates can be readily dried under vacuum without being removed from the flasks.
Vol. 17, No. 3
Bibliography 1-Banting and Best, J . Lab. Clin. M e d . , 1, 251 (1921-22). 2-Best and Scott, J . Bid. Chem., 67, 709 (1923). 3-DudIey and Starling, Biochcm. J . , 18, 147 (1924). 4-Dodds and Dickens, Brit. J . E z p f l . Path., 6, 115 (1924). 5-Doisy, Somogyi, and Shaffer, Am. J . Biol. Ckem., 60, 31 (1924). B-Macleod and Orr, Lab, Clin. M e d , , 9, (1g24), 7-Clough, Allen, and Murlin, A m . J . Physrd., 68, 213 (1924). 8-Moloney and Findlay, A m . J . Pkys. Chcm., 18, 402 (1924).
J.
Application of Enzymes to Beet Sugar Factory Control’ By H. S. Paine and R. T. Balch CARBOHYDRATE
LABORATORY, BUREAUOF
CHEMISTRY, WASHINGTON, D. C .
A
VAST amount of energy has been expended by a large sucrose and raffinose. The determination of sucrose is of even number of chemists in investigations designed to re- greater importance in beet sugar factory control than the duce the errors of the Clerget procedure for analytical determination of raffinose. determination of sucrose by polarization before and after If top yeast extract is added to a portion of the sample and inversion by acid. The literature of the subject is quite bottom yeast extract to a duplicate portion, sucrose is hyvoluminous. In suite of drolvzed in the first uortion to invert sugar and raffinose these efforts, the me’thod in A n analytical procedure involving the use of the enzymes to fructose and melibiose, its most approved form is invertase and melibiase has been adapted to practical use whereas in the second porgreatly lacking in precision for precise determination of the sugars sucrose and raffition sucrose is hydrolyzed to for many purposes, and nose in the chemical control of beet sugar factories. By invert sugar and raffinose to this is particularly true in comparing the data obtained with those resulting from dif r u c t o s e , d e x t r o s e , and the case of d e t e r m i n a rect polarization and acid hydrolysis, information is gained galactose; the difference betion of the sugars sucrose relative to the polarization and distribution of optically tween the corrected polariand raffinose in mixture by active nonsugar compounds at successive stages of the zations of the two portions the procedure of Creydt.* factory process. The comparison explains in part the after complete hydrolysis is The advantages of enzymes frequent decrease in factory operating efficiency toward a measure directly of the as hydrolytic reagents for the end of the campaign, and brings to light certain errors melibiose content and incertain analytical deterin sucrose accounting as now practiced. The data also directly of the raffinose conminations have been dishave a distinct bearing on the matter of “unaccountable” tent. The two stages of cussed by a number of insucrose losses and the extent to which these are only apthe hydrolysis may be exvestigators, but such proparent and are due to errors of polariscopic analysis. pressed as follows: cedures have not come into general use, owing largely to (invertase present) + invert sugar I/Sucrose the difficulty of securing sufficiently active enzyme prep\Raffinose (invertase present) -+ fructose melibiose arations. I1 Melibiose (melibiasepresent) -+ galactose dextrose I n connection with a beet sugar manufacturing problem unThe use of enzymes such as invertase and melibiase as hyder investigation in this laboratory, it was necessary to determine percentages of sucrose and raffinose with greater precision drolytic reagents in the determination of sucrose and raffinose than is possible by means of analytical methods depending has been rendered quite practicable by the ultrafiltration upon acid hydrolysis of these sugars. Recourse was there- method devised by F. W. Reynolds5 of this laboratory for fore had to the use of the enzymes invertase and melibiase. concentration of enzyme solutions. Invertase (top yeast Bau3has described a method in which top yeast, containing extract5) and invertase-melibiase (bottom yeast extract5) invertase, and bottom yeast, containing invertase and meli- solutions of high activity were prepared by this procedure biase, are used for the analytical determination of raffinose; from ordinary bakers’ yeast and from brewers’ yeast, rehis procedure, however, is time-consuming, since complete spectively, and constituted the hydrolysts employed in the fermentation of the sugars attacked by top and bottom investigation here described. The top yeast extract was yeasts is required. Hudson and Harding‘ developed this tested in order to make certain that it contained no melibiase. idea further by using purified extracts of top and bottom The analytical technic presented no difficulties and was simyeasts which they applied only to determination of raffinose in pler in some respects than that of methods involving acid mixture with certain other sugars in pure aqueous solutions. hydrolysis; the degree of precision obtained was much Their procedure was not elaborated to the point where it greater, and it is possible, if desired, to accomplish the enzymic could be used in a practical manner for determination of raffi- hydrolysis in as short a period as in the rapid form of the acid nose in beet sugar factory ,products. Furthermore, their inversion procedure. I n the work of this laboratory acid inversion methods for method does not provide for simultaneous determination of estimation of sucrose and of sucrose and raffinose in mixture 1 Presented by Mr. Balch under the title “Determination of Sucrose and have been abandoned and enzyme methods have been used Ra5nose in Beet Products by the Enzyme Method” before the Division of exclusively for over two years. By using a highly active Sugar Chemistry a t the 67th Meeting of the American Chemical Society, Washington, D. C., April 21 t o 26,1924. invertase solution concentrated by the ultrafiltration method 2 2. Vn.dcut. Zuckcrind., 81, 153 (1887); Ibid.. 40, 194 (1890). the writers have been able to operate the invertase inversion
++
4
I 4
Chcm. Zfg., 18, 1794 (1894). THISJOURNAL, 7,2193 (1915).
6
THISJOURNAL, 16.169 (1924).
