tivity was determined again to check the completeness of the extraction (Table
XI). The residual activity after digestion corresponds to a few per cent of the original activity. This residual activity was largely caused by mercury compounds taken up by the sample tube during previous contact with the radioactive seed sample. When the same meal bodies 11-ere counted in new. uncontaminated sample tubes, the average residual activity was only 0.7 = 0.2% of the original activity. This confirms that the mercury derivative is located in the surface layer and is extracted completely by the described procedure. The agreement between the analytical results obtained by the flame method and the radioactive method is demonstrated by the constancy of the ratio A / ( B - C). This ratio could also be determined in an independent way by counts on small samples of the original radioactive solution which was used in the treatment of the seed. Calibration in this manner gave the value of 21 x 10-5 y of mercury per counts per minute. n hich is in harmony with the average (20.1 =t0.8) x 10-5 y of mercury per counts per minute estimated from the data in Table XI. Data for A / ( B - C) in Table X I exhibit a coefficient of variation of 7%. This variation is evidently not caused by the variability of the radioactive determination or of the final flame assay of the aqueous solution of the extracted mercury compound, but is associated with the digestion step.
Analysis of Paper and Pulp. The technique for seed analysis was also used for mercury determinations in paper or in pulp. Twenty milligrams of pulp were heated with 0.05 ml. of concentrated nitric acid in a borosilicate glass tube for 30 seconds. The solubilized mercury compounds were eluated from the pulp by means of 10 ml. of degassed, distilled water. The solution was placed in the photometer setup. The qample size should not exceed 100 mg., because acid treatment in this manner of larger samples is difficult t o control. With the present technique, for samples containing more than 5 p.p.m. of mercury the lower limit is 2 mg. In this case, 3 ml. of water are used for the elution. Examples of this application are given in Table XII. The observed variation is not necessarily connected with the method of sample preparation. The distribution of mercury in commercially treated pulp is probably not homogeneous, because of the method of mixing the pulp with the preservative. ACKNOWLEDGMENT
This work has been supported by the manufacturers of the Panogen and Radam mercury formulations. The author wishes to extend his most sincere thanks to J. M. Blegvad of AB Casco, Stockholm, Sweden, for his kind interest and for permission to publish this invsstigation. The supporting interest of ilke Swensson and K. D. Lundgren
of the Department of Occupational Medicine a t the Carolina Hospital, Stockholm, is gratefully acknowledged. Particular thanks are due to Kajsa Weinar, who carried out the experimental R-ork with great skill and patience, LITERATURE CITED
( 1 ) Ballard, A. E., Stewart, D.
W.,
Kamm. W.0.. Zuehlke. C. W..AXAL. CHEM. 26,921 (1954). ’ (2) Ballard, A. E., Thornton, C. W.D., ISD. ENG.CHEW,ANAL. ED. 13, 893
(1941). (3) Campbell, E., U. S. Atomic Energy Comm.. ReDt. NLCO-595. 93-104 (Oct. 6, i955j. (4) Carlson, 0. T., Bethge, P. O., Svensk Papperstidn. 57, 405 (1954). ( 5 ) Cholak, J., Hubbard, D. hl., IND. ENG.CHEY.,ANAL.ED. 18, 149 (1946). (6) Johansson, A,, Uhmell, H., Acta Chem. Scand. 9, 583 (1955). ( 7 ) Lindstrom, O., J . Agr. Food Chem. 6, 283 (1958). (8) McBryde, W. T., Williams, F., U. S. Btomic Energy Comm., Rept. Y-1178 (h’ov. 20, 1957). (9) Miller, V. L., Svanberg, F., Jr., ANAL. CHEM.29,391 (1957). (10) Monkman, J. L., Maffet, Patricia A., Doherty, T. F., Ind. Hyg. Foundation Am. Quart. 17,418 (1956). (11) Reed,, G... J . Biol. Chem. 142, 61 (1942). (12) Stricks, W.,Kolthoff, I. M., J. Am. Chem. SOC.75, 5673 (1953). (13) Vesterberg, R., Sjoholm, O., Arkiv. Kemi, Mineral. Geol. 22A, S o . 22 (1946). (14) Weiner, I. M., Muller, 0. H., ANAL. CHEM.27,149 (1955). (15) Zuehlke, C. W., Ballard, A. E., ANAL.CHEM.22,953 (1950).
RECEIVEDfor review June 2, 1958. Accepted October 7, 1958.
Microdetermination of Water by Titration with Fischer Reagent E. L. BASTIN, HERBERT SIEGEL, and A. B. BULLOCK Shell Development Co., Emeryville, Calif.
A compact apparatus and convenient procedure have been developed for direct microtitration of 0.01 to 3 mg. of water with Fischer reagent. The method is a 100-fold reduction of conventional macromethods and maintains their proportions and concentrations of reactants, to have the same applicability. Water contamination from room air is prevented by a flow of dry gas through the titration cell, making it serve as its own dry box. This dry gas stream is also arranged as a barrier through which solid or liquid samples and solvents can b e introduced without simultaneous entry of moist air. Ampero-
metric measurement, utilizing the circuit and electrodes of the reverse dead-stop technique, and graphical location of the equivalence point eliminate titration error.
