Steven K. MacLeod Control Division The Upjohn Company Kalamazoo, MI 49001
An accurate and precise moisture determination is often essential in resolving issues where water is involved. Water has been identified as a contaminant limiting the reactivity of products (e.g., monomers, various resins and coating materials, prepregs), a reactant limiting the lifetime of a material (e.g., halocarbons, acetylsalicylic acid), a medium for reaction between components in a mixture (e.g., pharmaceutical formulations), and as a factor limiting the accuracy of other measurements (e.g., mass balance). Although a number of chromatographic, spectroscopic, electronic, thermal, and wet chemical methods have been used to determine moisture, the Karl Fischer titration, originally described in 1935 ( I ) , is the approach most widely used. This REPORT includes discussions of the reaction chemistry, side reactions, reagent composition, and experimental mechanics as well as the issues involved in assay development for the Karl Fischer titration. Reaction chemistry The Karl Fischer titration has been extensively reviewed (2, 3). I t has been suggested that different reactions occur, depending on the protic or aprotic nature of the medium (4,5>. 0003-2700/9 1/0363-557A/$02.50/0 0 1991 American Chemical Society
Titration in protic media. The Karl Fischer reaction in protic media (Le,, alcohol) is shown below.
+ SO, e RSO, + ROHg Solvolysis B + RSO, + ROHg +-+ BH+SO,R- + ROH Buffering H,O + I, + BH'S0,R- + 2B BH+SO,R- + 2BHI Redox
2ROH
(1)
(2)
(3)
Sulfur dioxide reacts with the alcohol to produce an alkyl sulfite in a buffered medium using a n appropriate base, B, to maintain the solution at the optimal apparent pH ( - 5 - 8 ) ( 3 , 6 ,7). Below pH 3, the overall reaction proceeds very slowly, and above pH 8 nonstoichiometric side reactions become significant. When water is present in the cell and iodine is added, a redox reaction occurs.
The method of adding iodine to the reaction differs according to the type of experiment, volumetric or coulometric. I n the volumetric experiment, the iodine is contained in a buret and metered out as required. The amount of iodine per volume is empirically determined during a standardization step, and the amount of water in the sample is calculated from this titer. In a coulometric experiment, the iodine is generated electrically from iodide present in the cell. The electri-
cal efficiency of this method is generally loo%, and the amount of water in the sample is calculated from the number of moles of electrons used in the iodine generation. There are two variants of the volumetric experiment. In the singlecomponent experiment a mixture of the alcohol, iodine, sulfur dioxide, and base is used as the titrant. A suitable organic solvent (usually containing a high percentage of alcohol) is used as the reaction medium to dissolve the sample. The two-component reagent system uses a solution of sulfur dioxide and base in alcohol as the reaction medium and a second solution of sulfur dioxide, base, and iodine in alcohol as the titrant. In a traditional single -component titration, the concentration of alkyl sulfite and the buffering capacity of the cell contents may be very low during the early part of the experiment, which will result in a slow overall reaction. As more titrant is added, t h e concentration of alkyl sulfite and buffering agent is i n creased and, eventually, the reaction goes to completion (8).This condition may readily occur if the reaction medium is very dry and requires only a small amount of reagent during the initial neutralization. Strongly basic or acidic samples may also overwhelm the buffering capacity in the single-component reaction vessel and cause the apparent pH to be shifted from the optimal region. The chemistry of the two-component titration is virtually identical to
ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991
557 A
REPORT that of the single-component titration, but it may be faster because of the higher initial concentration of alkyl sulfite and buffer in the reaction vessel. Samples containing trace amounts of water can be titrated more accurately using t h e twocomponent reagents because t h e buffering and concentration of alkyl sulfite are sufficient to promote rapid endpoint determination. Acidic or basic samples may benefit from the additional buffering capacity of the two-component reagents. However, the choice of reagents does not always favor the two-component variety because these reagents may exhibit greater susceptibility to side reactions involving noncomplexed sulfur dioxide than do single -component systems. Detailed mechanistic studies of the Karl Fischer reaction were per formed by Verhoef and Barendrecht (6,8-13)using various electrochemical techniques. They showed that the oxidizable species is a n alkyl sulfite; that pyridine plays no role in the reaction except as a buffering agent; that the reaction is first order in alkyl sulfite, water, and iodine; that elevated apparent pH (up to about 6) aids in the formation of the alkyl sulfite from the alcohol and sulfur dioxide, thus increasing the overall rate of reaction; and that the predominant reducible species is iodine (triiodide is only a secondary reactive species). Titration in aprotic media. Nonalcoholic Karl Fischer reagents were developed to increase the stability of the titrant and to overcome some undesirable side reactions involving alcohol. Because no alcohol is present to form the alkyl sulfite, other waterconsuming reactions are involved, but this technique is still often referred to as Karl Fischer titration. There is evidence that Fischer was attempting to stabilize the Bunsen reaction, which involves iodine and sulfur dioxide but no alcohol, when he discovered the reaction that bears his name (3). The unbuffered Bunsen reaction 2H,O
+ SO, + I, + H,SO, + 2HI
(4)
is the basis for the “universal” reagents (4)in which a n aprotic solvent is used. Two moles of water are consumed for each mole of iodine, in contrast to the 1:l molar ratio found in the protic Karl Fischer environment. The drawback of this reaction for water determination is the sensitivity of the titer to sample and solvent composition (4).
