The LCGC Blog: HPLC Diagnostic Skills II – Tailing Peaks

In HPLC Diagnostics Skills Part I we looked at baseline issues, and we continue here with HPLC peaks and in particular the skills required to identify tailing peaks, the causes of peak tailing, and most importantly, how to fix the issues that give rise to this peak deformation.

Tailing peaks create issues with resolution, quantitation (integration), and reproducibility. Peak shape is often the controlling factor when optimizing complex separations, especially when components are present in very different concentrations. The extent to which a peak is tailing can be assessed using Equation 1.

Figure 1: TF = (a) 1.24, (b) 1.42, (c) 1.58, and (d) overlay of a, b, and c.

When methods are designed, a limit is usually set on the level of tailing that is acceptable. If you work under regulatory guidelines, this may be set by the governing body. For in-house methods a reasonable guideline is TF ≤ 2.

How do you start troubleshooting a problem when it arises? Two great tools provided by CHROMacademy are our interactive HPLC (and GC) Troubleshooters. Therefore, we are going to look at the top five causes of peak tailing provided by the HPLC Troubleshooter. These results have been ranked, so the most likely causes of peak tailing and should, therefore, be your first consideration when you are faced with this problem. In total the Troubleshooter returns 25 possible causes of peak tailing, hence, this is often not a straightforward problem to fix.

Figure 2: CHROMacademy HPLC Troubleshooter used to diagnose problems with Peak Tailing – clicking on each causal factor will give tips to identify if this is the correct cause of your problems and recommendations for fixes (https://www.chromacademy.com/hplc_troubleshooting.html)

1. Analyte and Stationary-Phase Secondary Interactions

Unwanted secondary interactions between the analyte and stationary phase can lead to peak shape problems, typically peak tailing and loss of efficiency, which may ultimately lead to a loss of resolution and difficulty with peak integration.

The most common interaction is between polar or ionized analyte functional groups and uncapped (unreacted) silanol groups on a silica stationary phase surface (Figure 2).

Figure 3: Interaction between a charged basic analyte and an acidic surface silanol group.

There are several ways to avoid peak tailing due to analyte and surface silanol interactions:

1.  Choose a column with reduced secondary surface silanol species (end-capped, hybrid, or type B silica would be suitable)

2.  Choose a suitable pH to minimize secondary interactions: at low pH (~2.5) silanol species are non-ionized and the degree of peak tailing will reduce, at this pH acidic species will be less ionized and, therefore, peak shape will improve. With basic species at low pH, most bases will be protonated (ionized) but the suppression of surface silanol species will mitigate the degree of peak tailing

3.  Increasing buffer concentration (> 20 mM) or swapping the counter ion for one which is more highly surface active may also help to mitigate the degree of peak tailing

4.  A more traditional method for controlling peak tailing is the use of a sacrificial base (such as triethylamine) in the mobile phase (0.05 M is typical) which is sterically small and charged at low pH and will preferentially interact with the surface silanol species

5.  Certain analytes may chelate with metal ions on the stationary-phase surface (which may be included in the silica matrix or may have leached from the column internal surface or frit) – this secondary interaction will also affect peak shape and produce peak tailing

2. Incorrect or Non-Optimal Mobile Phase pH

Mobile-phase pH affects retention of ionizable analytes and the selectivity of separations in reversed-phase HPLC with some methods requiring a very precise pH to provide robust analyses. Low pH tends to increase retention of acidic analytes and decrease retention of basic analytes, whereas high pH tends to decrease retention of acidic species and increase retention of basic species.

Low pH (< 3) tends to improve peak tailing, especially for basic compounds due to the decrease in ionization of acidic silanol species on the stationary phase surface.

Figure 4: Surface silanol ionization

Buffers are used to prevent pH changes, and it is important that the correct buffer system is used to prevent pH shifts (usually due to the introduction of the analyte in a diluent that does not match the mobile phase pH). The pH should be measured using a calibrated pH meter on the aqueous portion of the mobile phase only, and accuracy to within ±0.05 pH units is recommended. The actual mobile phase pH may alter significantly (typically 1 to 1.5 pH units closer to neutral) in the presence of larger amounts of organic modifier. Mobile phase pH may alter on standing through the ingress of CO₂ (making the mobile phase more acidic) and via evaporation of the organic portion when pre-mixed mobile phases are used. The sensitivity of some detection systems (such as atmospheric pressure MS and fluorescence detection) may be affected by mobile phase pH changes which affect the degree of analyte ionization.

3. Void at Column Inlet

A void is caused by mechanical collapse of the stationary phase material which, under system pressure, compresses the bed materials, leaving a void at the head of the analytical column.

