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The Seven Deadly Sins of HPLC

Posted in: Analytical Chemistry and Chromatography Techniques
The Seven Deadly Sins of HPLC

If you work with an HPLC, then you know the frustration of going to use the machine and finding it in disarray. If you’re new to using an HPLC, then the machine can be intimidating to use and you might not know the ins and outs of using it. Here’s an article that has a bit for experienced users and novices: how use and maintain your HPLC.

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

1.  Failing to Maintain Your HPLC Column

 There are a few cardinal sins when it comes to column maintenance:

Running Your Column Dry

This is the absolute worst thing you can do! If you set up a large experiment to run overnight, make sure there is enough solvent for the whole run (and possibly a bit more). Otherwise the column will run dry and you will face a lengthy process of purging and resolvating the entire system the next day. In the worst case scenario, you might need to replace the column.

Using Corrosive Chemicals

Stay away from corrosive chemicals, such as strong acids and bases, because these will reduce the life of your column.

Not Fully Dissolving Your Sample

Running samples with precipitated material will lead to blockages in the column or elsewhere in the system and can actually destroy the column.

Not Cleaning the Column After Each Use

Have a standard protocol for cleaning the column before every HPLC session. Modern systems have an option to set up an automatic pre-wash program. Use it!

2.  Using Columns That Are Too Old

Remember that columns do get old and may need replacing if everything starts to go wrong at once.

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

Because HPLC does not give an absolute identification of your compound, include an authentic standard as part of your run. 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. When mixed with your sample, the standard should co-elute with your compound.

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. A blank run is, therefore, similar to a negative control in PCR. Some like to include blank runs between all samples and others run a blank at the beginning. Some have probably never run a blank (tut tut!), but you would never do that!

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

Retention times can shift from one run to the next – you need to know when it is ok and when it signifies a problem.

Small (i.e., seconds) shifts in retention time may be caused by air bubbles, column wear and tear over time, blockages in the column and elsewhere, and changes in the pumping pressure. This is normal within HPLC and doesn’t need to be a major cause for concern, especially if you include standards in your sample set.

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.

Even when the column is healthy and the user has kept everything constant, differences in retention time will almost certainly occur between different HPLC setups. This makes it difficult to compare data between collaborators. An authentic standard is a huge help when looking at a compound on different systems. You should also check absorbance profiles as this will confirm that you are looking at 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.

HPLC is not always the method of choice when it comes to quantification. However, with reliable standards and good working knowledge, HPLC can be used to robustly quantify a range of compounds. Depending on what you are quantifying, ELISA  might be a better option if antibodies and/or commercial kits are available.

Furthermore, due to retention shifts and variability between set-ups, it’s difficult to compare data from different labs. You might consider sending material to your collaborator or vice versa, so that all samples for a project can be run on the same system. This certainly makes it easier to convince the reviewers when you submit your paper.

Don’t forget that HPLC is only useful to analyze compounds that actually absorb light!

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

If you work in a lab where HPLC is routinely used to analyze bacterial metabolites (for instance), you probably have a set of protocols at hand for extracting compounds, running gradients, and analyzing data. However, bear in mind that what works for one compound may not work for the next.

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.

Originally published in 2014, republished in 2016.

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  1. Richa on December 1, 2019 at 5:42 am

    I have been doing HPLC since past 5 times but I am not able to get pass result sometime std agreement fails and she mentioned %assay.why does this happen?

  2. HPLC on August 20, 2018 at 11:05 am

    Over the past ten years that I have been heading the contract services business at our company, my team has grown from just 30 people to more than 300, couple that with the healthy attrition rate that we see in our industry, I have literally taken hundreds of interviews to build and maintain a strong, high-performance team.

  3. KaBe on June 28, 2018 at 1:01 am

    Good points to consider, although HPLC can use detectors other than UV/Vis light. And some setups do use strong bases (e.g. carbohydrate identification/quantification can use 1 M NaOH as an eluent, depending on the column used)

  4. Marian on March 13, 2017 at 6:43 pm

    If the HPLC column does dry out and you flush with solvent and do not observe bubbles, what is the next step to verify that it is working appropriately? I imagine running known samples and analyzing the observed peaks would be an indicator of the restoration of the column.

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