Spectrophotometric Determination of the Hydroxypropyl Group in Starch Ethers D. P. Johnson Product Development Center, R . J . Reynolds Tobacco Company, Winston-Salem, N. C .
THE principle employed by Jones and Riddick ( I ) to determine propylene glycol and polyoxypropylene, was adapted to the determination of the hydroxypropyl group in starch ethers. The method involves hydrolysis of the hydroxypropyl group to propylene glycol which in turn is dehydrated to propionaldehyde and the enolic form of allyl alcohol. These products are measured spectrophotometrically after they are reacted with ninhydrin to form a purple color (2). Two methods are described in this paper. Method No. 1 was designed for hydroxypropyl group concentrations of 1 or more, while Method No. 2 was intended for lower concentrations. EXPERIMENTAL
Method No. 1. Weigh 0.05 to 0.1 gram of hydroxypropyl starch into a 100-ml volumetric flask and add 25 ml of 1 N sulfuric acid. Prepare a sample of unmodified starch of the same source (Le., corn or potato) in the same manner. Place the flasks in a boiling water bath and heat until the samples are in solution. Cool and dilute the contents to 100 ml with water. If necessary, dilute the sample further to assure the presence of no more than 4 mg of hydroxypropyl group per 100 ml, and then dilute the blank starch in the same proportion. Pipet 1 ml of the solutions into 25ml graduated test tubes with glass stoppers and, with the tubes immersed in cold water, add dropwise 8 ml of concentrated sulfuric acid to each. Mix well and place the tubes in a boiling water bath for exactly 3 minutes. Immediately transfer the tubes to an ice bath until the solution is chilled. Add 0.6 ml of ninnydrin reagent ( 2 ) , carefully allowing the reagent to run down the walls of the test tubes. Immediately shake well, and place the tubes in a 25 "C. water bath for 100 minutes. Adjust the volume in each tube to 25 ml with concentrated sulfuric acid and mix by inverting the tubes several times. (Do not shake.) Immediately transfer portions of the solutions to 1-cm cells designed for a Beckman Model B spectrophotometer, and after exactly 5 minutes, measure the absorbance at 590 mp, using the starch blank as the reference. Prepare a calibration curve with 1-ml aliquots of standard aqueous solutions containing 10, 20, 30, 40, and 50 pg of propylene glycol per ml. Apply the factor 0.7763 to convert micrograms of the glycol to hydroxypropyl group equivalent . Method No. 2. Weigh 0.1 to 0.2 gram of sample into a 100-ml volumetric flask and add approximately 25 ml of dilute phosphoric acid (one volume of 85% phosphoric acid and one volume of water). Digest in a boiling water bath until the sample is in solution. Cool and dilute to the mark with dilute phosphoric acid. Mix and transfer 10 ml with a pipet into a 100-ml distillation flask equipped with a side arm. Add 2 or 3 boiling stones and attach to an air-cooled, gooseneck condenser. Pipet 10 ml of 5z sodium bisulfite into a 50ml volumetric flask and, with the flask in an ice bath, insert the delivery tube from the condenser to just below the surface of the bisulfite solution. Start a flow of nitrogen through the side arm, and adjust the rate to about 2 bubbles (1) L. R. Jones and J. A. (2) Ibid., 26, 1035 (1954).
Riddick, ANAL.CHEM., 29, 1214 (1957).
Table I. Effect of Starch on Determination of Propylene Glycol by Method No. 1 Propylene Starch glycol Hydroxypropyl added, added, gfouP mgn ma equivalent Absorbance 0 0 0 0 0 0 100 100 100 100
1.418 1.540 2.836 3.080 5.671 6.161 0 1.418 2.836 5.671
1.101 1.196 2.202 2.391 4.403 4.783 0 1.101 2.202 4.403
0.171 0.189 0.341 0.378 0.659 0.712 0.026 0.206 0.369 0.684
Represents the amount of starch and propylene glycol in 100 ml of solution. One-milliliter portions were processed for color.
per second as observed in the receiving flask. Using a heating mantel, heat the solution in the distillation flask to boiling and maintain the heat until intense white fumes form in the flask and the delivery tube becomes cool. Discontinue the heat but continue the nitrogen purge for 10 minutes. Remove the receiving flask from the ice bath and rinse the delivery tube with water, collecting the rinse solution in the flask. Adjust the temperature of the solution to about 25 "C. and dilute to the mark with water. Pipet 1 ml into a glass-stoppered test tube and continue as described in Method No. 1, beginning with the addition of 8 ml of sulfuric acid. Prepare a reagent blank for use as a spectrophotometric reference by processing 1 ml of water through the color development step along with the sample. Prepare a calibration curve by processing 10-ml aliquots of standard solutions containing 50, 100, and 200 pg of propylene glycol per milliliter in dilute phosphoric acid. Apply the factor 0.7763 to convert micrograms of propylene glycol to hydroxypropyl group equivalent. RESULTS AND DISCUSSION
Jones and Riddick recommended a heating period of 10 minutes at 70 "C to dehydrate propylene glycol with sulfuric acid, but those conditions were not adequate for hydroxypropyl starch. A time-temperature reaction rate study showed that the most consistent results were obtained for both propylene glycol and hydroxypropyl starch by heating for 3 minutes at 100 "C. At this temperature, the heating period must be controlled carefully to prevent loss of propionaldehyde and to avoid excessive discoloration of the solution. It is recommended, therefore, that a stop-watch be used for precise timing of the heating period. Although Method No. 1 is satisfactory for analyzing samples containing 1 or more of hydroxypropyl group, it is not recommended for lower concentrations. The large sample required for low concentrations produces a brown color upon heating in the sulfuric acid, and this color interVOL. 41, NO. 6,MAY 1969
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Table 11. Determination of Hydroxypropyl Group in Modified Starch Per Cent Hydroxypropyl Group Sample Method No. 2 Method No. 1 1 2 3 4 5 6
13.45 48.91 1.03
... ...
