DETERMINATION OF C A L C I U M A N D M A G N E S I U M I N M I L K BY

E.D.T.A. T I T R A T I O N 1

E. W. BIRD ANn JUNE WEBER

Department of Dairy and l%od Industry
C. P. COX ~ aND T. C. CttEN8
Department of Statistics
Iowa State University, Ames, Iowa

SUM?cIARY
Five procedures were compared for E.D.T.A. determination of calcium and mag-
nesium in milk. The method which combined Ling's method for removal of proteins and
phosphates by potassium metastannate, Flashka's indicator (Eriochrome blue S E) for
the calcium determination, his adjustment of pH in the magnesium determination, and
the method of Jenness (slightly modified) for magnesium yielded the best results. The
method is convenient, rapid, and recoveries for both calcium and magnesium were
essentially 100%.

Calcium and magnesium had been determined for some time in this laboratory
by J e n n e s s ' method (2). The end point in the calcium determination is some-
what difficult and, when slight amounts of acid have developed in the milk, the
samples filter slowly (after casein precipitation) and the whey is cloudy. E n d
points were sharper when but 0.05 g. Murexide and one drop of Eriochrome
Black T solution were used in place of the recommended amounts. The p H
values could be adjusted to that specified for the method if pilot runs were
made, thus aiding filtration. I t was likewise found that 0.1 N HC1 and N a O H
gave better results than the stronger solutions recommended.
A t about the time t h a t the above work was completed, L i n g (3) proposed
potassimn m e t a s t a n n a t e ( P M S ) to remove the proteins and phosphates simul-
taneously, in determining calcium in milk. He used a color standard for the
Murexide end point (the Ling end point). F l a s h k a et al. (1) introduced Erio-
chrome blue S E (Erio S E ) as an indicator for calcium; the end point is sharper
than that of Murexide. They suggested use of methyl red to adjust the p H of
the solution, p r i o r to the addition of buffer, and titration with Eriochrome
Black T, as indicator, in the ma~o-nesium determination.
Stannic acid, an intermediate in the PMS preparation, filters slowly and
causes PMS p r e p a r a t i o n to be long and tedious, and the exact titration of the
final p r e p a r a t i o n is difficult to achieve unless the stannie acid is well-washed.
The p r e p a r a t i o n of PMS was simplified b y substitution of eentrifugation for
filtration.

Received for publication August 22, 1961.

Journal Paper No. J-3935 of the Iowa Agricultural Experiment Station, Ames, Project
No. 1298.
2Visiting Professor from the National Institute for Research in Dairying, University
of :Reading, England.
Current address: C. D. Searle and Co., Division of Biological Research, Chicago, Ill.
1036
 C A L C I U M AND MAGNESIUI~[ I N I~IILK 1037

A method combining the procedures outlined in the preceding two paragraphs

with the Jenness procedure for magnesium has been compared with four other
methods and found to be superior from standpoints of accuracy and precision.

EXPERIIVIENTAL PROCEDURE
ri. Statistical. The relative merits of the various methods were examined
using two criteria. The first of t h e s e - - a c c u r a c y - - w a s used to compare the close-
ness with which the means of the observations on each method indicated the
known amounts of calcium and magnesium added. Secondly, since a method
which proved highly variable from one single determination to another would
be obviously inferior, the precisions of the methods were examined in terms of
the variances of the individual determinations. A slight complication arose here,
however, because it was suspected that these precision variances differed even
within a single method, at the different levels of calcium or magnesium added,
so that it was necessary first to examine the homogeneity of the variance within
each method before comparing different methods on the basis of precision.
Common statistical tests have been used throughout and the findings from
them are discussed in practical terms, so that familiarity with the technicalities
of the tests themselves is not essential. Explanations of the more frequently
used statistical terms and of the analysis of variance can, however, be found
with examples from chemical studies in (7) ; the following supplementary notes
may be helpful.
Degrees of freedom (d.f.), the variance values obtained to compare the pre-
cisions of the methods or to make tests of significance, are themselves subject to
variation or error and, as with a mean, this error is lessened when the number
of individual observations, from which the estimated variances are calculated,
is increased. In making comparisons, therefore, it is helpful to be aware of these
numbers; they are presented in the tables as the numbers of degrees of freedom
which are equal, in each case, to the number of basic observations less one.
The significance of any difference between the amount of calcium (or mag-
nesium) added and the amount recovered was examined by making the usual
" t " - t e s t (7). The accuracies (as defined above) of the different methods were
compared using the multiple range test (6). Homogeneity of the precision
variances obtained for each method at different levels of added calcium or
magnesium was examined by the M-test (4) or, as was possible when only two
variances were involved, by the simpler F-test (6). The precision variances
representative of the methods were then compared using the F max test (4).
The results of such tests are generally assessed in terms of the probabilities, P,
that values of t, F, and M, as large or larger than those calculated from the
observations, could have arisen by chance, so that, for example, P ~ 0.05 implied
that some factor other than chance is responsible unless an event has occurred
which, by chance, is likely in only five cases out of 100.
2. Analytical. Five methods were compared :
Method I. The composite procedure to be described.
Method II. Ling's method for calcium (Ling's end point) and Jenness'
method for magnesium were combined.
1038 E . W . UIRD ET AL

