Pharmaceutical Industry – Recommendations and Tips for Converting Your Manual Titration Procedures to Automatic Titration

Pharmaceutical Industry – Tips and Recommendations for Converting Manual Titration Procedures to Automated Titration Although various titrimetric assay methods contained in complementary pharmacopeial monographs have been converted to chromatographic methods and other quantitative techniques over time, titration continues to hold a highly important position within pharmaceutical analytical procedures and related processes. Applications such as distinctions between carbonate/bicarbonate or monobasis and dibasis phosphate salts can only be performed by the titration method, and this situation demonstrates the true importance of the said technique as a fit-for-purpose method. For example, Karl Fischer (KF) titration stands out as a highly selective water determination application technique that can be sensitive to the mg/L (ppm) level. Although modern KF titration instruments are currently being used in the pharmaceutical industry for selective and reliable water content analyses, many USP monographs still refer to manual visual endpoint titration methods for other applications. Visual detection with the aid of colored indicators is the oldest method of determining the equivalence point of a titration, and is still frequently used today and continues to be recommended within many different guidelines. Along with low cost and the need for minimal laboratory equipment, this technique is based on the principle of determining the endpoint by adding titrant drop by drop with a manual burette until the color change becomes stable, and in this context can be time-consuming. Another disadvantage of manual titration analyses performed with visual detection is that color perception differs among individual operators and is also dependent on lighting conditions. In addition to this situation, determining the endpoint in particularly dark-colored and/or turbid solutions can become quite challenging. All these factors result in reduced reliability of results along with increased susceptibility to human error. Another major disadvantage is that it is not possible to automate the visual method and therefore its validation is difficult and lacks data integrity. In this review article, we aim to provide detailed information about the steps involved in converting an existing manual titration procedure into a semi-automated or fully automated titration procedure. In addition to topics such as proper electrode and titration mode selection, we will be taking a close look at three different titrimetric analysis examples to make the content even more understandable.

Selected Example Titrations

Three different analysis examples have been selected to demonstrate possible changes between an existing manual titration procedure and an appropriate semi-automated or automated titration procedure. These are, in order:

1. Potassium Citrate: Potassium citrate assay testing is performed by means of acid-base titration in a non-aqueous medium using perchloric acid as titrant and crystal violet as indicator, in glacial acetic acid.[1]
2. Calcium Hydroxide: Calcium hydroxide assay testing is performed by means of complexometric titration using disodium EDTA (Na₂EDTA) as titrant and hydroxynaphtol blue as indicator. [2]
3. Potassium Bromide: Potassium bromide chloride limit testing is performed by means of back titration using silver nitrate as titrant, ammonium thiocyanate as back titrant, and ammonium ferric (III) sulfate as indicator. [3]

These examples above were specifically selected to encompass different titration reactions and analysis types (e.g. assay tests or impurities). Which Electrode Should Be Used? The first and most critical step in converting a manual titration into a semi-automated or fully automated procedure is the selection of an appropriate electrode that will detect the equivalence point. Using a sensor instead of visual endpoint detection means replacing subjective visual human perception with an objective detection tool. This makes it possible to easily automate and validate the said analysis. Electrode selection depends on the type of titration, the sample matrix, and the titrant. Acid-base titrations require the use of different electrode types than redox or precipitation titrations. In addition to this, the sample matrix can have different effects on the electrode. For example, combined pH electrodes used in non-aqueous acid-base titrations differ from those used in aqueous acid-base titrations. Table 1 lists the recommended relevant electrodes based on the titrant and the currently used indicator. Taking this table as a reference, the following electrodes have been selected for the three example analyses selected above:

1. Perchloric acid is used as titrant in potassium citrate assay testing [1]. For this titrant, regardless of indicator, a combined pH electrode suitable for non-aqueous titrations is recommended.
2. In calcium hydroxide assay testing, disodium EDTA is used as titrant. Two types of electrodes are recommended for this titrant depending on the indicator. Hydroxynaphtol blue indicator is used for calcium hydroxide [2] and in this context, a combined calcium electrode is recommended for use in semi-automated or automated titration methods.
3. The chloride limit test of potassium bromide uses ammonium thiocyanate as back titrant [3]. Generally, iron (III) ammonium sulfate is used only as indicator for this titration. However, electrode selection can also be directly affected by the sample itself. In this selected application, a partial precipitation titration is carried out with silver nitrate [3] and in this context, a combined silver electrode is preferred.

Other Adjustments Required for Method Conversion

Dilution Volume

With the completion of electrode selection, the most vital step of transition to semi-automated or automated titration has been completed. On the other hand, some additional adjustments may still be needed. One of the points to be considered is the amount of diluent (water or solvent) to be used in the titration. In order to obtain correct results, both the measuring and reference compartments of the electrode must be immersed in the solution at an appropriate level. Figure 1 presents an example visual showing the optimum immersion depth for a combined pH electrode. Electrode manufacturers typically specify the minimum immersion depth required for correct measurements.