March, 1925
INDUSTRIAL AND ENGINEERING CHEMISTRY
24 1
procedure6 for determination of sucrose in the absence of raffinose in fully as short a time as the rapid form of the acid inversion Clerget procedure. The technic of the invertase method-. g., control of temperature during inversion-was furthermore simpler than that of the acid inversion method.
cc. of top yeast extract and to the other 5 cc. of bottom yeast extract. The slight excess of ammonium dihydrogen phosphate present is ordinarily sufficient to produce approximately the optimum pH value for enzyme activity. Hydrolysis is allowed to proceed overnight a t atmospheric temperature (preferably not under 20" C.) and the solutions are then Determination of Sucrose and Raffinose in Beet Products diluted to volume at 20" C. and the polarizations determined by Use of Enzymes a t 20" C. If it is necessary to keep the solution for any considerable length of time a little toluene should be added as For determination of both sucrose and raffinose by the ina preservative. vertase-melibiase method, the procedure which we have deIf a rapid analysis is desired by the 50-100 cc. procedure, veloped is as follows: the flasks, after addition of 50 cc. of the de-leaded filt,rate and Weigh 39 or 78 grams of material, with the exceptions noted 10 cc. of the enzyme solutions, are placed in a water bath in Table I, and transfer to a 300-cc. volumetric flask. Add kept a t 50" to 55" C. The following test may be used for the quantity of 55" Brix basic lead acetate solution indicated botin Table I and dilute to volume a t 20" C. Mix thoroughly determining whether the invertase preparations (top and Dilute tom yeast extracts) employed are sufficiently active. and filter through fluted paper in a closely covered funnel. Discard the first 25 cc. of filtrate and de-lead with ammonium 1 cc. of the invertase solution to 200 cc. a t 20" C., place in a constant temperature bath at 20' C., and when the solution dihydrogen phosphate' in as small an excess as practicable. has attained this temperature add 20 to 200 cc. of a 10 per This condition is readily determined after a little practice by cent sucrose solution which has also attained a temperature of the appearance of the lead phosphate precipitate, which 20" C. in the same bath and the pH of which has been p r e flocculates and sediments rapidly in the presence of a slight viously adjusted to 4.3 to 4.6 by addition of glacial acetic excess of the salt. As an aid to filtration a small proportion acid. Mix well and note the time of mixing the invertase and of infusorial earth may be added. The first 25 cc. of filtrate sucrose solutions. Allow the mixture to remain in the conshould be discarded. Ammonium dihydrogen phosphate re moves more color from a molasses solution than the usual stant temperature bath and remove additional portions a t the end of 15, 30, and 45 minutes, render alkaline with sodium de-leading agents, and the direct polarization after de-leading carbonate and determine the polarization at 20" C. Calcumay usually be determined in a 4-dm. tube. Since invert the polarization of sugar is usually practically absent in beet products, and the late the initial polarization by multiplying the sucrose solution by 10/11. Correct all polarizations for error due to difference in the specificrotation of raffinose a t the concentrations employed for the direct and invert polariza- the polarization of the invertase solution and calculate the k tions is negligible, it is not necessary, even in very careful value corresponding to each of the polarizations subsequent to the initial polarization according to the formula: work, that the direct polarization be determined a t the same dilution as the invert polarization. The acid reaction due to the slight excess of ammonium dihydrogen phosphate is not sufficient to cause an appreciable error in the direct polariza- where k is the unimolecular reaction velocity constant; tion as a result of hydrolysis of sucrose, provided determina- t , the number of minutes elapsing from the time the invertase tion of the polarization is not unduly delayed. and sucrose solutions were mixed until invertase action was stopped by addition of sodium carbonate; Ro,initial polarizaT a b l e I-Quantities of S a m p l e and of R e a g e n t s Required for Clarifition; R m , final polarization after inversion is complete (calc a t i o n and De-leading culated by multiplying the initial polarization at 20" C. by Quantity of Basic lead Ammonium material acetate dihydrogen -0.32); and Rt, polarization after time t. I n solving for k per 100 cc. 55' Brix. phosphate MATERIAL Grams cc. Gram the substituted values are treated algebraically. Invertase 3 Cassettes' 13 Q.2 solutions of high activity prepared by the ultrafiltration Pulo 2to 4 100 cc.b 0.2 Lim'e cake or sewerc 26.5 1.5 . .d method are quite stable and, with a little toluene added, Thin juice 2 52 0 . 2 to 0 . 3 Thick juice have been kept for months a t laboratory temperature with4 26 0 . 3 to 0 . 4 White massecuite 30r 6 0 . 3 to 0.7 13 or 26 out appreciable loss of activity. The total time required for High wash sirup 13 or 26 0 . 3 to 0 . 7 3or 6 High green sirup 13 or 26 5 or 10 0 . 3 to 0.7 checking the activity of the invertase reagent is small. Raw or remelt massecuite 6 13 0 . 3 to 0 . 4 Raw or remelt sugar The constant k of the invertase solutions (top and bottom 3tO 4 26 0 . 3 to 0 . 4 2to 3 Sugar melter 26 0 . 3 to 0 . 4 yeast extracts), determined as described above, without corLow wash sirup 13 8 t o 10 0 . 4 to 0 . 5 Low green sirup or molasses 13 10 0 . 4 to 0 . 5 recting for dilution, should be at least 0.0005* for the rapid Saccharate cakes and milk (caranalytical procedure. The melibiase solution (bottom yeast bonated) 26 4 to 6 0 . 3 to 0.4 Steffen waste and wash watersc 78 or 50 cc. 2 t o 3 0.2 extract) should have such activity that, when 10 cc. are u Usual method of extraction, 26 grams in 201.2 CC. added to 100 cc. of a melibiose solution polarizing 20.0" V. in b Dilute t o 110 cc. c Neutralize-with acetic acid before adding basic lead acetate a 2-dm. tube a t 20" C., the polarization is reduced to 12.5' V. d Lime in solution will be partly precipitated by the phosphate, and it is necessary t o add sufficient phosphate t o complete the precipitation of both the lead and lime salts; hence no definite quantity can be specified.
