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Патент USA US2847358

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nited States Patent
[email protected]
2,847,349
Patented Aug. 12, 1958
2
in the diluted solution in an amount of 60 to 90 percent
by volume, and preferably 70 percent by volume. Dilu
tion in this range results in a clot very similar in appear
2,847,349
ance to a clot formed in human blood.
PLASMA PREPARATION
Undiluted oxa
lated bovine plasma may also be used.
In the preparation of the prothrombin-free horse, bo
Heron 0. Singher, Plain?eld, and Emanuel A. Swart,
Somerville, N. J., assignors to Ortho Pharmaceutical
Corporation, a corporation of New Jersey
vine, human or rabbit plasma extract for addition to
oxalated bovine or equine plasma to provide the standard
plasma preparation of this invention, horse, bovine, hu—
No Drawing. Application May 27, 1954
10 man or rabbit blood to which a heparin sodium solu
Serial No. 432,896
tion has been added to prevent coagulation, is centrifuged
in order to obtain the plasma. The plasma .is slurried
with two to ?ve percent, and preferably ?ve percenuof
barium sulfate, the barium sulfate being expressed in
grams, and the plasma in cubic centimeters. At ‘least
4 Claims. (Cl. 167-74)
This invention relates to a plasma preparation for use
as a standard in the determination of the prothrombin
time of a thromboplastic material. More particularly,
this invention relates to a plasma preparation obtained
by mixing oxalated bovine or equine plasma, with a
two percent of barium sulfate is required to remove
all the prothrombin, and the presence of more than ?ve
percent results in a mixture of such a consistency that
physical handling thereof is extremely di?icult. The
plasma fraction obtained from prothrombin-free rabbit, 20 slurry is stirred for one hour in order that the maximum
bovine, human or equine plasma.
amount of prothrombin be completely adsorbed, and is
Thromboplastic material obtained from rabbit brain
then centrifuged. It is preferred that the adsorption be
repeated to insure complete removal of prothrombin,
or lung tissue, or mixtures thereof, or from other sources
varies in activity with each preparation, and each prepara
preferably with the same amount of barium sulfate, and
tion must be standardized in order that prothrombin time 25 upon centrifugation after the second adsorption, the
measurements of di?ferent human blood specimens may
plasma is pro'thrombin free. Albumin and alpha-globu
lins are extracted from the prothrombin~free plasma by
be correlated. Heretofore, preparations having thrombo
plastic activity have been standardized by the use of
the addition of a ?rst buffered aqueous alcohol solution
and centrifugation. The alcohol may be ethanol, meth
rash, normal, oxalated, human plasma; however, the
prothrombin time of human plasma specimens may vary 30 anol, or a mixture of methanol and ethanol and may be
signi?cantly.
.
present in an amount of from about 15 to about 30 per~
‘
It is an object of this invention to provide a plasma
cent by volume, but it is preferred that the amount be
preparation suitable for use as a standard in the deter
mination of the thromboplastic activity of thromboplastic
material.
‘
Other objects and advantages of the invention Will be
apparent from the following descriptions and exemplary
disclosures.
V
The objects of this invention are accomplished by
plasma preparation prepared by the addition to oxalated
bovine or equine plasma of a prothrombin-free plasma
35
about 25 percent. if the amount is less than about 15
percent, a signi?cant amount of inactive substances re
main in the precipitate, and if the amount is greater than
about 30 percent a substantial amount of the desired
plasma fraction is present in the solution. The ?rst aque
ous alcohol solution is buttered within the pH range of
from 4 to 5, the preferred pI-l being 4.2 to 4.6. Buffering below a pH of v4 results in incomplete solution of
albumin in the extracting solution and buffering above
extract obtained by the extraction with a buffered aqueous
a pH of 5 results in a signi?cant amount of the desired
alcohol solution of prothrombin-free rabbit, bovine, hu
plasma fraction being lost in the extracting solution. in
general, any buffer system capable of maintaining the pH
man or equine plasma from which albumin and alpha
globulinsthave been substantially removed by extraction
of the mixture of the ?rst aqueous alcohol solution and '
with a ?rst buffered aqueous alcohol solution.
