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JANUARY 15, 1938 AN.


or of -log T into -log t values, may be made as explained in the coloring matter, and the writer's formula has been modi-
the preceding paper of this series (3). fied by substituting the new correction factor for their kC
term. It is thus possible to use the same correction factor
Summary for calculating either the absolute turbidity according t o the
A reexamination of previous data has shown that the cor- modified Sauer formula, or the coloring matter and turbidity,
rection factor for absorption, in Sauer's formula for the cal- expressed as -log T, by the revised formula of the writers.
culation of absolute turbidity, must be based on the concen- The new formulas have been checked against the experi-
tration of coloring matter alone, and not on the total light- mental data, and satisfactory agreement has been found.
absorbing material including the turbidity. I n all other re- Literature Cited
spects Sauer's formula is of the same form as that developed
by the writers, and if the correction factor, derived from (1) Landt and Witte, 2. mirtschuftsgruppe Zuckerind., 84,462(1934).
theory, is based on the coloring matter only, its numerical (2) Sauer, 2. tech. P h y s i k , 12,149 (1931); 2. Instrumentenk., 51,408
value checks that of the term kC in the original formula of (3) Zerban and Sattler, Ihm. ESQ.CHEM.,Anal. Ed., 9,229 (1937).
the writers, which was based on purely experimental evidence. (4) Zerban, Sattler, and Lorge, Ibid., 6,178 (1934).
Sauer's principle has therefore been accepted as a basis for cal- (5) Ibid., 7, 157 (1935).
culating the correction factor from only the concentration of 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, AND
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 Michi-
gan as a satisfactory reagent for the determination of iron in
at the outset of the experiment (Figure 1) which obviates
making up a standard simultaneously with the unknown.

