Photometric Determination of Glucose in Presence of Fructose FRED STITT, STANLEY FRIEDLANDER', HAROLD J. LEWIS2, and FRANK E. YOUNG Western Utilization Research Branch, Agricultural Research Service,
A method has been developed for measuring 0.05 to 0.5% of glucose in fructose. Sodium chlorite solution buffered to pH 4.0 is used to oxidize glucose at a much faster rate than fructose. Chlorine dioxide, a product of the reaction, is measured with a spectrophotometer or colorimeter. The difference in chlorine dioxide produced by oxidation of the test sample and of a suitable reference fructose sample is a measure of the glucose content. Recrystallization of fructose as dihydrate is shown to produce a suitable aldose-free reference material. The method can be used over the entire range of composition of glucose-fructose mixtures and it appears to be generally applicable to the measurement of aldoses in the presence of ketoses. Analyses by the spectrophotometric procedure of glucose-fructose samples of known composition showed standard dsviations of about 0.003% glucose for samples containing less than 0.5% glucose, about 0.03% glucose for samples containing 0.5 to 5% glucose, and about 1%of the glucose content for samples containing over 5% glucose. The corresponding standard deviation for results by the colorimetric procedure were greater by a factor of 1.5 to 2. These samples covered a range from 0 to 100 mg. of fructose and 0.05 to 1 mg. of glucose per ml. of the test solution.
D
URIXG an investigation of the use of fructose dihydrate in
thepurificationof fructose(I6,I 7 ) , a need arose for amethod by which small amounts of the principal impurities, glucose and mannose, could be determined in the presence of fructose. The usual methods (3, 5, 7 ) lacked the desired sensitivity and selectivity and, moreover, employed alkaline reagents which caused a portion of the fructose being analyzed to transform t o glucose and mannose during the analysis (3,7 ) . Notatin (glucose oxidase), which catalyzes the oxidation of glucose in acid solution, is said to be specific for glucose (IO), but it does not catalyze the oxidation of mannose ( I ) and appears t o be slightly less sensitive than the method reported here. Jeanes and Isbell (9) observed that chlorous acid oxidizes aldoses much more rapidly than ketoses, forming chlorine dioxide as one of the products. Launer, Wilson, and Flynn ( I S ) confirmed this observation and used chlorous acid as an oxidizing agent in developing both iodometric and photometric methods for determining glucose in the absence of ketoses. The authors have adapted their photometric method to the determination of smalI amounts of glucose in fructose. The glucose content is measured by the difference in chlorine dioxide produced in a definite period of time in a reaction mixture containing the sample of interest and in a similar reaction mixture containing standard reference fructose. The present work was primarily concerned with glucose, because mannose is likely to be present a t much lower concentration (14). Mannose, when present, wiII be included with glucose as aldohexose.
U.S. Department of Agriculture,
Albany 6, Calif.
dioxide-via., disproportionation of chlorous acid ( 2 , 9, 13, IS, I6), oxidation of glucose by chlorous acid (9, 13, IS), and oxidation of fructose by chlorous acid. Because the stoichiometry and kinetics of reactions involving chlorous acid have been found to vary with conditions (2, I;), kinetic studies n-ere made for the temperature and concentration ranges of interest to provide an adequate basis for choosing suitable reaction conditions for an analytical procedure. The results are briefly summarized here in so far as they supplement earlier kinetic aork. Reactions were followed by photometric measurement of the rate of formation of chlorine dioyide with either a colorimeter or a recording spectrophotometer at fixed wave length. Except where otherwise noted, reactions were carried out a t 25.0" C. in acetic acid-sodium acetate buffer solutions ivith total acetate concentration of 1.5X. Because the acid dissociation constant of chlorous acid is about a t 25" C. ( 2 ) , most of the chlorite is present as salt in the pH range of 3.7 to 1.7. The differencr in the rates of formation of chlorine dioxide in two reaction mixtures initially identical, except for the presence of sugar in one and not in the other, was used in studying the kinetics of oxidation of glucose and fructose by chlorous acid. Disproportionation of Chlorous Acid. This reaction is complex, but under conditions used in this investigation can probably be most simply approximated by the stoichiometric equation ( 2 ) :
At p H 4.3 the order of the reaction forming chlorine dioxide was found t o be 1.9 with respect t o total chlorite concentration when the latter was varied between 0.006 and 0 2 5 X . Slightly lower values were found a t pH 3.7 and pH 4.7, but these results are in essential agreement with second-order dependence found by Barnett (g) and by Launer, Wilson, and Flynn (IS). On the other hand the formation of chlorine dioxide appeared to be nearer to first order with respect t o [ H +], hydrogen ion concentration, than second order, the numerical values falling between 1.0 and 1.2. Experiments a t an initial chlorite concentration of C, = 0.075Mshowed approximately the same rate and dependence on pH when 1.534 citrate buffer was used in place of acetate buffer. Although a t fixed pH of 4.0 and 4.2, a change in acetate buffer strength produced marked changes in rate in the same direction, addition of sodium chloride ( 1 . O M ) or sodium sulfate (0.5M) to increase the ionic strength of 1.5L1f acetate buffer (pH 4.2) produced no rate changes not accounted for by the accompanying shift in pH. Rate data obtained a t 19.9", 25.0°, and 40.0" C., at pH 4.2, and C, of 0.075 and 0.025M gave an apparent activation energy of 20,000 calories per mole and confirmed the secondorder dependence on total chlorite concentration. Because this apparent activation energy is calculated from data for constant total chlorite concentration, it is a combination of the heat of dissociation of chlorous acid and the apparent activation energy for the reaction involving chlorous acid as a reactant. Oxidation of Glucose. Jeanes and Isbell (9) suggested the following equation for the oxidation of glucose by chlorous acid :
CHOICE OF REACTION CONDITIONS
When sodium chlorite, glucose, and fructose are brought together in acidic aqueous solution, a t least three simultaneous reactions occur which consume chlorite and produce chlorine 1 f
Present address, Applied Research Laboratories, Glendale, Calif. Present address, University of Minnesota, Minneapolis, Minn.
