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MICRODETERMINATION OF CHLORIDE IN BIOLOGICAL FLUIDS, WITH SOLID SILVER IODATE
II. TITRIMETRIC ANALYSIS
BY JULIUS SENDROY, JR.
(From the Hospital of The Rockefeller Institute for Medical Research, New York)
(Received for publication, May 8, 1937)
In Paper I of this series (Sendroy, 1937, a) a system of chlo- ride analysis based on the reaction
(1) NaCl + AgIOa * AgCl + NaIOa
soluble insoluble . insoluble soluble
was outlined in detail, with gasometric measurement of the iodate resulting from the reaction. In this paper titrimetric measure- ment of the iodate is described.
Titrimetric measurement of the iodate is directly applicable to the supernatant fluid in the AgI08 precipitation of chloride from salt solutions, urine, serum, and protein-free jiltrate of serum and of whole blood.’ For the titrimetric analysis of blood only protein- free filtrates may be used.
The measurement is carried out according to the classical reac- tion for iodate in acid solution in the presence of an excess of potassium iodide. The equation applicable to the present meas- urements is
(2) KIOs + 5KI + 6HsPO4 = 312 + 3HaO + GKHsPO,
The iodine evolved from the reaction of the iodate with acidified KI in excess is titrated against sodium thiosulfate with starch as indicator. Since 6 equivalents of iodine result from 1 of chloride, this reaction is 6 times as sensitive as the one on which the iodo-
1 The term “serum” is used here, as in the previous paper, for both plasma and serum.
405
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406 Titrimetric Chloride Determination
metric titration procedure of McLean and Van Slyke (1915), or that of Conway (1935), is based.2
What has been said of the advantages of the method in its gasometric form applies equally well to the titrimetric form, except that all titrimetric measurements must be compared against a known, standard iodate solution. In accuracy, convenience, and rapidity, the method in its titrimetric form is little different from the gasometric. Here also, samples of serum as small as 0.02 cc. may be used, with no loss of accuracy. By the reaction of Equation 2 about 3.2 cc. of 0.003 N NazSpOa are required for titration of the iodine in the supernatant fluid available from such a sample of serum. The sensitivity of the starch-iodine end- point makes titrations easily possible to within 0.01 cc.
DESCRIPTIVE
Reagents- Approximately 0.85 M, 0.34 M, 0.17 M, and 0.085 M Phosphoric
Acid Solutions. ~/15 Potassium Acid Phosphate Solution. Caprylic Alcohol. Silver Ioclate, Powder, C.P. Tungstic Acid Reagent (Folin-Wu, 1919). Zinc Hydroxide Reagent (Somogyi, 1930). The preparation of the above reagents is described in the cor-
responding section of Paper I (Sendroy, 1937, a). In addition, there are the following:
6 Per Cent Potassium Iodide Solution-This is freshly prepared every day. 5 gm. of KI are dissolved in 100 cc. of water.
2 Per Cent Starch Solution-Of Lintner’s soluble starch, 2 gm. are ground into suspension with a little water, then boiled in 100 cc. of saturated NaCl solution for 5 or 10 minutes. This reagent keeps indefinitely.
0.5 Per Cent Starch Solution-The 2 per cent solution is diluted 4 times with saturated NaCl solution.
Approximately 0.12 M (= 0.12 N) Sodium Thiosulfate Solution- About 30 gm. of NazBOe.5Hz0 are diluted to 1 liter with water.
* As far as the writer is aware, there has been no previous application of this reaction to the analysis of chlorides. See the foot-note to the title page of Paper I (Sendroy, 1937, a).
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J. Sendroy, Jr. 407
This stock solution and the weaker solutions made from it by simple dilution are kept in the dark, in amber bottles. If aged properly, they will become very stable. Without any precautions against COZ we have kept even 0.001 N solution unchanged for 5 days.
Approximately 0.03 N, 0.0075 N, and 0.003 N sodium thiosulfate solutions are made from the 0.12 N solution by approximate dilu- tion with water, to 4, 16, and 40 times volume, respectively.
0.05 M (= 0.6 N) Standard Potassium Biiodute fiolution- Exactly 19.498 gm. of KH(IO& are dissolved in water to make 1 liter of solution. This standard stock solution keeps indefinitely.
t!$tandard 0.03 N and 0.0075 N potassium biiodate solutions are made from the 0.6 N solution by dilution with 0.085 M HzPO4 solution to 20 and 80 times volume, respectively.
