Hi I'm Gerard Sharp. Welcome to the February 2011 issue of the monthly newsletter. This month the Tech Tip is on filtering and degassing the mobile phase in HPLC.

Dr Gerard Sharp...

Ardent Scientific has just launched a new Chromatography eLearning site full of interactive multi media training modules with voice overs explaining the content as you go along. There's lots of free content and if you want to follow on from this month's Tech Tip and see how a HPLC Degasser works, click here to view. Enjoy!



Tech Tip - Degassing the Mobile Phase

What grade of solvents solvents should I use?

Always use HPLC-grade solvents. These solvents have been pre-filtered and also have a specification for UV absorbancy. Analytical reagent grade solvents might not have been filtered so don't be tempted to use these if you run out of HPLC grade solvents.  

Should I degas my mobile phase off-line?

The recommendation is to degas off-line even if you have an on-line degasser. That said, I see lots of labs which don't filter and degas off-line and all seems well. The answer really depends on your application. Degassing removes both dissolved gases and oxygen. Dissolved gases can cause problems with the pump if not removed. The symptoms may be a noisy baseline and fluctuating back pressure. Oxygen can cause problems at low (<210 nm) UV detection and with electrochemical and fluorescence detectors. Degassing off-line followed by on-line degassing does seem to be more effective but you might only notice it if you doing trace level analysis.


Techniques for Off-line Degassing


The most common technique for degassing is vacuum filtration. The solvent is pulled through a 0.45um filter under vacuum and dissolved gas is then removed in the vacuum flask. Click on the picture on the left to navigate to the Ardent Scientific eLearning module 'HPLC Tips and Troubleshooting' and then click on the Pump Tips icon to see an animation of this.

Another less used but easy technique is to ultrasonicate the mobile phase for 5-10 minutes. You'll see the bubbles of gas rise to the surface and will be able to tell when most of the gas has been removed.

The third type of degassing is helium sparging. This was used a lot more in the past but has almost disappeared although it is the most effective of the three techniques. If you have an electrochemical detector you might have to use this technique. Helium is virtually insoluble in the solvent and the small helium bubbles push the dissolved air out of solution. Nitrogen is not effective as a sparging gas.


Online Degassing


An online degasser is very common in a modern HPLC. These work by removing dissolved gas under vacuum through a semi-permeable membrane. Click on the picture at left to see how they work.



What about filtering?


Filtering is every bit as important as degassing. Particulates will clog the column frit, wear down the pump seal and, block lines, In fact, as the trend continues towards small particle columns and with that the use of smaller inner diameter connecting tubing, removing particulates is even more important.



Chromatography eLearning is yet another way to enhance your chromatography knowledge. While not ever being able to replace a classroom course, eLearning does offer advantages on cost and multimedia content.  

Also, unlike a classrom course you can learn at your own pace and return to the content time after time.
Ardent Scientific offers varying levels of membership for viewing eLearning content. The Single Membership starts at $300 / year and corporate memberships are also available. View here for full details.


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