Determination of Moisture in Starch Hydrolyzates by Near-Infrared and Infrared Spectrophotometry Daniel W. V ~ m h o f ’ -and ~ James H. tho ma^^.^ National Bureau of Standards, Washington, D. C . 20234
Evidence is presented that the near-infrared method, employing either DMF or MezSOas the solvent, and the infrared method, with MeSO as the solvent, are quite applicable to the determination of the moisture content of starch hydrolyzates. The near-infrared method is superior in terms of both accuracy and precision to both the present vacuum-oven method and the infrared method. These methods do not seem to be influenced by the method of manufacture, the saccharide distribution, or the ash content. They are sufficiently rapid that they could be used for quality control both by manufacturers and users of corn syrups and solid sugars.
THIS PAPER REPORTS a study of the applicability of nearinfrared spectrophotometry to the determination of moisture in starch hydrolyzates. The accurate determination of the moisture (or the dry-substance) content of starch hydrolyzates has been the subject of several studies (I+, and is a continuing problem. The direct determination of moisture in these materials is impeded by the thermal sensitivity of the constituent saccharides, the formation of a glass at low levels of moisture, and the difficulty in handling materials that are viscous and adhesive. The most widely used direct method suitable for reference work in this regard involves a vacuum drying oven and an inert support such as diatomaceous earth. This method is quite lengthy and the occurrence of thermal degradation increases with increasing dextrose content of the hydrolyzate. Several of the major problems associated with both the direct and the indirect methods employed for the determination of moisture in starch hydrolyzates have been reviewed by Graefe ( 5 ) . The instrumentation and principles of near-infrared spectroscopy have been reviewed by Kaye (6) and, more recently, by Goddu (7). Spectrophotometric methods utilizing the 1.9-pm combination band for the quantitative determination of water in hydrazines (8), glycerol (9), and dried vegetables (10) have been reported. This technique has not been investigated for its applicability to starch hydrolyzates. Casu et al. ( 1 1 ) have reported an infrared method applicable to at least 1 Research Associate from the Corn Industries Research Foundation at the National Bureau of Standards, 1967-69. 2 Present address, Chief Chemist, U. S. Customs Laboratory, 610 S. Canal Street, Chicago, Ll1. 60607. a To whom inquiries should be directed. 4 Research Assistant from the Corn Industries Research Foundation at the National Bureau of Standards, 1968-69. 6 Present address: Washington Technical Institute, Washington, D.C. 20008 (1) J. W. Evans and W. R. Fetzer, IND.ENG.CHEM., ANAL.ED.,
13, 855 (1941). (2) J. E. Cleland and W. R. Fetzer, ibid., p 858. (3) Ibid., 14, 27 (1942). (4) Ibid., p 124. ( 5 ) G. Graefe, Staerke, 13,402 (1961). (6) W. Kaye, Spectrochim Acta, 6, 257 (1954); 7, 181 (1955). (7) R. F. Goddu, Aduan. Anal. Chem. Instrum., 1, 347 (1963). (8) H.F. Cordes and C. W. Tait, ANAL.-EM., 29,485 (1957). (9) D.Chapman and J. F. Nancy, Analyst, 83, 377 (1958). (10) B. R. Rader, J. Ass. Ofi.Anal. Chem., 50, 701 (1967). (11) B. Casu, G. Gaglioppa, and M. Reggiani, Staerke, 17, 386 (1965). 1230