prothrombin-free plasma within the range of 4 to 5 may
be used. The preferred buffer system is sodium acetate
acetic acid, however, buffer systems such as ‘sodium suc
More particularly, in preparing the standard plasma
preparation of this invention, the horse, bovine, human
or rabbit plasma fraction, and preferably a ‘fraction de
cinate-succinic acid, and sodium acid phthalate-phthalaic
rived from horse plasma, is dissolved in oxalated bovine 50 acid, have been found suitable.
plasma in an amount such that its concentration with
The volume of ?rst aqueous alcohol solution usedmay
respect to the bovine plasma is about two to ten milli
vary widely, but the most ef?cient removal of albumin
grams per milliliter, and preferably three milligrams per
and alpha-globulin from the prothrombin-free plasma is
milliliter. If the concentration of plasma fraction with
accomplished when the volume is three to ?ve times the
respect to bovine plasma is less than about two and 55 volume of the plasma. It is preferred that the volume
greater than about ten milligrams per milliliter, the pro
of the ?rst aqueous alcohol solution be about four times
thrombin time obtained with the standard plasma prepara—
the volume of the plasma.
tion is signi?cantly different from the time obtained with
It is necessary that the extraction and removal by cen-v
normal, oxalated, human plasma, when both are used in
trifugation of albumin and alpha-globulins in solution
the determination of the prothrombin time of the same 60 in the ?rst aqueous alcohol solution be accomplished at
* thromboplastic material.
One milliliter portions are
a low temperature in order that they be efficiently re
lyophilized to provide a stable, solid material which, upon
moved from the plasma. Extraction and removal at a
the addition of one milliliter of distilled water, provides
higher temperature results in denaturation of albumin
1 suspension having the same prothrombin time as the
and alpha-globulins by the alcohol and incomplete re
}-.verage, normal, fresh, oxalated human plasma when
moval thereof in the extracting solution. The pro
letermined by our modi?cation of the Shapiro-Weiner
thrombin-free horse, bovine, human or rabbit plasma is
nethod.
cooled/to 5 to 0° C., and preferably to 0° C.,,and the
The bovine plasma is prepared by centrifuging oxalated
?rst aqueous alcohol solution, which has been cooled to
bovine blood containing 10 percent by volume of 0.1
—5 to —‘1‘0° C., is slowly added with stirring and during
molar sodium oxalate solution. The supernatant from 70 the course of the addition, the temperature of the mixture
vthe centrifugation is diluted ‘with 0.85 ,percent sodium
is maintained between 0 and —5° C., and preferably at
about —5° C. After addition is completed the mixture
chloride solution such'that the bovine plasma is present
2,847,349
4
is stirred for about 30 minutes and during this time the
temperature of the mixture is maintained at 0 to ——5 ° C.,
and preferably at —~5° C. Immediately after stirring is
discontinued, the mixture is centrifuged and during the
'centrifugation the temperature is maintained at 0 to -5°
‘C., and preferably —5° C. The supernatant, which con
sists mainly of albumin and-alpha-globulins is discarded
which. had a volume of eight and one-half liters, was
cooled to 0° C. Thirty-four liters of an aqueous alco
hol solution buffered at a pH of 4.6 was cooled to a tem
perature of 0° C. and added to the plasma at such a
rate that the temperature of the mixture throughout the
addition was below 0° C.
The mixture was stirred for
30 minutes at —5° C. after addition was complete and
then centrifuged and the temperature of the mixture was
‘and the residue is extracted with a second buffered aque
maintained at a temperature of —5° during the cen
ous alcohol solution to obtain the desired plasma frac
trifugation. The supernatant, which contained substan
tion.
tially all the albumin and alpha-globulins of the plasma,
The alcohol in the second aqueous alcohol solution
was discarded. The aqueous alcohol solution containe
may be ethanol, methanol, or a mixture of ethanol and
per liter, 121/2 lml. of methanol, 2371/2 ml. of 95 per
methanol and the alcohol, or mixture of alcohols, is pres
cent ethanol, and 0.6 ml. of an acetate buffer solution
ent in an amount from 5 to 20 percent by volume and
preferably in an amount of about 16 percent by volume. 15 prepared by diluting a mixture of 20 ml. of 4 molar so—
dium acetate and 40 ml. of 10 molar acetic acid to 100
The second aqueous alcohol solution is buttered at a pH
ml. with water. The residue from the centrifugation
within the range of from 6 to 8, and preferably 6.8 to
was ?nely dispersed with a spatula in 34 liters of the
7.2. A solution buffered outside this pH range extracts
signi?cantly less of the desired plasma fraction from the
residue of the ?rst extraction. The second aqueous al
cohol solution contains alkali metal salt of an amino
acid having not more than six carbon atoms, such as
aqueous alcohol extracting solution buffered at a pH
of 7.
