acteristic of 1,l-disubstituted ethylenes. There is little or no overlap in this case, so no subtractive correction is necessary. To perform an analysis for 1,2-disubstituted ethylenes and 1,l-disubstituted ethylenes in a n a-olefin, it is only necessary to draw an integral of the region from 4.5 to 6.2 p.p.m. The steps in the integral are read off, and correction is made for the overlap around 5.4 p.p.m. Either the high-field or low-field signals may be used as a measure of the a-olefin. Normalization, keeping in mind the number of vinyl hydrogens in the various olefinic types, gives the mole per cent of each component type present. Some results obtained by this method on a set of standard samples are given in Table I. The results are somewhat in error for high concentrations of 1,2disubstituted ethylenes. Presumably this is caused by some low intensity signals in the 1,2-disubstituted ethylene spectrum that appear rather far out from the principal signal a t 5.35 p.p.m. No attempt was made to correct for this effect, since there was no interest in a-olefin samples with that much impurity. Trisubstituted ethylenes show vinyl hydrogen signals centered around 5.2 p.p.m. These signals would thus overlap ~ i t both h the high-field a-olefin and 1,2-disubstituted olefin signals. Trisubstituted ethylenes would be detected in the XblR spectrum, but their presence would render quantitative analysis quite difficult. Trisubstituted ethylenes were not present in detectable quantities in the samples for which this method was developed. Table IT compares some infrared and KMR data on a set, of unknown samples. The infrared results are by an unpublished method ( 5 ) . These samples
I S VALUES HO
-
Figure 3. Vinyl region of the NMR spectrum of 1-hexene with 6.18 mole per cent of 2-methyl-1 -hexene added
were all narrow distillation cuts, and thus each represents a single molecular weight. Trisubstituted ethylenes were not visible in the infrared sDectra. Where analyses on broad-range samples ivere required, the NMR method was used exclusively.
(4) Paulsen, P. J., C o o k
Palo Alto, Calif., urivate communicaI
LITERATURE CITED
(1) Bothner-By, A. A., Naar-Colin, C., J . Am. Chem. SOC.83,231 (1961). (2) C'hem. Eng. Xews 41,48 (1963). (3) Jungnickel, J. L., Forbes, J. W., AKAI,.CHEM.35,938 (1963).
w.D., Ibid., 36,
A. S., Smith, H. F. Continental Oil Co.. Ponca Citv. " , Okla.. Drivate communication. 1961. ( 6 j Saier, E. L., Cousins, L. R.,B a s h R., ANAL.CHEY, 35,,2219 (1963). ( 7 ) Shoolery, J. N., Varian Associates, 1713 Rosenberg, (1964).
_
tion, 1962: (8) Suatoni, J. C., ANAL. CHEM. 35, 2196 (1963). ( 9 ) Swalen, J. D., Reilley, C. A., J . Chem. Phys. 3 7 , 2 1 (1962).
RECEIVEDfor review August 12, 1965. Bccepted October 4, 1965.
Determination of the True Sucrose Content of Sugar Beets and Refinery Products by Isotope Dilution M. J. SIBLEY, F. G. EIS, and R. A. McGlNNlS Spreckels Sugar Co., Woodland, Calif.
b A general method is described for the determination of sucrose which is specific, accurate, and free of the bias inherent in most conventional analytical methods based on the measurement of optical rotation. Over a considerable range of sucrose concentrations, and in samples widely differing in types and levels of impurities, an accuracy of 0.1-0.2% of the amount of sucrose in the sample is realized. Applications to other sucrose-containing substances are suggested.
