Automated Water Content Determination of a Lyophilisate

Lyophilisation, commonly known as freeze-drying, is a process of dehydration generally used for the preservation of materials. Lyophilisation is performed by freezing the material and then decreasing the surrounding pressure to facilitate a process whereby the frozen solvent sublimates directly from the solid phase to the gas phase. Following on from this drying process, the eventual water content residual in the product is exceptionally low: around 1% to 4%.

To increase the shelf life of certain products including live virus vaccines, biologics, and other injectables, pharmaceutical companies typically use the lyophilization process. By separating the water from the material and then sealing the material in a glass vial, the contents can be stored, shipped, and later reconstituted effortlessly to their original form for injection.

For the purpose of this application, a coulometric C30S Karl Fischer titrator connected to an InMotion KF was utilized to automatically measure the water content of a lyophilized substance in septum bottles. This application can also be carried out using a T7 or T9 with a coulometer kit.

Automated Water Content Determination of a Lyophilisate

Image Credit: Mettler Toledo - Titration

Sample preparation and procedures

1. Gas-phase extraction principle: An air pump aspirates air and passes it through a drying unit filled with silica gel and subsequently through a drying unit filled with molecular sieve. The flow rate (typical 80 – 150 mL/min) that is defined in the titration method is regulated by a digital gas flow control. The water is extracted from the sample by the dry air and transferred into the titration cell.

2. Blank preparation: The sample vial contains atmospheric humidity as well as the water in the sample. This makes blank value determination necessary. Place three empty septum vials into positions 1, 2, and 3 of the InMotion KF.

3. Sample preparation: place the septum bottles containing the lyophilized substance on the InMotion KF rack. The vial height (in mm) has to be adapted under Setup > Hardware > Titration Stands > InMotion KF/1, depending on the height of the septum bottles.

4. Start of analysis: Place an empty septum vial into the drift position on the rack. Start M738. Pretitration will be performed first to remove excess water from the titration cell. Then it will go into the Standby modus. Until the online drift value is <5 µg/min, perform manual drift determinations. Then press ‘Start sample.’ Ordinarily, the method will start with the sequence: Drift – Blank – Sample. Before the sample loop, the drift is usually determined once. This drift value is used for all subsequent calculations. Sample determination and blank value determination need to be carried out using the same method parameters.

Automated Water Content Determination of a Lyophilisate

Image Credit: Mettler Toledo - Titration


Water, M = 18 g/mol.

ROH + SO2 + 3 RN + I2 + H2O = (RNH)•SO4 R + 2 (RNH)I


  • Chemicals: 100 mL HYDRANAL® Coulomat AG Oven.
  • Sample: lyophilized substance in septum bottles
  • Standard: 1% oven water standard

Instruments and accessories

  • Compact line titrator C30S, titration cell without diaphragm (30252662)
  • XPE205 Analytical balance (30087653)
  • Titration Excellence T7 (30252675) or T9 (30252676) with coulometric Karl-Fischer kit (30267113)
  • LabX software
  • Air pump AP200 (30407441)
  • InMotion KF Pro Oven Autosampler with 10 mL rack (30407502)
  • Spatula / Syringe

Important: The viability of a customer's particular septum vials necessitates testing in terms of feather strength and cutting out of material. A comprehensive list is available detailing tested septum vials. If the standard InMotion KF or feather strength is not up to standard, the InMotion KF Pro Oven Autosampler Custom (30407504) must be ordered.

Scan: The temperature scan demonstrated that water was discharged from the starting temperature of 40 °C. The sample decomposition commenced at approx. 90 °C – 100 °C. Thus, a temperature of 80 °C was selected for gas phase extraction.

Sample: The average water content of 1.07% is within the usual water content range of lyophilized substances, which ranges between 1– 4%.


Table 1. Source: Mettler Toledo - Titration

  R2: Water content Blank: [µg], Sample: [%]
Blank 1
Blank 2
Blank 3



By way of a double hollow needle, the gas is let into the sample. The needle’s length can be selected in line with the sample being analyzed. Typically, the recommendation is a short needle for solid samples to prevent any swirling of powder therefore preventing the needle from becoming clogged-up.

Water capacity of the HYDRANAL® Coulomat AG Oven is 700 mg H2O per 100 mL reagent as coulometric reagents have a limited water capacity.

Waste disposal and safety measures

Dispose of as an organic solvent

Measured values

Scan lypophilisate.

Scan lypophilisate. Image Credit: Mettler Toledo - Titration

Water content determination sample 1/5

Water content determination sample 1/5. Image Credit: Mettler Toledo - Titration

Table 2. Sample 1/5. Source: Mettler Toledo - Titration

Consumption mC Meas. value mV H2O µg Drift µg/min Time s
0.0 512.3 0.0 0.0 2
31.9 516.6 3.0 13.3 2
117.0 514.6 10.9 93.3 3
255.8 509.6 23.9 239.5 3
448.3 436.8 41.9 423.6 4
694.5 383.7 64.8 653.3 5
955.5 273.8 89.2 896.1 6
12915.2 101.9 1205.7 38.5 588
12916.7 103.9 1205.8 0.0 589
12919.7 103.7 1206.1 17.0 590
12927.3 103.0 1206.8 17.0 591
12933.4 100.1 1207.4 16.1 592
12933.4 104.6 1207.4 16.1 593
12940.7 103.9 1208.1 0.0 594
12943.8 101.6 1208.3 38.5 595
12943.9 104.4 1208.4 0.0 596
12950.7 106.5 1209.0 0.0 597
12964.3 104.5 1210.3 17.0 598
12968.8 100.0 1210.7 17.0 599
12970.5 101.4 1210.8 10.9 600
12970.5 101.4 1210.8 0.0 600


About Mettler Toledo - Titration

METTLER TOLEDO instruments are used in research, scientific and quality control labs, amongst many others in the pharmaceutical, chemical, food and cosmetics industries. They are a global market leader with the three instrument groups most frequently used in the laboratory, like balances, pipettes, and pH meters, and we are a pioneer in the field of Automated Chemistry.

METTLER TOLEDO industrial solutions cover the diverse steps in a host of manufacturing processes at many of the same customers as served for the laboratory. Solutions range from receiving raw materials, in-line process control and end-of-line packaging control, to logistics and shipping. Increasingly, these solutions are fully integrated into the customer's IT environment, helping automate their workflows.

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Last updated: Jan 22, 2021 at 6:40 AM


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