SOUTHWESTERN RADIOLOGICAL HEALTH LABORATORY
XM:RaLABORM?ORY TECHNICAL REPORT NUMBER 2
August 9, .1963
DETERMINATION OF STROKTI13M-89 AMD STROEPTSUM-90 IB .WHOLE MILK
PRECIPITATION AMD SEPARATION OF MILK
PROTEIN BY.TRICHLOROACETIC ACID
David L« Stevenson
SUMMARY
The precipitation and separation of protein from milk
by trichloroacetic acid: (TClU was examined to obtain
optimum .conditions for strontium recovery-- The variables
under consideration wares TCA concentration? TCA addition
rate, extent of mixing required* solution temperatures,
and the1 effect'of freesing the milk.sample.
" 1 •
It was - found that the present method meets raost
of "the requirements for maximum strontium yield if the
procedure is followed st.rict.ly. The one improvement which
might be. made is in pre-hea ting'the mi lie to speed up the
filtration step*
Additional information was obtained in the form of a
pE curve xtfhieh indicated that approximately 50 ail of the
TCA was lost after filtration.,
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PURPOSE
The purpose of this work is to minimise the strontium
loss across the TCA precipitation step without overcomplicating
the procedure..
1
The first step in a routine analysis of mil]?, for
radio-strontium is the precipitation (denaturation) of the
milk protein by trichioroacetic acid (TC&K The protein
solids may then be , filtered off leaving the desired strontium
in the filtrate liquor . . "
The effects of the following conditions were studied
with respect to the strontium recovery and ease of separation-.
-1 -TC& concentration . »
XI TCA addition rate
. Ill Temperature of TCA' and milk solutions
- ...IV Extant of missing required
V Previous freezing of milk
• . Because of its greater abundance' in milk and its
chemical similarity to strontium* the calcium recovery was
determined .instead of the recovery "of strontium.
Calc'iutn was determined by titrating aliquot s
2
of the sample %rith EDTh follcxired by back titration with
-standard calcium solution.
¥1 ,,As a inesns of determining the amount of TCA lost
V
in the precipitation step, 'a pH curve was made showing'
the pH of different TCA concentrations. The approximate
TCA concentration of a sample was determined by checking its
pH against the curve.
Bxichner funnel, 3 liter suction
flask o
pH meter j, Beekman zerohiatic.
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SS.ISMfiSSaS.tiiS^SS.M (^CA)j 50% W/¥, 500 -g TCA made
up to 1 liter with distilled HUG.
7
ten. .1/2 gallon containers of Anderson Dairy
milk were raised and preserved with 50 isl of 37%
formaldehyde .
I. To five 1000 ml aliquot s of milk .'were added 100 «
200, 300, 400., and 500 ml of 5056 TCA. After -thorough stirring
with a glass rod the samples were filtered through a Whatman
number 2 filter paper in a Buchner funnel*. The precipitates
were -washed with approximately 200 ml distilled water and
the total filtration and washing time for each sample was
noted*'- The filtrates obtained were made up to 2000 red with
distilled water* Aliquot s of this solution were used for
i
the calcium 'determination...
Ha To three 1.000 ml aliquot®' of milk were added 300 ral
portions of TC&« The TC& was added rapidly to the first sample
with vigorous hand stirring. 'The TC&- was added slowly to the
second saiaple with vigorous ^tir:eing.-> allowing approximately
5 minutes for the addition* A-separatory funnel was \ssed
to add the TCA dropwise to the third sample* allowing about
15 mimrtes for this addition* A mechanical ^tirrer was used
to mix the .-third sample during TCA addition. v
The samples were then, filtered, washed* and made up to
volume as in 1 «
111. Three 1000 ml aliquots of milk were heated to
20°C, 40DC, and 80°C respecti^-ely. To these samples were
added; 300 ml portions of .TCA. that had been heated to
raatching temperatures..' ....
The samples were then stirred by hand* filtered,,
washed"? and made up to volume.
-------
After addition of 300 ml TCAf three samples were
treated as follows?