foods, feces, blood stroma ( I ) , and other types of biological Reagents

materials. Since numerous requests have been made for the STANDARD IRON SOLUTION.Dissolve 1 gram of electrolytic
details of the procedure followed, it seems desirable to record iron in 10 per cent sulfuric acid and dilute t o 1 liter. Dilute 1 to
the method in full. 10 for use; 1 cc. of diluted standard corresponds to 0.1 mg. of
The small amount of iron present in some biological ma- iron.
HYDROQUINONE. Dissolve 1 gram of hydroquinone in 100 cc.
terials precludes the use of the classical gravimetric or titra- of sodium acetate-acetic acid buffer solution with a pH of 4.5 (3).
tion methods; various colored compounds of iron have Keep the solution in the refrigerator and discard as soon as any
therefore been adapted t o colorimetric determination. color develops.
Several of these procedures have been investigated by the SODIUM ACETATE. 0.2 M, M , and 2 M are convenient con-
centrations to have available.
authors, but have not been found satisfactory. The presence O-PHENANTHROLINE. Dissolve 0.5 gram of o-phenanthroline
of large amounts of phosphate in biological materials, es- monohydrate in 100 cc. of distilled water, and warm t o effect
pecially certain foods and feces, interferes with methods in- solution.
volving the ferric ion, and has necessitated the use of tedious
modifications t o avoid this interference. For this reason Procedure
attention has been turned to the color reactions of ferrous The material to be analyzed (foods and feces dried at 60" to
iron, which does not form a stable complex with pyrophos- 80" C. were used) is ashed overnight in an electric muffle furnace
phate. a t 450" to 500" C. The ash is dissolved in the smallest possible
The colored complex formed by ferrous iron with o-phe- 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 in-
nanthroline, which was orginally observed by Blau ( d ) , has complete, the paper and residue are reashed, after thorough
been used for the determination of iron in various types of washing with distilled water, and the ash is dissolved as before
materials. The orange-red color is quantitatively propor- and filtered into the same flask. The solution is then made to
tional to the concentration of iron within the p H range 2.5 volume, and an aliquot selected for analysis which will fall
within the range of accuracy of the colorimeter or the photelome-
to 8.0, and has been used therefore in both titration (7), and ter-i. e., 0.2 to 0.5 mg. or 0.01 to 0.70 mg. of iron, respectively.
colorimetric (6) methods. The colored complexes formed by Similar aliquots of the above unknown solution are measured
iron with cu,a'-dipyridyl (8, 4 ) and o-phenanthrcline have into both a 25432. volumetric flask and a test tube, and 2 M
much the same characteristics, with the advantage that the 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
latter reagent is less expensive and more readily available. (o- of La Motte indicator bromophenol blue. The unknown solution
Phenanthroline may be purchased from the G. Frederick in the 25-cc. volumetric flask is adjusted to pH 3.5, using the
Smith Chemical Company, Columbus, Ohio. The current same amount of 2 M sodium acetate, followed by the addition
price is about $1.75 per gram.) of 1 cc. of 1 per cent hydroquinone solution and 1 cc. of o-phe-
nanthroline solution. After thorough mixing, the solutions are
The method described herein was originally devised for use allowed to stand for 1 hour to assure complete conversion of the
in the colorimeter, but has since been adapted to the Cenco- iron to the ferrous o-phenanthroline complex, and then made to
Sheard-Standard photelometer ( 5 ) . The latter instrument volume and read in either the colorimeter or t'he photelometer.
possesses certain advantages over the colorimeter, since the 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
use of light filters widens the range of accuracy. The relation unknown. Since the color becomes yellow with dilution, it is not
of density of color to the concentration of iron is determined feasible to read lower concentrations in the colorimeter. Ac-
curate results are rarely obtained if the unknown varies from the TABLE
standard by more than 25 per cent; this confirms the observation AFTER ASHING
of other investigators (6). -."..
Sample Determined I n ash5 Recovered Added Recovered
The procedure for the determination of iron recorded herein MQ. Mg. My. .VQ. %
differs from that of Saywell and Cunningham (6) in several 1 0.335 0.234 0 101 0.100 101 0
2 0.250 0.165 0 085 0.0862 98 6
respects. The large quantity of calcium and phosphorus 3 0.300 0.165 0 135 0.135 100 0
present in metabolic materials necessitates a concentration of 4 0.435 0.236 0 199 0.200 99 5
5 0.296 0.194 0 102 0.100 102 0
acid sufficient to prevent the precipitation of calcium phos: 6 0,333 0.194 0 139 0.136 103 0
7 0.284 0.199 0 085 0.0862 98 6
phate, which carries down most of the iron as ferric phosphate. 8 0.330 0.199 0 131 0.135 97 0
Since the ferrous o-phenanthroline complex is not stable in the 9 0.374 0.167 0 207 0.200 103 5
10 0.370 0.199 0 171 0.172 99 4
presence of strong acid, the pH range 3.0 to 4.0 was selected; 11 0.254 0.167 0 087 0 862 100 9
12 0.339 0.167 0 172 0,172 100 0
this acidity permits maximum color development, yet pre- 13 0.293 0.196 0 097 0 100 97 0
vents any precipitation. A solution of hydroquinone in a 14 0.293 0.196 0 097 0.100 97 0
15 0.390 0.196 0 194 0.200 97 0
sodium acetate-acetic acid buffer of pH 4.5 has been used 16 0.400 0.196 0 204 0.200 102 0
effectively to reduce the iron to the ferrous state. Sodium 17 0.495 0.196 0 299 0.300 100 0
18 0,250 0.098 0 152 0.150 101 3
hydrosulfite was also tried as a reducing agent, but was 19 0,305 0.098 0 207 0.200 103 5
20 0.296 0.098 0 198 0 200 99 0
abandoned because it occasionally caused the solutions to 21 0 350 0,098 0 252 0 250 100 8
become turbid. Since accurate measurement of the hydro- 22 0.344 0.098 0 246 0.250 98 4
quinone solution can be made, and equal quantities used for a .4shes analyzed separately b y t h e same method.
Samples 1 t o 12: iron was added t o ash solution.
standard or unknown, traces of iron in the reagent do not Bamples 13 t o 22: iron was added t o food before ashing.
cause appreciable error.
Results Since the principal difficulty encountered in colorimetric
The solutions may be read with equal accuracy within the methods for iron in biological materials especially has been the
concentration range 0.2 to 0.5 mg. of iron for the colorimeter, interference of pyrophosphate, this action was investigated
and 0.01 to 0.07 mg. for the photelometer. The two instru- by adding amounts of sodium pyrophosphate representing
ments were used interchangeably in obtaining the results from 10 to 50 mg. of phosphorus to standard iron solutions.
presented in Tables I and 11. The color was developed in the usual manner, and, after
The smooth curves shown in Figure 1 were obtained by standing from 10 minutes to 24 hours, was read against iron
measuring the color developed by a series of standards in the standards containing no pyrophosphate The results shown
photelometer. Curve A is obtained from the concentration in Table I1 indicate that if the determinations are allowed to
range 0.01 to 0.16 mg. of iron, using a blue filter, when the stand 30 minutes, the error caused by fairly large amounts of
solutions are read against a solution of the reagents in sodium pyrophosphate is within normal limits.
acetate-acetic acid buffer pH 4.5 set a t 100. The effect of
traces of iron present in the reagents as impurity is eliminated TABLE
by using them as the standard instead of distilled water. OF PHOSPHORUS
Curve B is obtained in the same manner for the concentrations Time of Phosphorus Added
Standing 10 mg. 20 mg. 30. mg. 40 mg. 50 mg.
0.1 to 0.7 mg. of iron, using a green filter.
7 Iron Recovered
.MQ. .Mg . Mg . Mg. MQ.
R E L A T I ON O F COLOR I NTENS I T Y 10 min. 0 202 0.199
0 196
30 min. 0.200
3 hours
0 205
24hours 0.201 0.196 0.200 0 200 0 195
a -111 samples contained 0 . 2 mg. of iron.