The validity of this equation is established by the authors' results and by those of Launer et al. ( I d , IS). The reaction was studied over the same range of variables as the disproportionation reaction, The rate of formation of chlorine dioxide due to oxidation
V O L U M E 2 6 , NO. 9, S E P T E M B E R 1 9 5 4 of glucose a t pH of 3.7, 4.3, and 4.7, showed good agreement with first-order dependence on total chlorite, 0.006 t o 0.25M, and on glucose concentrations, the values of the latter being 0.5, 1, and 2 mg. per ml. On the other hand, the dependence on [H+] appeared to be definitely less than first order, the numerical values ranging from slightly less than 0.7 to nearly 0.8. The effect of changing buffer strength, adding salts to the buffer, or changing from acetate to citrate buffer were qualitatively the same as for the disproportionation reaction. An apparent activation energy of 16,500 calories per mole was found. Oxidation of Fructose. The stoichiometry of the oxidation of fructose by chlorous acid was not investigated, but approximately 3000 times as much fructose was required in a reaction mixture a t 25' C. to produce chlorine dioxide a t the same rate as a given concentration of glucose. The comparatively little kinetic data which were obtained on the fructose reaction showed approximately first-order dependence on fructose and total chlorite concentrations, the same dependence on p H as the oxidation of glucose, and an apparent activation energy of the order of 23,000 calories per mole.
325
315
495
WAVE LENGTH, p
Figure 1. Absorption Spectra A. B.
C.
1479 tion in the 18-hour reaction period of about 500 times as much chlorine dioxide due to oxidation of glucose as to oxidation of fructose for the same initial concentrations of the two sugars. ANALYTICAL METHOD
Reagents. Sodium chlorite solution, 0.60M, is prepared by dissolving the calculated amount of Mathieson "analytical" grade salt, allowing for the stated purity. The solution can be standardized precisely by iodometric titration, but need be within only about 1% of the nominal value. It is filtered through sintered glass, if necessary, and stored in the dark. Acetate buffer, 6M, is prepared by dissolving 590 grams of 99.5% (glacial) acetic acid and 302 grams of sodium acetate trihydrate in water and diluting to 2 liters. The p H should be 4.00 .t,0.05. Because slightly different oxidation and disproportionation rates have been observed with different buffers of the same concentration and pH, it is advisable where possible to prepare successive buffer solutions from the same stock reagents and to use the same buffer solution for all samples and controls in any one series of analyses. Size of Sample. The size of the sample is chosen so that the photometric measurement of the chlorine dioxide concentration can be made with reasonable accuracy. For samples containing less than 0.57' glucose, concentrations of 100 mg. per ml. are recommended for both the sample in the test solution and the reference fructose in the control solution. Thrse concentrations are each made 10 mg. per ml. if the glucose content is between 0.5 and 5%. No fructose is used in the control and 0.6 mg. of sample per ml. of test solution is recommended for samples concontents refer to solid taining over 5% glucose. These glucose samples on the dry basis. Procedure. To 12.5 ml. of 6M acetate buffer in a 50-ml. volumetric flask are added the sugar sample and water to a total volume of about 40 ml. When the reaction is to be started, 5.00 ml. of 0.60M sodium chlorite are added, the mixture is diluted to 50 ml. and well mixed. Reaction tubes for replicate specimens are filled immediately and placed in the dark in a constant temperature bath a t 25" C. (Colorimeter tubes, 15 nun. in diameter, modified to accept standard taper glass stoppers, are used as reaction tubes. Results are not affected if the tubes are not completely filled). The control solution is prepared in the same way except that the recommended amount of reference fructose is added instead of test sample. After the 18-hour reaction period, the chlorine dioxide concentrations are measured photometrically without opening the reaction tubes and with minimum exposure to light. Blthough results are very insensitive to changes of an hour or two in reaction period; this period must be the qame within a few minutes for each test solution and its associated control.
