1192
ANALYTICAL CHEMISTRY
Table I.
Definitions of Terms Referring to Performance of Precision Balances Sensitivity. General term to be used as “the ratio of the change in the response t o the change of the quantity measured” SENSIBILITY RECIPROCAL ( S R ) . The SR of a balance is the change in load required to change the equilibrium position-i.e., rest point-by one division at any load within the load limitations of the instrument. Unit. Mass units per division (width of division 1 mm. or more) Readability READABILITY. The readability of a balance is the smallest fraction of a division to which the index scale can be read with ease either by estimation or by use of a vernier. Unit. Divisions Precision. General term to be used as “the degree of agreement of repeated measurements of the same quantity” STANDARD DEVIATION.The standard deviation of a balance is the standard deviation, S , of repeated weighings of the same mass, a weighing being considered to involve the determination of two rest points (other than those used to determine the sensitivity). Unit. Mass units (usually gram, mg., or y) Equation.
was employed. Such a list might include ( a ) inequality of arms; ( b ) over-all adjustment, variation with load, and nonlinearity of a direct-reading scale; (c) errors of the chain due t o incorrect setting of the chain knife-edge, nonlinear distribution of mass along the chain, eccentricity or other defect of the drum on which the chain is wound, and the setting error or the “up-down” difference in the effective values of the chain when a position of the chain scale is carefully approached from below and from above; ( d ) errors in the total mass of the rider, in the positions of the rider notches, and in setting the rider in a notch (tilt error of wire rider, eccentricity error of cylindrical rider); ( e ) errors in the masses of the drop-on weights; and (f) errors resulting from change in zero rest point with change in temperature or relative humidity. Some of the errors listed above may under some circumstances be treated not as systematic errors but as random errors, and hence may be included in the “precision” of the balance. This is particularly true of the errors arising from setting the rider in a notch and the “up-down” error of a chain. The recommendations of the committee are summarized in Table I. LITERATURE CITED
Factors Influencing Accuracy (other than precision), Accuracy is to be used as ”the agreement between the result of a measurement and the true value of the quantity measured.” Some of the factors influencing accuracy are listed below. Definitions for these factors will be established in connection with test procedure. Inequality of arms Errors in masses of drop-on weights Position error of rider notch Nonlinearity of chain Error in mass of rider Nonlinearity of direct-reading scale
of a measurement and the true value of the quantity measured.” Unfortunately, we can only “guess” a t the true value of any quantity. This is done b y correcting for all known systematic errors. Thus accuracy is not a very useful performance parameter for the general characterizing of an instrument, It is more appropriate for evaluating a particular technique of measurement, such as weighing with standard and unknown in different pans, use of a “direct-reading” scale without calibration, etc. Under the general heading, “accuracy,” or “factors influencing accuracy,” should be listed the possible systematic errors present in the result of a measurement in which a particular technique
(1) Ainsworth and Sons, Inc., Wm., Denver, Colo., “Laboratory Balances and Weights,” Catalogue AW 12 (1948). (2) Eisenhart, C., Photogrammetric Eng., 18, 542 (1952). (3) Federal Specification for Analytical Balances, AAA-B-92, May 17, 1949, General Services Administration Regional Offices, or General Services Administration Regional Office Building, Seventh and D Sts. S.W., Washington 25, D. C. (4) Fisher Scientific Co., Pittsburgh, Pa., “Modern Laboratory Appliances,” Catalog 90 (1949). (5) Goldstein, S. W., and Mattocks, A. M., J . Am. Pharm. Assoc., 12, 293 (1951). (6) Indiana State Board of Health, Division of Weights and Measures, Indianapolis 7, “Questions Submitted by Members and Answered at Conferences of the Indiana Association of Inspectors of Weights and Measures,” Question S-121. (7) MacNevin, W. >I., “The Analytical Balance, Its Care and Use,” Sandusky, Ohio, Handbook Publishers, Inc., 1951. (8) Meek, R. E., “Information Concerning Prescription Scales and Prescription Weights,” Indiana State Board of Health, Division of Weights and Measures, Indianapolis 7, Ind. (9) Natl. Bur. Standards, Handbook 44, 92 (1949). (10) Rodden, C. J., Kuck, J. 4.,Benedetti-Pichler, A. A., Corwin, A. H., and Huffman, E. W. D., IND.ESG. CHEM.,ANAL. ED., 15, 415 (1943). (11) Youden, W. J., “Statistical Methods for Chemists,” p. 13, New York, John Wiley & Sons, 1951. RECEIVED for review April 7, 1954. .4ccepted M a y 27, 1954
Quantitative Method Using Paper Chromatography For Estimation of Reducing Oligosaccharides W. H. WADMAN, GWEN J. THOMAS, and ARTHUR B. PARDEE Department of Plant Biochemistry a n d Department of Biochemistry, University of California, Berkeley, Calif.
