Determination of the Diastatic Activity of Honey H. A. SCHUETTE AND R. J. PAULY,~ University of Wisconsin, Madison, Wis. T HAS been pointed out by others (4) that a high de-
I
gree of accuracy can be obtained in the determination of the diastatic activity of honey by the current German procedure (1, 2) only if attention be given to the pH of the reaction mixtures. Attempts to apply this newer technic unexpectedly brought forth the observation that still other factors enter the picture-the quality or age of the soluble starch which is employed as substrate and the interpretation of the starch-iodine end point. In this study five comb honeys were used, from which the honey itself was strained in the laboratory without the use of any external heat. Six soluble starch solutions, either personally prepared from crude starch or from commercial products, served as substrates. TABLEI. RECORDOF EXPERIMENTAL SOLUBLE STARCH SOLUTIONS STARCH 1. Mallinckrodt
2. Mallinckrodt 3. Merck
drops of 1 per cent phenolphthalein indicator solution and taking as end point that a t which the pink coloration persists for a t least one minute, and then treat with 6 cc. of 0.02 N hydrochloric acid to bring the honey solution back to approximately optimum conditions a t this point. (This volume of dilute hydrochloric acid will usually bring the p H of the solution within the limits 4.8 to 5.5, depending upon how accurately the end point of the titration has been determined in the presence of the phenolphthalein, and also, possibly, upon the nature of the honey itself.) Complete the volume of the whole to 100 cc., determine its pH with the aid of a quinhydrone electrode, and select the appropriate buffer solution for the reaction tubes on the basis of these measurements. TABLE111. PREPARATION OF TUBESFOR DETERMINATION OF DIASTASE VALUE
1% DE~CRIPTION SOLUTION 6.18 Of recent purchase. Boiled 3 min. Kept a t boiling point of water for 0.5 hr. 5.76 According t o Lintner." About 10 years 5.93
P I € OF
TUBE
5.17a 6.18
6. Lintner (3)
5.67
7. Small (6)
6.18
8. small (6)
6.27
.
CC
"Id
4. Kahlbaum 5. Kahlbaum
HONEY SOLUTION SUBSTRATE^ (10 Der cent) MIXTURE
cc.
STERIL, WATER (4. 8 . ad 16 cc.)
cc.
DIASTASRI VALUE
7.5b 0.5 6 7.5 1.5 7 7.5 3.0 9 7 ~n 1 4 A . i . .5-_1 5 3.6 7.5 4.9 14 7.5 5.7 18 6 2.8 7 2.2 7.5 6.3 23 7.5 6.8 29 8 1.7 7.5 7.2 39 9 1.3 10 1.0 7.5 7.5 50 This mixture consists of 5 cc. soluble starch solution 0.5 cc. 0.1 N NaCl solution, and 2 cc. of the 0.067 M phosphate buffer soldtion of appropriate
1 2 3
At-ie-ast 15 years old Preceding starch washed with 2% NHiQH soln. and then water, after which it was
1% HC1 concentration A solution made from starch taken from the upper layers in the bottle showed a p H of 4.22. a
8.0 7.0 5.5
"7
This tube may be omitted from the series if the honey under examination is an authentic one. If a lower limit must be set up beoause of a sample naturally poor in diastatic activity, the volume of substrate may be reduced.
Phosphate buffer solutions were made by mixing 0.067 A4 solutions of orthophosphoric acid and its salts in the proportions given in Table 11. The selection of that buffer mixture which is necessary to hold the pH of the various tubes near the optimum-5.26 to 5.30 as recommended by Lothrop and Paine (4)-will, of course, depend upon the pH of the honey solution already prepared as well as that of the substrate solution. If a careful selection of solutions is made, the 2 cc. of buffer solution which are added to each tube are sufficient to hold the p H of the series within a A0.03 variation. Should more exact control be desired, it will be necessary to vary the amount of buffer solution added to each tube in proportion to the amount of honey in each, but because of the time involved this is not recommended in routine control work.
