How do you go about recovering a column that you suspect has some problems? Or perhaps you’ve pulled a used column off the shelf and want to make sure that it will work OK. Do you just dust it off before trying it, or is there a better approach? This week’s HPLC Solutions looks at several aspects of how to best regenerate an HPLC column… and when to give up and dump it in the trash.
In one of the ‘Ask the Doctor” questions, a reader said that they heard that DMSO (dimethylsulfoxide) injections were a good way to regenerate a column. He wondered if this was true, because his column was showing signs of old age. There is no harm in injecting DMSO. Its ability to regenerate a column depends on several things: the nature of the column contamination, the amount of DMSO, and the age of the column. However, by the time you need to “regenerate” the column, it usually is past its useful lifetime anyway, so I discourage wasting your time at this. Usually a simple flushing with the strong solvent of the mobile phase (ACN or MeOH) at the end of a sample batch is sufficient. Column failure comes from a number of causes:
Particulate matter caught on the inlet frit
This can sometimes be removed by reversing the column and flushing to waste. An 0.5-µm porosity in-line filter will help to prevent this kind of problem, as will filtering or centrifuging your samples.
Loss of bonded phase
Some of this happens naturally over time and sometimes is accelerated by mobile phase pH outside the normal 2 < pH < 8 recommended operating conditions. If bonded phase loss occurs, there is no way to fix it.
Build-up of insoluble materials on the column
If these are indeed insoluble, they cannot be washed off. If they are just poorly soluble in the mobile phase, such as fats, waxes, etc, sometimes they can be washed off by reversing the column and after flushing with ACN or MeOH, flushing with 50 mL methylene chloride, then back through ACN or MeOH to the mobile phase. If you suspect the nature of the contaminant, sometimes a particular solvent can be chosen that will be most effective at removing it. For example, proteins might be removed by adding a chaeotrope, such as guanidine, to the mobile phase. I suspect that recommendations for DMSO fall in this category.
But each of these fixes only has a 25-30% chance of fixing the problem. In other words, it isn’t a very good investment of your time. Furthermore, the column after regeneration will never be as good as a new column, and often the selectivity can be significantly different. The bottom line: if you have between 500 and 2000 samples on your column, you’ve received your money’s worth and any repair efforts cost more in the value of your labor than you recover in column value.
And, as I’ve said before, if you are starting to use a new method, always start with a new column. Choosing to use a column that has been used for another method is just asking for trouble. If you amortize the cost of a column over >500 injections, you’ll see that it is a very small contribution to the overall cost of analysis. Don’t jeopardize your results by using a column that may not be performing at its best.
This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his research with Lloyd Snyder, which resulted in more than 100 technical publications and three books. If you have any questions about this article send them to TechTips@sepscience.com
