882
terniine the two dilution ratios and, thus, the concentration of
nickel carbon>-lin the calibrating gas. T h e final concentration,
X,is given by
n-here p = vapor pressure of nickel carbonyl a t 0" C .
P = atmospheric pressure
F1 = f l o rate
~ through first 08 Flonrat,or
F2 = f l o rate
~ through 01 Flowator
Fa = flox rate through second 08 Flonrator
I.'? = flow rate through 2L F l o w a t o r
The vapor pressure, p , of nickel carbonyl a t 0" C. is 134 nini.,
and F, and F , are held constant a t 10,000 cc. per niinute and
23,000 cc. per minute, respectively. If atmospheric pressure, P ,
is take:i as 760 mm., the eqiintion rednws to
-Y = 0.000iOS F,F:j p.p.m.
Carbon monoxide is used in the bubbler and for the first dilu-
tion t o prevent premature deconiposition of the nickel carbonyl.
Air is used for the second dilution to promote oxidation of the
deposit on the hot disk.
S o evidence of decomposition of nickel carbonyl in any part
of the system except on the hot disk was found.
DISCUSSIOS
From the basic principlea of operation, it is seen that the mini-
niiini concentration of nickel carbonyl that the instrunient can
detect depends upon the qu;tritity of deposit that is allowed
to build up on the surface of the disk. This, in turn, depends
upon the rate of sample flow aiitl npon the rate of rotation of the
Indicator for Titration of Calcium in Presence of Magnesium Using
Disodium Dihydrogen Ethylenediamine Tet raacetate
HARVEY DIEHL and JOHN L. ELLINGBOE
Department of Chemistry, lowa State College, Ames, lowa
A new indicator, designated calcein, has been prepared
for the titration of calcium in the presence of magne-
sium with disodium clihydrogen ethylenediamine
tetraacetate. No preliminary treatment is necessary
be>ond dissolving the sample and adjusting the pH to
a value of 12. Excessisel3 large amounts of sodium and
magnesium cause the results for calciuttl to be slightly
low. Interference 1)) copper and iron is obTiated by
the addition of c! ankle.
A SE\T- i.itlicatoi for the titration of calcium with disodium
dihydrogen ethylenediamine tetraacetate [the disodium salt
of (ethj-1enedinitrilo)tetraacetic acid] in the presence of mag-
nesium has been prepared by condensing irninodiacetic acid w t h
fluorescein. This is a procedure analogous t o t h a t employed by
Schwarzenbach and others for the preparation of the so-called
metal phthaleins (4). In highly alkaline solution t h e indicator is
brown and its calciuni complex is a yellow-green. At lower pH
values the free indicator is also yellow-green. Magnesium does
not form a complex with the indicator. T h e indicator m a y be
used for t h e determination of calcium in water, limestone, or
other calcium compounds. I t has been given the trivial name
calcein.
I n the analysis of limestone or water, the total calcium and
ANALYTICAL CHEMISTRY
disk. Large saniplr floir P, hon e w r , tend to cool the surface and
decrease the deposition efhieiicy, so that for given values of
temperature and rate of rotation, there exists a n optimum rate of
f l o ~ . T h e optimum rate Eo1 the iristrunient described in this
paper was found t o be about 500 cc. per n-incite. At this flow
rate, the instrument gives full-sc ale deflection a t a concentratioii
of approximately 4 p.p.m.
T h e sensitivity of the instrunLentcan be increased appreciably
by altering the position of the light beam mith respect to the
nozzle so t h a t a greater quantitv of material is deposited a t a
given point before the reflectance at that point is measured.
This, however, introduces a longer t h e lag beta een deposition
and measurement. K i t h a &minute time lag (which was not
considered excessive) the instrument registered a deflection of 1yc
of full scale for 0.2 p.p.m. A time lag of 10 minutes results in 1%
of full-scale deflection for 0.05 p.p.ni. The instrument is capable
of even greater sensitivity because, with the particular nozzle
which was used, the time of deposition a t any point on the pe-
riphery of the disk is approximately 40 minutes.
Although the instrument described in this paper n as designed
for the detection of nickel carbonjl, it is also sensitive t o iron
carbonyl and should be easily adapted to the detection of tetra-
ethyllead and other metallo-organic gases and vapors.
LITERATURE CITED
(1) Arch. I n d . H u g . and Occupational M e d . 9, 531 (1954).
(2) Chem. Processing (;lrnerican C o n f e r e n c e of G o v e r n m e n t a l Indus-
trial Hygienists) 15, 134 (1952).
(3) G a r r a t t , A. P., Thompson, H. K., J . Chem. SOC.(Lo7ldon) 1934,
1824.
R E C E I V Efor
D review 11s. 2 6 , 19.53. Accepted .Ianiiar~-28, 1956.
niagtiesiuni can be deterinilied b y using disodium dihydrogen
ethylenediamine tetraacetate as the titrant with Eriochronie
Black T as the indicator (1-3). Either the calcium or magnesium
must then be determined separately and the other calculated b y
difference. AIagnesium cannot be determined in the presence of
calcium because the formation constant of the calcium complex
of ethylenediamine tetraacetate is two orders of magnitude greater
than that of the magnesiwn complex. T o determine calcium di-
rectly using disodium dihydrogen et,hylenediamine tetraacetate
as the titrant, the pH is niade siifficiently high so that the mag-
nesium is largely precipitated as the hydroxide and an indicator is
used which combines with calcium only. Murexide is such an
indicator ( 5 , 6), b u t the end point with it is rather indefinite and
is made worse by increasing amounts of magnesium.
A sharper end point is obtained with calcein than with niurex-
ide, and larger quantities of magnesium may be present without
impairing the end point. T h e niagnesium may exceed the cal-
cium b y a factor of 20 to 30 without interference. Large amounts
of sodium salts-2 t o 3 granis of sodium chloride, for exaniple-
do not affect the titration. Strontium and barium interfere and
are titrated along with calcium; the end point with either alone
is t h e same as t h a t with calcium. Copper and iron interfere
with the end point, b u t such interference is easily obviated b y the
addition of cyanide. T h e titration of calcium may be performed
in the presence of chloride, nitrate, acetate. and sulfate.
V O L U M E 28, NO. 5, M A Y 1 9 5 6 883

