Chromatography Forum

hello all,
I am getting a peak split and difference retention time with same chromatographic conditions, (column, equipment) and same sample preparation but only difference vendor of sodium lauryl sulfate. What could be the reason?
Column: Lichrospher 100 RP-18; 5µm; 250*4,6 mm;
Detection (UV): 245 nm
Inj.volume: 10µl
Temp:25°C
Eluent: Buffer pH2,5 + ACN (60+40); Flow: 1,0ml/min.
Buffer pH2,5: 2,8g Natriumlaurylsulphate/1000 ml H2O + 11,0ml (H3PO4+Triethylamine 1:1) and the pH is adjusted to 2,5.
Figure 1:
http://tinypic.com/r/ws1qfb/5
sodium dodecylsulfate (Fluka); assay: ≥ 99,0 %. Rt of Terazosin at 13,1 min. and split.
Fluka: ƛ: 260nm: Amax.: 0,02
Figure 2:
http://tinypic.com/r/28wgdbo/5
sodium lauryl sulphate (Roth); assay: 99%. Rt of Terazosin at 17,9 min. and no split.
Roth: ƛ: 260nm: Amax.: 1,5


Thank.

Not very symmetrical peaks in both figures. In figure 1 I would say at the RT more than 1 compound is eluted. Also on figure 2 I would say it is another compound/form under your target compound peak.
Both SDS qualities are nearly the same, with only a difference in absorption.
It looks like there is a difference in equilibration on the column. How do you adjust the pH and how do you messure/control pH.
Did you use the same Lichrosphere column on different systems or different columns from different lots. All brand new columns? Or did you used these columns for another application before?

Thank you for the fast response.
How do you adjust the pH and how do you messure/control pH. The pH is adjusted exactly to 2,5 with H3PO4 85%
Did you use the same Lichrosphere column on different systems or different columns from different lots.
I use the same column on different lots of SDS
All brand new columns? Yes the column is brand new.
Or did you used these columns for another application before?
No, the column was used only for this application.

The spektrum of DAD shows the same apsorption of the first and second peak. I think that is only once compound.
http://tinypic.com/r/34sp2qp/5
http://tinypic.com/r/4vo6xy/5

Just to be sure: You can reproduce the "good" chromatogram when you use the "good" SDS?
In the pictures the axis values are not really readable - but the peak look veeeeery big. Are you overloading the column (and the detector)?
How does the peak (with the "bad" SDS) look like if you reduce the injection volume?

Just to be sure: You can reproduce the "good" chromatogram when you use the "good" SDS?
Yes
In the pictures the axis values are not really readable - but the peak look veeeeery big.
the axis value shows the run time (60min). The main peak eluted at 13 min. (fig.1) and 18 min (fig.2)
Are you overloading the column (and the detector)?
You mean fronting peak? It would seem with the bad SDS.
How does the peak (with the "bad" SDS) look like if you reduce the injection volume?
They run with the same equipment condition (all runs injected by 10µl).

As Gerhard already pointed out, even the "good" peak seems to show quite a bit of fronting. With this method and the original SDS, you might be quite at the edge of a working method. By using different SDS, whatever the difference is, you're pushing it over the edge and get those ugly peaks.

Just a few thoughts:
- fronting peaks and split peaks (as escalation of fronting) can be the result of mobile phase/solvent mismatch. What's the solvent in your case?
- IF mobile phase/solvent mismatch is the reason, usually peak shapes should get better if you inject less. What happens if you just lower the injection volume, keeping everything else the same.
- As already said, those peaks look very big. You're sure that you're not overloading the column?
->Lower the injection volume, what happens?
- Are those two grades of SDS really comparable? There are technical grades available that might contain considerable levels of C13-C16.