V O L U M E 2 4 , NO. 3, M A R C H 1 9 5 2 bromine caused late end points and correspondingly high results.
501 able the sample of redistilled aniline, and Paul S. Farrington for his cooperation during the experimental work.
CONFIRMATORY TITRATIONS
Table I contains data obtained from confirmatory titrations carried out as described. These data show a n average error of 0.5 microgram and an average deviation of 0.3 microgram for samples of from approximately 100 and 300 micrograms, and an average error of 0.2 microgram and an average deviation of 0.2 microgram for samples between approximately 10 and 50 micrograms. The factors limiting the accuracy of the above measurements are thought to be the preparations, the standardization, and the instability of the aniline solutions. I n the larger samples, there was considerably better agreement among titrations within one group than between the average titer of the group and the calculated value for the titer. The average “correction time” was 0.3 second of generation time. ACKNOWLEDGMENT
The authors wish t o thank Howard J. Lucas for making avail-
LITERATURE CITED
Brown, R. A,, and Swift, E. H., J. Am. C h a . Soc., 71, 2717 (1949). Meier, D. J . , master’s thesis, California Institute of Technology, 1948. hleier, D. J., Myers, R. J., and Swift, E. H., J . Am. Chem. Soc., 71,2340 (1949). Myers, R. J., and Swift, E. H., Ibid.,70, 1047 (1948). Pamfilov, A. V., 2.anal. Chmn., 69,282-92 (1926). Pamfilov, -4. V., and Kisselva, V. E., Ibid., 72, 100-12 (1927). Ramsep. W. J.. Farrington. P. S.. and Swift. E. H.. ANAL.C m x . 22,332 (1950). (8) Sease, J. W., Niemann, C., and Swift, E. H.,Ibid., 19, 197 (1947). (9) Wooster, W. S.,Farrington, P. S., and Swift, E. H., Ibid., 21, 1457 (1949). I ~ E C E I V Efor D review August 27, 1051. Accepted December 27, 1951. Contribution 1624 from the Gates and Crellin Laboratories of Chemistry, Califprnia Institute of Technology.
Estimation of Molecular Weight of Starch Polysaccharides Determination of Their Reducing End Groups SIEGFRIED NJSSENBAURI
AND
F.Z. ELASSID
Division of I’larit Biochemistry, College of Agriculture, University of California,Berkeley, Calif.
The methods for determining the molecular weight of amylodextrins, aniyloses, and amylopectins based on osmotic pressure or ultracentrifuge measurements are laborious and require large amounts of material. The existing colorimetric methods yield only relative molecular weights. The present method is based on the determination of the reducing group of the polysaccharide by an adaptation of the Folin and Malmros colorimetric procedure for the estimation of glucose. Alkaline ferricyanide in the presence of cyanide is used as an oxidizing agent. Comparison of the molecular weights of a number of amylodextrins, amylose starch fractions, synthetic polysaccharides, and amylopectin fractions of low molecular weight with those obtained from osmotic pressure and other measurements showed a fair agreement. The method allows rapid estimation of molecular u eights of some polysaccharides and requires little material.