M e t h o d I I I . E x c e p t f o r t h e use of J e n n e s s ' e n d p o i n t f o r t h e c a l c i u m d e t e r -
ruination, this was identical with Method II.
M e t h o d I V . T h i s w a s t h e m e t h o d e x a c t l y as d e s c r i b e d b y J e n n e s s .
M e t h o d V. T h e m e t h o d w a s i d e n t i c a l w i t h M e t h o d I V , e x c e p t t h a t L i n g ' s
m e t h o d of e s t a b l i s h i n g t h e e n d p o i n t w a s e m p l o y e d w i t h t h e c a l c i u m d e t e r -
mination.
Procedure for Method I: T r e a t m e n t of m i l k s a m p l e ( 3 ) . W e i g h a c c u r a t e l y
a b o u t 15 g. of m i l k i n t o a 250-ml. v o l u m e t r i c flask. A d d 90 ml. w a t e r 4 a n d t h ~ n
3 ml. (pipette) HN03 (one volume concentrated HN0a + 2 volumes HfO) with
c o n s t a n t r o t a t i o n o f t h e flask. M i x g e n t l y a n d a d d 30 ml. ( b u r e t t e ) o f p o t a s s i u m
m e t a s t a n n a t e s o l u t i o n , 5 w h i l e t h e flask is r o t a t e d c o n s t a n t l y . M a k e t o v o l u m e ,
m i x t h o r o u g h l y a n d , a f t e r s t a n d i n g f o r 5 m i n . , f i l t e r t h r o u g h a W h a t m a n No. 40
11-cm. f o l d e d filter. D i s c a r d t h e f i r s t 10 ml. of f i l t r a t e . T h e f i l t r a t e s h o u l d be
clear, a l t h o u g h a s l i g h t t u r b i d i t y does n o t i n v a l i d a t e t h e r e s u l t s .
A b l a n k c o n t a i n i n g 105 ml. of w a t e r is s u b j e c t e d to t h e s a m e p r o c e d u r e as
outlined for the sample.
The calcium determination (3). P i p e t t e 25 m l . of t h e f i l t r a t e i n t o a 125-ml.
E r l e n m e y e r flask. A d d a s q u a r e o f l i t m u s p a p e r (2 ram. o n e d g e ) a n d n e u t r a l i z e
w i t h 1.5 N N a O H . A d d a n a d d i t i o n a l 2 ml. of t h e N a O t t s o l u t i o n a n d s u f f i c i e n t
E r i o S E m i x e d i n d i c a t o r 6 (0.02 g.) to y i e l d a d e f i n i t e r e d c o l o r ( 1 ) . T i t r a t e
w i t h E D T A s o l u t i o n ( 4 g. d i s o d i u m d i h y d r o g e n e t h y l e n e d i a m i n e t e t r a a c e t a t e