The temperature of the extracting solution and
residue during the dispersion was maintained at —5°
C. and after dispersion was complete the mixture was
stirred for 30 minutes at a temperature of —5° C. and
alanine, glycine, proline, or serine. In general, other
centrifuged. The temperature of the mixture during the
amino acids are not su?iciently soluble in the second
extracting solution. The alkali metal salt acts as a sta 25 centrifugation was maintained at ——5° C. The super
natant was ?ltered and dialyzed against distilled water.
bilizing agent for the material to be extracted from the
The temperature of the supernatant during ?ltration and
plasma, as a solubilizer for beta- and gamma-globulins,
dialysis was maintained at 5° C. The dialysate was lyo~
and as an active part of the buffer system. The amino
philized and 350 grams of solid were obtained. The
acid is present in an amount within the range of 4 to 6
percent by weight, and preferably 5.4 to 5.8 percent by 30 solid had no demonstrable thromboplastic activity. They
aqueous alcohol extracting solution contained, per liter,
weight. At a concentration less than 4 percent by weight,
the solubilizing effect of the amino acid is signi?cantly
8 ml. of methanol, 152 ml. of 95 percent ethanol, 56
grams of glycine, 3.12 ml. of a solution of sodium gly
decreased; on the other hand, the solubilizing effect is not
cinate, prepared by dissolving 4.5 grams of glycine and
increased when the concentration is greater than 6 per
cent. Phosphates such as mono- or di-sodium phosphate 35 2.0 grams of sodium hydroxide in 100 ml. of water, 4
ml. of 0.5 molar disodium hydrogen phosphate, and 2.76
are also present in the second aqueous alcohol solution
ml. of 0.5 molar monosodium dihydrogen phosphate.
as part of the buffer system and the pH of the solution
Nine hundred milligrams of the fraction obtained from
is adjusted to the desired value with an alkali metal hy
horse blood were added to 210 ml. of oxalated bovine
droxide. Any buffer system may be used which is ef—
plasma which had been diluted with 90 m1. of 0.85 per
fective within a pH range of 6 to 8.
cent sodium chloride solution, and the mixture was lyo
The amount of the second aqueous alcohol solution
philized. An amount of the lyophilized material corre
used in the extraction of the albumin and alpha-globulin
sponding to 1/300 of the total amount was dissolved in 1.
free residue may vary widely but an amount of solution
ml. of distilled water to provide a reconstituted standard
three to ?ve times, and preferably four times, the volume
of the original plasma results in the most e?icient re~
plasma.
covery of the desired plasma fraction. The mixture of
residue and second aqueous alcohol solution is stirred at
Example II
a temperature of 0 to —5‘’ C., and preferably at -5°
Inv the preparation of the prothrombin-free plasma ex~
C., for about 30 minutes, and then centrifuged and the
tract from rabbit blood, three ml. of heparin sodium solu
residue from the centrifugation which consists mainly of
tion were added to 1200 ml. of rabbit blood and the mix
beta-globulins and ?brinogen is discarded. The super
ture was centrifuged for 30 minutes at 25 ° C. Six hun
natant, which contains the desired plasma fraction, is
dred twenty ml. of the supernatant plasma were slurried
?ltered, dialyzed against distilled water at a temperature
with 31 grams of barium sulfate at 25° C., stirred for one
of 1 to 5° C., and the dialysate is lyophilized. (The
term “dialysate” as used in this speci?cation designates
hour and centrifuged. The supernatant was slurried with
the material which has failed to pass through the dialysis
31 grams of barium sulfate, stirred for one hour at 25° C.,
membrane.)