The void often results in loss of efficiency and poor peak shape (fronting, tailing, and shouldering are typical).

Voids are typically related to silica collapse which occurs under high pressure or high pH conditions.

Figure 5: Schematic of flow path through an HPLC with a) normal flow b) flow disturbance due to a blocked inlet frit c) flow disturbance due to a column void (arrows indicate flow direction prior to separation as the sample plug enters the column) (Reproduced with permission of LCGC North America)

Avoid pressure shock - increase flow (pressure) gradually (some modern pumps can be programmed to achieve this automatically) and use ‘make before break’ injection valves when possible.

Operate at pH < 7.5 where possible, unless your stationary phase has been designed for use at high pH.

It may be possible to temporarily fix the problem by running the column in the reversed direction and then return to the normal flow direction, however, the void will ultimately return after extended operation.

Figure 6: Peak shape recovery after column reversal to unblock an inlet frit or repair inlet void

4. Large Extra Column Dead Volume

Extra column volume – the volume between the injection point and the detection point, excluding the part of the column containing the stationary phase. It is composed of the injector, connecting lines, frits, and the detector, and it is this that determines the extra column effects.

Extra column effects – the total band broadening effects of all parts of the chromatographic system outside of the column itself. Extra column effects must be minimized to maintain the efficiency of the column. Areas of band broadening can include the injector, injection volume, connecting tubing, end fittings, frits, detector cell volume, and internal detector tubing. The variances of all these contributions are additive.

W = bandwidth

The following are bandwidth contributions from

W_c = column

W_s = injector/autosampler

W_lc = lines and connectors

W_fc = detector flow cell

As long as the bandwidth contributions W_s, W_lc, and W_fc are each less than 0.3 W their effect on W can be neglected.

There are many aspects of the system which add dead volume, leading to loss of efficiency or resolution (Figure 5 and 6) – typically these include:

• Unnecessarily long tubing with i.d. larger than required

• Incorrect fitting type or poorly made connection

• Use of a guard column

• Use of unnecessary unions within tubing

• Large volume accessories or components (flow cell, injection loop, and others)

Therefore, using the correct fittings, minimizing tubing length and i.d., optimizing flow cell size for your instrument, and so on, will all help to reduce extra column volume. Extra column volume is of particular importance when working with UHPLC applications.

Figure 7: Effect of dead volume on HPLC resolution and separation. Left chromatogram generated with system of 10 µL extra column volume and right 100 µL extra column volume.

Figure 8: Effect of extra column volume on resolution and quantitation.

It is straightforward to measure the extra column volume of a system:

•   Remove the column and join the tubing with a zero-dead volume union

•   Inject 10 µL of 100% strong solvent (acetonitrile works with UV at 200 nm) or a solution of 1% acetone (monitor at 265 nm)

•   The apex retention time (tR) of the baseline perturbance due to the passage of the solvent gives the extra column hold up time of the system (expressed in minutes)

•   Multiply this time by the flow rate (in mL/min) to obtain the extra column volume (in mL)

5. Chelating Solute Interaction with Trace Metals in Base Silica

Trace metal content within or on the surface of the stationary-phase material can chelate with certain analytes causing peak tailing (Figure 8). The presence of surface metals can also increase silanol activity and interactions.

Figure 9: Surface silanol and metal moieties.

When you are analyzing analytes, which are capable of chelation the following will help to mitigate any peak tailing effect:

•   Use high-purity silica-based columns with low trace metal content

•   Add EDTA or another sacrificial chelating compound to the mobile phase which will be preferentially adsorbed to active sites to reduce analyte or stationary-phase secondary interactions (and mitigate peak tailing effects)

•   Consider the use of polymeric phases or those known to contain low levels of metals likely to create tailing effects

As with any troubleshooting problem, approaching it in a logical fashion and only changing one thing at a time will help you to quickly resolve the issue. An excellent troubleshooting tip is to have a bench marking method that is run on the system–column combination when they are functioning as expected. From this method parameters such as retention time, resolution, peak tailing, and so on should all be assessed to give you normal values. At the first sign of a problem, run the benchmarking method. If this method works well then there is a problem with the particular analysis being run, if the benchmarking method fails then it is most likely it is the column or instrument which is at fault. This will allow you to quickly focus on the correct part of the analysis, instrument, or method, and will greatly reduce your troubleshooting time.

Tony Taylor is Group Technical Director of Crawford Scientific Group and CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LC–MS and GC–MS for materials characterization, especially in the field of extractables and leachables analysis.