Hydrolysis may be accomplished according to either the 5&55 cc. or the 50-100 cc. method; 50 cc. of the de-leaded filtrate are diluted to 55 cc. in the former and to 100 cc. (after completion of hydrolysis) in the latter. I n either case two 50-cc. portions of clarified, de-leaded solution are transferred to 50-55 cc. or to 100-cc. flasks, and to one are added 5 6 This rapid form of the invertase Clerget method is described in the chapter on Sugars and Sugar Products of the "Tentative and Official Metho d s of Analysis" (new edition, 1925) of the Association of Official Agricultural Chemists. 7 The use of this reagent was suggested by M. S Badollet of this labora-tory
8 Invertase solutions standardized a t the k values 0.00075 and 0.0015 can be obtained from the Wallerstein Laboratories, 171 Madison Ave., New York City. Invertase solutions of lower activity are prepared by The Digestive Ferments Co., 920 Henry SL., Detroit, Mich., and the Industrial Research Laboratories, 220 West Ontario St.. Chicago, 111. These preparations have been placed on the market a s a result of industrial applications made by one of the writers [Paine, THISJOURNAL, 16, 513 (1924)l. These invertase solutions can be used as analytical reagents a s received, but i t is preferable to dialyze them and then concentrate in IJUCUO at low temperature (preferably not over 35' C.),or better still t o wash and concentrate by Reynolds' ultrafiltration method (loc. c i t . ) . By the two latter procedures salts and color may be removed and the solutions further concentrated as desired. The foregoing statements refer to invertase preparations suitable for determination of sucrase in the absence of raffinose. Arrangements are now being made for commercial production of standardized top and bottom yeast extracts suitable for determination of sucrose and raffinose in the presence of each other.
I.VDUSTRIAL A,VD ENGINEERING CHEMISTRY
242
(mutarotation completed by adding solid sodium carbonate) in 20 minutes at 20" C. when the pH of the solution is 4.3 and the polarization is corrected for the optics1 activity of the enzyme solution. Under such conditions hydrolysis by both enzymes in the case of beet molasses is complete in 30 to 40 minutes. For overnight hydrolysis it is also desirable that the enzyme solutions have the activities mentioned above, the proportions of enzyme solutions used being half of those employed in the rapid analytical procedure. Since melibiase is present in smaller quantity than invertase, hydrolysis by this enzyme lags considerably behind inversion by invertase. The time of hydrolysis may be materially reduced by further concentrating the bottom yeast extract. I n order to accelerate mutarotation the solutions are made
Vol. 17, No. 3
-32.139 a t 13 grams per 100 cc. sucrose concentration, the invert polarization of sucrose a t the concentration employed in the 50-100 cc. procedure is -0.3213 S. The value 0.514 is provisionally accepted as the ratio of the polarization of melibiose and fructose to the polarization of raffinose. Hence A = -0.3213 S (0.514 X 1.852)R = -0.3213 S 0.952 R (3) I Combining Equations 2 and 3 P - A - 0.900 R S=
+
+
(4)
1.3213
Substituting the value of R from Equation 1,there is obtained the value of S: P - 2.219 A 1.219 B S= (5) 1.3213
+
Table 11-Determination of Sucrose a n d R a a n o s e in Presence of Each Other 50-100 CC. M E T h O D 5 G 5 5 Cc. METHODINVERT POLARIZATION INVERT POL,ARIZATION Bottom Top Bottom TOP yeast yeast yeast Direct yeast extract extract polar- extract extract OS&7 - R ~ ~ ~ ~ ~ ~ S E - - (corr.) (corr.) ----SUCROSE--RAFFIN ization icorr.) (corr.) -SUCROSE(B) DeviaDevia(AI (B) DeviaDeviaZOO C. Found Taken tion Found Taken tion 20' C. 20' C. Found Taken tion Found Taken tion
-
Ramnose' (anhySucrose' drous) Grams/ Grams/ 100 cc. 100 c c . 15.600 0 . 4 6 8 15.600 0 . 1 3 6 13.000 0 . 3 3 1 10.400 0.163 9.100 0.195 7.800 0.312 7 . 8 0 0 0.195 6 500 0 . 2 2 1 a
ov.
63.37 60.95 52.35 41.14 36 34 32 16 31 33 26.56
-17.70 -18.95 -14.90 -12.31 -10.49 - 8 46 -5.84 --I
-19.06 -19.40 -15.80 -12.77 -11.04 -9.28 -9.35 11 - 7 . 7 3
%
%
%
60.07 60.01 50.04 40.11 35.03 30.08 30.03 25.00
60.00 60.00 50.00 40.00 35.00 30.00 30.00 2.i.00
+0.07
Nsrmal weight solution.
-A- v. .
+0.01 $0.04
+0.11 +0.03 +O.OR +0.03 0.00
4-0 , - . fl4R --
%
%
1.81 1.80 0.61 0 . 5 3 1 . 2 8 1.27 0.62 0.63 0.77 0.75 1.10 1.20 0.69 0.73 0.84 0.85
Av.
slightly alkaline to litmus paper with sodium carbonate before polarization. This is best done before diluting the solution to 100 cc. The results obtained by this method showed satisfactory agreement in the case of molasses with those obtained by overnight hydrolysis without addition of sodium carbonate. Formulas for Simultaneous Determination of Sucrose and Raffinose by Enzymic Hydrolysis
The difference between the two corrected invert polarizations measured in a 2-dm. tube at 20" C., when multiplied by the factor 0.352 (derived from the change in specific rotation resulting from hydrolysis of melibiose), gives the number of grams of anhydrous raffinose per 100 cc. of solution. If a normal weight of the material is taken for analysis, the percentage of raffinose (R)is given by the equation:
%
+0.01 +0.08 $0.01 -0.01 f0.02 -0.10 -0.06 -0.01 -0.007
Ov.Ov.
-17.37 -18.68 -14.64 -12.04 -10.38 -8.31 -8.77 -6.98
-18.65 -19.19 -15.58 -12.53 -10.94 -9.21 -9.33 -7.62
%
%
%
60.10 59.97 50.01 39.97 35.00 29.95 29.98 24.92
60.00 60.00 50.00 40.00 35.00 30.00 30.00 25.00
$0.10 -0.03
Av.
$0.01 -0.03 0.00 -0.05 -0.02 -0.08
-0.012
%
7 0 %
1.73 0.68 1.27 0.66 0.76 1.19 0.76 0.88
1.80 0.53 1.27 0.63 0.75 1.20 0.75 0.85
Av.
