Separation of Closely Eluting Impurities by Selecting Appropriate Stationary Phase

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Background

HPLC column is the heart of HPLC hence selecting the appropriate stationary phase is the most important part of method development. The selection of the stationary phase plays an important role in selectivity. There are numbers of regulatory queries on the inappropriate resolution between closely eluting impurity hence it is important to develop the suitable stability-indicating analytical method. There are more complex drug products hence analytical scientists need to develop the specific, accurate, precise and robust method to attain the optimum resolution between closely eluting impurities.  Adequate resolution and recovery between the closely eluting peaks are the definitive goals for good chromatography. The resolution, means the distance between the center of two peaks measured by the retention time or volume divided by the average width of the individual peaks.

USFDA recommends the resolution should be more than 2. If there are closely eluting peaks in the method then it is essential to give minimum resolution criteria between the two peaks.

Stationary Phase Physical Characteristics:

HPLC column is an important part of the analytical method. It is important to assess the resolution of closely eluting peaks by using a degradation sample. Also need to check the column lot to lot variation using the resolution solution.
It is essential to select the proper HPLC column based on Information of the Sample.

Column chemistry is evaluated with regards to the following properties:

  1. Bonded phase,
  2. Type of Bonding,
  3. End capping,
  4. Carbon load,
  5. Particle bed dimensions,
  6. Particle shape,
  7. Particle size,
  8. Surface area,
  9. Inconsistent packing,
  10. Column length,
  11. Column internal diameter and
  12. Pore size.

There are a number of stationary phases available which are silica, polymers, alumina, and zirconium. Silica is the most common medium for HPLC columns. Silica is chemically stable to most of the organic solvents and to low pH range.

Newly the use of core-shell technology shows higher sensitivity, better resolution, fast analysis, and improved peak size. Monolithic columns stationary phase is an incessant porous material that shows low back pressures, fast analysis time without losing resolution. 

Conclusion

Selection of stationary phase on the basis of sample chemistry and its physic selected as per the requirement of a method of analysis. To achieve the proper separation of closely eluting impurities complete study must be done and the resolution to achieve more than 2.

References:

  1. HPLC Method Development for Pharmaceuticals, Edited By: Satinder Ahuja, Henrik Rasmussen.
  2. Snyder, L.R., Kirkland, J.J., and Dolan, J.W. Introduction to Modern Liquid Chromatography, John Wiley & Sons, Inc., New Jersey, USA,
  3. 11 Majors, R.E.  Developments in HPLC/UHPLC column technology. LCGC Eur., Supplement,
  4. Snyder, L.R., Kirkland, J.J., and Glajch, J.L. (1997) Practical HPLC Method Development, 2nd edn, John Wiley & Sons, Inc., New York.
  5. Gritti, F. and Guiochon, G. The current revolution in column technology: how it began, where is it going? J. Chromatogr. A,
  6. L. R. Synder, J. W. Dolan, and P. W. Carr, J. Chromatography.

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