T
micromethod is applicable to a wide variety of materials, ranging from solids to volatile liquids. The measurable concentration of water ranges from 100 to 0.01% or less, with milligram to gram samples. The method is useful for determining water in small samples, and for conserving materials, particularly those whose HIS
preparation is expensive and time consuming. The precision (standard of deviation) of the data is to the water measured, or 3 y when less than 300 y of water are titrated. The procedure is simple and easily performed by operators having only minimum training in microanalysis. A complete analysis takes less than 30 minutes. The Karl Fischer reagent (4) is useful and convenient for titrating water in a wide variety of materials. It also serves in quantitative analyses of organic functional groups through measurement of the water produced or VOL. 31, NO. 3, MARCH 1959
467
1
GALVANOMETER AND POTENTIAL
I h%
( 7 -
\VIRE L E A D S
FEMALE PLUG
J + -
MICRO FLOWMETER 3 5 ML/MIN '
GAS I N L. E T
RESERVOIR 3-WAY SPRING-LOADED STOPCOCK CLAhlP SEALING DEVICE
r%."ib
RUBBER GASKET PLASTIC C A P
q D R Y GAS V E N T
G L A S S TUBING
(LINDE MOLECULAR SIEVE 4A OR P,Os)
I
I
hlODIFlED 2 DRAM VIAL
I
MICROMETRIC SYRINGE B U R E T
MAGNETIC STIRRER
Figure 1. Schematic diagram of apparatus determination of water by Fischer titration
for micro-
PLATIh'Uhl TIHRISG BAR S A L I P L E DISH
Figure
"0"RINGS 3 / l b " x 5 ! : a "
3.
Titration cell assembly
AND 3/a.'xl/Z''
1
SYRlNGE BURET
Female plug, Bakelite body with 17-gage stainless steel hypodermic tube connectors soldered to wire leads M a l e plug, Bakelite body with 20-gage stainless steel hypodermic tube connectors soldered to 0.020-inch platinum wire Dry gas inlet and vent, 16-gage stainless steel hypodermic tubing Platinum electrodes, 0.020-inch diameter platinum wire sealed in 3mm. diameter soft glass tubing; 1 mm. of platinum wire exposed. Platinum wires inside glass tubing insulated with thin-walled glass capillary
;/7/32":EiETER
T H R E A D SIZE 3/4"- IbNF
Figure 2. Sealing device for syringe buret
consumed in stoichiometric reactions with desired compounds. Mitchell and Smith ( l a ) have summarized the chemistry and uses of Fischer reagent. Peters and Jungnickel (14) extended its usefulness and developed a more stable reagent containing methyl Cellosolve instead of methanol. The developments described below are based on their reagent and titration solvents. Few procedures for measuring small amounts of water by titration with Fischer reagent have been published. Mitchell and Smith (16) described a small scale apparatus and procedure for determining 1 to 10 mg. of water, with a sensitivity of about 0.1 mg. Levy, Murtaugh, and Rosenblatt (10) titrated 1 to 25 mg. of water in a small vial sealed with a rubber serum stopper. Wiberley (90) developed an apparatus and procedure for measuring 1 mg. or less of water, primarily in liquid petroleum products. These techniques were not sufficiently sensitive or versatile to meet the needs of this investigation but served as useful guides. The main obstacle which had to be overcome before micro amounts of water could be determined quantitatively was contamination from atmospheric and other extraneous moisture; this was more acute than on the macro scale, which deals with much larger amounts. For example, 1 ml. of air (25' C., 50% relative humidity) contains 10 y of water, three times the 468
ANALYTICAL CHEMISTRY
sensitivity of this micromethod. Beside eliminating atmospheric contamination by a flow of dry gas through a specially designed titration cell, the dry gas stream f l o w out through the opening provided for introduction of a variety of sample containers and serves as a barrier against ingress of moist air during the operation. To capitalize on the experience and applicability of established macroprocedures the same reactant solutions, concentrations, and proportions are used in this micromethod; approximately 1,400 the quantities are employed. APPARATUS
Buret and Reservoir (see Figure 1). Fischer reagent is dispensed from a 0.5-ml. syringe buret graduated to 0.5 pl. (9) (Micro-Metric Instrument Co., Warrensville Heights, Ohio). The syringe was calibrated by weighing delivered quantities of mater ( 5 ) . A spacer was added to the mounting supplied t o prevent movement of the syringe in its clamp and t o ensure reducible positioning. The 100-ml. titrant reservoir has two stopcocks, the upper one closed when the apparatus is. not in use t o prevent entry of moist air. The reservoir is connected to the syringe through a short piece of Teflon tubing, '/*-inch in diameter, thin-walled (Sparta Manufacturing Go., Division of United States Ceramic Tile Co., Dover, Ohio) to provide flexibility and prevent breakage. Mounting supports and clamps for the reservoir and titration
cell fasten to a rod bolted onto the syringe buret base supplied by the manufacturer, so that all three components are secured in a fixed relationship to each other. This compact unit is readily moved without danger of breakage or change in alignment. -4special sealing device containing a rubber O-ring (Crane Packing Go., Morton Grove, Ill.) prevents ingress of moisture into the syringe buret through the annular space between the plunger and the barrel (Figure 2). Fischer reagent deteriorates the rubber O-ring slowly and it must be changed occasionally. A more resistant material would be desirable. The part having the male threads which surrounds the plunger must be a hard metal such as stainless steel, because a soft metal such as aluminum transfers metal t o the plunger and causes contamination and reduction of the reagent in the buret. To reduce the weight of the fitting, the outside piece mas aluminum. The 0ring on the syringe plunger is greased Kith a very thin film of Sisco 300 stopcock grease (Swedish Iron and Steel Corp., Westfield, N. J.) to permit the plunger to move smoothly. The O-ring a t the shoulder of the syringe barrel cushions the fitting against the glass tn avoid breakage. The buret tip on the third arm of the three way stopcock is drawn to a very fine internal capillary (about 0.1 mm.) to prevent diffusion and is bent toward the side of the titration cell as shown in Figure 3 to ensure immersion in the A swirling liquid during titration. standard medical-type hypodermic