Denaturation of the desired plasma frac
and centrifuged. The prothrombin-free plasma obtained,
tion is at a minimum when the temperature during dialy
which had a volume of 565 ml. was cooled to 0° C. Two
sis is within the range of 1 to 5° C. The solid material
thousand two hundred sixty ml. of an aqueous alcohol
obtained upon addition to thromboplastic material en 60 solution buffered at a pH of 4.6 was cooled to a tempera
hances its thromboplastic activity.
ture of 0° C. and added to the plasma at such a rate that
In order that those skilled in the art may better un
derstand how the present invention may be carried into
the temperature of the mixture throughout the addition
effect, the following examples are given by way of illus
tration and not by way of limitation.
was below 0° C. The mixture was stirred for 30 minutes
at —-5° C. after addition was complete and then cen~
trifuged and the temperature of the mixture was main
tained at a temperature of —-5° during the centrifugation
Example I
The supernatant, which contained substantially all th
albumin and alpha-globulins of the plasma, was discarde
In the preparation of the prothrombin-free plasma ex
tract from horse blood, ?fty ml. of heparin sodium solu
grams of barium sulfate at 25° C., stirred for one hour
The aqueous alcohol solution contained per liter, 121/2 m
of methanol, 2371/2 ml. of 95 percent ethanol, and 0.6 m
of an acetate buffer solution prepared by diluting a mi
ture of 20 ml. of 4 molar sodium acetate and 40 ml. 0
10 molar acetic acid to 100 ml. with water. The residu
from the centrifugation was ?nely dispersed with a spatul Y
and centrifuged. The prothrombin-free plasma obtained,
in 2260 ml. of the aqueous alcohol extracting solution buf
tion were added to 20 liters of horse blood and the mix
ture was centrifuged for 30 minutes at 25° C. Nine
liters of the supernatant plasma were slurried with 450
2,847,349
5
6
fered at a pH of 7. The temperature of the extracting
amples I and II and in seventeen seconds after addition of
solution and residue during the dispersion was maintained
at -5° C. and after dispersion was complete the mixture
the supernatant liquid to human plasma.
was stirred for 30 minutes at a temperature of —5° C.
ured by prothrombin time, were determined by our
modi?cation of the Shapiro-Weiner method for deter
mining prothrombin time of blood, which is described
in a book entitled: “Coagulation, Thrombosis and Dion
All determinations of thromboplastic activity, as meas
and centrifuged. The temperature of the mixture during
the centrifugation was maintained at —5 ° C. The super
natant was ?ltered and dialyzed against distilled water.
The temperature of the supernatant during ?ltration and
marol,” by Shapiro and Weiner, published in 1949 by
dialysis was maintained at 5° C. The dialysate was lyo
the Brooklyn Medical Press, Brooklyn, New York.
philized and eight grams of solid were obtained. The 10
It will be obvious to those skilled in the art that various
solid had no demonstrable thromboplastic activity. The
changes may be made without departing from the spirit
aqueous alcohol extracting solution contained, per liter,
8 ml. of methanol, 152 ml. of 95 percent ethanol, 56
of the invention and therefore it is to be understood that
the invention is not limited to what is described in the
speci?cation and examples but only as indicated in the
grams of glycine, 3.12 ml. of a solution of sodium glyci
nate, prepared by dissolving 4.5 grams of glycine and 2.0 15 appended claims.
grams of sodium hydroxide in 100 ml. of water, 4 ml. of
0.5 molar disodium hydrogen phosphate, and 2.76 ml. of
What is claimed is:
l. A process for the preparation of a plasma fraction
for use in the determination of the prothrombin time of
0.5 molar monosodium dihydrogen phosphate.
One thousand milligrams of the plasma fraction ob
thromboplastic material comprising the steps of: dissolv
tained from rabbit blood were dissolved in 100 milliliters 20 ing in oxalated bovine plasma two to ten milligrams per
milliliter of oxalated plasma of a plasma fraction prepared
of undiluted, oxalated, bovine plasma. One milliliter por
from prothrombin-free plasma, selected from the class
tions of the mixture were placed in glass vials and lyo
philized. The lyophilized material, in a vial, was recon
consisting of horse, bovine, human, and rabbit prothrom
bin-free plasma, by a process conducted at a temperature
stituted before use as a standard by the addition thereto
25 within the range of —5° to 0° C. throughout comprising
of one milliliter of distilled water.