Procedure
The general procedure with titrimetric analysis of the iodate consists of the same steps described in the “Procedure” of Paper I. For the treatment of samples up to the point where the solid AgI03 and AgCl are separated from the supernatant fluid, the reader is referred to the details in the corresponding section of that paper (Sendroy, 1937, a) from steps (1) through (4) to the be- ginning of the description of the “Gasometric determination of total dissolved iodate.”
Titrimetric Measurement
As in the gasometric measurement, the volume of the super- natant sample analyzed will depend on the size of the initial sample of original material and the extent of the dilution. Supernatant samples of from 0.5 cc. to 3.0 cc. volume are pipetted into a small test-tube (15 X 125 mm.).” 0.085 M H3POa is added to bring the volume to 3.0 cc. or 1.5 cc. Freshly prepared 5 per cent potassium iodide solution, in suitable amounts, is added and allowed to react for at least 30 seconds. The liberated iodine is then titrated with sodium thiosulfate solution. When the yellow color has been almost completely discharged, the starch indicator solution is
* Wherever the use of a cotton plug is indicated for withdrawal of samples of the supernatant fluid in the gasometric procedure, such a filter should be used in the corresponding procedure of the titrimetric analysis.
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408 Titrimetric Chloride Determination
added and the titration is carried on to an almost completely colorless, permanent end-point. The use of a constant light source, such as a daylight lamp, is desirable for this titration.
The thiosulfate solution used is in every case of a strength such that from 2.5 to 3.2 cc. are required for complete titration of the sample. The thiosulfate is delivered, drop by drop, from a Bang 3 cc. microburette (Peters and Van Slyke (1932) p. 13) calibrated in 0.01 cc. or 0.02 cc. intervals and tipped with a pointed capillary delivering 100 to 150 drops per cc. Increments of reagent of the order of magnitude of 0.002 cc. may thus be accurately delivered.
The standardization of the thiosulfate is carried out by titration of an equivalent amount of the standard iodate solution in place of the supernatant sample, in exactly the same way as described above. No blank analysis is made in the titrimetric procedure. It is necessary only to ascertain that the acid solution used to dilute the original sample is chloride-free, and that it develops no blue color with starch when potassium iodide is added to it.
In the analysis of urine samples the yellow color of the original sample does not interfere with the preliminary iodine titration before the addition of starch. The extent of dilution with phos- phoric acid, for dilute or for concentrated samples, is sufficient to produce an almost colorless solution. In the analysis of serum samples the same holds true. Upon the addition of the KI to the supernatant fluid of undeproteinized serum samples, the iodine will be observed to separate out, as if attached to protein particles. As the titration proceeds, however, this clears up, and the solution finally becomes almost entirely clear and transparent.
Specific directions for chloride determination in various kinds of materials, with titrimetric analysis of the.iodate, are given below. For details of the steps leading up to the actual titrimetric analysis of the supernatant samples, the reader is referred to the cor- responding sections in Paper I (Sendroy, 1937, a).
A. Determination of Chloride in Inorganic Material-Samples are diluted as described in the corresponding Section A of Paper I. Approximately 0.1 M neutral salt solutions or 0.1 N HCl solutions are diluted 10 to 30 times with 0.085 M HSPOJ, then analyzed as described for serum analyses below, except that no caprylic alcohol is necessary.
B. Determination of Chloride in Urine--As for gasometric
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J. Sendroy, Jr.
analysis, samples may be analyzed according to (a) a routine method used for all samples, or (b) a more accurate method.
Routine Method-The urine is diluted 20-fold with 0.17 M
HaPOd, as described in the corresponding Section B of Paper I. Samples of 0.5 cc. (from very concentrated, normal urines) to 3.0 cc. (from low chloride urines) of the supernatant fluid are taken, and 0.085 M HaPOd is added when needed to make the volume 3.0 cc. The analysis is completed as outlined in the previous section headed “Titrimetric measurement” by the addition of 1.0 cc. of 5 per cent KI, 1 drop of 2 per cent starch solution, and 0.03 N Na&&Oa delivered from a 3 cc. microburette. For the standardization, 2 cc. of 0.03 N KH(IO& plus 1 cc. of 0.085 M H8POd are titrated with 0.03 N Na&&Oa in exactly the same way.
