Help on: HPLC Dwell Volume and Dead Volume

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:
“In HPLC, how can I calculate dwell volume for my system, and what is the main difference between dead volume and dwell volume?”

Answer:
“The dwell volume for an HPLC system refers to the volume in the system from the point where the mobile phase is mixed to the inlet of the HPLC column. This volume is important when using gradient elution since there will be a delay between the time when a change in mobile phase is applied and when it actually reaches the column. The size of the dwell volume varies between different systems; it can be calculated as follows:

1. Remove the column from the system and use a short length of 0.010″ tubing to connect the injector directly to the detector.
2. For solvent A, use HPLC grade water; for solvent B, add about 0.1% acetone to water (methanol or acetonitrile can be used instead of water).
3. Set the detector wavelength to 265nm.
4. Run a typical gradient from 0 to 100% B (e.g. 0-100% in 20 minutes at 3 mL/min flow). Record the detector signal during this gradient.
5. Print out or display the "chromatogram" from the gradient run. It should look like Figure 1. Draw the best straight line fit to the flat portion at the beginning of the plot. Draw the best straight line fit to the linear ramp of the gradient. The time at which these two lines intersect is the dwell time (tD). The dwell volume is the product of the dwell time and the flow rate used for the test.

Figure 1

The term ‘system dead volume’ is used to refer to the volume in a HPLC system from the point of injection to that of detection. Think of it as the volume of mobile phase between these points. The ‘column dead volume’ is the volume of space in the column not taken up by stationary phase (sometimes also referred to as void volume), or you can also think of it as the volume of mobile phase that fits in the column. Typically the difference between the system dead volume and column dead volume is very small and so you may find the terms being used interchangeably. Unfortunately chromatographers can sometimes be a bit sloppy about using consistent terminology but the context should make it clear what is being referred to.”

Help on: HPLC Baseline Problem

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:
“The problem is the baseline looks lumpy (not steady) instead of being normal or steady. I have tried all the procedures and therefore eliminated the column, the mobile phase and detector. The system has been thoroughly flushed and purged but still my baseline is not as expected. I have tried the column on a different system under the same conditions (ambient temp, flow rate 1.0ml/mins, inj vol 2ul) and the baseline is normal as expected. I have attached a copy for you to see if you can help. Any ideas what could be happening on this system?”


Answer:
“The baseline in your chromatogram is very far from what you normally expect. The regular cycling pattern in the chromatogram makes me think that the problem is most likely related to the pumping of the mobile phase. Since you have ruled out the detector, and have transferred the mobile phase and column successfully to another system this means that the pump is the most likely source of the problem. Even though it is thoroughly flushed and purged there is still a small chance that it may be due to trapped air, but if not then a faulty check valve is likely. Try flushing and purging the pump with methanol (remove the column) and then with your mobile phase. If this doesn’t work try replacing the inlet check valve (this is the one which usually causes problems), or clean it by placing in a beaker and covering with methanol or isopropanol, then sonicate for about 5 minutes and replace.”

HPLC Training Video Shortlisted in Tutorial Competition

We are delighted to announce that the training video ‘A Brief Guide to HPLC Instruments’ has reached the shortlist of the Year of Education in Separation Science Tutorial Competition. The Year of Education in Separation Science is an initiative run in conjunction by separationsNOW.com (Wiley-Blackwell's separation science website) and Chromedia (an online community and database of tutorials and other educational content). The tutorial initiative is partnered by PITTCON 2011. The purpose of the competition was to facilitate and encourage the creation and sharing of educational material. The tutorials were judged on their content and how effective they are at communicating their message. The winner will be announced in the next few weeks.

MTS Recommends... How to Weigh Out Small Amounts