95696
M
OST CONVENTIONAL ANALYTICAL METHODS for the determination
of sucrose depend upon polarization measurements. These measurements are unreliable in the presence of optically active nonsucrose constituents (1-4). The desirability of a convenient analytical method specific for sucrose has been recognized for many years. I n 1959 Hoerning and Hirschmueller (6) described an application of the isotope dilution principle to the determination of sucrose in sugar beets. Their procedure requires 3-5 days t o
accomplish and, in addition to the mixing of the labeled sucrose with the sample and the final counting of the standards and samples, includes the following steps: freeze-drying a t -60' C., Soxhlet extraction over sodiumdried diethylamine, the formation of a sucrose-copper sulfate complex and a collidin-copper sulfate addition compound, recovery of the sucrose by crystallization from ethyl alcohol, and purification of the sucrose by repeated recrystallizations. Vnfortunately, the elaborate detail of their procedure VOL. 37, NO. 13, DECEMBER 1965
1701
limits its usefulness to specialized applications. The objective of this investigation was the development of a more convenient isotope dilution procedure, sufficiently general to apply to most sucrose-containing materials, and capable of routine application. The original analytical procedure, reported here, can be accomplished within a 24-hour period with accuracy of 0.10.2% of the amount of sucrose in any particular sample. The processing of the samples, intermediate between the mixing of the labeled sucrose with the samples and the counting, is accomplished in three straightforward steps: a simple barium precipitation and carbonation, purification by ion exchange treatment, and crystallization from ethyl alcohol. I n addition, the method of comparing standards and samples and of preparing planchets assures optimum counting conditions with respect to uniform geometry and absorption corrections. This procedure has been successfully applied to a variety of sucrose-containing materials, as well as sugar beets, and has proved to be reliable, precise, accurate, and acceptable for routine application. EXPERIMENTAL
Apparatus. COUNTER.A XuclearChicago automatic proportional beta counting system, consisting of a Model 447 gas flow detector, equipped with a n 8764 transistorized amplifier-discriminator and “micromil” window, a 1040 automatic sample changer, 8181 decade scaler, and 8430 printing timer, was used. T h e counting gas composition was 10% methane, 90% argon.
Table
I.
PLANCHET PRESS. il planchet press was designed which utilizes 1.0025 inch i.d. planchet retainers machined to i~0.0003inch in diameter and thickness. To permit counting at infinite thickness, 0.6 gram of sucrose per planchet is used. To prepare a planchet, the retainer is placed on the highly polished anvil of the press where it is held securely by the press body and cylinder. The sucrose sample is then transferred to the press cylinder and the piston is inserted. The sucrose is pressed into a retainer under a pressure of 5000 p.s.i for 1 minute. The resulting planchet has a precisely defined surface area which is in the plane of the retainer surface. When placed into specially designed planchet cups which are machined to equally close thickness tolerances, the requirement of essentially constant geometry is met, and the sample is ready for counting. Reagents. S U C R O S E - C ~ (uni~ formly labeled). Specific activity 5-15 mcuries/mmole, Nuclear-Chicago Corp., Des Plaines, Ill. Standards. The radioactive standard source is prepared by diluting 0.5 mcurie of sucrose-Cl4 (uniformly labeled) with 100 grams of reagent grade nonradioactive sucrose (sucroseC12). This dilution assures a n apparent count rate greater than 10,000 c.p.m. after recovery from the samples. The s u ~ r o s e - C and ~ ~ sucrose-C12 are dissolved in distilled water, the solution is concentrated on a rotary vacuum evaporator, and crystallized from absolute ethyl alcohol. As prepared, this standard sucrose is considered chemically pure but may contain a few parts per million of radioactive nonsucrose. Procedure. Accurately weighed portions of the standard are added t o the samples. The following mixing procedure has been successful for sugar beets. The sugar beets are