...
13.47, 13.38 48.30 1.14 20.06, 20.20 1.82, 1.82 2.03, 1.99
feres with the measurement of the ninhydrin propionaldehyde color. This interference is avoided in Method No. 2 by substituting phosphoric acid for sulfuric acid as the dehydrating medium and by distilling the propionaldehyde from the solution. The latter method, therefore, should be suitable for hydroxypropyl group concentration as low as 0.1 %. The ninhydrin color reaction is relatively specific for propionaldehyde in the presence of other aldehydes, but large
quantities of formaldehyde inhibit the color development. Although starch produces some formaldehyde under the conditions of both methods, the amount is not sufficient to interfere significantly. This is demonstrated by the data in Table I, which were obtained by processing known amounts of propylene glycol by Method No. 1 both in the presence and in the absence of corn starch. Because a starch derivative of known hydroxypropyl group content was not available, propylene glycol was used for calibrating the methods. The absorption of the color followed Beer's law for concentrations within the range specified for calibration, but some deviation was noted at higher concentrations. The reliability and the range of application of the methods were tested by analyzing starch derivatives of varying concentrations of the hydroxypropyl group. The results are contained in Table I1 and show good agreement between the methods. RECEIVED for review October 9, 1968. Accepted February 26,1969.
A Recommended Titrant: Anhydrous Perchloric Acid in Sulfolane J. F. Coetzee' and R. J. Bertozzi Department of Chemistry, University of Pittsburgh, Pittsburgh, Pa. 15213
BY REPLACING WATER with appropriate nonaqueous solvents, it is possible t o extend the acid-base scale both in the acidic and in the basic direction. In solvents that have lower basicities than water, higher acidities can be reached and weaker bases can be titrated than in water. In addition, in such solvents mixtures of relatively strong acids are resolved better than in a leveling solvent, such as water. Analogous advantages apply to solvents that have lower acidities than water. From the point of view of potentiometric titrations, the ideal solvent would be one which has negligible acid and base properties, but which nevertheless has a sufficiently high dielectric constant to avoid undue complications from solute association reactions and to allow conventional potentiometric apparatus. However, other factors also are important. For example, in such relatively inert solvents, formation of homoconjugate complexes A H . . .A- reduces the break in potentiometric titrations. This and other factors to be considered have been discussed extensively by Kolthoff and his coworkers ( I ) . Important practical considerations also apply. Purification and handling of the solvent should not be unduly difficult. Suitable titrants must be available. For potentiometric titrations, hydrogen ion indicator electrodes must be available; from the purely analytical point of view, it is not necessary that the response of such electrodes should be electrochemically reversible. 1
Please address all correspondence to this author.
(1) I. M. Kolthoff and M. K. Chantooni, Jr., J. Amer. Chem. Soc., 90, 5961 (1968). 860
ANALYTICAL CHEMISTRY
There still is a need for new acid and base titrants for relatively inert solvents. Tetraalkylammonium hydroxides dissolved in methanol-benzene mixtures or isopropanol (2), and the conjugate base of dimethylsulfoxide (dimsyl) in dimethylsulfoxide as solvent (3) are powerful titrants that have great practical utility and also are well suited to theoretical studies in the corresponding solvents. Anhydrous perchloric acid in methanol ( 4 ) or acetic acid as solvents has analogous advantages. However, these titrants have disadvantages for theoretical studies in more inert solvents or if full practical advantage is to be taken of the relative inertness of the solvent, because a more reactive solvent is introduced or generated by the titrant. We describe here the preparation and some of the properties of essentially anhydrous perchloric acid in sulfolane, which should have major advantages for titrations carried out in sulfolane and other relatively inert solvents. Even though our investigation is incomplete, we wish to report our preliminary results at this time to draw attention to the potentialities of this titrant. Sulfolane (tetramethylenesulfone) has become available in commercial quantities. It seems to be a highly inert solvent, with virtually negligible acidic properties and very low proton basicity, as indicated by a pKsH+ value of - 13 found from although it does exhibit Lewis acidity function studies (3, (2) D. H. Morman and G. A. Harlow, ANAL.CHEM., 39, 1869 (1967). (3) C. D. Ritchie and R. E. Uschold, J . Amer. Cl7em. SOC.,89, 1721 (1967). (4) C. D. Ritchie and P. D. Heffley, ibid.,87, 5402 (1965). ( 5 ) S. K. Hall and E. A. Robinson, Can. J . Chem.,42,1113(1964).