The presence of protic species in the sample tends to alter the stoichiometry of the Karl Fischer reaction to a n extent dependent on sample size a n d composition. Thus, t h e water:iodine stoichiometry can vary from 2:l to 1:l.A patent describing the use of alkylene carbonates as solvents (14)demonstrates that these solvents allow a conversion efficiency of only 90-95% and t h a t a small amount of alcohol must be present to m a i n t a i n high efficiency. Alkyl sulfites can be added directly to the reaction mixture. Aprotic reagents offer only marginal advantages in limited situations and have not been widely used in our laboratory or elsewhere. Volumetric versus coulometric chemistry. The reactions in the volumetric and coulometric applications are identical except that iodine in the coulometric titration is generated from the anodic oxidation of iodide. The nature of the cathodic half-reaction is somewhat uncertain, but the observation of small bubbles rising in the cathode cell during rapid titration suggests t h a t protons are reduced to hydrogen gas. The concentrations of sulfur dioxide and base are constant over the course of the coulometric titration, making it similar to the two-component, volumetric titration described above. The only suggested difference between the coulometric and volumetric systems ( 1 1 ) is that because the concentration of iodine is never high the concentration of triiodide is relatively low in the coulometric experiment; this is probably not significant, because the rate constant for triiodide is 4 orders of magnitude less than that for iodine (3). Interferingside reactions A number of reactions can occur between the components of the sample to be analyzed and the various species present in the Karl Fischer reagent (2,3,15-17).These undesired interfering reactions can result in the over- or underestimation of the water content of the sample as well as variability in the results. The magnitude of these side reactions can often be empirically estimat ed using the approach of Scholz (15), in which the progress of the titration is monitored as a function of time. These curves are available as outputs on several titrator models, and their shapes can provide insights into the nature of the side reactions. It should be pointed out that a n understanding of the chemistry occurring in each situation is equally as important as the
558 A * ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991
Figure 1. Initial and delayed titration curves for well-behaved samples.
shape of the curves, because these side reactions do not all yield unique titration curves. A well - behaved sample provides kinetic titration curves as shown in Figure 1. No water is generated by interaction of the sample and the reagent, and the curves come quickly to a plateau with a slope that approaches zero. Nondelayed and delayed titrations provide the same results. Some side reactions not involving water may go to completion during the course of the titration (17), and although this is rare, the observation of ideal curve shapes should not be taken as proof of a valid assay for moisture. Oxidation of iodide and bisulfite complex formation can cause underestimation of the amount of water in the sample. Iodide may be oxidized by a reducible species (e.g., Cu2+, Fe3+, NO,, Br,, Cl,, and quinones), and the resultant iodine can react with water present. As shown in Figure 2, this behavior should manifest itself in a curve in which the plateau of the delayed curve is lower than that of the nondelayed curve as a result of the “pretitration” of some water in the sample during the delay period. Elimination of this behavior is usually not possible in q typical Karl Fischer experiment because io dide would have to be excluded from the reaction. Bisulfite complexes formed from free water, sulfur dioxide, base, and carbonyl functions on aldehydes and ketones in the sample can also result in a n apparent reduction in the water content. During the time in which the sample dissolves, water can be tied up in this complex as shown below. R,C=O
+ SO, + H 2 0 + B H R,C(OH)SOiHB+ ( 5 )
The reaction can be reversible, and the shape of the titration curves depends on the stability of the complex. A stable complex (with respect to ti-
L
Moisture Titration with The MKC-210 Karl Fischer Titrator Quick and accurate measurement of low-moisture levels (1Opg to 100mg) is easy when you’re using the microprocessor-controlled MKC-210 Karl Fischer Titrator. The storage of 5 methods and 100 results in memory allows for automatic recall when working with varying conditions. Add the ADP-351 Moisture Vaporizer and you can measure difficult samples that cannot be placed in the cell. On command, the MKC-210 Karl Fischer Titrator will print the moisture -_ curve and data list for evaluation, -_ method development and/or sample recognition on its internal printer/ plotter. Like all KEM instruments, the MKC-210 Karl Fischer Titrator was designed with the needs of R&D, QC and QA analyses in mind . . . by the company that specializes in the manufacturing of measuring instrumentation for the analytical laboratory. Let KEM take you beyond basics. Ask us for more information on the MKC-210 or our other volumetric Karl Fischer titrators. For more information call us toll-free at 1-800-458-3168; in New Jersey, 201-379-9651. *-
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REPORT
Figure 2. Initial and delayed titratiort curves for samples that generate iodine.
