How to Develop HPLC Method for Acidic Compounds

Table of contents

• Introduction and Outcome
• Acidic Organic Compounds
• Principle for separation of Acidic compounds HPLC
• Selection of HPLC column and Mobile phase
• Gradient and Isocratic mode/Mobile phase elution mode
• Flow rate Optimization
• Sample Concentration Optimisation
• Injection Volume Optimisation
• Wavelength selection for Acidic compounds HPLC New Method
• Procedure
• Elution Order
• Conclusion

Introduction and Outcome; Acidic compounds HPLC New Method

Acidic compounds HPLC New Method development needs both knowledge and skills. In this article, I will discuss the procedure of stationary phase, mobile phase, buffers and solvents for effective method development of acidic compounds with a case study. After reading this article you will be able to independently develop an HPLC method for acidic compounds. This article will also enable you to answer several questions:

• What is the principle of separation of Acidic Compounds?
• How to select the HPLC column and Mobile Phase for the separation of Acidic Compounds?
• How to optimize chromatographic conditions for the separation of Acidic Compounds?
• How to optimize sample concentration and gradient?

Acidic Organic Compounds

Those organic compounds tending to donate the H⁺ ion (proton) and remain energetically stable after the loss of proton are called Acidic Organic Compounds. For example, compounds like Phenol and Benzoic acid are acidic.

Principle for separation of Acidic compounds HPLC

Like interact like principle will govern the separation. Here Molecules are acidic and therefore try to make them less polar/non-polar by suppressing the proton donation ability of the molecule. Keep the mobile phase pH acidic. Then these compounds can be separated in RPC mode using non-polar or moderately polar columns like C18 and C8.

Case Study: HPLC separation of Phenol and Benzoic acid

Following are the structure of Phenol and Benzoic acid.

Note: The above structures have been drawn using ACD/Chemsketch (Freeware)

Both compounds have ability to donate the proton and stabilize themselves due to resonance effect. Benzoic acid has greater tendency to donate protons than the Penol. Therefore Benzoic acid is more acidic compared to Phenol.

Selection of HPLC column and Mobile phase

Since Phenol and Benzoic acids are acidic compounds. Therefore the priority should be to suppress the protonation and make them make them less polar or nonpolar. The only solution is to keep the pH of the Mobile phase acidic ( preferably pH should be less than 4). Phosphate or acetate buffer at concertation between 0.01 M to 0.03 M and pH less than 4 can be tried for separation. Columns containing moderately polar stationary phases like C8 can be used. C18 columns with less end capping would also be suitable for the separation.

Selection of Organic Phase for Acidic Compounds HPLC separation

Methanol or Acetonitrile can be used. Considering the cost methanol should be the preferred choice.

Gradient and Isocratic mode

These compounds can be separated either in gradient or isocratic mode, but gradient mode should be the preferred choice during method development. The trial should be started with a higher aqueous phase of 78%. Based on the elution of each Benzoic acid and Phenol optimisation in the composition of the mobile phase should be done. Example:

• The above gradient table has been designed to explain this topic and it is not an actual experimental gradient.

Flow rate Optimization

Keep the flow rate between 0.5 to 1 ml/minute to get column pressure less than 2000 psi

Sample Concentration Optimisation:

Keep the sample concentration so that there should not be any column overloading and the peak should be sharp. Sample concentration can be increased or decreased based on requirement e.g sample concentration can be kept like 0.2mg/ml, 0.5mg/ml or 0.7mg/ml

Injection Volume Optimisation

Keep the Injection Volume in such a so that there should not be any column overloading and the peak

should be sharp e.g 5μl, 10μl or 20μl.

Wavelength selection for Acidic compounds HPLC New Method

Prepare the solution of each Benzoic acid and Phenol and scan in a UV spectrophotometer or PDA detector. Select a wavelength where each Benzoic acid and Phenol have almost equal response.

Procedure

Inject the standard solution of each Benzoic acid and Phenol and generate the chromatogram. Inject the sample mixture and generate the chromatogram. Based on the elution pattern optimise the mobile phase composition and chromatographic condition to get better separation.

Calculation

Based on the requirement use area % (area normalisation method) or external standard method to give the result.

Elution Order

Since Benzoic acid is more polar than Phenol. Therefore Benzoic acid will elute first and after that Phenol elute.

Conclusion

In addition to Benzoic acid and Phenol, the above HPLC septation tips can also be applied to other acidic compounds. Now you have now learned about column section, mobile phase selection and chromatographic condition optimization for separation of acidic compounds and I hope you can repeat it during method development. That is all about this post. Write your learnings/suggestions related to this post in the comment section.

FAQS

What is the principle of separation of Acidic Compounds?

To separate acidic molecules. Try to make these molecules less polar/non-polar by suppressing their proton donating ability. Keep the mobile phase pH acidic. These compounds can then be separated in RPC mode using non-polar or moderately polar columns such as C18 and C8.

How to select the HPLC column and Mobile Phase for the separation of Acidic Compounds?

Keep mobile phase pH acidic and use column like C8 and C18

Reference

• Practical HPLC – Veronika R Meyer, second edition, University of Berne, Switzerland

Abbreviations

• HPLC: High performance liquid chromatography
• C18: Octyl Decyl silane
• C8:Octyl silane
• M: Molarity
• UV: Ultraviolet
• RPC: Reverse phase chromatography