A flexible procedure for Karl Fischer microtitrations

MICROCHEMICAL

JOURNAL

10,

A Flexible

218-230

(1966)

Procedure

for

Karl

Fischer

Microtitrations’ JOHN Chemical

M.

CORLISS AND MARJORIE

F. BUCKLES

Research Division, U.S. Army Chemical Research and Development Lahovatories, Edgewood Arsenal, Maryland Received June 23, 1965

The nature of the Karl Fischer reagent, the analytical procedures based on its use, and the chemistry of the reactions involved have been thoroughly discussed by Mitchell and Smith (7). Improvements on the titrimetric determination of free water or water of hydration in organic compounds have been extensively reported: Peters and Jungnickel (9) contributed a stabilized reagent by substituting methyl Cellosolve for the methanol originally used as the solvent for the iodine-sulfur dioxide-pyridine system (5); Neuss et ~2. (8) recommended a more accurate primary standard for use with the reagent. In addition, numerous electrometric methods have been introduced to overcome difficulties normally encountered in visual observation at the end point. In the microdetermination of water by the Karl Fischer method, protection from atmospheric moisture is increasingly important. Several authors have described burets which adequately protect the hydrophilic titrants both in storage and in use, and titration cells which protect their contents from moisture contamination (1,4, 10). In general, these papers have been concerned with the water content of substances which are amenable to direct titration, being both inert and soluble in the Fischer reagent. Mitchell (7) has introduced procedures for classes of compounds which react nonstoichiometrically with one or more components of the Fischer reagent but which can be rendered inert by the proper reagents and also for the treatment of insoluble, but porous materials. These involve, either separately or in combination: (a) the addition of reagents for deactivation 1 Paper presented at the International Symposium on Microchemical 1965, held at The Pennsylvania State University, University Park, U.S.A., August 22-27, 1965. 218

TechniquesPennsylvania,

AN

APPROACH

TO

MICROAQUAMETRY

219

or extraction, (b) observation of a reaction period, and (c) reduced temperatures for the titration. In developing a micro method that incorporates these procedures and thus makes it applicable to a wide range of organic materials, it becomes necessary to minimize the effect of parasitic side reactions in the Fischer reagent, and to protect the materials being titrated from either gain or loss of moisture for extended periods of time. While a flowing, dry gas stream has been used successfully to protect the cell contents from moisture gain during direct titrations (I, 4), this would result in a loss of water content during a long reaction period owing to the appreciable vapor pressure of the water. Apparatus offering protection from the humid environment of the microanalytical laboratory cannot be of so rigid a nature as to block the introduction of samples or reagents. Ma (2) describes an ingenious adaptation of Wiberly’s apparatus (10) which surmounts this difficulty. Further ingenuity is needed to adapt Mitchell’s procedures to the micro scale. In an early paper, Levy et aE. (6) described a simple test-tube cell capped with a serum bottle stopper. This cell, with modifications designed in these laboratories, offers the required protection and an ease of access conducive to the flexibility of technique necessary for progress in Karl Fischer microtitrations. EXPERIMENTAL

Apparatus und reagents. Broadly, the microtitration equipment may be broken down into (1) a titrant dispensing unit, (2) an end-point indicating unit, and (3) the titration cell. .4uxiliary equipment includes a reagent addition apparatus for materials which cannot be immediately titrated, and protective equipment such as dry boxes for hygroscopic materials. The central item is the cell. ,4n essential feature of the titrant dispensing unit is protection of the Fischer reagent from deterioration and exposure to moisture. In the work described below, an assembly (Fig. 1) including gravity-filled 2-ml burets (graduated in O.Ol-ml increments), with overflow automatically returned to a reservoir at the top, is employed here to dispense Fischer reagent and standard methanol (3). Aquasorb (Mallinckrodt Chemical Works, St. Louis, Missouri) is used in the air-drying tube at the ports of the burets. The end points are observed by the ‘Ldead-stop” technique (3). The cell (Fig. 2) is essentially that of Levy (6) except that the exterior electrical connections terminate in 3.4G Littlefuse metal caps removed from the fuse

220

JOHN

M.