I
E T H E past, separation b y paper chromatography of oligo-
saccharides which contain more than four monosaccharide units has not been practicable oiving t o the low R, values of these substances. Bayly and Bourne ( 2 ) have recently overcome this difficulty by converting the sugars into the corresponding N-benzylglycosylamines. The increase in R, values is such that dextrins containing up to ten units are easily separated. French and Wild have also studied the chromatography of carbohydrates (S). Quantitative separations had been achieved before this b y use of charcoal columns. Whistler and Durso (8) have successfully separated sugars, the quantities used being of the order of 1 gram. Disadvantages of the method are that intermixing of adjacent compounds can beconsiderable and that small amounts of certain compounds may be overlooked.
Another quantitative method, described by Alm (I), involves application of a continuously increasing concentration of ethanol in water as eluting agent applied to a mixture of sugars on a carbon adsorbent which has been pretreated with stearic acid. I n this way, 5-mg. samples of hydrolyzed a-Schardinger-dextrin could be rapidly and quantitatively resolved. I n this report a method is described for rapid separation and estimation of oligosaccharides on a microgram scale, using a technique similar t o that of Bayly and Bourne. The sugars are allowed t o react with AT-( 1-naphthyl)-ethylenediamine and are then separated by paper chromatography. The amount of each sugar may be estimated from the brightness of fluorescence of its derivative under ultraviolet light.
V O L U M E 26, NO. 7, J U L Y 1 9 5 4 PROCEDURE
A portion of the mixture to be investigated, containing between 0.1 and 4% sugar, is added to an equal volume of a 10% solution of N-( 1-naphthyl)-ethylenediamine dihydrochloride (obtained from Eastman Kodak Co. j in triethylamine-ethanol-water (5-4-1) solvent. Five microliter aliquots are applied to Whatman S o . 1 paper; then the paper is heated a t 100" C. for 30 minutes. The chromatograms are run about 18 hours by the descending method in 1-butanol-ethanol-water-ammonia solvent (40-12-161) and dried a t room temperature. The uncombined amine is found immediately behind the solvent front and the positions of the sugar derivatives, which are easily located because of their fluorescence, are arranged in order of increasing size as well separated spots behind the amine. The sugars may be estimated directly on the paper or after elution. The most accurate determinations were made by cutting out the spots, eluting the fluorescent material with 5 ml. of 1% Xa3PO4.12H20and determining fluorescence in a fluorometer. In the present work the Beckman spectrophotometer with fluorescence accessory set was used. Known amounts of the sugars were run on the same paper to provide calibration values. EXPERIMENTAL
Various experiments leading to selection of the described procedure are presented here. Choice of Amine. Of a large number of amines tested, the following fluoresced on paper: p-aminobenzoic acid, l-naphthylamine, 3-aminophthalylhydrazide, anthranilic acid, and N-( 1naphthyl)-ethylenediamine. Only the last amine gave one compound of high R, with maltose, and because high R f is essential for success of method, this amine was used in all subsequent experiments. (It is sometimes referred to hereafter as the amine.) Anthranilic acid gave two spots, presumably the CY- and 8-glycosylamines, with each of twelve aldoses tested. The R/ of both spots was relatively small for maltose (0.17, 0.10) compared to the one spot obtained with maltose plus naphthylethylenediamine (R, = 0.6). Fluorescence of the Amine. The optimal exciting wave length for fluorescence was determined by placing pieces of paper