T
HE methods available for the estimation of molecular weights of polysaccharides based on the determination of the aldehydic end group, yield information only about the relative molecular size (6). Generally the values for molecular weights of polysaccharides obtained by these methods have not been compared with those obtained by accepted methods, such as osmotic pressure or ultracentrifuge measurements. Lansky, Kooi, and Schoch ( 5 ) investigated a number of such procedures for determination of molecular weight of starch fractions They found that some ferricyanide ( I , 4 ) , alkaline copper (IO),and alkaline 3,5-dinitrosalicylate (6, 7) reagents, even when fairly selective towards oxidation of the terminal aldehydic group, do not give a stoichiometric relationship between glucose and maltose. It therefore appeared that, when these methods are applied to higher polysaccharides, they reflect relative sizes only. In the present investigation it has been found that Folin and Malmros’ (2, 11) method for the determination of reducing sugars could be adapted for the estimation of molecular weights of polysaccharides with a considerable degree of accuracy. It is based on oxidation of the aldehydic group by ferricyanide, the addition of ferric sulfate to form Prussian blue, which is stabilized by gum ghatti, and the determination of the color intensity of the
Prussian blue formed. The relationship of the reducing values between glucose, maltose, and heptaose (seven-glucose-unitpolysaccharide) is stoichiometric, and the molecular weights of amylodextrins, amyloses, and the smaller amylopectin molecules obtained by this method are in close agreement with values obtained by osmotic pressure measurements. The applicability of this method to the determination of molecular mights of amyloses and amylopectins was tested on a number of samples available in this laboratory. The heptaose and 23- and 42-glucose unit amylodextrins were supplied by Dexter French and J. H. Pazur of the Iowa State College. Using chromatographic analysis these workers found the heptaose to be homogeneous and virtually free of impurities (3). They also determined the molecular weights of the nonhomogeneous amylodextrins by oxidation of the aldehydic end group and titration of the resulting acidity. Examination of the results presented in Figure 1 shows that the intensity of color developed by glucose, maltose, heptaose, and 23- and 42-unit dextrins is directly proportional to the number of moles of reducing groups present, and is independent of the chain length. Table I shows a fair agreement between the molecular weight values of amyloses obtained by osmotic pressure measurmenta
ANALYTICAL CHEMISTRY
502 Table I. Comparison of Molecular Weights of Amylose in Glucose Units by Colorimetric and Osmotic Pressure Measurement Methods Average No. of Glucose Units per Molecule Amylose Colorimetric Osmotic pressure 800 800" Corn 660 62W Eas'ter lily Modified corn I 410 390Q Modified corn I1 106 126 795 860a Wheat 595 ' 5600 Apple 1300a Tapioca 1220 Synthetic (potato phosuhorylase I) 820 848 Synthetic (potato phos1190 phorylase 11) 970 Synthetic (muscle phosphoryl423 ase) 470 Molecular weights obtained from osmotic pressure measurements reported by Potter and Hassid ( 9 ) . Those of other samples were determined by present authors.
Table 11. Comparison of Molecular Weight of Amylopectin by Colorimetric and Osmotic Pressure Measurement Methods Average No. of Glucose~-Units Amylopectin Colorimetric Osmotic pressure 236 19 1-253' Synthetic I 219 221b Synthetic I1 2820 3,080C Tapioca subfraction I 2200 37,OOOC Corn a Value obtained by John F. Tayler from the sedimentation constant using a spineo ultracentrifuge (see 8, p. 681). The sample has been synthesized in this laboratory. b Sample of iyntlietic amylopectin furnished by S. Peat, University of Bangor. North Wales. Its molecular weight was determined by present authors. Osmotic pressure molecular weight reported b y Potter and Hassid (9).
and by the present method. Table I1 indicates that this agreement also holds for amylopectins of relatively small molecular weight. This agreement, however, fails in the case of amylopect,ins of very large molecular weight, such as corn amylopect,in. REAGENTS
Potassium Ferricyanide, 0.8% Carbonate-Cyanide Solution. Eight grams of anhydrous sodium carbonate are dissolved in 50 ml. of water, to which 15 ml. of freshly prepared 1yo sodium cyanide are added, and the solution is diluted to 500 ml. Ferric Sulfate-Gum Ghatti Solution. Twenty grams of gum ghatti are soaked in 1liter of water for 24 hours. To this solution a mixture of 5 grams of anhydrous ferric sulfate, 75 ml. of 85% orthophosphoric acid, and 1OOm1. of water is added. Thesolution is then oxidized with 1% potassium permanganate, as described later. PROCEDURE
