Experimental Procedure for Determining Trace Moisture in Food by Karl Fischer Method

The Karl Fischer (KF) method, also known simply as the Fischer method or titration method, is an application of iodine-based titration in non-aqueous media. It is the most specific and accurate chemical method for moisture determination. The KF method was first officially adopted as an AOAC method in 1977.

The Karl Fischer method is widely used for determining trace moisture content in various liquids, solids, and some gases, consistently delivering satisfactory results. In many cases, this method is considered the standard analytical technique for trace water at the mg/kg level.

In food analysis, with appropriate precautions, the Karl Fischer method can be applied to samples with moisture content ranging from 1 mg/kg up to nearly 100%. It has been successfully used for moisture determination in flour, granulated sugar, margarine, chocolate, fats and oils, cocoa powder, molasses, dehydrated fruits and vegetables, confectionery, tea, milk powder, condensed milk, spices, and other food products.

For laboratories seeking precise trace moisture analysis in food and other materials, Alfa Chemistry offers professional Karl Fischer (KF) moisture testing services, utilizing both volumetric and coulometric methods to meet varying analytical needs.

The accuracy of moisture measurement by this method is superior to direct drying methods, and it is also the ideal method for determining trace moisture in fats and oils.

Instruments and Reagents

① Karl Fischer Titration Apparatus: Automatic or semi-automatic, equipped with a stirrer.

② Anhydrous Methanol: The water content must be below 0.05%. Measure about 200 mL of methanol and place it in a dry round-bottom flask. Add 15 g of clean magnesium ribbon (or magnesium filings) and 0.5 g of iodine. Attach a condenser; install anhydrous calcium chloride drying tubes at the top of the condenser and the branch of the receiver to prevent moisture contamination from the air. Heat under reflux until the metallic magnesium starts converting to white flocculent magnesium methoxide. Then add 800 mL of methanol and continue refluxing until the magnesium ribbon dissolves completely. Distill and collect the methanol fraction boiling at 64–65 °C using a dry suction flask as the receiver. During reflux and distillation, ensure calcium chloride drying tubes are installed at the condenser top and receiver branch. Methanol is toxic, so avoid inhaling its vapors during handling.

③ Anhydrous Pyridine: Water content must be below 0.1%. It can be dehydrated by the following method: take 200 mL of pyridine in a dry distillation flask, add 40 mL of benzene, and distill. Collect the pyridine fraction boiling at 110–116 °C.

④ Iodine: Dry solid iodine in a sulfuric acid desiccator for more than 48 hours.

⑤ Anhydrous Sodium Sulfate.

⑥ Sulfuric Acid.

⑦ Sulfur Dioxide: Use sulfur dioxide from a gas cylinder or produce it by decomposing sodium metabisulfite with sulfuric acid.

⑧ 5A Molecular Sieves.

⑨ Water-Methanol Standard Solution: Each milliliter contains 1 mg of water. Accurately pipette 1 mL of water into a pre-dried 1000 mL volumetric flask and dilute to the mark with anhydrous methanol. Mix well and keep for use.

⑩ Karl Fischer Reagent: Weigh 85 g of iodine into a dry 1 L brown glass reagent bottle with a stopper. Add 670 mL of anhydrous methanol, cover and shake until the iodine completely dissolves. Then add 270 mL of pyridine and mix well. Place the bottle in an ice-water bath and pass dry sulfur dioxide gas (60–70 g) through it. After gas passage, stopper the bottle and store it in a dark place for at least 24 hours before use.

Calibration of Karl Fischer Reagent:

Add 50 mL of anhydrous methanol to the titration vessel of the moisture analyzer. Turn on the instrument power and start the electromagnetic stirrer. First, add Karl Fischer reagent dropwise into the methanol to react with any residual trace moisture until the endpoint is reached, which corresponds to a fixed microammeter reading (45 μA or 48 μA) and remains stable for 1 minute (the reagent consumption at this stage is not recorded). Then, using a 10 μL micro-syringe, slowly inject 10 μL of distilled water [equivalent to 10 mg of water, which can be pre-weighed by balance or measured by gravimetric titration with a calibrated burette to 0.0001 g precision] into the vessel through the feeding port (sealed with rubber stopper). At this point, the microammeter needle shifts left close to zero. Titrate again with Karl Fischer reagent to the original endpoint and record the reagent consumption.

