New Indicator for Titration of Calcium with

(Ethy Ienedin itriI0) Tet raacetate

JAMES PATTON and WENDELL REEDER
Campbell Taggart Research Corp., Dallas, r e x .

A new in'dicator for calcium, 2-hydroxy-l-(2-hydroxy-4-
sulfo-l-naphthylazo)-3-naphthoicacid, has been syn-
thesized which permits an accurate titration of calcium
in the presence of magnesium with (ethylenedinitrilo)
tetraacetate. The indicator produces a sharp, stable
color change from wine red to piire blue. Calcium and
magnesium separations are not necessary. Titration
procedures for the determination of calcium and mag-
nesium in water, limestone, salt, and boiler scale are
described. The usual ammonia-ammonium chloride
buffer is replaced by an odorless monoethanolamine-
hydrochloric acid buffer containing coniplexed magne-
sium.
Tlirongh the synthesis of a new indicator, the authors have
made possible a direct titration of calcium in the presence of
magnesium. The new indicator produces a sharp color change
from wine red to pure blue xhen calcium is titrated with (ethyl-
enedinitrilo) tetraacetate a t p H values between 12 and 14.
Accurate determinations of calcium with no interference from
magnesium have been made on a variety of samples including
water, limestone, salt, blood serum, milk, and urine. Calcium
and magnesium separations were not necessary.
The indicator is 2-hydroxy-l-(2-hydroxy-4-sulfo-l-naphthyl-
azo)-3-naphthoic acid, which is synthesized by coupling diazo-
tized 1-amino-2-naphthol-lsulfonic acid with 2-hydroxy-3-
naphthoic acid. This indicator is commercially available from
Scientific Service Laboratories, P. 0. Box 175, Dallas 21, Tex.

1' H E determination of calcium and nidgnesium by titration

with standard solutions of disodium dihydrogen (ethylene-
dinitrilo) tetraacetate using Eriochrome Black T as the indicator
APPARATUS AND REAGESTS

A measuring spoon for dry indicators, approximately 0.1-gram

capacity, is available from the Hach Chemical Co., Ames, Iowa.
has been investigated thoroughly and widely accepted. As a titration light an ordinary reflector-type lamp, fitted r i t h
Following the original work of Biedermann and Schwarzen- a 100-watt screw-base bulb, was used.
bach ( d ) , Diehl, Goetz, and Hach ( 5 ) utilized the titration for Total Calcium and Magnesium Indicator. Triturate 0.2 gram
the separate determination of calcium and magnesium by pre- of Eriochrome Black T (F 241) and 50 grams of reagent grade
potassium chloride wit,h a mortar and pestle until all the dye is
cipitating the calcium as the oxalate and then titrating the mag- evenly distributed on the surface of the potassium chloride
nesium after filtration. Banewicz and Kenner (2) further stud- crystals.
ied the oxalate separation method for calcium and magnesium Calcium Indicator. Diazotized l-amino-2-naphthol-4-sulfonic
in dolomites and limestones. More recently, Gehrke, .Iffsprung, acid is so readily decomposed by light that it has been used in
photogra hic printing fapers ( 1 1 ) . The diazotization process
and Lee (8) adopted a sulfite separation method for the deter- describezby Woodwar (9) produces good results when the re-
mination of magnesium r h e n calcium is present. Schwarzenbach, action takes place in the absence of light. A photographic dark
Biederniann, and Bangerter (13) showed that calcium can be room is an ideal place t o carry out the reaction. If a dark room
titrated in the presence of magnesium when ammonium pur- is not available, the reaction may be carried out at, night or in a
light-tight container.
purate is used as the indicator. Betz and No11 (3) studied the Stir a mixture of 23.9 grams of l-amino-2-naphthol-4-sulfonic
calcium det,ermination, using ammonium purpurate as the acid and 100 ml. of water at 20" C. in a 400-ml. beaker. -4dd 0.2
indicator, and found the tit,rat,ion acceptable for use in m t e r gram of cupric sulfate pentahydrate and 23 ml. of a 307; solution
analysis. Calcium has been determined in biological fluids with of sodium nitrite to the mixture, Continue stirring the mixture
for 45 minutes and filter the solution into a dropping funnel. .-Z
(ethylenedinitrilo) tetraacetate, using ammonium purpurate as dark room safe light may be used t o aid in the manipulations.
the indicator (6, 7). Pre are a suspension of 18.8 grams of powdered 2-hydroxy-3-
The main objections to the determination of calcium or mag- naphtIoic acid and 50 ml. of water in a 600-ml. beaker. .kid
nesium separately by the compleximetric methods offered to with stirring 35 ml. of a SOYo solution of potassium hydroxide.