spotted with amine in the light path of the Beckman spectrophotometer. A filter which cut out light below 390 mp was placed in front of the photocell to absorb the exciting beam. (This filter was supplied as part of the fluorescence accessory set.) Under these conditions the exciting wave length which gave maximum readings for emitted light was found to be 355 mp, although it varied from 335 to 370 mp, depending on amine concentration. Also, the wave length must be influenced by output of the light source and photocell sensitivity in this region. I n subsequent experiments light of 355 mp was used. The fluorescence of the amine was maximal in basic solution, in which a readily measurable reading \vas obtained with 0.05 microgram (y). Acid caused an irreversible loss of fluorescence. Coupling of Amine and Sugar. Bayly and Bourne ( 2 ) coupled benzylamine and sugars by spotting one above the other on paper. They reported that about 80% of the sugar reacts under their conditions. I n the present work it was found that reaction with naphthylethylenediamine was not so complete, as shown by the presence of spots corresponding to free sugars, made visible by panisidine spray of the chromatogram. To improve coupling, solutions of maltose and amine in various solvents were placed on paper, heated 30 minutes a t 100" C., and chromatographed. The fluorescence of the maltose spot was compared with fluorescence of the amount of amine equivalent to the maltose, in order to determine the efficiency of coupling. The highest coupling (60%) was obtained when a solution containing 1 gram of amine dissolved in 10 ml. of triethylamine-ethanol-water (5-4-1) was combined with an equal volume of maltose. In the presence of triethylamine, the most effective of several amines and other basic compounds tested, the fluorescence reading was 63 as compared to a reading of 20 with water present. It was found that the concentration of naphthylethylenediamine is critical and that as high a concentration as possible should be used.
1193 The efficiency of coupling was also checked using radioactive glucose. The radioactivity of the glycosylamine spot was 61% of that of the applied glucose. The effect of various types of heating was studied and it was found that 30 minutes a t 100" C. on the paper gave maximum coupling of amine and sugar. Preliminary experiments indicated that heating amine-sugar mixtures in solution was not as effective. Spotting sugar and amine separately on the paper gave fairly good coupling in preliminary experiments and this method would conserve material. A large excess of amine must be added. Paper. Whatman KO. 1 paper was used, Schleicher and Schull No. 597 gave similar results. Whatman iVo. 4 gave faster movement of spots and hence may be superior for separation of higher oligosaccharides. Prewashing the paper with 1o/o trisodium phosphate reduced the blank 5Oy0 and improved resolution slightly, but this procedure was not usually used because the blank with unwashed paper was not seriously large. Preloading the paper with trisodium phosphate is advantageous for separation of higher oligosaccharides because RJ is increased. Development. Of 17 solvents tried, butanol-ethanol-waterammonia (40-12-16-1) was found to be best, since well-formed spots were obtained and trailing was reduced to a minimum. It was used in all experiments. Equilibration of paper in the cabinet was not necessary. Elution of Spots. The most effective eluent found was 1% sodium phosphate dodecahydrate. ,411 fluorescence was removed by the first milliliter upon chromatographic elution. Hydrochloric acid (0.2AV)also appeared to remove the amine spot but actually caused irreversible loss of fluorescence. Mixtures of various alcohols and water were not effective.