Figure 3. Initial and delayed titration curves for samples that form moderately stable bisulfite complexes. tration time) may exhibit curves similar to those in Figure2, whereas a less stable complex may exhibit the behavior shown in Figure 3. Resolution of this issue can take different courses. Solvent systems should allow rapid dissolution and immediate titration. Because t h e strength (and reversibility) of the bisulfite complex depends on the pK, of the base in the reagent, substitution of weaker bases (e.g., pyridine) for stronger bases (e.g., imidazole) may lessen the problem. Elimination of sulfur dioxide is not generally a viable alternative in the Karl Fischer titration, but the use of methanolic reagents reduces the concentration of free sulfur dioxide by efficient complex formation (3). A large number of side reactions can result in the overestimation of moisture in a sample. Reduction of iodine by oxidizable species (e.g., ammonia, thiols, Tl’, Sn2+, In+, thiosulfite, ascorbic acid, and hydroxylamines) results in the consumption of iodine, which may be erroneously interpreted as high moisture content in the sample. Phenolic derivatives, including hydroxy and aminophenols and naphthols, are also oxidized (18) with corresponding reduction of iodine. This effect is illustrated in Figure 4, where the rising curves demonstrate the continuous consumption of iodine. Moisture determination 560 A
may be possible after oxidation of the offending functionalities (3, 16). I n the case of the phenols, a solvent system with a reduced apparent pH may decrease the magnitude of the problem (18). Water may be produced in the reaction cell in a variety of ways to produce titration curves similar to those in Figure 4. Reaction of esterifiable acids with alcohols can produce a mole of water per mole of acid; esters and acid salts do not react. Resolution of this problem involves eliminating the alcohol, substituting another- alcohol t h a t r e a c t s at a n insignificant rate, or raising the apparent pH of the reagent (this is generally a n acid-catalyzed process). Conversion of strong acids to amine salts prior to titration has been suggested (16),although the uncertainty in the determination may rise as a result of the presence of a n additional component. Ketones and aldehydes can react with alcoholic solvents to form ketals and acetals with production of a mole of water. The rates of these reactions vary, depending on the compound as well as the alcohol and apparent pH (15). This problem can often be solved by substituting another alcohol that reacts at a n insignificant rate or by increasing the apparent pH of the reagent. Experiments have been done in which the alcohol was systematically varied (15) to study the effect on rate of water generation for some probe molecules. Scholz used a common base, but it is not clear that any attempt was made to control the apparent pH of the medium. Because of this, the results may represent a combination of alcoholrelated effects having to do with steric hindrance in the ketal formation and the effects of apparent pH. I t may also be possible to avoid reaction with the solvent by forming cyanohydrin derivatives of the compound prior to analysis (2).