-0
07
+ O 15 0 4-0 +0 -0
+o +o +o
00 03 01 01 01 03 019
As the concentration of raffinose is usually relatively small, it is unnecessary to correct for the change in specific rotation resultingfrom change in concentration of the fructose produced from this sugar. I n deriving the equation for S the value 0.514,1° determined for acid hydrolysis, was used for the invertase hydrolysis coefficient of raffinose. Some preliminary observations indicate that the invertase value is a little higher than this. However, the data given in Table I1 show that the resulting error is very small, as is also the error resulting from any inaccuracy in the other basic constants employed. The writers are now determining with a considerable degree of precision the invertase inversion constants for both sucrose and raffinose, and these data together with any necessary modification of the formulas, will be reported in the near future. The general formulas (including concentration corrections) that have been used for calculation of percentage of sucrose a t 20" C . are as follows for the procedures indicated: (50-55 cc. method) :
where A and B denote the polarizations after hydrolysis by top and bottom yeast extracts, respectively. The signs are treated algebraically, the polarization after action of bottom yeast extract being more negative than that after action of top yeast extract when raffinose is present. The direct polarization ( P ) in a 2-dm. tube at 20" C. of a solution containing 26 grams of a mixture of pure sucrose and raffinose per 100 cc., expressed in degrees Ventzke is P =S
+ 1.852 R
(2)
where S represents percentage of sucqoRe, and 1.852 =
,-'
the ratio qf the normal weights of sucrose and anhy-
drous raffinose. The corrected polarization after hydrolysis by top yeast extract is due to invert sugar and to fructose and melibiose, the produotsi of hydrolysis of raffinose by invertase. Acceptifiq tentatively the valhe of' the nekative constituent of the Clerget divisor for invertase inversion as
(P - 2.219 A + 1.219 B ) 100 s = 132.92 - 0.0131 [132.92 - (P - 2.219 A + 1.219 B ) ]
(50-100 cc. method) :
(P - 2.219 A + 1.219 B) 100 s = 132.13 - 0 00716 [132.13 - (P - 2.219 R + 1219 B ) ]
The concentration correction factors have been derived in the customary manner upon the basis of change in polarization resulting from enzymic hydrolysis. However, Vosburghl' 9 Determined by the authors in an investigation which will be described in a fo&tcoming publication. Zerban, in a paper entitled "The Rotation of Invert Sdgar and the Clerget Divisor" presented before the Division of Sub$ Chemistry a t the 68th Meeting of the American Chemical Society, September 8 t o 13, 1924, reported the value 132 09 obtained by calculation f r w the data of Tollens on the specific rotation ot degtrosq and the data of Uqsburgh o a t h e specific rotation of fructope. Zerban, in a Rqrsonal commumc&t$n to t$e authors, later reported the valve 132.10 determined experimentally. I 14Browne and Gamble, THIS JOURNAL, 13,793 (l9.21). I * J . A m . Chcm. Soc., 48, 219 (1921).
.
I,VD U S T R I A L A N D ENGINEERING CHEMISTRY
March, 1925
has shown in the case of binary mixtures of certain sugars that the net specific rotation of the two sugars at a given total concentration is not the sum of the specific rotations of the sugars a t their partial concentrations, but is one-half the sum of the specific rotations of the two sugars a t the total sugar concentration. BrowneI2has pointed out the significance of Vosburgh's conclusions when applied to methods of sugar analysis and has stressed the importance of water concentration in evaluating physical constants such as specific rotation. Zerban'3 has furnished experimental evidence in the case of invert sugar and sucrose mixtures which supports this position. While a recalculation of the writers' analytical data with a concentration factor based upon total solids (or indirectly upon water concentration) would cause a certain change in percentage values of sucrose and raffinose, such manner of recalculation would similarly influence the acid hydrolysis data. Since such a recalculation would in no wise affect the conclusions derived from their data and hereinafter discussed, the writers have allowed the data to remain as originally calculated. Furthermore, they deem it advisible to test the general validity of the extension of Vosburgh's rule in the case of mixtures of sucrose, raffiose, and molasses nonsugar substances, and have such an investigation in view. The formulas for calculation of S and R were tested by applying them to mixtures of known quantities of sucrose and raffinose. The data obtained are shown in Table 11. The difference between the quantities of sucrose and raffinose added and determined analytically was quite small and was usually within the limits of error of polariscopic observation. 1'
Louisiana Planter, 67, 44 (1921).
Report as Associate Referee on Polariscopic Methods, Association of Official Agricultural Chemists, October, 1924. 18
Small proportions of raffinose did not appreciably affect the sucrose inversion constant. For most purposes it would be unnecessary to complicate the calculation of percentage of sucrose by introducing into the formula concentration corrections for raffinose and its hydrolytic products. The influence of molasses nonsugar substances on the sucrose inversion constant was investigated. For this purpose diluted beet molasses was fermented by repeated inoculation with yeast and the absence of sucrose was established. The liquor was filtered and evaporated in vucuo until free from alcohol and then concentrated to a definite weight. To known quantities of recrystallized sucrose were added weights of fermented, clarified, and de-leaded molasses which contained the quantities of nonsugar substances present in a sample of original molasses taken for analysis by the 50-55 cc. procedure. I n every instance 6.5 grams per 100 cc. of sucrose were used, as this was the approximate quantity present in 13 grams of molasses. The constant was calculated from
cg,
MAT5RIAL Cossettes
v.
v.
-4.67 -4.45 -5.22 -5.10 -4.16
(Av. Colorado)
15.15
-4.70
-4.78
(Av. Idaho)
16.60
-5.05
(Av. Nebraska)
14.62
-4.42
Pulp
2.10 1.58 1.73 1.56 2.09 1.69 60.35 59.89 52.90 80.74 85.00 80.50 60.71 67.75 66.80 57.16 62.00 65.62 69.94 76.40 72.00 49.28 54.60 58.90 56.20 49.00
-
+
The data ob-
tained are given in Table 111. Table 111-Influence of Beet Molasses Nonsugar S u b s t a n c e s u p o n Invertase Sucrose Inversion C o n s t a n t Inversion Direct Invert constant polarization polarization = P - A (100) (P) (A Sa Sample v. v. 1 24.52 -8.50 132.1 2 24.06 -8.89 131.8 3 23.65 -9.31 131.8 4 24.09 -8.87 131.8 5 23.71 -9.28 132.0 6 24.10 -8.81 131.6 7 24.64 -8.34 131.9 Av. 131.9 a S 25.00 per cent.