I
I
('0 70
aJ u C 0
60
e
50
.$C
40
0
30
20 10
0 IO
20
30
40
Maltose 6 Figure 1. Fluorescence of Various Amounts of Maltose
__ Fluorescence in solution after elution - - _Fluorescence o n the paper Estimation of Amines. The most reliable method of estimation requires elution of the spot and determination of fluorescence in solution. A calibration curve obtained with known amounts of maltose is shown in Figure 1. Good linearity was obtained with up to a t least 40 y of maltose. Fluorescence may also be read directly on the paper. A strip is cut from the center of the spot and placed in front of the photocell of the Beckman spectrophotometer, I t may be placed in the cell holder if desired or fastened with rubber bands in front of a hole in a wooden block, as close to the photocell as possible. A filter to absorb the exciting light (355 mp) and to permit passage of light above 400 mp is between paper and photocell. With slit a t 2.0 and sensitivity a t maximum, fluorescence is read on the T scale. Figure 1 s h o w a curve for varying amounts of maltose obtained in this way. A reasonably good estimate may be obtained for up to a t least 30 y of maltose. If the spot size varies considerably, the entire spot may,be illuminated (if not too large) by detaching the photocell housing of the spectrophotometer and moving i t as far as possible from the body of the instrument. A wooden box connecting the two parts keeps out stray light. With this device, spots are placed before
1194
A N A L Y T I C A L CHEMISTRY
the photocell, next to the filter, and are read as above. A linear plot of log 2' versus amount of maltose was obtained for 20 t o 200 y. One may, alternatively, elute the spot and read the optical density a t 340 mp in the conventional way. Less sensitivity is obtained than when flaorescence is determined and the blank is much higher. However, 20 to 200 y of maltose can be estimated readily. It is possible that some other amine could be found which couples well with sugars and has a higher extinction coefficient than naphthylethylenediamine. APPLICATIONS
Analysis of Hydrolyzates of Complex Saccharides. When an acid hydrolyzate of amylose was chromatographed in the manner described, dextrins containing up t o eight sugar units were separated after 24hour development, the pattern being similar to that obtained by Bayly and Bourne (Table I). A linear relation was obtained when log R. was plotted against the number of glucose units up t o five. The resolution was improved by using paper which had been pretreated with a 1% solution of sodium phosphate. The R, values could also be increased by using Whatman No. 4 paper.
Table I. lMovement of Various Oligosaccharides on a Chromatogram Dietance,'Cm. 39 4 35 2
dmine Glucose 2
Ro"
26
loo 0.89 0 66
16 10.2 7.5
0.41 0.26 0.19
~~
3 4
5 6
0 09
3.4
Ro equals the ratio of distance moved by the spot to the distance moved by free amine. a
loo
80
r -
70 0,
0
C 0
.-
i
6050
5c
40
E I-
30
-
-
g201 / 10
100
200
Counts Figure 3.
300
400
500
600
per m i n u t e
Comparisonof Fluorescenceand Activity of Radioactive Maltose Spots
with the Shaffer-Hartmann reagent ( 7 ) . Similar results were obtained by the two methods (Figure 2). Determination of Specific Activity of Labeled Compounds. Radioactive maltose was isolated by paper chromatography from the products of action of a-amylase on radioactive starch. Different weights of maltose were treated with the amine and chromatographed. The maltose-amine spots were eluted, diluted to 5 ml. with water, and portions were concentrated to dryness on copper disks for counting, while the remainder was used for determination of fluorescence. Standard maltose spots were also applied to the paper. In view of the resulting linear plot of count minus background versus fluorescence (Figure 3), it is suggested that this method may have an important application in the determination of the specific activities of the various metabolic products formed after a labeled sugar has been introduced into a plant or animal. Both the quantity and activity of any simple reducing sugar can be measured either directly on the paprr chromatogram or after elution. DISCUSSION
c Y)
0
-u
60
-
40
-
20
-
ln 0
a0
=aJ 0
8
0
0
I
2
3
4
5
6
7
Hours Figure 2.