I3elow p H 12, both the indicator and its calcium complex have a
yvllow-green color. Above p H 12 the indicator is brown, but
tkir d c i u m coniplex has the same yellow-green color. T h e
titration is carried out a t a p H above 12 so t h a t the end point is
nisrked by a change from yellox-green to brown. T h e indicator
can be added either as the solid (one part of indicator mixed with
100 parts of potassium chloride) or as a 27, solution in dilute
sodium hydroside. A better end point is obtained if t h e solid
indicat,or contains some charcoal (1 part of indicator, 10 parts of
charcoal, and 100 parts of pot,assiuni chloride). T h e calcium
complex appears much greener xhen this is doqe and, when large
amounts of iron are present, the end point is much better. K h e n
thc iric!icat,or is used alone or in conjunction with charcoal, it,
is completely reversible. T h e end point is better in diffuse light
tliaii in illumination of high intensity.
SOLID VTTH CHARCOAL. Grind together 1 gram of the indicat,or,
10 grams of charcoal (Sorite A is satisfactory), and 100 grams of
potassium chloride.
It is convenient t o measure out eit,her of t.he solid indicators
with a small metal scoop t,hat holds about 0.07 gram of the mix-
ture.
Calcium in Water. Pipet a 50-ml. sample into a conical flask.
Add 1 or 2 drops of 2% indicator solution (or a scoop of a 1%
solid mixture, about 0.07 gram) and 5 ml. of 1.W sodium hydroxide
containing 1 gram of sodium cyanide per 100 ml. Titrate with
0.02.V disodium dihydrogen ethylenediamine tetraacetate until
the color changes from yellow-green to brown. Vigorous stirring
is necessary throughout the titration. Do not carry out the
titration under a fluorescent lamp or in light of high intensity.
Standardize the disodium dihydrogen ethylenediamine tetra-
acetate solution against Iceland spar or a primary standard grade
of calcium carbonate.

