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HPLC Troubleshooting Guide to Changes in Selectivity, Retention, and Resolution

The Problem

This is definitely a problem which most chromatographers will be faced with at some point in their career; a change in selectivity, retention, and resolution of peaks.

Comparison of the chromatograms shown in Figure 1 shows that the selectivity of the peaks have changed, the retention times have decreased, and there is a loss of resolution between the peaks.

The method was used to assess the purity of a mildly hydrophobic (log P = 1.0) NSAID.  It had been recently developed and validated, however, upon installation of a new column the analyte retention, selectivity, and resolution changed.

Split Peaks

 

 

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

HPLC

Changes in Selectivity, Retention
and Resolution »

Cycling Baselines and Pressure Fluctuations »

Solving HPLC Peak Splitting Problems »

GC

Broad Peaks and Loss of MS Sensitivity »

Broad, Double/Split, Fronting Peaks »

Peak Area Reproducibility and Reduction
of Sensitivity »

Figure 1: NSAID purity analysis. (Top) ‘Good’ chromatogram. (Bottom) ‘Bad’ chromatogram showing change in selectivity, loss of resolution, and decreasing retention times.


Column: Modern alkyl-phenyl consisting of Type B silica and endcapped.  50 x 2.1 mm, 1.9 μm

Mobile Phase A: Aq. buffer pH 7.6:MeCN, 95:5

Mobile Phase B: Aq. buffer pH 7.6:MeCN, 5:95

Time (minutes) Mobile phase A (%) Mobile phase B (%)
0 100 0
8 40 60
44 0 100
48 0 100
48.1 100 0
30 100 0

Sample diluent: 100% Aq

Injection volume: 40 μL

   
 

The CHROMacademy HPLC Troubleshooter

The troubleshooter is unique in that it lets you combine multiple chromatographic and instrument symptoms. Our chromatogram is showing changes in selectivity, loss of resolution, and a decrease in retention time. All three of these symptoms can be entered simultaneously into the Troubleshooter to give a much more precise list of possible causes (Figure 2).

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

 

With very little effort, in two simple steps we can now access possible answers to our problem (Figure 3).  There are 32 possible causes of our symptoms, indicting just how complex chromatographic troubleshooting can be.  However, the troubleshooter reports problems with a star rating showing the number of symptoms which relate to that cause.   The causes are also ranked by how common they are as well as the likelihood that this particular combination of symptoms could lead to a particular problem. 

This ranking, based on our unique and proprietary algorithm, allows you to work through the list one item at a time in the knowledge that the causes are common and, therefore, you are not wasting your time on improbable solutions.

Once you have clicked on the cause the solution box displays information on how the cause may have resulted in your problem, how to solve it, and helpful practical tips on avoiding the problem in the future.  Further resources from within CHROMacademy or provided by our partners LCGC or vendors add to the information already provided to give a well-rounded insight into the problem at hand.

Figure 3: CHROMacademy HPLC Troubleshooter results page, including causes of changes in selectivity, loss of resolution,
and decreasing retention times, solutions to each individual cause, and further resources for resolving the problem.

 

For some golden rules of troubleshooting see:

Chromatography Troubleshooting Tips »
 

The Solution

Although this looks like a very complex problem at first sight (and believe us when we tell you that it could have been) we were able to solve the problem by only investigating the first two possible causes.

The first cause suggested was ‘incorrect/non-optimal mobile phase pH’. This problem does relate to our analytes as they are ionizable. If the pH of the mobile phase is close to the pKa of an analyte it can cause changes in peak shape as well as altering the retention characteristics of the analyte.

Small fluctuations in the pH when it is very close to the pKa of the analyte will have a profound effect on the retention, selectivity, and resolution of ionizable analytes. The pH of the mobile phase can vary if it is not precisely prepared each time, through evaporation, or even through ingress of CO2. The pH of the mobile phase (7.6) was confirmed to be optimum for this separation as it was far enough removed from any pKa values. In order to ensure that there were no problems with preparation of the buffer, evaporation etc. the mobile phase was re-prepared. With fresh eluent the problem persisted, hence, this cause could be ruled out as the possible source of the problem.

The second cause in the list is ‘column contamination - analytes or sample components irreversibly bound to the stationary phase’. It was noted when the problem was submitted to CHROMacademy that the problem had occurred when a NEW column was installed. Now, we could immediately jump to the conclusion that the new column is the problem. However, when validating a method it is always advisable to assess, as a minimum, different batches of the column, this can also be extended to included columns of the same nominal stationary phase type from different manufacturers in order to evaluate any changes in chromatography.

This had not been carried out during this method validation, and in fact, when other new columns from different batches were obtained from the manufacturer they produced the same chromatography as the new column. It turned out that the column which had been used for method development and validation was an old column with an unknown history. If a column has been used with ion-pair reagents, at extremes of pH, or highly hydrophobic analytes have been analyzed and have irreversibly bound to the stationary phase, these can all modify the column creating a unique stationary phase and surface which can never be fully emulated. This was demonstrated by the fact that the original chromatogram could not be reproduced at all on the new column and the method had to be completely redeveloped and validated.

  try our hplc troubleshooter

Let this be a cautionary tale to all chromatographers, if you are developing a new method use a new column (or at least one that you definitely know the history of).

In some cases if the column is contaminated it can be regenerated by washing and this is a very viable approach that can be tried before purchasing a new column. How the column is washed will depend on the stationary phase type. The user guide supplied with the column will often detail how to regenerate it and it is worth consulting this document.

Nevertheless, prevention is often better than cure and there are certainly ways to avoid column contamination:

  • Use a guard column to protect the analytical column.  It will preferentially scavenge the contaminating species and can be changed more frequently and less expensively than the analytical column.  However, care should be taken that the guard column does not irreversibly adsorb analyte species or affect the efficiency of analyte peaks by adding extra column volume.
  • If gradient elution is being used a wash at high organic concentrations (if appropriate) can be used to remove any strongly retained components.
  • Use a dedicated column for ion-pair analyses.

 

For further causes of selectivity, resolution, and retention changes see the following CHROMacademy pages:

Retention Time Variability in HPLC »

HPLC Troubleshooting Separations Retention Time, Efficiency and Peak Shape »

Selectivity, Resolution and Baseline Issues »

 

Have you ever had a problem with the following symptoms?

Erratic baseline

 

Leaks around the check valves or fittings

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

HPLC troubleshooting

 
 

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