lleasurement of Hydrolysis of Cellibiose by Two Methods
The conversion of wheat amylopectin into maltose was determined by three methods. After 24-hour digestion with crystalline 8-amylase at pH 4.8, aliquots Tvere drawn for the determination of reducing power, maltose (by paper chromatography), and iodine stain. The conversion into maltose determined by the reducingpower method was56% and by paper chromatography it was 59%. The absorption value of the iodine-stained polysaccharide was 55% of the original value. The hydrolysis of cellobiose by emulsin has been followed by measuring the disappearance of disaccharide with time, determined by chromatography of the digest. At the same time the increase in reducing power was determined by difference titration
The method may be useful in the study of hydrolysis of polysaccharides and the metabolism of radioactive mgars, owing to the ease with which analyses may be performed and the small amounts of sugar required. The method might also be suitable for the detection and rapid estimation of those trape sugars which are formed during the initial stages of the enzymatic hydrolysis of certain disaccharides (6). The reaction between the sugar and amine is not complete, as shown by the fact that uncombined sugar may be detected close to the origin by spraying with p-anisidine. Measurements have shown that 60% of the sugar reacted with the amine under the conditions described. Attempts t o increase coupling, such aa further heating of the reactants on the paper and heating the solution before applying to the paper, were not beneficial. Both autoclaving the paper and heating the reaction mixture a t 100" C. in a sealed tube led to the formation of multiple spots. A high concentration of amine in the solution was important and the concentration chosen was the highest that could be prepared. The addition of triethylamine to the reaction had a marked effect. It is felt that the incomplete reaction between the sugar and amine is not a drawback to the use of the method if the appropriate standards are included on the same paper because the presence of one sugar does not interfere with estimation of a second sugar. Thus, a constant ratio was obtained for maltose and maltotriose obtained by the action of salivary a-amylase on different amounts of amylose. The procedure described here is not entirely satisfactory for the estimation of monosaccharides, although glucose may be de-
V O L U M E 26, NO. 7, J U L Y 1 9 5 4 tected qualitatively. The reason for this is that the R , value of the glucose-amine complex is very close to that of the free amine. (Little advantage is gained by prolonging the development because the size of the spots increases correspondingly.) Solvent systems have been found in which the distance traveled by the glucose-amine spot is half of that traveled by the amine. However, when such systems were used there was marked elongation of the spots together with a fluorescent streak all the way down the paper. When monosaccharides were treated with anthranilic acid rather than with naphthylethylenediamine, all the hexoses and pentoses investigated were separated, their R j values being far removed from that of the amine Anthranilic acid was not, however, adopted for general use since each sugar was found to give two fluorescent spots. The method of McFarren et al. ( 4 ) is available for quantitative separation and estimation of monosaccharides by chromatography. Improved chromatographic separation can be achieved when an additional group is added to members of a homologous series h t first glance it might appear that differences in the series would become less important and therefore a poorer separation would result. However, it can be shown theoretically that this is not
1195 necessarily so ( 5 ) and that R, differences should be increased by making the proper derivatives. ACKNOWLEDG.M ENT
The authors wish to express their gratitude to the Corn Industries &search Foundation for its financial support, to W. Z. Hassid for encouragement of this work, and to E. W. Putman for preparing the Cl4-labeled starch. LITERATURE CITED
(1) Alm, R.
S.,Acta Chem. Seand., 6 , 1186 (1952).
(2) Bayly, R. J., and Bourne, E. J., S u t u r e , 171, 385 (1953). (3) French, D.. and Wild. G. M.,J . Am. Chcm. Soc.. 75. 2612 (1953). (4) McFarren, E. F., Brand, K., and Rutkowski, H. R., ANAL.C H E ~ . , 23, 1146 (1951). (5) Martin, A. J. P., Biochem. Soc. Symposia (Cambridge, Engl.), 3, 4 (1949). (6) Roberts, H. R.. and McFarren, E. F., Arch. Biochem. and Biophys., 43, 233 (1953). (7) Shaffer, P. A., and Hartmann, A. F., J . B i d . Chem., 54, 365 (1921). ( 8 ) Whistler, R. L., and Durso, D. F., J . Am. Chem. Soc., 72, 677 (1950). RECEIVED for review October 27, 1953. Accepted rebrriary 23, 1954.