Plasma preparations prepared according to Examples
adding thereto three to ?ve volumes of a ?rst aqueous
alcohol solution buffered at a pH of 4—5 and containing
I and II may be used as a standard for the determination
about 15-30 percent by volume of an alcohol selected
of the prothrombin time of thromboplastic materials in
from the class consisting of methanol and ethanol and
place of fresh, normal, oxalated, human plasma and the '
prothrombin‘time of a given thromboplastic ‘material is
substantially the same with the standard preparation as
mixtures thereof, stirring and centrifuging the mixture,
separating the supernatant from the residue; and adding
with fresh, normal, oxalated human plasma.
to the residue three to ?ve volumes of a second aqueous
A thromboplastic material for use in thromboplastic ac
tivity determinations using the standard plasma prepara
tion and fresh, normal, oxalated, human plasma was pre
pared as follows:
Seventy-six grams of frozen rabbit brain and 1440
alcohol solution buffered at a pH of 6—8 and containing
5-20 percent by volume of an alcohol selected from the
35 class consisting of methanol and ethanol and mixtures
thereof and 4-6 percent by weight of an alkali meta-l salt
of an amino acid having not more than six carbon atoms,
stirring and centrifuging the mixture, ?ltering the su
pernatant, dialyzing the ?ltered supernatant against dis~
4:0 tilled water, and lyophilizing the dialyzed supernatant.
solution containing, per liter, 150 ml. of an alcoholic
2. A process for the preparation of a plasma prepara
solution prepared by adding 7.5 ml. of methanol, 15
_ grams of frozen rabbit lung were homogenized at 5° C.
for one minute in the presence of 7600 m1. of an aqueous
grams of glycine, 4.8 ml. of one molar aqueous sodium
acetate solution, and 2.6 ml. of one molar aqueous acetic
acid solution to 142.5 ml. of 95 percent ethanol. The
homogenate was stirred for two hours at 5° C. and cen
trifuged at —5° C. for thirty minutes. The supernatant
liquid was ?ltered, dialyzed against distilled water at 5° C.
tion for use in the determination of the prothrombin
time of thromboplastic material comprising the steps of :
dissolving in oxalated bovine plasma diluted with an
amount of 0.85 percent aqueous sodium chloride solu
tion such that 60-90 percent by volume of the plasma is
present ‘in the diluted solution, two to ten milligrams per
milliliter of diluted ‘bovine plasma of a plasma fraction
prepared from prothrombin-free plasma at a tempera
grams of thromboplastic solid material were obtained.
50
ture of 5° to 0° C., selected from the class consisting of
The thromboplastic activity, as measured by the pro
and lyophilized.
Forty-four and forty-six hundredths
thrombin time, of the thromboplastic material prepared
horse, bovine, human, and rabbit prothrombin-free plas
ma, by adding thereto at a temperature of —5° to —10°
C., about four volumes of a ?rst aqueous alcohol solu
A calcium-thromboplastin suspension was prepared in
a test tube by adding 25 milligrams of lyophilized solid 55 tion bu?ered at a pH of 4.2-4.6 and containing about
25 percent by volume of 2an alcohol selected from the
to 5 ml. of 0.85 percent aqueous sodium chloride solu
class consisting of methanol and ethanol and mixtures
tion, admixing by inverting the tube three or four times
thereof,
stirring and centrifuging the mixture, and sepa
until a uniform suspension was obtained, keeping the
rating the supernatant from the residue while the tem
suspension in a water bath at 46—5 0° C. for twenty min
utes, centrifuging, cooling the supernatant to room tem 60 perature is maintained at 0° to 5° C. and adding at a
as above was determined as follows:
perature, adding 0.1 ml, of 0.25 molar calcium chloride
solution to 4 inl. of the suspension, mixing as above, and
centrifuging again. Two-tenths ml. of the slightly turbid
supernatant liquid was added to 0.1 ml. each of the re
constituted standard plasmas prepared according to Ex
' amples I and H and to 0.1 ml. of fresh, oxalated human
plasma which had been prepared by the addition of 0.1
molar aqueous sodium oxalate solution to fresh human
‘*blood in the proportion of one part sodium oxalate solu
tion to nine parts of blood and centrifugation of the oxa
lated blood. The mixtures were agitated at 37° C. by
j‘tilting the test tube back and forth and timing the ?rst
lj'appearance of a ?brin clot. Clot formation was detected
in 16.8 and 16.5 seconds, respectively, after addition of
temperature of 0° to —5° C. to the residue at a tempera
ture of 0° ‘to —5° C., three to ?ve volumes of a second
aqueous alcohol solution buffered at a pH of 6.8 to 7.2
and containing about 16 percent by volume of an alcohol
selected from the class consisting of methanol and ethanol
65 and mixtures thereof and 5.4-5.8 percent ‘by weight of
an alkali metal salt of an amino acid having not more
than six carbon atoms, stirring the mixture while the
temperature is at 0° to —5‘’ C., centrifuging the mixture,
?ltering the supernatant, and dialyzing the filtered super
natant against distilled water while the temperature is at
1° to 5° C., and lyophilizing the dialyzed supernatant.
