Spectrophotometric Titration of Spinal Fluid Calcium and Magnesium BENNIE ZAK, W. M. HINDMAN, and E. S. BAGINSKI Department o f Pathology, W a y n e University College o f Medicine, and Detroit Receiving Hospital, Detroit, M i c h .
A simple procedure for the spectrophotometric titration of calciuni and magnesium in cerebrospinal fluid involves the separate determination of the individual cations of the same sample instead of the use of the difference between total divalent cation and either constituent. The several phases of the titration have been investigated: dje variance, constitution of the titrant, spectral studies, and accuracy and precision of the determination.
C
ALCIPLI and magnesium, two of the more commonly det,erniined metals, have been quantitatively determined by a number of comparatively recent complexometric procedures. Some of these hal-e been concerned with visual titrimetry involving the iise of the indicators murexide, Eriochrome black T, or phthalein complexon ( 1 , 2! 4,10, 12, 18,21),while several spectrophotometric titrations employing t,he same indicators for either one of the two cation3 or total cations appear to have been successful1)- investigated (3, 6-8, 13-15, 1 9 ) . The employment of individii:~l spectropliotometric titrimetry for small volumes (1 to 2 ml.) of Ion- rwiicentration mixtures of calcium and magnesium within the fraction of a milliequivalent range (0.001 to 0.005 nieq. per sample anal>-zed) for both metals incorporated within the sitme medium has seen but little investigation. This is the circiinistance v-hen a physiological medium such as spinal fluid is the material being examined. Most spectrophotometric titrations appear to have been involved with the determination of serum calcium, using murexide as the indicator. T-isual titrations, however, have been much more commonly emplol-ed. Sobel and Medoff ( 2 0 ) developed a visual microtechnique for calcium and magnesium by titrating the t,otal divalent c.;itions present using Eriochrome black T, then adding oxalic acid and excess nickel ions, and titrating the liberated magnesiuni :rnd residual nickel t,o get the calcium value. The magnesium n-;ts then calculated by difference. Friedman and Rubiii ( 5 ) . iising a modification of Diehl's procedure ( 5 ) , visually titrated the total divalent cations of serum and spinal fluid, using Eriochrome black T as the indicator. Magnesium was then determined in the supernatant fluid of a second sample after calcium had been prpcipitated as the oxalate. The difficulty involved in objectively seeing the end point change, when an aliqiiot of a solution of but several milliequivalent,s per liter is 1 1 4 , can be overcome by spectrophotometric siibstitiition for the visual means of quantitation ( 7 , 16, 19). K h e n the veq- dilute titrant required t o get a satisfactory titer is employed, 0.001.1/ or less, the end point creeps up and usually appears too early. The Fork reported here represents a study of the phases of the titration as well as a presentation of a relatively simple procedure for determining calcium and magnesium in spinal fluid, where each component from the same starting sample is analyzed individually. REAGENTS
-411chemicals ivrw~of reagent, grade, unless otherwise specified. Calcium Carbonate Standard Solution. Weigh out 250 mg. of dried analytical reagent grade calcium carbonate, transfer to a 1-liter volumetric flask containing about 300 ml. of distilled water, and dissolve hy adding a minimum amount of concentrated hydrochloric ncid. Dilute to the mark of t,he flask with mising.