Water in this study was prepared from distilled water, to ]8 liters of which 15 ml. of
a solution, containing 5% H_~SO, (v/v) and 5% K_oC~O7 (w/v), were added. This mixture
was distilled; the first and last 500 ml. of a 5-liter batch were discarded.
5Place 15 g. of 30-mesh granular tin (reagent grade), 71.5 ml. of water, and an egg-
shaped (No. 8445, Cole-Parmer Instrument and Equipment Co., Chicago, Ill., 1% in. long)
stirring bar, into a flat-bottomed, 250-ml. centrifuge bottle (Corning No. 1280), and place
on a magnetic stirrer. With constant stirring, add 65 ml. cone. tINO3, in 5-ml. portions, at
3-min. intervals. The fourth 5-ml. portion is ~tdded in two 2.5-ml. portions, because the
addition of the entire 5 ml. causes a violent reaction; the second 2.5-ml. portion is added as
soon as the reaction from the first portion subsides. When all of the acid has been added, cool,
remove the stirring bar, dilute with about 100 ml. of water, disperse the precipitate, fill to
the shoulder of the bottle with water, and mix well. I f the correct titration is obtained for
the final product, the precipitate must be completely dispersed in the washing procedures.
Centrifuge for 10 min. at 1,500 r.p.m. (International Size 2 centrifuge). Decant the super-
natant and wash three additional times in the same manner. After the last wash, distribute
the precipitate in 20 ml. of water, add 5 ml. KOtI solution by pipette, and dilute to 250 ml.
(volumetric flask). The concentration of KOH s~ggested by Ling is 56.0 g. analytical grade
KOH dissolved in 44 g. water. This concentration was stated to yield a metastannate, 10 ml.
of which required 11.6 ml. of 0.1 N HC1 to the pheno]phthaleln end point. When a reagent
grade of KOH (85.1 to 85.7%) was used it was found that 41 g. dissolved in 44 g. water
gave the specified titration. In establishing this fact, it was found that no difference was
obtained in the calcium or magnesium assay if the ratios were varied from 3 2 : 4 4 to 56:44
and that the filtrates all were negative for phosphate.
6 One-tenth gram Eriochrome Blue S E (Geigy Dyestuffs Division, Geigy Chemical Corp.,
Saw Mill ]~iver Road, Ardsley, N. Y.) and 0.I7 g. Naphthol Green B (Coleman and Bell,
Norwood, Ohio) are ground to a fine pow(ler with 20 g. NaC1 (1). The authors are indebted
to the Geigy Dyestuffs Division for furnishing the Eriochrome Blue S. E. dye.
 CALCIUM AND MAGNESIUM IN ~[ILK 1039

dissolved in and made to one liter with water), until the red color is changed to
a clear blue. This titration is Tsl; the blank titration, TB.
Calcium plus mag,nesium determination (2). Pipette a second 25 ml. of the
filtrate into a 125-ml. Erlenmeyer flask. Add one drop of methyl red indicator
(1) (0.1 g. methyl red in 500 ml. 95% ethanol), then 0.5 N N a O H dropwise
until the indicator changes to yellow. A d d I ml. borate buffer solution 7 (2)
(to maintain a p H of 8 to 10), two drops of Eriochrome Black T indicator
[1 g. in 30 ml. water containing 1 ml., 1 N Na2COa; make to 100 ml. with 2-
propanol (2)]. Titrate immediately from the pink color to a light-blue end
point. This titration is Ts ; the blank titration, TB.
2 2
Correction for precipitate volume (3). The titration of the filtrate aliquot
of a sample of milk is compared with a filtrate aliquot from an identical sample
of the same milk to which (after weighing) a definite volume of standard
calcium solution s has been added. F r o m the titrations obtained, a factor (f)
which corrects for precipitate volume can be calculated as in the following
example: I f 25 ml. of milk filtrate are equivalent to 4.09 ml. E D T A 9 and the
titration of 25 m]. of filtrate from the same milk, to 15 g. of which 15 ml. of
standard calcimn solution were added, is 6.38 ml., the difference, 2.29 ml., is
equivalent to 1.5 ml. standard calcium solution. B y direct titration, 5 ml. of
standard calcium solution are assumed equivalent to 7.43 ml. E D T A . The
correction factor is:
f = [(7.43 × 1.5)/51/2.29 = 0.9734
The milliliters of E D T A employed for the calcium and the calcium plus mag-
nesium titration are nmltiplied by f to correct for precipitate volume.
Calculations. F o r Methods 2, II, and III, the following calculations were
employed :
100
rag. Ca/100 g. milk = (Ts~ -- T B ) × f × (mg. Ca/ml E D T A ) ×
g. milk ~ - Ts
rag. Mg/100 g. milk = [ ( T s 2 - - TB.) -- (Ts~-- TB )] × f × (rag. M g / m l E D T A )
× 100
g. milk ~ TS

F o r methods I V and V, f was eliminated in making the calculations.

