[3] Troubleshooting HPLC- Tailing Peaks

Patterns in your HPLC chromatography can exhibit telltale signs that point toward probable sources of error.  In this brief series of posts, we will look at possible scenarios that you may encounter in the laboratory and how you might approach resolving those difficulties.

If you are having difficulty with tailing, it is likely that you are also having an issue with loss in response

. For this reason, you may also find the previous two posts in this series helpful:

[1]Troubleshooting HPLC-I'Loss in Response for All Analytes

[2]Troubleshooting HPLC-Loss in Response for Some, but Not All Analytes

As in the previous posts for this series, these suggestions assume that you are working with an established method and had successful results in the past. If a method is rugged, it is developed to minimize tailing by using a stationary column phase that is base-deactivated as well as appropriate modifiers and pH adjustment in the mobile phase as needed.  Keep in mind that if the method is not as rugged as it should be, change(s) in the method might be advised if tailing continues to be an ongoing problem.

With troubleshooting, as always, it is critical to note when the change occurred and how it might correlate to changes in the system.  Equally important is to change one thing at a time to identify the source(s) of difficulty. The following are examples of things that could contribute to tailing. I suggest investigating these in this order:

1. Issues with mobile phase- This could be a problem with preparation of new solution or degradation of an existing solution.  Particularly if you notice some retention time changes and/or interferences that weren’t present earlier, this could be the cause. Try preparing some fresh mobile phase and see if your tailing gets better.  Pay special attention to which peaks are tailing and whether the issue might be pH-related.  Lack of proper pH adjustment for the mobile phase could be a factor. For more on this topic, please see the earlier posts on Buffers, When should you use a buffer for HPLC, how does it work and which one to use? and How much buffer to weigh and what’s the best way to prepare my buffered mobile phase?. 
2. Obstructed or dirty guard cartridge- Put simply, if you are using a guard cartridge, try replacing it with a new one. Replace the cap frit also if you are using a guard holder that accommodates the frit (such as catalog #27474). A buildup of sample material on the frit or adsorbed on the cartridge could create tailing that would most likely affect all of your analytes.
3. Using an inappropriate guard cartridge- This is usually easy to correct and diagnose.  If using a guard cartridge, it should contain packing material that is the same as the analytical column or one that is very similar. Especially if you have just recently started using one of these, make sure that it contains the right packing material with the right bonded phase.  Use caution if you are using a Restek guard cartridge with an analytical column from another vendor.  This is addressed in the FAQ section of our website.  If there is a significant difference between the guard and analytical column packing, there will be two competing modes of separation, which could create split or distorted peaks or tailing.
4. Increase in dead volume- This is most critical for columns with small ID and tailing is usually more evident with early eluting peaks. However, if it is bad enough, you might see an effect to some degree on all analytes.  If you have changed tubing lately, make sure you aren’t using a larger ID or longer length than previously.  Also make sure the tube fitting at the inlet of the column is fully seated and that you are using the proper fitting and ferrule. For instance, connection to a Waters column or Waters/Rheodyne parts sometimes requires alternate fitting parts due to deeper seated threads. Please see the LC FAQ pertaining to this for more information.  Our Universal PEEK connectors and new EXP fittings accommodate for all of the common fitting styles.  If you are using a guard holder that accommodates a cap frit, make sure that a cap frit is installed; otherwise, dead volume will result. If you are using a Trident Direct guard holder with an analytical column that is not from Restek, make sure you are using the appropriate PEEK tip. An alternate tip is offered for Waters-style column end fittings. Be sure to check out our video "Minimizing System Volume".
5. Mass overloading- Injecting too much sample material, that is, a solution that is too concentrated in terms of sample material weight (including matrix), can create tailing, particularly for basic analytes. Try injecting a less concentrated solution to see if tailing decreases.
6. Volume overloading-Injecting too large of a volume causes peak broadening, along with fronting and/or tailing. You can determine if this is the problem by injecting a smaller volume. Some general guidelines as far as suggested volumes can be found in the FAQ section on our website.
7. Interfering coelution- If you have tailing only for one or two of your peaks, there may be an interfering contaminant that coelutes.  If that is the case, you might see the tailing come and go, and a shoulder may appear at times. If you think this might be happening, try using a slower gradient or changing your organic/water ratio and see if the peak separates into two.
8. Coating or obstructions on column frit or particles of column packing- This is due to sample matrix and very common.  This usually affects all of the analytes and is often accompanied by an increase in column backpressure.  It can best be prevented by filtering sample extracts prior to HPLC and by using sample cleanup techniques such as SPE. In some cases, flushing with various solvents may help. Please see our LC Cleaning Recommendations.  If cleaning does not help, your column probably has been damaged chemically and should be replaced.
9. Physical damage to column- This does not happen frequently but is a disturbance in terms of how the packing material is arranged inside the column. It could be related to a void, fracture, open channel, or non-uniform compression.  These can result from a physical shock in terms of pressure or flow.  The phenomenon may explain tailing on columns which have had no or very little sample matrix injected.  The tailing would occur for all analytes and is likely to be accompanied by a change in column pressure. A column with this type of damage should be replaced.
10. Loss of inertness in tubing, injector parts or associated valves- If switching to a new column doesn’t help, it may actually be an issue with tubing or instrument parts.  This is a tough one to diagnose, but it can and has happened.  Symptoms of this tend to mimic a similar problem with the column but may not be affected by efforts to clean the column.  This is caused by accumulation of certain components from sample matrix over time.  To remedy this, tubing should be replaced, and valves physically cleaned or replaced. Please see your manufacturer’s instructions to do this.

Be sure to also check out the following videos we have available in our library:

LC Troubleshooting: Optimizing Your Injection to Improve Peak Shape

LC Troubleshooting: All of My Peaks Are Tailing! What Should I Do?

Thank you for reading.  Don’t forget to check for the next post in this series on troubleshooting.