needle, of stainless steel and brass and chromium-plated, was unsuitable for long-term use as a buret tip because Fischer reagent standing in it for days corroded the metal slowly and the water-reacting capacity of the reagent therein was reduced. Titration Cell Assembly. The titration cell is shown in Figure 3. The plastic cap is drilled to fit snugly and t o position the buret tip, electrode assembly, and gas inlet tube; these are placed off-center in the titration cell to permit as much free space as possible for the introduction of stmplc and solvents. The gum rubber g sket, 1 mm. thick, restricts passage of room air into the cell but is not gas-tight. The glass cell is equipped with a side arm, 8 mm. in internal diameter, positioned t o permit introduction of solvent and samples into the assembled apparatus by opening directly into the free space left by this off-center positioning when the cell is screwed in place. A rubber serum stopper (No. 15, Catalog KO. 16201, Braun-Knecht-Heiman Co., San Francisco, Calif.), modified by cutting off the inner plug, caps this side arm. A 15mm. section of 16-gage stainless steel hypodermic tubing inserted through this cap provides an outlet for the flowing gas stream. The stirring bar is made by sealing iron wire into 1.3-mm. diameter glass tubing 11 mm. long. The length is critical, because it must rotate freely without hitting the buret tip and electrodes. A magnetic stirrer centered directly under the titration cell rotates the stirring bar. The model sold by Arthur H. Thomas Co., Philadelphia, Pa., is satisfactory; two others were tried and did not maintain a constant rate with such a small stirring bar. The electrode pair is made with unalloyed platinum wire, sealed into a piece of 3 X 60 mm. long soft-glass tubing. The top of the electrode assembly is confined by a metal loop and rod to hold the electrodes in a fixed position in the titration cell. The electrodes and buret tip are adjusted in the plastic screw cap t o dip into 250 pl. of swirling titration solvent in the assembled titration cell without interfering with rotation of the stirring bar. The gas inlet tube should be located as indicated in Figure 3. -4Fisher Elecdropode (Fisher Scientific Co.) was suitable for the entire external circuit. It has a well damped, sensitive galvanometer (0.008 pa. per mm. scale deflection), a series of shunts to reduce galvanometer sensitivity as desired, and a circuit for applying external potentials across the two platinum electrodes. The gas stream flowing through the apparatus is metered by a microflowmeter (Technical Equipment Co., Berkeley, Calif.) and dried in a Schwartz drying tube containing a suitable desiccant. The ground stoppers on the drying tube are closed to protect the desiccant when the apparatus is not in use. Stainless steel hypodermic tubing conducts the dry gas into the titration cell. As indicated in Figure 1, a rubber hose connected to a vacuum line removes pyridine vapors and other compounds
m-hich emerge from the titration cell in the flowing gas stream.
I
REAGENTS
Fischer Reagent. This reagent is prepared as described by Peters and Jungnickel (14) using methyl Cellosolve as solvent. A freshly prepared solution has a water equivalency of approximately 6 mg. water per ml. of reagent. It is standardized by titrating weighed amounts of water by the procedure given below. Dry Gas Stream. A continuous stream of dry gas flows through the titration cell t o exclude atmospheric moisture. Kitrogen was used in most of the experiments because it was convenient and fairly dry. Air was also used successfully. The preferred drying agent is Molecular Sieve 4A (Linde Co., Division of Union Carbide Corp.). Phosphorus pentoxide or a liquid nitrogen-cooled cold trap also was satisfactory. Anhydrous calcium sulfate (Drierite), anhydrous magnesium perchlorate (Dehydrite), or Fischer reagent was not successful. Titration Solvent Mixtures'. Ethylene glycol-pyridine, 4 to l by volume, and pyridine-methanol, 4 t o 1 by volume, mixtures are prepared from dry reagent-grade chemicals. Dry ethylene glycol is also used alone. When more than 0.1 weight % water is present the solvents should be dried by distillation (14). PROCEDURE
To assemble the apparatus, the stopcocks of the clean dry reservoir are first greased with Sisco 300 stopcock grease. Fischer reagent is drawn into the unmounted reservoir through the Teflon tubing. The apparatus is then assembled as shown in Figure 1, omitting the glass titration cell. The syringe buret and tip are rinsed and filled with titrant, and the apparatus is tipped nearly 90" to eliminate air bubbles. The glass titration cell is rinsed with acetone, dried by evacuation a t room temperature, and positioned on the apparatus; the side arm is capped and the vent tube inserted. Dry air or nitrogen is passed continuously through the titration cell a t 35 ml. per minute during the entire procedure. The titration cell is purged for 10 minutes to remove the surface water from its inside walls before insertion of any solvents. A potential of 25 mv. from the Fisher Elecdropode is applied across the platinum electrodes; galvanometer sensitivity is set a t 1X. Rinsing the buret tip with titrant and purging the titration cell for 10 minutes are sufficient after the apparatus has been inactive. General Procedure. About 250 pl. of 4 to 1 ethylene glycol-pyridine titration solvent are placed in the assembled and purged titration cell using a 1ml. turberculin syringe and hypodermic needle through the momentarily uncapped side arm. The magnetic stirrer
8 ' d
I
P
Y G rl
m 3 u
0.