Magnesium Stock Standard Solution. Weigh out 1 gram of pure magnesium and dissolve in distilled water with the addition of a minimum amount of concentrated hydrochloric acid. Dilute to the mark of a 1-liter volumetric flask with mixing. Magnesium Working Standard Solution. Pipet 5 ml. of the stock standard into a 100-ml. volumetric flask and dilute to the mark with mixing. Buffer Solution, Precipitating. Weigh out 1.5 grams of ammonium oxalate and dissolve in a 250-ml. volumetric flask containing about 200 ml. of distilled water. Add 0.35 ml. of concentrated ammonium hydroxide and 3.5 grams of ammonium chloride. Dilute t o the mark of the flask and mix well. Buffer Solution, Titrating ( 5 ) . Weigh out 67.5 grams of ammonium chloride and dissolve in a 1-liter volumetric flask containing 2 to 300 ml of distilled water. Add 570 ml. of concentrated ammonium hydroxide, dilute to the mark of the flask with mixing, and store in the refrigerator. Ethylenediaminetetraacetic Acid Titrant (EDTA). Weigh out 100 mg. of dried analytical reagent grade ethylenediaminetetraacetic acid [ (ethylenedinitri1o)tetraacetic acid, EDTA] and transfer to a 1-liter volumetric flask containing several hundred milliliters of distilled water with four sodium hydroxide pellets dissolved in it. Add 10 ml. of the magnesium stock solution and dilute to the mark of the flask with mixing after the ethylenediaminetetraacetic acid has gone into solution. Eriochrome Black T Indicator. Weigh out 50 mg. of Eriochrome black T dye, dissolve in distilled water, and dilute to the mark of a 50-ml. volumetric flask. This dye is satisfactory for the titration for 2 neeks, Tvhen it is kept refrigerated. Ammonium hydroxide, 2 % Perchloric acid, 727& APPARATUS
Coleman, Jr., spectrophotometer, Model 6A. Cuvettes, 19 by 150 mm., or borosilicate glass test tubrs of the same size. PROCEDURE
Standardization of Calcium. Pipet 1.0 ml. of the calcium standard solution into a cuvette and add 1.0 ml. of distilled water and 2.0 nil. of the precipitating buffer solution. Mix well and allow to stand for 1 hour. Centrifuge a t 3000 r.p.m. for 15 minutes and carefully decant the supernatant fluid. Add 4.0 ml. of the 2%, ammonium hydroside solution, mix well, and recentrifuge at 3000 r.p.m. for 15 minutes. Decant the supernate and drain. Add 0.05 ml. of perchloric acid and put in a hot sand bath for 10 minutes. The tube fills with dense fumes of perchloric acid during this period of time. The temperature is approximately in the vicinity of 200" C. Cool, and add 3.0 ml. of water, 3.0 ml. of the titrating buffer, and 0.2 ml. of Eriochrome black T . Set the instriiment to read 0.5 to 0.6 at 660 mp and titrate in the spectrophotometer, mixing ~vellafter the addition of each increment and then recording the absorbances until the upper plateau range has been reached. The intersection of the steeply ascending portion of the curve and the upper plateau represents the end point,. KOblank is used in the t,itration, because dilution does not affect the end point. During the short period involved in the titration no drift was caused by instability of the indicator, vhich is fairly stable, or the battery-operated spectrophotometer. Standardization of Magnesium. Pipet 1 .0 ml. of the magnesium working standard into a test tube and add 1.0 nil. of distilled xater and 2.0 ml. of the precipitahg buffer solution. Mix well and then pipet a 3.0-ml. aliquot into a cuvette or test tube. Add 3.0 nil. of the titrating buffer and 0.2 ml. of Eriochromr black T solution, set the instrument at 0.05 to 0.10 with the sample at 660 nip, and titrate as for calcium. Again, no blank is required, as the sample acts as its o w 1 blank. Cerebrospinal Fluid Analysis. Pipet 2.0 ml. of spinal fluid int,o a cuvette, add 2.0 ml. of precipitating buffer, and allow the precipitate of calcium oxalate to form for I hour. Centrifuge a t 3000 r.p.m. and carefullj- decant the supernatant fluid into a clean container. Pipet 3.0 ml. of the supernate into a cuvet,te, add 3.0 mi. of the titrating buffer and 0.2 ml. of dye, and titrate