some starch hydrolyzates; it utilizes the 0 - H band at 1640 cm-l. The requirements for a suitable direct method for the determination of moisture in starch hydrolyzates are that the method be accurate, free from interferences due to the saccharides types and nonsaccharides, usable at, or near, room temperature (to minimize thermal degradation), and reasonably rapid. This investigation shows that both the near-infrared and the infrared method meet these requirements. Furthermore, the accuracy and precision obtained by these methods are compared with the Corn Industries Research Foundation (CIRF) vacuum-oven filter-aid method (12) which is the present standard method used by the corn wet-milling industry. EXPERIMENTAL
Apparatus. Near-infrared measurements were made on a Cary Model 14 spectrophotometer (Certain commercial equipment, instruments, or materials are identified in this paper in order to adequately specify the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for the purpose.) operated in the absorbance mode. The 1.00-cm absorption cells, fitted with polytetrafluoroethylene stoppers, had a volume of 3 ml. Infrared measurements were made with a Perkin-Elmer Model 257 grating instrument by use of absorption cells of 0.30-mm path and Irtran-2 windows. The ground-glass stoppered flasks were oven-dried prior to use. The “normal” slit program and medium scan speeds were used. Reagents. Reagent grade N,N-dimethylformamide (DMF) and methyl sulfoxide (Me2SO) were used without further purification or drying. The water content of the solvents was less than 0.1 weight per cent. The reagent containers must be kept tightly stoppered to prevent gradual uptake of atmospheric moisture. Dextrose was NBS Standard Reference Material 41a. Starch hydrolyzates were supplied by member companies of the Corn Refiners Association, Inc. Procedure. Calibration standards are prepared on a weight basis by the addition of water to the solvent. At least three standards should be prepared which bracket the expected water concentration of the samples. The flasks are kept tightly stoppered between transfers. Similarly, solutions of the sample material are prepared by the addition of syrup or solids to the solvent on a weight basis. Transfer of syrups is accomplished by means of a medicine dropper with the tip removed. The quantity of sample taken is determined by differential weighing of the sample-solution flask. The amount of sample is taken to yield a solution containing about 0.5 weight per cent of water. The stoppered flasks may be warmed to facilitate dissolution of the sample. If this is done, however, the cooled flasks should be reweighed to assure that no loss of solvent has occurred. (12) “Standard Analytical Methods,” Corn Industries Research Foundation, 1001 Connecticut Avenue, N.W., Washington, D. C. 20036.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
Solvent DMF
Me80
Table I. Near-Infrared Calibration Data for Moisture in DMF and MeBO Set 11 Set I Water in Net Absorbance/ Water in Net solution, wt % absorbance, mm concentration solution, wt absorbance, mm 0.2705 0.5012 0.7912 1.115 0.2112 0.4485 0.7497 0.9783
102.5 190.0 300.0 423.0 67.0 141.2 239.0 311.0
0.3460 0,5455 0.8331 1.257 0.4403 0.7820 0.8491
379.0 379.2 379.2 379.2 317.2 315.0 318.8 318.0
Table 11. Near-Infrared Method, Sensitivity with MezSO
Factor
= =
z 4 m
Detection limit is the level at which a substance must be present in order to have a 95 chance of being detected. * Determination limit is the level at which the relative standard deviation reaches a sufficiently low value, here taken to be 0.01. Q
372.8 372.1 372.7 372.1 333.8 331.2 332.0
SUCROSE/ D M F S Y R U P / DMF
-
0.8
-
0.6
332 mm/wt % H20 1.322 AU/wt HIO
129.0 203.0 310.5 467.7 147.0 259.0 282.0
L
Blank standard SB = 0.002 AU = 0.507 mm deviation Ln = 4.65 S g = 2.36 mm = 0.00711 wt % HzO Detection limit0 Determination LQ = S~j0.01= 50.7 mm = 0.153 wt H20 limit*
Absorbance] concentration
-
-
f 0.4 4
.