CORLISS

AND

MARJORIE

P.

BUCKLES

body and cemented to the cell with Sauereisen Adhesive No. 2. Mixing action during titration or extraction is obtained by means of a magnetic stirring motor (Fig. 3) mounted sideways. The cell is approximately 8 ml in volume and is capped with a sleeve-type needle-puncture red rubber stopper (serum-bottle stopper). Since these stoppers desorb moisture during titrations, extraction in a mixture of methanol and Karl Fischer reagent for at least a week prepares the stoppers for use. Single-solution stabilized Fischer reagent is diluted so that 1 ml reacts

FIG.

1. Karl Fischer microtitration

apparatus.

AN

APPROACH

TO

MICROAQUAMETRY

221

with 2-3 mg of water. The reagent, diluent, and water standard in methanol (standard methanol) are the commercially available products. To enhance solubility and complete reaction in the minimum solution for a small cell

FIG. 2.

FIG. 3.

Titration

Titration

cell.

cell, mounted.

222

JOHN

M.

CORLISS

AND

MARJORIE

F.

BUCKLES

volume, the back-titration method is preferred. Under these circumstances, it is necessary to obtain a comparison between the Fischer reagent and the standard methanol, a standardization of one of these solutions, and a titer on the material being analyzed. In addition, titers or blanks on reagents must be known. Titrants are added to the cell either through metal syringe needles or through Teflon needles, where needle flexibility is desirable. Procedure. Briefly, the procedure is as follows: A sample is added to a preconditioned Levy cell, followed by reagents for extraction or deactivation, as necessary. A waiting period based on prior experimental work with the given material is observed. Fischer reagent is added and the excess is titrated with standard methanol reagent. A clean, dry Levy cell capped with a desorbed stopper is prepared for a Fischer microtitration by admitting less than 1 ml of Fischer reagent, shaking vigorously so that all interior surfaces are contacted by the reagent, and back titrating, as below, with standard methanol. At this end point, the cell is shaken vigorously again to wash down unreacted reagent, and again brought to the end point by the addition of standard methanol. In weighing a sample, an attempt is made to take a quantity containing between 2 and 3 mg of water. Transfer may be made under dry air in polyethylene glove bags (procurable from Instruments for Research and Industry, Cheltenham, Pennsylvania) to Roth or Benedetti-Pichler weighing bottles which are small enough so that the weighing vessel can be deposited directly into the cell. After placing a soluble sample in the cell and capping, the cell is vented with a thin gage, short syringe needle since seating the cap induces a slight positive pressure in the cell. Sufficient Fischer reagent is added to react with any water present plus enough to give a standard methanol back titer of l-2 ml. Stirring and the addition of the back titrant are commenced. In the small cell, a visual check should be made to insure that the sample mass is freed from the weighing vessel. For addition of reagents, the cell must be vented (through Aquasorb) to equalize pressure inside the cell. When Teflon needles are to be used for this addition, a means of introducing the needle through the cap is necessary. A large gage steel needle through which the thinner gage Teflon needle may be led serves this purpose as well as that of a vent. For reproducible end points, the standard methanol is added slowly (about 0.25 ml per minute) to the end point. Flow can be controlled either by valve adjustment or selection of a suitable length of thin-gage syringe needle to result in proper rate.

AN

APPROACH

TO

MICROAQUAMETRY

223

Reagents necessary for water extraction from, or deactivation of materials being analyzed are added through the stopper from plastic micrometer syringes. The reagents themselves are stored in loo-ml serum bottles, stoppered and kept in desiccators to insure protection from moisture pickup between blank and sample determinations. Insoluble but porous materials, which may need to be ground before weighing, are deposited in the cell, and 2 ml of anhydrous methanol is admitted. Vigorous stirring is required to disperse the material. An extraction period of 20 minutes proved sufficient for the work described here. Spent Fischer reagent for amine deactivation is prepared in a IOO-ml serum bottle. The bottle is filled with full strength Fischer reagent, and water is added until the solution is very near the equivalence point. Re-

FIG.