3. Experimental design. A large sample of raw milk was obtained from the
Market Milk Department at Iowa State University. This milk was centrifuged,
in 250-ml. lots, for 30 rain. at 1,500 r.p.m. (Size 2, International centrifuged),
to separate the fat. The samples were chilled (4.4 ° C.) to solidify the cream

Dissolve: (a) 4.0 g. e.p. Na2B40~ • 10 I-I~Oin 80 ml. of water, and (b) 1.0 g.c.p. NaOtt
+ 0.5 g.c.p, sodium sulfide in 10 m]. of water. Mix the two solutions, and make to 100 ml.
SDisso]ve about 2.0 g. dried (100°C.) primary standard grade of CaCO3 (exactly
weighed) in a minimum volume of HC1 (1: 10; v/v) in a 250-ml. beaker. Dilute to 100 ml,
boil gently to expel CO_%transfer to a l-liter volumetric flask, and make to volume at 20 ° C.
Measure aliquots at this temperature. One milliliter contMns: g. CaCO~ weighed X(40.08/
100.09) rag. calcium.
10~0 E . W . BIRD ET AL

layer. The skimmilk was removed and all batches were combined and thor-
oughly mixed.
The mixed skimmilk sample was divided into three portions (Samples I, I I ,
and I I I ) . Samples I I and I I I were frozen a n d held at --28.9 ° C. until used.
Sample I was used immediately. When Samples I I and I I I subsequently were
employed, they were completely thawed at 4.4 ° C.
F o r each sample, ten aliquots were weighed (20 ° C.). No calcium or mag-
nesium was added to two of the aliquots. S t a n d a r d calcium 9 or magnesium ~o
solution (20 ° C.) were added to the other aliquots in such m a n n e r t h a t the
added calcium was a p p r o x i m a t e l y half that of milk, in a second pair of aliquots
and roughly equivalent to t h a t of milk in a third pair.
The added magnesium was a p p r o x i m a t e l y t h a t of milk in a f o u r t h pair of
aliquots and 1.5 times t h a t of milk in the fifth pair. A f t e r the additions indi-
cated, the samples were equilibrated for 20 hr. at 4.4 ° C.
A set of ten such aliquots was p r e p a r e d and analyzed by each of the five
analytical procedures described above.
The data for each of the three samples constituted a run.

TABLE 1
Within-level variances of calcium determination. Degrees of f r e e d o m in parentheses
Method
Calcium a d d e d
rag/100 g. milk I II III IV V
41.56 0.2532 (3) 0.0728 (3) 0.4836 (3) ....
62.35 1.2828 (7) 0.8612 (7)
62.38 0.3241 (11) 1.7270 (11) 1.0731 (11)
93.5~ 8.1365 (7) 7.5996(7)
103.99 0.0926 (7) 0.2'30() (7) 0.15:1S (7)
124.69 1.6398 (3) 1.2532 (3)
155.89 12.1080 (3) 6.1978 (3)
Significant hetero-
geneity, P ~ 0.05 .... ~ ~ ....
Combined precision
variance 0.2368 (21) 0.9917 (21) 0.6818 (21) 5.3589 (20) 4.0789 (20)

RESULTS AND DISCUSSION

1. Precision of the methods. As noted under experimental methods, t h e
extent to which the variation among individual determinations differed at the
different levels of added calcium or magnesium was tested by examining the
homogeneity of these within-level variances. Estimates of these variances, and
the results of the tests, are given in Tables 1 and 2. They show that significant

