Approach for separating target compound from impurities
Nov 07 2018 Read 2921 Times
Preparative chromatography is run to: isolate or purify a substance from a mixture for subsequent studies, such as product and impurity characterisation, using mass spectrometry (MS) or Raman spectra (NMR), supply material for Phase I, II and III studies in the pharmaceutical industry, and for large scale manufacture of drugs or APIs. Contrary to analytical chromatography where the eluent is discarded, when purifying from a mixture, the eluent is no longer waste because it contains the chemical of interest. Therefore there is a need for fraction collection.
The scale of the experiment depends on the amount of substance to isolate. It is therefore possible to carry out preparative chromatography in any column size, depending on need. With the loading capabilities of Kromasil stationary phases, it is possible to purify material in a 4.6 mm ID column for substance characterisation as well as in large production facilities using for example a 80 cm diameter column for the production of medicines. Method development becomes a significant activity in the purification and commercialisation of given substances and there is a need to optimise factors such as loadability, purity and yield.
Traditionally, choose a few analytical columns packed with preparative stationary phase material for method development. In this initial step, it is very important to choose analytical columns from a vendor that can provide the stationary phase in large quantities, defined by the scale of the project. Switching vendor during a scale-up process is not recommended since stationary phase properties can vary considerably and therefore selectivity and resolution between critical peaks can be a challenge.
Carry out method development by screening the selected columns under different mobile phase conditions (including buffer systems, pH and organic modifiers). At first, focus on selectivity under analytical conditions. Selectivity is the most important parameter for any preparative separation, and production rate increases rapidly with increased selectivity. Depending on the interaction with the stationary phase it is important to consider the mobile phase composition during the run. Choose isocratic, gradient or step gradient conditions at this point. Generally, the need for gradient conditions increases with the size of the molecule. Continue with overloading experiments of the most promising stationary phases and evaluate the results by collecting and analysing fractions. Tune in your method by optimising important scale-up parameters such as flow rate, particle size, bed length and loading amount.
For more information on preparative chromatography request our Kromasil Guide.
Do you like or dislike what you have read? Why not post a comment to tell others / the manufacturer and our Editor what you think. To leave comments please complete the form below. Providing the content is approved, your comment will be on screen in less than 24 hours. Leaving comments on product information and articles can assist with future editorial and article content. Post questions, thoughts or simply whether you like the content.
In This Edition Articles - Translating Proteomic MALDi Tissue Typing to Clinical Pathology - Fusion Protein Complexes Analysed by CG-MALS - The Separation and Purification of Glutamine Deri...
View all digital editions
Nov 25 2019 Beijing, China
Dec 05 2019 New Delhi, India
Dec 13 2019 Kathmandu, Nepal
Jan 25 2020 San Diego, CA, USA
Jan 27 2020 Dubai, UAE