0.2
I
For the samples and calibration standards, the same lot of solvent must be used as is used for the reference cell. Near-Infrared Method. After the instrument has been adjusted to zero with solvent in both beams, the watercontaining solution is scanned over the region from 2.0 to 1.8 pm, depending on the solvent used, and a minimum is found at 1.80 pm. The net absorbance of the water band is taken as the difference in absorbance between the peak maximum and the minimum at 1.80 pm. The precision of measurement is improved if the net peak-height is determined with a millimeter rule graduated in 0.5-mm divisions, rather than in terms of chart graduations. The sample size is adjusted, when practicable, to give a net peak-height of at least 100 mm. Accuracy is improved when the calibration standards are interspersed among the samples. Infrared Method. Only MQSO is a suitable solvent in this case. After the instrument has been adjusted to 100% T with solvent in both beams, the aqueous solution is scanned over the region from 1818 to 1429 cm-l. The net peakheight of the 1640-cm-' band is determined by taking the 1786-cm-l absorbance as the base line. In this case, the transmittance values must be converted into absorbance units prior to obtaining the net peak-height. The accuracy is increased when the calibration standards are interspersed among the samples. In either method, the sample cell should be rinsed once with fresh solvent and twice with the next sample. Calculations. In both cases, the water content of the sample solution may be calculated either from a calibration curve or by use of the Beer-Lambert equation and an averaged absorptivity. However, the best accuracy is obtained by employing Equation 1 AxC.9 -AS Cx Cp
x
102 = Ch
(1)
where A S = net absorbance of a water standard containing about the same proportion of water as the sample of interest, A x = net absorbance of the sample solution, CS = weight per cent of water in the standard, CX = weight per cent of water in the sample solution, C, = weight per cent of hy-
1.80
1.90
WAVELENCTH
- pm
2.00
Figure 1, Near-infrared spectra of water and saccharides in DMF drolyzate in the sample solution, and Ch of water in the hydrolyzate.
=
weight per cent
RESULTS AND DISCUSSION Near-Infrared Method. The near-infrared absorption spectra of water, sucrose, and a corn syrup in D M F are shown in Figure 1. Similar spectra are obtained with Me2S0 as the solvent. The saccharide hydroxyl groups are the major contributors to the absorbance in the region from 2.0 to 2.5 pm. The minimum at 2.0 pm is dependent on both the saccharide and the water content of the solution. For this reason, it is preferable to take the absorbance value at 1.80 pm as the base line value in determining the net absorbance of the water peak. For better precision of measurement, the peak heights were measured with a millimeter rule, rather than by the chart graduations. [Chart divisions can be estimated to about 0.002 absorbance unit (AU) the equivalent of 0.5 mm on the chart paper used. A millimeter rule graduated in 0.5-mm units can be read to about 0.2 mm (equivalent to 0.0008 AU). Thus, when the peak height exceeds 100.0 mm the uncertainty is in the fourth significant figure, resulting in increased accuracy and improved precision.] The Beer-Lambert relationship is followed over the water concentration range of interest. Typical calibration data for D M F and MezSO are presented in Table 1. Each set was obtained on a different day. It may be seen that for improved accuracy, a calibration curve should be obtained each day. These differences are not as apparent when the peak height is measured in absorbance units, because of the decreased precision. The sensitivity of' this method, as defined by Currie (13), is summarized in Table 11. (13) L. A. Currie, ANAL.CHEM., 40, 586 (1968).
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Table 111. Saccharide Solution Moisture Recovery by the Near-Infrared Method Water, wt % Recovery, Sugar/ Sugar, Present Found % Ha0 wt % 0.463 0.653 0.898 0.528 0.520
0.628 1.91 4.25 4.23 6.01
0.465 0.651 0.887 0.518 0.526
100.5 99.7 98.8 98.1 101.1
1.36 2.93 4.73 8.02 11.5
Solvent : methyl sulfoxide.
