Peak tailing is a crucial attribute in chromatography. It directly influences the integration of peaks, which, in turn, affects the quantification of compounds. The significance of peak tailing is emphasized by the fact that regulatory bodies like the United States Pharmacopeia (USP) and the European Pharmacopoeia (EP) have specific requirements for acceptable peak symmetry (0.8 to 1.8). In this article we will delve into the underlying phenomena causing peak tailing, the associated symptoms, and corrective measures.
1. Wrong combination of sample and column packing:
Example:
So in the chromatogram at the bottom, you will see that the C8 stationary phase is used and these two compounds are basic and they have a significant amount of tailing. The late eluting compound has more tailing. Again there is an impurity present in the tailing of the second compound which does not have a good amount of response and because of that, its recovery can get compromised.
However, if the same sample is analyzed onto the alkyl amide stationary phase, you will find the peak tailing has been improvised, and the response for the impurity can be seen. So the recovery for this impurity can be better as compared to the last chromatogram.
To achieve symmetric peaks, it's essential to select a stationary phase with properties that match the compound in the sample. Polar compounds should be paired with polar stationary phases, while nonpolar compounds are better suited for nonpolar stationary phases. Mismatched combinations may lead to tailing, lower plate counts, suppressed peak heights, increased peak widths, and compromised quantification.
2. Mixed Retention:
Peak tailing can result from mixed retention, particularly in size exclusion chromatography, bonded phase chromatography, and liquid-liquid chromatography. Adding high molecular weight polar compounds or salts to the mobile phase can help in size exclusion chromatography. Normal-phase liquid chromatography may require a polar modifier, such as methanol or water, while reverse-phase liquid chromatography can benefit from a buffer system to suppress sample ionization. Using end-capped columns can also improve peak symmetry.
Example:
Bonded Phase Chromatography (BPC) in which the silica surface is only partly covered by the organic phase (e.g. C8). The resulting column could then allow retention by both the normal BPC mechanism with the organic coating and by adsorption onto the bare silica surface
3. Extra column Effect:
Peak tailing tends to be more pronounced for early eluting compounds due to extra column effects. To mitigate this, use lower inner diameter tubing to reduce the extra column volume. Employ detectors with lower volumes or consider using larger columns with more significant column volumes.
Example:
Different chromatograms demonstrate the impact of tubing's inner diameter on peak shape.
4. Poorly packed column:
Properly packed columns are essential for achieving symmetric peaks. Poorly packed columns can lead to peak tailing and distortion.
5. Unbuffered system:
In the absence of buffering, the degree of sample ionization varies with sample concentration. As sample concentration varies across the sample band - the degree of sample ionization also varies across the sample band.
This can result in either a fronting or tailing band, depending on conditions.
The severity of band asymmetry, in this case, is greatest for bands that are about half-ionized (i.e., pH ≈ pKa)
6. Sample-solvent effect:
The choice of sample diluent is crucial for obtaining symmetric peaks. If the sample solvent is stronger than the mobile phase, peak distortion or tailing can occur.
Example:
Caffeine and salicylamide analysis reveals the impact of sample diluents on peak shape.
Conclusion:
Understanding the causes and symptoms of peak tailing is essential for successful chromatography. By addressing the underlying phenomena and implementing appropriate corrections, chromatographers can achieve more symmetric peaks, improving quantification accuracy and overall analysis quality. If you have additional insights or experiences with peak tailing, please share them in the comments. Thank you for your attention.