The document outlines the determination of water content using a Karl Fischer (KF) autotitrator, detailing the principles of KF titration, instrument specifications, and standardization procedures. It includes precautions for accurate measurement, such as ensuring proper connections and cleanliness of equipment, as well as methodologies for different sample types. Additionally, it specifies documentation requirements and calibration procedures to ensure reliable results in pharmaceutical analysis.

1. TITLE: DETERMINATION OF WATER BY KF AUTOTITRATOR

2. KF AUTOTITRATOR
Contents
1- Basics of Karl Fischer titration.
2- Details of Karl Fischer’s instrument.
3- Pre-checks
4- Standardization of Karl Fischer reagent.
5- Determination of water content.
6- Calibration
7- Documentation
8- Precautions.
9- Past History
10- Electrode Storage & Maintenance.

3. BASICS OF KF TITRATION

4. BASICS OF KF TITRATION Cont..
KARL FISCHER REAGENT
• Pyridine-free reagent
• Imidazole is the Base
• One component Reagent  Iodine, Sulfur dioxide & Imidazole dissolved in an Alcohol
• KF titrations are in the pH range of 5.5 to 8
SAMPLE DILUENT SOLVENT
• Methanol is commonly used
• Other Alcohols e.g. Ethanol, 2-Propanol , Methoxyethanol, Diethylene Glycol, etc. can be used
KARL FISCHER REACTIONS
• Iodine and Water react in the ratio of 1:1 if Methanol (Alcoholic solvent) is used as a solvent
• Iodine and Water react in the ratio of 2:1 if Dimethylformamide (non- Alcoholic solvent) is used
as a solvent
• Methanol-free solvents shall be used for samples of Ketones & Aldehydes

5. BASICS OF KF TITRATION Cont..
ALDEHYDES & KETONES – KF TITRATIONS
• Aldehydes (R-CHO) and Ketones (R-CO-R) form acetals and ketals when titrated with standard
methanol-containing reagents.
• Additional water is produced and titrated at the same time, leading to higher water contents and
a vanishing endpoint.
• Special methanol-free KF one-component reagents such as e.g. HYDRANAL® are used to prevent
this problem.
• The titration takes a slightly longer time than with the standard KF reagent.
• Amines also react with Methanol during routine KF titrations
• One-component reagent HYDRANAL®
• The titrant contains iodine, imidazole, sulfur dioxide and 2-methoxy ethanol
• The solvent contains 2-chloroethane and trichloromethane

6. DETAILS OF KF INSTRUMENT
1- Instrument Name : - KF Autotitrator
2- Instrument ID : - SMLJ/QCD/227 and SMLJ/QCD/383.
3- Make : - Metrohm
4- Model No: - 881, 905
5- Operating Software : - Tiamo 2.4 & 2.5.
6- Electrode type : - Double platinum KF electrode.
7- SOP No.: - SOP-QCD-OPC-119 & SOP-QCD-OPC-158.

7. PRE-CHECKS
• Check all the cleaning of the instrument and workbench, if not clean the instrument and
workbench.
• Check the calibration status of the instrument.
• Check all the tubes for proper connection.
• Check the color of the molecular sieves, if the color is dark brown then replace the
molecular sieves.
• Ensure that no air bubbles shall be present in the tubing before initiation of analysis.
• Cleaning of the electrode with white toothpaste by brush and washing with methanol.
• Titration vessel should be clean and dry.
• Waste bottles should be empty.
• After filling of KF reagent in the reservoir, mix well and wait for stabilization.
• Ensure that the minimum level of the KF reservoir is maintained at 1/3 of capacity.
• Electrode cleanliness, performance, damage, etc. shall be checked before the start of
the analysis.

8. STANDARDIZATION OF KF REAGENT
• Karl Fischer reagent used for Water estimation
• KF Titrations need to be standardized daily for knowing the factor
• Disodium tartrate or Water can be used for KF reagent standardization.
 If the sample contains less than 1% water content then “Disodium tartrate” shall
be used for KF reagent standardization.
 If the sample contains more than 1% water content then “Water” shall be used for
KF reagent standardization.
a) Standardization with Purified water-
Weigh accurately ~ 25 to 250 mg of water and transfer it immediately to the titration vessel.
Close the stopper and start the titration. Repeat the procedure in triplicate and estimate the
‘Average and RSD values.
KF Factor (mg/ml) weight of water taken (mg)/Titrant consumed (ml)
b) Standardization with Sodium Tartrate Dihydrate –
Weigh accurately ~ 25 to 125 mg of Sodium Tartrate Dihydrate and transfer it
immediately to the titration vessel. Close the stopper and start the titration. Repeat the
procedure in triplicate and estimate the ‘Average and RSD values.
KF Factor (mg/ml) weight of water taken (mg)/Titrant consumed (ml) x (36.04/230.08)

9. DETERMINATION OF WATER CONTENT
• Pharmaceutical substances have ‘water ‘ in the form of hydrates or in adsorbed form.
• Water in the form of hydrates is estimated by –
a) Method I (Titrimetric – Direct or Residual or Coulometric Titration)
b) Method II (Azeotropic )
c) Method III (Gravimetric)
• Analytical method for each type of sample shall be specified in individual monograph or STP
Steps involved in analysis initiation ?
 Sample receipt / storage
 Allocation/ method reference
 Instrument setup / Factor determination
 Sample preparation
 Sample analysis
 Result recording
 Specification conformance
 Documentation

