04 Feb 2022
MythBusters: You can just regenerate a reverse phase column
The column is the heart of our HPLC separation, but it’s delicate and something we have a duty to protect to maintain optimal performance. As such a commonly used item, it has become shrouded in all sorts of hearsay over the years. We often get asked for advice on regenerating columns that no longer deliver the required chromatography.
The problem with this request is, can it actually be done? We’ve had a look into this for you.
First, let’s look at two main types of problems that occur with use:
1. Physical Damage
Although this includes voids and fissures, these are rare on modern columns and the most common issue we see is physical blockage at the top of the column, with either the frit or the packing interstitial spaces becoming clogged by particulate contamination. This results in increased backpressure.
2. Chemical Damage
This can be either:
- Direct damage, such as removal or permanent chemical modification of the stationary phase ligand
- Indirect damage where contamination becomes strongly/permanently bound to the stationary phase, changing column selectivity.
This type of problem results in a decrease in column performance, usually poor peak shape (tailing, fronting, chairing or splitting), a decrease in resolution and a change in retention time. It can often occur without a significant increase in backpressure.
Can we fix it?
If column performance has degenerated to a noticeable level the answer is (almost always) no. If you have already written the column off, there’s no harm in trying the following desperate measures:
1. Reverse the column
If a column is considered unusable, reversing the column is well worth trying – particularly with columns showing high backpressure. Modern columns are more tolerant to being used in reverse, although there is still a risk that reversing will cause permanent damage. If you’re going to dispose of the column anyway, it’s well worth a try.
2. Flush the column with a strong organic wash for an extended period.
This would usually mean switching the column to a water: acetonitrile gradient. Start at a high concentration of water (e.g. 90%) to remove any residual buffers that may precipitate in high organic. Ramp the gradient up to 100% acetonitrile and hold it there for several hours. Many users are reluctant to use 100% organic on their columns – don’t be, columns love it!
3. Perform several large injections (e.g. 100uL) of dimethylsulphoxide (DMSO)
Generally speaking, column regeneration is a myth! The real solution to regenerating a column is to not let it degenerate in the first place. As such, we have put together a list of 10 ways that you can protect your column.
Top 10 Tips for Column Protection
In all cases though, we ideally should be using columns in a way that prevents damage from occurring in the first place, rather than trying to repair after the damage has been inflicted. Monitor performance, using system suitability (even if just read and record rather than pass/fail) and intervene early.
The top 10 tips for column protection are:
1. Use a gradient that extends to 100% organic. Although dependent on the buffer in your mobile phase, this is one of the best tactics in column protection. This removes chemical contamination with each injection, preventing the build-up that causes permanent damage.
2. Consider your sample preparation. Samples containing damaging matrix components which employ minimal sample prep present a high risk for column damage. More complex sample prep (e.g. SPE) can significantly increase cost and time, whilst decreasing robustness and quantitative ability. In many cases, the costs associated with high column attrition rates are much lower than those of more complex prep.
3. Post sequence flush. If validation doesn’t allow you to modify your gradient profile to a higher organic content you could employ a column flush with high organic content. This can either be post sequence or at several points during the sequence. This makes good use of the additional lines available in quaternary mobile phase systems, is easily automated and can make a significant improvement to column longevity.
4. Dilute your sample as much as possible. This dilutes out contamination and reduces the load onto the column. This is especially important for semi-solid substances, such as gels, which cannot be filtered out and can be difficult to remove using sample prep.
5. If necessary consider alternative methods of detection to increase sensitivity and allow you to use lower sample loadings.
6. Check the pH rating of your column. Unless otherwise stated, silica-based columns are rated for use with mobile phases between pH 2 – 8.
7. If you see column pressure increasing, this is likely to be particulates and there’s a good chance you can remove these by filtration. Initially try an inline filter between the autosampler and column, but if this doesn’t work try filtering the samples using a small pore (0.2um) membrane filter of appropriate chemistry.
8. Use a larger particle size column. They are much more robust to both particulate and chemical contamination.
9. Use a guard column. Newer products have no detrimental effect on efficiency, although they can be expensive.
10. Filter your mobile phase. You should do this for all mobile phases, but it is especially important for columns using smaller particle sizes.