Turbidity in Sugar Products VI. Generalized Method and Formulas for

Turbidity in. Sugar. Products. VI. Generalized Method and Formulas for the Determination of Color and. Turbidity in Colored Media. F. W. ZERBAN AND LO...
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JANUARY 15, 1938

ANALYTICAL EDITION

phosphate, pyrophosphate, fluoride, tartrate, citrate, and oxalate ions, all of which exhibit a strong tendency to form stable complexes with ferric iron. For the analysis of materials which contain phosphates, the method is especially to be recommended. A number of metals interfere, but some of these are seldom found in appreciable amounts with iron. A more serious fault is the use of an alkaline solution, which precipitates many metals. The limiting amounts of interfering ions are specified for a volume of 100 ml. and an iron content of 0.10 mg. With smaller amounts of iron, the apparent interference will be greater for some metals, thus lowering the amount that can be present without serious interference.

Conclusions The effect Of the common cations and anions On the mercapt,oacetic acid method for iron has been studied, as well as

9

the general conditions affecting such methods. The lack of interference by nearly all anions and the reproducibility and sensitivity of the color reaction make the method superior to various other colorimetric procedures for iron.

Literature Cited Andreasch, Ber., 12, 1391 (1879). Burrnester, J . Biol. Chem., 105, 180 (1934). Cannon and Richardson, Biochem. J., 23, 1242 (1929). Claesson, Ber., 14, 412 (1881). Hanzel, Proc. SOC.Ezptl. Bid. ‘Wed., 30, 846 (1933). Leave11 and Ellis, IXD.ENO.CHEW,Anal. Ed., 6 , 46 (1934). Lyons, J. Am. Chem. Soc., 49, 1916 (1927). Snell and Snell, “Colorimetric Methods of Analysis,” Vol. I, p. 298, New York, D. Van Nostrand Co., 1936. (9) Swank and hfellon, IXD.Exo. CHEhI., Anal. Ed., 9, 406 (1937). (10) Tompett, Biochem. S.,28, 1536 (1934). (1) (2) (3) (4) (5) (6) (7) (8)

RECEIVED October 7, 1937. Abstracted from a portion of a thesis submitted by H. W. Swank t o the Graduate School of Purdue University in partial fulfillment of the requirements for the degree of doctor of philosophy.

Turbidity in Sugar Products VI.

Generalized Method and Formulas for the Determination of Color and Turbidity in Colored Media F. W. ZERBAN AND LOUIS SATTLER, New York Sugar Trade Laboratory, New York, N. Y.

THE

recent work of the writers (3) on the turbidity and color of white sugars has shown that the absolute turbidity, calculated according to Sauer’s system (d), is directly proportional, within the limits of error of the method, to the turbidity found by the method of the writers with this type of sugars. This fact suggested a reexamination of the data obtained with raw sugars, where no such proportionality had been observed ( 5 ) . The writers have therefore calculated the absolute turbidity of the 21 sirup mixtures containing known by the formula of proportions of color and turbidity (4, Sauer : Absolute turbidity (8) = A f k D t (1)

coefficients and for varying thickness has been published by Landt and Witte (1). When the logarithms of fk are plotted against the extinction coefficient k a t constant thickness, a nearly straight line is obtained, starting a t f k = 0, and k = 0, and satisfying approximately the equation fk

=

(3)

mk

where m is a constant showing slight fluctuations. The values of Sauer’s f k , a t a thickness of 2.455 mm. and corresponding to the extinction coefficients of the 21 mixtures, are shown in Table I, column 5, and the absolute turbidities, S, calculated by Equation 1, in column 6. Three of the mixtures-viz., those highest in turbidity-show absolute turbidities well above unity, which is an impossibility because the intensity of the Tyndall beam cannot be greater