reduced t o a fine-particle-size pulp by being passed through a multibladed circular saw. =Iccurately weighed, 15- t o 20-gram portions of the finely divided, well mixed pulp are placed into half-pint Mason jars equipped with Waring Blendor stirring attachments. To each jar is added 2 t o 3 grams of the accurately weighed standard sucrose, 0.5 gram of Ca(OH)2, 2.0 grams of filter aid, and 100 ml. of distilled water. The contents of the jars are mixed on the Waring Rlendor at high speed for 5 minutes. This procedure assures homogeneous mixing of the standard sucrose with the sucrose in the samples and a good extraction of the mixture. The remaining steps in the procedure are identical for all materials. The mixture is filtered through a 150ml. capacity, medium-porosity sinteredglass funnel. Barium hydroxide is added to the filtrate a t 60’ C. For samples containing approximately 4 to 6 grams of total sugar in 100 ml., 20 grams of Ba(OH), are sufficient to produce a good precipitation. The precipitate usually appears after 3 minutes’ elapsed time, and the precipitation is allowed to continue for a t least 15 minutes. The barium saccharate precipitate is separated by filtration, washed tryice with small amounts of hot, saturated Ba(OH), solution, and the filtrate is discarded. The precipitate is made into a slurry with about 75 ml. of distilled water, brought to 80’ C., and carbonated with CO, to a phenolphthalein end point. This step removes the barium as the barium carbonate precipitate. The clear sucrose solution is separated from the barium carbonate precipitate by filtration through a 150ml. capacity, medium porosity-sintered glass funnel. The barium carbonate precipitate is washed with about 10 ml. of distilled water and discarded. The described precipitation with
Determination of Percentage Sucrose in Sugar Beets and Refinery Products
Sample Material analyzed
No. 1
No. 2
No. 3
KO. 4
R
14.04 i4.09 14.07 f uz = 14.07 =t0.02
14.45 14:40 14.38 14.41 i 0.03
15.48 15.47 15.53 15.50 =I= 0.02
13.94 13.99 13.97 13.97 i 0.02% sucrose
r7
9.20 9.19 9.21 f uz = 9.20 i 0.01
8.56 8.58 8.53 8.56 =I= 0.01
9.13 9.18 9.14 9.15 k 0.02
9.35 9.43 9.36 9.38 f 0 . 02% sucrose
Sugar beets
Sugar beet crowns
13.03
Thin juice
12.98 r? f uz = 13.01 i 0.02
Molasses (beet)
0
11.99
16.50
12.00 =t 0.01
16.48 16.49 =t 0.01
14.60 -
54.46 54.41 54.44 i 0.06
50.58 50.53 50.56 f 0.05
51.95 52.03 51.99 i 0.037, sucrose
8 i %c
-
2 f 6,
65.26 65.11 65.22 65.09 = 65.17 i 0.04
61.78 61.61 61.82 61.58 61.70 i 0.07% sucrose
ANALYTICAL CHEMISTRY
-
-
14.62
12.00 -
54.94 54.98 = 54.96 f 0.03
High remelt syrup (cane sugar)
1702
-
-
-
~
14.61 =!= 0.0170 sucrose
barium hydroxide followed by carbonation is repeated on this filtrate. At this point, the sucrose solution is treated with activated carbon, Korit SG-1, approximately 2% of the total amount of sucrose in the sample, and filtered through a 0.45-micron HA Millipore filter. The sucrose solution so obtained is cooled to less than 20" C. and passed through an ion exchange column consisting of 50-ml. bed volumes of Duolite -1-7 and Amberlite IRC-50 separated by a cotton plug. The flow rate in the ion exchange column is regulated to about 5 ml. per minute. The first 50 ml. of the effluent is discarded, and the remainder collected and washed by passing deionized water through the column until the refractometric dry substance (r.d.s.) of the effluent becomes less than 0.29?,. The effluent from the ion exchange column is concentrated to a thick syrup, a t least 70 r.d.s., on a rotary vacuum evaporator, and the sucrose in the concentrated solution is recovered by crystallization from absolute ethyl alcohol. After filtering and washing with small amounts of absolute ethyl alcohol and acetone, the purified sucrose obtained is repurified by repeated recrystallizations from absolute ethyl alcohol. After one crystallization and two recrystallizations, an average yield of about 507, of the original total sucrose has been obtained. This high-purity sucrose is dried in a vacuum oven for 4 hours a t 50" C. The dried sucrose is then placed in a vacuum desiccator to cool. Once cooled to room temperature, the sucrose is made into planchets, as described previously, and counted. Purification by recrystallization is ( > o n h u e duntil, on repeated counts, a constant count rate is achieved on the sample (within allowable statistical error limits). I n addition to processing the samples through the above-described procedure, a set of counting standards is similarly processed in which the ratio of the radioactive sucrose to nonradioactive sucrose is adjusted to be equal to the ratio expected in the sample. This is accomplished easily by, first, estimating the sucrose content of the sample by conventional methods-e.g., polarization-and then diluting the standard source with the appropriate amount of nonradioactive sucrose. Thus, the counting rate of both the standard and sample are sufficiently close to minimize errors due to background and absorption corrections. By this expediency, the sucrose recovered from the standard is processed similarly to the sucrose recovered from the sample. ilfter plancheting, the amount of C14 radioactivity in the samples and counting standards is determined in the counter. Sufficient counts are taken on both standards and samples to assure the precision desired.