When complete solution is obtained, cool the mixture to about
date are indistinct end points and/or the necessity of separating 20" C. Add the diazotized l-an1ino-2-naphthol-4-s~1lfonic acid
the metals prior t o titration. Hoffman and Shapiro (10) used solution dropwise with constant stirring. When all of the
a n automatic photometric tit,rator to obtain improved end points. diazonium salt has been added, stir for an additional 15 minutes.
T h e advantage of a method which gives a good visual end point' The mixture now may be exposed to ordinary light intensitj-.
Add about 50 grams of cracked ice to the dark blue solution
and permits a rapid titration of calcium in the presence of mag- and acidify with 50 ml. of concentrated hydrochloric acid slowly
Iiesiiim is apparent'. added with constant stirring. Filter the solution by suction and
wash the dye with lOy0hydrochloric acid ilntil the filtrate is light
red in color. Dry the 2-h~-drox~-1-(2-hydrosy-4-srilfo-l-naph-
thylazo).3-naphthoic acid on the steam bath until it can be re-
duced to a fine powder. Dry the material in an oven at 110' C.
Table I. Determination of Calcium and RIagnesiunl in for 3 to 4 hours.
Water Samples The yield is 30 to 32 grams or about 7 5 S of the theoretical
Calcium. P.P.M. a s Magnesium! P.~P.I\l.as .IIgCOsy
amount.
CaCOa - Oxalare
Triturate 0.5 gram of 2-hydroxy-l-(2-hydroxy-4-sulfo-l-
Sample Direct separation BY naphthylazo)-3-naphthoic acid and 50 grams of reagent grade
No. AOAC method method difference sodium sulfate until the dye is evenly distributed on the sodium
141 140 29 sulfate.
69 08 07 Standard Magnesium Chloride. Dissolve 2.03 grams of re-
68 68 52 agent grade magnesium chloride hexahydrate in a small volume
81 83 36
31 32 11 of water and dilute to 1 liter.
29 30 10 Standard Calcium Chloride. Transfer 1.000 gram of pure
61 61 5 calcium carbonate (special lo^ in alkalies and heavy metals) to
8.56 854 23
a 1-liter volumetric flask with the aid of a powder funnel. Rinse
0 Average of triplicate determinations the powder funnel and the neck of the flask with distilled water.
.4dd 250 ml. of 0.100N hydrochloric acid and swirl the contents
1026
V O L U M E 28, NO. 6, J U N E 1956 1027

indicator and titrate to the pure blue end point with standard
Table 11. Recovery of Calcium from Known Solutions of (ethylenedinitrilo) tetraacetate solution. Record the volume as
Calcium and Magnesium the amount of standard solution required for total calcium and
magnewum.
Magnesium Calcium, P.P.M. a s CaCOla
Make a blank titration, replacing the sample with distilled
Present, P.P.?J.
as hlgCOs Present Found water.
Calcium. Pipet duplicate aliquots of the prepared sample
into 250-ml. wide-mouthed Erlenmeyer flasks and dilute to about
50 ml. Add 4 ml. of 8N potassium hydroxide solution to the
first flask and mix. (A precipitate of magnesium hydroxide may
be noted here.) Allow the flask to stand for 3 to 5 minutes with
occasional sn-irling. Add about,30 mg. each of potassium cyanide
a Average of triplicate determinations. and hydroxylamine hydrochloride and swirl the flask until the
reagents have dissolved. -4dd one spoonful of calcium indicator
and titrate with standard (ethylenedinitrilo) tetraacetate solution
Table IIT. Determination of Calcium and Magnesium in to the pure blue end point. Refill the buret and put into the
Salt Samples second flask a n amount of titrant e ual to the amount required
Calcium a s CaSOd, 7%" Magnesium a s hIgS04, %" to reach the end point less 1 ml. I d d 4 ml. of Si\- potassium
Direct BY hydroxide and mix the contents thoroughly. Continue the titra-
AOAC titration AOAC difference tion procedure as n-ith the first sample. Record the volume as
0.80 0.80 0.13 0.13 the amount of standard (ethylenedinitrilo) tetraacetate required
0.19 0.19 0.01 0.01 for calcium.
