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HPLC Troubleshooting Guide to Peak Splitting Problems

The Problem

The analysis of an α2 adrenergic agonist and its main impurity are showing peak splitting that was not previously seen (Figure 1).  A moderate increase in system backpressure has also been observed.
The analyte is fairly hydrophobic with a log P value of 2.0.  The analyte possesses a number of basic nitrogen groups within its structure; however, the only significant pKa is at 7.49. 

Split Peaks

Figure 1: HPLC analysis of an α2 adrenergic agonist and its main impurity.  Chromatogram shows peak splitting and shouldering of both the analyte and impurity peaks.

This page is part of our series of 6 free Chromatography Troubleshooting Guides.


Changes in Selectivity, Retention
and Resolution »

Cycling Baselines and Pressure Fluctuations »

Solving HPLC Peak Splitting Problems »


Broad Peaks and Loss of MS Sensitivity »

Broad, Double/Split, Fronting Peaks »

Peak Area Reproducibility and Reduction
of Sensitivity »

Column: Modern C18 consisting of type B silica and endcapped.  250 x 4.6 mm, 5 µm
Mobile Phase A: Aq. buffer pH 3.6:MeCN, 95:5
Mobile Phase B: Aq. buffer pH 3.6:MeCN, 30:70

Time (minutes) Mobile phase A (%) Mobile phase B (%)
0 95 5
10 80 20
22 44 56
45 44 56
46 95 5
55 95 5

Sample diluent: Mobile phase B
Injection volume: 10 µL


The CHROMacademy HPLC Troubleshooter

The program is built on a database of problems, symptoms, and remedies contributed by over 70 of the world's leading HPLC experts.
The troubleshooter uses a proprietary algorithm to compute the likely causes when a number of symptoms are combined. This approach is unique and there are no other troubleshooting programs of this type available.

Choose from a vast array of chromatographic and instrument symptoms to form the combination which best defines your problem and obtain the most accurate assessment of the cause and remedy. 

Up to 3 chromatographic symptoms can be selected at one time; in this case peak shouldering (one or more peaks) and peak splitting (one or more peaks).  Each option has a roll-over which illustrates the chromatographic problem in question in order to clarify the selection (Figure 2).

Figure 2: Input of chromatographic symptoms into the CHROMacademy HPLC Troubleshooter.


Two instrument symptoms can also be selected concurrently.  For the problem being discussed here this is an increased backpressure (Figure 3). 
This means that we can combine both chromatographic and instrument symptoms, a first for online chromatographic troubleshooting.

Figure 3: Input of instrument symptoms into the CHROMacademy HPLC Troubleshooter.


Figure 4: CHROMacademy HPLC Troubleshooter results page, including causes of peak splitting,
solutions to each individual cause, and further resources for resolving the problem


The possible causes of the problem are rated using a star system based on the number of symptoms which are matched and the likelihood that your particular combination of symptoms is caused by that particular problem. 

They are then ordered by how likely that cause is to have resulted in the problem being investigated. 

By clicking on the suggested cause, useful information is displayed in the Solution window to instruct you how to remedy the problem and also measures which can be taken to avoid the problem in the future. 

Finally, there are further resources listed for extra reading to help increase your troubleshooting abilities.  These resources come from the CHROMacademy multi-media learning database, LCGC magazine and our many instrument vendor partners and there are over 1million separate resources for you to learn from.

The Possible Causes list can be investigated in turn until the problem is remedied, however, remember also to use some common sense.  If a cause is suggested that is highly unlikely – i.e. a problem with a refractive Index detector when you are using UV, this is unlikely to be the answer you are looking for.

For some further great troubleshooting tips see the following CHROMacademy pages:

Chromatography Troubleshooting Tips »

  try our hplc troubleshooter

The Solution

Armed with the results from the CHROMacademy HPLC Troubleshooter we were able to begin to try and resolve the problem of the split peaks.

The first possible cause suggested was 'column end frit(s) contaminated/blocked'.  Column end frit blockages can lead to problems with increased backpressure (as we have seen in this problem) but can also affect peak shape leading to tailing, shouldered, and split peaks as the analyte is not deposited onto the head of the packing material in a contiguous plug.  Typically the problem can be resolved by reverse flushing the column (disconnected from the detector first) in a solvent that will either flush away or dissolve the trapped particulate materials or contaminants, in which case one must choose a flushing solution in which the suspected contaminants are highly soluble. 

If the column inlet frit is blocked then on reversing the column and re-running the analysis the peak shape should improve (Figure 5).

Figure 5: Analysis performed with the HPLC column reversed to improve peak shape. 


We know that there may be some concerns about reversing an HPLC column and the possible damage that may be done. 
If you are concerned about this course of action please see HPLC Column Abuse »

For our problem reversing the column did improve peak shape.  Therefore, the inlet frit was cleaned by washing the column to resolve the problem and return the chromatography to normal (Figure 6).

Figure 6: HPLC analysis of an α2 adrenergic agonist and its main impurity.

The occurrence of this type of problem can be mitigated by some very simple measures such as filtering mobile phases to remove particulate matter, using an appropriate in-line filter to remove particulate matter, using a guard column to remove contaminants prior to the column, and employing more effective sample preparation techniques, such as solid phase extraction, to selectively remove unwanted sample matrix components.
For more details on this problem see the following CHROMacademy page:

HPLC Troubleshooting - Autosampler, Column & Detector Issues »


Have you ever had a problem with the following symptoms?

Cycling Baseline (short term)

Pressure fluctuation

Use the CHROMacademy HPLC Troubleshooter to see what could be causing the problem and how to resolve it.

HPLC troubleshooting


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