I I
l
l
62
63
64
4h
1
1
1
1
,
456
457
458
459
460
VOLUME OF FISCHER REAGENT, MICROLITERS
Figure 4. Amperometric titrations of solvent and sample with Fischer reagent is adjusted so that the solution swirls rapidly and evenly. To pretitrate the water in the solvents, Fischer reagent is added slowly until a steady current of about 0.5 pa. is indicated on the galvanometer. One-half microliter (one buret division) increments of titrant are added and the current measurements are plotted against the corresponding volume readings as shown in Figure 4. Without resetting the buret, the weighed sample is added through the momentarily uncapped side arm of the titration cell. The sample should not normally exceed 100 mg. nor contain more than 3 mg. of water. Fischer reagent is immediately added, rapidly a t first, and slowly near the end point as temporary current fluctuations occur. When a steady current of about 0.5 pa. is indicated, 0.5-fil. increments of titrant are added and the current measurements are plotted against the corresponding volume reading as before. A straight line is extrapolated through these points t o zero current to obtain the volume of titrant a t the equivalence point. The difference between this graphic amperometric end point and that obtained in pretitration of solvent is the amount of Fischer reagent required to react with the water in the sample. This water content is calculated from the standardization value of the titrant. Small amounts of solid and viscous liquid samples (less than 15 mg.) are weighed in a small round platinum dish (made from 0.006-inch platinum sheet about 6 mm. in diameter and 2 mm. deep). The dish and sample are inserted through the uncapped side arm of the titration cell and placed under the stirring bar in the rounded bottom of the titration cell as shown in Figure 3 by manipulation with forceps. Larger portions of solids are weighed into the titration cell by difference. VOL. 31, NO. 3, MARCH 1959
469
Table I. Comparison of Quantities Used in Micro and Macro FischerWater Titration Methods
Item Titration cell size, ml. Solvent volume, ml. Fischer reagent strength, mg. water per ml. Optimum amount of water in sample, mg. Titration volume of above amount of sample, ml. Buret volume, ml. Buret graduations, ml.
Macro 250 15-30
Micro 5 0.25
5-6
5-6
100-200
1-2
20-40 50
0,2-0.4 0.50
0.1
0.0005
HYPODERMIC TUBING V E S T CAPILLARY
TITRATION SOLVENT CELL
Volatile liquid samples are transferred into the pretitrated titration solvent from a small glass bulb having a capillary stem by applying heat (Figure 5). Alternate heating and cooling several times moves solvent in and out of the capillary, rinsing the sample into the solution. Fischer reagent is added until a steady reading of about 0.5 pa. is maintained during additional rinsings. Increments of titrant complete the titration. Less volatile liquids are weighed by difference from a modified Lunge weighing bottle (Braun-Knecht-Heiman Co., San Francisco, Calif.). The lower portion of the weighing bottle, cut off just above the lorn-er stopcock, is used. The delivery tip is drawn to a small internal diameter and the last 3-mm. section is bent about 30" from the axis. Immediately after titration, the mixture is removed from the titration cell by sucking it out through a section of hypodermic tubing connected to a suction trap, without disconnecting the titration cell from the apparatus, and with the stream of dry gas flowing. The apparatus is then ready for the next analysis. To remove the platinum dish, the titration cell is disconnected from the apparatus. When this is done, the cell is cleaned, dried, reassembled on the apparatus, and purged with dry gas as described before the next titration. Standardization. Fischer reagent is standardized by weighing 2 t o 3 me. of distilled water in a small glass bulb with a capillary stem and titrating by the procedure for volatile liquids Special Procedures. When ketones or organic acids are being titrated for water with Fischer reagent, the reaction mixture must be kept near 0" C. to minimize side reactions (14). This is conveniently done by sandwiching a shallow glass dish containing an ice slurry between the titration cell and the magnetic stirrer. To titrate the water content of basic nitrogen compounds with Fischer reagent they must be neutralized to avoid high results (12) by adding 150 pl. of dry acetic acid to 150 pl. of the titration solvent in the cell before pretitration of the water in the solvents. The solution is kept near 0' C. during pretitration and titration of the sample. 470
ANALYTICAL CHEMISTRY
Figure 5. Technique for introducing volatile liquids into titration cell
:r " 0
100
100
100
400
SOP
bo0
100
APPLIED POTENTIAL. MILLIVOLTS
Figure 6. Current-voltage curve of Fischer reagent in ethylene glycolpyridine titration solvent
An established method for determining the small amounts of water present in hydrocarbons is extraction from the relatively large sample required into a small volume of ethylene glycol, followed by titration of the glycol phase with Fischer reagent (12, 14). Th'is was successfully carried out in the authors' apparatus, using 280 pl. of ethylene glycol and as much as 1 ml. of benzene. A methanol-pyridine (1 to 4 by volume) mixture \vas used as the titration solvent for epoxy resin samples. Using this quantity of methanol, the temporary galvanometer deflections which occur shortly before the end point are sufficiently enhanced to be troublesome. Reducing the galvanometer sensitivity by a factor of 2 compensates this behavior adequately. EXPERIMENTAL FACTORS
Reactant Concentrations and Proportions. The micromethod was given
the same applicability as established macroprocedures (12, 14) by using the same reagents in smaller amounts in