9 Standardize by t i t r a t i n g 5-ml. aliquots of s t a n d a r d calcium solution to which 20 ml.

water had been added. E x p r e s s concentration as milligrams calcium or magnesium per
milliliter E D T A .
~oWeigh accurately about 10 g. analytical r e a g e n t grade MgSO4 • 7 tt,O. Make to 1 liter
with water. Determine the exact M g concentration by p r e c i p i t a t i n g the M g as the pyrophos-
phate (5). Dilute 50 ml. to 250 ml. for use as the standard.
 CALCIUM AND MAGNESIUM IN MILK 1041

TABLE 2
Within-level variances of the magnesium determinations. Degrees of freedom in ]parentheses
Method
Magnesium added,
rag/100 g. milk I II III IV V
19.41 0.1651 (11) 0.082~5(11) 0.4092(11) 2.0074 (11) 1.9223 (11)
29.10 4.2562' (11) 2.4131 (11)
32.35 0.1097 (11) 0.1754-(11) 0.2927 (11) ........
Combined preci-
sion variance 0.1374 (22) 0.1289(22) 0'.3510(22) 3.1318 (22) 2.1677 (2'2)

differences b e t w e e n t h e v a r i a t i o n of t h e d i f f e r e n t levels of a d d i t i o n o c c u r r e d
o n l y a m o n g t h e c a l c i u m d e t e r m i n a t i o n s m a d e b y M e t h o d s I I , I I I , a n d V. I n -
v e s t i g a t i o n of t h e o r i g i n a l d a t a i n d i c a t e d a g e n e r a l t r e n d of d e c r e a s i n g v a r i a t i o n
f r o m R u n 1 to R u n 3 f o r b o t h c a l c i u m a n d m a ~ l e s i u m d e t e r m i n a t i o n s . T h i s
s u g g e s t e d t h a t t h e i n t e g r a t i o n of the l a r g e n u m b e r s of s a m p l e s a n d t h e e x p e r i -
m e n t a l t e c h n i q u e h a d i m p r o v e d d u r i n g t h e course of t h e s t u d y .
To e x a m i n e this p o i n t f u r t h e r , s e p a r a t e s t a t i s t i c a l a n a l y s e s w e r e c a r r i e d o u t
on t h e r e s u l t s f o r R u n 3 alone. B e c a u s e of t h e f e w e r o b s e r v a t i o n s , t h e v a r i a n c e
e s t i m a t e s ( T a b l e s 3 a n d 4) a r e b a s e d on b u t t h r e e d e g r e e s of f r e e d o m . D e s p i t e
this f a c t c o m p a r i s o n w i t h T a b l e s 3 a n d 4 shows thai; t h e v a r i a t i o n in R u n 3 was
g e n e r a l l y s u b s t a n t i a l l y l o w e r t h a n t h a t f o u n d f o r a l l t h e r u n s combined. I n

TABLE 3
Within-level variances of the calcium determinations (Run 3 only). Three degrees of
freedom in all cases
Method
Calcium added
mg/100 g. milk I II III IV V
62.35 1.6111 0.6163
62.38 0.1083 0.0'771 0.2'362
93.53 o.17i~ 1.3o~6
103.99 0.054,2 0.24i9 0.05~2 . . . . . . . .
Combined preci-
sion variance
(6 df) 0.0812 0.1595 0.1452 0.8914 0.9620

TABLE 4
Within-level variances of the magnesium determinations (Run 3 only). Three degrees of
freedom in all cases
Method
Magnesium added,
mg/lO0 g. milk I II III IV V
19.41 0.00'72 0.0274 0.14~1 0.1153 0.1803
29.10 0.2054 0.4715
32.45 o.oo~ o.o25~ o.o5~ . . . . . . . .