3. A plasma fraction for use in the determination of
the prothrombin time {of thromboplastic material pre
pared by the steps of: dissolving in oxalated bovine
the supernatant liquid to the standard plasmas of Ex 75 plasma two to ten milligrams per milliliter of oxalated
2,847,349
7
8
?rst aqueous alcohol solution buffered at a pH of 4.2-4.6
and containing about 25 percent by volume of an al
cohol selected from the class consisting of methanol
plasma of a plasma fraction prepared from prothrombin
free plasma, selected from the class consisting of horse,
bovine, human, and rabbit prothrombin-free plasma, by
and ethanol and mixtures thereof, stirring and centri~
fuging the mixture, and separating the supernatant from
a process conducted at a temperature Within the range
of —5° to 0° C. throughout comprising adding thereto
"the residue while the temperature is maintained at 0° to
5° C. and adding at a temperature of 0° to -—5° C. to
three to ?ve volumes of a ?rst aqueous alcohol solution
bu?ered at a pH of 4-5 and containing about 15-30 per
cent -by volume of an alcohol selected from the class con
the residue at a temperature of 0° to —5° C., three to
?ve volumes of a second aqueous alcohol solution but
stirring and centrifuging the mixture, separating the 10 fered at a pH of 6.8 to 7.2 and containing about 16 per
cent by volume of an alcohol selected from the class con
supernatant from the residue; and adding to the residue
sisting of methanol and ethanol and mixtures thereof
three to ?ve volumes of a second aqueous alcohol solu
sisting of methanol and ethanol and mixtures thereof,
and 5.4-5.8 percent by weight of an alkali metal salt of
tion buttered at a pH of 6-8 and containing 5-20 percent
an amino acid having not more than six carbon atoms,
by volume of an alcohol selected from the class consist
ing of methanol and ethanol and mixtures thereof and 15 stirring the mixture while the temperature is at 0° to
——5° C., centrifuging the mixture, ?ltering the super
4-6 percent by Weight of an alkali metal salt of an amino
natant, and dialyzing the ?ltered supernatant against dis
acid having not more than six carbon atoms, stirring and
tilled water While the temperature is at 1° to 5° C., and
centrifuging the mixture, ?ltering the supernatant, dialyz
lyophilizing the dialyzed supernatant.
ing the ?ltered supernatant against distilled water, and
lyophilizing the dialyzed supernatant.
References Cited in the ?le of this patent
Wintrobe: Clin. Hematology, 2nd ed., 1946, Lea and
4. A plasma fraction for use in the determination of
the prothrombin time of thromboplastic material pre
pared by the steps of: dissolving in oxalated bovine plas
Febiger, Philadelphia, Pa., pp. 207-209.
sodium chloride solution such that 60-90 percent by vol
25 pp. 3448-3450.
Cohn: Ann. Int. Med, vol 26, No. 3, March 1947, pp.
341-352 (pp. 346, 347, 349-352 relied on). ,
Eds?l: Advances in Protein Chem., vol. 3, 1947, Aca
ume of the plasma is present in the diluted solution, two
to ten milligrams per milliliter of diluted bovine plasma
‘of a plasma fraction prepared from prothrombin-free
plasma at a temperature of 5° to 0° C., selected from
> demic Press Inc., N. Y. C., pp. 440-445.
the class consisting of horse, bovine, human, and rabbit 30
prothrombin~free plasma, by adding thereto at a tem
perature of —5° to —10° C., about four volumes of a
'
Surgenor: J. A. C. S., vol. 74, No. 13, July 5, 1952,
ma diluted with an amount of 0.85 percent aqueous
Callaham: Chem. and Metallurg, Eng, June 1946, pp.
101-103.
Hardy: Chem. Abst., vol. 45, March 1951, p. 2046a.
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