Table 11. Analysis of Limestone and Gypsum

(Disodium dihydrogen ethylenediamine tetraacetate standardized against
Table 1. Analyses on Calcein Iceland sparu)
Seutralization h-eutralization Standard Sample

Pwpnra-
Equivalrnt,
h-aOH in
c
Sitrogen,
Seutralization
Equivalent.
Equiralent,
HClOi in la
NBS
88 1099
co.
1100 Seleniteb
tiun" Water Iijeldalil Bromination Acetic Acid
CaO reported, % 41.32 30 49 54.28 30.49 32.57c
RIgO reported, % 2 . 1 9 21.48 0.85 21.83 b
CnO found, "0 41.31 30 48 54.33 30.34 32.53
41.37 30 3 i 54 17 30 48 32.46
41.2.5 30 41 54.31 30 53 32.51
41 26 30.46 54.30 30 56 32.54
f> 207 3 38 41.21 30 44 54.23 30 4 3 32.64
I 187 4 16 88 194 41.36 30 5 6 54.18 30 38 32.50
4 1 21 30 44 54.26 30 44 32.47
0 T-arioui preparations ohtained from numerous attempts a t purification. 41.20 30 50 54 20 30 51 32.54
41.18 32.5''
32.60
32.54
32 46
Av. 41.26 30.4ci 54.25 30.45 32 5 2
Range 0.19 0 19 0.16 0.22 0 18
Owing to the high p H at which the titration is performed, AT. d e r . 0.056 0 044 0 054 0 061 0.039
Std. der-. 0 070 0 059 0.065 0.075 0.054
some calcium is precipitated as the hydroxide at the beginning.
a Transparent crystals; magnesium content determined spectrographically
T.igorous stirring is necessary to dissolve the hydroxide as the t o be 40 p.p.iii,
titration progresses. If the stirring is slow, false end points are b Transparent crystals of gypsum (selenite variety from Freedoni, Okla.) :
niagnesiiim content determined spectrographically t o be 20 p . p . i n
ohtained, the color returning after each change as the stirring is C Theoretical CaO content for Cas04 2HzO.

coiitiniietl.

PREPAR.ATION O F I N D I C A T O R
Limestone. Keigh a sample of about 0.3 gram into a 400-ml.
llis 100 grams (0.3 mole) of fluorescein, 300 ml. of ethyl alco- beaker. Add 20 ml. of 1 to 1 hydrochloric acid and evaporate to
hol, 150 nil. of distilled xater, and 90 ml. of 307, sodium hydrox- dryness. Redissolve the sample in 5 ml. of 1 t o 10 hydrochloric
ide. Add with stirring 87 grams (0.M mole) of iminodiacetic acid, acid and then dilute t o 100 t o 200 ml. with distilled water. To
dissolved in 105 ml. of 30% sodium hydroxide plus 120 ml. of this add 1 to 2 drops of the indicator solution or a scoop of the
distilled n.ater. Cool the mixture to 10" C. in an ice bath. Add solid indicator mixture and about 5 ml. of 1OM sodium hydroside
dropwise 74 ml. (0.75 mole) of 3 i % formaldehyde, stirring vigor- containing 5 grams of sodium cyanide per 100 ml. Titrate with
ously. After ail of the i'ormddehyde has heen added, heat the 0.LV disodium dihydrogen ethylenediamine tetraacetate under
mixture to 60' to 70" C. for 0 to 7 hours, stirring continuously. the same conditions as for calcium in water.
rlllow the solution to cool, then dilute to 3 liters. Add 1 to 1
h?-tirochloric acid, precipitating the indicator as the free acid.
Filter and A-ash with distilled water. Redissolve the material in 3
liters of \\-ater containing 120 grams of sodium acetate. Precipi-
tate it again with hydrochloric acid, filter, and wash. Transfer Table 111. Titration of Calcium in Presence of Large
the material into 2 liters of ethyl alcohol, stir for 1 hour, and filter. Amounts of Magnesium and Sodium Salts
Repeat the ethyl alcohol xvdiing: then dry the materid in a
Sodium Calciuin
vaciium. The product is bright yellow. When heated, it begins Calcium, Gram Magnesium, Chloride, Recovered,
to clccompose slowly a t ahout 185' C. It is apparently a mixture, Taken Found Grains Grams %
with a compound predominating which contains t x o imino- 0.1188 0,1188 1.0 100 00
diacetic acid residues, because analyses performed on materials 0.1188 0 1191 3.6 100 29
ol)t:iinetl from various purification processes gave variable results 0.1188 0.1178 3.0 99 18
0.1188 0.1190 100 19
(Tai)le I). T h e titration with sodium hydroside gave a titration 0.1188 0.1173 98 74
curve having a single sharp break with the end point around a p H 0,0833 0.0827 99 32
of 7.5. .Ittempts to determine the molecular weight. were un- 0.1176 0.1162 2.b 98 91
0 0595 0,0585 3 0 98 38
surcessful. 0 0.59: 0,0589 98 99
Though admittedly not a p\ire product, the material so pre-
p : i d functions well as an indicator. It is also available from the
G , I2reclericl; Smith Chemical Co., Columbus, Ohio.

