Early eluting peak that merges with void?

[Ma08873]

I have an early eluting peak that merges with void in some Alliance HPLC system. I also ran it on Agilent 1100 systems. It did not coelute with void on any Agilent system.

This compound is very polar and I had to use a very strong diluent. The mobile phase starts with isocratic hold with mobile phase A (5% acetonitrile in water with 0.05% of TFA. The injection volume is 10. The column is a C18 column. The flow rate is 1 ml/min.

What is the reason that this peak elutes earlier in some Alliance systems? How to fix the problem?

Thanks for any ideas.

[danko]

It's probably due to system volume differences. The Alliance has a smaller dwell volume which means when using it, the gradient starts earlier than is the case with the Agilent 1100. The solution is as follows:
Alter the grdient method so that the hold up part of it is a minute (a half may be enough) longer f.x. 3 min. isocratic instead of 2, when using the Alliance system.
Best Regards

[juddc]

Danko's likely correct as to the difference between the two, however we can't possibly have any hope of telling you how to "fix it" unless you give more information. From what lfew details there are, I'd guess you have a reversed phase column and your molecule is basic. If you want your analyte to be retained to a greater extent, lower the % acetonirile at the beginning of your gradient or perhaps use methanol instead, lower the column temperature, use a more retentive column (C18 versus C8, higher carbon loading, etc...), use more TFA or a higher MW ion-pairing agent (heptafluorobutyric acid) or a different ion pairing agent (alkyl sulfonates - be careful with these, they can be tricky).

To really help you we need a few details!
- Column type (and dimensions)
- Mobile phase and gradient details
- Analyte (class may suffice) with pKa data if available
- Column temperature
- Injection volume
- What you've tried before

Cheers!
Chris

[Ma08873]

Thanks Chris and Dancho for your suggestions. Please see below for the details of the method conditions.

Column: phenomonex NX C18' 4.6 x 150 mm, 3 micro
Mobile phase A: water : acetonitrile : TFA 95:5:0.05
Mobile phase B: water : acetonitrile : TFA 5:95:0.05
Gradient table:
Time. MP A. MPB
0. 100 0
3. 100. 0
20. 45. 55
35 10. 90
40 10 90
41. 100. 0
46. 100. 0

Analyte is a strong base. PKa is greater than 10.
Column temp is room temperature
Injection volume is 10 microliter
I have tried polar embedded column and mix mode column.
For polar embedded column, the peak shape is bad maybe due to the strong diluent (MeOH:water 75:25) when running 100% aqueous mobile phase.
For mix mode column, I tried Primesep 100 and 200. Both gave poor resolution for my critical pair.

Thanks for your suggestions.

Yan

[kopkromfer]

I think you have a typical "solvent front breaking through" due to a mismatch of sample solvent and the initial composition of the mobile phase. I would suggest you to dissolve/dilute it in a solvent containing as much aqueous as possible. Since your analyte is very polar, you should have a good chance to do it with very little methanol. As a rule of thumb in reversed phase LC, whenever possible, always try to make samples using solvent that is weaker than mobile phase.

Good luck.

[XL]

You can consider using Acclaim Trinity P1 (http://www.dionex.com/en-us/webdocs/707 ... 239-02.pdf) if reversed-phase colulmns fail to provide you desirablke result. It is a strong cation-exchange, weak anion-exchange and reversed-phase trimode column, designed for charged pharmaceutica molecules and counteriion analysis. Its selectivity can be adjusted by changing mobile phase organic solvent, pH and buffer concentration, for optimal result. If you need to know more details, please read the document from above link and feel free to contact me at xiaodong.liu@dionex.com.