4.

Adaptation

for sample release.

224

JOHN

M.

CORLISS

AND

MARJORIE

F.

BUCKLES

action products, insoluble in stabilized Fischer reagent, are allowed to settle for several hours, and the liquid is decanted from the bottle which is then filled with anhydrous methanol. The small amount of residual water in the methanol produces a very slight excess of water in the spent reagent. Two ml is added to the sample in the cell. A minute of stirring in the case of soluble materials prepares for the titration and final step. Other liquid reagents for deactivation may be added to the cell in a similar manner. If one should desire to blank out the cell contents immediately prior to a water determination, as in Ma’s modification (2)) an arrangement is suggested (Fig. 4) whereby a weighed sample is suspended by a wire from the Levy cell stopper. At the desired moment, pulling the wire releases the sample into the body of the cell. Removal of spent reagents can be effected without stopper removal by use of a hypodermic syringe with a long needle through the stopper of the closed cell. RESULTS

Tables l-3 list research materials submitted for analysis on which water was ultimately determined by Karl Fischer microtitration. Calculated values are italicized; experimental values appear in conventional type. Our custom is to analyze materials as received since further studies are TABLE ANALYSIS

1

OF 9-AMINO-1,2,3,4-TETRAAHYDROACRIDINE

HYDROCHLORIDE

Sample Ia

% H,O C H Cl N 0

Calcd. (dry) 0.0 66.5 6.4 15.1 11.9 0.0

Found 2.4 65.6 6.7 14.9 11.2 1.6d

(27) (15) (15) (20) (28)

Sample IIIC

Calcd. (on basis water found) 2.4 64.9 6.6 14.7 11.6 2.1

Calcd. (as monohydrate)

Found

Calcd. (on basis water found)

7.1 61.8 6.8 14.0 11.1 6.3

6.6 61.6 7.0 13.9 10.9 6.ld

6.6 62.1 6.7 14.1 11.1 5.9

parenthetical

numbers

Sample IIb

Found 7.0 61.5 7.0 14.0 11.1

6.4d

a Prepared from pure acridine base on 2nd of month; indicate day of month determination made. b Purchased material. c Purchased material, purified and resubmitted. d By difference.

AN

APPROACH

TO

225

MICROAQUAMETRY

made on samples of the same materials. Often, prior to a water determination, a calculation was made on the basis of an integer hydrate as a justification for the experimental elemental values. When hydrogen and oxygen values are observed to be higher then theoretical, and all other elements TABLE ANALYSIS

2

CALCULATED

TO HYDRATES

Composition

0

z

C,aHa,IN,O, ; research compound, as the sesquihydrate. Calcd. (dry) 0.0 39.4 5.2 34.6 Found 6.6 38.4 6.0 31.1 Calcd. (wet) 6.6 38.4 6.0 31.1

7.6 7.0 7.0

13.1 17.7 17.7

100.0

C,sH4,Br,N,0,; Calcd. (dry) Found Calcd. (wet)

research compound, as the monohydrate. 0.0 56.2 5.5 19.7 2.4 55.3 5.7 18.9 2.2 54.9 5.6 19.2

6.9 6.9 6.7

11.8 13.2 13.5

C3,H2aBrzN,0,; Calcd. (dry) Found Calcd. (wet)

research compound, as the dihydrate. 0.0 57.8 4.5 5.7 54.6 4.9 5.8 54.4 4.9

-

5.5 10.3 10.3

H,O

C

H

x

(%) ix

Compound

100.2

100.0 100.0 100.0

100.0

-

lower, the analyst naturally looks for the presence of water in an otherwise pure sample. Table 1 illustrates an approach to this problem. The three materials, designated as Samples I, II, and III, were separately submitted by the same chemist. The quaternized material had been prepared from the pure acridine base at the beginning of a month. The progress of this material in absorbing water may be followed by noting the dates of the individual determinations. Later a sample of this material was purchased as a hydrate and proved to be the monohydrate (Sample II). A purification was performed and the material was resubmitted (Sample III). One may conclude from these results and calculations that, to prepare a dry sample of the acridine hydrochloride, it is better to quaternize the dry acridine than to purify the monohydrate. Protection from humid air is essential. All of the materials in Tables l-3 were soluble in Karl Fischer reagent and methanol and also nonreactive so that water could be determined immediately. Table 2 presents the analysis on materials whose experimental water content corresponded to the content of a hydrate of the compound postulated. Formulas given are for dry material. Table 3 continues this

226

JOHN

M.