Combined preci-
sion variance
(6 dr) 0.0072 0.0~2'66 0.0982 0.1604 0.3259
1042 ~. w. BIRI) ET ~L

addition, the homogeneity test showed that the significant differences between
the within-level variances had disappeared in Run 3, s u p p o r t i n g the suggestion
that complete stability in the techniques was not achieved in the first two runs.
Next, to examine the over-all variability of the methods, characteristic pre-
cision variances were calculated by combining the values obtained at the
separate levels. The results a p p e a r as the bottom row of figures in Tables 1 and
2, for calcimn and magnesium, respectively. I t can be seen that the two methods,
I V and V, which employ J e n n e s s ' procedures for removing proteins and phos-
phates, were considerably more variable than Methods I through I I I , in which
the Ling method for simultaneous removal of proteins and phosphates is used;
the objective statistical test confirms the significance of the difference between
the two groups. The indication is, therefore, that the removal of proteins and
phosphates, accompanied b y the simultaneous liberation of calcium and mag-
nesium from the proteins and phosphates, was involved.
The results of testing the significance of the differences within these two
groups of methods showed that, for the calcium determinations, there was no
significant difference between Methods I I and I I I or between Methods I V and
V. Method I was, however, superior (P ~ 0.05) to all the other methods, and
since it differed f r o m Methods I I and I I I only in the indicator employed, it is
considered that the superiority to these methods is attributable to the fact that
the Erio S E mixed indicator gives a sharper and more reproducible point
than does Murexide.
F o r the magnesium determinations no sig~dfieant differences were detected
within either of the two groups.
When, for completeness, similar tests were made on the data for Run 3
alone, the results agreed with those just described in that the groups of Methods
I, II, and I I I were significantly less variable than Methods I V and V. No
differences were found between methods within these two groups.
Since there were but three runs, there is no absolute assurance that the
experimental techniques had completely stabilized in Run 3. Nevertheless,
since the results for Run 3 exhibit no heterogeneity within methods, it seems
reasonable to suppose that the differences detected among methocLs do reflect
real differences in precision, in support of the general findings t h a t Method !
has greater precision than Methods I I and I I I which, in turn, have greater
precision than Methods I V and V.
2. Accuracy of the methods. As was indicated under E x p e r i m e n t a l Pro-
cedures, the accuracy of the methods was judged by the difference (±Ca) be-
tween the amonnt of calcium recovered and t h a t added, and b y the similar
difference (±Mg) for magnesium.
The results for calcium (Table 5) show that among Methods I, I I , I I I , there
is no evidence that ACa deviated significantly f r o m zero. I t can be considered,
therefore, that these methods are accurate by the criterion chosen. W i t h
Methods I V and V, ACa deviated significantly f r o m zero. These methods under-
estimate the quantity of calcium added to milk, although no correction is made
for precipitated proteins.
 CALCIUI~f AND MAGNESIUM IN hlILK 1043

TABLE 5
Mean values f o r 2~Ca (Ca r e c o v e r e d - - C a added)
Method
Calcium added
rag/100 g. milk I II III IV V
41.56 0.18r8 0.0122 -0.2r9r
62.35 0.90gr o.s6~i
62.38 -0.16~5 -0.3~iJ5 -0.4688
93.53 --2.5750 --0.5226
103.99 0.6358 0.60aa -0.1796
12'4.69 . . . . . . . . . . . . --9.0706 --9.0706
155.89 . . . . . . . . . . . . --3.8989 --3.5766
Mean 0.1605 0.0388 --0.2217 --2.7180 ~ --2.7602 ~
S.E. ( m e a n ) 0.0993 0.1867 0.1867 0.4425 0.442'5
d.f. 21 42 42 40 40
~ P ~ 0.01.

TABLE 6
Mean values f o r ~ M g ( M g r e e o v e r e d - - M g added)
Method
M a g n e s i u m added,
rag/100 g. milk I II III IV V
19.41 0.0208 0.4000 0.2017 0.9167 0.9392
29.10 1.3687 0.96,12
32.35 0.46i~ 0.88g~ 1.06~ ........
Mean 0.2413 ,~ 0.6~21 ~ 0.6321 ~ 1.1427 ~ 0.9502 ' ~
S.E. ( m e a n ) 0.0,926 0.0'926 0.0926 0.3324 0.3324
d.f. 60 60 60 40 40
P<0.05; ~P<0.01.

