Moisture Determination Using Karl Fischer Τitrations - Analytical

Moisture

Determination Using Karl Fischer Τitrations Steven Κ. MacLeod Control Division The Upjohn Company Kalamazoo, Ml 49001

An accurate and precise moisture de­ termination is often essential in re­ solving issues where water is involved. Water has been identified as a con­ taminant limiting the reactivity of products (e.g., monomers, various res­ ins and coating materials, prepregs), a reactant limiting the lifetime of a ma­ terial (e.g., halocarbons, acetylsalicylic acid), a medium for reaction between components in a mixture (e.g., phar­ maceutical formulations), and as a factor limiting the accuracy of other measurements (e.g., mass balance). Although a n u m b e r of c h r o m a t o ­ graphic, spectroscopic, electronic, thermal, and wet chemical methods have been used to determine moisture, the Karl Fischer titration, originally described in 1935 (1), is the approach most widely used. This REPORT includes discussions of the reaction chemistry, side reac­ tions, reagent composition, and ex­ perimental 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). It has been suggested that different reac­ tions occur, depending on the protic or aprotic n a t u r e of the medium (4, 5). 0003-2700/91/0363-557A/$02.50/0 © 1991 American Chemical Society

Titration i n protic media. The Karl Fischer reaction in protic media (i.e., alcohol) is shown below. 2ROH + S 0 2 RSOi + ROHJ

Solvolysis (1)

Β + RSOà + ROHJ ο B H + S 0 3 R - + ROH

Buffering

(2)

+

H 2 0 + I 2 + B H S 0 3 R - + 2B -> B H + S 0 4 R - + 2BHI Redox (3) Sulfur dioxide reacts with the alcohol to produce an alkyl sulfite in a buff­ ered medium using an appropriate base, B, to maintain the solution at t h e optimal a p p a r e n t pH (- 5 - 8 ) (3, 6, 7). Below pH 3, the overall re­ action proceeds very slowly, a n d above pH 8 nonstoichiometric side reactions become significant. When water is present in the cell and iodine is added, a redox reaction occurs.

REPORT The method of adding iodine to the reaction differs according to the type of experiment, volumetric or coulometric. In the volumetric experi­ ment, the iodine is contained in a bu­ ret and metered out as required. The amount of iodine per volume is em­ pirically determined during a stan­ dardization 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 gener­ ally 100%, 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 volu­ metric 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 con­ taining a high percentage of alcohol) is used as the reaction medium to dissolve the sample. The two-compo­ nent 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 experi­ ment, which will result in a slow overall reaction. As more titrant is added, the concentration of alkyl sulfite a n d buffering agent is in­ creased and, eventually, the reaction goes to completion (S). This condition may readily occur if the reaction me­ dium is very dry and requires only a small amount of reagent during the initial neutralization. Strongly basic or acidic samples may also over­ whelm 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-compo­ nent titration is virtually identical to

ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991 · 557 A

REPORT that of the single-component titra­ tion, but it may be faster because of the higher initial concentration of alkyl sulfite and buffer in the reac­ tion vessel. Samples containing trace amounts of water can be t i t r a t e d more a c c u r a t e l y u s i n g t h e t w o component r e a g e n t s b e c a u s e t h e buffering and concentration of alkyl sulfite are sufficient to promote rap­ id 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 al­ ways favor the two-component vari­ ety because these reagents may ex­ hibit greater susceptibility to side reactions involving noncomplexed sulfur dioxide than do single-compo­ nent systems. Detailed mechanistic studies of the Karl F i s c h e r r e a c t i o n w e r e p e r ­ formed by Verhoef and Barendrecht (6, 8-13) using various electrochemi­ cal techniques. They showed that the oxidizable species is an alkyl sulfite; t h a t pyridine plays no role in the re­ action except as a buffering agent; t h a t 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 di­ oxide, t h u s increasing the overall rate of reaction; and that the pre­ dominant reducible species is iodine (triiodide is only a secondary reactive species). Titration i n aprotic media. Non­ alcoholic Karl Fischer reagents were developed to increase the stability of the titrant and to overcome some un­ desirable side reactions involving al­ cohol. Because no alcohol is present to form the alkyl sulfite, other water consuming reactions are involved, but this technique is still often re­ ferred 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 2 H 2 0 + S 0 2 + 1 2 -> H 2 S 0 4 + 2ΗΙ

(4)

is the basis for the "universal" re­ agents (4) in which an aprotic solvent is used. Two moles of water are con­ sumed for each mole of iodine, in con­ trast to the 1:1 molar ratio found in the protic Karl Fischer environment. The drawback of this reaction for wa­ ter determination is the sensitivity of the titer to sample and solvent com­ position (4).