Fill ten test tubes, rather than twelve as recommended by Gothe (a), with the mixture as shown in Table 111. Thoroughly mix the contents of each tube, place the entire group in a water bath maintained a t a temperature of 45' to 47" C. for one hour, gently agitating the tubes every 10 minutes, after which cool them rapidly by immersing in a bath of cold water. Immediately add 1 drop of 0.1 N iodine test solution, mix thoroughly, and note the colors obtained. Finally, because it is sometimes difficult to reach a decision as to the end point, add a second drop of the iodine solution, shake, and again note the colors. Select the last tube which shows a true purple color changing to a red upon the addition TABLE11. VOLUME COMPOSITION OF PHOSPHATE BUFFER of the second drop of iodine solution as the basis for calculating the diastase value, or select that tube having a true SOLUTIOKS purple color immediately preceding the first one in the series PH &PO4 KHZPOI NanHPOa. 12Hn0 in which a true blue color was developed with 1 drop of the 1 10.0 1 5.14 1 7.5 1 5.17 iodine solution, as lilac and violet colors often appear before 1 5.0 1 5.33 1 2.0 1 5.50 purple. These can be eliminated only by the addition of a 1 1.0 1 5.63 second drop of iodine, whereas the true blue color of iodoAfter several variations of procedure were experimented starch is easily discernible from wines, lilacs, and purples. with throughout this investigation, the method given below TABLEIV. SELECTION OF STARCH-IODIDE END POINT was adopted as leading to the best reproducible results with c COLORATIONS OBSERVED a minimum of exacting technic. It embodies several deEXPERIMENT 4 EXPBRIMENT 17 EXPERIMENT 25 TUBE Drops Iodine Drops Indine Drops Iodine partures from the original Gothe method (a). 1 2 1 2 1 2 Dissolve a 10-gram sample of honey in approximately 70 cc. 4 ... Brown Red of freshly boiled, and then cooled, distilled water. Neutralize 5 Bk&n B&vn Yeiiow Brown Light wine Red 6 Wine Red Light brown Brown Wine Red the resulting solution with 0.05 N sodium hydroxide, using 2 7 Purple Dark wine Light wine Brown Purple Red
--.
8 9
1 Present address, American University of Beirut, Beirut, Lebanese Republic.
10
53
Blue Blue Blue
Dark purple Wine Blue Purple Blue Blue
Red Violet Purple
Blue Blue Blue
Purple Blue Blue
ANALYTICAL EDITION
54 VALUE TABLE v. DIASTASE
Vol. 5, No. 1
HONEYAS AFFECTEDBY KIND values, as well as the interpretation of the end point of the reaction when expressed in terms of quantity of iodine test DIAETAEB VALUB solution used, are so obvious as hardly to require comment. HONBY AB SHOWN BY REACIDITY IODINB TEST ACTIOS However, in analyzing the data statistically, it appears that a FLORAL (Calcd; SOLUTIONADDED T U B E @ minimum variation of 64 per cent and a maximum of 100 were EOERCE as formic) METHOD STARCH"1 drop 2 drops pH range found in the diastase value of the honeys under examination. % Clover 0.'185 Lothrop-Paine 4 39 .. 5.32-5.81 Quantitatively these figures have littIe significance because Pauly 2 29 39 5.28-5.29 Gothe 1 23.8 .. 5.89-4.71 the survey is probably insufficientwith respect to number of Range 23-39 honeys to which the modiiied technic has been applied. Buckwheat 0 , 2 1 2 Lothrop-Paine 1 23 23 5.29-5.31 Qualitatively, however, they show that the character of the Pauly 1 23 29 5.25-5.27 Gothe 1 14 5.38-4.76 soluble starch which serves as substrate is a factor which Range 14-23 23-29 may easily nullify the results of a determination of the 29 29 5.32-5.32 Tulip-poplar 0 , 1 8 3 Pauly 2 14 18 5.26-5.25 8 diastatic activity of a honey which has otherwise been carried 18 6.61-4.75 Gothe 1 Range 14-29 18-29 out with meticulous attention to detail. A standard starch 2 39 50 5.31-5.33 Alfalfa 0 . 1 2 2 Pauly is obviously necessary for this work. 8 23 23 5.26-5.29 39 5.24-5.26 i 29 OF
OF
SOLUBLESTARCH USED
5 1
Clover
0 . 1 8 8 Pauly
Gothe 5
28
39
23 a w e 23-39 50 2 29 S 39 1 Range 29-50
23-50 50+ 29 29-50+
5.24-5.26 5.42-4.58
5.28-5.30 5.33-5.25 5.76-4.49
Numbers in this column refer t o starch solutions recorded in Table I.