where A is the relative Tyndall beam intensity, measured with the Pulfrich Dhotometer. and equals 0.01 R in the system used by the writers. D is a factor varying with the thickness of the absorption cell, and equals 6.6395 for the 2.455-mm. cells used; t is the abTABLEI. COMPARISON BETWEEN TURBIDITY DATA solute turbidity of the standard glass block of According t o Sauer’s system and the system of Zerban and Sattler, for mixturea known proportions of turbidity and coloring matter. the instrument, in this case 0.00282 for the 1 2 3 4 5 green flter. The factor fk is a function of the 6, 7 “ B&d B&id extinction coefficient (-log T for 1-em. thickComposition of on fk;; on ness) of the solution measured. KO. Mixtures N C fk fk fk’ The relation between f k and k has been de5 U O F O W 0.5155 0.4276 13.288 2.2724 3 . 3 6 7 0.5758 4 U: 1 F : 0 W 0 . 4 3 7 8 0 . 4 1 7 0 10.450 1.5636 3 . 2 6 8 0 . 4 8 9 0 rived by Sauer from theoretical considerations, 8 . 2 0 5 0.7573 3 . 6 9 2 0.3408 3 U, 2 F, 0 W 0.3051 0.4601 6 . 0 6 3 0.4176 3 . 4 8 2 0.2398 2 U 3 F O W 0.2147 0.4395 and is expressed (1) by the following formula: 1 U: 4 F: 0 W 0 . 1 1 7 9 0 . 4 5 2 8 4 . 8 2 6 0 . 1 7 5 8 3 . 6 1 6 0.1317 kd fk

10

-kd

(d- 1) X {1 -

10-kd

2.30269

(42

- l)1

(2)

where d is the depth of layer, in centimeters. The form of this equation would seem to indicate that fk equals 0 when k equals 0. It must be remembered, however, that k is a logarithm; hence log fk equals 0 for k equal to 0, and f k itself equals 1 under that condition. A table of fk values for varying extinction

11 12 13 14 15 16 17 18 19 20 21

containing 9

,_I

Jli

OU,5F,O W 4U,OF,lW 3 U 1F 1 W 2 U: 2 F: 1 W 1 U, 3 F , 1 W

0.0287 0.4175 0,2932 0.2105 0.1259

0.4245 0,3495 0.3764 0.3684 0.3545

3.497 8.245 6.323 4.935 3.768

0.0336 1.3872 0.7113 0.4077 0.1936

3.341 2.764 2.911 2.846 2.737

0.0321 0.4664 0.3275 0.2351 0.1406

from Sauer’a Formula 3.259 3.166 3.563 3.370 3.493 3.219 2.629 2.831 2.770 2.666

OU 4 F 1 W 3 U’OF’2W 2 U’ 1 F ’ 2 W 1U12F’2W O U ’ 3 F ’ ZW 2 U ’ O F ’ 3W 1 U: 1 F : 3 W 0 U, 2 F , 3 W 1 U, 0 F , 4 W 0 U, 1 F , 4 W 0 U, 0 F, o TV

