Help on: Retention Time Shifts in NP-HPLC

MTS HELPDESK

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

Question:
“I am using NP-HPLC, my analyte needs 100% hexane. I am using pure material with un-known RT. The RT shifts to the right with every injection, something like 2-2.5 min for each injection. I started at 9 min and get to 16 min in 6 injections. Then I decided to clean the column overnight with 100% hexane and now I started from 27 min and it's shifting ... Any advice will be helpful. The temperature is constant, there is only one peak for my analyte.”

Answer:
“The most common problem when using NP-HPLC is retention time variability. This is normally due to the presence of very polar molecules such as water in the mobile phase. A small amount of water can significantly alter retention times. Once your column has equilibrated with the water content in your mobile phase any alteration in this amount will cause shifts in retention time. Reaching full equilibration is very time consuming in normal phase systems and can take anything from a few hours to a few days.

It sounds like your original injections had drifting retention times because the column had not reached equilibration. A strategy of running the mobile phase through the column overnight should help to achieve equilibration but since the RTs have not settled down either the column has not yet equilibrated or perhaps the mobile phase was altered. Is the mobile phase 100% hexane?

Controlling the water content of the mobile phase is desirable for reproducible results. Working with dry solvents is usually difficult and results in very long equilibration times. Using solvents which are fully saturated with water causes water to build up in the pores of the column. A common approach is to use ‘half-saturated’ solvents. Add 1 or 2 mL of water to 500mL of dry solvent and stir for 30 minutes, then remove excess water and combine with 500mL of dry solvent. Use this as your mobile phase and your equilibration should be quicker (although it may still take a few hours). If you can dedicate the column to your method then equilibration should be quicker when you use the column again in the future.

A possible solution for shifting retention times and equilibration problems that I have heard works well (but have not personally tried) is to use a magnetic stirrer to stir the mobile phase gently throughout the run, making sure not to place it close to the detector since it may cause baseline ripples.

Another potential source of water is your sample, if you suspect it may contain water then it is a good idea to dry it with anhydrous sodium sulfate of a high purity grade.”

Lab Tech Guy on a Delicate Problem

Lab Tech Guy on a Delicate Problem

View more presentations from Oona McPolin.

Help on: Investigating Out of Specification (OOS) Results

MTS HELPDESK

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

Question:
“If any sample fails in testing, i.e. OOS or OOT, then what sort of thing does an analyst have to do, and what is basic difference between OOS &OOT?”

Answer:
“The difference between out of specification (OOS) and out of trend (OOT) results was discussed in a previous MTS helpdesk enquiry. I recommend that you refer to the FDA Guideline ‘Investigating Out-of-Specification (OOS) Test Results for Pharmaceutical Production’ for a detailed description of the procedure for investigation of an OOS or OOT result.”

Tips to Enhance your Learning Experience

LEARNING AT WORK

When you attend a training course you hope to improve your knowledge and skills on the subject matter in question so that you can use your newfound competence to do your job better, and potentially enhance your career prospects. No matter how good training is, there are ways in which you can make the most out of the learning experience. The following tips are based on my experience of delivering and evaluating training. They should help you to retain what you learn on your training course and thus enable implementation of the learning in your day to day tasks.

1. Pre-training preparation
It is worth taking some time before you go on the course to think about exactly why you are going on the training and what you hope to get out of it. This means that you arrive at the training with clear expectations and can make sure that these are met. It also gets you thinking about the subject matter in terms of what you already know, providing a good starting point for improving your knowledge. Even if you have been ‘volunteered’ for the course you will still want to get the most out of the time you invest in attending so some preparation will pay off.

2. Take notes in your own words
How many times have you attended a presentation where the presenter says ‘Don’t worry, you don’t need to write anything down, there are handouts’? Research has shown that making some notes in your own words will help you to retain what you have learned. This doesn’t mean that you have to write down everything that you hear. Simply take down some quick notes about the main points of the presentation. The important thing is that it is in your words. This will help you to retain what has been said. Also, reading through these notes later will help you to remember the content.

3. Ask questions
Throughout your training it is important that you have a clear understanding of the content, so don’t be afraid to ask questions. You are in a learning environment so you are not expected to have the answers. Most trainers will encourage questions since it gives an opportunity for interaction and allows them to gauge how well their training is working.

4. Participate fully in the training
Most training events will include some type of session where you get a chance to do something other than just listen to the trainer. This may be in form of exercises, discussions, case studies, workshops, group exercises etc. These sessions allow you to apply what you have learned so try to throw yourself into them to get the most out of the experience. If you find the session is a bit uncomfortable (role-play springs to mind) then try to focus on what you want to get from the training.

5. Review what you have learned
At the end of the training you will probably summarise what you have learned with the trainer and may have an assessment to measure the learning. This review of the main points of the training is very useful for helping you to retain your new knowledge and skills. You should also use this review to visualise how you will apply your learning when back in work. If you can plan how you are going to use the training then it is more likely to happen.

6. Apply learning as soon as possible
When you get back to work look through all the notes associated with the course and remember the material that was covered. Then try to apply the learning. This will reinforce the learning so that you are less likely to forget.

MTS Recommends... QbD for Chromatography

‘Practical Implications of Quality by Design to Chromatographic Method Development’ by Mike McBrien in Chromatography Today, Volume 3, Issue 2, June 2010

A good discussion of the practical implications of applying Quality by Design to chromatographic methods.

MTS Recommends... Headspace GC for Pharmaceutical Analysis

'Experimental Considerations in Headspace Gas Chromatography' by Laila Kott, Hong Ming Chen in Pharmaceutical Technology, Volume 34, Issue 5, pp. 76-79 May 2, 2010

In this article a case study of amines is used to consider the important experimental parameters when developing a GC headspace method.