1* The first sample was stirred lightly by
hand and filtered.
2o The second was stirred vigorously for
about 5 minutes before filtering..
3. Sample number 3 was stirred mechanically
foic about 15 minutes followed by filtering.
These samples were washed and brought up to volume
as before*
V. A 1000 Esl milk sample was frozenf thawed out» and.
then precipitated with 300 nil TCA. The solution was then
filteredf washed/ and made up to 2000 ml*
¥2c pH curve for EGA.
A standard filtrate solution was prepared by precipitating
a 1000 ml roilk sample with 150 ml of 6 N HC1. The solution
then filtered and washed with approximately 50 ml 3 H
. After neutralising the filtrate with NaOB, it was
made up to 2000 ml vol«me«
The pH curve xi?as made by taking a 200 ml aliquot of
the neutral filtrate? adding 20 ral of 5 M KOHf and titrating
with 50% TCA sol«tion« A pH raster v;as used and the pH
plotted against the corresponding volume of TCA used*
The TCA concentration of a 200 ml sample aliquot could
then foe determined by adding 20 ml of 5 IS KOH/- reading the
pH on the meterf and finding the corresponding TC& concentration
on the graph»
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TC& CQNCSHTR&TJOW
filtration
Time, Min.
100
so.
60 •
40 '
20-
0 •
_~£i~- -~"€)
03. ^^^^^^^r^^
V^<*^/^r*"^
jS^X
-100
- 95
V \
S- —
I 4 I £ 5
" 85
- 80
- 75
12345
% Ca
Recovery
Increasing TC& Concentration ~->
Q Filtration Time in Minutes
A Calcium Recovery from Filtrate Digested in HMO.,
j
O Calcium Recovery from Untreated Filtrate Samples
PREC2PITAT2OH
FILTRATION
r~
ml TCA
Sample Used
X-I 100
X~2 200
1-3 300
1-4 400
1-5 500
Filtrate
Appearance
Cloudy
Slightly
Cloudy
Clear
Clear
Clear
Addition of Excess
TCA to Filtrate
More Precipitate
Formed
Mo
Precipitate
Ho
Precipitate
Mo
Precipitate
Ho
Precipitate
Filtration
and Washing
Time s Min .
90
75
45
40
30
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BDTA~Ca TITRATION - UNTREATED FILTRATE
Sample
! 10 ml
| Blank
20 ml
Blank
1-1
1-2
•1-3
1-4
| -1-5
ral
Used
1.41
i
3.20
3. 12 .
3.10
2.96
2.36
. 2.88
ml Ca
So lu t ion Us ed
0.40
2.18
2.68
2,12
-1.19
: o'.eo
0.80-
0
Ca Percent
g/1 Recovery
1.15)
Vl.14
1.13' •
1.04
1.09.
1,12
- 1=13 •
1.14
100
,
\
91
96
98 ]
99 • !
100 I
BDTA-Ca T1TEKP1OH - IN- HBO-
j Sample
! 10 mi
i Blank
!
1 10 ml
Blank
•1-1
t
; 1-2
1-3 '
1-4
1-5
ml
Used
1.40
1.40
2.61
2«75
3»02
3 = 35
2 . 90 .
ml Ca
Solution Used
0.40
0.48
Oo81
. 1 . 34
1.72
3.02
1.04
'Ca
g/i .
1.15
-
1.14
1.02 '
Io03
1.10
1.10
1.12
Percent
Recovery
101
100
90
91 '
97
- 3?
98
Calculations
10 ral samples (ml X 0,848} - (ml Ca Solution X 0=1)
~ Ca g/I
20 ml samples (ml .X 0.424) - (ml Ca Solution'X 0.1)
= Ca g/i
-------
-S-
TC& RS.T!
Filtratibsn
Time
-------
EDTA-Ca
[ Sample
j 'Blank
j Blank .
1 II-I
21-1
1 1-2
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III. TCA - MILK TEMPERATURE
Filtration
Time, Min.