The remarks made in Paper I concerning the accuracy of the routine method for urine apply equally well to the titrimetric analysis. The correction for AgIOa solubility in the case of low chloride samples is also used for titrimetric analyses. However, the use of thiosulfate of a single strength (0.03 N) for all samples necessarily limits the accuracy of the measurements in the case of low chloride urines, The resulting errors are, nevertheless, much less than those obtained by the usual Volhard titration by differ- ence (Sendroy, 1937, b), especially when protein is present. For analysis by 20-fold dilution the titration with 0.03 N NazSz03 will range from 0.3 cc. for a 3 cc. sample of the supernatant fluid from a urine 10 mM in chloride to about 2.4 cc. for a 1 cc. sample of supernatant fluid from a urine 240 mM in chloride.
Precise Method-Dilution with 0.17 M HsP04 is made as de- scribed in the corresponding Section B of Paper I (Sendroy (1937, a) Table I). Samples of 2 cc. of the supernatant fluid are diluted with 1 cc. of 0.085 M H8POI and 1 cc. of 5 per cent KI solution. Titration is carried out with 0.03 N NaZSZOs, 1 drop of 2 per cent starch being added near the end-point. Standardiza- tion is carried out as for the routine procedure.
C. Determinution of Chloride in Plasma or Serum, without De- protein&&on-The method of preparing samples, from 1.00 cc. down to 0.02 cc., by dilution with 0.085 M HaPOd, is the same as that described in the corresponding Section C for the gasometric determination (Sendroy, 1937, a). The use of samples of various
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410 Titrimetric Chloride Determination
volumes and titration with Na&&Oa of different, suitable concen- trations make possible the maintenance of a high degree of ac- curacy even for 0.02 cc. samples (Sendroy (1937, a) Table XXI).
Macrodetermination-A sample of 0.5 cc. of serum is diluted 20 times and prepared for iodate analysis, as in the gasometric method. Of the supernatant fluid, 3 cc. are taken, usually with- out the necessity of a cotton plug in the pipette. Then 1 cc. of 5 per cent KI is added, and the titration is carried out with 0.03 N Na&0~ 1 drop of 2 per cent starch being added near the end- point. For the thiosulfate standardization 2 cc. of 0.03 N
KH(IO& plus 1 cc. of 0.085 M H3P04 plus 1 cc. of 5 per cent KI are-titrated with 0.03 N NazSz03, 1 drop of 2 per cent starch being added near the end-point.
Microdetermination-Samples of 0.2 cc. or 0.1 cc. of serum are diluted and prepared for iodate analysis, as in the gasometric method. Of the supernatant fluid, samples of 1 cc. are taken with a pipette equipped with a cotton plug. Then 2 cc. of 0.085 M
HsP04 and 0.3 cc. of 5 per cent KI are added. The titration is carried out with 0.0075 N NazSz03, 1 drop of 0.5 per cent starch solution being added near the end-point. For the thiosulfate standardization 2 cc. of 0.0075 N KH(IO& plus 1 cc. of 0.085 M
H3P04 plus 0.3 cc. of 5 per cent HI are used. Ultramicrodetermination-Samples of 0.05 cc. or 0.02 cc. are
diluted and prepared for iodate analysis, as in the gasometric method. For the 0.05 cc. anaZysis, the titration of a 1 cc. sample of the supernatant fluid is carried out exactly as above for micro samples.
For the 0.02 cc. analysis, the sample of supernatant fluid is with- drawn in a 0.5 cc. pipette, calibrated between marks. A cotton plug filter is used in the pipette. To the sample are added 1.0 cc. of 0.085 M H3P04 and 0.1 cc. of 5 per cent KI. The titration is carried out with 0.003 N NazSz03, 1 drop of 0.2 per cent starch solution being added near the end-point. For the thiosulfate standardization 1 cc. of 0.0075 N KH(IO& plus 1 cc. of 0.085 M
H3P04 plus 0.1 cc. of 5 per cent KI are used. D. Determination of Chloride in Protein-Free Filtrates of Pto*sma,
Serum, or of Whole Blood-These filtrates are prepared for analysis as described in the corresponding Section D (Sendroy, 1937, a) on gasometric analysis.