0.0015M ClOz in water solution
0.06M NaClOz No. 42 filter
D. 0.00073iM KzCrOa (in 0.05.M NaOH)
Choice of Reaction Conditions. By varying temperature, pH, chlorite concentration, buffer strength, and reaction time, the relative amounts of chlorine dioxide produced by each of the three reactions discussed can be varied over wide limits. A reaction temperature of 25 O C. was chosen because the advantages of working near room temperature outweigh the relatively small improvements obtainable a t other temperatures in the proportion of chlorine dioxide produced by oxidation of glucose. Because the chlorite dependence of the rate of the disproportionation reaction is higher than that of the rates of sugar oxidation reactions, chlcrite concentration was chosen as low as seemed consistent with other requirements. A convenient reaction period corresponding to practically complete oxidation of glucose, ample buffer capacity, suitable excess chlorite capacity, and chlorine dioxide concentrations suitable for photometric measurement are the other criteria which were applied in picking 25.0' C., 1.5M acetate buffer of pH 4.00, C, = 0.060M sodium chlorite, and reaction time of 18 hours for standard reaction conditions. Under these conditions the disproportionation reaction produces chlorine dioxide a t an initial rate of about 0.000143M per hour, the period for Soyo oxidation of glucose is approximately 2 hours, and 100 mg. per ml. of fructose produces chlorine dioxide a t an initial net rate of about 0.000132-W per hour. This corresponds to produc-
Spectrophotometric Measurement of Chlorine Dioxide Concentration. A Beckman Model DU spectrophotometer was used in this investigation. Aqueous solutions of chlorine dioxide show an absorption maximum at 358 mp ( A , Figure I ) , but because of the absorption of sodium chlorite ( B , Figure 1) in this region, a higher wave length should be used for spectrophotometric measurement of chlorine dioxide in reaction mixtures In order to eliminate errors in wave length calibration and shift in wave length a t a fixed wave length dial position due to changes in temperature of the the monochromator, much of the development work was done with the 435 8 or 404.7 mp lines of a mercury arc source such as is available for wave length calibration of the instrument. However, the convenience of the tungsten source, resulting from its greater stability, led to its use at 436 mp in later work. When the tungsten source is used, the calibration should be checked against the mercury arc, and the wave length dial should always be set precisely and from the same direction. Use of either the 435.8 mp mercury line or the tungsten source of 436.0 mp is recommended. A t these wave lengths, slit widths up to 1.0 mm. for the mercury line or up to 0.5 uith the tungsten source can be used without introducing errors in the absorbance measurements. The absorption coefficients of chlorine dioxide in water solution at 25 O C. were determined a t various wave length. and concentrations and were in accord with Beer's law. Values of 113.5, 115.3, and 495 liter per mole-cm. were found for the molar absorption coefficients a t 436.0, 435.8, and 404.7 mp, respectively.
ANALYTICAL CHEMISTRY
1480
Figure 2.
in the reaction tube and in m absorption cell of known thicknesa. For all tubes used in this study p was approximately 1.29 cm. The tubes should be matched in both blank reading and in path length or suitable corrections applied. Colorimetric Measurement of Chlorine Dioxide Concentrations. A Klet&Summerson colorimeter was used in this study, but other instruments should be satisfactory if properly calibrated. A No. 42 filter is used, the absorbance of which is shown by cwve C of Figure 1. The instrument is operated from a constmtvoltage transformer to eliminate detectable changes in reading due to line voltage changes. The calibration curves for oolorimeter reading versus chlorine dioxide concentration in aqueous solutions, illustrated in Figure 3, should he determined for the instrument used. These curves can be obtained by iodometric titration of chlorine dioxide solutions, or indirectly, by measuring the same solutions on the colorimeter and a spectrophotometer, obtaining concentrations from the spectrophotometric data. Because the readings of the colorimeter may vary with the refmotive index of the solution measured (with round tubes), it is necessary t o determine calihration curves in the presence of buffer and fructose ooncentrations typical of the controls to be used. The magnitude of this effeotis indicated by the separation of the curve8 of Figure 3. A correction, ahout three scale units on the instrument used here, for the absorption of sodium chlorite should also be determined and added t o the scale readings in plotting the calibration curves. Check measurements with potassium chromate as the ahsorbing medium showed that this correction is nearly additive in scale units over the scale range used. Because the calibration curves of Figure 3 deviate markedly from Beer’s law, the test and control solutions are each measured separately against a reference containing water. If the tubes are not matched in hlmk readings or path lengths, corrections for these should he applied by the following equation:
Tube Compartment for Spectrophotometer
The second of these values agrees reasonably well with the value found hy Launer et al. (18) who prepared chlorine dioxide by acidifying sodium chlorite solution. The authors’ chlorine dioxide solutions were prepared by the method of B r w ( 4 )in which 150 Erama of oxalic acid, 40 mama
Ihlorine, throigh glass wool to remove spray, and into ice water containing a small amount of acetic acid. The apparatus should be all glass. The chlorine dioxide content is determined iodometrically. In order t o permit measurement of chlorine dioxide roncentrations in the reaction tubes, a vertical light-tight extension of the 1-cm. cell compartment was constructed for the spectrophotometer. This is fastened by screws to the regular compartment of the instrument and accommodates the standard cover (Figure 2). The reaction tubes fit into the four positions of the I-em. square cell carrier so that test and control reitation tubes can be pared readily either with each other or with erenee tube containing water.
