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KARL FISHER APPARATUS AND ITS PERFORMANCE VERIFICATIONR ICK J AIRAM, R OBERT M ETCALFE, P H.D., AND Y U-H ONG T SE, P H.D. GlaxoSmithKline Canada, Inc.14.1 INTRODUCTIONThe Karl Fisher titration is one of the most common and most sensitive methods used in the analytical laboratory. The titrimetric determination of water is based on the quantitative reaction of water with an anhydrous solution of sulfur dioxide and iodine in the presence of a buffer that reacts with hydrogen ions. This titration is a two-stage process:SO2 + MeOH + RN→(RNH)SO3Me (14.1)(RNH)SO3Me + I2 + H2O + 2RN→(RNH)SO4Me + 2(RNH)I (14.2)where RN is a base, typically pyridine or imidazole. Reaction (14.1) reaches equilibrium and produces methylsulfite as the reaction intermediate. Reaction (14.2) the redox process, is very rapid. From equation (14.2) the direct relation between water and iodine consumption can be seen, which enables the amountof water to be determined. Complete esterification of the sulfur dioxide with the alcohol, and the ability of the base to neutralize the methyl sulfurous acid, arethe key requirements for the reaction above to be stoichiometric.Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan, Herman Lam, Y. C. Lee, and Xue-Ming ZhangISBN 0-471-25953-5 Copyright 2004 John Wiley & Sons, Inc.221222 KARL FISHER APPARATUS AND ITS PERFORMANCE VERIFICATIONPyridine was used in the beginning of the development of the method. The reaction was slow and the endpoint unstable because of weak basicity of pyridine. The pyridine system buffers at about pH 4. A stronger base, imidazole, has been used to replace pyridine since it gives a faster response and has the advantages of lower toxicity and decreased odor. The optimal pH range for the SO2 imidazole buffer is at pH 6. It is important that the pH of the Karl Fisher reaction be maintained within the range 5 to 7. Outside this recommended pH range, the endpoint may not be reached.There are two types of Karl Fisher titrations: volumetric and coulometric. Volumetric titration is used to determine relatively large amounts of water (1to 100 μg) and can be performed using the single- or two-component system. Most commercially available titrators make use of the one-component titrant, which can be purchased in two strengths; 2 mg of water per milliliter of titrant and the 5 mg of water per milliliter of titrant. The choice of concentration isdependent on the amount of water in the sample and any sample size limitations.In both cases, the sample is typically dissolved in a methanol solution. Theiodine/SO2/pyridine (imidazole) required for the reaction is titrated into the sample solution either manually or automatically. The reaction endpoint is generally detected bivoltametrically.Coulometric titration is used to determine relatively low concentrations ofwater (10 μg to 10 mg) and requires two reagents: a catholyte and an anolyte (the generating solution). The iodine required for the reaction is generated in situby the anodic oxidation of iodide.2I−→I2 + 2e−(14.3)The iodine then reacts with the water that is present. The amount of water titratedis proportional to the total current (according to Faraday’s law) used in generating the iodine necessary to react with the water. One mole of iodine reacts quantitatively with 1 mol of water. As a result, 1 mg of water is equivalent to 10.71 C.Based on this principle, the water content of the sample can be determined bythe quantity of current that flows during the electrolysis. For this reason, the coulometric method is considered an absolute technique, and no standardizationof the reagents is required.14.2 SCOPE OF CHAPTERThe Karl Fisher instrumentation and its performance verification are discussed in this chapter. The instrumentation, calibration practices, and common difficulties that are encountered are presented. Neither method validation nor method specific problems are discussed.14.3 INSTRUMENTATIONKarl Fisher apparatus has to be designed to exclude moisture, deliver titrant, andto detect the endpoint. The air in the system is kept dry with a suitable desiccant, INSTRUMENTATION 223and the titration vessel may be purged by means of a stream of dry nitrogen orair. For endpoint detection, most commercially available units use a bivoltametric method to indicate that the endpoint has been reached. For this method a constant current of about 20 μA is applied across a pair of platinum electrodes that are about 2.5 mm apart. As the titration proceeds, water reacts with iodine and is consumed. When the endpoint is reached and all the water is consumed, thereis a buildup of free iodine in solution. The free iodine causes ionic conductionin the solution. As a result, the voltage must be reduced to keep the polarization current constant [1,2]. When the voltage drops below a defined value, the titrationis stopped. Figure 14.1 shows a schematic diagram of a typical KF titrator.In some cases (see below) a KF drying oven is required to get the water froma sample into the titration vessel. For these special cases, a solid sample (usually)is placed into a specially designed KF oven where the sample is heated, and the water goes into the vapor phase. A stream of dry carrier gas (usually, N2 orair) sweeps the liberated moisture into the reaction vessel, where it is titratedby either the coulometric or the volumetric method. It is critical that the carriergas is dry and that there are no leaks along the pathway to the reaction vessel. Passing the carrier gas over