Karl Fischer Titration

Unlike gravimetric measurements , which are indirect methods that assume that all volatiles removed are water, the Karl Fischer titration is a direct method that is almost specific for water. The method is especially useful for low moisture levels (<1%), and levels <0.01% are achievable. The Karl Fischer titration method is especially useful for samples that may be high in sugar, or high in both reducing sugar and protein, which may decompose in the gravimetric methods. The method is a titration of water with an anhydrous methanol solution containing iodine, sulfur dioxide and excess pyridine. The titration is based on the reaction between iodine and sulfur dioxide that will occur only if water is present:

I2-Pyr + SO2-Pyr + Pyr + H2O SO3-Pyr + 2PyrH+I

where Pyr represents pyridine. The product, SO3-Pyr reacts further with the methanol to form the methylsulfate anion:

SO3-Pyr + CH3OH PyrH+CH3SO4

From these equations it can be seen that each mole of water requires one mole of I2. In a visual endpoint Karl Fischer titration, a sample is titrated with the Karl Fischer reagent until a permanent iodine color (indicating that all water has been reacted) is observed. Because of other reaction products, the color change is usually from a yellow to a brownish color, which may be difficult to detect visually. Highly colored samples may affect the visual end point as well. A much sharper end point, known as the dead stop end point, can be obtained if the titration is done electrometrically. Here, two small platinum electrodes dip into the titration cell, a small constant voltage is impressed across these electrodes, and any current that flows is measured with a galvanometer. At the end point of the titration the current either goes to a minimum or else increases suddenly from nearly zero. Commercially available Karl Fischer instruments incorporate semiautomatic mi- croprocessors based on this principle. Only the volumetric Karl Fischer titration (see Basic Protocol) will be discussed in this unit. In addition, it is assumed that the sample being tested is a low-moisture solid, where it is necessary to weigh out the sample, add it to a sealable flask of anhydrous methanol to extract the water, and measure the amount of extracted water. Materials Karl Fischer reagent, Water standard Karl Fischer solvent: anhydrous methanol (dried using molecular sieves) Sample for analysis, homogeneous Karl Fischer titration assembly, automatic (Metrohm 701 or 758, Brinkmann or equivalent) 1-ml syringes (or 10-ml for low-moisture samples) Dried, clean 10- and 50-ml volumetric flasks Balance capable of measuring 0.1 mg

Standardize Karl Fischer reagent 1. Set up titration assembly per manufacturers instructions. Most titrators have a sealed titration vessel where liquid samples are injected and the titrator adds the Karl Fischer reagent until a stable endpoint is achieved. This protocol assumes such a case where the endpoint is indicated as that volume of Karl Fischer reagent required to react with all water present. No matter which volumetric titration instrument is used, it is necessary to standardize the Karl Fischer reagent and run blanks on the Karl Fischer solvent (methanol). Commercially available Karl Fischer reagent is stabilized and can be obtained free of pyridine, which is toxic. It can also be prepared in the laboratory; however, thisrequires the use of SO2, a poisonous, liquified gas. 2. Add dried Karl Fischer solvent to the titration assembly vessel so that electrodes are submerged, and seal vessel. Remember to reseal the container containing the Karl Fischer solvent immediately after removing the amount needed. The vessel will allow many titrations before it has to be emptied, cleaned, dried and reassembled. 3. Run titrator, without adding any sample, to neutralize any water from the Karl Fischer solvent or moisture in vessel. 4. Accurately weigh 125 mg of water and dilute to volume in a dried, clean 10-ml volumetric flask with dried Karl Fischer solvent. 5. Draw up and discard an aliquot of water standard in a syringe to remove any moisture in the syringe. Repeat. Remember to reseal flask immediately upon removing any sample. 6. Inject 1-ml aliquot of the water standard into the vessel and titrate to determine conversion factor. This standard must be run daily or whenever a new batch of Karl Fischer reagent or solvent is being used. 7. Draw up and discard an aliquot of the Karl Fischer solvent to remove any moisture in the syringe. Repeat. 8. Inject a 1-ml aliquot of dried Karl Fischer solvent into the vessel of the titration assembly and titrate to determine blank correction. Duplicates are recommended. 9. Using measured blank result, correct readings for Karl Fischer solvent and determine standardization concentration for the Karl Fischer reagent (mg H2O/ml Karl Fischer reagent). Most commercial Karl Fischer reagents run 5 mg H2O/ml reagent. Extract and measure sample moisture 10.Weigh, to the nearest 0.1 mg, a quantity of the homogeneous sample containing 100 mg water into a dried, 50-ml volumetric flask. Dilute to volume with dried Karl Fischer solvent.