0.0264 0.3200 0.2086 0.1161 0.0303 0.2224 0.1154 0.0151 0.1035 0.0115 0.0036

0.3371 0.2581 0.2609 0.2699 0.2440 0.1510 0.1941 0.1729

2.733 4.924 3.657 2.828 2.138 2.808 2.355 1.684 1.700 1.299 1.000

0.0310 0.8456 0.4060 0,1752 0.0362 0.4542 0.1750 0.0174 0.1530 0.0131 0.0040

2.603 2.081 2.098 2.092 2.001 1.535 1.734 1.635 1.284 1.267 1.000

0.0295 0.3574 0.2330 0.1297 0.0338 0.2484 0.1289 0.0169 0.1156 0.0128 0.0040

2.541 2.044 2.060 2.054 1.966 1.521 1.713 1.615 1.277 1.258 1.000

0.0880

0.0827

0.0000

IKDUSTRIAL AXD ENGINEERIIL’G CHEMISTRY

10

than that of the incident light. The relative turbidities of mixtures 1, 7 , 12, 16, and 19, which are known to be in the proportion of about 5 : 4 : 3 : 2 : 1, are found to be in the proportion of 14.86 : 9.07 : 5.53 : 2.90 : 1. I n other words, the discrepancies between the turbidity present and that found increase rapidly with a n increase in known turbidity. This proves conclusively that the correction factors fk, if based on k , are erroneous. The following considerations will indicate what the values of the correction factors should really be for the 21 mixtures. I n the work on white sugars the ratio between the absolute turbidity, S , and that expressed in terms of S (as -log T, 2.455 mm.), was found to equal 1.117. By substituting 1.117 N for S in Equation 1, the correction factor, which will be designated by fn,, may be calculated for the 21 mixtures:

”’

1.117 X = 0.01 R X 6.6395 X 0.00282

(1)

The values of S,calculated by the writers’ formulas ( 4 ) , in terms of -log T for 2.455-mm. thickness, are shown in Table I, column 3, and those of fk, in column 7. Examination of these fk, values disclosed the fact that they are numerically equal to 6’ (C represents coloring matter) in the formula established by the writers:

R

VOL. 10, XO. 1

=

(5)

a1Vb-C

or

R = -a N

(6)

bC

where C is also expressed as -log Tfor 2.455-mm. thickness; b has been substituted for the symbol k used originally, to avoid confusion with Sauer’s k. It is thus clearly shown that the correction factor is a function of the coloring matter alone, and not of k , which expresses the total absorbency due to coloring matter plus turbidity. The formula of Sauer, Equation 1, and that of the writers are of the same general form: S = 0.01 R X

A’ = R

X

bC

fkj

X

x l/a

D

X t

(7) (8)

and, since fk, equals bC, the only difference between the two formulas is in the constants, 0.01 D X t in one case, and l / a in the other. This difference is due to the fact that in Sauer’s formula the turbidity is expressed as a fraction of the intensity of the incident light, while in that of the writers it is expressed as -log T for 2.455-mm. thickness. The ratio between the two constants, 0.01 D X t divided by l / a , or 0.01 D X t x a, represents the ratio between S a n d 1Y. Substi-

ANALYTICAL EDITIOX

JASUARY 15, 1938

tuting the nunierical values of D, t, and a, we obtain 0.066395 X 0.00282 x 5963.7 = 1.1166, which checks the figure found experimentally and used in Equation 4. If fkf is now calculated for the 21 mixtures, on the basis of C (as -log T,1 cm.), instead of k , by Sauer’s formula forf,, Equation 2, the figures shown in Table I, column 9, are obtained. These values check closely with bC and with the f,,values Calculated from Equation 4. This result may be interpreted in two ways. Either log fL, is really a linear function of C, as found by the authors’ and C experiments, or else the true relationship between is correctly expressed by Sauer’s formula based on theory. It is logical to accept the latter alternative and to ascribe the strictly linear relationship found experimentally to permissible error. Sauer’s formula is thus found to be correct in form, but the correction factor must be based on the color alone. The original formulas of the writers must then be modified as follows: By substitutingf,, for bC in Equation 8, we obtain f,?

11

A graph covering the entire range of C, -log T , and R values of raw sugars is shown in Figure 1. A blueprint of the large working graph, in which increments of 0.1 C and 0.1( -log 2‘) are made equal to 50 mm., nill be gladly sent to anyone expressing a desire for it. From the graph the C corresponding to given values of -log T and R can be read off directly with close approximation to the third significant figure. If more exact results are desired, interpolation for R is carried out by means of Table 11,on the basis of the approximate value of C read from the graph. The use of the table is best explained by an example. A raw sugar sample gave -log T = 0.57807, and R = 917.1, for the green filter. A glance a t the graph shows that C lies between 0.25 and 0.30. The value of Rjc/a for I R , a t C = 0.25, is 0.0003351; hence that for 917.1R is 0.0003551 X 917.1, or 0.30732, which added to 0.25 C gives -log T = 0.55732. Similarly, the value of Rfc/a for 1R, a t C = 0.30, is 0.0003847, and that for R = 917.1 is 0.35280, which added to 0.30 gives -log T = 0.65280. Then the difference, x, between the required value of C and the value 0.25 is found from the following equation : (Z