The data obtained in the application of the above-described mode of procedure for the determination of the diastase values of honey are shown in Table V. The influence exerted by the quality of the soluble starch, upon the observed diastase
LITERATURE CITED
(1) Fiehe, J., and Kordatzki, W., 2. Untersuch. Lebensm., 55, 163-9 (1928). (2) Gothe, F., 2.Nahr. Genussm., 28,286-321 (1914). (3) Lintner, C.J., J. prakt. Chem., 142,378-94 (1886). (4)Lothrop, R. E., and Paine, H. S., IND.ENQ.CHZM.,23, 71-4 (1931). ( 5 ) Small, J. C., J . Am. Chem. Soc., 41,114-20 (1919). RBCBIVBDAugust 20, 1932. Presented before the joint session of the Divisions of Agrioultural and Food Chemistry and Biological Chemistry at the 84th Meeting of the American Chemical Society, Denver, Colo., August 22 to 26, 1932.
A Device for the Calibration of the Variable-Deviation Spectroscope CLARENCE F. GRAHAM, 493 Western Ave., Albany, N. Y.
1
THE calibration of the variable-deviation spectroscope can be simplified by the device shown in the accompanying illustrations, which applies to the spectroscope the registration system of the mirror galvanometer and allows the
j'* FIGURE 1
telescope deviations to be read off on a long illuminated scale in place of the arc of a graduated circle. Figure 1shows a bar clamped to the telescope of a spectroscope, with its end brought forward over the prism. A mirror about 2 ern. square is mounted perpendicular to the bar in such a position that the axis of rotation of the telescope lies in the plane of the mirror. A projector casts upon the mirror the vertical image of a hair, which is reflected to a scale a t a convenient distance from the spectroscope. The
Se.1. i
\\
II
\\ \\ ??bJedm
\
\ \
\ \ \ /
/
FIGURE2
projector, spectroscope, and scale are fastened rigidly in relation to one another, as shown in Figure 2, and calibration is carried out on the scale in the usual way. The deviation readings of the telescope can be spread out on the scale to any desired extent by increasing the distance between mirror and scale, and a t the same time the compression of the red end of the spectrum can be counteracted by arranging the parts of the system so that the beam from the mirror strikes the scale at right angles when the telescope is a t the extreme violet end of the spectrum. With a flat scale, the readings will vary as the tangent of twice the angle of telescope deviation, and will widen out rapidly as the beam strikes the scale more obliquely. The projector can easily be made from a 6-volt tail-light FIGURE3 bulb, the condensing lens of a flashlight, and a small reading lens. A 6- to &volt bell-ringing transformer will supply sufficient current to operate the bulb at full capacity. The image of the hair on a bright circle of light is produced by a hair stretched across an 8-mm. round hole in a small piece of sheet brass, and cemented a t its ends to the brass. The lighted circle surrounding the image of the hair allows the adjacent numbers on the scale to be read, as shown in Figure 3. The expense of the whole device is nominal, and it has proved a great convenience in quickly picking up desired lines in the spectroscope. REOBXVBDSeptember 29, 1932.