Some metal oxides and hydroxides can react with hydroiodic acid to produce water, causing the water content of the sample to be overestimated. Because the solution involves elimination of iodide from the cell, t h i s problem cannot be resolved within the typical Karl Fischer experiment. Silanols and cyclic siloxanes also can react with alcohols to produce ethers and water. Remedies involve the use of a hindered alcohol, reduction of the alcohol concentration, or the use of a buffer with a higher apparent pH. A cooled titration cell approach using extrapolation to zero time to resolve the slower side reaction from the Karl Fischer reaction also has been successfully employed (19). For aldehydes and ketones, the bisulfite re act ion and acet a l k et a1 formation reactions often occur together. Low levels of residual solvents with carbonyl functions (e.g., acetone) can result in unexpected molar equivalent amounts of apparent water in the sample. This combination of reactions results in titration curves similar to those shown in Figure5. Because of the severity of these reactions with aldehydes, it has been suggested that water cannot reliably be determined in aldehydes via Karl Fischer titration (15). However, we have had considerable success using the approaches mentioned above. Other problems can be related to properties of the samples. If the sample can displace the apparent pH of the medium outside the optimum range, changes in the stoichiometry and kinetics can produce erroneous results. These samples can be neutralized prior to titration or added to a heavily buffered medium (e.g., 1 : l methanol/pyridine or 10% w/v imidazole in methanol). At sufficiently high concentrations, any sample can effectively reduce the availability of the active Karl Fischer
8
Figure 4. Initial and delayed titration curves for samples that consume iodine or generate water.
ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991
10
Figure 5. Initial and delayed titration curves for samples that form water and bisulfite complexes.
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REPORT components to a level where titrations are sluggish and results are no longer predictable. Large samples or samples dissolved in large amounts of solvent should be avoided for coulometric work; volumetric titrations can often handle large volumes of sample if a well-buffered medium is used. If solubility limitations cause slow release of moisture and result in sluggish titrations, appropriate cosolvents can be added to assist in dissolving analytes. Reagent composition A wide variety of compounds have been used in protic and aprotic Karl Fischer reagents (see box below). Based on the chemistry of the Karl Fischer reaction and the nature of side reactions, it is useful to classify these reagents into three broad categories: methanolic reagents, reagents based on other alcohols (generally 2 -methoxyethanol), and nonalcoholic reagents (see box above). This division is useful because the nature of side reactions that result from interactions with the base solvent can be avoided or minimized by switching to another solvent type. The apparent pH of the reagent is also useful in overcoming side reactions and can be used for classification. Those reagents with a low apparent pH are generally based on pyridine or its derivatives, whereas high-apparent-pH reagents are most
Methanolic reagents
I
I I
iodine
Other al Low apparent pH: 2-Methoxyethanol, pyridine or substituted pyridine dioxide (methyl sulfite), iodine dioxide (methyl sulfite), iodir?
I
Nonalcoholic reagents
commonly based on imidazole. (However, because “pyridine-free” r e agents are made using either a substituted pyridine to decrease vapor pressure while maintaining the ap parent pH of pyridine or a nonpyridyl base at a significantly different apparent pH, the pyridine-free designation is not a substitute for knowledge of the apparent pH.) Solubilitymodifying co-solvents do not affect the Karl Fischer chemistry directly and are therefore not used as a basis for classification of reagents. The qualitative composition of some reagents as determined by capillary GC and GCMS is shown in the box above. Coulometric cath-
I
I Reagents used in Karl Fischer titrations
I
Protic syst Alcohol: Methanol, 1-propanol, 2-propano1, -butanol, 2-butano1, tert tert-pentyl alcohol, propylene glycol, 2-methoxyethanol, et 2-ethoxyethanol, 2-butoxyethanol, benzyl alcohol, 2-chlor methanol, diphenylcarbinol, trinkenylcarbinol, cyclohexanol Bases Pyridine, methylimidazole, aiemanoi acetate, guanidinium benzoate
’
Pyridine, imidazole aceta
1
Modifier-
562 A
ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15,1991
ode solutions are similar to those shown in the box above with addition of chloroform and/or carbon tetrachloride in most cases. Anode solutions are usually qualitative matches to cathode solutions, but may lack iodine. Quantitative differences are often significant. Addition of several percent water to these reagents does not shift the apparent pH, which is similar to that expected from pK, values for each base, by more than 0.1 units. The ideal reagent. The choice available to the formulators of Karl Fischer reagents is fairly broad, and commercially available reagents are generally formulated to provide acceptable results to a wide range of users. An ideal reagent should have proper stoichiometry (1:liodine to water), no side reactions, high solubility for a variety of samples (e.g., oils, salts, polymers, sugars, and proteins), stable endpoints, rapid reaction kinetics, lack of bisulfite complex formation or at least formation of labile complexes, lack of odor, lack of toxicity, and ease of availability. Unfortunately, no reagent fills all these needs for all samples. Solvents that have ideal stoichiometry and lack of bisulfite formation with free water are limited to those based on alcohol. Side reactions involving iodine and sulfur dioxide cannot be avoided, but the concentrations of iodine and sulfur dioxide can be reduced (20) and the concentration of free sulhr dioxide can be minimized through the choice of alcohol (3). Side reactions involving the alcohol can be minimized by reducing the alcohol level or using a hindered alcohol. Substituted pyridines can be used as bases to provide stable, odorless reagents t h a t should have labile bisulfite complexes. Modifiers can be included to allow enhanced solubility