1661
ANALYTICAL CHEMISTRY
1662 as for the magnesium standard. Wash the precipitate in the first cuvette with 2y0 ammonium hydroxide and proceed as for the calcium standard. DISCUSSION
Previous work appearing in the literature has shown that the end points in a spectrophotometric titration of calcium or magnesium, when EDTA is used as the titrant along with metal specific indicators, are sharper and more accurate a t the wave length where the absorbance difference is greatest between the metal-containing indicator form and the metal-free indicator form (8, I S ) . Figure 1 shows the spectral curves in the visible range for several of the media encountered in magnesium and calcium determination-Le., water, spinal fluid, and serum. The water solution used contains calcium, magnesium, ammonium phosphate, and sodium and potassium chlorides in the physiological range expected. Absorbance maxima were obtained on the one hand by the addition of a fixed amount of dye to the buffered medium and on the other hand by the addition of a small amount of solid EDTA to the same system, to remove the divalent cations from the dye complexes. In the work of Karsten and associates (13) the wave length of 530 mp was chosen because it best suited their macro system and their instrumentation. But in view of the fact that a greater change in absorbance is obtained beyond 600 mp, it was felt that a greater sensitivity would enable a more effective titration when small amounts of calcium and magnesium are involved. The spectral curves are not quite the same for the different media shown here, although water and spinal fluid, as might be expected, resemble each other closely. -4s Eriochrome black T works well with magnesium and very poorly with calcium (6, l 7 ) , it was necessary t o incorporate magnesium into the EDTA titrant. The amount of magnesium added was varied from 0 t o 30 y per milliliter of titrant, whereas the uncomplexed EDTA was held fairly constant a t 400 mg. per
liter. Curve A of Figure 2 shows the poor data plot graphed for a calcium titration with no magnesium present, while B to E show the curves obtained for 2, 7.5, 10, and 30 y per milliliter of titrants, respectively. -4standard solution containing 10 y of magnesium and 0.4 mg. of EDTA per milliliter was decided upon as most suitable for the titration. This corresponded roughly to about a 0.001M solution of uncomplesed EDTA.
“OOr
G9@t 080t 0.7CJ-
0.00
0
Figure 2.
I
C
1.0
2 .o
Milliliters of Titrant
4.0
Effect of varying magnesium concentration of EDTA in calcium titrations D. 1 0 y p e r m l . E. 30 y perml.
A . No magnesium B. 2 yperml. C. 7.5 y per ml.
.oo
3.0
0.80
64-
z
0.60
2-
a
m LT 0
8
v)
m
a
1.0-
0
g2 0 . 8 -
0.40
a
0.60.20
0.4 -
0.2 -
0 .oo 400
500
600
700
Woveleng t h in M i l l irn i c r o n s
Figure 1. Spectral curves in the visible range for metal-containing and metal-free complexes
Milliliters of Titront
Figure 3.
Effect of variation in dye concentration for magnesium ( l e f t ) and calcium ( r i g h t ) A . 50 y B . 100 y
c.
2007
D . 3007
1663
V O L U M E 2 8 , NO. 1 1 , N O V E M B E R 1 9 5 6
.
Table I. Determination of Absolute Amounts of Calcium and Magnesium in Their Mixtures (lfilliequivalents per liter)
t
0.91-
0
660 m p
et
660 miL
I
U
tn
I
L
Calcium Present
Calcium Found
1.28 2 50 3 75 5.00 2.50 L25 2.50 2.50 2.50 3.75
1 27 2.49 3.65 4 95 2.54 1.23 2.48 2.40 2.42 3.58
Nagnesium Present 2 2 2 2
10 10
10 10
1 OB 2 10 3 15 4 20 2 10 3 15
lfaqnesium Found 2 2 2 2
12 10
10 10 1 17 2 12 3 14 4 18 2 02 3 28
5 4 0 rnk .cy.