-
Analyses of Beet Sugar Factory Products by E n z y m i c a n d Acid Hydrolysis POLARIZATIONBottom yeast extract Acid d U C R 0 5 S -RAFFINOSESucro?e (1) Enzyme Acid Enzyme Acid inversion (B) Z?'C. 2O'C. Sea Sob Rea Rab constant by Method of invertase inversion v. ov. % % 73 % -4.87 -4.64 15.07 14.79 0.27 0.30 131.26 0.12 -4.50 -4.56 14.41 14.31 0.07 131.26 0.25 -5.30 -5.00 16.50 16.06 0.11 131.26 -5.16 -5.10 16.29 16.06 0.08 0.16 131.26 -4.38 -4.30 13.98 0.30 14.01 0.2s 131.26
15.35 14.53 16.53 16.35 14.50
-0.59 -0.51 -0.60 Steffen waste water -1.22 (total) -1.10 -1.27 Thick juice -19.87 -18.16 -16.31 White massecuite -25.04 -25.64 -24.72 High wash sirup -19.23 -20.14 -19.56 High green sirup -18.02 -17.54 -19.06 Raw massecuite -21.41 -21.14 -21.04 Molasses -16.19 -16.41 -13.90 -15.18 -14.72 A 1.219 B ) o s e - (p- 2.219constant
- SA ) loo,where S = 25.00 per cent.
C = ('
...... ...... T a b l e IV-Typical -INVERT Too Direct polariyeait extract zation (A) 20 c. ZOOC.
243
REUARS5
0.5124 P-I
o.s31
-4.67
15.06
14.82
0.10
0.18
131.26
50-100 cc. So =
-5.09
-5.15
16.44
16.27
0.06
0.18
131.26
50-100 cc.
Sa =
-4.53
-4.47
14.39
14.26
0.15
0.20
131.26
50-100
Ss = 0'5124
.. .. .. .. .. ..
... ...
0.01 0.22 0.75 0.58 1.25 1.27 1.03 1.03 0.84 1.49 0.99 1.76 1.57 1.62 2.68 2.27 1.04 2.25 4.31 3.30 1.23
0.04 0.44 0.35 1.01 0.87 0.83 0.53 0.84 0.87 0.76 1.61 1.63 1.64 2.54 1.81 0.73 5.00 4.20 2.80 1.23
... ... ...
... ...
... ... ...
-1.34 -1.15 -1.31 -20.03 -19.64 -18.71 -1X.88 -16.74 -16.74 -25.96 -24.80 -26.58 -26.40 -25.48 -25.00 -19.99 -19,OO -20.75 -20.82 -20.66 -20.37 -18.75 -17.50 -18.84 -17.75 -20.22 -18.90 -22.61 -20.30 -23.12 -21.00 -22.72 -20.70 -16.96 -14.96 -18.07 -14.72 -17.08 -12.72 -17.62 -14.00 -15.70 -14.10 100 ; R e = 1.354 (A
...
0.29 0.23 0.25 0.33 0.40 0.37 60.52 58.74 52.16 79.37 83.21 79.27 59.99 66.27 64.66 56.42 59.29 63.32 68.25 72.32 69.18 48.92 52.30 52.33 61.93 47.47
- 23)
...... ... 0.03 ... ... 0.01 60.27 59.08 52.26 78.87 83.38 78.96 59.72 66.20 65.19 55.76 59.02 62.60 66.91 71.69 65.64 47.93 50.89 51.13 51.01 46.73
... ... ... ...
bso
CC.
131.20 50-100 cc 131.20 50-100 cc: 131.20 50-100 cc. 131.20 50-100 CC. 131.20 50-100 cc. 50-100 cc. 131.20 132.23 50-55 CC. 131.74 50-100 cc. 131.67 50-100 CC. 131.86 50-55 cc. 131.69 50-100 cc. 131.56 50-100 cc. 131.70 50-55 cc. 131.50 50-100 cc. 131.49 50-100 cc. 131.66 50-55 cc. 131.47 50-100 cc. 131 49 50-100 CC. 131.71 50-55 cc. 131.53 50-100 cc. 131.52 50-100 cc. 131.90 50-55 cc. 131.90 50-55 cc. 131.70 50-100 cc. 131.70 50-100 cc. 131.70 50-100 cc. 0.5124 P I = ; Ra = o,83g
-
= l4.Q6%
0'5ziA-1-
18.43%
14.3Q%
0.831 Apparent sucrose = 0.307 Apparent sucrose 0.23d Apparent sucrose 0.25% Apparent sucrwe 0 267 Apparent sucrose = 0:35$ Apparent sucrose = 0.28
0.54 (P
- S)
--
244
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Vol. 17, No. 3
The effectof these nonsugar constituents is in most cases to the consequent presence in the factory liquors of an increased decrease the direct polarization to a smaller extent than the percentage of objectionable nonsugar compounds. However, invert polarization is increased ; hence, the inversion constant this decrease in efficiency can be explained in part on the basis is greater than that for sucrose in pure aqueous solution. of the progressive increase in the “apparent sucrose” (POThe average value of the inversion constants was 131.9, larization of a normal weight solution) as compared with perwhereas for sucrose alone the constant at this concentration centage of sucrose in the cossettes. A greater quantity of is 131.54 according to Browne’s’4 data, which include the sucrose than exists in the cossettes is therefore charged value -32.00 for the negative constituent of the Clerget into the factory. The progressive change in the character of divisor a t 13 grams per 100 cc. sucrose concentration. Fur- the beets during the campaign as indicated by the difference ther investigation is required in order to determine how typ- between direct normal weight polarization and percentage of ical these constants are for beet molasses. According to the sucrose by the enzyme method was more pronounced during data so far obtained, the constant 131.9 represents an average 1923 than in 1922. for the 50-55 cc. procedure and 131.7 for the 50-100 cc. probetween Direct Normal Weight Polarization cedure. Of the other beet sugar-house products cossettes are Table V-Comparison a n d Percentage of Sucrose i n Cossettes of most importance from the standpoint of factory control. Idaho Nebraska Colorado I n view of the relatively low concentration of nonsugar sub(1923) (1923) (1922) 14.3g5 13.06 stances in the digestion liquor obtained from cossettes, ac- Per cent sucrose by enzyme method 16.443 S 0 . llg 0.285 0. 1g6 curacy of estimation of sucrose is determined by error of Direct rafinose polarization, ’V. polariscopic observation rather than by influence of nonsugar Sum of sucrose and raffinose polarizations, V. 16.56, lf1.25~ .. 14.68, substances on the inversion constant. O
I
Application of Enzyme Analytical Methods to Beet Sugar Factory Control The enzyme method for the determination of sucrose and raffinose as described was applied by the writers to factory control in a beet sugar factory in Colorado in 1922 and in factories in Idaho and Nebraska in 1923.15 Analyses were made by the following three methods and extraction statements were prepared on the basis of the first and third: (1) by the enzyme method for determiiiation of sucrose and raffinose; (2) by the Herzfeld-Creydt acid inversion method;lG and (3) by direct polarization. Some striking conclusions were derived as a result of these comparisons, and they serve to explain certain mystifying results frequently obtained in controlling factory operation by direct polarization and acid inversion. They also furnish more exact information regarding sucrose extraction and sucrose losses than has heretofore been available. Table IV shows comparatively some typical analytical data selected from several hundred” determinations made by the enzyme