- 0.25) : (0.30 - 0.25)

=

(0.57807

Combining Equations 9 and 11 gives

c=

-log T

-

This value for C is now substituted for kd in Sauer’s equation. Since Sauer’s k is now expressed as -log T for 1em., while C is -log T for 0.2455 em., C must first be divided by 0.2455 to reduce it to I-em. thickness, and the quotient must then be multiplied by d = 0.2455 em. The net result is C in place of k d , and the symbol for the correction factor on the basis of C for 0.2455 cm. is now changed to fc. The formula for fc thus becomes fc =

iI -log T 10

-

[-log

I])?(

(F)[ ( 4 2

T -

11

-

10

- 1) X 2.30259

-[-log T -(Y)(./s - 1) /

r (13)

There remains some uncertainty about the true value of constant a, which is derived from the writers’ experimental data and is subject to the same sources of error as constant b. But since there appears to be no theoretical correlation between D X t and a, the value found experimentally nill be accepted for the present. The physical significance of constant a may be derived from Equation 12. Xhen C equals 0 and consequently fc is equal to unity, a equals R, (-log 5”). This relationship makes it possible to check a experimentally, and also to tell whether coloring matter is present in significant amounts, because in its absence the ratio between R and -log T should not vary. In order t o calculate C and -1-from R and -log T , Equation 13 is solved for varying values of C, which is the -log T (Rfca ) term, and a tabulation of corresponding fc values is thus obtained. Substitution of these C and fc values a t specified increments of R in Equation 12 yields a table from which C may be found for any pair of -log T and R values. Practically, C is found from curves based on the table, and S is obtained by subtraction from -log T.

- 0.55732) : (0.63280 - 0.55732)

The result for xis 0.0109, which added to 0.25 gives a value of 0.2609 for C. *V equals 0.57807 - 0.2609, or 0.3172. The above interpolation assumes linear relationship between Rfclaand C for the small traject between 0.25 C and 0.30 C. The curve shows that this is not quite correct. The aberration, however, is so slight that the result obtained is well within the limits of error of the photometric data. If the interpolation is made on the basis of the more exact linear relationship between log Rfc/aand C, the result for C is 0.26 11. T.4BLE

c

11.

INTERPOLATIOS TABLE FOR FINDINGAND fc FROM -LOG T AND R

C’

J’C

0,0000 0.0010 0.0020 0,0030 0.0040 0.0050 0.0060 0 0070 0.0080 0.0090

1 . 0000

1.0027 1.0055

1.0083 1.0110 1.0138 1.0166 1.0194 1.0222 1,0250

Rfc

R A

a

a

for

for

C 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 0.4000 0.4500 0.5000 0.5500

1.3200 1.5160 1.7406 1.9986 2.2941 2.6331 3.0214 3.4664 3.9757 4.5598

R = l 0.00022l3 0.0002542 0.0002919 0.0003351 0.0003847 0.0004416 0.0005066 0. 005813 0.8006667 0.0007646

0.0001723 0.0001728 0.000 1733 0.0001742 0.0001738

0,6000 0.6500 0.7000 0.7500 0.8000 0.8500 0.9000 0,9500 1,0000 1,0500

5.2281 5.9937 6.8699 7.8727 9.0199 IO.3329 11.8342 13,5520 15.5154 17.7609

0008767 00l0050 0011520 0013201 0015125 0017327 0019844 0022724 0026017 0 0029782