Table 11. Determination of 4bsolute Amounts of Calcium and Magnesium in 3lixtures in the Presence of Koriiial Amounts of Sodium, Potassium, and Phosphate (,llilliequi\alents per liter)
V+vXA
v
Calcium Piesent 1 25
0 01
0
! 0
20
I
I
3.0 4.0 1.0 Miliiliters of Titrant
I
2.0
I
3.0
I
4.0
Figure 4. Titration curves for calcium at 540 and 660 rnlr uncorrected ( l e f t ) and corrected ( r i g h t ) for dilution with titrant
2 50 3 75
5 00 2 50 2 50
2 50 2 50 2 50 3 75
Calciuni Found 1 2 3 4 2 2 2 2 2 3
28 46 77
98
46
48 S6
48 50 67
3Iagnes:um Present
31agnesium E ound
2 10 2.10 2 10 2.10 1.05
2 15 2.10 2.13 2.15 1.10 2.13 3.50 4.42 2.20 3.08
2.10 3 15 4 20
2.10 3,l5
portion of the curve with the upper plateau represents the end point of the titration. Yisually, i t
ANALYTICAL CHEMISTRY
1664 differences between the two dye forms, one in the 600- t o 700-mp range, and the other in the 480- t o 560-mp range. It was decided to compare the two areas to see if both were favorable. Figure 4 shows the curves obtained for calcium a t 540 and 660 mp and Figure 5 shows the curves for magnesium a t 520 and
0
Figure 6.
I
I
I
I
1.0
2.0
3.0
4.0
Milliliters of Titront Titrimetric curves for calcium at 660 mp Meq. per liter of spinal fluid
660 mp. The absorbance break is much steeper a t 660 mp. When all curves were corrected for the dilution of absorbance by the titrating solution ( I I ) , those obtained a t 520 and 540 mp. still did not exhibit very large absorbance differences for the two dye forms, while the 660-mp curves were increased by a milch larger factor. However, the end points a t 660 mp for dilutioncorrected 01 -uncorrected data are easy to plot and give the same results, eo that for the purpose of titrating in the low milliequivalent ~ a n g eof normal and pathological spinal fluids 520 or 540 nip appear to be somewhat less desirable. Recovery studies were then carried out to test the accuracy of the procedural techniques involved These include four phases of investigation. 1. Mixtures of calcium and magnesium alone were analvzed according to the procedure described. For several samples, the calcium content v a s kept constant while the magnesium content m-as varied, and then the reverse procedure was followed (Table I ) . The analytical accuracy appears to be excellent. 2 . A second set of mixtures of pure calcium and magnesium solutions contained both cations in the same proportions as in the experiments reported in Table I, but with noimal amounts of sodium, potassium, and phosphate. Recoveries viere made and the results are shorn in Table TI. The accuracy was not decreased by addition of these constituents, which are always preaent in cerebrospinal fluid. 3. -4third set of mixtures was prepared, sindar to those described in Table 11, but t o these were added comparatively large amounts of proteins in the form of human albumin and gammaglobulin, obtained through the Red Cross. The recoveries made are shown in Table 111. 4. A number of pooled spinal fluids rrere tested and to each separately was added a fixed amount of calcium and then magnesium. Each sample was analyzed three times-once for calcium and magnesium t o which no addition had been made, a second time for calcium after calcium had been added, and a third time
A. 1.25 E . 2.50
c.
D.
3.75 5.00
Table 111. Determination of Absolute Amounts of Calcium and Magnesium in Mixtures with Sodium, Potassium, Phosphate, and Proteina
1.0
0.9
(Milliequiralents per liter)
7 P
Calcium Present
Calcium Found
1.25 2.50 3.75 5.00 2.50 2.50 2.50 2.50 2.50 3.75
l,25 2.48 3.79 5.00 2.44 2.51 2.44 2.51
0.8 0.7 Ill
z U
0.6
llagnesiuni Present
2 14 2 05 2 02 2 07 1 05 2 10 3 24 4 18 2 10 3 25
10 10 10 10 05 10
2.44
2 2 2 2 1 2 3 4 2
3.80
3 15
a140 mg. of albumin and 80 me. of Cross were dissolved in the solution.
IIagnesiuiii Found
13
20 10
ilin obtained through the Red
J
0.5 0.4
Table IV. Recoveries of Additions of Calcium and Magnesium Made to Pooled Spinal Fluid" (hlilliequivalents per liter)
0.3 0.2
Sample
Present
A A
3.61
iB
0.I
B
0.0
2.0
1.0
3.0
C C C D D D E E E
4.0
Milliliters of Titrant
Figure 7.