0.89 0.88 0.10 0.11
0.19 0.19 0.10 0.10 Make a blank titration, replacing the sample with distilled
0.26 0.26 0.01 0 01 water.
0.14 0.14 0.01 0 01 One milliliter of 0.02005(ethylenedinitrilo j tet raacet ate solu-
0 19 0.17 0.03 0 03
0 31 0.31 0.04 0 04 tion is equivalent to 0.4008 mg. of calcium.
-4verage of triplicate dctcrminations.
Magnesium. The volume of standard (ethylenedinitrilo)
tetraacetate solution equivalent to magnesium is obtained by
subtracting the volume required for the calcium from the volume
required for total calcium and magnesium on equal aliquots.
One milliliter of 0.0200N (ethylenedinitrilo) tetraacetate is
until solution is complete. Add 40 ml. of 0.1005 sodium equivalent to 0.2432 mg. of magnesium.
hydroxide] cool bhe contents to room temperature] and dilute to
the mark. RESULTS
Standard Disodium Dihydrogen (Ethylenedinitrilo) Tetra-
acetate Dihydrate. Dissolve 3.9 grams of the analyt,ical reagent The results of analysis of water, common salt, limestone! and
material in distilled water and dilute to 1 liter. St'andardize boiler scale samples showed good agreement between the values
against the standard calcium chloride solution using Eriochrome obtained for calcium by the direct titration, using 2-hydroxy-l-
Black T as the indicator. (2-hydroxy-4-sulfo-l-naphthylazo)-3-naphthoic acid as the in-
Buffer (14). Add 55 ml. of concentrated hydrochloric acid to
400 ml. of distilled water and mix thoroughly. Slowly pour 310 dicator, and the official .IOAC method (1). Indirect magnesium
nil. of redistilled monoethanolamine with stirring into the mix- tieterminat,ions made by subtracting the direct calcium titration
ture. from the total calcium and magnesium titration were also in good
Cool the solution t o room temperature. Titrate 50.0 ml. of
standard magnesium chloride solution n4th standard (ethylene- agreement with the direct magnesium determinations volumetri-
dinitrilo) tetraacetate solution using 1 ml. of the monoethanol- cally and gravimetrically, following an oxalate separation of
amine-hydrochloric acid solution as the buffer. Use the method calcium. Direct magnesium deterniinat,ions were made volu-
described below for total calcium and magnesium determination. metrically with (ethylenedinitrilo) tetraacetate, using Eriochrome
.Idd 50.0 ml. of magnesium chloride solution t o the amount of Black T as the indicator, and gravimetrically by official .40AC
(ethylenedinitrilo) tetraacetate required t o sequester the magne-
sium exactly, pour the mixture into the monoethanolamine- methods.
hydrochloric acid solution, and mix well. Dilute the mixture to 1 The results of analyses of water samples are given in Table I.
liter. The maximum variation in the values obtained for calcium and
Potassium cyanide, reagent grade. magnesium is 2 p.p.ni. as calcium carbonate and 1 p.p.m. as
Hydroxylamine hydrochloride, reagent grade.
Pot,assium hydroxide, 8 N , made with reagent grade potassium magnesium carbonate.
hydroxide.
PREPAR.4TION OF SAMPLES
Table IV. Determination of Calcium and 3Iagncsium in
Salt Samples. Transfer 25 grams of a representative sample Limestones and Boiler Scales
to a 250-ml.volumetric flask Tvith the aid of a powder funnel. .~~Calciiim Oxide?,C/o" Magnesium Oxide,
Wash the powder funnel and the neck of the flask with small Sample Present Detd. Present Detd.
poTtions of distilled water. Add 100 ml. of 0.100N hydrochloric
NBSlimestone l a !1.32 41.44 2.19 2 13
acid to the flask and warm the contents on the steam bath until X B S dolomite 88 JO.49 :io 1 3 21.48 21 37
complete solution is obtained. Add 50 ml. of 0.1OOiV sodium Boiler scale 1 3 7 .32
!J 3 7 .'I 5 Il.68a 11.79
hydroxide to the flask, cool the contents t o room temperature, Boiler scale 2 4,j,Ci7!, -12 .i8 1.4Rh 1 .4.3
then dilute to the mark. Average of trililicatc rlrtrrminations.
