Friday 24 September 2010

Help on: Retention Time Variability


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 have a question regarding retention time variability between different types of HPLC systems. An experiment was performed on an Agilent 1200 HPLC and on a Waters 717 instrument using a gradient profile. On the Agilent, the retention time was 14.7 minutes, and on the Waters, the retention time was 19.6 minutes. The same mobile phase was used for each system along with the same sample preparations, which seems to point to a problem with the Waters instrument (the Agilent gives typical results). What are some issues that could cause such variability with the retention times?”

“The most probable reason for the difference in retention time that you are observing is that the dwell volume for the two HPLC systems being used is different. The dwell volume relates to the time taken for a change made in the mobile phase proportions to reach the column. The dwell volume for modern instruments like the Agilent 1200 is quite small (typically less than 1 mL) but for older instruments it can be in the region of 2 to 3 mL or larger. The Waters 717 is an autosampler but I assume that it is connected to other Waters components of a similar age. It is likely that the dwell volume for this system is greater than that for the Agilent and thus is the cause of the larger retention time.

You can easily check if the dwell volumes are different by determining this value for each system. I have provided directions for this in a previous blog post. You may also be interested in the reply to another MTS Helpdesk question which was similar to yours where I discuss further the reasons for differing retention times for different systems.“

Monday 13 September 2010

'How to Troubleshoot HPLC' - New External Training Course

Sponsored by
MTS is delighted to announce a series of training dates for a new course ‘How to Troubleshoot HPLC’. These one day external training courses are sponsored by Phenomenex. The available dates are as follows:

Thursday 18th November
Louis Fitzgerald Hotel, Naas Road, Dublin
Tuesday 23rd November
De Vere Wychwood Park, Weston, Crewe, Cheshire

Tuesday 30th November
The Lensbury, Broom Road, Teddington, West London


Learn how to find solutions for problems encountered when running HPLC analysis by diagnosing symptoms and implementing appropriate preventative measures. This course is ideal for those who have experience of using HPLC and now want to develop their skills further.

  • Overview of the HPLC and how it works:
    Mobile phase, pumps, injectors, columns, detectors and connections
  • Common problems and preventative measures
  • Problem solving strategy:
    Assessing the symptoms
    Making diagnosis
    Finding the appropriate solution


This course will enable you to go back to your lab with a full understanding of why problems may arise with your HPLC system and give you the skills and knowledge to both prevent and resolve those problems. In addition you will be able to:

  1. Understand how HPLC works and the role of each component in an HPLC system
  2. Understand how problems can arise in the individual components of an HPLC system
  3. Implement measures which prevent problems occurring
  4. Use a systematic problem-solving approach to HPLC troubleshooting
  5. Diagnose and resolve problems associated with HPLC
The course costs £195 which includes the full day of training (including post training assessment), course literature, technical brochures, lunch & refreshments. To register click here or contact Phenomenex and quote course number SS0-7449.
Telephone: 01625 501 367 (UK) or 01 247 5405 (Eire)
Or email:

Monday 6 September 2010

Help on: Peak Purity Assessment


Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

“While performing peak purity assessment (or doing force degradation studies), if any unknown impurity with similar spectra co-elutes with the peak of interest, then software will show that the peak is pure. But is there any trick or any method to get correct results...?”

“Peak purity assessment software can only interpret the information that is collected from the UV diode array. Unfortunately organic impurities often produce very similar spectra to analytes of interest because the difference in the molecules does not relate to the functional groups which are producing the UV response. Although software may report that a peak is pure you can never reach this conclusion when investigating peak purity by this method. All you can conclude is that you have not found any impurities co-eluting with the peak of interest. To differentiate between analytes of interest and their impurities where the structure is very similar is not within the capability of this UV technique so there are no tricks or methods to make it possible. Mass spectrometry may also be used for peak purity assessment but it also suffers from some limitations.”