HPLC Peak Tailing

[amithg3]

Hi,

The problem is related to a new method under development. The method is to separate and quantify a fatty acid amide in a sample. the sample contains 0.1% of this active and almost 79% ethanol. I am using a Luna c8 column 250mm*4.6mmID.
I am using solution A in mobile phase as 0.05% acetic acid in DI water and Solution B as 0.05% in ACN. I have a gradient for the mobile phase and the peak for the analyte elutes at around 9.3 minutes when the composition of the mobile phase is 5% solution A and 95% solution B...I am observing excessive peak tailing for this analyte peak in sample injections but not in standard injections. Can anyone help me how to optimise and avoid peak tailing.Thanks...

[t_morris]

What is the diluent and strength of your sample and standard?

I'm guessing that your sample might require a dilution into mobile phase to prevent poor peak shape, whereas your standard is composed of a different diluent...if that is the issue. I think that because your standard isn't poorly shaped, but sample is...

What is your tailing factor?

[ABAdam]

Are the peak areas comparable between standard and sample?

[M Farooq]

When the sample solvent is very different (in concentration/ or in composition) from the mobile phase, it is common to see distorted peak shapes. You probably have the same issue because the sample is 79% ethanol. Are the standards present in ethanol as well?
Also ensure that the sample is not overloading the column given that standards are behaving perfectly.

[amithg3]

The standards are made in 79% ethanol. The sample as is 79% ethanol with the active which is further diluted to 1 gm in 10 ml of 79% ethanol. Tried reducing the injection volume but didn't solve the tailing. The tailing is around 4. The area when compared to the standard is same. But really poor peak shape because of this excessive tailing.

[tom jupille]

As has been pointed out, using a diluent which is stronger than the initial mobile phase is always risky. If this were a case of the strong diluent effect, however, I would expect to see the same problem with both samples and standards. Soooo, what's different? You're using the same diluent. Are you injecting the same volume? What else is in the sample?

[amithg3]

I am using the same injection volume for the standards and samples.. the sample also contains some quaternary ammonium compounds, making most of the actives in the sample basic in nature. I think that might be the reason for the peak tailing. should i try using some buffer in the mobile phase. But afraid of precipitation because my gradient has a 95% ACN for almost 5 minutes. What else can i do?..

[tom jupille]

To steal from Andy Alpert (http://www.nestgrp.com/protocols/polylc/hilic_op.shtml):

Solubility of salts can be a problem with mostly organic mobile phases, but sodium perchlorate works well and is transparent at low wavelengths (Fisher sells an HPLC grade). Buffer salts with reasonable solubility in 80% acetonitrile include triethylamine phosphate (TEAP) and sodium methylphosphonate (from methylphosphonic acid). Isocratic retention is typically several times greater with TEA salts than with the corresponding sodium or potassium salt. With 80% acetonitrile, concentrations of 75 mM (pH 5.0) or 100 mM (pH 2.8 ) TEAP are attainable.

For reversed-phase you probably don't need much above 25 mM or so (but "your mileage may vary").

[shadow]

what is the buffering range for sodium perchlorate and TEAP? Is sodium perchlorate suitable for LCMS? Thanks.

[tom jupille]

I

s sodium perchlorate suitable for LCMS?

No. Neither is TEAP.
Anything going into your MS must be volatile. Perhaps just formic acid would suffice.