the same proportions. Thus the equilibria and rates of reactions involved are not changed in the scaledown. Table I compares the quantities involved in the micromethod with those of an established macromethod used in this laboratory (14). The scale factor is 1/100. Less concentrated Fischer reagent, corresponding to 1 to 2 mg. of water per ml. has been used for measuring small amounts of water (12, 16, 20). The stoichiometry of the reaction with water is reported to be unchanged with Fischer reagent as dilute as 0.15 mg. of water per ml. (11). It seems likely that the sensitivity and scope of this micromethod could be extended to smaller amounts of water by using more dilute Fischer reagent, although end point location and prevention of contamination by extraneous moisture would require further attention. The principles and techniques set forth are not restricted to the scale of operation described here. For routine use by operators not trained in micromanipulations, a reduced-scale apparatus and procedure, scaled-up fivefold from that described here, has been convenient in this laboratory. Amperometric E n d Point. The first trials showed that in this scale of work, a visual end point was impractical. The color change was very gradual and a relatively large overtitration was necessary before the orange-red of excess Fischer reagent became visually discernable in the small volume being titrated. Accordingly a n electrometric end point, similar in principle to the reverse dead-stop technique frequently used by others (12, IC), was developed. As described under apparatus and illustrated in Figure 3, two small platinum electrodes in the solution being titrated have a low potential applied across them. Any current produced is measured with a sensitive galvanometer. To achieve sensitivity and eliminate titration error (8) the equivalence point of the direct titration of water with Fischer reagent is located graphically as in other amperometric titrations ( 7 ) . The currents produced by the addition of successive small increments of excess titrant are measured and plotted against the corresponding volumes. Extrapolation to zero current, as illustrated in Figure 4, determines the titrant volume at the equivalence point; no measurable current flows before the end point. The dead-stop end point technique is a special case of amperometric measurement of concentration of one of the species involved in the titration (1, I , 6, 17). I n the titration of water with Fischer reagent, the effective couple for electrode reactions are the 12-1species (as their pyridine complexes). Because iodine is reduced to iodide
by the reaction with water, the solution contains no iodine as long as there is water present. With no iodine present there is no cathode reaction, and hence no current flows. As soon as unreacted Fischer reagent is present, I t and I- are both present, the electrode reactions start, and produce current. Parallel to observations on other systems with dead-stop electrodes (1, 6, 15, 18), the current produced immediately after the end point varies linearly with the concentration of excess Fischer reagent, The current-voltage curve of a very dilute solution of Fischer reagent in ethylene glycol-pyridine titration solvent (about ‘? pi. in 250 pl. of solution) was measured using the platinum electrodes and apparatus described above, and is shown in Figure 6. In the region from 75 to about 550 mv. the current produced is limited by the rate of diffusion of I 2 to the cathode; a t higher potentials other electrode reactions occur. Thus the maximum current per unit volume of excess Fischer reagent will be obtained a t 7 5 mv. or more applied potential. Measurements of end point sensitivity, defined here as the slope of the current-excess titrant concentration graph illustrated in Figure 4, a t various applied potentials were in accord with the current-voltage curve. Although maximum end point sensitivity would be obtained in this system at 7 5 mv. or more applied potential, 25 niv. were selected for practical use because galvanometer readings were steadier. In this connection Bradbury ( I ) found from theoretical considerations that the pquivalence point would be measured more accurately with lower rather than nith higher applied potentials. In common nith other amperometric riieasurements ( 7 ) ,the current produced by a small excess of Fischer reagent Iaried n ith the rate of stirring and n ith movement of the electrodes. It was necessary to mount the electrodes rigidly and to rotate the magnetic stirring bar rapidly a t approximately constant speed to obtain steady current measurements. In the reverse dead-stop method for detecting the end point in the direct titration of n-ater nith Fischer reagent, temporary drifting deflections of the galvanometer are frequently observed liefore the end point. These are ascribed to a slower than instantaneous rate of reaction of Fischer reagent with small concentrations of water (12, 14). The addition of excess Fischer reagent and back-titration with a standard solution of water in methanol are frequently recommended to avoid this (12, 19). These temporary drifting galvanometer deflections in the direct titration can be minimized by reducing the current generated in the titration cell, reducing the sensitivity of the
