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A non-mercurimetric automated method for serum chloride
247
CLINICA CHIMICA ACTA
CCA 5207
A NON-MERCURIMETRIC
BEN JAMIN
AUTOMATED
METHOD
FOR SERUM CHLORIDE*
FINGERHUT
ofPathology, Kings County Hoqbital Center, Brooklya, N.Y.
Institute (Received
May 25.
II~
03 (U.S.A.)
1972)
SUMMARY
A new automated method for serum chloride is presented for use with the Technicon AutoAnalyzer II system. It is based on the reaction between ferric perchlorate and chloride ion in dilute perchloric acid. Results for 30 sera agreed within 95% confidence limits with a manual mercurimetric titration method and averaged 1.6 mequiv/l higher than the AAI chloride procedure. The S.D. for 50 duplicate samples was 0.65 mequiv/l. Recoveries ranged from g6 to IOO~/~. The proposed method offers the advantage of greater specificity and eliminates the mercury pollution that the currently widely used automated chloride method contributes to theenvironment. An additional advantage is that the method yields a direct relation between recorder chart reading and chloride concentration, while the standard AA11 chloride method is non-linear and requires a linearizer to provide a linear response.
INTRODUCTION
There has been little attention focused upon minimizing pollutants that the clinical chemistry laboratory contributes to the environment. In this report, a new automated procedure for serum chloride is presented based on the reaction between ferric perchlorate and chloride in dilute perchloric acidl. Since mercuric salts are not used in this procedure, the proposed method eliminates the mercury pollution current automated procedures contribute to the environment. EXPERIMENTAL
Reagents Fe(ClO,), (non-yellow, K and K Labs. Plainview, N.Y.). HC10,--60~~ (Mallinckrodt). (3) Fe(ClO,),-HClO, working solution. Dissolve 75 gFe (ClO,), H,O. Add 375 ml 60% HClO, and dilute to a liter and H,O. (I)
(2)
* Portions of this report presented orallyon N.Y.C., N.Y.
in 500 ml
June rq, 1972 at the Technicon International C&n. Chinz. Acta, 4’
(1972)
Congress,
247-253
FINGERHUT
248
(4) 0.2 N acetate buffer (pH 6.0). To 60 ml 0.2 N acetic acid add g4o ml 0.2 N sodium acetate. (5) Stock I N chloride solution. 58.45 g NaCl per liter H,O. (6) Working chloride standards. 8.4, 9.2, 10.0, 10.8, 11.6 and 12.4 ml stock chloride diluted to IOO ml with H,O to give 84, 92, IOO, 108, 116 and 124 mequiv/l chloride respectively.
AutoAnalyzer II system consisting of a pump III, sampler IV, manifold module with 12” dialyzer, AA11 single channel calorimeter with r5-mm flow cells and 352nm interference filters, and a recorder. Procedure
The flow diagram in Fig. I indicates the steps in the procedure. Since the chloride concentration in the range 84 to 124 mequiv/l is the area of interest for serum, it is necessary to adjust the calorimeter apertures so that this range appears on the recorder chart paper. This is done by adjusting the apertures on the calorimeter while the chloride (84 mequiv/l) standard is passing through the flow cell. On the AA11 recorder chart paper (type number 011-0173-01) the reading should be between 20 and.30 units in order to obtain an on-scale reading for the 124 mequiv/l chloride standard, when the standard calibration helipot is set at I. The 124 mequiv/l standard will usually read between 75 and 85 units. Standards and sera are sampled at the rate of 40/h with a sample-to-wash ratio of 2 : I. A standard is run after every tenth specimen to check for drift. The calorimeter mode switch is set at damp position z to provide more easily readable recorder peaks of samples. RESULTS AND DISCUSSION
Fig. 2 shows tracings given by a series of standards and some sera. Construction of a calibration curve (Fig. 3) from these readings yields a linear relation between recorder chart units and chloride concentration. In contrast, the current AA11 chloride method is a non-linear test2. CHLORIDE
MANIFOLD Scd.tPLERI”
WASTE
8 TURNS
)$ORN/YLW
M2MMM
(0.161 SAMPLE
nORN/WHT023)AlR ~WHT/WI~T~O.~O~I~~O XWHT/W~~T(O~O)FERRIC
TO F/C PUMP TVBE
WASTE N TO SAMPLER IV ~ WASH RECEPTACLE
Fig. I. Flow diagram for automated Clia
uWHT,WHT(0.60) c> nGRN’6RN (2.00)
Chim. Acta, 41 (1972) 247-253
chloride procedure.