and acid hydrolysis methods and illustrates the error inherent in the latter. The Herzfeld-Creydt method in its customary form was chosen as the acid inversion procedure in order that comparison might be made with certain data obtained by the factory control laboratories. I n the various tables the polarizations are given upon the basis of normal weight solutions unless otherwise indicated. Table V gives a comparison between the direct polarization and per cent sucrose in cossettes by the enzyme method. In the case of Idaho beets, the average difference for the first two weeks was 0.09 per cent, which rapidly increased to 0.20 per cent and remained practically constant at that value. The difference between the final averages of the two series of values was 0.15 per cent. The difference between the average direct polarization and average percentage of sucrose determined by the enzyme method a t the Nebraska factory during the last 2 weeks of the campaign was 0.22 per cent. It is well known that factory performance efficiency frequently decreases as the campaign progresses. This is due in part to the working of a greater proportion of stored beets and J . Assoc. Ofictal Agr. Chcm , 2, 134 (1916). Appreciative acknowledgment is made of courtesies extended by The Great Western Sugar Company and the Utah-Idaho Sugar Company. 16 The acid inversion method wns used in the form customarily employed in the control laboratories of American beet sugar factories and as described in the “Methods of Analysis” of The Great Western Sugar Company. 17 These data are shown i n extenso in Report I11 (1923) and Report IV (1924) of chemical investigations of this laboratory relating t o beet sugar manufacture. A limited number of copies of these reports can be obtained from the writers upon request. 14
16
Observeddirect polarization, V. Direct polarization of optica!y active nonsugar substances, V.
16.5g5
+0.033
14.619 -O.Ofil
15 153 -0
lo1
In Table V the direct polarization of raffinose (calculated from per cent raffinose) is added to the per cent sucrose ( r e garded as normal weight polarization) and the sum compared with the observed direct polarization. The difference r e p resents the net polarization of optically active nonsugar substances, assuming, as is almost invariably the case in beet products, that the amount of any invert sugar present is negligible. The average direct polarization of optically active nonsugar substances was positive in the Idaho beets and negative in the Nebraska and Colorado beets. I n molasses the net direct polarization of the optically active nonsugars is negative. However, it is quite possible that the influence of lime, high temperature, and other factors may transform a positive nonsugar polarization in cossettes into a negative polarization in molasses and intermediate products. Furthermore, certain observations in connection with the ‘isteffenization” of molasses indicate that positively rotating nonsugars are also present in molasses and that it is merely the net polarization of this group of compounds which is negative. The direct nonsugar polarization of the cossettes may be compared with that of the molasses on the assumption that this polarization merely becomes concentrated in relation to sucrose and is not otherwise modified. It is assumed, as a typical value, that the nonsugar polarization in relation to sucrose polarization is about six times as great in molasses as in cossettes, the ratio of total nonsugars to sucrose being about 1 : 9 in the thick juice and about 4: 6 in the molasses. Molasses “worked” was chosen as the basis of comparison, since it contains a larger percentage of molasses direct from beets than does molasses “produced.” I n the case of the Colorado cossette data the average direct nonsugar polarization -O.lO1’ is equivalent to -0.67. per 100” sucrose polarization. The value -0.67” multiplied by 6 equals -4.02’ as compared with -3.19’, the average direct nonsugar polarization per 100’ sucrose polarization in molasses worked. Similarly with the Nebraska cossette data, the average direct nonsugar polarization -0.061 is equivalent to -0.42 ’ per 100’ sucrose polarization, which, when multiplied by 6, equals -2.52’ as compared with -3.21’, the average direct nonsugar polarization per 100sucrose polarization in molasses worked. In the case of t h e Idaho cossette data, the average direct nonsugar polarization +o.0330 is equivalent to +0.20’ per 100’ sucrose O
I N D U S T R I A L A N D ENGINEERING CHEMISTRY
March, 1925
245
polarization, and this value multiplied by 6 equals t 1 . 2 0 ” tent for the decrease in nonsugar acid invert polarization as as compared with -2.71”, the average direct nonsugar po- compared with nonsugar direct polarization. It is possible larization per 100” sucrose polarization in molasses worked. that there is also a change in the polarization of other opticHowever, even fairly close agreement need not be expected, ally active nonsugar constituents. since there is considerable magnification of error in making b l e VIII-Nonsugar Direct a n d Nonsugar Acid Invert Polarizations the foregoing calculations and, moreover, there appears to be TofaCalifornia Molasses C o m m o n l y P r e s u m e d t o C o n t a i n No Raffinose no justification for the assumption that the nonsugar polari- (Polarizations refer t o sugar scale degrees, normal weight solutions, and 2-dm. tube) zation simply becomes concentrated in molasses without (1919) (1923) the occurrence of any other change. The action of lime direct polarization 47.53 49.00 alone wodd be expected to exert great influence on the Observed Per cent sucrose by Herzfeld-Creydt formula with 0.836 divisor 48.71 50.10 nonsugar polarization. Per cent sucrose b y Herzfeld-Creydt formula with Table VI-Nonsugar
Direct a n d N o n s u g a r Acid Invert Polarizations of Cossettes (Values gi\ en are averages of all available data) Idaho Nebraska Colorado
(1923)
(1923)
(1922)
16.5g5
14.61g
15.133
16.562 +0.033
14.680 -0.061
15.25 4 -O.lO1
-4.489 -4.470 +0.Ol9
-4.748 -4.669 +0.079
v.