current sensing device, and damping the galvanometer movement. Peters and Jungnickel (14) found that temporary galvanometer deflections in advance of the end point were less in ethylene glycol-pyridine titration solvent than in methanol because the current generated is smaller in the former. In the authors’ experiments relatively large currents were readily obtained with electrodes having moderate areas, but the transitory currents before the end point were too large. The small electrodes with minimum area described above and s h o m in Figure 3 together with the sensitive, adequately damped, galvanometer in the Fisher Elecdropode n-ere a good compromise, giving adequate end point sensitivity with tolerable temporary deflections before the end point. Current readings after the end point were stable and were readily distinguished from the drifting deflections iyhich occur just before the end point. The apparatus and procedure were designed primarily for use with the ethylene glycol-pyridine titration solvent. Mixtures containing methanol are titrated simply by reducing the galvanometer sensitivity; there is a series of shunts on the Elecdropode for this purpose. This compensates for the distinctly enhanced end point sensitivity and concurrent enlarged transistory currents before the end point which occur in the presence of methanol. Extension to other solvent mixtures may require other adjustments of galvanometer sensitivity. The graphical end point adopted has no inherent titration error, as do the reverse dead-stop end points commonly employed, because these depend upon the presence of sufficient excess titrant concentration to cause a measurable current in the sensing device. Confirming Peters and Jungnickel’s findings ( I d ) , the end point sensitivity, in current per unit volume of excess titrant, varies distinctly with the composition of the solution a t the end point. The sensitivity is greater in methanol than in ethylene glycol-pyridine mixtures and is the least in ethylene glycol. Thus the titration error varies n-ith solvent composition as well as volume when reverse dead-stop end points are used; graphical extrapolation to zero current eliminates this variation. Figure 4 illustrates the change in end point sensitivity that occurs during a typical microtitration due to changes in volume as well as solvent composition; the latter occurs because Fischer reagent has a different composition than the titration solvent. Prevention of Contamination with Water. Water is present in significant amounts in the atmosphere and in or on nearly all materials, including the surfaces of laboratory glassware. Be-
cause very small quantities of water are measured in this micromethod, special precautions are necessary to exclude atmospheric moisture during the titration. Also a variety of kinds and sizes of sample have to be introduced into the titration cell without concurrent contamination with moist air, which can contain 10 y or more of mater per ml. These objectives are accomplished by maintaining a flow of dry gas through the titration cell via the stainless steel tubing shown in Figure 3. As in the dry box described by Fiebig, Spencer, and McCoy (S), the stream of dry gas flowing out the momentarily opened side arm provides a barrier against the ingress of moist air. The gas stream is adjusted to a rate that prevents any significant amount of moisture from entering the titration cell when the side arm is uncapped briefly to introduce samples or solvent through the barrier of flowing dry gas. In this apparatus, a flow of 35 ml. per minute was necessary; at this rate less than 3 y of water entered the cell through the uncapped side arm in 30 seconds. At slower rates of gas flow measurable amounts of water entered the cell-for example, about 10 y entered in 30 seconds with a gas flow rate of 10 ml. per minute. The amount of moisture contamination was determined by starting with a solution containing a small excess of Fischer reagent vr-hich gave an upscale galvanometer reading of 1 to 1.5 pa. Any water entering the solution from the uncapped side arm consumed Fischer reagent and decreased the galvanometer reading. The amount of titrant required to return the galvanometer to its original reading measured the amount of water that entered. With the present apparatus, a continuous flow of gas is necessary even when the side arm is closed completely with a solid rubber cap. If the gas flow is stopped, moisture leaks into the cell a t a significant rate, presumably around the buret tip and electrodes, because these are not tightly sealed into the titration cell cover. Moisture contamination is not detected if the gas flow rate is reduced from 35 t o 10 ml. per minute after capping the side arm of the titration cell, Future development will be aimed a t a gas-tight cell which passes a stream of dry gas only when the side arm is opened to admit samples or solvents. Although water has an appreciable vapor pressure, an insignificant amount is w e p t away during titration of a sample by this flowing gas stream. Fischer reagent is added rapidly at the beginning of the titration and most of the water is consumed in 1 minute or less; when the titration rate is slotTed down near the end point the vapor pressure of the remaining water has become VOL. 31, NO. 3, MARCH 1959
471
Table II.
Comparison of Micro and Macro Fischer-Water Titration Methods
Microtitration Sample Titration] size, mg. PI.
Material Adipic acid
N-( Hydroxyethy1)diethylenetriamine, commercial grade Allyl alcohol-propyl alcohol-water mixture Ethylene glycol-water mixture Epoxy resin Benzene-water (Water present = 0.0359 wt. Yc)
Table 111. Precision of Standardization of Fischer Reagent by Microtitration of W a t e r
Water Weighed, Mg.
Date 9-29
Fischer Reanent ConcenTitratration, tion, mg. H,O/ml. PI.
2.235 2.683 2.737 3.137 2.328 2.721 1.395 2.350 3.152 4.500 2.103 2.357 1.478 3.240
360.5 431.0 9-30 443.5 510.5 10-3 373.0 442.0 10-7 235.0 388.0 11-4 564.5 808.0 11-10 388.5 437.5 11-18 268.5 588.0 Pooled std. dev. = 3=0.7%
6.20 6.23 6.17 6.14 6.24 6.16 5.94 6.06 5.58 5.57 5.41 5.39 5.50 5.51
50 70 45 22 8.4 1.8 37 24 34 23 1 ml. 1ml.