FROM Hz0
RE*GENT F/C
NON-.MERCURIMETRIC
METHOD FOR SERUM CHLORIDE
249
b
tn m
i
;i !I
/
i
I
~
Fig. a. Recorder tracings for aqueous chloride standards and some sera
OF!, CHLORIDE
140 CONCENTRATION
tmeq/l)
Fig. 3. Relation between chloride concentration and recorder reading.
Table I lists mean values for 30 sera analyzed by three methods, as well as the deviation (S.D.) of the difference between these methods. Calculation of the statistic t indicates agreement (95% confidence limits) between the proposed automated and the manual lnercurimetric titration method3. However, t values greater standard
Clin. Chim.
Acta,
41 (1972) 247-253
FINGERHUT
250 TABLE
I
RESULTS
BY
AUTOMATED
AND
MANUAL
NIETHODS
AA Hg (CNS),+Fo(NO,)z
roq.4
AA Hg (CNS),+Fe(NO,), and manual fIg(NO,),
30
AA Fe(ClO&
106.0
AA
30
Manual H&NO,),
105.6
I.69
3.93
Fe(CI0,) and manual HgjNO,),
1.59
I.58
AA Hg(CNS),+Fe(NO,), and AA Fe(CIO,),
1.7~
5-34
than 2 indicate significant difference at the 95% confidence level between automated ferric perchlorate and automated mercurimet~c methods”, as well as between the automated mercurimetric and manual mercurimetric procedures. Within-run reproducibility for 50 sera analyzed by the proposed method yielded a SD. of 0.65 mequiv/l Cl. Day-to-day reproducibility for 50 sera gave a S.D. of 0.85 mequiv/l Cl. Recovery experiments yielded a mean value of 98% (range 96-100%). Chloride levels for 32 apparently heahhy adults ranged from 98-110 mequivjl. This is similar to previously reported normal value.+. The use of a buffer as a sample diluent was found advantageous for three reasons : (I) The marked variation of serum chloride results with a diluent pH, as seen in Table II. (2) Aqueous chloride standards instead of protein-containing standards can be used. (3) The effect of the variable pH of different sera is minimized. The apparent chloride values listed for the three sera and Versatol, based on a calibration curve obtained with aqueous standards, increased as much as 30% as the pH of sample diluent rose from x.4 to II .a. Apparently the pH influences the dialysis rate of serum chloride differently from aqueous chloride solutions. The rate of chloride dialysis from aqueous standards appears to vary only slightly with diluents of different acidity. This is evident from the values in recorder chart units per mequiv/l chloride (zk sensitivity) listed in the last column on the right in Table II. The use of water as sample diluent gave the highest serum chloride results although the pH was close to that of the acetate buffer. Possibly diluent ionic strength as well as pH influences chloride dialysis rate. It is of interest to note that the mercurimetric AutoTABLE II EFFECT OFDTLUEi’X pH
OX
a.
CHLORIDE
RESULT
Versatol N Semwz a (label CI wsequivil Cl value log mequiv/l)
Serum b mequivjl Cl
mequivjl Cl
78 99 104
75 98 104
85 101 106
Sr 99 105
I.65
acetic acid 3-o 0.2 M acetate buffer 6.0 0.2 M sod. acetate 0.1 M Na,CO,
105 ‘07
105
107
109
108
III
112
x.75 1.84
III
II2
112
112
r.57
PH
sanzple diluent
0.1
0.1
N HNO,
I.4
M
7.6 II.2
Hz0 C&n.
