Fronting, cause and remedy?

[Mattias]

Hi!

I have a peak that has a quite large fronting problem. The peak has a asymmetry factor of 0.6 and looks more or less triangular.

I inject 15 µl of sample on a 4.6 mm i.d. column, which I don't believe is an overload of the column (The peak height is approx 0.3 AU). The sample is dissolved in mobile phase (start composition of the gradient).

Other settings
Temp 60 °C
Flow 1.2 ml/min
Column: C18 from a large company...

I think the conditions looks more or less perfect for good chromatography, but still there is something wrong?

[Daren]

What is the concentration of your analyte? overloading can be either by volume or by absolute mass of the analyte

[Mattias]

It is 0.5 mg/ml of a peptide (10 amino acids).

[Rafael Chust]

Dear Daren:

Usually fronting is caused by column overloading.

Dilute your sample by 10 times and check if there is still fronting.

Other problems might be related to column (too old) or temperature (too cold).

I hope this helps.

[JMB]

Mattias,

1. Repeat the chromatography on a C18 from different manufacturers (better endcapping; higher phase loading ??)

2. Depending upon the structure of your analyte, and the mobile phase, you may have some chemical interconversions occurring.

K. Brogle et al. [J. Pharm. Biomed. Anal. 19 (1999) 669-678] describe severe peak fronting in the H2O/MeOH chromatography of oxycodone at 30 C; found that it was due to gem-diol and hemiketal formation with water and methanol, respectively, at the C-6 ketone. Resolved to two peaks at 0 C, or a single, symmetrical peak at 60 C.

Since you are running at 60 C, suggest to lower to 0 C to check for resolution into two (or more) peaks.

JMB

[Daren]

Mattias,

I would try diluting the sample and seeing how that helps, 7.5 ug on column tyipically wouldn't cause fronting but won't hurt to double check.

From a chromatography standpoint, what is your mobile phase? It may be a pKa thing . I would make sure you have a very low pH, use TFA 0.1%. If that doesn't work or you are already there, then go to a new column. I like the waters hybrid columns (Xterra, Xbridge) for peak symmetry issues myself.

[tom jupille]

Only about seven things can cause fronting. Some of them have already been discussed, but just for the sake of completeness:

• - Partially plugged inlet frit and/or column headspace. Tailing is more common, but depending on the flow dynamics at the inlet, the peak shape problem can also show up as fronting or split peaks. Diagnostic is that all peaks in the chromatogram show a similar problem. If only one peak fronts, it's more likely to be chemistry-related.
  
  - An unresolved peak. Always a possibility. Particularly troublesome for peptides because the chromophores are very similar (so spectral information is not very useful). You could try coupling two columns together (to double the plate count). That should improve resolution by 40%; the presence of a second peak should then become apparent.
  
  - Slow equilibration between two forms of the analyte. As suggested by JMB. This has actually been discussed quite a bit here on the Forum in the past few months. This is quite common with proteins where multiple conformers can be in equilibrium. A decapeptide is fairly short for this, but depending on the specifics, it is possible. As JMB suggested, at lower temperature, you should either resolve into two peaks (by slowing the interconversion) or get a single peak (if the equilibrium shifts so that one conformer predominates).
  
  - Too strong a diluent. Peak shape can be anything. You've already eliminated this possibility.
  
  - Inadequate buffering. If you don't have enough buffer capacity, then the peptide may itself act as a buffer. The result can be different pH values at the beginning, top, and end of the peak. If the top of the peak is retarded as a consequence, you will get fronting (if the top accelerates, you will get tailing).
  
  - Analyte aggregation. This is essentially an overloading issue. If the analyte has a greater affinity for other analyte molecules than it does for the stationary phase, then an overload will make the top of the peak more retained, resulting in a front. This is relatively rare, but it does happen.
  
  - Limited solubility. This is also essentially an overload effect. We usually think of overloading as affecting the stationary phase (i.e., "no more room on the surface") which makes the top of the peak come out early, and results in a tail. If the analyte has limited solubility in the mobile phase, however, things can go the other way, with the top of the peak coming out later.

[HW Mueller]

At 0.5mg/mL and 15µL injection my bet would be on overload.