Peak distortions are a common and frustrating problem when performing chromatographic experiments, and peak tailing in high-performance liquid chromatography (HPLC) is one of the most frequent distortions that can occur. One of the challenging aspects of tailing peaks is that they can be caused by a range of different factors, so accurately diagnosing the problem as well as mitigating risks across your entire process are necessary to prevent further distortions. If your HPLC peaks look more like skateboard ramps than the nice Gaussian curve we all like to see, follow these tips to help identify the root of the problem and restore symmetry to your chromatograms:
1. Buffer up and bring down the pH
One of the main culprits of peak tailing in reversed-phase LC, in cases where only one or a few peaks are distorted, is secondary interactions between basic analytes and residual silanol groups on the stationary phase. One of the best ways to resolve this issue is to lower the pH of the mobile phase to about 3.0 or lower, which will protonate the ionisable silanol groups and prevent secondary retention.1 Utilizing low pH additives and buffer salts such as 0.1% formic acid and ammonium formate or ammonium acetate can help you achieve this effect and are compatible with LC-MS.2 Because there is a risk of silica dissolution at pH levels below 3.0, be sure to use a column designed to withstand these acidic conditions.
Even at a higher (neutral) pH, smart use of buffers can improve peak shape and mask silanol interactions. For example, increasing phosphate buffer concentration from 10 mM to 25 mM at pH 7.0 reduces peak tailing for LC-UV applications by increasing ionic strength of the mobile phase.3 However, buffers used for LC-MS should be kept at concentrations below 10 mM to prevent ion suppression, and higher buffer concentrations used for LC-UV could lead to buffer precipitation depending on the solvent used and the ratio (%B). Pay attention to the solubility of your buffer in your solvent and try a solvent like methanol rather than ACN or THF for better results when increasing phosphate concentration.
Another type of additive that has been used to prevent secondary silanol group interactions is competing bases like triethylamine (TEA), which would work by interacting with the silanol groups, thus preventing analytes from doing the same. However, advancements in silica stationary phase design have made the use of competing bases less necessary, as explained in our next tip.
2. Use end-capped/base-deactivated columns for analyzing basic compounds
Another simple way to reduce peak tailing from secondary silanol group interactions is to choose a column with a stationary phase designed to prevent these interactions. These columns may be referred to as end-capped or base-deactivated silica (BDS) columns, and are widely available from most column manufacturers. The packing in these columns is deactivated by bonding free silanol groups with an agent such as trimethylsilyl (TMS), which can also protect the silica from dissolution. When you know you will be working with basic analytes, choosing end-capped columns is a proactive method of preventing peak tailing due to secondary interactions. Using columns with high-purity silica is also advisable to prevent interactions between analytes and contaminants such as trace metals.
3. Lose the dead volume
While secondary silanol interactions and other chemical quirks are the likely cause of peak tailing that occurs for one or handful of analytes, when all of your peaks are tailing, you’ll need to examine some other aspects of your setup and method. One cause of peak tailing across your entire chromatogram could be dead volume, either in the form of a void in your stationary phase or extra-column effects due tubing that is too large or improperly fitted. One sign that dead volume might be the culprit is more pronounced tailing at earlier eluting peaks as well as band broadening in addition to asymmetry. This problem is also amplified when using smaller diameter columns.
Silica column voids can often occur at the inlet end where the silica becomes compressed or collapsed due to high pressure or pH conditions.4 This can be prevented by increasing the flow gradually to avoid pressure shock on the stationary phase, using a guard column, and ensuring your columns are suitable for the pH conditions you plan to operate with. In some cases, a void can be repacked with the proper material, but often the best solution is to replace the deformed column.
Excessive extra-column volume can be prevented by decreasing the tubing length and diameter, but if this does not fix the problem, you may be dealing with improper fitting between the tubing and column. If the fitting is improperly installed, or if you are using a column end fitting that is mismatched with the ferrule and stem length of the tubing, you can end up with a gap of dead space between the tubing and column, as well as additional dead space around the ferrule that can cause leaks.5 One reason this problem can occur is that parts produced by different manufacturers may have different dimensions, so using fitting parts from the same manufacturer or ensuring that all your fitting dimensions match up can prevent this issue. If you’re sure that a fitting mismatch isn’t the problem, make sure you’re properly tightening your fitting when installing a column.
4. Filter, flush and backflush to bust blockages
Another problem that can be occurring either inside your column or at the inlet frit is blockage, which can alter flow through the column and cause peak distortions for all analytes. This problem could be occurring if you notice a change in pressure. One way to prevent this issue is to use an in-line filter that catches particulates before they can plug the frit. To rule out contaminants and blockages inside the column, flushing with 5-10 column volumes of buffer-free mobile phase followed by 10-20 volumes of strong solvent, or another suitable washing procedure, can remove most build-up.6
If washing does not solve the problem, backflushing the column, if permitted according to the manufacturer’s instructions, can remove blockages of the inlet frit. Disconnect the column from the detector, reverse it and run at least 10 column volumes of strong solvent directly to waste.
In addition to filters, guard columns can prevent contaminants from making their way into the analytical column, but blocked or defective guard columns can also be a source of peak tailing. Before you go to wash your column back and forth, you can rule this out by removing the guard column and running a standard to see if the problem persists.
5. Don’t overload your column!
While column overloading is typically more associated with peak fronting or “shark fin” shaped peaks, both mass overload and volume overload could also cause peak tailing across the chromatogram.7 To test for mass overloading, simply dilute your sample to see if the peak tailing lessens or resolves. For volume overload, try injecting less sample. Another option is to use a column with increased capacity to lessen the risk of overloading altogether.
Lastly, be sure to always prepare your mobile phases and samples with proper care and cleanup, and perform regular maintenance to keep your setup and peaks in tip-top shape. Happy separating!
References
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"Peak Tailing in HPLC," Crawford Scientific. https://www.crawfordscientific.com/chromatography-blog/post/peak-tailing-in-hplc
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"A Guide to HPLC and LC-MS Buffer Selection," ACE HPLC Columns. https://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf
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"The Secrets of Good Peak Shape in HPLC," Agilent. https://www.agilent.com/cs/library/eseminars/Public/secrets%20of%20good%20peak%20shape%20in%20hplc.pdf
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"The LCGC Blog: HPLC Diagnostic Skills II — Tailing Peaks," LCGC. https://www.chromatographyonline.com/view/lcgc-blog-hplc-diagnostic-skills-ii-tailing-peaks
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"Fittings and Connections for Liquid Chromatography—So Many Choices!," LCGC North America. https://www.chromatographyonline.com/view/fittings-and-connections-liquid-chromatography-so-many-choices
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"Washing Reversed-Phase Silica-Based Columns," LCGC. https://www.chromatographyonline.com/view/washing-reversed-phase-silica-based-columns
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"Troubleshooting HPLC- Tailing Peaks," Restek. https://www.restek.com/en/chromablography/chromablography/3troubleshooting-hplc--tailing-peaks/