R: where
= gARz
- 8,)
(4)
Ri and R, are the corrected and observed readings for
E
~
Because precision of the net chlorine dioxide urement is increased by differential absalpurvu measurements between the test and control solutions, this procedure is recommended. I n addition, t h e absorbance of the control solution is measured as a eheok on gross deviations from standard condit ionssuch as major temperature fluctuations-durii ig the reaction period. If the chlorine dioxide cone entrations of the test and control solutions are d e>.A-w m u by B , and B,, then the difference in the se coneentrar tions (the net chlorine dioxide concentration) is
Bt
Ae) - Be = (At CP
5 b
2
x ’
9 POO
(3)
where A , and A , are the absorbances of the test and control solutions, t is the molar absorption coefficient of chlorine dioxide a t the wave length used and p is the effective path length in centimeters of the reaction tube& The value of p is found by compitrison of the absorbances at m y convenient wave length of a suitable reference solution-such as potassium c h r o m a t e
0
0
...,,.--
~~~~~
E composition as in A hut 3
= 1.341
le composition a8
with fructose umoentra-
in 4, but with no frvcfosei no =
V O L U M E 2 6 , NO. 9, S E P T E M B E R 1 9 5 4
1481
Table I. Analyses of Prepared Mixtures of Glucose and Fructose Sample Set Ae
BI
Concentrations Present in Test Samples Fructose Glucose mg./ml. lIg,/ml. %b 100.0 0 050 0.050 0.100 0.100 0,330 0.329 1,000 0.991 10.0
0,050
0,100 0.330 1.000 0.300
0,050
0.99 3.2 9.1 4.8
Glucose Found (Mean Value), .\Ig./Ml. 18 hr.C 19 hr.d 0 049 0.049 0 , l O O ~ 0.100; 0,330 0,331 0.966 0.969 0,0492 0.1000
0.330 0.981 0 300h 0.301h 0 299h 0.301
0.0502 0.1016
0.328
0.979
Error in Mean Value&, Mg./Ml. Glucose 18 hr,c 19 hr.d -0.0002 -0.0001 0.0000 +0.0003 0,000 -0,034
f0.001
-0.0008 0.0000 0.000 -0,019 0.000h t0.001h -0.001h
+0.0002 +0.0016 -0.002 -0.021 -0.002h
Standard Deviation from Mean (Mg./Ml. Glucose) 18 hr C 19 hr.d 0.001 0 002 0.003
0,003 0 010 0.003 0.003 0.001 0,005
-0.031
0.005 0.005
0.015 0.005
0.003 0.005
0.009 0.004 0,003
0.001 6.0 0.298h +O.O03h 0.001 3.0 0,300 Q.1 0.303h -0.001h 0.003 0.003 0.299h 1.0 0.300 23.1 0.002 0.002 +0.002 0,302 fO.OO1 0.0 0,300 100.0 a Glucose found minus glucose present. b Percentage of sugar content which was glucose. C Spectrophotometer. d Colorimeter. e Controls containing 100 mg./ml. fructose shoved 426 X 10-534 ClOz a t 18 hours, 442 X 10-5.11 ClOz a t 19 hours. f Controls containing 10 nig./ml. fructose shoned 239 X lo-5.M ClOz a t 18 hours 250 X 10-6.M C1Oz a t 19 hours. 0 Controls containing no fructose showed 226 X 10-634 ClOz a t 18 hours, 237 X IO-5.M ClOz a t 19 hours. h Corrected for C102 evolred by fructose in sample. C3
solution in tube X ; S, is the reading of tube X filled with water, and g. is an effective path length correction factor given by (5) where K Oand K , are readings for the reference tube and tube X when filled with 0.00200M potassium chromate solution. The corrected colorimeter readings for the test and control solutions Rt' and Rd, are converted into the corresponding chlorine dioxide concentrations, B t and B,, by use of the appropriate calihration curve for the colorimeter. Because the use of Equation 4 for making colorimeter tube corrections may be open to question, g was shoiw to be essentially constant by determining it (Equation 5 ) with various concentrations of potassium chromate solution in several poorly matched tubes. Potassium chromate solution was used here for effrctive path-length measurements because of the similarity of its absorption curve to that of chlorine dioxide in the pertinent spectral region (D,Figure 1). I t has been extensively studied as a spectrophotometric standard solution (6). A tube filled with 0.00200.W potassium chromate solution Tvas found useful as a rapid check on the constancy of the colorimeter scale calibration. Calculation of Glucose Concentration. The difference in chlorine dioxide concentrations of the test and control solutions must be corrected for two effects before it represents the amount of chlorine dioxide produced by the oxidation of glucose. One of these is a slow reaction consuming chlorine dioxide (13), presumably hydrolytic disproportionation (4). This effect was estimated by measuring the rate a t which chlorine dioxide disappeared in 1.5X acetate buffer solutions of pH 4.00 at 25" C. when initially present in amounts varying from 150 to 1200 x lO-5M. A loss of 7.8 =I= 0.9% (mean deviation) was found in 16.5 hours for nine samples with no significant dependence on initial concentration. Corresponding figures for 22 5 and 90 hours were 8.9 i 0.7 and 14.9 =t2.5%, respectively. Because no chlorine dioxide is initially present in reaction mixtures, a correction of about 4.5% is estimated as applicable to net chlorine dioxide concentrations for the authors' standard reaction conditions. The other correction is required because the chlorite concentration is not the same in test and control solutions as the reactions proceed. Launer (12, I S ) has derived a simple and apparently adequate expression t o correct for this effect for test solutions containing glucose and control solutions containing no sugar. The derivation of the corresponding expression for the case
where fructose is present a t the same concentration in both test and control solutions is given below. This expression may be written as a correction factor by which the net chlorine dioxide concentration should be multiplied-namely
C,
-
CO 1.5 qBc
where C, is the initial total chlorite concentration, B, is the chlorine dioxide concentration in the control solution, and q is a factor which reduces to unity for no fructose present and which is somewhat less than unity for appreciable amounts of fructose. Values calculated for expression (6) corresponding t o observed values of B, of Table I11 for an 18-hour reaction period for controls containing 0, 10, and 100 mg. per ml. of fructose are 1.017, 1.017, and 1.057, respectively. Combining these figures with a 4.5% correction for loss of chlorine dioxide formed gives corresponding over-all correction factors, d, to the observed net chlorine dioxide concentration of 1.063, 1.063, and 1.105. These are in good agreement with the experimentally determined values 1.064, 1.073, and 1.108 based on stoichiometry of Equation 2. The concentration of glucose in the test solution is then 90.1 d ( B , - B c )mg. per ml., where ( B t B,) is the observed net chlorine dioxide concentration and d is the appropriate theoretical correction factor just discussed. iin additional correction is made for impurity of the fructose used in the control solution if the latter is not glucose-free.