lIlO0O 1.1500 1.2000 1,2500 1.3000 1.3500 1.4000 1,4800

20,3270 23.2604 26.6117 30.4416 34.8136 39.8110 45.5150 .52.02SO 59.4610 67,9469

0 0 0 0 0 0 0

0 0100 0.0110 0.0120 0.0130 0.0140 0.0150 0.0160 0,0170 0 0180 0.0190

1.0278 1.0306 1.0335 1,0363 1 0391 1.0420 1.0448 1.0477 1.0535

0.0001747 0.0001782 0.0001757 0.0001762 0.0001767

0 0200 0 0220 0 0240 0 0260 0 0280 0,0300 0 0350 0 0400 0 0450 0 0500

1.0564 1.0622 I . 0680 1,0739 1,0798 1.0862 1.1023 1.1175 1,1332 1.1488

0 0001771 0,0001781 0 0001791 0 0001801 0 0001811 0 0001821 0 0001848 0 0001874 0 0001900 0.0001926

0.0550 0.0600 0.0650 0,0700 0.0750 0 0800 0.0850 0.0900 0,0950

1.1647 1.1814 1,1978 1,2146 1.2316 1.2491 1.2661 1.2838 1.3017

0 0001953

1.0506

fc

R = l 0.0001677 0.0001681 0.0001686 0,0001691 0.0001695 0.0001700 0.0001705 0.0001709 0.0001714 0.0001719

0.0001981 0.0002008 0.0002037 0.0002065 0.0002095 0.0002123 0.0002153 0 0002183

1,5000

1.5500

1.6000 1.6500 1.7000 1.7500 1.8000

1.8500 1.9000 1.9500 2 0000

77.6286 88.6764 101.1057 115.6513 132.0390 150.7322 172 0380 196.3277 224 0030

0 0 0 0 0 0 0 0 0

0034085 0039004 0044623 0051045 0058376 0066756 0076321 0087242

0 0099706 0 0113936 0 0130170 0 0148696 0.0169537 0.0193928 0 0221408

0.0252753 0.0288479 0.0329209 0.0375616

INDUSTRIAL AND ENGINEERING CHEMISTRY

12

VOL. 10, NO. I

-109 T FIQURE 2.

ENL.4RGED

GRAPHO F LOWERPORTION

The absolute turbidity can also be readily calculated by means of Table 11. By interpolation of column 2 in this table, the fc for C(kd) equal to 0.2609 is found to be 2.0630. By substituting this figure in Sauer’s Equation 1, with A (0.01 R) = 9.171, D = 6.6395, and t = 0.00282, we iind the absolute turbidity to equal 0.3542. The ratio between this and the N found previously (0.3172) is again 1.117. OF CALCULATED AND FOUND VALUES TABLE 111. COMPARISON OF

C, N, C

T,AND R

FOR

21 SIRUPMIXTURES T T R

R

N

N

Calcd.

Found

Calcd.

Found Calod. Found

0.4355 0 4368 0.4355 0.4384 0.4355 0.4465 0.4355 0 4392 0.4355 0,4525 0.4355 0.4399

0.5200 0.5163 0.4187 0.4164 0.3173 0.3187 0.2160 0.2150 0.1147 0.1182 0.0133 0.0133

11.08 13.99 17.67 22.31 28.17 35.58

11.40 13.97 17.17 22.17 26.87 35.22

8 9 10 11

0.3484 0.3484 0.3484 0.3484 0.3484

0.3687 0.3663 0.8671 0,3629 0.3339

12 13 14 15

0.2613 0.2613 0.2613 0.2613

16 17 18

No.

C Calcd.