The presence of protic species in the sample tends to alter the stoichi ometry of the Karl Fischer reaction to an extent dependent on sample size a n d composition. T h u s , t h e water:iodine stoichiometry can vary from 2:1 to 1:1. A patent describing the use of alkylene carbonates as sol­ vents (14) demonstrates t h a t these solvents allow a conversion efficiency of only 9 0 - 9 5 % and t h a t a small amount of alcohol must be present to m a i n t a i n h i g h 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 else­ where. Volumetric v e r s u s coulometric chemistry. The reactions in the vol­ umetric and coulometric applications are identical except that iodine in the coulometric t i t r a t i o n is generated from the anodic oxidation of iodide. The nature of the cathodic half-reac­ tion is somewhat uncertain, but the observation of small bubbles rising in the cathode cell during rapid titra­ tion suggests t h a t protons are re­ duced to hydrogen gas. The concentrations of sulfur diox­ ide and base are constant over the course of the coulometric titration, making it similar to the two-compo­ nent, volumetric titration described above. The only suggested difference between the coulometric and volu­ metric systems (11) is that because the concentration of iodine is never high the concentration of triiodide is relatively low in the coulometric ex­ periment; this is probably not signif­ icant, because the rate constant for triiodide is 4 orders of magnitude less t h a n t h a t for iodine (3). Interfering side reactions A number of reactions can occur be­ tween the components of the sample to be analyzed and the various spe­ cies present in the Karl Fischer r e ­ agent (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 reac­ tions 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 an understanding of the chemistry occurring in each sit­ uation is equally as important as the

558 A · ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991

Time (min) Figure 1. Initial and delayed titration curves for well-behaved samples.

shape of the curves, because these side reactions do not all yield unique titra­ tion 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 re­ agent, and the curves come quickly to a plateau with a slope that approach­ es zero. Nondelayed and delayed ti­ trations 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 under­ estimation of the amount of water in the sample. Iodide may be oxidized by a reducible species (e.g., Cu 2 + , Fe 3 + , N 0 2 , Br 2 , Cl 2 , and quinones), and the resultant iodine can react with water present. As shown in Fig­ ure 2, this behavior should manifest itself in a curve in which the plateau of the delayed curve is lower t h a n that of the nondelayed curve as a re­ sult of the "pretitration" of some wa­ ter in the sample during the delay period. Elimination of this behavior is usually not possible in a 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 an apparent reduction in the wa­ ter content. During the time in which the sample dissolves, water can be tied up in this complex as shown be­ low. R 2 C=0 + S 0 2 + H 2 0 + Β ο R 2 C(OH)SO;HB +

(5)

The reaction can be reversible, and the shape of the titration curves de­ pends on the stability of the complex. A stable complex (with respect to ti-

REPORT

Time (min) Figure 2. Initial and delayed titration curves for samples that generate iodine.

Time (min) Figure 3. Initial and delayed titration curves for samples that form moder­ ately stable bisulfite complexes. tration time) may exhibit curves sim­ ilar to those in Figure 2, 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 t i t r a t i o n . Because t h e strength (and reversibility) of the bisulfite complex depends on the pKa of the base in the reagent, substitu­ tion of weaker bases (e.g., pyridine) for stronger bases (e.g., imidazole) may lessen the problem. Elimination of sulfur dioxide is not generally a vi­ able alternative in the Karl Fischer titration, but the use of methanolic reagents reduces the concentration of free sulfur dioxide by efficient com­ plex 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., am­ monia, thiols, Τ Γ , Sn 2 + , 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 io­ dine. This effect is illustrated in Fig­ ure 4, where the rising curves dem­ onstrate the continuous consumption of iodine. Moisture determination