activated molecular sieve prior to the sample will ensure that the gas is dry.The KF reaction depends on free water available for titration. Instruments maybe designed to homogenize the sample to release water prior to the titration. In Magnetic StirrerDouble Pt electrodeMethanol solutionTo pumping systemTubing to deliver titrantI = 20 m AV = variableFigure 14.1. Schematic diagram of a typical Karl Fischer titrator.224 KARL FISHER APPARATUS AND ITS PERFORMANCE VERIFICATIONthis case, a high-speed blender may be used directly in the titration vessel. Thisis a particularly useful accessory for determination of water in tablets or certain food products that are difficult to prepare for analysis.14.3.1 Performance VerificationThe performance verification of Karl Fisher apparatus should include checksfor the accuracy and precision of the instrument. The linearity of the instrument should be determined at installation. The first step is to standardize the instrument (see Section 14.3.2); pure water is sufficient for this purpose. Sodium tartrate dihydrate standard (water content 15.66 ±0.05%) can be used to assess the accuracy, precision, and linearity of the instrument. Typically, one would measure the water content of at least five samples, over the intended instrument user range. For example, the water content of sodium tartrate dihydratesamples that were 65 mg (ca. 10 mg H2O), 195 mg (ca. 30 mg H2O), 325 mg (ca. 50 mg H2O), 455 mg (ca. 70 mg H2O), and 650 mg (100 mg H2O) could be determined. Calculate the percent water to assess the instrument’s accuracy. The results should be within 98 to 102% of 15.66% water. Determine the % RSD of the percent water found to assess the precision of the instrument. The % RSD should be less than or equal to 1%. Finally, plot the expected water content versus the percent water content to assess the linearity of the instrument’s response.A correlation coefficient (r) value of 0.999 or greater is acceptable.14.3.2 StandardizationStandardization should be performed daily, since the titrant will absorb moisture over time. The standardization is typically performed using one of three standards: (1) disodium tartrate dihydrate (15.66% water, w/w), (2) commercially available water standards with a certified concentration of water (typically, 10 mg of water per gram of standard), or (3) pure water. In all of these cases, a suitable balance should be used, preferably one that gives at least ±0. l mg of resolution.When disodium tartrate dihydrate is used, samples ranging from 50 to 120 mg are dissolved in methanol, and the concentration of the KF reagent is determined. Based on volume of titrant used, the weight of the sample and the percent waterin the disodium tartrate dihydrate (15.66% w/w), the standardization factor can be calculated. One pitfall with this method is the solubility of the disodium tartrate dihydrate in methanol. It is recommended that the disodium tartrate dihydrate be finely divided and that a suitable extraction time be given for the solids to dissolve. If a water standard is used, samples of the water standard ranging from 1.0 to1.5 g are injected into the methanol via syringe. This method is best performedby opening the ampoule that contains the water standard, rinsing out a 10-mL syringe with about 1 mL of the standard, and then filling the syringe with the remaining contents of the ampoule. Aliquots of approximately 1 mL are then injected into the vessel, and the true weights are determined by difference. Standardization of the KF titrant using purified water is widely accepted and probably the most commonly used in analytical laboratories. Using a suitable COMMON PROBLEMS AND SOLUTIONS 225glass syringe, a known volume of purified water can be added in the titration vessel, and the KF value for the titrant can be determined. This method is highly operator dependent and is not recommended for measurements where the accuracy of the determination is critical. The preferred method would be to add aknown quantity of water by weight.In the coulometric method, standardization is not necessary, since the current consumed can be measured absolutely. However, a standard with known water content should be checked periodically to ensure that the system is functioning properly. In this case, a certified water standard is generally used, and the amount of water is determined and compared with the amount that is certified to be present. Some coulometric titrators are equipped with an oven for liberating the moisture from samples that are either insoluble in methanol or that react with I2, methanol, or one of the other reagents. Solid standards (e.g., potassium citrate monohydrate) are available for checking the oven, and this check is performed after the coulometer function has been verified.14.3.3 Measurement of SamplesThe handling of the sample, its storage, and the amount of sample used are very important to consider when making a KF measurement. When taking a sample from the bulk material, great care must be taken to exclude atmospheric moisture, which is one of the most common sources of error. If the sample absorbs or desorbs water during its handling, the true moisture content can no longer be determined. The sample must also contain the average amount of water that is contained in the material as a whole. This is a particular problem when dealing with nonpolar liquids such as fats or oils where the water is floating on the top or settled to the bottom of the container. In this situation the liquids must be mixed thoroughly just prior