Soluble samples such as sugars, amylopectins, and some amylodextrins are dissolved in water. One- or 2-ml. samples of Liolution containing from 20 micrograms to 50 mg., depending on the molecular weights of the polysaccharide, are pipetted into graduated test tubes or centrifuge tubes. One-half milliliter of an 0.8% solution of potassium ferricyanide and water, if necessary, is added to each tube to bring the volume to 2.5 ml. After 0.5 nil. of the carbonate-cyanide solution is added, the contents are mixed and the tubes immediately heated in a boiling water bath for exactly 8 minutes. The tubes are then cooled in running cold water, 5 in]. of ferric sulfate-gum ghatti solution are added to each tube, the contents are mixed, and the tubes are allowed to stand for 5 minutes. Enough water is then added to dilute the volume to 10 ml. A green filter (520 m r ) is inserted into a KlettSummerson photocolorimeter and the apparatus is adjusted to give a zero reading when the tube containing the blank is examined. Blanks are prepared by substituting water for the polysaccharide sample. Standard samples of glucose or amylodextrin of known molecular weight are run simultaneously with each determination. .4mylopectin solutions are usually cloudy, but in most cases the cloudiness disappears after the samples have been heated with ferricyanide. 11it persists, a correction can be made by means of a special blank. Such a blank is prepared by heating a mivture of
the usual volume of ferricyanide and carbonate-cyanide solution, cooling, and adding gum ghatti and the proper aliquot of amylopectin solution and water to make e volume of 10 ml. The molecular weight of the polysaccharide is calculated from the reducing value in terms of anhydroglucose residues. The following procedure has been found convenient for the determination of amyloses :
A 25- to 100-mg. sample of the dry, finely powdered mmple is weighed into a 10-ml. volumetric flask and thoroughly wetted with 1 to 2 ml. of petroleum ether. Two milliliters of approximately 1 N sodium hydroxide are added and the contents are mixed. The sample is allowed to stand until the amylose is completely dissolved, which may require from 5 to 60 minutes, A previously determined amount of 0.5 N hydrochloric acid, sufficient to neutralize the alkali, is then added from a buret. The pH of the solution may be further checked with the aid of strips of indicator paper. The petroleum ether is eliminated a t this point by gentle warming of the solution. The volume is made up to mark and the solution is used for analysis aa soon as possible. FACTORS AFFECTING ACCURACY O F METHOD
Certain precautichs must be observed, in the event extraneous substances are present in the solution containing the polysaccharides or sugars. When the reducing values of carbohydrates are determined in enzymatic digests, ammonium sulfate, citrate, acetate or other buffers are usually present. These constituenta must be kept a t a fairly low level; othern-ise a considerable error will result. Table I11 shows the effect of various buffers a t pH 7 on the reducing value of standards of known concentration. It can be seen that when 0.5 millimole of citrate or 0.05 millimole of ammonium sulfate is added to the carbohydrate sample, the error becomes so large that the determination is worthless. The results are also affected if the sample is not carefully neutralized. In analyzing the samples, it is desirable to use duplicate aliquots of differentsizes. If large amounts of inorganic salts or other interfering constituents are present, no proportionality in results is obtained when different size aliquots of the same carbohydrate concentration are used. The volume of the sample a t the time of heating with ferricyanide is critical and must be carefully controlled.
A A
301 v)
(3
i 4L P y
c
POI
2 a
2
U
10 P
PO
10
0
MICROMOLES X 102
Figure 1. Reducing Values 0 Glucose
A Maltose
0
Heptaose 23-unit dextrin
A 42-unit
dextrin
30
503
V O L U M E 2 4 , NO. 3, M A R C H 1 9 5 2 ACKNOWLEDGMENT
Table 111. Effect of Various Buffers (pH 7) Acid and Alkali on Colorimetric Determination Reading Klett-Sunimerson Photocolorimeter Divisions Standard (30 y maltose) Standard 0.5 mmol. citrate Standard 0.2 mmol. citrate Standard 0.1 mmol. ammonium sulfate Standard 0.05 mrnol. ammonium sulfate Standard 0.025 mmol. ammonium sulfate Standard 0.1 mmol. sodium hydroxide Standard 0.3 mmol. hydrochloric acid
+ + ++ ++ +
The authors are grateful to the Corn Industries Research Foundation for support of this work. LITERATURE CITED
96
55 95 0 12 96 85
0
-
Enough 1% permanganate solution must be added to the gum ghatti-phosphoric acid solution so that one additional drop of the oxidizing agent will impart to gum ghat,ti solution a pink color that mill remain stable for at' least 24 hours. The blank should rc9ni:iin yellon- in color. 9green color indicates the presence of reducing impurities and makes accurnte analysis difficult, even if the hlank is used to determine an arbitrary zero point. If the gum ghatti solution has been standing for a long time in the acid medium, hydrolysis is apt, to liberate further reducing groups which must be reoxidized wit.h permanganate. Improperly oxidized gum ghatti solutions cause large blanks.