The sucrose content of the sample is calculated from the expression: Percentage of sucrose in sample where W*
TY, E?,
8, y
=
W,
the weight, in grams, of the standard source added to the sample = the weight, in grams, of the sample = the average counting rate of the counting standards, c.p.m. = the average counting rate of the sample, c.p.m. = the weight ratio of the counting standard to the standard source =
RESULTS AND DISCUSSION
Listed in Table I are typical results for the determination of sucrose in five materials of interest which differ considerably in impurity levels and in types of impurities. The accuracy of the isotope dilution procedure, as measured by the standard deviation, is quite high and amounts to a consistent 0.1-0.2% on the percentage sucrose in the sample. To test the isotope dilution procedure for lack of bias due to the presence of other interfering sugars, incomplete chemical uniformity, etc., portions of a molasses sample containing approximately 2% raffinose and a typical sugar beet sample were analyzed with and without known amounts of added sucrose. The results of this test are given in Table 11. For the samples with added sucrose, the percentage sucrose values are reported after subtracting the weight of added sucrose. I n addition to the criterion of purity mentioned previously-Le. , achieving constant counts on repeated recrystallizations-the purity of the final sucrose was determined by polarizing a carefully prepared normal solution (26.00 grams of sucrose diluted to 100 ml. with distilled water) of final product sucrose remaining from isotope dilution determinations on several different materials in a 200-mm. tube jacketed a t 20' C. On 10 visual readings an average value of 99.95" polarization was obtained. Allowing for slight traces of moisture and ash which are always present and the accuracy of the polarimeter, this test indicated that the final product sucrose possessed high purity. Although the isotope dilution procedure described above was developed for sugar beet and refinery products, it is sufficiently general so that it can be
Table II.
Results on Samples with Added Sucrose
Sample State Molasses Original with 27, Original plus raffinose 10% added content sucrose Original Original plus 20y0 sucrose Beet brei Original Originalplus 7y0added sucrose
% Sucrose A u; 51.2 & 0 . 1 51.1 j=0 . 1 51.4 i 0 . 1 51.5 i 0 . 1
11.8 i 0.02 11.9 rt 0.03
readily adaptable to the determination of sucrose in many other sucrose containing materials of interest-e.g., sorghum, palm sap, sugar maple sap, etc. The conditions which must be met for analyzing other sucrose containing materials are: the original sample must contain enough sucrose to yield sufficient material after isolation and purification for radiochemical analysis, and a method must be devised for homogeneous mixing of the radioactive standard source and the sucrose in the sample. For most sucrose-containing materials, the first condition may be met by simply taking a sufficiently large sample. The second condition may be met by utilizing sufficiently vigorous mixing equipment. Provided no unusual interfering constituents are present, the remaining steps for processing other sucrose-containing materials should remain identical. ACKNOWLEDGMENT
The authors thank W. 0. Bernhardt, Research Associate, Spreckels Sugar Co., Woodland, Calif., for the design of the planchet press, J. J. Brodie for technical assistance, and the Spreckels Sugar Co. for permission to publish this paper. LITERATURE CITED
(1) Bates, F. J., NBS Bulletin CWI (1942). (2) "Beet Sugar Technology," R. A. McGinnis, ed., p. 518, Reinhold, New
York, 1951. (3) Cane Sugar Handbook, G. P. Meade, ed., 9th Ed., p. 402, Wiley, New York, 1963. (4) Hirschmueller, H., Hoerning, H., 2. Zuckerindustrie 84, 389 (1959).
(5) Hoerning, H., Hirschmueller, H., Zbid., 84, 499 (1959).
RECEIVED for review July
Accepted September 27, 1965.
VOL. 37, NO. 13, DECEMBER 1965
26,
1965.
1703
