Moisture Determination with Karl Fischer Reagent UtiIizing a n Ent ha Ipimet ric Te mperat ure PuIse Sir: Moisture content measurements of organic solvents, of solids and of gases, described in the relevant literature (4,68, 14), have been based upon principles of nucleonics, capacitance, conductivity, photometry, chromatography, and coulometry. The predominant routine analytical approach is titrimetry with the Karl Fischer Reagent (KFR). The empirical stoichiometry of the Karl Fischer reaction (1, 6, 6), and the difficulty in getting a clearly defined end point in some systems prompted the present study. Conspicuous by its absence in the literature is any reference to the use of an end point depending on the heat of the Karl Fischer reaction. This is surprising, because the very pronounced exothermic heat of the Karl Fischer reaction (- 16.1 kcal. per mole of water) is qualitatively evident whenever water is added to Karl Fischer reagent ($,I@. The purpose of this communication is to alert analytical chemists to a new and simple method based on the utilization of a temperature pulse in an adiabatic system as a direct linear measure of moisture content. The met,hod described below represents an application of the principles of “Direct Injection Enthalpimetry” (DIE) (2, 13). A three-step procedure was used. EXPERIMENTAL
Temperature pulses were recorded with the aid of a Model 4-8350 AMINCO TitrsrThermo-Mat (10) equipped with a special accessory for handling 1- or 2-ml. sample volumes (2). An air operated buret (11) was used to deliver water in highly reproducible increments of 10 or 20 pl. (3). Step I (Blank). A single-phase liquid mixture consisting of 1,000 ml. of K F R plus a 1.000-ml. aliquot of a dry sample (e.g., a miscible alcohol) was pipetted into an adiabatic
Table 1.
-11
I
20+
2.O0c
Water, mg.
Found
III
ANALYTICAL CHEMISTRY
0 W
rc W
n L
z 0 W
15 sec Time Figure 1.
Typical enthalpimetric temperature pulses
Excess woter (20PI.) injected into 1000 MI.of KFR plur: 1. 1000 MI. of blank; 11. 1000 MI.of moist “unknown”; 111. 1000 MI. of solution with known amount of water
reaction cell and allowed to attain temperature equilibrium. The temperature was monitored with the aid of a thermistor bridge, which had the sensitivity of a 250-junction thermccouple, in which the unbalance potential ( = l o mv. per degree) varied linearly with temperature. The titer of the KFR corresponded to approximately 5 pl. (mg.) of water per ml. of KFR. Pure water, 20 pl. (i.e., a fourfold excess) were then rapidly injected into the adiabatic cell. The unbalance potential of the thermistor bridge, corresponding to the temperature pulse, was recorded yielding the plot illustrated in Figure 1, Curve I.
4 3 1 1 1.5 0.5 1 0.5
The temperature increment, identified in the figure as AT”, represents a measure of the amount of water, with which the KFR in the adiabatic cell is capable of reacting. Under the prevailing experimental conditions (virtually invariant heat capacity and excess of water) the integral heat evolved, Q0 a ATo, represents a linear measure of the effective titer of the KFR (IS). Step I1 (Measurement of the Unknown). Evervthing else being eaual. Step I’was repeated-using a 1.306-ml: aliquot of a moist sample whose water content was t o be determined. The corresponding temperature pulse is plotted in Figure 1 as Curve 11. The temperature increment, ATz, is correlated as follows with the amount of water, W,, present in the unknown:
Relative moisture
W , = k (ATo - AT,) (1) where W , is expressed in micrograms
0.030 0.043 0.061 0.150 0.165 0.20 0.225 0.234
and k is a proportionality constant. Step I11 (Standard Addition). Another temperature pulse was recorded under conditions identical to those described in Steps I and I1 above, except that the 1.000-ml. aliquot sample added to the K F R in the adiabatic cell contained a known amount of water, yielding Curve I11 in Figure 1. This known amount of water is correlated with the corresponding temperature increment AT8,
content,
* Calculated from: Milligrams water added and weight of solution titrated. (Density of KFR = 1.15 grams/ml.; density of methanol = 0.79 gram/ml.) 1750
t
a
Precision,a % Accuracy, %
0.46 0.90 1.20 2.90 3.10 3.85 4.50 4.60 Standard deviation of 3-5 replicates. 0.42 0.85 1.18 2.85 3.20 3.90 4.40 4.55
E
.-0
W
Precision and Accuracy in a Representative Series of Determinations
Added
0
f - - - - - r*
yob
by an analog of Equation 1, viz.:
W , = k (ATo - AT,)
(2)
where the subscript s identifies the standard or “known” amount of water. Eliminating k from Expressions 1 and 2 and solving for W , yields
- AT, w,= w,ATo ATo - ATa
(3)
Typical performance data are presented in Table I. RESULTS A N D DISCUSSION
The method described in this communication has two distinctive features. It correlates moisture with a direct temperature signal measured as the deflection of a recording millivoltmeter; and the measurement is performed in the presence of excess water. This is a signal advantage. This investigation has revealed that in the presence of excess water the reaction proceeded rapidly to virtual completeness. The enthalpimetric temperature pulse method for determination of water described in this communication is of quite general applicability. For instance, moisture in gases can be determined by measuring a heat pulse similar to those illustrated in Figure 1, subsequent to the injection of a gaseous sample directly into a solution of K F R in the adiabatic cell. The water content of immiscible liquids, solid samples, and slurries can
similarly be determined by equilibrating these samples in an adiabatic cell with KFR, and measuring the temperature pulse obtained upon injection of excess water. Measurement of moisture content is made more direct by calculating the reciprocal, l / k , of proportionality constant shown in Step 111, Equation 2. This is expressed as millivolts per microliter of water reacted. This constant is experimentally determined by obtaining a series of enthalpographs using known standard solutions, and a plot of millivolts vs. water reacted is made. Moisture content of the unknown sample is determined from a single experimental data point which is referenced to the curve or the proportionality constant. Experimental data indicate for Ilk: 16.1 mv. per pl. water reacted for a 1-ml. test solution and 13.5 mv. per pl. for a 2-ml. test solution. A more detailed study using up to 100 ml. of solution to extend the dynamic range of moisture content together with data on synchronous addition of water to KFR will be reported in the future. LITERATURE CITED
(1) Fisher, K., Angew. Chem. 48, 394
(1935).