18 24 450 225 800 170 395 257 36 27 54 53
0.19 0.18 5.38 5.48 52.2 52.7 5.01 5.04 0.50 0.52 0.0361 0.0354
0.22 5.45 52.3 5.02 0.50 0.0364
rather than Drierite would probably improve the stability of the reagent. The inner glass surfaces of titration vessels have been dried before use by various means (10, 20,ZI). With glassware stored in room air or dried with acetone, the flowing stream of dry gas (35 ml. per minute) in this apparatus dried the surface sufficiently in about 10 minutes; a solution containing a very small excess of Fischer reagent put in thereafter gave a steady upscale galvanometer reading. Any residual moisture on the cell walls above the solution after the 10-minute purge was swept away by the gas stream before it reacted with the Fischer reagent. The flowing gas stream also keeps moisture out of the cell during removal of titrated mixtures in preparation for the next analysis. STANDARDIZATION OF FISCHER REAGENT
small. However, appreciable amounts of water were lost if the solution of sample in titration solvent stands untitrated in the flowing dry gas stream (35 ml. per minute) for 5 minutes. The results in Table I1 show that under the conditions of the procedure adopted losses of water are too small to be measured. If a gastight cell were developed, this source of water loss would be minimized because it should not be necessary to purge such a cell continuously. The apparatus for storing and dispensing Fischer reagent illustrated in Figure 1 was devised to eliminate contamination from moisture. The stopcock a t the top of the reservoir, which is closed when reagent is not being dispensed, was suggested by Wiberley (20). I n this apparatus, stored titrant deteriorated no faster (Table 111) than the rate expected from macro experience with Fischer reagent made with methyl Cellosolve (14). However a drying tube containing either Molecular Sieve 4A or phosphorus pentoxide 472
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ANALYTICAL CHEMISTRY
O’Brien, and Frediani ( I S ) , using sodium tartrate dihydrate as primary standard, was tried, but the solubility of the salt in ethylene glycol-pyridine titration solvent was low and titration was prolonged. Standard solutions of water in alcohol, either methanol or ethylene glycol (12, d o ) , were used satisfactorily. Because these solutions require prior quantitative preparation and/or analysis and do not lead to more precise results, direct standardization with distilled water n.as preferred.
Fischer reagent is not entirely stable and loses its strength by side reactions a t an appreciable rate and must be standardized frequently (18, 14). To have a direct and independent standardization in the micromethod, and in conformance with established practice in this laboratory ( I C ) , a technique for titrating weighed samples of water was developed. Two to 3 mg. of distilled water are weighed in a small glass bulb having a capillary stem several centimeters long. The water is transferred to the pretitrated solvent and titrated with Fischer reagent as described in the procedure. The standardization history of one batch of Fischer reagent, stored in the apparatus depicted in Figure 1, is tabulated in Table 111. Duplicate standardizations were performed on 7 different days over a period of 6 weeks; the standard deviation of an individual measurement in this group is *0.7%. Other techniques for standardizing the Fischer reagent were also considered. The method published by Keuss,
RESULTS
To test this procedure for the determination of water, several different materials were analyzed by the micromethod and also by established macroprocedures used in this laboratory (14). The results are tabulated in Table 11; in all cases good agreement was obtained. The 1%-atercontent varied from less than 0.1 to about 50%. The synthetic water in benzene solution was prepared to contain 0.0359 weight 70water. Ethylene glycol was used as titrant solvent (14); stirring the two immiscible layers extracted the water into the glycol, where it reacted with Fischer reagent. The results in Table I1 demonstrate the success of this procedure. The amine sample was titrated in a solvent containing excess acetic acid to neutralize the amine and the mixture was chilled during the titration to avoid high results (12). The epoxy resin analyzed is more soluble in a mixture containing methanol than one containing ethylene glycol; accordingly the alternate solvent mixture, methanol-pyridine (1 tc 4 by volume) was used with this sample. The data in Table I1 show, as expected, that this micromethod is applicable to a wide range of materials now being routinely analyzed on a macro scale; only about 1/100 as much sample is required. The data in Tables I1 and I11 show that results by the micromethod have a standard deviation of about i l % of the water measured, or 3 y when less than 300 y of water are titrated. ACKNOWLEDGMENl
The authors are indebted to R. K. McCoy for design of the sealing device used on the syringe buret. LITERATURE CITED
(1) Bradbury, J. H., Trans. Faraday. SOC. 49, 304 (1953); 50, 959 (1954). (2) Delahay, Paul, ‘(New Instrumental
Methods in Electrochemistry,” Inter-
science, New York, 1952. (3) Fiebig, E. C., Spencer, E. L., McCoy, R. N., ANAL.CHEM.29, 861 (1957). ( 4 ) Fischer, Karl, Angew. Chem. 48, 394 (1935).
(5) Kirk, P. L. “Quantitative Ultramicroanalysis,” diley, New York, 1950. (6) Kolthoff, I. M., ANAL. CHEM. 26, 1685 (1954).
(7) Kolthoff,’I.M., Lingane, J. J., “Polarography,” 2nd ed., Vol. 11, p. 887, Interscience, New York, 1952. (.8.) Kolthoff, I. M., Steneer. V. il.. “Volumetric Analysis,” 2Gd ’ ed., Vol: I, Chap. VI, Interscience, New York, *n>n
lY4L.
(9) Lazarow, Arnold, J. Lab. Clin. M e d . 35, 870 (1950). (10) Levy, G. B., Murtaugh, J. J., Rosen-
blatt, Maurice, ISD.ENQ.CHEM.,ANAL, ED. 17, 193 (1945).
(11) Mitchell, John, Jr., E. I. du Pont de Nemours & Co., Inc., Wilmington
Del., private communication.