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J. Sendroy, Jr. 411
(a) Tungstic acid filtrates of plasma or serum. When enough filtrate is available, samples of 1.5 cc. or 1.0 cc. of the supernatant fluid are taken in a pipette without a cotton plug. There are then added 1.5 or 2.0 cc. of 0.085 M HsP04, respectively, followed by 1.0 cc. of 5 per cent RI. Titration is carried out with 0.03 N NazSz03 and 1 drop of 2 per cent starch solution. Standar‘diza- tion is carried out with 2 cc. of 0.03 N KH(IO&, as described above for the macrodetermination without deproteinization.
If there is a scarcity of filtrate, samples of 0.5 cc. of the super- natant fluid, taken in a pipette equipped with a cotton plug, may be used. There are added 1.5 cc. of 0.085 M HsP04, 0.3 cc. of 5 per cent KI, and 0.0075 N NazSz03. Near the end-point 1 drop of 0.5 per cent starch is added. Standardization is carried out with 2 cc. of 0.0075 N KH(IO&, as described above for the micro- determination without deproteinization.
(b) Tungstic acid jiltrates of whole blood are made, and prepared for analysis as described for the gasometric method. The usual Folin-Wu filtrate may not be used. Of the supernatant fluid from the macro (1 cc.) or the micro (0.2 cc.) determination, 1 cc. samples are taken. There are added 2.0 cc. of 0.085 M H3P04 and 0.3 cc. of 5 per cent KI. Titration is carried out with 0.0075 N
Na&O, standardized as described above for the microdeter- mination without deproteinization.
(c) Zinc hydroxide jiltrates of plasma or serum are made, and prepared for analysis as described for the gasometric method. Samples of 1.5, 1.0, or 0.5 cc. of the supernatant fluid are analyzed as described above (a) for tungstic acid filtrates of serum.
Calculations
From theoretical considerations (Sendroy, 1937, a) and experi- mental results, 1 mole of iodate found by analysis corresponds to l/K mole of chloride originally present. In the titra- tion 1 mole of iodate is equivalent to 6 moles of thiosulfate. Hence, the general equation for the calculation of titrimetric results is :
(3) [Clli = Y x n X d X 1000
6XKXs
where
[Cl]i = rnM chloride per liter of original sample V= cc. volume of thiosulfate used for titration of the supernatant
sample
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TAB
LE
I 4
Fact
ors
for
Cal
cula
tion
of
Titri
met
ric
Chl
orid
e A
naly
ses
by I
odat
e M
etho
d is
Th
e ch
lorid
e co
ncen
tratio
n of
the
or
igin
al
sam
ples
is
fou
nd
by m
ultip
lying
th
e pr
oduc
t (ti
tratio
n vo
lum
e,
V, X
st
anda
rdiz
ed
norm
ality
, n)
of
the
N
asSI
O,
by
the
fact
or
f, ac
cord
ing
to
Equa
tion
4, [
Cl]
= s
X n
X f.
-
i
1
-
1
.-
-i-
-- _-
_-
.- -
-
(
--
i =
dilu
- io
n fa
ctor
If
orig
inal
S
Slll
plC
l in
mpt
x-
nata
nt
liqui
d
f =
f&or
by
whi
ch
to m
ultip
ly
(titra
tion
volu
me,
9, X
sta
nd-
ardi
md
norm
ality
, n)
of t
he
Na&
Oa.
to
obt
ain
vo:&
,, A
ppro
xi-
of s
uper
- m
ate
nor-
nsts
nt
mal
ity
of
liqui
d iU
Udy
53d
“a&
20:
Y =
rat
ic
m LC
U
Mat
&al
A
naly
sis
-
(
_ _ _.
-
;m.
N&
l pe
r lit
er
maa
Cl
per
liter
0.99
8 6,
680*
0.
998
3,34
0 0.
998
1,11
3
0.99
0 0.
990
0.. 9
90
0.98
5 0.
985
0.98
5 0.
985
0.98
5
6,73
4*
3,36
7 1,
122
1,12
8 4,
408
5,25
4 5,
254
10,5
40
1.01
7 1.