(2) Jordan, J., Henry, R. A., Wasilewski, J. C., Microchem. J . 10, 260 (1966). (3) Marinenko, G., Taylor, J. K., J . Res. Natl. Bur. Std. 70c No. 1, Jan.-
March 1966.
(4) Meyer, A. S., Jr., Boyd, C. M., ANAL.CHEM.31, 215 (1959). (5) Mitchell, J., Smith, D. M., “Aquametry”, Interscience, New York, 1948. (6) Mitchell, J., Jr., “Treatise of Analytical Chemistry,” I. M. Kolthoff and P. J. Elving, eds., Part 11, Vol. 1, pp. 69-206, Interscience, New York, 1961. (7) Schwecke, W. M., Nelson, J. H., ANAL.CHEM.36, 689 (1964). (8) Swensen, R. F., Keyworth, D. A., Ibid., 35, 863 (1963). (9) Wasilewski, J. C., Reprint N o . 233, American Instrument Co., Inc., Silver Spring, Md. (10) Wasilewski, J. C., (to American Instrument Co.,) U.S. Patent 3,160,477 (Dec. 8, 1964). (11) Wasilewski, J. C., Dorman, H. E., (to American Instrument Co.) U. S. Patent 3,180,527 (April 1965). (12) Wasilewski, J. C., Miller, C. D., Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, 1966; Abstract No. 22, pp. 64-5. (13) Wasilewski, J. C., Pei, P. T-S., Jordan, J., ANAL. CHEM. 36, 2131 (1964). (14) Wickett, J. Allan, Chem. Processing, 25, No. 15, p. 20, (1962). J. C. WASILEWSEI~ C. DAVIDMILLER American Instrument Co., Inc. 8030 Georgia Ave. Silver Spring, Md. RECEIVEDfor review July 18, 1966. Accepted September 2, 1966. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., February 1966. 1 Present address, FMC Corp., Chemical Research & Development Center, Princeton, N. J. 08540.
Gas Chromatographic Separation of Some Iodine Compounds of Serum SIR: The accurate determination of thyroid hormones is of great importance in biomedical investigations and in the pharmaceutical industry, and there is much interest in improving methods. At present, determination of these materials generally involves the use of paper, column, or thin layer chromatography and final quantitation is almost exclusively performed by the cericarsenite color reaction first described by Sandell and Kolthoff (3). This reaction, although extremely sensitive, has in practice the serious shortcoming of being nonspecific, thus interferences by both organic and inorganic substances are frequent even when preliminary separations are performed. The present study was undertaken to develop a method whereby the higher resolution and greater ease of separation by gas chromatography could be utilized. Because of the low vapor pressure of iodinated tyrosines and thyronines, it
was necessary to work with derivatives. Satisfactory results have been obtained with N,O-bistrifluoroacetyl methyl esters, which were prepared by modifying a procedure employed for amino acids (4). Details are given in the following paragraphs. EXPERIMENTAL
Derivatives of individual compounds and of a composite mixture were made for 3-iodotyrosine (MIT) ; 3,5-diiodotyrosine (DIT) ; 3,5-diiodothyronine (Tz) ; 3,3’,5-triiodothyronine (T3) and 3,3’,5,5‘ - tetraiodothyronine (TJ. These materials were all obtained from Sigma Chemical Co. Suspensions containing 10 pmoles of the respective compounds (10 pmoles of each component for the mixture) in 10 ml. of anhydrous methanol were treated with dry HCl gas for several minutes to ensure saturation. Each solution was then stirred for 30 minutes and taken t o dryness under reduced pressure. After storing under vacuum with so-
dium hydroxide and phosphorus pentoxide for 2 hours, each residue was redissolved in 1 ml. of trifluoroacetic anhydride, 0.9 ml. of dichloromethane and 0.1 ml. of dimethylformamide. After stirring for 30 minutes, 1- t o 3-pl. aliquots were taken for gas chromatography. Precautions to maintain anhydrous conditions were observed during these preparations. Studies using infrared spectrophotometry indicated quantitative conversion to the intermediate methyl ester hydrochloride and to the final derivative for DIT, TI, and T 4 (8). Comparable studies were not made for M I T and Tz. Gas chromatography was performed with an F & M Model 402 equipped with a flame ionization detector and Moseley 1-mv. recorder. A 4-foot glass column packed with 3.8% SE 30 on 8O/lOO mesh Diatoport S (F & M) was used with helium carrier a t 75 ml./ minute flow (40 p.s.i.g. inlet pressure). Operating temperatures were: injector, 270’ C.; column, 250’ C.; detector, 250’ C. VOL 38, NO. 12, NOVEMBER 1966
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