(12) Mitchell, John, Jr., Smith, D. M.,
“Aquametry,” Interscience, New York,
1948. (13) Neuss, J. D., O’Brien, M. G., Frediani, H. A., ANAL. CHEM. 23, 1332 (1951). 114) Peters. E. D.. Junenickel. J. L.. ‘ Ibid., 27,’450 (1955). (15) Potter, E. C., White, J. F., J. ’ A p p l . Chem. 7 , 309 (1957): (16) Roth, C. F., Mitchell, John, Jr., A N A L . CHEM. 28, 1502 (1956).
(17) Stock, J. T., Metallurgia 55, 48 (1957). (18) Stone, K. G., Scholten, H. G., ANAL. CHEM.24, 671 (1952). (19) Wernimont, Grant, Hopkinson, F. J., IND. ENQ.CHEM., ANAL.ED. 15, 272 (1943). (20) Wiberley, J. S., ANAL. CHEM. 23, 656 1951). (21) Jakubik, M. G., J . Chem. Educ. 35, 5(1958).
RECEIVEDfor review May 2, 1958. Accepted September 19, 1958. Division of Analytical Chemistry, 133rd Meeting, ACS, San Francisco, Calif., April 1958.
Microtitration of Calcium to Visible End Point in Presence of Magnesium Sodium (Ethylened initri1o)tetra aceta te as Titrant SIDNEY J. SOCOLAR’ and JAMES 1. SALACH College and Deparfmenf o f Physiology, The University o f Chicago, Chicago, 111. ,Calcium of the order of 1 y or less can b e determined by titrating to a visible end point with sodium (ethylenedinitrilo)tetraacetate, obviating the need for photometry. The procedure employs calcein indicator and is therefore applicable in the presence of magnesium. The standard deviation of an individual determination is.about 0.03 y of calcium.
S
procedures have been described for the determination of microgram and submicrogram quantities of calcium (4-6). All are photometric. One of these ( 4 ) is a n adaptation of the well known method wherein the end point of a titration with (ethylenedinitri1o)tetraacetic acid (EDTA) is indicated by murexide dye. The others (6, 6) employ organic dyes whose colors are sensitive functions of calcium ion concentration over a considerable range. Volumetric titration to a visible end point is often more convenient than photometry. Such a procedure has been developed and applied to quantities of calcium as small as 0.2 y . It appears to be simpler and, for many applications, more rapid than at least two of the previous methods (4, 6). Within broad limits it avoids the problem of magnesium interference. The procedure reported here employs calcein, the end point indicator described by Diehl and Ellingboe ( 2 ) . They noted that calcein in highly alkaEVERAL
Present address, 430 West 118th St., Kew York 27, K . Y.
line solution is normally brown, but assumes a yellow-green color in the presence of calcium ions, which jt complexes. At high p H calcein binds magnesium far less strongly than it binds calcium. [The color of the calceincalciuni complex has since been ascribed to fluorescence, largely absent a t high p H if the calcein is free ( 7 ) . ] The procedure described here does not discriminate between strontium or barium and calcium; hon-ever, interference by copper or iron is prevented by the addition of cyanide ( 2 ) . Neither sodium nor potassium interferes. APPARATUS AND REAGENTS
Titration Vessels. The titration is done in a 4-ml. white porcelain crucible (Coors No. 0000, high form). Crucibles used concurrently should not differ perceptibly in color, as a slight variation in whiteness of sample and blank crucibles interferes seriously with determination of the end point. Glassware Treatment. Crucibles and glassware, except for silicone-coated volumetric pipets (used for all but strongly alkaline solutions), are cleaned in the conventional manner, then soaked for 6 hours in 2N nitric acid, and rinsed with metal-free water until the effluent rinse is neutral to methyl red (1). Titration Light. The titration is done by the condensed light from a tungsten source passed through a triple thickness of Kodak Wratten filter No. 78 AA. The source used by the authors was the lamp-type G. E. G161/2-29 (100 watts, 120 volts), in a microscope illuminator. This light provides good contrast between the colors of the calcium-free
blank and the calcium-containing solution. Buret. A Gilmont ultramicroburet (Emil Greiner Co.) of the steel piston in mercury type was used. The instrument has a 0.1-ml. capacity and its dial is graduated in 0.1-pl. divisions. Magnetic stirring is provided during the titration. Water. Metal-free water is prepared by passing singly distilled water over Amberlite MB-3 (analytical grade) ion exchange resin. Titrating Solution. A solution 0.002M in EDTA is prepared from the disodium salt according to Buckley et al. (I) and standardized against Iceland spar. Hydroxide-Cyanide Solution. A single solution is prepared 1N in sodium hydroxide, 0.7N in potassium cyanide. Indicator. Calcein indicator is purchased in powder form from the G. Fredrick Smith Chemical Go., Columbus, Ohio. For use, 1 drop of a 2% stock solution (2) is diluted with water to 25 ml. Observed color changes show that the dissolved indicator is unstable in ordinary light; therefore, solutions are kept in lighttight (foil-covered) containers. This instability is more readily noticed in dilute solution. The titrant, 2% indicator stock solution, and hydroxide-cyanide solution are stored in polyethylene bottles. Iceland Spar. The Iceland spar used in this work was supplied by the J. T. Baker Chemical Co. and was designated “for standardizing.” PROCEDURE
Into a 4-ml. crucible are pipetted 0.5 ml. of sample, 0.3 ml. of hydroxidecyanide solution, and 0.5 ml. of diluted VOL. 31, NO. 3, MARCH 1959
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