Obser\-ed direct polarization Direct polarization due to sucrose and raffinose Nonsugar direct polarization
Acid invert polarization due to hydrol-5,234 ysis of sucrose and raffinose Obsened acid invert polarization Nonsugar acid inbert polarization
-5.148 +0.086
v.
a
v.
In Table VI the acid invert polarizations due to hydrolysis of sucrose and raffinose have been calculated by applying the sucrose and raffinose hydrolysis constants of the Creydt formula as given by Browne and Gamblelo to the direct polarizations of these sugars calculated from the enzyme data. The nonsugar direct polarizations and nonsugar acid invert polarizations are thus obtained, and represent the amounts by which the direct and invert polarizations in the usual acid hydrolysis method vary from the polarizations due solely to sucrose and raffinose and their hydrolytic products. The nonsugar acid invert polarization is positive in all cases and the nonsugar direct polarization is negative in two instances and positive in the third. Table VII-Nonsugar
Direct a n d N o n s u g a r Acid Invert Polarizations of Molasses Produced (Values given are averages of all available data) Idaho Nebraska Colorado
($923) V.
Observed direct polarization
~56.75~
Direct polarization due to sucrose and raffinose
(1923)
v.
(1922)
v.
55.415
53.64 4
58.071
57.178
Nonsugar direct polarization -1.3l3 Acid invert polarization due t o hydrolysis of sucrose and raffinose -13.645 Observed acid invert polarization 13.579
-l.763
55.49 7 -1 852
-
Nonsugar acid invert polarization
+0.066
-14.075
-15.1l5
- 14.loo
- 15.103
-0.OZj
+O.0l2
Table VI1 gives similar data for molasses produced. In molasses the nonsugar direct polarization was always found to be negative. The nonsugar acid invert polarization is quite small and is positive in two instances and negative in the third. A number of investigators’* have given attention to the probable influence of the optically active amino acids of the sugar beet on the precision of determination of sucrose in beet sugar factory products. Certain of these amino acids in the presence of an acid such as hydrochloric are known to exhibit a considerable degree of dextrorotation, whereas, when alone in water solution, their polarization may be practically nil, slightly dextrorotatory, or even levorotatory. This change in polarization probably accounts to a great ex18
Ehrlich, Z . Ver. deut. Z u c k e r i n d . , I S , 809 (1903); Andrlik and Stanek.
Z.Z u c k e r i n d . Bohman, 31, 417 (1906-7); Smolenski, Z.Ver. deut. Z u c k e r i n d . , 60,1215(1910); 6 2 , 791 (1912); Browne, THISJOURNAL, 13,793 (1921).
0.839 divisor” 48.54 49.92 Per cent sucrose by Browne and Gamble (Creydt) formula with 0.8396 divisor 48.61 50.00 Per cent sucrose by enzyme method with 131.7 inver48.87 50.42 sion constant Per cent raffinose b y enzyme method 1.02 1.28 Per cent raffinose by all acid formulas Nil Nil 48.87 50.42 Direct sucrose polarization 1.89 2.37 Direct raffinose polarization Direct sucrose and raffinose polarization 50.76 52.79 -3.23 -3.79 Nonsugar direct polarization Acid invert polarization due t o hydrolysis of sucrose -14.94 -15.20 and raffinose Observed acid invert polarization -16.37 -16.78 -1.43 -1.58 Nonsugar acid invert polarization As directed in “Methods of Analysis,” The Great Western Sugar Company.
Table VI11 shows results of analysis of 1919 and 1923 residual molasses from a California beet sugar fact,ory. The 1923 sample represents final molasses from four years’ operation of the Steffen desugarization process without discarding. It will be noted that, contrary to prevalent opinion, California molasses contains an appreciable amount of raffinose. The presence of raffinose is not indicated by the acid formulas, because the direct polarization is smaller than the indicated per cent sucrose, this being due to the unusually large negative value of the nonsugar direct polarization. Furthermore, while the nonsugar polarization value decreases considerably in the acid invert polarization, it by no means approximates zero as was the case with the Idaho, Nebraska, and Colorado molasses. It is also noted that both the direct and acid invert nonsugar polarization are higher in the 1923 than in the 1919 molasses. The long uninterrupted operation of the Steffen process in this California factory with accompanying accumulation of substances producing a negative direct polarization indicates that the substances which are responsible for the nonsugar polarization-or a t least for the negative constituent thereof-play little or no part in the necessity for discarding molasses which is frequently encountered in the operation of the Steffen process. As nearly as can be calculated from the analytical results based upon products from a factory using the Steffen process where both “foreign” and “home” molasses are worked, the greatest proportion of substances producing nonsugar direct polarization which are eliminated passes into the Steffen waste water. The average nonsugar direct polarization of the waste water was -0.0680 for Idaho, -0.1030 for Nebraska, and -0.1220 for the Colorado data. These values vary in the same order as the average nonsugar direct polarizations of molasses “produced.” Possibly a small proportion of these substances is present in the first carbonation lime cake. A portion of the substances which produce nonsugar direct polarization is returned to the process with the hot and cold lime saccharates. That this is due primarily to inefficient washing is very doubtful, for the increase in organic nonsugar compounds is more apparent the more highly a molasses is ‘kteffenized”-i. e., the greater the number of Steffen cyclesand the character of the waste water changes in such a manner as to indicate a decrease in organic nonsugar compounds, especially those that are optically active. The following tabulation shows the average nonsugar direct polarization of carbonated saccharate milk:
INDUSTRIAL AND ENGINEERING CHE.VISTRY
246
O
Direct sucrose polarization Direct rafFnose polarization
Observed direct polarization Nonsugar direct polarization
v.
