How to Develop HPLC Method for Nonpolar Compounds

Table of Contents

• Introduction and Outcome
• Non-Polar Molecules
• Principle of separation
• Selection of HPLC column
• Selection Mobile Phase
• Gradient Optimization
• Flow rate Optimization
• Sample Concentration Optimisation
• Injection Volume Optimisation
• Wavelength selection
• Procedure
• Elution order
• Conclusion

Introduction and Outcome

HPLC method development plays a vital role in Pharmaceuticals’ development. It needs a high degree of analytical expertise. In this article, I will share skill-based knowledge on the HPLC method development for Nonpolar compounds. After reading this article you will be able to develop the HPLC method for nonpolar compounds independently. You will be also able to answer several questions:

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

Non-Polar Molecules

When there is no separation of charge on the molecule then the molecule is called a Nonpolar molecule. Typical examples are Benzene, Naphthalene and Anthracene.

Principle of separation

Like interact like principle will govern the separation. Here Molecules are nonpolar and non-polar HPLC columns will be the suitable choice for the separation.

Case Study: How to Separation of Naphthalene and Anthracene?

Both Naphthalene and Anthracene are non polar compounds.

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

Selection of HPLC column

Since Naphthalene and Anthracene are non-polar compounds therefore, column containing nonpolar stationary phase like C18, C8 will be suitable for the separation.

Selection of Mobile Phase

Selection of Aqueous phase

Since compounds are nonpolar and hence water can be used as an aqueous phase. If you want you can also use any buffer with neutral pH.

Selection of Organic Phase

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

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 80%. Based on the elution of each Naphthalene Anthracene optimisation should be done. Example:

• Modify the composition of A and B to get best elution pattern
• The above gradient table has been designed to explain the this topic and it is not actual

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 in such so that there should not be any column overloading and the peak should be sharp. Sample concentration can be increased or decrease based on requirement e.g 0.2mg/ml, 0.5mg/ml, 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, 20μl.

Wavelength selection

Prepare the solution of each Naphthalene and Anthracene and scan in a UV spectrophotometer or PDA detector. Select a wavelength where each Naphthalene and Anthracene almost equal response

Procedure

Inject the standard solution of each Naphthalene and Anthracene 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

The molecular weight of Anthracene is higher than Naphthalene and consequently, Anthracene is comparatively more nonpolar than Naphthalene. Therefore Naphthalene will elute first and after that Anthracene elute.

Conclusion

You have learned column section, mobile phase selection and chromatographic condition optimization for nonpolar compounds HPLC method development. I hope now you can develop the Analytical method for nonpolar compounds independently. Write your learnings/suggestions related to this post in the comment section.

FAQs

What is the principle of separation of Nonpolar Compounds?

To separate non-polar compounds select non-polar columns like C18, C18 with high carbon load and a simple mobile phase like a mixture of water and organic solvent can be used.

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