6.3
Chim. Acta, 41
(1972)
2.$7-253
Ser%*z. G
Calibvation cuwe as recorder ckavt lmits mey& 11Cl I.jj 1.60
NON-MERCWRIMETRIC
METHOD FOR SERUM CHLORIDE
251
Analyzer II chloride method2 recommends the addition of albumin to the aqueous chloride standards for reliable results. The optimal concentrations of perchloric acid and ferric perchlorate to use was investigated in order to determine the reaction conditions which would give the greatest sensitivity. In Figs. 4 and 5 the variation of recorder units per mequiv/l I.8
*“\a
I.6
0/
\
0
/
0
0.4
0.2
0
0
-I
100
200 ml/l
300. 9M
400
f60%1
500
600
700
HCIO,
Fig. 4. Variation of recorder units per mequiv/l chloride with perchIoric acid concentration. 1.6,
MOLARITY
OF
Fe K10415
Fig. 5. Variation of recorder units per mequiv/l chloride with ferric perchlorate concentration.
FINGERHUT
252
chloride with acid and iron concentration, respectively, are represented. For both of these reagents there is a maximum which affords the greatest sensitivity. Fig. 6 shows the relation between absorbance and wave length of reagent blank %rs.H,O (curve I), sample us. H,O (curve 2) and sample US.reagent blank (curve 3), obtained using a Bausch and Lomb 505 recording spectrophotometer. The blank and sample solutions used were collected from the calorimeter flow-cell outlet. From these curves the maximum difference in absorbance between curves I and 2 is in the range of 353 to 354 nm. Filters close to this wave length (352 nm) were used in the present study. Since chloro complexes of ferric ion display a strong absorption band in the area of 340 nm, it is considered that the proposed procedure measures a chloro complex of ferric ion’. The use of perchloric is not hazardous1 in the concentration used and is advantageous because ferric perchlorate complexes have a low stability6a7and exhibit little energy absorption at the wave length measuredl. -
1.0
.9
I
.8
I 440.5
430.5
420.5
410.5
400.5
390.5
WAVE
380.5
LENGTH
370.5
360.5
35Q5
34Q5
330.5
lnm)
Fig. 6. Relation between absorbance and optical density of reagent blank vs. F&O (curve I), sample 8s. II,0 (curve 2) and sample vs. reagent blank (curve 3). TABLE
III
EFFECT OF ADDED Anion
added
Fluoride Bromide Iodide Phosphate Sulfate
ANIONS
Gown. added
of a&n
Intevfemnce
20 mequivjl 30 mequiv/l 20 mequiv/l 20 mg% 20 mg% _.
C&n. Chim. Acta,
41 (1972)
as nzeq&~/l CZlunit cont. of added anion
247-253
-
AA Hg(CNS),-;F~(No,), method
Manual Hs(NWz method
--0.05
-0.04
+o.o3 +o.51 none detected none detected
fw8 +*.75 none detected none detected
none detected +1.00 +o.7o none detected none detected
AA Fe(ClO,), method
_
NON-MERCURIMETRIC
mated
The specificity mercurimetric
253
METHOD FOR SERUM CHLORIDE
of the proposed method was compared to the manual and autoprocedures. Results presented in Table III indicate less inter-
ference in the proposed method from bromide and iodide than manual or automated mercurimetric procedures. The concentration of added anions was several times above that normally present in sera. In the proposed method only iodide interferes appreciably to elevate the serum chloride level 0.5 mequiv/l for every mequiv/l of added iodide. ACKNOWLEDGEMENTS
The author is grateful for the Norris and Mr. Henry Miller.
assistance
of Mrs. Roslyn
Zirkind,
Mr. John
REFERENCES P. W. WEST AND H. COLL, Anal. Chem., 28 (1956) 1834. Technicon method AAII--o~, Dec. rg71, Technicon Instruments Corp., Tarrytown, N.Y. xo5gr. 0. SCHALES AND S. S. SCHALES, J. Biol. Chem., 140 (1941) 879. Technicon method N-zIb/II, Technicon Instruments Corp., Tarrytown, N.Y. 10591. R. J. HENRY, Clinical Chew&try: Principles and Techniques, Harper and Row, New York, 1964, p. 408. 6 R. BASTIAN, R. WEBERLING AND F. PALILLA, Anal. Chem., 25 (1953) 284. 7 J. SUTTON, Nature, 169 (1952) 71.