DETERIIIN.ATIOS OF CALCIUM

Preparation of Indicator. SOLCTIOS. Dissolve 2 grams of the RESULTS

indicator in 25 nil. of lL\- sodium hydroxide and dilute to 100 ml. Two limestone samples from the Bureau of Standards, two
with distilled water. Use 1 to 2 drops of this solution.
SOLID. Grind thoroughly 1 gram of the indicator with 100 samples of limestone from the Standard Sample Co., h m e s ,
grams of potassium chloride. Iowa, and a sample of selenite (transparent variety of gypsum 1,
884
obtained from the G. Frederick Smith Chemical Co., were an-
alyzed for calcium. T h e limestones were analyzed b y the proce-
dure given above. T h e selenite was dissolved in a n excess of
standard disodium dihydrogen ethylenediamine tetraacetate at a
pH of 12, the stirring being continued for 4 t o 6 hours, Excess
standard calcium chloride was then added and t h e solution
titrated with disodium dihydrogen ethylenediamine tetraacetate.
The results reported were obtained using both the indicator
solution and the solid indicator with and without charroal.
T h e results on the samples from the Bureau of Standards were
correctedfor the strontium present (0.0570 in sample l a and O.OlyG
in sample 88). T h e results are given in Table 11.
Titrations of k n o m amounts of calcium in the presence of
magnesium and sodium chloride were carried out. T h e results of
Fluorometric Micromethod for Determination of Tryptophan
GERALD D. MILLER and JOHN A. JOHNSON
Department o f flour and Feed M i l l i n g Industries, Kansas State College, Manhattan, Kan.
BYRON S. MILLER
Federal Hard W i n t e r W h e a t Quality Laboratory,
The measurement of fluorescence intensity of sub-
stances formed by the reaction of glucose and trypto-
phan is suggested as a method for the quantitative
determination of microquantities of tryptophan.
Tryptophan is separated from the other amino acids on
a resin column of Dowex-50 (sodium form), and is made
to react with glucose under optimum standard condi-
tions. These conditions include the heating of 20 y or
less of tryptophan with 0.8 gram of glucose at pH 1.38
and a temperature of 118' C. for 4 hours. There is a
linear relationship between tryptophan concentration
and fluorescence intensity which is read at pH 1.80.
The standard error is *3qG for 12 y of tryptophan.
T HE determination of ti yptophan, particularly in materials
containing carbohydrates, requires special analytical tech-
niques as well as hydrolysis in an alkaline rather than in an
acid medium. Both chemical and biological methods may be
used for the analysis of tryptophan in pure protein.
The many available chemical methods for the determination
of tryptophan involve reaction with oxidizing agents, condensa-
tion with aldehydes, or diazotization reactions. These methods
have the common limitation t h a t they are not sensitive enough
to determine microquantities of tryptophan which occur in some
biological materials. T h e ninhydrin reaction ( I O ) which has been
used for assay of many amino acids is not sufficiently sensitive
t o give satisfactory results for assay of tryptophan in the low
concentrations present in flour.
Portner and Hog1 ( 1 2 ) reviewed the chemical methods used in
the determination of tryptophan and concluded t h a t the most
advantageous method is t h a t of Spies and Chambers (14-17).
This method involves condensation of tryptophan and p-di-
methylaminobenzaldehyde followed by oxidation with nitrous
acid. While the Spies and Chambers method is satisfactory
for tryptophan assay of proteins, i t is not adaptable t o the assay
of flour and similar materials.
T h e limitations of available methods for the analysis of trypto-