to sampling to disperse the water evenly.Once a sample is taken, it is best to determine the water as quickly as possible.If a sample must be stored, it is best to keep it in a small, tightly sealed glass bottle with a screw cap. Glass is better than plastic since plastic is not water vapor–tight. The size of the bottle should be as small as possible to minimizethe gas space that is above the sample. A smaller gas space will mean lessmoisture vapor.The size of the sample to be used depends on the expected water content andthe degree of accuracy that is desired. If a high level of accuracy is required, a large sample size should be used to minimize the effect of atmospheric moisture. For titrimetric KF, the United States Pharmacopoeia recommends using a sample that will contain between 10 and 250 mg of water. The minimum amount of water is generally agreed to be 10 mg.14.4 COMMON PROBLEMS AND SOLUTIONSAtmospheric moisture is a major cause of error in Karl Fischer titrations. Moisture can enter the sample, titrant, and titration vessel. The apparatus and titrant must 226 KARL FISHER APPARATUS AND ITS PERFORMANCE VERIFICATIONbe sealed against atmospheric moisture and situated away from high-humidity areas of the laboratory. Desiccants and dry purge gases can be used to reduce moisture ingress. Proper conditioning of the apparatus is required to remove moisture present in the apparatus before the titration. Moisture will, however, enter the apparatus during introduction of the sample in a normal lab environment. With high-speed blending, this introduction of moisture can cause bias due to homogenizing air containing water into the sample. To reduce bias when highspeed blending is used, it is necessary to apply background correction or purgethe sample compartment to exclude moisture.The titrant delivery system can also be a source of error. The delivery system needs to be precise in the number of steps required to titrate a fixed amount of water. The delivery of titrant is typically controlled by either a peristaltic pumpor an automatic buret. Both are acceptable methods for the delivery of titrant; however, the peristaltic pump relies on the contact between the pump rollersand the tygon tubing, which is pressed into position by a platen. Therefore, the quality of the tubing that is used is critical to the accuracy of the instrument, so this tubing should be examined frequently to make sure that it is in good working order (free of blockages and not pinched to the point that flow is restricted).The pump rollers should also be examined to make sure that they are operating well, and the platen should be examined to make sure that it holds the tubing tightly against the rollers. The automatic buret is actually a syringe driven bya metered servomotor. For this type of delivery system the key concerns arethat the servomotor is operating properly (i.e., delivering the correct amount of liquid) and that there are no leaks, blockages, or air bubbles in the lines or in the syringe itself.Depending on the samples being titrated, electrode contamination may bean issue. The electrodes may become coated when substances such as oils and sugars are titrated. This results in delayed endpoint detection and an overtitration.A dark brown color on the electrodes indicates that they should be cleaned. The coatings can usually be removed by polar organic solvents or by cleaning the platinum physically.Side reactions can cause major problems in the determination water [3]. Some chemical species react with methanol to produce water that will lead to higherwater results, some react with water (low results), and some react with iodine (high result). Aldehydes and ketones react with methanol to produce acetalsand ketals. Water is a by-product of this reaction, and its production leads to erroneously high water contents:Acetal formation: CH3CHO + 2CH3OH →CH3CH(OCH3)2 + H2O (14.4)Ketal formation : (CH3)2CO + 2CH3OH →(CH3)2C(OCH3)2 + H2O (14.5)This can be remedied by using commercially available one-component reagents that contain 2-methoxyethanol and 2-chloroethanol rather than methanol. In the presence of SO2 and base, aldehydes, undergo what is called the bisulfite addition. REFERENCES 227This reaction consumes water.Bisulfite addition: CH3CHO + H2O + SO2 + NR →HC(OH)SO3(HNR)(14.6)The kinetics of this type of reaction are slow, so this can be avoided by starting the reaction immediately after sample addition.Many samples have redox potentials such that they can be oxidized by iodine. Therefore, the iodine in the titrant may be consumed by readily oxidizable samples that will give a false high value for the water content. Some common substances that can be oxidized by iodine are ascorbic acid, arsenite (AsO2−),arsenate (AsO43−), boric acid, tetraborate (B4O72−), carbonate (CO32−), disulfite(S2O52−), iron(II) salts, hydrazine derivatives, hydroxides (OH−), bicarbonates(HCO3−), copper(I) salts, mercaptans (RSH), nitrite (NO2−), some metaloxides, peroxides, selenite (SeO32−), silanols (R3SiOH), sulfite (SO32−), tellurite(TeO32−), thiosulfate (S2O32−), and tin(II) salts. For situations such as thesewhere the material under analysis reacts with iodine, an oven can be used to liberate the moisture from the sample, which is then carried into the reaction vessel and titrated without interference.REFERENCES1. United States Pharmacopoeia, USP 25, Chapter _921_, Water Determination.2. Fundamentals of the Volumetric Karl Fischer Titration, Mettler Toledo Application Brochure 26.3. Riedel-deHaen Hydranal Manual.。