I 2 3 4 5
Clin. Chim
Acta,
41 (1972) 247-253
CLINICA CHIMICA ACTA
CCA 5207
A NON-MERCURIMETRIC
BEN JAMIN
AUTOMATED
METHOD
FOR SERUM CHLORIDE*
FINGERHUT
ofPathology, Kings County Hoqbital Center, Brooklya, N.Y.
Institute (Received
May 25.
II~
03 (U.S.A.)
1972)
SUMMARY
A new automated method for serum chloride is presented for use with the Technicon AutoAnalyzer II system. It is based on the reaction between ferric perchlorate and chloride ion in dilute perchloric acid. Results for 30 sera agreed within 95% confidence limits with a manual mercurimetric titration method and averaged 1.6 mequiv/l higher than the AAI chloride procedure. The S.D. for 50 duplicate samples was 0.65 mequiv/l. Recoveries ranged from g6 to IOO~/~. The proposed method offers the advantage of greater specificity and eliminates the mercury pollution that the currently widely used automated chloride method contributes to theenvironment. An additional advantage is that the method yields a direct relation between recorder chart reading and chloride concentration, while the standard AA11 chloride method is non-linear and requires a linearizer to provide a linear response.
INTRODUCTION
There has been little attention focused upon minimizing pollutants that the clinical chemistry laboratory contributes to the environment. In this report, a new automated procedure for serum chloride is presented based on the reaction between ferric perchlorate and chloride in dilute perchloric acidl. Since mercuric salts are not used in this procedure, the proposed method eliminates the mercury pollution current automated procedures contribute to the environment. EXPERIMENTAL
Reagents Fe(ClO,), (non-yellow, K and K Labs. Plainview, N.Y.). HC10,--60~~ (Mallinckrodt). (3) Fe(ClO,),-HClO, working solution. Dissolve 75 gFe (ClO,), H,O. Add 375 ml 60% HClO, and dilute to a liter and H,O. (I)
(2)
* Portions of this report presented orallyon N.Y.C., N.Y.
in 500 ml
June rq, 1972 at the Technicon International C&n. Chinz. Acta, 4’
(1972)
Congress,
247-253
FINGERHUT
248
(4) 0.2 N acetate buffer (pH 6.0). To 60 ml 0.2 N acetic acid add g4o ml 0.2 N sodium acetate. (5) Stock I N chloride solution. 58.45 g NaCl per liter H,O. (6) Working chloride standards. 8.4, 9.2, 10.0, 10.8, 11.6 and 12.4 ml stock chloride diluted to IOO ml with H,O to give 84, 92, IOO, 108, 116 and 124 mequiv/l chloride respectively.
AutoAnalyzer II system consisting of a pump III, sampler IV, manifold module with 12” dialyzer, AA11 single channel calorimeter with r5-mm flow cells and 352nm interference filters, and a recorder. Procedure
The flow diagram in Fig. I indicates the steps in the procedure. Since the chloride concentration in the range 84 to 124 mequiv/l is the area of interest for serum, it is necessary to adjust the calorimeter apertures so that this range appears on the recorder chart paper. This is done by adjusting the apertures on the calorimeter while the chloride (84 mequiv/l) standard is passing through the flow cell. On the AA11 recorder chart paper (type number 011-0173-01) the reading should be between 20 and.30 units in order to obtain an on-scale reading for the 124 mequiv/l chloride standard, when the standard calibration helipot is set at I. The 124 mequiv/l standard will usually read between 75 and 85 units. Standards and sera are sampled at the rate of 40/h with a sample-to-wash ratio of 2 : I. A standard is run after every tenth specimen to check for drift. The calorimeter mode switch is set at damp position z to provide more easily readable recorder peaks of samples. RESULTS AND DISCUSSION
Fig. 2 shows tracings given by a series of standards and some sera. Construction of a calibration curve (Fig. 3) from these readings yields a linear relation between recorder chart units and chloride concentration. In contrast, the current AA11 chloride method is a non-linear test2. CHLORIDE
MANIFOLD Scd.tPLERI”
WASTE
8 TURNS
)$ORN/YLW
M2MMM
(0.161 SAMPLE
nORN/WHT023)AlR ~WHT/WI~T~O.~O~I~~O XWHT/W~~T(O~O)FERRIC
TO F/C PUMP TVBE
WASTE N TO SAMPLER IV ~ WASH RECEPTACLE
Fig. I. Flow diagram for automated Clia
uWHT,WHT(0.60) c> nGRN’6RN (2.00)
Chim. Acta, 41 (1972) 247-253
chloride procedure.
