493
V O L U M E 2 2 , NO. 3 M A R C H 1950 num chloride, and although no amino acids present in gelatin were found to give the interfering reaction, cysteine and tryptophan were recorded as interfering with the sulfur dioxide test. The tryptophan trace reaction is, however, very slow. Enediols like ascorbic acid and reductone and o-dihydroxybenzenes like adrenalin, catechol, and ethyl hydrocaffeate, were found to give strong but slow to very slow color reactions with the reagent. These reactions, unlike the quick but fading thiol reaction of cysteine, are not inhibited by mercuric or platinum chloride, and it can therefore be assumed that untreated fruit juices or proteins which give the color reaction very distinctly, even in the presence of platinum or mercuric chloride, contain ascorbic acid,
an enediol, or o-dihydroxybenzene derivatives. The reaction with proteins needs further investigation. The presence of anionic wetting agents can also interfere nrith all the reactions which are given by acid-bleached fuchsinformaldehyde (sulfur dioxide, thiols, enediols, o-dihydroxybenzenes, sulfinic acids), but the anionic wetting agents are easily detected because they react slowly with Stock Solution I, and do not depend on the presence of formaldehyde. , LITERATURE CITED
(1) Grant, w. M., AX.41.. C H E Y . , 19, 346 (1947).
RECEIVED April 21, 1949.
Microdetermination of Iodine in Plant Material FORREST G. HOUSTON K e n t u c k y Agricultural Experiment S t a t i o n , I;exin,g:lort, Ii?.,
IIE simplicity inherent in the spectrophotometric procedure rused by Gross, Wood, and McHargue (1) for the determination of iodine in biological materials makes it well suited for the routine examination of agricultural crops. However, certain modifications which make it possible to determine smaller amounts of iodine extend greatly the usefulness of this procedure. 7
Table I.
Determination of Iodine Yo Transmittance
Iodine, 7 / 5 Ml. 0.10 0.25 0.50 1.00 2.50 5 00
a t 575 mp 96.7 92.0 84 0 70.0 41.0 li.O
E X P E R I Vw v r . k L
l3y decreasing the final volume from 50 to 5 ml., 0.1 micrograiii of iodine can easily be detected when a Model DU Beckman quartz spectrophotometer is used n-ith standard Corex cells having a 1-em. light path. However, the color developed is not suitably stable if the concentrations of the reagents are maintained a t the level used by Gross, Wood, and McHargue. The chief tlifficulty appears to be associated with a slow decomposition of tlir: potassium iodide, resulting in a gradual increase in the amount of starch-iodide chromogen formed. This difficulty is partially overcome by greatly decreasing thc amount of potassium iodide used. Further improvement is obtained by substituting phosphoric acid for sulfuric acid in order to maintain the pH of the solution at approximately 2.5. These changes do not significantly decrease the. sensitivity of the starch to iodine and the peak of maximum light absorption remains a t 575 mp. The color is stable for an indiifiriite time if the solution is kept in a bath of ice water until transriiittitiice readings are ready to be made. Cooling also increases the sensitivity of the starch iodide reaction, especially a t thc l o w r concentrations of iodine. Potato starch appears to be slightly bettc.1. than arrowroot starch, because the sol is less opalescent.
Collect the distillate in a 50-ml. Pyrex beaker containing 1 ml. of 0.2 A; sodium hydroxide. Five milliliters of 30oJ,phosphorus acid are sufficient to reduce the excess chromium trioxide completely and ensure recovery of the iodine present. Collect exactly 35 ml. of distillate. After the distillate has been evaporated to 5 or 10 ml., prepare a reference solution in another 50-ml. Pyrex beaker by adding 1 ml. of 0.2 N sodium hydroxide and 5 to 10 ml. of water. Oxidize the iodides as described by Gross, but use 1 or 2 drops of 0.2 AI potassium permanganate followed by 3 drops of 28% phosphoric acid, and continue the evaporation to a final volume of 2 or 3 ml. Transfer the contents of the beakers to small graduated cylinders or test tubes marked a t 5 ml. Immerse them in a beaker of ice water for 2 minutes. Add one drop of 5% potassium iodide, mix, and add 10 drops of 0.25% potato starch. Mix thoroughly, dilute to the 5-ml. mark, mix again, and immerse in the ice water. illlow the tubes to remain in the ice water a t least 2 minutes before reading the transmittance of the unknown against the reference solution. Make readings a t 575 mM in a cell having a 1-cm. light path. A curve prepared in the following manner indicates that the starch-iodide chromogen follows Beer’s law over the range of 0.1 to 5.0 micrograms of iodine. Standard solutions of potassium iodate containing from 0.10 to 5.00 micrograms of iodine and 1 ml. of 0.2 N sodium hydroxide are treated as described above for an unknown, except that distillation and the treatments prior t o distillation are omitted. When the micrograms of iodine per 5 ml. are plotted against the logarithm of the per cent transmittance a straight line is obtained which passes through 100% transmittance a t zero microgram of iodine. Table I shows the results obtained in this laboratory with a Model DU Beckman spectrophotometer.
Table 11.
Kecoberj and Keproducibility Tests
.\faterial Standard iodine solution Standard iodine solution 10 ml. of 10 h’ CrOs 30 ml. of 10 M CrOs Wheat straw I , 5-gram sample Wheat straw I , 1-gram sample Wheat straw 11, 1-gram sample
Knon n Iodine Content
I.ound by Thls Metllod
Y
Y
P.p.m
0.25 0.50
0.24 0.4(1 0.18 0.55 1.32 0.25 2.85
...
..
.. .. ..
... ,..