51.115 2.630
-
53. 745 53. 333
-0.412 or 0.81 per cent
on sucrose polarization as compared with 3.19 per cent for molasses worked.
Analysis by the enzyme method shows that the direct polarization of the waste water from Steffen molasses more nearly approaches the actual percentage of sucrose the greater the number of Steffen cycles represented in the molasses. In the Idaho factory, where there was an excess of molasses worked, the average ratio of apparent sucrose (direct polarization) to actual percentage of sucrose in Steffen waste water was 1:1.14, whereas in Nebraska (and in Colorado in 1922), with an excess of molasses produced, the average ratio was approximately 1:1.33. Quite different results were obtained in both Idaho and Nebraska in 1923 than were found in Colorado in 1922, when the “unaccountable” sucrose losses during factory operation were exceptionally low and practically the same by both the apparent and true purity methods, which indicated that the portion of unaccountable losses due to analytical variation in the polarization of nonsugar substances present in the factory liquors was practically negligible. I n 1923, with a much greater difference between percentage of sucrose and “apparent” sucrose in cossettes, a greater ratio of raffinose to sucrose, and larger unaccountable losses, the difference between the unaccountable losses calculated by the two methods was likewise considerably greater. The difference between the unaccountable losses by the apparent and enzyme true purity methods is, in the writers’ opinion, due primarily to variation in the polarization of the optically active nonsugar substances, which may be present to a variable extent, depending upon the character of the beets; the latter may vary considerably from season to season even in the same region. The writers feel justified in assuming that the technical operation of the three factories mentioned was so well standardized that the differences discussed above may be attributed primarily to differences in the character of the beets. Table IX summarizes the unaccountable sucrose losses based upon apparent and enzyme true purities for three periods of the Colorado 1922 data and “final-to-date” extraction statements covering the Idaho and Nebraska 1923 data. T a b l e IX-Unaccountable Sucrose Losses Calculated on Basis of Apparent P u r i t y a n d E n z y m e M e t h o d T r u e P u r i t y Colorado, 1922 Per cent on beets Per cent on sucrose I Apparent purity 0.08 0.51 Enzyme method true purity 0.02 0.11 I1 Apparent purity 0.15 0.96 Enzyme method true purity 0.10 0.66 I11 Apparent purity 0.05 0.34 Enzyme method true purity 0.13 0.89 Idaho, 1923 Apparent purity 0.40 2.39 Enzyme method true purity 0.31 1.90 Nebraska, 1923 2.92 Apparent purity 0.42 Enzyme method true purity 0.24 1.60
In all cases except the third period of the Colorado data the unaccountable loss was less by the enzyme true purity than by the apparent purity method. In the third Colorado period, sucrose in process at the end of the campaign was determined by acid inversion instead of direct polarization in agreement with the procedure followed by the factory laboratory; this fact may be of some significance in connection with the relation between unaccountable losses by the two methods during this period.
Vol. 17, No. 3
There has been much discussion regarding the extent to which unaccountable sucrose losses in beet sugar factory operation are real and the extent to which they are the r e sult of errors in sampling and analysis. Since the writers’ comparative determinations were in each case made upon the same sample, errors of sampling are eliminated from the comparison. I n comparing unaccountable losses by the direct polarization and acid inversion methods on the one hand and the enzyme method on the other, it is to be kept in mind that the difference in unaccountable losses arises from the variation in the error of the direct polarization and acid inversion methods and not from the absolute error. Comparing these losses by apparent purity and enzyme true purity as given in Table IX, and considering the fact that analytical errors due to variation of nonsugar polarization during analysis are eliminated by the enzyme method, the writers conclude that variation in the polarization of nonsugars and variation in the difference between degrees Brix and total solids by drying accounted for 10 to 40 per cent of the unaccountable losses based on apparent purity. This conclusion is based on data obtained during two campaigns a t three factories. Under other conditions, especially with regard to character of the beets, this percentage may conceivably vary to a great extent; the effect of storage on the beets is apparently of great importance in this respect. Conclusions
It is hoped that greater attention will be given to the use of enzymes for analytical determination of such sugars as sucrose, raffinose, maltose, and lactose. If only a fraction of the time expended in more or less futile elaborations of acid inversion methods had been devoted to a study of enzymes as analytical hydrolysts, the status of hydrolytic methods of sugar analysis would be greatly in advance of their present-day position. The specificity of enzymes is far superior to that of acids as hydrolytic agents and they do not influence the o p tical activity of compounds other than those upon which they act as specific hydrolysts. There is no danger of decomposition of fructose during invertase and melibiase hydrolysis, and the tedious, painstaking control of temperature required in rapid acid inversion methods is not necessary. Invertase as an analytical reagent can now be purchased in quantity in the United States as readily as hydrochloric acid. Even from the standpoint of rapidity of action, results comparable to acid hydrolysis can be obtained, as has been shown in the rapid forms of the invertase and invertase-melibiase methods. If similar preparations of the enzymes maltase and lactase were available, they, in conjunction with invertase, melibiase, and diastase, would make possible the analysis of even complex sugar and starch mixtures with a considerable degree of precision. Note-The following comments are added by way of explanation of certain points involved in the foregoing discussion. In the writers’ calculations no correction has been made for volume of lead precipitate, owing t o the fact that their primary purpose was to compare the enzyme method with the acid inversion method as ordinarily used. For work of higher precision, it is of course necessary to make this correction, and this precaution should be included with the various factors affecting the accuracy of the method which have already been discussed. Evidence exists that some optically active nonsugars are eliminated by the basic lead acetate clarification. In the enzyme method both the direct and invert polarizations were preceded b y clarification with basic lead acetate, and were made at the same degree of acidity, Since, in the acid inversion method, a basic lead acetate clarification precedes the direct but not the invert polarization, part of the error of this method may be due to this factor as well as to the influence of the varying reaction on the polarization of optically active amino acids as already discussed.
Import Duty on Gasoline, Benzene, Naphtha, Etc., IncreasedThe duty on refined petroleum oils, such as gasoline, benzene, naphtha, and benzoline, imported into the Fiji Islands from all sources has been increased from 2 d. t o 3 d. per gallon.