The titration factor T of Karl Fischer reagent (mg/mL) is calculated by:

Where:

T = Titration factor of Karl Fischer reagent, mg/mL

M = Mass of water, mg

V = Volume of Karl Fischer reagent consumed for titrating water, mL

Experment Steps

For solid samples, crush them as finely as possible to achieve uniformity. Samples that are difficult to pulverize can be chopped. For products such as candies, pre-grinding to a uniform powder is necessary. Depending on the moisture content of the sample, it is generally appropriate to have 20~40 mg of water per portion of the sample tested. Accurately weigh 0.30 to 0.50 g of the sample and place it into the sample weighing vial.

Add 50 mL of anhydrous methanol to the titration vessel of the moisture analyzer, ensuring the electrodes are fully submerged. Use Karl Fischer reagent to titrate the trace moisture present in the 50 mL methanol until the microampere meter needle deflection matches the deflection observed during the calibration of the Karl Fischer reagent. Maintain this reading for 1 minute without recording reagent consumption. Then, quickly add the weighed sample to the titration vessel through the sample addition port, immediately seal the vessel with a rubber stopper, and start the electromagnetic stirrer to fully extract the moisture from the sample into the methanol. Continue titrating with Karl Fischer reagent until the originally set endpoint is reached and maintain it for 1 minute. Record the volume of reagent consumed (mL).

Expression of Analysis Results

Where:

T — Titer of the Karl Fischer reagent for water, mg/mL;

V — Volume of Karl Fischer reagent consumed during titration, mL;

m — Mass of the sample, g.

When the moisture content is ≥ 1 g/100 g, the result is reported with three significant figures; when moisture content is < 1 g/100 g, the result is reported with two significant figures.

Precision

The absolute difference between two independent determinations under repeatability conditions shall not exceed 10% of their arithmetic mean.

Notes and Precautions

① For samples that are difficult to dissolve, the titration cup should be heated or supplemented with a solvent of known moisture content to assist dissolution before titrating to the endpoint with Karl Fischer reagent. It is recommended that when using the coulometric method, the water content in the sample should be greater than 10 μg; for the volumetric method, greater than 100 μg. For some samples requiring longer titration times, the drift volume must be deducted.

Determination of drift volume:

Add the same solvent as the test sample into the titration cup and titrate to the endpoint. After resting for no less than 10 minutes, titrate again to the endpoint. The volume change per unit time between the two titrations is the drift volume (D).

Calculation of results:

Water content in solid samples is calculated using formula (3), and in liquid samples using formula (4):

Where:

X — Water content in the sample, g/100 g;

V₁ — Volume of Karl Fischer reagent used for sample titration, mL;

T — Titer of Karl Fischer reagent, g/mL;

M — Sample mass, g;

V₂ — Volume of liquid sample, mL;

D — Drift volume, mL/min;

t — Titration time, min;

ρ — Density of liquid sample, g/mL.

② This method is for determining trace moisture in food. If the food contains oxidizing agents, reducing agents, basic oxides, hydroxides, carbonates, boric acid, etc., they may react with components of the Karl Fischer reagent and interfere with the measurement.

③ The particle size of solid samples should be about 40 mesh to ensure complete extraction of moisture. It is preferable to use a crusher rather than a grinder to prevent moisture loss. Additionally, ensuring uniform moisture distribution during grinding is key to obtaining accurate results.

④ 5A molecular sieves should be loaded into drying towers or drying tubes for drying nitrogen or air.

⑤ Anhydrous methanol and anhydrous pyridine should be stored with anhydrous sodium sulfate.

⑥ Ambient humidity affects the measurement. Outside air must not enter the reaction chamber.

⑦ Water easily adsorbs on glass instrument walls, so all glassware used must be thoroughly dried.

⑧ Tests show that the moisture determined in candy samples by Karl Fischer method equals the moisture by oven drying plus the residual moisture determined by Karl Fischer in the oven-dried samples. This indicates that the Karl Fischer method measures not only free water but also bound water, and thus more objectively reflects the total moisture content of the sample.