In a previous post entitled "TroubleshootingLC / HPLC Systems – Tailing Peaks" peak tailing due to bad columns and sample overload was explained and methods were proposed to fix it.
Peaks with strange shapes represent one of the most common problems that can arise in the LC/HPLC laboratory. It should be first explained why tailing is bad :
- Peaks with tailing can be hard to integrate
- The precision and the reliability of assay methods involving distorted – peaks with tailing – is often poor when compared to good chromatography
- Resolution is low when peaks show tailing
Another cause of peak tailing is when wrong solvent is used to dissolve the sample.
Wrong Solvent for Sample
Peak tailing is observed when the volume and the kind of solvent used to dissolve the sample is not appropriate. Ideally a small volume of sample (dissolved in the mobile phase) should be injected. However, when a relatively large volume of a solvent stronger than the mobile phase is injected with the dissolved sample there is a severe effect in the chromatogram. This is shown in Fig. 1a where 30 μL of a two-component sample dissolved in acetonitrile is injected in 18% acetonitrile/water mobile phase. Two tailing and distorted peaks appear. When the same sample was dissolved in 18% acetonitrile/water two normal peaks appear (Fig. 1b).
 |
| Fig. 1: a) 30 ml injection of sample in acetonitrile. Reversed-phase separation with 18% acetonitrile/water as mobile phase b) same as (a) but the sample is dissolved in the mobile phase and 2 normal peaks appear. |
From the above discussion it appears that the following procedure should be used for choosing the solvent for dissolving the sample:
- The best option is to dissolve the sample in the mobile phase and inject 10-50 μL
- Alternatively, a larger volume of weaker solvent can be injected i.e. 100-500 μL of sample dissolved in water for the case of reversed-phase LC. A main disadvantage is that a larger baseline upset at the beginning of the chromatogram appears.
- Larger volumes (100-500 μL) of sample dissolved in the mobile phase can also be used but the resolution may suffer especially for the early-eluting peaks
- In case that the above do not work and as a last resort 10-25 μL of a stronger solvent than the mobile phase can be used.
Extra-Column Effects
For well-designed LC systems, the major contribution to band tailing due to extra-column volume is usually the detector flowcell. Some detectors allow the substitution of smaller-volume flowcells, which reduces band tailing, but also reduces detector sensitivity. In some cases, the connecting tubing may be too long or its internal diameter is too large. It may be replaced by 0.007 inches i.d. tubing to fix the problem.
Band Fronting
Band fronting in LC (Fig. 2) may be caused by column temperature problems particularly in ion-pair chromatography at ambient temperature. In cases like this repeating the separation at higher temperature (~45 C) usually eliminates the problem. Temperatures of 40-50 C are generally favored for ion-pair chromatography, because narrower bands and better separation result.
 |
| Fig. 2: Peak fronting in liquid chromatography (LC) |
Another source of fronting bands in ion-pair chromatography is the use of sample-solvent other than the mobile phase. In general, in ion-pair chromatography the sample -25 to 30 μl - should only be injected as a solution in the mobile phase to avoid fronting bands and other problems.
Strong Retention Sites
Separations by normal-phase or ion-exchange chromatography involve the binding of sample molecules to specific sites on the surface of the column packing. For example, the sulfonate (-SO3-) groups on a cation exchanger or the silanol (Si-OH) groups on silica. Often sites like these are not all equivalent. Some sites are favorably situated for strong interaction with sample molecules. The strongest retention sites will be preferred by sample molecules, and these sites will be used first. Because these strong sites often are present in low concentration, they are quickly occupied by the sample, so that only weaker sites are then available.
Another feature of strong retention sites is that they generally attract strongly retained sample molecules. This means that premature column overloading occurs mainly for later-eluting bands in the chromatogram. Therefore, it is the last bands in the chromatogram that show band-tailing as a result.
This band-tailing in ion-exchange or normal-phase LC as a result of strong retention sites can sometimes be reduced by using less sample. A more effective approach is to increase the strength of the mobile phase.
References
L.R. Snyder. J.J. Kirkland, “Introduction to Modern Liquid Chromatography”, 2nd edition, Wiley, 1979
A. Weston and P. Brown, “HPLC and CE Principles and Practice”, Academic Press, 1997
J.W. Dolan, L.R. Snyder, “Troubleshooting LC Systems”, Humana Press, 1989
Hiç yorum yok:
Yorum Gönder