Limestone and Scale Samples. Grind representative samples b Determined hy .%O.-\C iiictliods.
to a fine powder with a mortar and pest,le, then pass through a No.
80 sieve. Dry the samples to constant weight in an oven a t 100"
to 105" C. Ignite 1-gram samples in platinum crucibles over
the full heat of a RIeker-type burner. Bring the samples into The determinations of calcium in solutions containing kr1on.n
solution as described by Banewicz and Kenner ( 2 ) and dilute to a quantities of calciiini and magnesium are given in Table 11.
final volume of 250 ml. The direct calcium titration gave good results in the presence
Water Samples. Filter the samples through a retentive fluted of magnesium a t concentrations froin 0 to 839 p.p.ni. as mag-
filter paper such as Whatman S o . 12.
nesium carbonate.
The results of comparative analyses of common salt samples are
ANALYTICAL PROCEDURE
given in Table 111. The maximum deviation from the average
Total Calcium and Magnesium. Pipet an appropriate aliquot of the determinations nas =k0.0107c for calcium sulfate and
of the prepared sample into a 250-ml. wide-mouthed Erlenmeyer =!cO.O05%l,for magnesium sulfate.
flask and dilute with distilled water to about 50 ml. Add 5 ml.
of buffer solution and mix by swirling. rldd about 30 mg. each The results of analyses of National Bureau of Standards Dolo-
of potassium cyanide and hydroxylamine hydrochloride and mite 88 and limestone l a and of two typical carbonate boiler
again mix. ildd one spoonful of the total calcium and magnesium scales are given in Table IV. Calcium and magnesium were
1028
determined gravimetrically on the boiler scale samples by the
AOAC methods for soils and are given in the table as calcium
oxide and magnesium oxide present. The maximum deviation
from the average for the determinations of both calcium oxide
and magnesium oxide in limestones and boiler scales was i O . O O % , .
Corrections were made on the determinations of calcium and
magnesium in Kational Bureau of Standards limestone l a in
order to account for the titration of strontium 1%-ith(ethylenedi-
nitrilo) tetraacetate.
DISCUSSION
The success of the titration of calcium with (ethylenedinitrilo) the calcium titration. The interference from small amounts
tetraacetate using 2-hydroxy-l-(2-hydroxy-4-sulfo-l-naphthyl- of lead may be eliminated by adding an excess of sodium di
azo)-3-naphthoic acid as the indicator is made possible by two
facts: (1) The p H of the solution to be titrated is sufficiently
high to assure the quantitative precipitation of magnesium as
magnesium hydroxide; (2) calcium forms a stronger complex
with (ethylenedinitrilo) tetraacetate than does magnesium ( 1 2 ) .
Although magnesium hydroxide precipitate is present in the
titrating flask, the chelating solution nil1 not react with mag-
nesium until all free calcium and calcium complexed by the
indicator have been chelated by (ethylenedinitrilo) tetraacetate.
If the indicator is added before potassium hydroxide, an end point
may not be reached because magnesium salts form a lake with the
indicator as the p H increases. The magnesium-indicator lake
is coprecipitated with magnesium hydroxide.
The colored complex formed with the indicator and calcium
is absorbed on any magnesium hydroxide precipitate, b u t a sharp
end point is possible because the chelating agent has contact
with the colored complex. A sharper end point is obtained in
the presence of magnesium hydroxide precipitate.
A tungsten light source placed near the titration flask improves precipitate is a gelatinous m-hite 11-ith no red-brown color.
the end point, of the direct calcium titration principally because
the red complex of the indicator and calcium is greatly intensified.
T h e monoethanolamine-hydrochloric acid buffer ( 2 4 ) has a
buffering capacity equal to the ammonia-ammonium chloride
buffer n-hich is commonly recommended for the titration of
calcium and magnesium with (ethylenedinitrilo) tetraacetate.