Looking at the Three Example Analyses:

1. For potassium citrate, 25 mL of glacial acetic acid is used as solvent and no beaker size is specified [1]. If a 100 mL beaker is used, the 25 mL of glacial acetic acid will not be sufficient for the electrode immersion level. For this reason, it is recommended to increase the solvent volume to, for example, 50 mL, and of course it is advisable to make the same increase in the blank analysis as well.
2. For calcium hydroxide, the total volume in the beaker before titration is 165 mL and again no beaker size is specified [2]. If a 250 mL tall-form beaker is used, since the volume is sufficient for electrode immersion, there will be no need to adjust the diluent volume.
3. For potassium bromide, the chloride limit test contains 56 mL of solution in the flask after sample preparation [3]. A wide-mouth flask will be needed for the electrode to be easily immersed during titration. If a 100 mL wide-mouth flask is used, the electrode will not be able to immerse deeply enough. For this reason, it will be necessary to increase the amount of water used in the titration (e.g. 75 mL).

As seen in the above examples, the choice of container used for titration plays an important role in determining the amount of diluent needed. The selection of titration vessel is also affected by the amount of titrant added during titration. In this context, sample volume emerges as another important point to be evaluated when converting a manual titration into a semi-automated or automated titration.

Sample Volume

Automatic or semi-automated titrators are typically equipped with 10 mL or 20 mL burettes. However, many manual titration methods have endpoints exceeding 30 mL and even 40 mL. Since refilling the burette during titration would introduce systematic error, reducing sample volume emerges as a requirement. Reducing sample volume also brings with it the advantage of less titrant consumption during titration and thus less waste generation. Generally, it is recommended that the equivalence point of a titration fall between 10% and 90% of the total burette volume. When an optimum sample volume is taken, it is typically aimed to result in titrant consumption of about half the total burette volume. When using a 20 mL burette for semi-automated or automated titration, optimum titrant consumption of around 10 mL can be expected. Again, looking at the above three examples:

1. In potassium citrate assay testing, 200 mg sample and 0.1 N perchloric acid are used [1]. According to this combination, when a 100% assay result is obtained, the endpoint comes at approximately 19-20 mL. For semi-automated or automated titration using a 20 mL burette, 100 mg potassium citrate can be evaluated as the ideal sample volume.
2. In calcium hydroxide assay testing, 1.5 g sample is used to prepare a 500 mL stock solution. 50 mL of this stock solution is used for titration, thus this aliquot corresponds to 0.15 g sample [2]. When 0.05 M disodium EDTA titrant is used and a 100% purity result is obtained, the endpoint will correspond to approximately 40 mL. In order to achieve an endpoint around 10 mL, using a sample amount of 0.375 g in the stock solution would be an appropriate solution.
3. In potassium bromide chloride limit testing, no more than 1.7 mL of silver nitrate titrant should be used for the test to pass [3]. For this reason, there is no need to adjust the sample volume. On the other hand, since 1.7 mL consumption falls below the recommended minimum 10% burette consumption level for the equivalence point, using a 10 mL burette instead of a 20 mL burette would be an appropriate choice.

Following the evaluation of the above details, there is one final step remaining to be performed in the process of converting the said manual titrations to semi-automated or automated titrations: the selection of titration mode and relevant parameters.

Selection of Titration Mode

Similar to how different titrations require different titrant and electrode selection, the selection of titration mode and titration parameter settings can have a significant effect on the accuracy and precision of results. For this reason, automatic and semi-automated titrator suppliers provide default pre-defined titration methods for various titration type, titrant, and sensor combinations. Titration mode defines the principle of titrant addition (e.g. monotonic or dynamic addition) and the manner of recording the titration curve (endpoint or complete titration curve). The most common titration modes stand out as endpoint titration, monotonic titration, and dynamic titration. Below you can find brief explanations of these titration modes. For more information about different titration modes and basic examples related to semi-automated and automated titrations, you can access literature provided by titrator suppliers.

Endpoint Titration

Endpoint titrations are equivalent to manual titrations. Titrant addition continues until the indicator changes color and shows the endpoint. The basis of endpoint titrations is based on the principle that the indicator always reliably and reproducibly changes color at the same endpoint (e.g. at a predefined pH value). For automatic titrations, this principle is applied identically; titrant addition continues until the sensor detects the endpoint. Acid-base titrations can easily be converted to endpoint titrations. As a typical example, USP <301>Acid-neutralizing Capacity [4] can be taken. Because they are easy and fast, endpoint titrations are generally used for routine analyses. In cases where the detection signal is not stable enough to provide a reliable and reproducible endpoint titration, when more information about the sample is desired, or when multiple analytes need to be detected within the same titration, the complete titration curve must be recorded. When recording the complete titration curve, two different dosing principles can be applied for titrant addition: monotonic or dynamic titrant addition. Figure 2 shows these two different dosing principles.

