Failing to Maintain Your HPLC Column

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Here’s an article that has a bit for experienced users and novices.

Don’t be a sinner!  Avoid these sins and get the most from your HPLC run.

1.  Failing to Maintain Your HPLC Column

Running Your Column Dry

If you set up a large experiment to run, make sure there is enough solvent for the whole run. Otherwise the column will run dry and you will face a lengthy process of purging and resolvating the entire system.

Don’t use Corrosive Chemicals

Not Fully Dissolving Your Sample

Not Cleaning the Column After Each Use

2.  Using Columns That Are Too Old

You can extend the life of your column by using a pre-column. These are short columns that are fitted in front of the real HPLC column, and they catch a lot of the ‘junk before it has a chance to ruin your column. Pre-columns are available at a fraction of the price of a full column.

3.  Not Using Standards

An authentic standard is a pure preparation of your compound of interest that will allow you to follow your compound throughout the run.The inclusion of the standard will permit you to compare retention times and DAD profiles. Well known compounds (e.g., aflatoxin, vitamin D) can be bought as pure preparations from most chemical suppliers.

Standards are used to:

• Correctly identify the compound
• Sort out retention time shifts between runs
• Quantify your compound by using a known concentration of standard or by setting up a standard curve

4.  Forgetting Your Blanks

A blank HPLC run is one where you only inject the solvent that your samples are dissolved in.Running a blank can be costly in terms of time and solvent use, but you should always perform a blank run:

• At the beginning of each HPLC session (you never know what the previous user left behind)
• Between samples when your compound is very sticky (apolar) to help clean the column
• Between samples when you are working with new compounds

5.  Not Understanding Retention Time Shifts

Big changes in retention time (for instance >15 seconds) could be a hint that your column is dying. Have a look at the other compounds in your chromatogram (if you are not working with a single compound that is). If everything is shifting from how it usually appears, then either the gradient has been altered or there is something wrong with the column. Check the health of your column by running an authentic standard of your compound.

6.  Not Understanding Your HPLC Data and Its Limitations

As with every lab technique, HPLC has its strengths and weaknesses. You must recognize the limitations of HPLC, so that you don’t spend a lot of time gathering data that you can’t use.

The chromatogram profile and retention time can help you identify issues with the column, so that you can address them early. Watch out for peak tailing, where peaks tail off broadly (like a skateboard ramp), because this can be a sign of a dying or overloaded column. To troubleshoot peak tailing, try re-running with a smaller injection volume, and see if the peaks are sharp again.

7.  Not Looking ‘Outside The Box’ When It Comes to New Projects

If you are embarking on a new project to find new compounds, you should use a broad range of extraction mixtures and play around with gradients to maximize your chances of seeing all your compounds.

Now, you can be HPLC sin-free. If you can think of other HPLC sins, don’t be shy to let us know

Compiled By Charan