Thursday 24 November 2011

USP Chromatographic Columns

A resource for chromatographers

If you have ever followed an HPLC method in the United States Pharmacopeia, then you have probably already encountered the problem of selecting a suitable HPLC column for the analysis. Columns are designated by a letter and number which identifies the stationary phase, e.g. L1 refers to ‘Octadecylsilane chemically bonded to porous silica or ceramic micro-particles, 1.5 to 10 μm in diameter, or a monolithic rod’. Unfortunately there are hundreds of columns that fit this description and due to selectivity differences they may not all give similar results.

The outcome of the USP Working group on HPLC Columns is that you can now look up the chromatographic column which was used to validate the procedure. The free online database provides a cumulative listing of columns referenced in gas- and liquid-chromatographic methods related to revisions made to USP–NF since January 1980. USP Chromatographic Columns can be accessed via the USP website, you will need to register to access the database, then search for the monograph in question. Once you know the actual column which was used for the method you can either use this or an equivalent. The USP Column Database provides a tool which can be used to find equivalent columns.

Friday 11 November 2011

MTS Recommends... ‘HPLC Columns’ by Uwe Neue

‘HPLC Columns - Theory, Technology, and Practice’ by Uwe D. Neue, Wiley- VCH, 1997

Although this book was published some time ago in 1997, it is still a fantastic resource for chromatographers. Neue managed to combine a thorough overview of the theoretical background relating to column technology with a practical guide, all in a very readable style.

Wednesday 9 November 2011

Help on: Assessing Peak Purity Data

Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.
“I am developing a HPLC indicating assay for a hormonal drug. I am using the peak purity option to analysis the purity of my peak once the sample has been subjected to 20% degradation under various conditions. The purity results show that my peak is not pure although its 5 overlaid spectrums are more or less identical. The resolution of my drug from its degradation products is greater than 2. I need to know how I can accurately interpret my peak purity results in order to ensure that no other degradation product is co-eluting at the same time as my main peak. I would appreciate any suggestions with regards to this matter.”
"I assume that the results you are referring to are from the software that you are using for peak purity analysis. The purpose of this software is to compare the spectra obtained at time points across the peak and detect differences which cannot be observed by eye. Therefore it is possible that there are spectral differences which may be explained by the presence of another peak eluting at a similar time as your drug. When a drug is degraded, the degradation products are often very similar in structure leading to similar retention times and UV spectra. This means that the spectral differences observed during peak purity analysis may be very small.
To investigate whether your purity result is actually due to another peak or is just a result of the way the spectra have been compared by the software requires knowledge of how the purity result was generated. I recommend that you read through any available information on how the software compares the spectra.
Things to consider:
  • What reference spectrum is being used, particularly important if it is a gradient method;
  • Compare the peak purity plot observed for your forced deg sample with that for a pure reference standard;
  • Loss of linearity can result in spectral differences which are not due to another peak, try diluting your sample and compare the peak purity plot obtained with the original.
A final note, peak purity can never be proven. You can only gain confidence that no other peaks have been detected. This is because the UV spectrum for similar compounds may be identical, also if the apex of both co-eluting peaks is at the same time then the spectra across the peak are likely to be identical.”

Thursday 3 November 2011

Free HPLC Calculator for Method Development

A resource for chromatographers

The MTS free HPLC calculator now includes a method development tool for HPLC. The tool is based on a scouting gradient approach for determining suitable initial conditions for a mixture of analytes, where a single gradient analysis is used to decide whether isocratic or gradient elution is most suitable, and to select promising mobile phase composition. This will be familiar to delegates on MTS training courses and you should be able to start using the calculator straightaway. I plan to post more details in the new year on how to use the tool for those of you who have not attended one of my courses.