FROM Hz0
RE*GENT F/C
NON-.MERCURIMETRIC
METHOD FOR SERUM CHLORIDE
249
b
tn m
i
;i !I
/
i
I
~
Fig. a. Recorder tracings for aqueous chloride standards and some sera
OF!, CHLORIDE
140 CONCENTRATION
tmeq/l)
Fig. 3. Relation between chloride concentration and recorder reading.
Table I lists mean values for 30 sera analyzed by three methods, as well as the deviation (S.D.) of the difference between these methods. Calculation of the statistic t indicates agreement (95% confidence limits) between the proposed automated and the manual lnercurimetric titration method3. However, t values greater standard
Clin. Chim.
Acta,
41 (1972) 247-253
FINGERHUT
250 TABLE
I
RESULTS
BY
AUTOMATED
AND
MANUAL
NIETHODS
AA Hg (CNS),+Fo(NO,)z
roq.4
AA Hg (CNS),+Fe(NO,), and manual fIg(NO,),
30
AA Fe(ClO&
106.0
AA
30
Manual H&NO,),
105.6
I.69
3.93
Fe(CI0,) and manual HgjNO,),
1.59
I.58
AA Hg(CNS),+Fe(NO,), and AA Fe(CIO,),
1.7~
5-34
than 2 indicate significant difference at the 95% confidence level between automated ferric perchlorate and automated mercurimet~c methods”, as well as between the automated mercurimetric and manual mercurimetric procedures. Within-run reproducibility for 50 sera analyzed by the proposed method yielded a SD. of 0.65 mequiv/l Cl. Day-to-day reproducibility for 50 sera gave a S.D. of 0.85 mequiv/l Cl. Recovery experiments yielded a mean value of 98% (range 96-100%). Chloride levels for 32 apparently heahhy adults ranged from 98-110 mequivjl. This is similar to previously reported normal value.+. The use of a buffer as a sample diluent was found advantageous for three reasons : (I) The marked variation of serum chloride results with a diluent pH, as seen in Table II. (2) Aqueous chloride standards instead of protein-containing standards can be used. (3) The effect of the variable pH of different sera is minimized. The apparent chloride values listed for the three sera and Versatol, based on a calibration curve obtained with aqueous standards, increased as much as 30% as the pH of sample diluent rose from x.4 to II .a. Apparently the pH influences the dialysis rate of serum chloride differently from aqueous chloride solutions. The rate of chloride dialysis from aqueous standards appears to vary only slightly with diluents of different acidity. This is evident from the values in recorder chart units per mequiv/l chloride (zk sensitivity) listed in the last column on the right in Table II. The use of water as sample diluent gave the highest serum chloride results although the pH was close to that of the acetate buffer. Possibly diluent ionic strength as well as pH influences chloride dialysis rate. It is of interest to note that the mercurimetric AutoTABLE II EFFECT OFDTLUEi’X pH
OX
a.
CHLORIDE
RESULT
Versatol N Semwz a (label CI wsequivil Cl value log mequiv/l)
Serum b mequivjl Cl
mequivjl Cl
78 99 104
75 98 104
85 101 106
Sr 99 105
I.65
acetic acid 3-o 0.2 M acetate buffer 6.0 0.2 M sod. acetate 0.1 M Na,CO,
105 ‘07
105
107
109
108
III
112
x.75 1.84
III
II2
112
112
r.57
PH
sanzple diluent
0.1
0.1
N HNO,
I.4
M
7.6 II.2
Hz0 C&n.