phan in concentrations that occur in foods suggested t h a t a
new approach t o the problem might be profitable. Tryptophan
is known t o react with reducing carbohydrates ( 7 ) t o produce
ANALYTICAL CHEMISTRY
these titrations are given in Table 111. S o interference n i t h
the end point could bp detected.
LITER4TURE CITED
(1) Biedermann, W.,Schwarzenbach, G., Chimia. (Swift.) 2, 56
(1948).
(2) Diehl, H., Goetz. C. A , Hach, C. C., J . 4711.Water Works Assoc.
4 2 , 4 0 (1950).
(3) Schwarzenbach, G., Ackerinann. H., Helz. Chim.-4cta 30, IT98
(1947).
(4) Schwarzenbach, G., Anderegg, G., Flaschka, H., Salliman It.,
Ibid., 37, 113 (1954).
(5) Schwarzenbach, G., Biedermann, W., Bangerter, F., Ibid., 29,811
(1946).
(6) Schu-arzenbsch, G., Gysling, H., Ibid., 32, 1314 (1949).
RECEIVED
f o r review d u g u s t 15. 1955. Accepted Fehruary 20, 1956.
U. S. Department o f Agriculture, Manhattan, Kan.
fluorescent material (2, 4,5 ) . .Ilthough moJt of the amino acids
react with glucose t o form fluorescent compounds, the fluorescence
intensity of compounds resulting from the reaction of glucose
and tryptophan is much greater than t h a t for the other amino
acids (6). This reaction, therefore, appeared t o have several
advantages over other methods. The object of the present
study was to define the conditions affecting the development of
fluorescent compounds resulting from the ieaction of tryptophan
with glucose and t o apply the reaction to the quantitative esti-
mation of tryptophan. The application of this method t o the
determination of tryptophan in biological materials \vi11 be pub-
lished later.
APPARATUS AND MATERI.iLS
A Coleman electronic photofluorometer, Model 12C, \vas ustd
for t h e fluorometric analyses. I t was equipped with a S o . 5 8 i 4
Corning filter which transmits the 365-nip mercury line, and
another filter consisting of tv-o Corning filters (Sos. 3398 and
4308) which absorbs below 425 mp. A reference standard con-
sisting of a solution of 0.1 y per milliliter of sodium fluorescein
in mater was used t o adjust t h e instrument to a given sensitivity.
All values m r e calculated on the basis of a fised value of 40 for
sensitivity. Dilutions of thp reaction niistures used to estahlish
the standard curve were made with the buffer in order to adjust
them t o the reading range of t h e photofluoronieter and to adjust
the p H to permit reading at maximum fluorescence. Commei,-
cially available chromatographic columns (10 X 300 mm.) wew
fitted into condenser jackets and used to remove tryptophan from
other amino acids according to the procedure of Moore ailti
Stein (11). One-ounce prescription bottles v i t h TeAon inserts
in the caps were used as containers for t h e solutions containiiin
tryptophan and glucose, which were heated in the autoclave to
develop fluorescent compounds.
A Beckman Model GS pH meter was used to measure the pH
of all solutions. A saturated solution of potassium acid tartratc'
(6) was used as a standard buffer (pH 3.57). A Coleman standaid
buffer (pH 2.0) also was used.
Tryptophan. Analytically pure b t r y p t o p h a n was obtaincd
from Sutritional Biochemicals Corp., Cleveland, Ohio. .I sample
of DL-tryptophan obtained from the Dow Chemical Co., Mid-
land, hlich., ~ a recrystallized
s twice from water and ethyl :I]-
coho1 followed by a rinse with anhydrous ethyl ether. Tlir
purified DL-tryptophan was used as a standard.
Sources of Other Amino Acids. All of the amino acids othcr
than tryptophan normally found in food products were obtained
from Xutritional Biochemicals Corp. and v-ere used without
additional purification.