Titrimetric curves for magnesium at 660 mp Meq. per liter of spinal fluid A . 1.05 B.
2.10
C. 3.15 D . 4.20
4
..
2:i1
..
2:52
..
2:kl
..
2:42
..
..
Calcium Added
Found
1:i5 ..
5'00
2:io
5:iz
2:io
4:70
1:i5
3:s3
2:i o
5'
..
..
..
..
..
'
Present 2 20
..
3:68
..
2:50
.. ..
io ..
..
2:i9
..
2'72
Ilagnesiurn Added Found
..
2'
.. io
4.20
1'03
4.63
2: io
4'i4
1'05
3:60
1:03
3 65
..
.. ..
..
..
.. , .
..
..
Random cerebrospinal fluid samples taken from clinical laboratories
V O L U M E 2 8 , N O . 11, N O V E M B E R 1 9 5 6
1655
for magnesium after magnesium had been added. I n other words, all analyses were made but once, but in effect each mixture was analyzed in triplicate. These iesults are shown in Table IV.
Diehl, H., Goeta. C. A,, Hach, C. C., J . A m . Water W o r k s Assoc. 42, 40 (1950). Eldjarn, L., NygaiErd, D., Sveinsson, S. L., Scand. J . Clin. Lab. Inrest. 7, 92 (1955). Fales, F. W., J . Bid. Chem. 204, 577 (1953). Fortuin, J. R.1. H., Karsten, P., Kies, H. L., Anal. Chim. Acta 10,
K i t h the exception of an occasional determination, the analytical results for the recoveries using calcium or magnesium appear to be eqiially good, regardless of xhether pure calcium and magnesium mixtures alone or other mixtures were analyzed. Figure G s h o w the titrimetric curves obtained at 660 mp for varying concentrations of calcium between 1.25 and 5.0 mey per liter of spinal fluid. The intersection of the tangent to the steep ascending slope of the titration curve to a line parallel to the abscissa and tangent to the upper portion of the cuive indicates the end point. A >imilar study ip shoxn in Figure 7 , where increasing concentrations of concentration of magnesium in the range of 1.05 to 4 20 meq. per liter of spinal fluid are titrated a t 660 mp. The end point is graphically determined in the same manner a.; io1 calciiim.
356 (1954).
Friedman, H. S., Rubin, AI. A,, Clin. Chem. 1, 125 (1955). Gehrke, C. W., Affsprung, H. E., Lee, R . C., ASAL.CHEM.26, 1710 (1954).
Goddu, R.F., Hume, D. N., Ibid., 26, 1740 (1954). Greenbiatt, I. J., Hartman, S., Ibid., 23, 1708 (1951). K a r s t e n , P., Kies, H. L., Van Engelen, H. T. J . , DeHoag, P., A n a l . Chim. Acta 12, 64 (1955). Kenny, A. D., Tovernd. S.U., A x . 4 ~ C . H E ~ 26, I . 1059 (1951). Kibrick, A. C., Ross, AI., Rogers, H. E., Proc. Sac. Exptl. B i d . and X e d . 81, 353 (1952). Lehman, J., Scand. J . Clin. Lab. Incest. 5 , 203 (1953). llartell, A. E., Calvin, lI.,“Chemistry of the Metal Chelate Compounds,” Prentice-Hall, New York, 1952.
Pribel, R., “Complexometrie,” Chemapol, Prague, Czechoslovakia, 1954. Shaviro. R.. Brannock. W.W.. AKAL.CHEM.27. 725 (1955). Sobel, A. E., Xedoff, S., Abstracts, 127th Meeting, A\IERICAN CHEMICAL SOCIETY, Cincinnati, 1955, p. 21C. Wilson, A. A , , J . Comp. Pathol. Therap. 63, 294 (1953); 65, 285
LITERATURE CITED
(1) Banexvirs, J. L.. Iiriiiier, c‘. T., ANAL.CHEM.24, 1186 (1952). ( 2 ) Betz, J. D., Sol!, C . .I..J . Am. Wuter W o r k s Assoc. 42,49 (1950). ( 3 ) Buckner. B., Shively, J. -4., Am. J . Med. Technol. 21, 269 (1955). (1) C’heng, K. L., Kurtz, T.. Bray, R. H., ANAL.CHEX 24, 1040 (1952).