of a given sample type. For example, formamide reportedly has increased reaction kinetics through enhancement of the iodineltriiodide ratio (20). Methanolic reagents, or those containing methyl sulfite, are considered the most rapid, as are those adjusted to higher apparent pH values (3). Complete safety cannot be achieved because of the need for sulfur dioxide and iodine in the reagent, but amines with vapor pressures lower than that of pyridine (such as diisopropylpyridine or imidazole) are considered safer for the analyst. Inclusion of a surfactant film may decrease atmospheric absorption of moisture (21). Mechanics of the Karl Flscher titration Although we have repeatedly dis cussed the equivalence of coulometric and volumetric titration results, there are differences in the practice of these experiments. In the coulometric titration, small amounts of iodine can be added accurately to fixed solution composition, resulting in the ability to titrate minute quantities of moisture. Standard deviations of 5-10 pg of water are typical in coulometric systems versus 150 pg of water in typical volumetric systems using commercial reagents, and the amount of water titrated is practically limited to about 3 mg in a coulometric system versus 250mg in a volumetric system. Titration of small quantities of water in coulometric systems requires correction for atmospheric moisture entering the system. The leakage of moisture into a reaction cell (typically 5-15 pg/min) is generally compensated by circuitry designed into coulometric systems. When the sample cell is opened to introduce a sample, several milliliters of moisture-laden air also enters the cell, and this wat e r (typically 15-50 pg) must be measured and subtracted from the final result. Neither of these sources of moisture is significant for the typical volumetric experiment. Care should be taken not to plug the frit or coat the exchange membrane in coulometric cells with insoluble materials as this slows the titration and may reduce t h e coulometric efficiency. Volumetric cells can handle particulates, a l though concerns regarding the lack of total release of moisture in the absence of dissolution should be addressed. Endpoint detection. The potentiometric detection system is based on the presence of the iodineliodide cou-
ple a t a pair of electrodes with a small applied current. When operating in a “wet” solution, the absence of the oxidized form (iodine) results in a large potential difference between the sensing electrodes. During the titration, iodine is added and reduced to iodide in the presence of moisture. At the end of the titration, no more water is present and the concentration of iodine rises rapidly, resulting in a rapid decrease in the voltage difference between the two electrodes. The amperometric detection system also depends on the presence of both members of the reversible couple in order for c u r r e n t t o flow through a pair of electrodes with a small applied potential. As pointed out above, iodine is not present prior to the endpoint, so current will not flow. At the endpoint, both species are present, the current rises, and the endpoint is sensed. Amperometric endpoint detection has been referred to as a “dead stop” technique, but, in view of the nature of the current flow, a more precise description might be a “kick-off” endpoint (2,22). The current trend is toward potentiometric endpoint detection, a l though a number of amperometric systems are still in use. In coulometric work, the trend is toward the use of a pulsed detection system a r ranged such that the detection pulse does not coincide with the pulsed coulometric currents. This arrangement may result in a more noise-free detection system because of greater discrimination between detection current and noise from the generator current. An additional parameter called the ‘(persistence of endpoint” has been included in some volumetric titrators and is intended to ensure that all the water in the sample has been titrated. Modern instruments may use an analogous measurement of the rate a t which iodine must be added to the cell to compensate for moisture leakage into the cell and to maintain the equivalence point. The rate of iodine addition during a titration is compared with the rate a t the beginning of the titration to determine the endpoint of the titration. Visual endpoints, based on the color of iodine in the vessel a t the endpoint, have been used (2). Instrumental methods for colorimetric endpoint detection have only rarely been employed, and the potential advantages of such systems are not clear (22). Calibration. A variety of materials have been proposed as primary or
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secondary standards for water determination (3). The principal requirements of these materials are t h a t they contain a stoichiometric amount of moisture that is stable over a wide range of temperatures and humidity, solubility i n t h e Karl Fischer reagents, ease of handling and storage, availability, and uniformity. Secondary water standards a r e sold by various vendors. Although some efforts have been made to render these solutions less hygroscopic, the water content of the solution changes after piercing the septum over the solution several times. Sodium tartrate dihydrate is a primary standard with many useful characteristics, but it is not easily dissolved in many Karl Fischer reagents ( 3 ) and questions about uniformity of supplies have been raised (22). Water is a very good calibration reagent when delivered by weight. For the coulometric titrator, small quantities can be delivered using microliter capillary tubes used to spot TLC plates. The tube can be tared, then filled with water and reweighed. The contents are expressed from the tube into the titrator, and the weight of t h e water is compared