10. DETERMINATION OF WATER CONTENT Cont….
• KF solution containing Iodine is added using a motorized burette piston
• Suitable for samples containing Water content 100 ppm – 100 %
• Three types of KF Reagents are available for analysis
a)5 mg/ ml for samples with a Water content of 1000 ppm – 100 %
b)2mg/ml for samples with Water content less than 1000 ppm
c) 1 mg/ ml for samples with Water content less than 200 ppm
ROH + SO2 + 3 RN + I2 + H2O → (RNH)•SO4 R + 2 (RNH)I
• End point determination -
The addition or generation of iodine must be controlled and should be as quick as possible
The reaction of addition or production of Iodine should be terminated exactly at the endpoint
Accuracy required for Water % determination

11. DETERMINATION OF WATER CONTENT Cont….
FACTORS AFFECTING KF TITRATION
• Atmospheric humidity
• Solvent used for Sample
• Reactions between Sample and KF Reagent
• Moisture ingress – Desiccant effectiveness
• Analytical method for each type of sample shall be specified in individual monograph or STP
Proper mixing is essential for accurate KF Titrations. Mixing is influenced by –
 Stirring speed – Optimum speed is achieved when a vortex is visible . If speed is slow , Titration addition shall be
slow & irregular. High speed shall result in bubble formation and lead to false end points
 Point of titrant addition – Titrant addition shall be at the point of turbulence to ensure proper mixing. Titrant
addition shall be away from the sensor
 Shape of the Titration vessel – The vessel shall be of conical shape and the stirring bar be small for effective
turbulence.

12. CALIBRATION
Calibration procedure for KF Titrator
A-Burette volume Accuracy Test:
• Burette calibration shall be done for all the available burettes used for the instrument.
• 5 ml Burette shall be calibrated for 1 ml, 2 ml, and 5 ml set volume.
• 10 ml Burette shall be calibrated for 1 ml, 5 ml, and 10 ml set volume.
• Take a previously weighed 10mL volumetric flask and insert the burette tip into the flask,
then set 1 mL volume and press the ‘DOS’ key. Collect the 1 mL of water into a flask
delivered from the Burette, weigh the flask, and calculate the volume as follows.
• Volume = Weight of the water delivered x Conversion factor (Z). Conversion factor (Z) for 1
g of water at various temperatures:
• Acceptance Criteria: The difference between the delivered volume and the actual volume
should be ± 2.0% of the set volume.

13. CALIBRATION
Calibration procedure for KF Titrator
B-Verification of Precision and Accuracy :
• Weight and transfer about 100 mg of Di-sodium tartrate dihydrate in to titration vessel
containing previously neutralized Methanol.
• Enter the weight of the sample and then press OK.
• After completion of the time set for stirring, the KF titration will automatically start.
After completion of titration, the report is generated automatically and it will be
displayed in the report window. For taking print out go to Database, click on menu item
Tools and Report template, and then print.
• Repeat the above procedure another time and report the average of 6 replicates.
Acceptance Criteria:
• Accuracy: The averagemoisture content of six
• Repetitions should be 15.66 % ± 0.5 (15.16 to 16.16).
• Precision: The percentage relative standard deviation (RSD) of six repetitions for
moisture content should not be more than 2.0%.

14. CALIBRATION
Calibration procedure for KF Titrator
C-Verification of Linearity:
• Transfer about 100 mg of Di-sodium tartrate dihydrate into a titration vessel containing previously
neutralized Methanol.
• Enter the weight of the sample and then press OK.
• After completion of the time set for stirring, the KF titration will automatically start and completes the
titration, report is generated automatically and will be displayed in the report window. For taking print
out go to Database, click on menu item Tools and Report template, and then print.
• Perform the moisture content procedure for remaining weight as 150mg; 200mg; 250mg; and 320mg.
Take the print and report the average results. Similarly, calculate the volume of KF reagent consumed
in mL.
• Plot the linearity graph for the weight of DST versus the volume of KF reagent consumed in mL and
calculate the correlation coefficient.
Acceptance Criteria:
• Linearity: The correlation coefficient should not be less than 0.99

15. DOCUMENTATION
1- Before starting the operation of the instrument, verify the logbook and make a “Start” entry in
the logbook.
2- Document the all relevant information in the worksheet (i.e. Methanol grade, quantity, KF regent
details, sample weight) online simultaneously.
3- After completion of the analysis, the analyst shall take the printout of the results, and document
it in the worksheet, update the “End” time in the logbook.
4- The analyst shall record the result in LIMS.
5- The analyst shall hand over the complete document along with instrument prints to the reviewer
for further review of reports.
6- The reviewer shall review the hard copy document as per checklist SOP/QAD/GEN/059 and
electronic data as well as “Audit trails” in the instrument.
7- After review of the report by the QC-Reviewer, the report shall be handover to AQA (analytical
Quality Assurance) for further review.

16. PRECAUTIONS
1- Do not use the general grade of “Disodium tartrate” for factor determination.
2- Do not start the weighing until the instrument shows “Condition OK”.
3- After weighing sample should be transferred to the titration vessel immediately to avoid moisture
gain from the environment.
4- Transfer the sample very carefully, the sample does not spill or stick to the titration vessel.
5- Check all the tubes for proper connection.
6- Ensure that no air bubbles shall be present in the tubing before initiation of analysis.
7- Determination of trace amounts of water (less than 1%), use reagent with a water equivalency
factor of not more than 2.0.
8- The titration vessel lid should be closed immediately and properly after sample transfer in the
vessel.
9-Sample diluent (methanol) shall be changed intermittently.
10- The sample weight shall be as per the STP.

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