may be possible after oxidation of the offending functionalities (3, 16). In the case of the phenols, a solvent sys­ tem with a reduced apparent pH may decrease the magnitude of the prob­ lem (18). Water may be produced in the re­ action cell in a variety of ways to pro­ duce 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. Resolu­ tion of this problem involves elimi­ nating the alcohol, substituting an­ o t h e r alcohol t h a t r e a c t s a t a n insignificant rate, or raising the ap­ parent pH of the reagent (this is gen­ erally a n acid-catalyzed process). Conversion of strong acids to amine salts prior to titration has been sug­ gested (16), although the uncertainty in the determination may rise as a result of the presence of an addition­ al 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 a p p a r e n t pH (15). This problem can often be solved by substituting another alco­ hol t h a t reacts at an 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 t h a t any attempt was made to control the apparent pH of the medi­ um. 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. It may also be possible to avoid reaction with the solvent by forming cyanohydrin derivatives of the compound pri­ or to analysis (2).

Some metal oxides and hydroxides can react with hydroiodic acid to pro­ duce water, causing the water con­ tent of the sample to be overestimat­ ed. Because the solution involves elimination of iodide from the cell, t h i s problem c a n n o t be resolved within the typical Karl Fischer ex­ periment. Silanols and cyclic siloxanes also can react with alcohols to produce ethers and water. Remedies involve the use of a hindered alcohol, reduc­ tion of the alcohol concentration, or the use of a buffer with a higher ap­ parent pH. A cooled titration cell ap­ proach using extrapolation to zero time to resolve the slower side reac­ tion from the Karl Fischer reaction also has been successfully employed (19). For aldehydes and ketones, the bisulfite reaction and acetal/ketal formation reactions often occur to­ gether. Low levels of residual sol­ vents with carbonyl functions (e.g., acetone) can result in unexpected molar equivalent amounts of appar­ ent water in the sample. This combi­ nation of reactions results in titra­ tion curves similar to those shown in Figure 5. Because of the severity of these reactions with aldehydes, it has been suggested that water can­ not reliably be determined in alde­ hydes via Karl Fischer titration (15). However, we have had considerable success using the approaches men­ tioned above. Other problems can be related to properties of the samples. If the sam­ ple 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 neu­ tralized prior to titration or added to a heavily buffered m e d i u m (e.g., 1:1 m e t h a n o l / p y r i d i n e or 10% w/v imidazole in methanol). At sufficiently high concentrations, any sample can effectively reduce the availability of the active Karl Fischer

Time (min)

Time (min)

Figure 4. Initial and delayed titration curves for samples that consume iodine or generate water.

560 A · ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991

Figure 5. Initial and delayed titration curves for samples that form water and bisulfite complexes.

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 r e a g e n t s (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

Composition of common Karl Fischer reagents Methanolic reagents Low apparent pH: Methanol, pyridine or substituted pyridine, sulfur dioxide, iodine High apparent pH: Methanol, imidazole or diethanolamine, sulfur dioxide, iodine Other alcoholic reagents Low apparent pH: 2-Methoxyethanol, pyridine or substituted pyridine, sulfur dioxide (methyl sulfite), iodine High apparent pH: 2-Methoxyethanol, imidazole or diethanolamine, sulfur dioxide (methyl sulfite), iodine Nonalcoholic reagents Propylene carbonate, pyridine, ethanol (low level), sulfur dioxide, chloroform, iodine

commonly based on imidazole. (However, because " p y r i d i n e - f r e e " r e agents are made using either a substituted pyridine to decrease vapor pressure while maintaining the apparent 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 q u a l i t a t i v e composition of some reagents as determined by capillary GC and GC/MS is shown in the box above. Coulometric cath-