with t h e amount titrated. (The inherent volumetric accuracy of these tubes is not sufficient to allow for direct use in calibration.) We have found that lincomycin hydrochloride monohydrate is stable in methanolic reagents, does not change water content over a wide range of relative humidities, and is quite stable. The availability of this material is unclear because it is a prescription pharmaceutical, but a lincomycin hydrochloride monohydrate standard for water determination is being developed as a n in-house standard. Assay development and validation. Sample size can limit the choice of instrument used for titration with smaller, more valuable, or more toxic samples, favoring the coulometric method. Solubility limitations also favor coulometric titrations because the smaller sample size is typically dissolved in a volume larger than that used in the volumetric experiment, although a greater flexibility to use co-solvents exists with volumetric systems. Care should be taken not to decrease the alcohol concentration below about 50% because the iodine:water stoichiometry may be affected. The addition of a co-solvent to the reaction medium should not result in a change in the titer of the reagent. (In our laboratory, dimethylformamide and dimethyl sulfoxide have been problematic a t high con-
centrations and therefore a r e not used as co-solvents.) Because of the many compromises involved, there is no ideal choice for a reagent, but certain guidelines can apply. Generally, t h e traditional methanol-pyridine- based reagents should be initially examined to ensure the greatest ease of assay transfer from development laboratory to production environment. If problems are found to exist with side reactions, an alternate apparent-pH buffered medium (such as 10% imidazole in methanol for volumetric systems) should be examined. Several different levels of accuracy validation can be envisioned for different times in the development of a product, but it is important to remember that often it is not just the Karl Fischer chemistry but the entire process of sample handling, titration, and data analysis that must be validated within some acceptable range of precision. Early in the development process, limited supplies, the high cost of materials, and infrequent samples may suggest the use of manual, coulometric techniques. If the cost of the material is lowered, volumetric techniques become more favorable and as sampling becomes more frequent, automated methods are preferred. The issue of hygroscopic sample-handling techniques may become convoluted into the evolution of methodology as well. The validation of t h e e n t i r e method should be reexamined as the methods of analysis are changed. Several different strategies can be used for validation, including verification of results by two independent methods (e.g., chromatography, moisture evolution analysis, thermogravimetric analysis, Karl Fischer oven techniques, normalization of elemental composition, o r spectroscopic methods), demonstration of water levels not significantly different from zero in a dried sample, demonstration of the lack of significant side reactions a t elevated concentrations in the titration cell, and demonstration of the same results in different Karl Fischer reagents. The independent methods a p proach is often the most rigorous method of validation, but, depending on the uncertainty of the methods used, the confidence may be low. The use of dried samples is a very sensitive method for determining bias but requires the use of careful hygroscopic sample-handling approaches. It is important to note that storage of a “dried” sample in a desiccator over silica gel exposes the sample to a rel-
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ative humidity of about 9% a t room temperature, and significant water uptake can result when these dried samples are subsequently assayed. In addition, many samples are difficult to dry a n d o r may decompose, making this approach impossible. Explicit examination of the side reactions using the approach of Scholz (15)works well for most systems but does not demonstrate that all the water has been determined, nor does it work well for very rapid side reactions. The use of different Karl Fischer reagents is a low confidence approach but may provide supporting data when other approaches cannot be used. Validation of a coulometric assay deserves special mention with regard to the fact that the solution is not changed between samples, and replicate samples that show increasing water content should be viewed with great suspicion. Such results may indicate that there is a side reaction t h a t increases in magnitude with each increase in analyte concentration. In a t least one case (18),after analysis of several samples, the recovery of water from a sample was found to be greater than 10096, indicating a lack of efficiency in the iodine generation step of the coulometric system. Solution of this problem can be verified by a calibration check after several samples are run. Sample handling can have a significant impact on the results of a titration a s a result of gain or loss of moisture between sampling and analysis. This problem becomes over whelming when one considers that no sample is in equilibrium with every environment throughout the year and around the world. To reduce t h e s e e r r o r s , sample containers should be impervious to moisture and the headspace should be small to minimize the partitioning of moisture. Samples should be weighed by difference from sealable weighing vessels. When other assays may be strongly influenced by moisture levels, samples for those procedures should be weighed a t the time of the moisture determination. Extremely hygroscopic samples may be handled using vacuum line transfer techniques (17). A thorough understanding of the chemistry of the Karl Fischer titration and a few simple experiments can help to assure the quality of moisture measurements. The choice of reagent, co-solvent, titration method, a n d sample handling procedure should be based on the demonstrated ability to produce valid results.
