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  • Separation of Closely Eluting Impurities by Selecting Appropriate Stationary Phase


    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 is a number of regulatory queries on the inappropriate resolution between closely eluting impurity hence it is important to develop a suitable stability-indicating analytical method. There are more complex drug products hence analytical scientists need to develop a 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 the Information from the Sample.

    Column chemistry is evaluated with regard 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 organic solvents and to a low pH range.

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


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    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 and, 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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