Reagents used in Karl Fischer titrations Protic systems Alcohols Methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, ierf-butyl alcohol, fert-pentyl alcohol, propylene glycol, 2-methoxyethanol, ethylene glycol, 2-ethoxyethanol, 2-butoxyethanol, benzyl alcohol, 2-chloroethanol, trifluoroethanol, diphenylcarbinol, triphenylcarbinol, cyclohexanol Bases Pyridine, methylimidazole, diethanolamine, diisopropylpyridine, imidazole, acetate, guanidinium benzoate Modifiers Chloroform, formamide, carbon tetrachloride, dimethylformamide, dimethyl sulfoxide Aprotic systems Solvents Pyridine, benzene, chloroform, dimethylformamide, propylene carbonate, acetic acid Bases Pyridine, imidazole, acetate Modifiers Chloroform, carbon tetrachloride

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 t e t r a chloride 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 t h a t expected from pKa values for each base, by more than 0.1 units. The i d e a l r e a g e n t . 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 (1:1 iodine 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 sulfur 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 r e a g e n t s t h a t should h a v e 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 enhance­ ment of the iodine/triiodide ratio (20). Methanolic reagents, or those containing methyl sulfite, are consid­ ered the most rapid, as are those ad­ justed to higher apparent pH val­ ues (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 surfac­ tant film may decrease atmospheric absorption of moisture (21). Mechanics of the Karl Fischer 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 coulo­ metric titration, small amounts of io­ dine can be added accurately to fixed solution composition, resulting in the ability to titrate minute quantities of moisture. Standard deviations of 5-10 μg of water are typical in coulo­ metric systems versus 150 μg of wa­ ter in typical volumetric systems us­ ing commercial reagents, and the amount of water titrated is practical­ ly limited to about 3 mg in a coulo­ metric system versus 250 mg in a volumetric system. Titration of small quantities of wa­ ter in coulometric systems requires correction for atmospheric moisture entering the system. The leakage of moisture into a reaction cell (typical­ ly 5-15 μg/min) is generally compen­ sated by circuitry designed into cou­ lometric systems. When the sample cell is opened to introduce a sample, several milliliters of moisture-laden air also enters the cell, and this wa­ ter (typically 15-50 μg) must be measured and subtracted from the fi­ nal 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 mem­ brane in coulometric cells with insol­ uble materials as this slows the ti­ t r a t i o n a n d may r e d u c e t h e coulometric efficiency. Volumetric cells can handle particulates, al­ though concerns regarding the lack of total release of moisture in the ab­ sence of dissolution should be ad­ dressed. Endpoint detection. The potentiometric detection system is based on the presence of the iodine/iodide cou­

ple at a pair of electrodes with a small applied current. When operat­ ing in a "wet" solution, the absence of the oxidized form (iodine) results in a large potential difference between the sensing electrodes. During the ti­ tration, 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 concentra­ tion of iodine rises rapidly, resulting in a rapid decrease in the voltage dif­ ference between the two electrodes. The amperometric detection sys­ tem also depends on the presence of both members of the reversible cou­ ple in order for current to 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. Amperomet­ ric endpoint detection has been re­ ferred to as a "dead stop" technique, but, in view of the nature of the cur­ rent flow, a more precise description might be a "kick-off" endpoint (2,22). The current trend is toward potentiometric endpoint detection, al­ though a number of amperometric systems are still in use. In coulomet­ ric work, the trend is toward the use of a pulsed detection system ar­ ranged such that the detection pulse does not coincide with the pulsed coulometric currents. This arrange­ ment 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 titrat­ ed. Modern instruments may use an analogous measurement of the rate at which iodine must be added to the cell to compensate for moisture leak­ age into the cell and to maintain the equivalence point. The rate of iodine addition during a titration is com­ pared with the rate at the beginning of the titration to determine the endpoint of the titration. Visual endpoints, based on the col­ or of iodine in the vessel at the endpoint, have been used (2). Instru­ mental methods for colorimetric endpoint detection have only rarely been employed, and the potential advan­ tages of such systems are not clear (22). Calibration. A variety of materi­ als have been proposed as primary or