The influence of some of the elements which occur in traces

in biological materials was investigated. Two milligrams
w . each of lead, zinc, aluminum, mercury, arsenic, fluorine, and
iodine, 0.6 mg. of tin, and 0.2 mg. of copper were added to
standard iron solutions, and the iron was determined in the
usual manner. Since copper reacts with o-phenanthroline in
amounts over 0.2 mg. and tin precipitates a t a pH of 3.5 if
more than 0.6 mg. is present, quantities which rarely occur in
biological materials, smaller concentrations Qf these elements
were used. No interference was observed.
Preliminary experiments have shown that o-phenanthroline
may be used for the determination of available iron in foods
in much the same manner as a,a'-dipyridyl ( 4 ) ; it does not
L form the colored complex when added t o purified hematin.
04'o I 02 03 04 0'5 06 07
o-Phenanthroline has been used in the quantitative deter-
Table I shows the recovery of known amounts of iron added mination of iron in biological materials such as foods, feces,
to ash solutions. I n order to check the accuracy of the dry- and blood. I n the photelometer 0.01 to 0.70 mg. of iron can be
ashing method, in some cases the iron was added to the food determined with an accuracy of 3 per cent; the colorimeter
before ashing. The recoveries are for the most part within can be used with equal accuracy for quantities from 0.2 to 0.5
the accuracy assigned to colorimetric methods, and dry ashing mg. of iron.
gives satisfactory results. The method is free from interference by pyrophosphate if

sufficient time is permitted for complete conversion of the (3) Clark, W. M.,“Determination of Hydrogen Ions,” 3rd ed.,
iron to the ferrous o-phenanthroline complex. Baltimore, Williams & Wilkins Co., 1928.
(4) Hill, R., Proc. Roil. SOC.(London), B107, 205 (1930).
‘One Of the found in traces in ( 5 ) Sanford, A. H., Sheard, C., and Osterberg, A, E,, Am, J . Clin.
except copper in amounts over 0.2 mg. and tin in amounts Path.. 3. 405 11933).

over-0.6 mg. interferes I+-iththe determination of iron by this (6) Saywell, L. G., and Cunningham, B. B., IND.ENG.CHEW,Anal.
method. Ed., 9, 67 (1937).
(7) Walden, G. H., Hammett, L. P., and Edmonds, S. M., J. Am
Literature Cited Chem. SOC.,56, 350 (1934)
(1) Bernstein, S.S., Jones, R. L., Erickson, B. N., Wlliams, H. H., RECEIVEDOctober 2 7 , 1937. Presented before the Division of Biological
Amin, I., and Macy, I. G. (in press). Chemistry a t the 94th Meeting of the American Chemical Society, Rochester,
(2) Blau, Monatsh., 19, 647 (1898). pi. T.,
September 6 t o 10, 1937.