100-
30-
60"
40-
20'
O.j
e_~-~— ~t ~— e
-v
X
•N
N
X
V
-IU-P | • / | , ^
123
- 100
" 95
' 90
• 85
80
L 75
Sample
% Ca
Recovery
Increasing Temper a tu.re —>
D Filtration Time, Minutes
O Caicitun Recovery, SDTA Titration
A Calcium Recovery, Flame Analysis
PRECIPITATION AND FILTRATION
i fSample
i
. ml TCA Used
Temp. Milk TCA
Precipitate
Appearance
Precipitate
Appearance
Filtration and
Washing Time
322-1
300
20°C
Medium
Grain
Clear
45 Min.
IIS-2
300
40 °C
Very porous
Granular
Clear
IS Min.
. III-3
300
80°C
Very porous
Granular
Clear
12 Min.
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-9-
&DTA-Ca TlT
iGM AMD, FLAME! ANALYSIS
Santple
Blank
ii2-i
1XX--2
122-3
Blank
ml EDTA
Used
1.46
1.44
1*49
1,42
Flame
; t-. ml Ca
Solution Used
0.45
0.64
0.80
' 0.38
Ca
g/i
1.16
1.13
1.15
1,14
1.20
Percent
Recovery
100
. 97
99
93
100
IIX-1
12S-2
£11-3
Photometer
Flame ;
Photometer
Analysis
Flame
Photometesr.
Analysis
Flame
Photometer
Analysis
1.15
1.15
1.15
96
96
Calculafcioti
(si SDTA X 0«828) - (ral Ca Solution X 6.1) = Ca g/i
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-10-
Filtration
Min*
IV. MIXING OF MILK - TC& SOMJTIQM
100 . -
80-
i
60 "
40 -
20 -
0 -
„«_-—• — '~'®'
. Jar^ ,m
'
a*
*'
B.....-B-'
-
am^faKMUa~aa^n'»H. M • -- «. i». -rWKeoaoaugeK^t^a^*^ •'• •• «" «««^-^«»rt«»-^*«^^**---^'^^
3V-I SV-2 IV-3
'100
" 95
" 90
- 85
" 80
r 75
Increased Mixing ~*
d Filtration Time? Minute
O Calcium Recovery
Kecoverv
Saftiple
SKEGIPITATIOK AH
Sample
f sal TCA-Used
Stirring-
Precipitate
Appearance
«-. ™ v •«
300
Slight
Stirring
Glass Rod
Gelatinous
Fine Grain
IV-2 {
300
Vigorous
Stirring •
Glass Rod
Medium
Grain
• IV-3
300 v
Mechanical
Stirring
15 Min.
sfc
Appeesrance
Filtration and
Washing 'Time
Cloudy
45 Min
Clear
45 Min
*
•;clasufficient milk to run this sample- Sample 12-3 is
shown on graph due to similarity of sample treatment.
-------
-11-
EDTA-Ca TITR&TIOW AMD Fl-AME ANALYSIS
Sample
Blank
IV-1
1V--2
Blank
IV-1
I XV-2
ml EDTA
' Used
1.46
1=40
1.44
Flame
Analysis
Flame
'Analysis
Flame
Analysis
ml Ca
Solution Used
0.45
0.70
.0.64
Ca
9/1
1.16
1.09
"1.13
;U20
1.10
1.15
Percent
Recovery
100
94
98
100
92
* 96
|
Calculation
(ml EDTA 3C 0*828) - (ml Ca Solution X 0*1) = Ca g/1
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-12-
FROZEN MILK
PRECIPITATION. MTO FJLTHATIOJJ
1 Sample
!•« • ' .- •
i V-l'
ml TCa
used
300
Sample
Appearance
Partially
Denatured
Precipitate
Appearance
Dry,
. Porous
Filtj-ate
Appearance
Clear .