Found

933 751 569 387 206 23.9

916 738 559 381 202 23.5

0.4168 0.3983 17.17 0.3193 0.3023 21.49 0.2218 0.2118 26.90 0.1244 0.1175 33.67 0.0268 0,0296 42.15

17.10 906 21.40 688 26.37 482 33.08 267 67.3 43.30

857 657 457 257 69.9

0,2644 0.2580 0.1552 0.2490

0,3134 0.2178 0.1221 0.0264

0.3137 26.56 0.2115 33.18 0.1208 41.36 0.0253 51.56

26.42 33.92 42.07 53.17

907 612 349 73.2

898 624 350 75.7

0.1742 0.1742 0.1742

0.1543 0.1843 0.1764

0.2100 0.1102 0.0125

0.2191 0.1252 0.0116

41.29 51.95 65.06

42.33 49.03 64.87

806 461 42.7

851 447 42.1

19 20

0.0871 0.0871

0.0883 0.0828

0.1070 0.0121

0.1032 0,0114

63.96 79.58

64.35 80.50

483 53.4

481 54.1

21

0.0000

0.0000

0.0036

0,0036

99.17

100.00

21.5

21.6

1 2

3 4 5 6

7

OF

FIGURE 1

The new method of calculation has been applied to the 21 sirup mixtures mentioned before (Table I), and the calculated values of C, N , T, and R are compared with those found experimentally in Table 111. The agreement between found and calculated values is satisfactory if it be considered that thorough mixing of the heavy constituent sirups was a difficult matter because agitation tends to disturb the colloid equilibrium. For products which are low in both color and turbidity, the graph shown in Figure 2 is used. If the scale is such that C and -log T are plotted a t 50 mm. = 0.001 unit, it is possible to read C and -log T values accurately to the fourth decimal place. A blueprint of this graph is also available. I n this range linear interpolation is accurate. For such computational purposes Table I1 is used in exactly the same way as in the previous case. C and N have been calculated in this manner for the reh e d sugars previously examined (3). As was to be expected, the results obtained checked with those calculated by the original formulas of the writers within one unit of the fourth decimal place. With this type of sugars, representing a practically colorless medium, it is permissible to calculate the absolute turbidity directly by the use of Sauer’s formula and the correction factor based on the -log T of the turbid solution rather than on C, and to find N , expressed as -log T, by dividing by 1.117. C then equals -log T - N . Conversion of -log T and of R values determined a t one thickness into the corresponding figures a t another thickness,

JANUARY 15, 1938

AN.4LYTICAL EDITION

or of -log T into -log t values, may be made as explained in the preceding paper of this series (3). Summary A reexamination of previous data has shown that the correction factor for absorption, in Sauer's formula for the calculation of absolute turbidity, must be based on the concentration of coloring matter alone, and not on the total lightabsorbing material including the turbidity. I n all other respects Sauer's formula is of the same form as that developed by the writers, and if the correction factor, derived from theory, is based on the coloring matter only, its numerical value checks that of the term kC in the original formula of the writers, which was based on purely experimental evidence. Sauer's principle has therefore been accepted as a basis for calculating the correction factor from only the concentration of

13

the coloring matter, and the writer's formula has been modified by substituting the new correction factor for their kC term. It is thus possible to use the same correction factor for calculating either the absolute turbidity according t o the modified Sauer formula, or the coloring matter and turbidity, expressed as -log T, by the revised formula of the writers. The new formulas have been checked against the experimental data, and satisfactory agreement has been found. Literature Cited (1) Landt and Witte, 2. mirtschuftsgruppe Zuckerind., 84,462(1934). (2) Sauer, 2. tech. P h y s i k , 12,149 (1931); 2. Instrumentenk., 51,408 (1931). (3) Zerban and Sattler, Ihm. ESQ.CHEM.,Anal. Ed., 9,229 (1937). (4) Zerban, Sattler, and Lorge, Ibid., 6,178 (1934). (5) Ibid., 7, 157 (1935). RECEIVED October 21, 1937.

Determination of Iron in Biological Materials The Use of o-Phenanthroline FR4NCE.S COPE HUMRIEL, Children's Fund of Michigan, Detroit, H. H. WILLARD, University of Michigan, Ann Arbor, RIich.