0:264 0.250 2.850
PROCEDURE
.I sample of dry plant material weighing 1 gram will be sufficient i n most cases. Oxidize the sample in the usual manner, using 10
ml. of 10 M chromium trioxide solution and 50 ml. of concentrated sulfuric acid. After cooling the digestion mixture add 50 ml. of distilled water and 2 or 3 large chips of porous tile or alundum.
If commercial chromium trioxide is used in this procedure, it is necessary to make a blank correction or to prepare 8, reference curve by carrying the standard iodine solutions through the same
ANALYTICAL CHEMISTRY
494
procedure used for an unknown. The latter procedure is preferable, for in this case the iodine standards and unknown receive identical treatments. The reference curve obtained in this manner will deviate from the one shown in Table I, depending on the amount of iodine in the digestion and distillation reagents. A new curve should be prepared with each new batch of chromium trioxide solution. However, this is not inconvenient, because 2 liters of.this reagent are enough for 200 determinations. If commercial chromium trioxide sufficiently low in iodine cannot be obtained, the procedure used by Matthews, Curtis, and Brode (2) for preparing this reagent is recommended.
The average error in 20 determinations (each performed in triplicate) was *0.02 microgram and was independent of the amount of iodine in the range of 0.10 to 5.00 micrograms, LITERATURE CITED
(1) Gross, W. G., Wood, L. K., and MoHargue, J. S., Bx.4~.CHHM., 20, 900 (1948). (2) Matthews, N. L., Curtis, G. M.,and Brode, W. R., IND. ENO. CHEM.,ANAL.ED., 10, 612 (1938). RECEIVED March 28, 1949, Work done in connection with a project of the Kentucky Agricultural Experiment Station and published by permission of the director.
Estimation of Vitamin A in the Presence of Interfering Materials W. A. MCGILLIVRAY Massey Agricultural College, University of New Zealand, Palmerton North, ,V. Z . S T H E estimation of vitamin A by measurement of the absorp-
I tion a t 325 mp, a correction must usually be made forathenumber pres-
ence of other substances which absorb in this region, and of methods have been introduced to make allowance for this irrelevant absorption. Morton and Stubbs ( 1 ) have recently investigated the nature of the correction required for various fish oils and have introduced a correction procedure based on measurements of the extinction coefficients at Xmsx. and a t two other wave lengths such that, for pure vitamin A, E is 8 / 7 of Em=. From the difference between these two extinctions and the extent to which r
I
0.3-
tors affect both the position of Amax. and the shape of the absorption curve of the vitamin. The application of the correction procedure to vitamin Aalcohol plus interfering materials in ethyl dcohol is a typical case and is illustrated in Figure 1. In the inset graph the dotted line represents an idealized curve for the interfering substances which together with the vitamin present give the a b sorption curve for the solution shown as a full line. Readings, giving extinction coefficients denoted by the letters A , B , and C, respectively, are taken a t wave lengths 325,310, and 340 mp-Le., A,,., Amax. - 15 mp, and . , , ,A 15 mp. The absorption due to the pure vitamin A present a t these three wave lengths is A - (z y) a t 325 mp, B - (z 2y) a t 310 mp, and C - z a t 340 mp. For pure vitamin h alcohol in ethyl alcohol the extinction ratios E3,0/E326 mp and E340/E325mp have been calculated in this laboratory as 0.846 and 0.771, rpspectively. I t follows therefore that:
+
+
B Q r-
50.2 -
,
+
- (X + 2 y ) = 0.846 [ A - (Z + y ) ] C - z = 0.771 [ A - (X + y ) l
(1) (2)
Adding Equations 1 and 2 gives thr expression
+ C - 2 (Z + y) = 1.617 [A - + y ) ] (3) which in terms of z + w, the irrelevant absorption a t 325 mp, reduces to x + y = 2.60 (B + C - 1.617 A ) (4)
I
B
V
+
m 0.1
0.2
0.6 INTERFERENCE ( X + Y )
0.4
0.8
Figure 1. Application of Correction Procedure
(2
The actual amount of this irrelevant absorption may conveniently be read directly from a graph as shown in Figure 1 and subtracted from reading A to give the extinction due to vitamin -4. Alternatively, Equation 4 may be rewritten to give A - (z y), the extinctioii due to the vitamin A present, directly
+
A they deviate from 6 / ~ of Emax.,the irrelevant absorption a t Am*.. may be calculated. A similar method, but one which lends itself to a simpler calculation, has been in use for some time in this laboratory for detecting and estimating vitamin A in the presence of partially oxidized carotene. This method, in which readings are taken a t Amax. and a t two other wave lengths equidistant above and below it, is capable of extension to other examples of superimposed irrelevant absorption, provided the points corresponding to the three wave lengths on the curve for the interfering substances alone lie in a straight line. Over the small wave band considered, this generally applies and the proviso is common to all such three-point methods. The wave lengths a t which readings are taken and the interpretation of these readings will depend on the solvent employed and on whether the vitamin is free or esterified, inasmuch as these fac-
- (Z + y)
= 2.60 [2A
- (B + C ) ]
(325 mp) for vitamin A alcohol in ethyl ahand taking hol as 1780 this becomes Concentration of vitamin A in test solution = 14.6 [2A Equations similar to these but with new constailts can be derived for other solvents snd for the vitamin in esterified forms. Although a graphical solution could be applied to Morton and Stubbs’s method, it is considered that the selection of wave lengths equidistant from Amax. in the present method renders it simpler and therefore more readily applicable to routine work. LITERATURE CITED
(1) Morton, R. A., and Stubbs,A. L., Biochem. J.,42, 195 (1948) RECEIVED December 10, 1948.