The accuracy of this method for the present application was studied with both D M F and Me2S0 by the determination of water in known sugar solutions. The results in MezSO are presented in Table 111. Similar results were obtained with D M F as the solvent. Neither the amount of water present nor the ratio of sugar to water appeared to influence the determination of the water. However, the base line at 1.80 pm does shift from zero in the presence of a sample. The accuracy is improved, therefore, by calculating the net peakheight as the difference between the values at 1.93 and 1.80 pm, instead of as the absolute height at 1.93 Fm. Infrared Method. As with the near-infrared method, the saccharide hydroxyl groups do not interfere with the determination of moisture by this method. However, the base line does shift slightly with the introduction of sample into the solvent. For this reason, it is necessary to use as the base line the absorbance at 1786 cm-1, not the instrument base line at 1640 cm-’, in the calculation of net absorbance. The results in Table IV indicate a linear relationship between moisture content and net absorbance over the usable range of concentration. The sensitivity of this method, as defined by Currie (13), is summarized in Table V. The change in absorbance for a unit change in water content is only about one fourth that of the near-infrared method. The accuracy of this method in the determination of the moisture in carbohydrate solutions was studied with solutions of known content of saccharide and water. The results, calculated using Equation l , are presented in Table VI. Neither the amount of water present nor the sugar-to-water ratio seemed to adversely influence the determination. The major source of error in this method is in the conversion of the estimated transmittance value, taken from the chart, to absorbance units. Obviously as the water content increases, the magnitude of the measurement error increases in terms of absorbance units. By using variable-path cells, the 0.30-mm absorption cell path-length was found to be the maximum length for which a linear increase in absorbance was observed for a linear increase in path-length, at constant content of water. Cells having a length of 0.25 to 0.30 mm are satisfactory. Application to Starch Hydrolyzates. Further indication of the accuracy of these methods, as well as estimates of the
Water in solution, wt 0.2112 0.4485 0.7491 0.9783
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Z
Table V. Infrared Method, Sensitivity with MeSO Blank standard deviation S B = 0.0023 AU Detection Ln = 4.65 SB = 0.0054 AU = 0.0162 wt B O limit. Determination LQ = S ~ l 0 . 0 1= 0.2300 AU = 0.692 wt % H20 limitb Factor = 0.3325 AU/wt HIO a Detection limit is the level at which a substance must be present in order to have a 95% chance of being detected. Determination limit is the level at which the relative standard deviation reaches a sufficiently low value, here taken to be 0.01. Table VI. Saccharide Solution Moisture Recovery by the Infrared Method Sugar wt
Water, wt % Present Found
0.997 3.82 4.25 4.23
0.557 1.21 0.898 0.528
0.561 1.24 0.919 0.533
Recovery,
z
Sugar/ SO
100.7 102.0 102.4 100.9
1.79 3.15 4.73 8.02
Solvent : methyl sulfoxide.
overall precision of each method, was obtained in the analysis of commercial hydrolyzates. Corn syrups and corn syrup solids obtained from member companies of the CRA were analyzed for moisture content by both of the spectrophotometric methods and by the CIRF vacuum-oven, filteraid method. Information on the characteristics of these hydrolyzates is summarized in Table VII. Combined estimates of the precision of each method (14) are given in Table VIII. The precision estimates are based on observed differences within three series of determinations of the hydrolyzates in classes 111-VII. These series extended over a two-week period. In the case of syrups, as with most materials submitted for analysis, it is not possible to know absolutely that all of the constituent sought has been determined. However, when the same value is obtained by more than one method, each of which is based on a different physical or chemical principle, the accuracy both of the result and of the method in question can be accepted. In Table IX, the moisture values obtained for a range of representative hydrolyzates, as determined by the vacuum-oven and spectrophotometric methods, are compared. Each value reported is the mean of at least three separate determinations. From these results, it may be seen that the methods are direct and specific for moisture. They are not influenced by ash content, manufacturing process, or saccharide distribu(14) W. J. Youden, “Treatise on Analytical Chemistry,” Part I, Vol. 1, I. M. Kolthoff and P. J. Elving, Ed., John Wiley & Sons, New York, 1959, p 51.
Table IV. Infrared Method Calibration Data for Moisture in MeSO Set I1 Set I Net A bsorbancel Water in Net concentration solution, wt absorbance, AU absorbance, AU
z
0.0862 0.1805 0.3056 0.3979
0.408 0.403 0.408 0.407
0.4403 0.7820 0.8491 1.413
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
0.1549 0.2596 0.2840 0.4673
Absorbance1 concentration 0.352 0.332 0.335 0.331
Hydrolyzate class
6
b
DE I 11.6 I1 17.0 111 29.0 35.5 IV 43.7 V 53.4 VI 62.7 VI1 68.1 A = Acid hydrolysis, AE = = Dried granular product.