Determination of Air and Carbon Dioxide

in Beer
PHILIP P. GRAY AND IRWIN 51. STOKE, Wallerstein Laboratories, 180 RIadison Ave., New York, IV. Y

I S YIEW of the relation of air content to beer stability,

the determination of the amount of air present in pack-
aged beer has, in recent years, assumed an importance a t least
the pressure method and supplied the principles for car-
rying out the calculations, but did not give in detail the ac-
tual method for making the air determination.
equal to that of the determination of carbon dioxide. Such Experience with this method has indicated that more ac-
methods for determining air as those of Murray ( 5 ) , Helm curate results are obtained when all pressure determinations
and Richardt (4),and Siegfried ( 6 ) ,while no doubt giving ac- are carried out a t 25” C. rather than at much lower tempera-
curate results, are cumbersome and unwieldy. tures. At this temperature, which is generally easy to main-
The chief attribute of the pressure method for determining tain, the pressure reading is high, minimizing any gage er-
carbon dioxide, which has been in use for many years in the rors; the air is readily evolved from the beer; and the se-
carbonated beverage industry, is its convenience. I n view lection of a single temperature reduces any errors due to dif-
of the importance attached to air determination, any modi- fering solubility-temperature coefficients or deviations from
fication of the pressure method which results in the determina- gas laws which might enter into the results when determina-
tion of both air and carbon dioxide a t the same time upon tions are carried out, sometimes at one temperature and some-
the same package in a satisfactorily accurate manner deserves times a t another. For example, with the much lower pres-
consideration. sures prevailing a t temperatures close to cellar temperatures
I n a previous paper (Z), the authors worked out a precise in the brewery, a given amount of air will naturally exert,
chemical method for determining carbon dioxide in bottled proportionately, a greater effect on the total pressure; in
beer and carbonated beverages, based on the use of the whole fact, evidence has accumulated that, for the same samples,
bottle as a sample, a foam suppressant, and an evolution slightly higher carbon dioxide results are to be expected if the
regulating material. Complete liberation of gas was ob- pressures are measured a t these lower temperatures.
tained by boiling, the liberated gas being absorbed in alkali The authors have also adopted, based on considerable ex-
and the alkali then being differentially titrated. This method perience with a large number of samples, a revised value for
was adopted as tentative by the Association of Official Ag- a (per cent of carbon dioxide per pound pressure) a t 25” C.-
ricultural Chemists ( 1 ) . namely, 0.00965 instead of 0.0095 (3)-embodying an adjust-
I n the same paper, advantage was taken of the availability ment for such errors as are always involved in assuming an
of this precise chemical method to study and evaluate the “average beer,” and correcting for experimental errors on rou-
errors inherent in the customary pressure methods for deter- tine samples. The value has been found to give results which
mining carbon dioxide. The authors were able to establish are quite as satisfactory as the precise chemical methods.
that, if the influence of the variable amounts of air present I n the present paper, details are given for determining both
during the pressure reading is taken into account, accurate “air” and carbon dioxide by the pressure method. The
carbon dioxide results may be obtained by the pressure results of special experiments are also presented, carried out
method, and that solubility of carbon dioxide in beer, under to determine the extent to which the procedure is capable of
varying temperature and pressure conditions, can be satisfac- recovering all the air present in the package. While there is
torily predicted on the basis of publishedHenry’s law constants no need, either from the standpoint of controlling air con-
for carbon dioxide, assuming that the small amount of alco- tent or as regards the accuracy of the carbon dioxide results,
hol does not affect the total solvent properties of the com- to ensure 100 per cent recovery of the free and dissolved air
bined alcohol and water in beer, and that extract has no other present, these experiments indicate substantially complete
effect on solubility than as an inert diluent. recovery beyond amounts Tv’hich might be anticipated on the
Thus, there was made available an accurate, simple, and basis of solubility.
convenient pressure procedure once the equipment is a t hand. Another important factor having a bearing on the accu-
Results presented in the previous paper, as well as experience racy of carbon dioxide results by the pressure method, which
with the test in the authors’ laboratories since that time, has heretofore not been touched upon, is the mechanical dif-
amply justify the conclusion that, for most purposes the pres- ficulties and errors inherent in the usual type of pressure gage.
sure method, correcting for air and using solubility of carbon The usual gage, based on mechanical spring action, is fre-
dioxide in beer as a basis, yields satisfactory, accurate, and quently found to lag and stick and give erratic results after
reproducible data for carbon dioxide. The paper ( 2 ) outlined some use. In view of the frequency with which this occurs,