F

OR several years o-phenanthroline has been used in the Research Laboratory of the Children's Fund of Michigan as a satisfactory reagent for the determination of iron in foods, feces, blood stroma ( I ) , and other types of biological materials. Since numerous requests have been made for the details of the procedure followed, it seems desirable to record the method in full. The small amount of iron present in some biological materials precludes the use of the classical gravimetric or titration methods; various colored compounds of iron have therefore been adapted t o colorimetric determination. Several of these procedures have been investigated by the authors, but have not been found satisfactory. The presence of large amounts of phosphate in biological materials, especially certain foods and feces, interferes with methods involving the ferric ion, and has necessitated the use of tedious modifications t o avoid this interference. For this reason attention has been turned to the color reactions of ferrous iron, which does not form a stable complex with pyrophosphate. The colored complex formed by ferrous iron with o-phenanthroline, which was orginally observed by Blau ( d ) , has been used for the determination of iron in various types of materials. The orange-red color is quantitatively proportional to the concentration of iron within the p H range 2.5 to 8.0, and has been used therefore in both titration (7), and colorimetric (6) methods. The colored complexes formed by iron with cu,a'-dipyridyl (8, 4 ) and o-phenanthrcline have much the same characteristics, with the advantage that the latter reagent is less expensive and more readily available. (oPhenanthroline may be purchased from the G. Frederick Smith Chemical Company, Columbus, Ohio. The current price is about $1.75 per gram.) The method described herein was originally devised for use in the colorimeter, but has since been adapted to the CencoSheard-Standard photelometer ( 5 ) . The latter instrument possesses certain advantages over the colorimeter, since the use of light filters widens the range of accuracy. The relation of density of color to the concentration of iron is determined

AND

at the outset of the experiment (Figure 1) which obviates making up a standard simultaneously with the unknown. Reagents STANDARD IRON SOLUTION.Dissolve 1 gram of electrolytic iron in 10 per cent sulfuric acid and dilute t o 1 liter. Dilute 1 to 10 for use; 1 cc. of diluted standard corresponds to 0.1 mg. of iron. HYDROQUINONE. Dissolve 1 gram of hydroquinone in 100 cc. of sodium acetate-acetic acid buffer solution with a pH of 4.5 (3). Keep the solution in the refrigerator and discard as soon as any color develops. SODIUM ACETATE. 0.2 M, M , and 2 M are convenient concentrations to have available. Dissolve 0.5 gram of o-phenanthroline O-PHENANTHROLINE. monohydrate in 100 cc. of distilled water, and warm t o effect solution. Procedure The material to be analyzed (foods and feces dried at 60" to 80" C. were used) is ashed overnight in an electric muffle furnace a t 450" to 500" C. The ash is dissolved in the smallest possible amount of dilute hydrochloric acid (1 t o 3) and the solution is

filtered into a 100-cc. volumetric flask; if the first ashing is incomplete, the paper and residue are reashed, after thorough washing with distilled water, and the ash is dissolved as before and filtered into the same flask. The solution is then made to volume, and an aliquot selected for analysis which will fall within the range of accuracy of the colorimeter or the photelometer-i. e., 0.2 to 0.5 mg. or 0.01 to 0.70 mg. of iron, respectively. Similar aliquots of the above unknown solution are measured into both a 25432. volumetric flask and a test tube, and 2 M sodium acetate solution is added from a buret to the test tube until the color corresponding to pH 3.5 is reached, using 5 drops of La Motte indicator bromophenol blue. The unknown solution in the 25-cc. volumetric flask is adjusted to pH 3.5, using the same amount of 2 M sodium acetate, followed by the addition of 1 cc. of 1 per cent hydroquinone solution and 1 cc. of o-phenanthroline solution. After thorough mixing, the solutions are allowed to stand for 1 hour to assure complete conversion of the iron to the ferrous o-phenanthroline complex, and then made to volume and read in either the colorimeter or t'he photelometer. If the colorimeter is t o be used, a series of standards containing from 0.2 t o 0.5 mg. of iron is prepared simultaneously with the unknown. Since the color becomes yellow with dilution, it is not feasible to read lower concentrations in the colorimeter. Ac-