Table VII. Characterization of Starch Hydrolyzates Studied Sugar (dry basis) Ash, DPi DPz sulfated 1.6 2.3 1.06 4.1 3.6 0.20 6.8 9.9 0.43 14.9 9.2 0.22 8.3 33.5 0.01 28.6 17.6 0.08 31.0 25.1 0.27 40.4 37.3 0.18 acid-enzyme hydrolysis.
tion. Furthermore, they are free from interference resulting from thermal degradation of the saccharides. Sources of Interference. The most common source of error, or interference, is extraneous moisture either on the walls of the flask or absorbed from the atmosphere during sample preparation, or both. Moisture within the flask can be readily eliminated by using glassware that is oven dried. Absorption of moisture from the atmosphere has not been a problem under the conditions of these experiments (20 "C, 50 to 6 5 x R.H.). The rate of moisture uptake by 25 grams of either D M F or Me2S0 in an open, 125-ml Erlenmeyer flask, as determined in this laboratory by using the NIR method as the monitoring procedure, has been less than 0.02 weight per cent of moisture per hour (20"C, 50% R.H.). During the preparation of a sample, the flask is open to the atmosphere for about 5 minutes. Thus, the effect is quite small. As a consequence, we have not found it necessary to employ a dry box at any time. Such precautions may, however, prove necessary at higher temperatures and relative humidities. The effect of change of temperature is small over a 5 "C range. The effect can be eliminated by either maintaining the samples at the temperature of the cell compartment or always following the same time sequence with regard to the length of time the sample is kept in the cell compartment, L e . , a high scanning rate or a 5-minute wait between placement of the sample cell in the compartment and recording of the spectrum. With the exceptions noted, sugars do not interfere with these methods. In some cases, the presence in foods of such functional groups as the amine and the alcohol group may interfere in the near-infrared method (7, 8). These are generally not present in starch hydrolyzates and other processed carbohydrates. Provided that the samples are tightly sealed to prevent uptake of atmospheric moisture, sample solutions remain stable for at least three days with respect to the moisture content. Solvent Selection. Although, for the near-infrared method, both D M F and Me2S0 are satisfactory from the analytical standpoint, Me2S0 is to be preferred from the standpoint of solvent properties. Several samples of dried hydrolyzates having low dextrose equivalence (DE) values were only dissolved in D M F with difficulty. One sample (17 DE) would not dissolve at all, even after heating at 80 "C for 1 hour. On the other hand, with Me2S0, all hydrolyzates studies were completely dissolved within 15 minutes at 70 "C. Only
DPa 3.1 3.2 10.3 9.2 19.2 12.3 9.7 4.7
Process= AEb Ab Ab AE AE A AE AE
Table VIII. Combined Estimates of Precision of the Spectrophotometric and Vacuum-oven Methods (14) Method vo NIR IR Degrees of freedom 24 21 12 Standard deviation 0.034 0.135 0.046 Table IX. Moisture Content of Starch Hydrolyzates as Determined by the Spectrophotometric and Vacuum-oven Methods Method .DE vo NIR IR (wt mean of 3 determinations) 11.6 6.199 6.170 17.0 ... 5.053 ... 29 21.27 21.29 21.32 36 19.23 19.34 19.47 19.33 44 19.33 19.26 53 19.57 19.60 19.32 16.22 62 16.29 16.29 17.49 68 17.73 17.79 26.34 26.39 Table syrup Blackstrap 25.37 25.36 molasses fO. 041 99% C.L. zk0.019 zk0.083
z,
Me2S0can be used in the infrared method since D M F absorbs in the 1640-cm-' region. Rapidity of Analysis. The time required for a spectrophotometric determination is about 30 minutes with Me2S0 as the solvent, in comparison to the 24 hours required for the vacuum-oven method. The average time per sample is, of course, lessened considerably when a series of samples is studied. The time required for calculations is dependent on the degree of accuracy desired and on the frequency and extent of use. ACKNOWLEDGMENT
The authors thank R. Schaffer and J. K. Taylor of the National Bureau of Standards and R. J. Smith of the Corn Products Company for several helpful discussions. RECEIVED for review May 26, 1969. Resubmitted June 19, 1970. Accepted June 19, 1970. Presented, in part, before the Division of Carbohydrate Chemistry, 156th Meeting, ACS, Atlantic City, N. J., September 1968.
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