-
Derivation of Correction. Correction factor for difference in rate of consumption of chlorite in test and control solutions containing fructose. Assume
where B = chlorine dioxide concentration, C = total chlorite concentration, G = glucose concentration, F = fructose concentration, a, g, f = rate constants for the respective reactions: disproportionation of chlorous acid, oxidation of glucose, oxidation of fructose, a , y , 4 = number of moles of chlorine dioxide produced per mole of chlorite consumed in the respective reactions. y = 2/3. Further, assume a t time t chosen large enough so that oxidation of glucose is virtually complete
+ 1/2 4fF(CO + C,) t Bt = a~CoCtt+ I / 2 +fF(Co + Ct)t + 2Go B, = aaCoCet
(11) (111)
where subscripts 0, c, t refer to initial value, control solution, and test solution, respectively. When t is eliminated in combining I1 and 111,we can write the result as
ANALYTICAL CHEMISTRY
1482 Table 11. Response of Various Sugars and Other Substances to Chlorous .4cid Oxidation Procedure Substance
Concentration, M g ./ M 1.
Moles of ClOn per Mole of Substances 2 00 1 90 1 98 1.92 1.98 0 0034 0 0035 0 0038 0 0047 0 00019 1 44 0 013
Glucose 0.1 Galactose 0.1 Mannose 0.1 Lactose monohydrate 0.1 Maltose monohydrate 0.1 Fructose 100 Sorbose 50 Sucrose 50 Raffinose pentahydrate 50 E t h y l alcohol 50 Isobutyraldehyde 0.158 Betaine hvdrochloride 5 Lysozymeb 2 9 5 Galacturonic acid monohydrate 0.2 2 01 a Corrected for hydrolytic loss of chlorine dioxide and difference in chlorite concentration in test and control. Except for glucose and fructose these figures are the mean results of duplicate determinstiona. b Molecular weight 14,600.
agreement with the earlier work (9, 13). The disaccharides lactose and maltose do not appear to be significantly hydrolyzed under the conditions of the analysis. Thus the chlorine dioxide evolved by these two sugars per milligram is half of that evolved by the aldohexoses. REFERENCE FRUCTOSE
Fructose esssentially free of glucose can be prepared by crystallization as dihydrate. Material prepared in this manner can be used as a reference standard in cont,rol solutions for determining the aldose (glucose-equivalent) content of a good grade of commercial fructose. The latter can then be used in controls for other analyses. Preparation. A cold (0' C.) aqueous solution (about 65% by weight,) of the best fructose commercially available is seeded with fructose dihydrate crystals (16) which are crushed in the solution and well dispersed. After crystallization a t 0" for 12 hours or more, the crystals are freed of mother liquor so far as possible by use of a Biichner funnel (without paper), a sintered glass filter, or a centrifuge. The crystals arc then washed by intimate mixing with eit,her undersaturated solution of previously purified fructose or cold water, in the latter case using about one fift,h the volume of the original solution. The n-ashed crystals are separated as before. The product may be air dried a t 0" C. or stored below 10' C. as wet crystals (about 80% fructose), or as a sirup (diluted to about, 70%). Fructose content can be found from refractive index measurement (8) or by dichromate oxidation (11).
For standard reaction conditions, identical values of q were found for the two assumed stoichiometries-via., CY
=
1/2,
= 1/2 and a =
1/2, #J = 2/3.