AMg differed significantly (Table 6) f r o m zero for all five methods and in
each case the amount of magncsimn was overestimated. Tile multiple-range test
showed t h a t the mean AMg was nearer to zero for Method I t h a n were the means
for Methods I I and I I I and t h a t the means f o r Methods I I and I I I were nearer
to zero t h a n were the means for Methods I V and V.
The indicator employed for the calcium plus magnesium titration was the
same for all five methods. The errors t h a t existed in this titration should,
therefore, have been the same for all methods. Because magnesiu!m was deter-
mined by difference in all of the methods, a M g could have been dependent
largely on the acem'aey of the calcium determination. The means of Table 5
show t h a t Methods I and I I slightly overestimate calcium, Method I I I slightly
underestimates calcium, and Methods I V and V considerably underestimate
calcium. I n comparison (Table 6), Method I slightly overestimates magnesium,
Methods I I and I I I overestimate it to a greater degree, and Methods I V and V
yielded the greatest overestimation. There does seem, therefore, to be an inverse
correlation between the underestimation of calcium and the overestimation of
magnesium.
Again, a eomparison of Methods I, I I , and I I I with Methods I V and V sug-
gests t h a t the method of removing proteins and phosphates is v e r y important.
W h e t h e r or not it is pertinent, the p H at the time of filtration is 1.6-1.7 with the
1044 E.W. BI~D E~ ~L

L i n g p r o c e d u r e a n d 4.0-4.2 w i t h the J e n n e s s method. T h e differences b e t w e e n

Method I a n d Methods ] I a n d I I I m a y r e s u l t f r o m the differences i n i n d i c a t o r
employed i n c a l c i u m d e t e r m i n a t i o n .
To o b t a i n f u r t h e r comparisons, p a r t i c u l a r l y b e t w e e n methods, a n a l y s e s of
v a r i a n c e s were c a r r i e d out for a C a i n those cases, discussed i n the n e x t section,
for which no h e t e r o g e n e i t y was detected a m o n g the p r e c i s i o n variances. No
differences i n ACa were f o u n d ( T a b l e 7) b e t w e e n Methods I I a n d I I I or b e t w e e n
Methods I V a n d V. Table 5 c l e a r l y shows t h a t the m e a n s for Methods I V a n d
V are so m u c h g r e a t e r t h a n those for Methods I, I I , a n d l I I , t h a t n o statistical
test is r e q u i r e d .
TABLE 7
Calcium determinations. Analysis of variance for each homogeneous group
Homogeneous
group Variation d.f. M.S.
Method I Levels "2 1.5427"*
Error 21 0.2368
Methods 1 0.8112
Methods II, I I I Levels 2 2.9803*
Methods X levels 2 0.0911
Error 42 0.8368
Methods I 0.0213
Methods IV, V Levels 3 174.1&97"*
Methods × levels 3 0.2200
Error 40 4.6993
*P<0.05; *~P<0.01.

A l l of the a n a l y s e s of v a r i a n c e show t h a t there were s i g n i f i c a n t differences

b e t w e e n the m e a n s for ACa at the v a r i o u s a d d i t i o n - l e v e l s of calcium. The
m e a n s d i d not, however, change p r o g r e s s i v e l y i n one d i r e c t i o n as the a d d e d
c a l c i u m i n c r e a s e d ( T a b l e 5).
F o r the m a g n e s i u m d e t e r m i n a t i o n , the h o m o g e n e i t y of v a r i a n c e was such
t h a t pooled a n a l y s e s were possible for Methods I t h r o u g h I I I , a n d for Methods
I V a n d V ( T a b l e 8). T h e s i g n i f i c a n t differences a m o n g the first t h r e e methods,
i n d i c a t i n g the s u p e r i o r i t y of M e t h o d I over Methods I I a n d I I I , have a l r e a d y

TABLE 8
Magnesium determinations. Analysis of variance for each homogeneous group
Homogeneous
group Variation d.f. M.S.
Methods 2 1.2541~*
Methods I, II, I I I Levels 1 6.3791 ~*
Methods × levels 2 0.3202
Error 66 0.2058
Methods 1 0.4447
Methods IV, V Levels 1 0.6.74.2
Methods × levels 1 0.5547
Error 44 2.6513
~ P < 0.01.
 CALCIUM AND MAGNESIUM IN MILK 1045

been noted (Table 6). Methods II and I I I do not differ from each other. No
differences, either between levels or methods, appeared for Methods IV and V
in the magnesium determination.
As a result of the suspicion that the experimental technique improved during
the course of the experiment, it was considered advisable, for thoroughness, to
submit the data of Run 3 to separate examination. The appropriate analyses
of variance are given in Tables 9 and 10 for ACa and AMg, respectively. The
findings generally confirm those presented above, except that the increased