Sharp end points are obtainable and the strong ammonia odor
is completely eliminated. The buffer has excellent keeping
qualities.
Biedermann and Schwarzenbach (4)pointed out that magne-
sium must be added to obtain a sharp end point when relatively
pure samples of calcium are titrated using Eriochrome Black T as
the indicator. Hence, magnesium is usually added t o the
(ethylenedinitrilo) tetraacetate solution prior to the standardiza-
tion. Magnesium in the chelating solution prevents a sharp
end point when calcium is titrated using 2-hydroxy-l-(2-hydroxy-
4-sulfo-l-naphthylazo)-3-naphthoic acid as the indicator. Addi-
tion of romplexed magneeium to the buffer eliminates the need
of two standard (ethylenedinitrilo) tetraacetate solutions and
assures an adequate amount of magnesium t o give a sharp end
point, even when small amounts of pure calcium are titrated.
INTERFERENCES
Ions which interfere in the usual titration of calcium and mag-
nesium using Eriochrome Black T as the indicator also interfere
U n
K Y
ANALYTICAL CHEMISTRY
in the titration of calcium when 2-hydrosy-l-(2-hydroxy-4-
sulfo-l-naphthylazo)-3-naphthoic acid is used as the indicator.
Diehl, Goetz, and Hach ( 5 ) have made a rather complete study
of the more common interfering ions and have indicated the
proper measures to be taken to eliminate the interferences.
When calcium is titrated directly a t high pH values, barium,
strontium, lead, and zinc will titrate as calcium. Barium inter-
ference may be eliminated by adding exress sulfate ions to the
hot slightly acid aliquot prior to the addition of potassium hy-
droxide. Strontium ions cannot be separated from calcium bj-
simple means; therefore, any strontium present will increase
ethyldithiocarbamate before the indicator is added. Small
amounts of zinc, copper, cobalt, nickel, and manganese do not
interfere n-hen potassium cyanide and hydroxylamine hydro-
chloride are added as in the recommended procedure.
I n the presence of sufficient magnesium, small amounts of
phosphate and iron do not interfere. Fifty parts per million of
magnesium eliminates the interference from 40 p.p.m. of phos.
phate and over 20 p.p.m, of iron.
The manner in which magnesium removes the interference
from iron and phosphate is not completely understood. The
authors assume that the interfering ions are coprecipitated with
magnesium hydroxide in such a manner that the outer surface
of the resulting precipitate is almost completely composed of the
very insoluble magnesium hydroxide. If the iron and phosphate
precipitates are coated with magnesium hydroxide, they are not
readily available to the chelating agent and indicator. It has
been noted that when ferric ions are precipitated with potassium
hydroxide in the presence of sufficient magnesium the resulting
LITERATURE CITED
. h o c . Offic. d g r . Chemists, “Official Methods of Analysis.”
7th ed., 1950.
Banewica, J. J., Kenner, C. T., A s a ~CHmr.. 24, 1186 (1952).
Beta, J. D., Noll, C. A , , J . Am. Water Works Assoc. 42, 49
(1950).
Biedermann, W.,Schwarxenbach, G., Chimia (Prague) 2, 56
(1948).
Diehl, H., Goeta, C. A , , Hach, C. C., J . Am. Water Works Assoc.
42, 40 (1950).
Elliot, W.E., J . Biol. Chem. 197, 641 (1952).
Fales, F. W., Ibid., 204, 577 (1953).
Gehrke, C. IT., ilffsprung, H., Lee, Y., SAL. CHEW 26, 1944
(1954) I
Groggins, P. H., “Vnit Processes in Organic Synthesis,” 2nd
ed., p. 136, XIcGraw-Hill, Kew York and London, 1938.
Hoffman, W.h l . , Shapiro, H., J . Assoc. Ofic. Agr. Chemists 37,
966 (1954).
Schmidt, Julius, Maier, W., Ber. 64B, 767 (1931).
Schwarzenbach, G., dckermann, H., Hela. Chim. Acta 31, 1029
(1948).
Schwarzenbach, G., Biedermann, W.,Bangerter, F., Ibid., 29,
811 (1946).
Thies, H., Kallinich, G., Biochem. Z . 324, 485 (1953).
D review > f a y 1 3 , 1955. Accepted RIarch 29, 1956
R E C E I V Efor