RESULTS WITH PREPARED SAMPLES
The analytical method applied to glucose-fructose samples of known composition gave the results shown in Table I. In this series of analyses, chlorine dioxide concentrations were measured with the spectrophotometer a t 18- and 20-hours reaction time and with the colorimeter a t 19 hours. The 20-hour data are not included as they are almost identical with those a t 18 hours. The samples were prepared from stock solutions of National Bureau of Standards "dextrose" (Standard Sample No. 41) and of fructose dihydrate containing 0.021% ' glucose-equivalent impurity oxidizable under the present analytical conditions. Test solutions in each set and the appropriate control were run in duplicate simultaneously. The mean of the controls was then used for calculating the glucose content of each test solution. Because duplicate analyses for each set were repeated on two additional occasions, each value in Table I is the mean of six analytical results. The average values found for glucose concentrations in the test solutions are seen to be in error by less than 2% of the glucose present except for the test solutions which contained 1 mg. per ml. of glucose. For the latter solutions, average values found are 2 to 3% low and the standard deviations from the means are larger, This effect is apparently caused by the excessive consumption of chlorite due t o the high glucose concentration and can be avoided by the use of a smaller sample when the glucose content is high. INTERFERING SUBSTANCES
The behavior of a number of substances added as sample impurities in test solutions in the analytical procedure is indicated by the exploratory results shown in Table 11. The almost complete oxidation of the aldoses and the similarity in'lthe behavior of fructose, sorbose, sucrose, and raffinose indicate that themethod can easily be adapted to serve as a general method for the determination of aldoses in the presence of ketoses, as well as sucrose, raffinose, and other similar sugars. These results are in
0.00
0
50
100
150
TIME, MINUTES
Figure 4. I.
11.
Initial Rates of Formation of Chlorine Dioxide
Control solution, 0.0600.M NaClOz, 1.5M acetate buffer (pH 3.97), 25O C. Same as I , with 100 mg. per m l . aldose-free fructose artrled _____
111. Curve calculated for 0.02170 glucose impurity added to the fructose of I1 IV. Same a8 I , with 100 mg. per ml. fructose (preparation
D) added
Purity. To obtain a fructose assumed to be completely free of aldose, fructose dihydrate was crystallized from fructose solution which had been subjected to oxidation by chlorous acid under conditions somewhat more drastic than employed in the analytical procedure-via., 24-hour reaction time a t 25 ' C., p H 3.7 to 4.0, Co = 0.1M sodium chlorite. After recrystallization, this material was used as an aldose-free standard n-ith which were compared other dihydrate preparations. Column 4 of Table I11 shows the aldose contents of various fructose preparations as determined by the spectrophotometric procedure referred to the chloritetreated preparation as aldose-free fructose. The glucose-equivalent contents of the commercial materials were reduced from values as high as 0.7 to 0.03% or less by the dihydrate crystallization procedurk.
V O L U M E 2 6 , NO. 9, S E P T E M B E R 1 9 5 4
1483
Some of the preparations of Table I11 were also compared with the chlorite-treated preparation with reppect to the initial rate a t which chlorine dioxide is formed in a test solution containing fructose under reaction conditions of the analytical procedure. Figure 4 shows typical results obtained using 1-cm. spectrophotometer cells as reaction vessels. The pronounced curvature of curve IV for the test solution containing preparation D contrasts with the negligible initial curvature of curve I1 for the test solution containing the chlorite-treated fructose preparation. That this curvature cannot be accounted for as due to glucose impurity is seen by comparison of curve IV with curve 111. The latter curve would have been obtained if all of the oxidizable impurity, found for preparation D by the analytical procedure, were glucose. The much greater initial curvature of curve IV over that of I11 is interpreted as evidence for the presence of nonglucose impurity which is oxidized much more rapidly than glucose. As a rough measure of the amount of such impurity the authors have used the zero-time intercept of the straight line determined by the points of curve IV a t times greater than 60 minutes. Thus, this intercept exceeds that for the control solution (curve I) by an amount equivalent to the chlorine dioxide expected for the oxidation of 0.024% of glucose. Estimates by this intercept method of the content of impurities oxidized more rapidly than glucose are included as the last column of Table 111. From these figures it was concluded that most of the oxidizable impurity in preparation D was not glucose.
Table 111. Oxidizable I m p u r i t i e s in S i x Purified Fructose Samples
Preparationb A B C Df E F a
Source of Original Fructose Xd
Xd
Y 1’
z Z
Oxidizable Impuritiesa Original fructose, Purified Fructose photometric Photometric Rate curve method method intercept C 6 0 10 - 0 004 0 0 0 0
10 18 18
+o
70
0 70
005 0.008 0 021 0 031 0 039
c
0 0 0 0
009 024 016 019
Calculated as per cent glucose in anhydrous fructose.
b Preparations 4 , B, C, and F were each washed twice with cold water, and E once. D wns washed with fructose solution. T h e volumes of water used in washing E and F were relatively small. C
d
Interpreted a s nonglucose oxidizable impurity. Different lots from same comiriercial source.