CIRCLE 64 ON READER SERVICE CARD
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991
The author is grateful to Larry Beaubien, Fumie Block, Phil Bowman, Daryl Chestney, Jack DeZwaan, Lois Marquardt, Dawn McDaniel, John Nanos, Tore Ramstad, Bill Schinzer, Alyssa TenHarmsel, and Dale Wieber for their contributions to this work. The Control Division management is acknowledged for providing the resources to pursue this work.
References (1)Fischer, K. Angew. Chem. 1935,48,394. (2) Mitchell, J., Jr.; Smith, D. M. Aquametry: Part III (The Karl Fischer Reagent); John Wiley and Sons: New York, 1980. (3) Scholz, E. Karl Fischer Titration-Detemination of Water-Chemical Laboratory Practice; Springer-Verlag:New York, 1984. (4) Sherman, F. B. Talanta 1980, 27, 106772. (5) Cedergren, A. Talanta 1978,25,229-32. (6) Verhoef, J. C.; Barendrecht, E. J. Electroanal. Chem. 1976, 71, 305-15. (7) Popovych, 0.; Tomkins, R.P.T. Nonaqueous Solution Chemistry; John Wiley and Sons: New York, 1981. (8) Verhoef, J. C.; Barendrecht, E. Anal. Chim. Acta 1977,94, 395-403. (9) Verhoef, J. C.; Barendrecht, E. J. Electroanal. Chem. 1977, 75, 705-17. (10) Verhoef, J. C.; Kok, W. Th.; Barendrecht, E. J. Electroanal. Chem. 1978, 86, 407-15. (11)Verhoef, J. C.; Barendrecht, E. Electroanal. Chem. Interfacial Electrochem. 1975, 59, 221-25. (12) Verhoef, J . C.; Cofino, W. P.; Barendrecht, E. J. Electroanal. Chem. 1978, 93, 75-80. (13) Verhoef, J. C.; Barendrecht, E. Electrochimica Acta 1978, 23, 433-38. (14) German Patent DE 3,040,474 A l , May 14, 1981. (15) Scholz, E. Anal. Chem. 1985, 57, 2965-71. (16)Hydranal Water Reagent According to Eugen Scholz for Karl Fischer Titration; 3rd id., Riedel-de Haen. (17) Brumleve, T. R. Anal. Chim. Acta 1983, 155, 79-87. (18) Scholz, E . Fresenius 2. Anal Chem. 1988, 330,694-97. (19) Kellum, G. E.; Smith, R. C. Anal. Chem. 1967,39, 1877-79. (20) Nordin-Andersson, I.; Cedergren, A. Anal. Chem. 1987,59, 749-53. (21)U.S. Patent 3 656 907, April 18, 1972. (22) Beasley, T. H.; Ziegler, H. W.; Charles, R. L.; King, P. Anal. Chem. 1972, 44, 1833-40. ,
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Steven K. MacLeod is a principal investigator at Upjohn responsible for development of pharmaceuticals for human use. He received a B.S. degree from Bates College and a Ph.D. from Colorado State University. His research interests include moisture determination, the theory and practice of chromatography, and computer simulation of analytical systems.