11. Add a small stir bar, seal flask, and stir for 10 min. Let sample settle. 12. After solids have settled out, draw up and discard an aliquot of the solvent containing the extracted water, to remove any extraneous moisture from the syringe. Repeat. 13. Inject a 1-ml aliquot of the solvent containing the extracted water into the vessel and start titration. Use larger syringe to inject larger volumes for low moisture samples. Duplicates are recommended. 14. Calculate percent moisture using the equation: % Moisture = [(M B)(C/1000/W) 50] 100 where M is ml of Karl Fischer titrant from the 1-ml injection in step 13, B is ml ofKarl Fischer titrant for the blank in step 8, and C is the standardization amount (mg H2O/ml Karl Fischer titrant) in step 6, W is the weight of sample in grams in step 10,1000 converts mg to g, 50 is a dilution factor (50 ml total volume, 1 ml aliquot used),and 100 converts to percentage. COMMENTARY Background Information The Karl Fischer titration, nearly specific for water, is very important when looking at very low levels of moisture. It is especially useful for the determination of water in organic com- pounds and for some samples that are partially decomposed at temperatures used in the gravi- metric methods. Unlike gravimetric methods, the Karl Fischer titration does not provide a dried sample that may be used for further tests. Because it is a sensitive method, contact with any moisture, even from the sur- rounding environment, must be eliminated as much as possible. Commercial instruments are available with allglass joints and seals, which keep moisture contamination to a minimum. In addition, these instruments can be fitted with openings into the reaction vessel where sam- ples can be added directly. The disadvantage of direct addition of samples is the presence of particulates and decomposition materials in the reaction vessel, which eventually will have to be removed more frequently. Commercial instruments can be classified into either coulometric or volumetric Karl Fis- cher titrators. In coulometric Karl Fischer measurements, one measures the amount of water that will undergo a reaction at an elec- trode with 100% efficiency by measuring the amount of electricity (in coulombs) necessary to effect complete reaction (in this case, be- tween water and the Karl Fischer reagent). The advantage is that standard solutions are unnec- essary here. Sensitivity is very high (10 g water) and is very useful for trace level deter- minations. In the volumetric Karl Fischer titra- tion, the analysis is based on the measurement of the volume of a standard solution (Karl Fischer reagent) that must be added to react with the water present. From the volume of the reagent of known concentration that has been added to reach the end point, the concentration of water may be determined. Volumetric Karl Fischer titrators require that the Karl Fischer reagent be standardized, but moisture levels from 10 ppm up to 100% can be measured. Which type of titrator to choose depends on individual needs (trace or high-level measure- ments) and budget as coulometric Karl Fischer titrators generally

cost more than volumetric Karl Fischer titrators. Though not as sensitive at very low amounts of moisture compared to coulometric Karl Fischer titrators, volumetric Karl Fischer titrations allow one a larger dy- namic range to measure. Though the protocol was written for a dry solid, it should also be mentioned that liquid samples can also be run (Firestone, 1998). Liq- uid samples can be added directly to the vessel itself or can be treated as a solid with an extrac- tion step in the Karl Fischer solvent as pre- sented in the protocol. The preference for mois- ture analyses is the gravimetric method largely due the higher costs of equipment and the use of chemicals in the Karl Fischer method. Currently one observes very few ap- plications of the Karl Fischer method on food products published by the AOAC for nutritional labeling purposes. In particular, these food products are cocoa, cocoa products, confec- tionery coatings, and molasses (Sullivan and Carpenter, 1993). Critical Parameters and Troubleshooting Like the gravimetric methods, the Karl Fis-cher titration method requires only a very small amount of sample (between 1 and 10 g depend- ing on the moisture level). Therefore, sample homogeneity is very critical and special care must be given to sample blending before sub- sampling for analysis. The method used for homogenization will depend on the type of sample being analyzed. Blenders, homogeniz- ers, mincers, food processors, and graters are currently available for dry, moist, and very wet samples. Because these devices produce heat, it is very important not to overhomogenize, thereby causing moisture to be lost. Once a sample has been properly homogenized, it is from 10 ppm up to 100% can be measured. Which type of titrator to choose depends on individual needs (trace or highlevel measure- ments) and budget as coulometric Karl Fischer titrators generally cost more than volumetric Karl Fischer titrators. Though not as sensitive at very low amounts of moisture compared to coulometric Karl Fischer titrators, volumetric Karl Fischer titrations allow one a larger dy- namic range to measure. Though the protocol was written for a dry solid, it should also be mentioned that liquid samples can also be run (Firestone, 1998). Liq- uid samples can be added directly to the vessel itself or can be treated as a solid with an extrac- tion step in the Karl Fischer solvent as pre- sented in the protocol. The preference for mois- ture analyses is the gravimetric method largely due the higher costs of equipment and the use of chemicals in the Karl Fischer method. Currently one observes very few ap- plications of the Karl Fischer method on food products published by the AOAC for nutritional labeling purposes. In particular, these food products are cocoa, cocoa products, confec- tionery coatings, and molasses (Sullivan and Carpenter, 1993). Anticipated Results

Levels of moisture <0.01% are measurable and one can expect a relative standard deviation of 1%.

Time Considerations The Karl Fischer titration requires 20 to 45 min (depending on sample being tested) to complete calibration, blank determination, sample equilibration (extraction in Karl Fischer solvent), and final testing. Key References Cunniff, P. (ed.) 1998. Official Methods of Analysis of the Association of Official Analytical Chemists, 16th ed., 4th rev. AOAC International, Gaith- ersburg, Md. Mitchell, J. and Smith, D.M. 1977. Aquametry, Part I. John Wiley & Sons, New York. Textbook devoted largely to analytical applications of the Karl Fischer method.