017
1.01
7
1.00
0 l.o
oo
1,09
3 1,
639
3,27
8
4,16
7 4,
167
6,59
9 3
25,7
g7
30,7
36
El
30,7
36
5’
61,6
59
z -*
6,39
4 g
9,m
19
,176
24,3
77
24,3
77
cc;
0.5
0.03
1.
0 0.
03
3.0
0.03
~-
0.5
0.03
1.
0 0.
03
3.0
0.03
--
3.0
0.03
1.
0 0.
0075
1.
0 0.
0075
1.
0 0.
0075
0.
5 0.
003
1.5
0.03
1.
0 0.
03
0.5
0.00
75
-- 1.
0 0.
0075
1.
0 0.
0075
Nor
mal
ur
ine
Rou
tine
20
390.
23*
195.
4 65
.1
393.
9*
197.
0 65
.6
Albu
min
ous
urin
e R
outin
e 20
Seru
m,
with
out
depr
otei
niza
tion
Mac
ro
Micr
o
Ultr
amicr
o
20
26.0
5 31
.05
31.0
5 31
.15
Seru
m,
tung
stic
ac
id
filtra
te
10
10
10
_- 25
25
W
hole
bl
ood,
tu
ngst
ic
acid
fil
trate
M
acro
M
icro
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J. Sendroy, Jr.
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414 Titrimetric Chloride Determination
n= standardized normality of the thiosulfate d= dilution factor for the number of times original sample was diluted,
with HaPOa or protein precipitant, before shaking with AgIOt s = cc. volume of supernatant sample used for titration
K= maI - = empirical reaction yield factor expressing the ratio of the [Cl1
concentration of iodate, found by analysis, to the concentration of chloride originally present, per liter of original sample
For convenience in calculating, Equation 3 can take the form
(4)
When results in terms of gm. of NaCl per liter, or mg. of NaCl per 100 cc. are desired, the values off for mM of Cl per liter are multi- plied by 0.0585, or 5.85, respectively.
In Table I are given values for the factor f, under different conditions of analysis. When the product (titration volume, V, X standardized normality, n) of the NazSz03 is multiplied by f, the result in terms of mM of chloride per liter, gm. of NaCl per liter, or mg. of NaCl per 100 cc. is found.
Example-A sample of serum was diluted to 29 times the original volume (0.5 to 10 cc.) before addition of silver iodate. Of the centrifuged super- natant liquid, 3 cc. were then analyzed., the titration requiring 2.97 cc. of the “0.03 N” NazSzOS, the standardized normality of which was found to be 0.03039 N. (A 2 cc. sample of exactly 0.03 N KH(IO& required 1.974 cc. of the NazSzOs at the end-point.) From Equation 3 the chloride content of the original sample was calculated to be
2.97 x 0.03039 x 20 x 1000 = 6 X 0.985 X 3 101.8 rnM per liter
Calculated according to Equation 4 and Table I, the result was found to be 2.97 X 0.03039 X 1128 = 101.8 mM of chloride per liter, or 2.97 X 0.03039X 6599 = 595.5 mg. of NaCl per 100 cc.
Correction for Soluubility of Agl’03 in the Routine Method for Urine-In the routine analysis of urine, when the calculation according to Table I gives a result less than 60 rnr+r of chloride per liter (or 3.51 gm. of NaCl per liter), a solubility correction for
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J. Sendroy, Jr. 415
AgI03 must be applied, exactly as for the gasometric method, according to Fig. 2 of Paper I (Sendroy (1937, a) p. 363).
However, in the routine analysis of such low chloride urines, when the titration is bss than i .8 cc., instead of calculating according to Table I, and subtracting the correction for AgI03 solubility, we may calculate the corrected chloride concentration graphically,
TABLE II
Table for Direct Graphical Calculation of Titrimetric Results for the Routine Analysis of Low Chloride Urines
For supernatant samples of 3 cc. titrated with approximately 0..03 N NazSeOa, the titration volume, Y, of the approximately 0.03 N NakLOa actually used for the titration, must first be corrected to the corresponding volume, II’, of exactly 0.03 N NazSeOa, by multiplying by the standardization factor, n/0.03.
P’ = volume of axmtly 0.03 N NazStOs used
cc.