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REPORT secondary standards for water determination (3). The principal requirem e n t s of t h e s e materials are t h a t they contain a stoichiometric amount of moisture t h a t is stable over a wide range of temperatures and humidity, solubility in t h e Karl Fischer r e agents, ease of handling and storage, availability, and uniformity. Secondary w a t e r s t a n d a r d s a r e sold by various vendors. Although some efforts have been made to render these solutions less hygroscopic, t h e w a t e r content of t h e 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 w a t e r is c o m p a r e d w i t h 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 s t a n d a r d for water determination is being developed as an in-house standard. A s s a y d e v e l o p m e n t a n d validat i o n . Sample size can limit the choice of instrument used for titration with smaller, more valuable, or more toxic samples, favoring t h e coulometric method. Solubility limitations also favor coulometric titrations because the smaller sample size is typically dissolved in a volume larger t h a n t h a t 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 iodinerwater 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, dimethyl formamide and dimethyl sulfoxide have been problematic at high con-

c e n t r a t i o n s and therefore are not used as co-solvents.) Because of the many compromises involved, there is no ideal choice for a reagent, but certain guidelines can apply. G e n e r a l l y , t h e t r a d i t i o n a l m e t h a n o l - p y r i d i n e - b a s e d reagents should be initially examined to ensure the greatest ease of assay t r a n s fer from development laboratory to production environment. If problems are found to exist with side reactions, an alternate a p p a r e n t - p H buffered medium (such as 10% imidazole in m e t h a n o l 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 r e member 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 v a l i d a t i o n of t h e e n t i r e m e t h o d 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 m e t h o d s (e.g., c h r o m a t o g r a p h y , moisture evolution analysis, thermogravimetric analysis, Karl Fischer oven techniques, normalization of elemental composition, or spectroscopic methods), demonstration of water levels not significantly different from zero in a dried sample, demonstration of the lack of significant side reactions at 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 t h a t storage of a "dried" sample in a desiccator over silica gel exposes the sample to a rel-

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REPORT ative humidity of about 9% at room temperature, and significant water uptake can result when these dried samples are subsequently assayed. In addition, many samples are diffi­ cult to dry and/or may decompose, making this approach impossible. Explicit examination of the side re­ actions using the approach of Scholz (15) works well for most systems but does not demonstrate that all the wa­ ter has been determined, nor does it work well for very rapid side reac­ tions. The use of different Karl Fis­ cher reagents is a low confidence ap­ proach 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 repli­ cate samples t h a t show increasing water content should be viewed with great suspicion. Such results may in­ dicate that there is a side reaction t h a t increases in m a g n i t u d e with each increase in analyte concentra­ tion. In at least one case (18), after analysis of several samples, the re­ covery of water from a sample was found to be greater than 100%, indi­ cating a lack of efficiency in the io­ dine generation step of the coulomet­ ric system. Solution of this problem can be verified by a calibration check after several samples are run. Sample handling can have a signif­ icant impact on the results of a titra­ tion as 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 these errors, sample containers should be impervious to moisture and the headspace should be small to minimize the partitioning of mois­ ture. Samples should be weighed by difference from sealable weighing vessels. When other assays may be strongly influenced by moisture lev­ els, samples for those procedures should be weighed at the time of the moisture determination. Extremely hygroscopic samples may be handled u s i n g vacuum line transfer tech­ niques (17). A thorough understanding of the chemistry of the Karl Fischer titra­ tion and a few simple experiments can help to assure the quality of moisture measurements. The choice of reagent, co-solvent, titration method, and sample handling procedure should be based on the demonstrated ability to produce valid results.

CIRCLE 64 ON READER SERVICE CARD

566 A · 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 con­ tributions 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—Determi­ nation of Water—Chemical Laboratory Prac­ tice; 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. /. Electroanal. Chem. 1976, 71, 305-15. (7) Popovych, O.; Tomkins, R.P.T. Non­ aqueous 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. /. Electroanal. Chem. 1977, 75, 705-17. (10) Verhoef, J. C; Kok, W. Th.; Baren­ drecht, E. /. Electroanal. Chem. 1978, 86, 407-15. (11) Verhoef, J. C; Barendrecht, E. Elec­ troanal. Chem. Interfacial Electrochem. 1975, 59, 221-25. (12) Verhoef, J. C; Cofino, W. P.; Baren­ drecht, E. /. 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 Al, 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 éd., Riedel-de Haen. (17) Brumleve, T. R. Anal. Chim. Acta 1983, 155, 79-87. (18) Scholz, E. Fresenius Z. 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.

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.