Live Long and Prosper-How to make your HPLC columns last longer

We frequently get questions about how long an HPLC or UHPLC column should last. To borrow a phrase from my colleagues, “Your mileage may vary.”  Columns are like cars, so their lifetime depends how they are used and how well they are maintained. That being said, some phases are known to be more rugged than others. A C18 or aqueous C18 column generally lasts the longest, while something like an amino phase usually has shortest lifetime.

Here are the most common LC column killers:

  1. Particulates– These usually come from unfiltered sample matrix, but can also come from unfiltered mobile phases.
  2. Reactive material in samples– The most obvious of these is derivatizing agent, but can also come from sample matrix. Unless you have detailed info of your sample matrix, there are likely to be components that are unexpected and uncharacterized. This can even be present in matrices that appear to be fairly clean, such as urine.
  3. Harsh pH conditions– Suggested operating range for our columns is 2.5 – 8.0.  A pH that is too low may remove the bonded phase from the silica, while a pH that is too high will start to hydrolyze the silica itself.
  4. Incompatible mobile phase or flow conditions– Be aware that only certain phases are designed to be used with more than 95% water in the mobile phase.  The bonded phase in a traditional C18 column will collapse under these conditions. (You should be using an Aqueous C18 column if this is a possibility.) This is one example of this type of column damage.

So… to help your columns live long and prosper, you should do the following:

1.  Make sure your flow rate and pressure are appropriate for your column dimensions and particle size. Here is a useful table from our current catalog:

If you have recently reduced your column ID, please ensure that you reduce the flow rate accordingly.  Click here to reference a note on the subject.  As far as pressure goes, generally, it’s best to stay below a backpressure of 5000 psi for HPLC and below 12000 psi for UHPLC (particle size below 3 um).

2.  Pre-filter all samples prior to LC injection,

either by using a syringe filter or Thomson™ Single Step filter vial.                    

If sample volume is too low for either option above, make sure you use a cap frit with your guard cartridge.

If doing UHPLC, you will need to use one of our inline filters instead, either the

UltraShield UHPLC Precolumn Filter or the Ultra UHPLC InLine Filter.                    

3.  Use a guard cartridge for HPLC to minimize damage from reactive and adsorptive material. If you’ve had prior issues with short column lifetime, a longer cartridge might help. The 20 mm length should be used for any samples potentially containing reactive or highly adsorbed components. For details regarding our guard cartridges and holders, see our LC FAQs, which will be accessible through the troubleshooting page on our website.

Be sure to also check out our YouTube video on this topic, “Do I Need an LC Guard?”.

4. Pre-filter mobile phases if they contain buffer salts or any modifiers that come from a weigh-out of solid material. Also make sure you are using solvents that are HPLC grade or better. Check out our filtration supplies for this.

5.  If using buffer in your mobile phase, don’t make drastic changes from high to low aqueous content while pumping your column. If your intent is to switch to 100% organic solvent, make sure you flush out the buffer first using a mix that is at least 50% water for at least 7 column volumes. Failure to do this may result in salts falling out of solution, clogging frits and increasing backpressure.

6.  Store your column in the appropriate solvent. When storing for longer term intervals (more than 2 days), it is always best to consult the column handling instructions for a given column. Most reverse phase columns (including C18, aqueous C18 and IBD) are best stored in a mixture of methanol or acetonitrile and no more than 50% water. Some of our phases, such as the biphenyl, PFP Propyl and Amino are better stored in 100% acetonitrile.

7.  Avoid incompatible mobile phases. We’ve already talked about highly aqueous mobile phases.  Other things to watch for are materials that are known to be reactive to specific phases. This includes:

  • Aldehydes and ketones with amino phase (this includes acetone!)
  • Sulfur containing compounds with PFP propyl phase
  • Significant percentages and/or inconsistent amounts of water with bare silica

8.  Check the pH of your sample (and mobile phase if acids or bases are used). Most analysts use a pH meter, but you can do it old school with litmus paper if you want. If you are using a meter, make sure it is calibrated properly and frequently. Although we do not sell litmus paper, you can purchase it through just about any distributor that sells lab or chromatography supplies.

9.  Keep it clean (this is a family show). If normally running a gradient for reverse phase LC, give it an occasional flush with mobile phase that contains slightly more water than your starting conditions and no buffer salts, not to exceed 90% water for traditional C18 phases. Likewise, make sure you flush occasionally with mobile phase that contains slightly more organic than you usually use. For example, if your gradient goes up to 90% organic, flush it with 95% organic occasionally.  About 7 column volumes for each of these, done once a week or so should do the trick for this. (You will probably be doing some of this anyway prior to storage, but it doesn’t hurt to mention it!)  You may find that one of these two procedures is more beneficial than the other, depending on what is in your sample that tends to settle out or be absorbed. You can alter your maintenance as needed according to this. You might consider programming a flush cycle (with gradient that holds the concentration at highest and lowest points) at the end of each day or at regular intervals if it seems to help. If you are running isocratic, you can still do something similar.

If you notice increasing backpressure, you may need to do more extensive flushing in a backward direction. For reverse phase columns, please see our technical note for LC Cleaning Recommendations.

If you feel like any of these are a hassle, remember the needs of the many outweigh the needs of a few, so you may as well bite the bullet and take preventative measures. Feel free to contact Tech Services if you need further information. May you and your LC columns live long and prosper.

Many thanks go to Star Trek for the inspiration.


Tags: , , , , , , , , ,

Leave a Reply