CORLISS

AND

MARJORIE

F.

BUCKLES

listing of research compounds but is restricted to materials whose water content were not integer hydrates. Table 4 presents a listing of pure hydrated materials which were chosen TABLE ANALYSES

3

OF RESEARCH NONINTEGER

Composition Compound

HYDRATES

(%)

H,O

C

H

X

N

0

z

0.0

64.3 62.8 63.3

5.9 6.1 6.1

18.1 17.6 17.8

3.6

8.2 9.5 9.4

--

C,,H,,CINOa ; two preparations 68.7 0.0 Calcd. (dry) 3.8 66.6 Found, 1st 3.9 66.5 Found, 2nd 3.9 66.1 Calcd. (wet)

7.0 7.3 7.3 7.2

8.8 8.7 8.7 8.5

-

3.3

11.9 15.2 14.4 14.9

-

8.1 8.4a 7.9

6.2 7.9 8.1

-

8.9

100.0

Calcd. (dry) Found Calcd. (wet)

1.5

1.5

4.1”

3.5 3.5 2.2" 3.1”

C,,H,,Br2N,O,

Calcd. (dry) Found Calcd. (wet) (kKdW~O4 Calcd. (dry) Found Calcd. (wet)

0.0 2.3 2.3

46.3 45.4 45.2

8.7 8.9 8.8

30.8 29.5 30.0

0.0

50.1 49.2 49.2

7.0 6.9 7.0

22.2

53.7 52.7 53.2

7.3 7.2 7.3

22.3

1.5

69.3 68.0 68.2

7.3 7.4 7.4

0.0 8.0 8.0

71.6 66.2 66.0

6.0 6.4 6.4

1.7

1.7

11.7 11.3 11.5

10.3

99.5 100.0

22.1

-

-

-

-

-

11.5 12.3 12.6

21.8

21.8

10.2

C32H52Br2N404

Calcd. (dry) Found Calcd. (wet)

0.0 1.0

1.0

21.6

C,,H,&INO,

Calcd. (dry) Found Calcd. (wet)

0.0 1.5

C,,HI,N,O2

Calcd. (dry) Found Calcd. (wet) a By difference.

10.4 9.3 9.6

11.9

100.0

17.6

99.5 100.0

17.9

for their variety of action with Karl Fischer reagent. Compounds l-4 were all soluble in the reagent but with decreasing coloration of solution. A carbon and hydrogen determination on the maltose indicated a larger water content than monohydration; however, these analyses are discussed

AN

APPROACH

227

TO MICROAQUAMETRY

further below. Compounds 5 and 6 were insoluble in the reagent and were subjected to extraction before titration. Water analyses on these two materials as well as the standard, sodium tartrate, resulted in appreciably low values when titrated without extraction. Compound 7 reacted with the TABLE ANALYSES

4

OF SELECTED MATERIALS

Water (%) Compound 1. 2. 3. 4. 5.

l,lO-Phenanthroline 2,4-Dinitrosoresorcinol Maltose Sodium acetate (Ethylenedinitrilo) tetraacetic acid, disodium salt 6. Adenine sulfate 7. Hi&dine monohydrochloride

Hydrate

Calcd.