1.789 1.491 1.193 0.895 0.596 0.537 0.477 0.418 0.358 0.298 0.239 0.209
- I Chloride concentration of original sample. corrected for
solubility of AgIos in the supernatant
mab Cl per liter Gm. NaCl per liter
59.76 3.497 49.62 2.903 39.44 2.307 29.16 1.706 18.62 1.089 16.42 0.961 14.20 0.831 11.92 0.697 9.62 0.563 7.14 0.418 4.39 0.257 2.86 0.167
directly from the volume of Na2S203 used for the titration. Thus, if Equation 4 be recast for this purpose,
(5) [Cl] = (eXnXf)--S
where [Cl]’ = [Cl] - S = the concentration of chloride in mM per liter, in the original sample, corrected for the solubility of AgIOs in the supernatant liquid, and S = the variable solubility cor- rection from Fig. 2 of the preceding paper (Sendroy, 1937, a).
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416 Titrimetric Chloride Determination
Then we may write for the routine urine analysis of 3 cc. samples of supernatant liquid, using the average K value of 0.994,
(6) [Cl]’ = (V’ x 33.53) - s
where v’ = v X (n/0.03) = volume v of approximately 0.03 N
NazSzOa used for titration, corrected to a corresponding volume of ezucl~y 0.03 N Na&&Os.
Table II, calculated from Equation 6, may be used to draw curves with [Cl] ’ and o’ as coordinate axes for the direct reading of corrected chloride concentrations from titration volumes of exactly 0.03 N Nas&Os. It is desirable that the graphs be drawn on a fairly large scale, so that there may be no great loss of ac- curacy in the readings of low chloride concentrations.
Examples-Two samples of urine, Samples A and B, were analyzed by the routine method. The supernatant samples of 3 cc. required 2.50 cc. and 0.481 cc., respectively, of 0.03 N Na2S208, the standardization factor of which was 0.992. The analysis of Sample A was calculated according to Table I to be 2.50 X 0.02976 X 65.1 = 4.84 gm. of NaCl per liter. The analysis of Sample B was calculated according to a chart based on Table II, whereby 0.481 cc. X 0.992 = 0.477 cc. was found to correspond to a value of 0.831 gm. of NaCl per liter. Calculated according to Table I, Sample B showed 0.481 X 0.02976 X 65.1 = 0.932 gm. of NaCl per liter. With the correction for AgIOa solubility from Curve B, Fig. 2, of Paper I, the final result was 0.932 - 0.104 = 0.828 gm. of NaCl per liter.
The theoretical aspects of the reaction of Equation 1, whereby chloride is precipitated by AgIOa, have already been discussed in Paper I (Sendroy, 1937, a). The experimental results obtained by titration have been included there and require no further dis- cussion here.
SUMMARY
The present paper contains an outline of a titrimetric procedure for the measurement of the iodate in the chloride method outlined in the preceding paper. The 103~ is determined by its reaction with acidified KI, with starch as an end-point indicator: KIOa + 5KI + 6H3POa = 31z + 3Hz0 + 6KHzP04. Thus, as in the gasometric method, the hexavalent oxidative power of IOa- results in 1 equivalent of Cl- being represented by 6 equivalents of free iodine.
The accuracy and rapidity of this procedure are about the same as
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J. Sendroy, Jr. 417
of the gasometric procedure. Here, likewise, no removal of proteins, either by precipitation or digestion, from urine, or plasma or serum, is required, although protein-free filtrates of serum and of whole blood may be used for analysis.
BIBLIOGRAPHY
Conway, E. J., Biochem. J., 29,222l (1935). Folin, O., and Wu, H., J. Biol. Chem., 38, 81 (1919). McLean, F. C., and Van Slyke, D. D., J. Bio2. Chem., al,.361 (1915). Peters, J. P., and Van Slyke, D. D., Quantitative clinical chemistry,
Methods, Baltimore (1932). Sendroy, J, Jr., J. Biol. Chem., 120, 335 (1937, a); 120,441 (1937, b). Somogyi, M., J. Biol. Chem., 86, 655.(1930).
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Julius Sendroy, Jr.TITRIMETRIC ANALYSIS
WITH SOLID SILVER IODATE: II.CHLORIDE IN BIOLOGICAL FLUIDS,
MICRODETERMINATION OF
1937, 120:405-417.J. Biol. Chem.
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