Farley, E. F., a n d Hixon, K. )I., IND. FNG. CHEM.,ANAr.. ED., 13, 616 (1941). Foliii, O., a n d lfalmros, H., J . Biol. Chem., 131, 211 (1929). French. D.. Levine. M. L., a n d Pazur. J. H.. J . A m . Chem. SOC., 71, 356 (1949). Gore, H. C., a n d Steele. H. K., IXD.E ~ G CHEY., . ANAL.ED.,7, 324 (1935). L a n s k y , S.,Kooi, >I., a n d dchoch, T. S., J . Am. Chcm. Soc., 71, 4066 (1949). hIeyer, K. U., iYoelting!. G , and Bernfeld, P., Helv. Chzm. Acla, 31, 103 (1948). Koeltinn. G . , a n d Bernfeld, P., I t i d . , 31, 286 (1948). Xussenbaum S. and Hassid, I T . Z.,J . Bzol. Chem., 190, 673 (1 951). P o t t e r , A. L., and Hassid. \t. 2.. J . Am C'hem. S o c , 70, 3774 ,19381. Richardson, K.A,. Higgjiibotham, R. S., a n d Farrow, F. D., J . Yestile Inst., 27, 131. r m b r e i t . R'. IT., Burris, K. H . , and Stauffer, J . F., "hlanometric Techniques a n d Related M e t h o d s for the S t u d y of Tissue hIetabolisrn." p. 103. hlinneapolis, M i n n . , Burgess Publishing Co., 1945. R E C H IE D for review October 26, 1951. .\ccepted Decelnber 26, 1961. I
_
Organoleptic Panel Testing as a Research Tool L. C. CARTWRIGHT, CORNELIA T. SNELL, 4 N D PATRICIA H. KELLEY Foster D . Snell, Inc.. New York, N . Y. Organoleptic panel test methods can be utilized by any laboratory group having 5 to 15-preferably 15people available for organoleptic training. The method is suitable for solving problems or answering questions concerning foods and beverages, not susceptible f solution by other analytical procedures. The principal practical applications deal with detecting the source of a disturbing off-quality in flavor or taste, the effect of a specific ingredient added to improve quality, and probable consumer preference of competing products similar in general character
T
HE human seiises are capable of distinguishing a tremendous
number of distinct impressions. The senses of most humans can be rendered much more acute and the memory of sensory perceptions can be highly developed through training (1). Herein lies a powerful tool for evaluation of those properties of matter that affect the senses, but serious errors may result from its careless use. In the first place, there are great differences in the reactions of individuals to the same sensory stimuli (21). Recognition of this fact led to the use of carefully selected and trained professional tasters for beverages, and of odor experts in the perfume and essential oil industries (6, 7 ) . A less widely recognized, but perhaps more serious source of error is the almost continual, sometimes extrenie, variation in the sensory acuity of even the most carefully selected expert ( 1 ) . Errors from this source can be reduced by having the expert re-evaluate each sample several times, but this is not always feasible. A more effective nieans of detecting and niininiizitig such errors is through the use of an organoleptic panel of carefully selected and trained nienibers. Individual errors then tend to be compensating. Much attjention has been given in recent years to the improvement and refinement of techniques of organoleptic panel t,esting,
but differing somewhat in flavor. Various aspects of such problems are discussed in terms of the experi-
ence of the group; examples illustrate different types of applications drawn from many industrial problems. Methods of scoring, the training of panel members, and statistical treatment of results are discussed. A summary of data i8 given, based on the evaluation of eight samples of a particular product. T-alues for standard error of the mean demonstrate the accuracy of evaluation and show the relative quality of the different samples.
and many published papers attest to the effectiveness of this method as a research tool (9, 13, 14, 16-20.). Although organoleptic panel testing has been used most extensively in the evaluation of odor and flavor in foods and beverages, there are numerous eunigles of its application to other problems. Most of the basic principle are directly applicahle to the evaluation of any sensory stimulus SELECTION O F SYSTEM OF EVALUATION
First the property or properties of the product to be evaluated must, be determined. These may be aroma, flavor, appearance, consistency, or any other property affecting sensory response. It must tic decided whether these are to be evaluated separately, each as :in entity, or each as a composite of several factors; and whether :in over-all evaluation of the product or material is to be atteniptc>d,involving some weighted coinbination of the ratings of various factors. Then it must be decided whether evaluation is to be mnde on the basis of preference ratings of samples or on the basis of some effectively quantitative rating of each property, and whether results will be expressed as direct numerical scores or