Detection of Adulteration of Butter with Vegetable Oils by Means Of the Tocopherol Content J. H. MAHON and ROSS A. CHAPMAN Food and Drug Laboratories, Ottawa, Canada
T
HE tocopherol level of butter oil is low (0.002 to 0.005%)
while that of most vegetable oils, with the exceptionof coconut oil, is considerably higher. I n most cases, therefore, the addition of vegetable oils to butter will result in a sigriificant increase in the tocopherol content of the adulterated butter oil. Thus, the tocopherol content of butter oil should be a valuable index of adulteration with vegetable oils other than coconut. In order to take advantage of this fact, a rapid procedure for the determination of total tocopherol in butter oil has been developed employing a modification of the Emmerie and Engel ( 4 ) colorimetric method. Because of the nonspecificity of the ferric chloride plus 2,2’bipyridine reagents, a number of substances present in commercial butter oil are capable of contributing an “apparent tocopherol” value. Such interfering substances include the carotenes, vitamin A alcohol, and the synthetic food colors, 1-phenylazo-2-naphthylamine (Oil Yellow AB) and 1-o-tolylazo-2-naphthylamine (Oil Yellow OB). Bird et al. ( 1 , 6 )employed the tocopherol content of butter as an index of adulteration with vegetable oils and removed interferences by saponification and chromatography of the resulting nonsaponifiable residue. In this investigation, the synthetic butter colors and vitamin A alcohol are removed by extraction of the fat solution with 60 volume yosulfuric acid. Carotene which is not removed by this procedure must be estimated and a correction applied. DEFELOPMEYT OF ANALYTICAL METHOD
Therefore, if butter oil is subjected to this treatment, interference due to vitamin A, Oil Yellow A4B,and Oil Yellow OB should be eliminated. However, carotene is not affected by this extraction procedure and therefore constitutes the chief interference i n the acid-extracted solution of butter oil. Quaife et al. (9) employed the absorbancy of a fat solution a t 460 mp as the basis of a correction for the error due to carotene. Employing a petroleum ether solution of crystalline 90% @-lo% or-carotene it was found essentially that equal absorbancies were obtained in an Evelyn colorimeter fitted with either a S o . 440 or a No. 470 filter. A No. 440 filter was employed in this investigation. I n order to ascertain the appropriate correction factor for carotene in butter oil, solutions of crystalline 90% @ 10% or-carotene were prepared in petroleum ether (60’ to 100” C. boiling range). These solutions were extracted for 20 seconds with one fifth their ’ sulfuric acid and washed free of acid with volume of 60 volume % distilled water. Using these solutions, a calibration curve was prepared for carotene in petroleum ether solution employing a KO. 440 filter. A second calibration curve was prepared after reaction with the ferric chloride plus 2,2’-bipyridine reagents employing a No. 515 filter. Under these conditions it was found that the absorbancy with a No. 515 filter after reaction with the ferric chloride plus 2,2’-bipyridine reagents was equal to 26% of the absorbancy of the original carotene solution measured with a Xo. 440 filter. Therefore, the interference due to carotene can be eliminated as follows:
Fox and Mueller ( 5 )state that all the vitamin A is removed by Absorbancy with a No. 515 filter - (0.26 X absorbancy with a S o . 410 filter) = corrected absorbancy extracting a fat-petroleum ether solution with 60 volume % sul(1) furic acid. Chapman and Campbell ( 3 )report that the extraction of a mixture of tocopherols in petroleum ether solution with 60 The corrected absorbancy is employed to calculate the tocopherol volume sulfuric acid does not destroy any of the tocopherols. content of the oil sample. Investigations in this laboratory have shown that a single extracIf another type of colorimeter or spectrophotometer is emtion of butter oil in petroleum ether solution with 60 volume yo , ployed, a new correction factor for interference due to carotene sulfuric acid extracts all of the Oil Yellow AB and Oil Yellow OB. must be determined for that instrument.