fer solutions, and minor fluctuations of temperature of the solutions during the reaction period. Minimum exposure of the reaction mixtures to light is recommended, because chlorine dioxide solutions are known to be photosensitive, but the brief exposure incident to transfer of tubes from the bath and photometric measurement was found to produce no significant change. Errors due to light exposure or other procedural details can be detected by check analyses using Sational Bureau of Standards “dextrose” as a test sample. The sensitivity of the method for measuring glucose as an impurity in fructose is ultimately limited by the reproducibility of replicate control and replicate test solutions run simultaneously. It is seen from Table I that a standard deviation of about 0.003 mg. per ml. of glucose is to be expected if the chlorine dioxide is measured spectrophotometrically, indicating that glucose impurity as low as 0.01% could probably be detected and estimated with considerable uncertainty provided a suitable fructose standard were available. Suitahle aldose-free standard fructose can be prepared by recrystallization of fructose dihydrate without resort to chlorite treatment as shown by the fact that preparations A and B of Table I11 did not differ significantly from chloritetreated fructose in their yields of chlorine dioxide produced in the analytical procedure. Fructose, purified by recrystallization as the dihydrate to the point where successive crystallizations produce no change in response to the analytical procedure, is accordingly thought to be equivalent to fructose freed from aldose by treatment with chlorous acid. The results of Table I11 further show that reference fructose containing less than 0.03% of glucose-equivalent can be prepared by a single recrystallization of commercially available material as dihydrate provided the product is adequately washed. Although this work has not been extensive enough to evaluate the method fully, the results of Table I indicate that it can be used to determine glucose in mixtures of glucose and fructose over the entire range of composition. For the spectrophotometric procedure, approximate standard deviations in the percentage glucose are 0.003, for samples containing less than 0.5% glucose, 0.03 for samples containing from 0.5 to 5 % glucose, and about 1% of the glucose content for samples containing more than 5% glucose. For the colorimeter procedure the corresponding standard deviations are larger by a factor 1.5 t o 2. The accuracy of the results for glucose contents below a few per cent is determined by the accuracy with which the glucose content of the reference fructose employed in the method is known. ACKNOWLEDGMENT
DISCUSSION AND CONCLUSIONS
The analytical method presented has the advantages and disadvantages characteristic of most difference methods. Thus about as much chlorine dioxide is formed in the control solution containing no fructose as is produced by the oxidation of 0.2 mg. per ml. of glucose. Similarly, the chlorine dioxide produced in control solutions containing 100 mg. per ml. of fructose is approximately equivalent to that resulting from 0.4% of glucose impurity in a fructose sample. This emphasizes the importance of obtaining maximum precision in the photometric measurements, especially if a colorimetric procedure is used where the difference in chlorine dioxide concentrat’ions of the test and control solutions cannot be measured directly in a single measurement. The comparison of test and control solutions prepared from the same reagents and subject to the same temperature history practically eliminates, as sources of error, the slight differences in absolute reaction rates observed for different buffer preparations, the slight differences in the small amount of chlorine dioxide produced immediately upon mixing the chlorite and buf-
The authors wish to thank H. F. Launer for suggesting the uee of sodium chlorite in acid solution as a suitable oxidizing agent and for making his manuscript ( I S ) available in advance of publication. The authors also benefited from discussions with H. F. Launer and Yoshia Tomimatsu, who have recently extended the sensitivity of determining glucose alone by oxidation with sodium chlorite in acid solution ( I d ) . LITERATURE CITED
(1) Baldwin, R. W., Campbell, H. A., Thiessen, R., Jr., and Lorant,
G. J., Food Technol., 7, 275 (1953). (2) Barnett, Benjamin, Ph.D. dissertation, University of California. Berkeley, 1935. (3) Bates, F . J., and associates, Natl. Bur. Standards, Circ. C440, 208 (1942). (4) Bray, William, 2. physik. Chem., 54, 569 (1906). (6) Browne, C. A., and Zerban, F. W., “Physical and Chemical Methods of Sugar Analysis,” 3rd ed., p. 895, New York, John Waey & Sons, 1941. (6) Haupt, G. W., J . Research Natl. B u r . Standards, 48, 414 (1952) (RP 2331). (7) Hodge, J. E., and Davis, H. A . , U. S. Dept. Agr., Bur. Agr. Ind. Chem., AIC 333, 42, 46 (1952.) (8) Jackson, R. F., and Mathews, J. A., J . Research Natl. B u r . Standards, 8 , 412 (1932) (RP 426).
ANALYTICAL CHEMISTRY
1484 Jeanes, Allene, and Isbell, H. S., J . Research Natl. Bur. Standurds, 27, 125 (1941) (RP 1408). Keilin, D., and Hartree, E. F., Biochem. J . , 42, 221 (1948). Launer, H. F., and Tomimatsu, Y.,ANAL. CHEM.,25, 1767
(16)
Young, F. E., and Jones, F. T., U. S. Patent 2,588,449 (March
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Young, F. E., Jones, F. T., and Lewis, H. J., J . Phys. Chem., 56,
1 1 , 1952). 738 (1952).
(1953). (12)
Launer, H. F., and Tomimatsu, Y., J . Am. Chem. SOC.,76, 2591 (1954).
F., Wilson, W. K., and Flynn, J. H., J . Research Natl. Bur. Standards, 51, 237 (1953) (RP 2456). (14) Sowden, J. C., and Schaffer, R., J . Am. Chem. Soc., 74, 499 (13)
Launer,”. (1952).
(15) White, J. F., Taylor, M . C., and Vincent, G. P., Ind. Eng. Chem., 34,782 (1942).
RECEIVED for review December 21, 1953. Accepted June 7, 1954. Presented before the joint sessions of the Divisions of Analytical and Carbohydrate Chemistry. Symposium on Analytical Methods and Instrumentation Applied to Sugars and Other Carbohydrates at the 124th RIeeting of the AMERICAN CHEMICAL SOCIETY, Chicago, Ill. Mention of products by specific manufacturers does not imply that they are endorsed or recommended by the Department of Agriculture over others of a similar nature not mentioned.
