Dear Sir,

The chromatographic problem I wish to submit to your attention concerns an RP – HPLC analysis of a standard mixture of herbicides at a 0.1 mg/L concentration level. The separation of the components has been accomplished with the same apparatus on two different C-18 reverse phase HPLC columns, which characteristics are:

- Purospher RP – 18e; l = 125 mm; i.d. = 3 mm; dp = 5 µm + guard column C18; l = 20 mm; i.d. = 4.6 mm.

- Zorbax C 18 Extend; l = 150 mm; i.d. = 4.6 mm; dp = 3.5 µm + guard column C18; l = 12.5 mm; i.d. = 4.6 mm.

The analytical condition, as reported in table 1, are the same for both columns apart from the flow rate.

Table 1

Mobile phases: A: ammonium formate 5 mM
B: acetonitrile
Gradient: B% 20 40 60 90 90
0 5 18 20 26

A flow rate of 0.6 mL/min was used for Purosphere column, while a 0.9 mL/min was chosen for the Zorbax column.

In optimising sensitivity of the method two injections of 10 µL and 20 µL were performed. With surprise an evident heading was observed after injecting 20 µL of standard in the Zorbax column; such a pattern was not encountered with the Purosphere column. I have non explanation for such a behaviour. For quantitative evaluation the symmetry factors for atrazine that elute in the middle portion of the chromatogram are reported in table 2


Asimetry factor 10 µL 20 µL

Purosphere 0.86 0.89
Zorbax 0.85 1.26

See the discussion "pH of sample-split peaks". It could be that your sample solvent is too strong and that the Zorbax has a lower pre-bed volume (simply: not as much mixing takes place so that some sample transfers too fast into the column).