Filtration & \
Washing Time j
15 Min.
EDTA-Ca TITRATIOH AND FLAME AJIALYSIS
I Sample:
Blank
V-l
V-l
Blank
ml EDTA
Used
1.46
1.44
1.46
Flame
Analysis
Flame
Analysis
sal Ca -
Solution Used
"0.45
0^58
0.91
Ca
g/i
1,16 -
.1 . 13
1.12
•1.20
1 . 14
Percent |
Recovery
• 100 |
1
98
97
100
95 .
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-13-
V3U pH '- TCA COSCEM'RATXOSJ
pH AND TGA CONCENTRATION OS5 STANDARD SOLUTIONS
. TCA Cone. pH TCA, Cone*
; ml/1 Milk _ ml/1. Mi Ik
'.. ; 0 13 a4
100 • ' 13.1
150 12,9
200 12*5
240 lliS
260 ' 11.2
270
280
j .
290
300
310
320
TCA . COHCEMTRATfcON
ml ' Milk
Sample .: Used
.1 1000
2 1000
3 1000
4 1000
5 1000
**f.wa* ««.m:;^g*tret'*«a^^«ivtci*ra«.^.Mtt*^^
ml TCA
Added
100
200
300
400
500
pH . • TCA -Cone. pH
: . . , ' ml/1 Milk
i ' • , . ; ' : ' i , • 1 • =
10.3
7-3
5.7
3«9
2.4
1.9
530 1.7
350 1.4
400 1.0
450 0.8
500 6.7
IH MIIIK FILTRII^E
' pH of
Filtrate
13.3
12,9
11.3
3. »2
0«8
ml 3?CA ml TCA
; Found O'sed '
50 .50
150 50
258 * 42
375 25
450 50
-------
pH - TC&
PH
100
200
300
400
500
ml 50% TGA/IOOO ml 'Mi BE
-------
-15-
Considering the cost of reagents as. well as the caleiura
recovery and filtering efficiency/ the optimum trichloro-
aeetic acid |TC&) concentration appears to be about 300 mi
of 50% TCA per liter of milk, increasing the TCA concentration!
by 605& (200 ml) over this amount only increases the calcium
recovery by 2% and decreases filtering time by 15 minutes.
Decreasing the TCA boneentration by 60% lowers the calcium
!
yield by as much as 8% and increases the filtering time by
45 minutes. ' .
Slow addition of TCA solution to the milk increases the
ce.lci.mn recovery slightly {2%} but increases pouring and
filtering time to 60 &inutes.
;; Heating the mill? and TC& adds littlet {2%\ to the calcium
recovery but significantly reduces the time required for
filtration. ' A simple method of pre-heating the samples might
be worthy of further investigation.
The calcium recovery is somewhat dependent on the TCA-milk
solution being missed completely. Vigorous stirring with a
glass rod is necessary for a good recovery.
Previously frozen milk filters much faster than a
regular milk sample. This is due to the protein being
partially denatured by the freezing process. The calcium
recovery doesn't appear to be significantly altered by freezing.
The loss of approximately 50 ml of TGA across the*.
precipitation-filtration step is probably due to occlusion
in the precipitate but the possibilty exists of a reaction
occur ing which -would account for the loss*
1
The current method resets nearly ail of the requirements
for a maximum calcium—strontium yield if the procedure is
followed strictly; The one improvement which might be made
is in pre-heating the milk to speed up the filtration step.
-------
CD ^"Determination of Strontiuia-89 and StroatIusa-90
In Whole Milk*, Nitric Acid Procedure » M - (August 14 * 1962}
p. 2? SWRBL* .Las Vegas* Nevada..
{2} % Rapid Determination of Calcium In Milk Using .
Ethyienediaminetetraaeetate {S)I«.Sodium) as a Tifcrant."
F. B. Johns « M« Knox* D, Modenff and E. Halker. Division
of Radiological Health, U. S* Public Health. Service
(October 1962 }tt .
and E. Cooley* Advice on thejory and methods
E« Raws OB. and 'Jo • Dillon j Spectrophotometric Analyses
P* Adkins'' and 'F« 'Jakubowski t Technical assistance.
'• . . V
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