Conductometric Standardization of Solutions of Common Divalent Metallic Ions Using Disodium Salt of Ethylenediaminetetraacetic Acid JAMES L. HALL, JOHN A. GIBSON, JR., PAUL R. WILKINSON, and HAROLD 0.PHILLIPS W e s t Virginia University, Morgantown,
W. Va.
An effort has been made to evaluate the use of conductometric methods for end-point determinations in the titration of solutions of the disodium salt of ethylenediaminetetraacetic acid and divalent metallic ions. Conductance methods may be used for accurate standardization of solutions of copper(II), zinc, lead, nicltel(11), cobalt, calcium, barium, strontium, magnesium, manganese, cadmium, iron(IL), and mercury(I1) in the concentration range from 0,001 to 0.5M, before dilution in the titration vessel.
R
ECENTLY the disodium salt of ethylenediaminetetraacetic acid (Versenate, Sequestrene, Complexone 111) has been proposed as a standard for establishing the concentrations of solutions of certain divalent cations ( 2 ) . The stability constants of the complexes are great enough t o make precise end-point determinations possible ( 1 7 , 18, 20). The stoichiometric relations for the reactions between the metallic ions and the reagent have already been determined by several methods with reported accuracies within 0.05 to 2.0%. Metal ion concentrations have been determined potentiometrically (1, 10, 11, 19),by use of indicators ( I ! 4 , 5 , 9 , 1 6 , 19), spectrophotometrically (12, I S , 22, 23), polarographically (6, 14,1.5,21),and by a specialized high-frequency technique ( 3 ) . The present work shows that conventional conductometric methods may be used for the standardization of solutions of several common cations. The accuracy compares favorably with the best previously described methods. REAGEVTS
Disodium Versenate. Standard solutions of the reagent were prepared from the analytical reagent (disodium Versenate dihydrate, manufactured by the Bersworth Chemical Co.) and from Versenate purified by the method of Blaedel and Knight (2). All solutions mere standardized with electrolytic copper, dissolved in a minimum amount of 6 S nitric acid. The end points were determined conductometrically as described below. Titration in either acidic or basic solution yielded the same molarity. Solutions O.lOOlM, 0.04724M, O.O1001M, and 0.001004.11 were prepnred. In weighing the copper and disodium Versenate for these solutions, the weight of the I’ersenate was corrected for the difference in density between the Versenate and the brass weights. Rascd on a density of 1.8 for the Versenate, this correction was 0.06% relative to the metallic copper. Cation Solutions. Solutions of cupric nitrate, cupric per-
chlorate, nickel nitrate, cobalt nitrate, lead nitrate, zinc sulfate, manganese sulfate, cadmium chloride, ferrous sulfate, magnesium sulfate, strontium nitrate, calcium chloride, barium nitrate, mercuric acetate, lanthanum nitrate, and cerium nitrate were prepared a t various concentrations from Baker’s analyzed or Mallinckrodt reagent grade chemicals. In addition, copper, zinc, and nickel nitrates were prepared by dissolving metal of known purity in 6n’ nitric acid. Konconductometric standardizations were made for most of the solutions; the purity of the calcium carbonate from which the calcium chloride solution was made, the strontium nitrate, and the barium nitrate was established by gravimetric analyses ( 2 4 ) . The normality of the manganese(11), cadmium, lead, zinc, magnesium, and mercury(I1) salt solutions was determined with Versenate using the indicator method of Schwarzenbach ( 1 ). The solutions Ivere made in concentrations from 0.001 to 0.2;2f. Ammonia. Where ammonia was required, the C.P. product proved to be satisfactory for solutions of metal ion concentrations of 0.1.V or greater. At lower concentrations, errors introduced by impurities became appreciable and distilled ammonia was necessary. The ammonia was distilled into conductivity water to a concentration of 3M and was stored in polyethylene bottles. Water. Whenever the available distilled water was used, a correction equivalent to 0.4 ml. of 0.01M Versenate per 1000 ml. of aater was required. Twice distilled water was preferable for all solutions 0.lM or less. Acid Buffer. Twenty-five grams of Baker’s analyzed sodium hydroxide and 65 ml. of glacial acetic acid were dissolved in water, mixed, and diluted to 250 ml. The p H of this buffer was 5.1. KO difference in end-point ratio was found for 0.01M copper(I1) solution titrated with and without this buffer. The use of U.S.P. sodium acetate for the buffer yielded a result 295 in error. APPARATUS
The most precise measurements were made a t 2000 cycles using a Leeds & Xorthrup Type 1553 ratio box and Type 4754 decade resistance with recommended oscillator and amplifier. A 50ppf. variable capacitor, and decade capacitors to provide a total caparitance up to 1 uf.,were connected in parallel with the known resistance. The null point was determined by observing the output wave on an oscillograph. Used in this way, the apparatus has a range of 0.01 to 10,000 ohms with a maximum error of 0.03yo at 10 ohms or greater. .A dip-type conductivitv cell with platinized platinum electrodes and a cell constant of 0.0964 wa8 used. Titrations werr made at room temperature. Additional conventional conductance measurements were made using a Model RCZI15 Serfase direct-reading conductance bridge. This instrument gave satisfactory results for work a t concentrations of 0.01.11 or less. Many of the determinations were also performed using two high-frequency instruments ( 7 , 8). Thefie instruments were satisfactory for routine work in the more dilute solutions but did not contribute any new or more useful results. Kumerical data are not included for these high-frequency determinations.