(1955). RECEIVED for review January 7 , 1956. Accepted July 18, 1956. Division of Biological Chemistry, 128th Meeting, ACS, Minneapolis, Minn., September 1955. Supported in part by a Grant-in-Aid from the Receiving Hospital Research Corp.
Photometric Determination of Chlorides in Water DAVID M. ZALL, DONALD FISHER, and MARY Q. GARNER U. S.
N a v a l Engineering Experiment Station, Annapolis,
A method has been devised for the photometric determination of small amounts of chloride in water. The method is based on the displacement of thiocyanate f r o m mercuric thiocy-anate by chloride ion and the subsequent reaction of the liberated thiocyanate with ferric iron to form the colored complex [Fe(SCN)]++, which is measured either visually or in a spectrophotometer. Concentrations of chloride as low as 0.05 p.p.m. can be determined.
T
HE literature on the determination of chloridesisvoluminous.
\Yhether present as a required constitumt or as an impuiity, the chloride ion is usually determined by either gravimetric or volumetric methods. The oldest and the classical method is the gravimetric, in which the chloride ion is evaluated as silver chloride. ..inother method frequently employed is the volumetiic, several variations of which are available. The Volhard method, oiiginated by Carpentier ( 7 ) , described by T‘olhard (38), and later improved by Lundbak (65) and others (26), is more accurate than the Mohr method ( 2 7 ) . A comparatively recent method is the mercurometric method, which was developed in 1933 by Dubskj. and Trtilek (9, 10). Diphenplcarbohydrazide v a s used as an indicator in the titration n-ith mercuric nitrate. Other 13-orkers (1, 5 , 8, 20, 62, 29, 32, 54) later adopted this method with some modifications. ill1 these methods, homeever, are not always suitable for the detei mination of micro quantities of chloride. The present investigation was the result of a need for a simple colorimetric method for the determination of less than microgram quantities of chloride in condensate. Luce, Denice, and ilkerlund ( 6 4 ) determined small amounts of chloride turbidimet-
Md.
1 ically. This method, however, lacked the required precision. Other methods (2, 4, 6, 14, 15, 17, 81, 28, 55, S 9 ) for the determination of small amounts of chloride either required special apparatus or lacked the desired simplicity. The method presented here is a modification of that proposed b!. Vtsumi (36, 37) and followed up by Imasaki (16). This modified procedure has been greatly improved and broadened in ita application. The use of ferric perchlorate instead of ferric ammonium sulfate eliminates a variable inherent in the latter iragent. Use of an aqueous instead of an alcoholic solution of inei curie thiocyanate minimizes the glaring blank when visual color comparisons are made. The improved sensitivity thus obtained and the adaptability to either visual or spectrophotometric comparison made it useful for a more varied application. Although designed for the determination of chloride in condensate, it can also be applied in other fields.
REAGENTS
Mercuric thiocyanate, saturated water solution (0.07%). Ferric perchlorate. Dissolve 6 grams in 100 ml. of LV perchloric acid. This reagent may also be prepared by dissolving 14.0 grams of pure iron wire in dilute nitric acid. Upon dissolution of the iron, add 120 ml. of perchloric acid and heat the solution until it fumes. Continue heating until the solution turns purple, then cool and dilute t o 1 liter. PROCEDURE
Place 10 ml. of sample in a 50-ml. volumetric flask, add 5 ml. of (30% perchloric acid, 1 ml. of mercuric thiocyanate, then 2 ml. of ferric perchlorate. Make up to volume and mix well. Allow t o stand for 10 minutes, then read the transmittancy on a spectrophotometer a t 460 mp. The color can also be compared visually with suitably prepared standards.