MonoMonoMonoTri-

9.09 9.68 5.00 39.71

DiDiMono-

9.68 8.91 8.59

Found 8.95, 9.85,

5.67, 39.0, 9.54, 8.97, 8.44,

8.99 9.79

5.73 39.2

9.55 9.00 8.31

reagent and required a deactivation reaction to overcome its strong amine qualities. Values obtained by immediate titration on this material were high and variable. All results in Tables l-3 were based on sodium acetate trihydrate as primary standard; the determinations in Table 4 were based on the more rigorous standard, sodium tartrate (8). Sodium acetate is very soluble in the stabilized reagent and is convenient to use in daily work, where it meets looser accuracy requirements; the sodium tartrate is insoluble and requires the additional extraction step before titration. The sodium acetate in Table 4 was found to be 1.5% lower than calculated. When this factor is used as an experimental correction in the preceding tables negligible changes are produced in all cases. Results here indicate sodium acetate loses water (after grinding), while Neuss et al. (8) experienced a gain. DISCUSSION

The Levy cell is attractive for use in microtitrations because of its small volume, simplicity, and flexibility. The necessity of preconditioning both cell and stopper was observed early in this work. In a single experiment, 17 mg of water was extracted from a stopper by anhydrous methanol in 7 days. Sequential comparisons with 0.800-ml aliquots of Fischer reagent, in which no pains were taken to contact the inner surface of the stopper with the reagent, gave titers of 1.231, 1.398,

228

JOHN

M.

CORLISS

AND

MARJORIE

F.

BUCKLES

1.470, 1.522, 1.580, 1.613, 1.641, and finally, 1.642 ml of standard methanol. Water was slowly being desorbed from the stopper. After conditioning by the procedure detailed above, immediate duplicate comparisons obtained were 0.492 and 0.492 ml Fischer reagent per milliliter standard methanol with one pair of solutions, and 0.520 and 0.523 with another pair. In a test tube containing a methanol-Fischer reagent mixture, the slow desorption of water by the serum bottle stopper can be observed readily. Overnight the mixture in the bottom of the test tube adjacent to the stopper will decolorize. After mixing again, the local decolorization will not return for several days, but observable desorption will continue for about 10 days. The cell itself requires no predrying at elevated temperatures or in vacua as the initial preparation of the cell neutralizes all inner surface moisture. After treatment, a stoppered cell will protect a solution from appreciable gain or loss of moisture for longer than 2 hours. The stoppers used in this work were not preselected and showed no chemical or physical reaction after immersion in Fischer reagent and methanol for a period of a month. Occasionally, anomalous behavior at end points resulted from earlier reaction of material with the electrodes. High titers and poor end points were experienced. In all cases, this behavior was corrected by cleaning the cell interior with nitric acid to reactivate the electrodes. A most adaptable procedure for retaining dry cell conditions while introducing a sample involves transfer in a glove bag under dry air. Thus moisture pick-up by the cell is held to a minimum. The ambient air of the microanalytical laboratory with 50% relative humidity contains of the order of 10 l.tg of water per cubic centimeter. In even a rapid transfer into a prepared cell, relatively large quantities of moisture are absorbed by those surfaces covered by anhydrous titrants. If titration proceeds immediately after such a transfer, no error is observed. However, waiting periods before titration of greater than 5 minutes introduce observable error, rising in direct proportion to time. After 30 minutes an error equivalent to 0.3 mg of water was observed. Evidently, on closing the cell, a short period occurs before drainage from the upper surfaces of the cell and the stopper becomes appreciable, In the work here, with ideal nonreactive and soluble materials, transfer was made without use of the glove bags. Another source of error may arise from premature addition of stabilized Fischer reagent to methanol solutions. Fischer’s original reagent is unstable