TABL]~ 9
Calcium determinations. Analysis of variance for each homogeneous g r o u p ( R u n 3 only)
Homogeneous
group Variation d.f. ]~I.S.
Method 2 0.4543
Level 1 5.9254 ~
Methods I, II, III
Methods × level 2 0.346.8
Error 18 0.1286
Method 1 0.4761
Level 1 51.7536
Methods I V , V
Method × level 1 0.2.970
Error 12 0.9183
~ P ~ 0.01.

T A B L E 10
Magnesimn determinations. Analysis of variance f o r each homogeneous g r o u p ( R u n 3 only)
Homogeneous
group Variation d.f. M.S.
Method 2 0.9143 ~
Level 1 2.6676 ~
Methods I, II, III
Method × level 2 0.0228
]~rror 28 0.0439
Method 1 0.0033
Level 1 23.0410 ~
Methods I V , V
Method × level 1 0.0077
Error 12 0.24.31
~ P < 0.01.

sensitivity of the statistical analysis resulting from the reduction of the error
variance has now revealed significant differences for Methods IV and V in the
AMg at different levels of added magnesium.

SUMMARY AND CONCLUSIONS

Five methods for determining calcium and magnesium in milk using EDTA
titrations were compared. Method I was a combination of Ling's potassium
metastannate (PMS) precipitation of proteins and phosphates (3), the use of
Erio S E mixed indicator [Flashka et al. (1)] for the calcium titration, and
Jenness' method for magnesium (2), except that pH adjustment, prior to the
addition of buffer, involved the use of methyl red. Method II employed Ling's
1046 E . W . BIRD ET AL

method for calcium and Jenness' procedure for magnesium. Method I I I was
identical with II, except that Jenness' method of establishing the end point in
the calcium determination was employed. Method IV was the method for calcium
and magnesium as described by Jeancss (2). Method V was identical with IV
except Ling's method of establishing the end point in the calcium determination
was used.
From a convenience standpoint, the end point o~ the Erio S E mixed indi-
cator is preferred to that of Murexide and the PMS procedure for removal of
protein and phosphate (Ling) was found superior to the acid precipitation of
proteins followed by ion-exchange removal of phosphates (Jenness).
The precision (reproducibility) of Method I was found superior to that of
Methods II and I I I which, in turn, were significantly better than Methods IV
and V, for both calcium and magnesium.
The accuracy (recovery of added calcium or magnesium) of Method I
ranked above that of Methods II and III, which again had significantly greater
accuracy than Methods IV and V, for both calcium and magnesium.
Method I is considered to be an excellent, simple, and rapid method for
determining cMcium and magnesium in milk.

REFERENCES
(1) FLASHKA, H., ABD EL RAHE~M, A. A., AND SADZK, F. Erio S E als neuer Indikator zur
komplexometrischen Bestimmung yon Calcium (und Magnesium) im Serum. Z. physiol.
Chem., 310: 97. 1958.
(2) J~NNESS) R. Titration of Calcium and Magnesium in Milk with EDTA. Anal. Chem.,
25: 966. 1953.
(3) Lm~, E. R. The Determination of Calcium in Milk and Whey. Analyst, 83: 179. 1958.
(4) P~A~SON, E. S., AN]) HAUtbOY, tI. O. Biometrika Tables for Statisticians. VoL 1, Cam-
bridge University Press, London, England. 1956.
(5) SCHLITZ, B., TRF~ADW~LL AND ItALL. Analytical Chemistry. Vol. II. 6th ed., p. 78.
John Wiley and Sons, Inc., New York, N. Y. 1924.
(6) SN~ECOa, G. W. Statistical Methods. 5th ed. Iowa State College Press, Ames. 1956.
(7) ¥o~-D~, W. J. Statistical Methods for Chemists. John Wiley and Sons, Inc., New York,
N . Y . 1951.