Monotonic Titration

When fixed volume steps are used for titrant addition, this titration is called monotonic titration. Monotonic titration mode is generally recommended for titrations with slow reaction kinetics (e.g. slow-reacting complexometric titrations) or analyses with low expected titrant consumption (e.g. blank determinations). Monotonic titration is also used in cases where the titration curve does not have an S shape (e.g. redox titrations or titrations using a photometric sensor). In such analyses, dynamic titrant addition generally results in over-titration of the sample. The disadvantages of monotonic titration are the low data density around the equivalence point (See Figure 2 and Figure 3) and the length of the analysis.

Dynamic Titration

During a dynamic titration, as its name suggests, titrant is added dynamically based on the slope of the titration curve. For example, if the signal shows little change during several additions, the volume of subsequent additions will increase, and similarly, if the signal change increases after any addition, the volume of subsequent additions will automatically decrease. Dynamic titration resembles manual titration in this respect. Because the analyst speeds up or slows down the rate of titrant addition depending on the appearance of color changes. The greatest advantage of this method is the ability to obtain high data density around the equivalence point (See Figure 4) and thus achieve higher resolution, higher repeatability, and faster titration. Typically, acid-base titrations, precipitation titrations, and complexometric titrations are performed in this titration mode. Table 2 provides a general overview of the most common titration modes and principles, and also lists the most common uses for different titration modes. All three titration examples in our review – potassium citrate, calcium hydroxide, and potassium bromide – can be titrated using dynamic titration mode. On the other hand, since low blank values are expected, it is recommended that blank determinations for potassium citrate and calcium hydroxide analyses be performed in monotonic titration mode. Since potassium bromide chloride limit assay is a back titration, it is possible to select dynamic titration mode for the blank analysis.

Conclusion

When important points such as electrode and titration mode selection are evaluated correctly, it is possible to successfully convert a manual titration method into a semi-automated or automated titration method. Adapting the titration method provides important opportunities in terms of evaluating method optimizations such as titrant consumption and related waste management. Looking at the three analysis examples we have covered in our review article, the following changes are seen to be necessary to convert these manual titrations to semi-automated or automated titration methods.

1. Potassium Citrate Assay Test

• Use of a combined pH electrode suitable for non-aqueous titrations instead of crystal violet indicator.
• Increase in glacial acetic acid volume to enable proper electrode immersion.
• Reduction of sample volume from 200 mg to 100 mg.
• Use of dynamic titration mode.

2. Calcium Hydroxide Assay Test

• Use of a combined calcium ion-selective electrode instead of hydroxynaphtol blue indicator.
• Reduction of sample volume from 1.5 g to 0.375 g.
• Use of dynamic titration mode.

3. Potassium Bromide Chloride Limit Test

• Use of a combined silver electrode instead of ammonium ferric sulfate indicator.
• Increase in water volume to enable proper electrode immersion.
• Use of dynamic titration mode.

The said changes require validation of the resulting semi-automated or automated titration methods. USP General Chapter <1225>Validation of Compendial Procedures provides a framework for which parameters need to be tested during method validation [5]. For previously established general procedures such as titration, suitability for use must be verified by conducting accuracy, precision, and specificity (absence of possible interference) determinations. When sample volume is changed, linearity testing should also be performed. As Metrohm, with our more than 75 years of experience in the titration field and our unique technologies that set industry standards, we offer a wide range of instrumentation and application options to help you advance your pharmaceutical manufacturing, quality control, and R&D processes. With our analytical solutions that are 100% compliant with international pharmacopeias, we are with you for all your application needs spanning from active pharmaceutical ingredients (API), excipients, impurities, starting materials, and finished pharmaceutical products (FPP).

References

1.Metrohm White Paper WP 063EN_2021-03 "Recommendations for Converting a Manual Titration Procedure into an Automated Titration Procedure" [1] USP. Potassium Citrate. In: USP 42–NF 37. Rockville, MD: USP; 2020:3613. [2] USP. Calcium Hydroxide. In: USP 42–NF 37. Rockville, MD: USP; 2020:701. [3] USP. Potassium Bromide. In: USP 42–NF 37. Rockville, MD: USP; 2020:3600. [4] USP. Acid-neutralizing Capacity. In: USP–NF. Rockville, MD: USP; May 1, 2019. [5] USP. Validation of Compendial Procedures. In: USP–NF. Rockville, MD: USP; May 1, 2019