AN

APPROACH

TO

MICROAQUAMETRY

229

due to parasitic side reactions occurring during storage. Similar reactions are evident when mixing the stabilized reagent with methanol. From this source of potential error, a loss in titer ranging from 30 yg of water for a mixing time of 30 minutes, to 150 pg for 2 hours was observed. A third source of error, perhaps related uniquely to the apparatus used here, was the loss in titer due to the reaction of iodine with the stainless steel syringe needles. In our procedure, this was of the same order of magnitude as the error due to side reactions. Both of the last two errors were circumvented by delaying the addition of Fischer reagent until after any reaction or extraction periods, and by flushing buret tips and needles with several tenths of a milliliter of reagent before use. Use of electronically actuated burets in these titrations has the advantage of releasing the operator and also of titrating water as it is freed in the solution. Thus a cell protected by a flow of dry gas may be used with such burets without water loss, even for extractions. With compounds which react with Fischer reagent, a deactivation step and reaction time is observed, during which time the water to be titrated is free in the solution and present in both liquid and vapor phases. A flow of dry gas through a cell would sweep out water vapor, but in a static system as described here, this water vapor is retained. In the research compounds tabulated above, the water usually was present as only a few parts per hundred. The Karl Fischer microtitration was used as a final proof for the existence of the hydration that the analyst had postulated from elemental analyses. In these analyses the less reliable acetate hydrate possessed sufficient accuracy for this quantitative proof so that in a sense the water determination was less sensitive than various elemental analyses. However, in Table 4 the water content of the maltose was found to be appreciably higher than expected. No elemental analysis could clarify this picture completely since the difference in elemental content was just within the range of experimental error for the determinations. Again, infrared spectra were of no value due to the added absorbance of the hydroxyl groups in the compound itself. Therefore, the Fischer microtitration gave more information than any other method conceived. In applying the Karl Fischer microtitration to the range of materials covered by macro methods, three additional steps have been mentioned. The two most challenging for adaptation to the micro scale, i.e., reagent addition and reaction period, have been successfully used in this work.

230

JOHN

M. ~0~~188

AND MARJORIE

F. BUCKLES

SUMMARY The focal point of an effective Karl Fischer microtitration apparatus lies in the design and handling of the titration cell. The simple Levy cell with a pre-extracted serum bottle stopper as a closure has been shown to protect its hydrophilic contents from gain or loss of moisture in the humid microanalytical laboratory environment. This protection has been experimentally established by a number of successful determinations, some after long periods of extraction or reaction. Furthermore, flexibility of the method with this cell is shown. Reagents may be added, samples released, or spent reagents removed without compromising the protection from moisture exchange. Thus water determinations on reactive and insoluble materials may be completed without loss of accuracy. ACKNOWLEDGMENT The authors wish to thank Mr. Frank DeCesare of the Analytical Research Branch for incorporating certain design features into the titration cell which have added to its utility and ruggedness. REFERENCES E. L., SIEGEL, H., AND BULLOCK, A. B., Microdetermination of water by titration with Fischer reagent. Anal. Chem. 31, 467-473 (1959). Group Analysis,” 696 pp. 2. CHERONIS, N. D., AND Ma, T. S., “Organic Functional Wiley (Interscience), New York, 1964. 3. CORLISS, J. M., AND BUCKLES, M. F., Water determination in pure chemical agents via Karl Fischer microtitration. In preparation. 4. DIRSCHERL, A., AND ERNE, F., Zur Mikrobestimmung von Wasser in Organischen Substanzen nach Karl Fischer. Mikrochim. Acta 1962, 794-802. 5. FISCHER, K., A new method for the analytical determination of the water content of liquids and solids. Anger. Chem. 48, 394-396 (1935). 6. LEVY, G. B., MURTAUGH, J. J., AND ROSENBLATT, M., Microdetermination of water. Ad. Chem. 17, 193-195 (1945). 7. MITCHELL, J., AND SMITH, D. M., “Aquametry,” 444 pp. Wiley (Interscience), New York, 1948. 8. NEWS, J. D., O’BRIEN, M. G., AND FREDIANI, H. A., Sodium tartrate dihydrate as a primary standard for Karl Fischer reagent. Anal. Chem. 23, 1332-1333 (1951). 9. PETERS, E. D., AND JUNGNICKEL, J. L., Improvements in Karl Fischer method for determination of water. Anal. Chem. 27, 450-453 (1955). 10. WIBERLY, J. S., Improved procedure for Karl Fischer microtitrations. Anal. Chem. 23, 656-659 (1951). I.

BASTIN,