6.3
Chim. Acta, 41
(1972)
2.$7-253
Ser%*z. G
Calibvation cuwe as recorder ckavt lmits mey& 11Cl I.jj 1.60
NON-MERCWRIMETRIC
METHOD FOR SERUM CHLORIDE
251
Analyzer II chloride method2 recommends the addition of albumin to the aqueous chloride standards for reliable results. The optimal concentrations of perchloric acid and ferric perchlorate to use was investigated in order to determine the reaction conditions which would give the greatest sensitivity. In Figs. 4 and 5 the variation of recorder units per mequiv/l I.8
*“\a
I.6
0/
\
0
/
0
0.4
0.2
0
0
-I
100
200 ml/l
300. 9M
400
f60%1
500
600
700
HCIO,
Fig. 4. Variation of recorder units per mequiv/l chloride with perchIoric acid concentration. 1.6,
MOLARITY
OF
Fe K10415
Fig. 5. Variation of recorder units per mequiv/l chloride with ferric perchlorate concentration.
FINGERHUT
252
chloride with acid and iron concentration, respectively, are represented. For both of these reagents there is a maximum which affords the greatest sensitivity. Fig. 6 shows the relation between absorbance and wave length of reagent blank %rs.H,O (curve I), sample us. H,O (curve 2) and sample US.reagent blank (curve 3), obtained using a Bausch and Lomb 505 recording spectrophotometer. The blank and sample solutions used were collected from the calorimeter flow-cell outlet. From these curves the maximum difference in absorbance between curves I and 2 is in the range of 353 to 354 nm. Filters close to this wave length (352 nm) were used in the present study. Since chloro complexes of ferric ion display a strong absorption band in the area of 340 nm, it is considered that the proposed procedure measures a chloro complex of ferric ion’. The use of perchloric is not hazardous1 in the concentration used and is advantageous because ferric perchlorate complexes have a low stability6a7and exhibit little energy absorption at the wave length measuredl. -
1.0
.9
I
.8
I 440.5
430.5
420.5
410.5
400.5
390.5
WAVE
380.5
LENGTH
370.5
360.5
35Q5
34Q5
330.5
lnm)
Fig. 6. Relation between absorbance and optical density of reagent blank vs. F&O (curve I), sample 8s. II,0 (curve 2) and sample vs. reagent blank (curve 3). TABLE
III
EFFECT OF ADDED Anion
added
Fluoride Bromide Iodide Phosphate Sulfate
ANIONS
Gown. added
of a&n
Intevfemnce
20 mequivjl 30 mequiv/l 20 mequiv/l 20 mg% 20 mg% _.
C&n. Chim. Acta,
41 (1972)
as nzeq&~/l CZlunit cont. of added anion
247-253
-
AA Hg(CNS),-;F~(No,), method
Manual Hs(NWz method
--0.05
-0.04
+o.o3 +o.51 none detected none detected
fw8 +*.75 none detected none detected
none detected +1.00 +o.7o none detected none detected
AA Fe(ClO,), method
_
NON-MERCURIMETRIC
mated
The specificity mercurimetric
253
METHOD FOR SERUM CHLORIDE
of the proposed method was compared to the manual and autoprocedures. Results presented in Table III indicate less inter-
ference in the proposed method from bromide and iodide than manual or automated mercurimetric procedures. The concentration of added anions was several times above that normally present in sera. In the proposed method only iodide interferes appreciably to elevate the serum chloride level 0.5 mequiv/l for every mequiv/l of added iodide. ACKNOWLEDGEMENTS
The author is grateful for the Norris and Mr. Henry Miller.
assistance
of Mrs. Roslyn
Zirkind,
Mr. John
REFERENCES P. W. WEST AND H. COLL, Anal. Chem., 28 (1956) 1834. Technicon method AAII--o~, Dec. rg71, Technicon Instruments Corp., Tarrytown, N.Y. xo5gr. 0. SCHALES AND S. S. SCHALES, J. Biol. Chem., 140 (1941) 879. Technicon method N-zIb/II, Technicon Instruments Corp., Tarrytown, N.Y. 10591. R. J. HENRY, Clinical Chew&try: Principles and Techniques, Harper and Row, New York, 1964, p. 408. 6 R. BASTIAN, R. WEBERLING AND F. PALILLA, Anal. Chem., 25 (1953) 284. 7 J. SUTTON, Nature, 169 (1952) 71.
I 2 3 4 5
Clin. Chim
Acta,
41 (1972) 247-253