“How do you calculate the gradient program in HPLC method development?”
“The aim of an HPLC method is to enable the separation of a mixture of components. This may be achieved by selecting a suitable mobile phase composition for a particular column which results in a peak for each component that is separated from other peaks, and is retained at a suitable retention time. The composition of the mobile phase may be isocratic, where it is held constant throughout each injection, or gradient, where the amount of the stronger solvent is increased throughout each injection. Whether you use isocratic or gradient conditions depends on the nature of your mixture of analytes. For example in reversed phase HPLC a mixture of analytes which have widely differing hydrophobicity is likely to require an unfeasibly long run time under isocratic conditions and will need to be analysed under gradient conditions.
For RP-HPLC, I suggest that you run your test sample using a full range gradient, e.g. 5 to 95 %B. An inspection of the resulting chromatogram will indicate whether the method is suitable for isocratic analysis (if the difference between the first and last peak is less than 25% of the gradient time) and if not, provides a starting point for gradient method development. Changing the gradient time, tG will make the gradient more or less steep, corresponding to stronger and weaker mobile phase composition in isocratic analysis.
For a detailed discussion on gradient method development you may wish to attend one of my training courses. ‘How to Develop HPLC Methods’ will be held next week in London and Milton Keynes. Future events will be publicised on this blog.”
Friday, 20 May 2011
Thursday, 19 May 2011
There are two more dates for this course next week, in London on Tuesday and in Milton Keynes on Wednesday. If you are interested in attending, contact Phenomenex (course code SS0-5947) to check if there are any places left. Tel: UK: 01625 501367 or email: firstname.lastname@example.org
Learn how to select appropriate method conditions and perform suitable investigative experiments to obtain a set of method parameters which enables the desired separation for mixtures of analytes.
This course is ideal for those who have experience of running HPLC methods and now want to learn how to develop new methods.
Developing an HPLC method using a 5-step strategy:
Step 1: Setting suitable objectives for method development
Step 2: Assessing all available information
Step 3: Selecting suitable samples
Step 4: Performing scouting experiments to select suitable initial conditions
Step 5: Optimising the method to define method parameters which achieve the desired separation
This course focuses on reversed phase mode separations.
Practical Skills Acquired
This course will enable you to take a strategic approach to developing HPLC methods with an understanding of the factors which can be adjusted to manipulate the retention time of analytes. In addition you will be able to:
1. Define the objectives for the development of a HPLC analytical method.
2. Effectively assess all the available relevant information relating to the desired method, e.g. pKa of the analyte.
3. Select and prepare a suitable sample or samples to be used for the method development.
4. Select suitable scouting conditions to find a suitable column and mobile phase system.
5. Optimise the chromatographic conditions to result in the best possible separation.
Course price is £195 + VAT per delegate. The price includes: Full day training (including post training assessment), course literature, technical brochures, lunch and refreshments.
A follow up course: ‘How to Develop HPLC Methods for Challenging Separations’ (course code SS0-5946) is planned for September, at Crewe on the 13th and Milton Keynes on the 20th. The aim of this course is to enable development of methods for problem samples and analytes, examples being very polar molecules and samples containing numerous analytes.
Tuesday, 3 May 2011
MTS HELPDESKIn a forced degradation study a molecule is subjected to extremes of heat, humidity, pH, light, etc. so that the molecule degrades. This study is commonly performed for pharmaceutical active pharmaceutical ingredients and formulations and is typically done for one of two reasons:
1. It provides information on the possible degradation pathways of the molecule and thus an assessment of its stability under different types of conditions. The information obtained is usually included in the marketing dossier for the drug.
2. It provides samples which can be used to develop a stability indicating method, i.e. a method which can detect and quantify degradation products. This method is used to assess shelf life during stability studies.
A problem with forced degradation studies is that the extreme conditions used may not be representative of the degradation under ‘normal’ conditions. For this reason the aim is to degrade the molecule of interest by no more than 10%.