HPLC PEAK Fronting and Tailing, Common Reasons For It

All users of HPLC need to know and be familiar with the correct terms used to describe non-Gaussian shaped peaks. Two of the most common undesirable peak shapes, peaks that show "Fronting" and peaks that show "Tailing" indicate problems with the HPLC method.  A quick refresher on why you may observe an HPLC peak front or tail on the chromatogram follows. 

Peak FRONTING: First, let us define what peak fronting looks like. The leading edge (front) of the peak is vertical, straight up and non-Gaussian in shape. This sharp increase in signal is easy to spot. 

Common Reasons for Peak FRONTING:

• Poor sample/peak capacity. In other words, too low a K prime (not enough retention on the HPLC column) resulting in no chromatography taking place. To solve this problem you must develop a proper HPLC method which first retains the compound(s) of interest, holds them long enough to obtain an acceptable K prime and resolve them away from other peaks, then elutes them off the column.

• Injection Solution Too Strong:Your sample(s) should be dissolved in the mobile phase and not in a solution that is "stronger" in elution strength than the mobile phase. Example: If you method is 100% aqueous, do not inject the sample in a solution with organic solvent. Follow fundamental good chromatography guidelines.

• Column Fouling / Overloading of sample. When the HPLC column is overloaded with sample, the peak shape will show fronting. Decrease the injection volume and/or concentration, as appropriate, in 10x graduations until the peak shape is normal.

• Saturation of the Detector: Just as with overloading the column the peak shape may change, overloading the detector's measuring range may also result in saturation of the signal and loss of accuracy. Decrease the injection volume and/or concentration, as appropriate, in 10x graduations until the peak shape is normal and back on-scale.

Peak TAILING: First, let us define what peak tailing looks like. The trailing edge (tail) of the peak slowly drops off towards the baseline and  is non-Gaussian in shape. For those with GC experience it appears similar to a peak that "bleeds" and continues to interact with the column for an extended period of time.

Common Reasons for Peak TAILING:

• Flow path Diffusion (from extra-delay volume). Poorly swaged fittings/connectors, a column with a void, incorrectly sized capillary connection lines may all contribute to peak tailing. Optimize the flow path, column and connections.

• pH dependence for ionizable compounds. If the sample is easily ionized and the difference between the pka of the sample and the mobile phase is less than 2 pH unit, tailing may result. Being sure to work within a safe pH range for your column, increase or decrease the mobile phase pH to be > 2 pH units away from the sample's pka to reduce tailing.

• Type 'A' silica or heavy metal contamination of the support. Many older style column supports did not use ultra-pure, heavy metal free packing material. These material often interacted with the sample on the column resulting in changes in retention, The use of more modern type 'B' or 'C' packings has eliminated many of these problems.

• Residual silanol groups present on support. As with the earlier type 'A' supports, non fully end-capped supports with residual silanol groups often resulted in secondary, extended retention effects. Use of more modern, fully end-capped, ultra-high purity packing materials (and/or mobile phases which better address these residual groups) often allow Gaussian peak shapes without the need for many additives.

• Column Fouling / Overloading of sample. When a column is not washed of all retained material after each analysis, it may build up over time and change the surface chemistry of the support. This may lead to changes in retention, especially delays in both binding and elution. Wash, regenerate or replace the column to solve.

You may also be interested in reading a related article; "Two Common HPLC Problems and their Causes (Sudden changes to either the HPLC Backpressure or Peak Shape)".

HPLC Peak Tailing - Some of the Most Common Reasons For it

Three easy ways to minimize chromatography peak tailing:

(1) Tailing often results from using “Type – A” HPLC silica. Type-A silica often contains more acidic silanol groups and metal impurities than Type-B. To improve peak shape, use modern “Type – B” silicas which are of higher overall purity, have less metal contamination and feature minimal silanol ionization under higher pH conditions.

(2) Minimize ionic interactions and utilize a buffer or ion pairing agent (e.g. TFA 0.02%) in your mobile phase. Select a buffer that is at least 2.0 pH units away from your sample's pKa and use the smallest concentration or amount that gets the job done. For LC/MS or MS/MS applications, remember to only use volatile buffers and avoid the use of ion pairing agents unless absolutely necessary (and if used, use at the lowest possible concentration to avoid source contamination).

(3) Always use a freshly washed and equilibrated column. Is the column fouled or the inlet frit dirty? If the head of the column is fouled from sample overloading or from a failure to wash off strongly retained compounds from many runs (much more common problem), then your peak shape and reproducibility will suffer. Incorporate a washing step in between your analysis methods which utilizes a solvent which is stronger (in concentration) than your mobile phase to wash off any strongly retained material after each run. For example, if you normally end a method with an 80% concentration of ACN, utilize a separate wash method which has 95% ACN in it. Allow enough wash time for this work.

Two Common HPLC Problems and their Causes (Sudden changes to either the HPLC Backpressure or Peak Shape)

   Let's take a quick look at two different problems which you may encounter when operating an HPLC system. We start with the basic observation and then look at the most likely causes so we can begin the troubleshooting process and repair the problem. An automated HPLC system's flow path typically consists of: The Solvent Pickup Filters (in the mobile phase reservoirs); The Pump(s); AutoSampler; AutoInjector; Column and one or more Detectors.*You should have a good understanding of this flow path before you proceed to diagnose the problem(s).

 *A gradual increase of pressure for the same method over time is often due to column fouling or a dirty inlet frit (e.g. PTFE frit). This article specifically focuses on the causes of a sudden change, not a slow change over time.

   Sudden System Back Pressure Changes: We will assume that you have been running the same method for some time or at least several times without a problem and then suddenly notice that the back pressure has changed from what is normally seen. The problem must lie within the flow path of the system.

   Excessive High Pressure: Typical reasons for this are:

1.      A fouled or plugged column;
2.      Wrong flow rate (higher than normal);
3.      Inlet frit/filter plugged or restricted;
4.      Plugged line;
5.      Wrong mobile phase composition.

   Large Drop in Pressure: Typical reasons for this are:

1.      A leak at a fitting, column or line (Number one reason);
2.      Wrong flow rate (lower than normal);
3.      Wrong mobile phase composition. 

• Start by checking the method parameters to insure that they have not changed (i.e. flow rate, mobile phase composition). Check for leaks or plugs. If the column is suspect, replace it with a zero dead volume union (ZDU) and restrictor and flush the system. Replace the column with a new one or wash the current column according to the column manufacturer's guidelines.

   Sudden Peak Shape Changes: We will again assume that you have been running the same method for some time or at least several times without a problem and then suddenly notice that the peak shape of one or all of the peaks has changed from what is normally seen. *The key thing to keep in mind is that the change occurs all of a sudden, not because of poor initial method development.

   Typical reasons for this are:

1.      Tailing or Split Peaks: Sample overload, change in flow rate, mobile phase composition (e.g. composition or pH), void formation, dirty frit, injection solvent too strong or a fouled column.
2.      Fronting: Commonly seen when overloading sample on column.
3.      Ghost Peaks: Usually due to a contaminated mobile phase, contaminated sample vial or contaminated injector (e.g. rotor seal).
4.      Broad Peaks: Large sample injection volumes or extra column volume (bad connections with the system or tubing) are usually to blame. Try reducing the injection volume by a factor of 10 and see if the problem goes away. You may also want to wash the column as it may be fouled with sample.

   These are just two common problems we see when using HPLC systems. Note that a dirty or fouled column can cause many of these problems so take care of your columns and wash and test them regularly to insure they are in compliance. There are many other commonly seen problems besides these. If you would like to see a specific problem featured on this blog, then please send me a request.