Chromatography Forum

Extraction Buffer: 70% ethanol (EtOH), water, sodium acetate trihydrate (NaOAc), and beta-mercaptoethanol (BME)
Solvent A: water +0.1% trifluoracetic acid (TFA)
Solvent B: acetonitrile + 0.1% TFA
C18 column

I am using an HPLC to determine maize zein compositions (alcohol-soluble proteins). Unfortunately the only peaks I am getting are the injection peaks. Everything after that noise is flat, flat, flat. I've remade my solvents and buffer, changed columns, heated the column to a higher temperature, used various maize samples, and adjusted flow and percentage of solvent in the gradient, but nothing has produced peaks after the injection peaks.

Can anyone suggest other steps to try or another standard I could use that would give similar peaks as a zein?

Any ideas would be greatly appreciated.

Would you please describe the gradient? And please state the composition of the samples you're injecting. Finally, did you inject a blanck solution (i.e. the extraction solvent) following the sample injection? If not, try that and follow with an injection of pure water. Then tell us what you see.
Btw, what is the detection wavelenght?

Best Regards

Gradient
Time %A %B
0 100 0
1 65 35
2 65 35
34 25 75
35 25 75
36 100 0

The samples are whole corn kernels, ground into a fine flour. They are mixed with the exactraction buffer, shaken, centrifuged, and filtered. The detection wavelength is 200 nm.

I see many problems with the protocol you're using, but the main one and probably the one that's causing the biggest trouble here is the wavelenght.
TFA absorbs hugely UV light at 200 nm. So, you'll need to change the detection wavelenght to 215 nm at which the TFA's absorbance is acceptable and any protein highly detectable.
Another problem could be the extaction solvent which is also a strong solvent/eluent compared to the initial mobile phase composition which is 100 % aquaeous. If you inject a large volume it might be causing rapid elution of the analyte/s.
Finally, if you inject samles right after "time 36 min" the column is not equilibrated at that time which might be causing rapid elution too.

Hope the above is helpful for a start.

Best Regards

In addition to danko's suggestions, you might try starting your gradient a 0%B and running up to 75% at 1%/min (i.e., 75-minutes). That will confirm or eliminate the possibility that your proteins are all eluting as one peak when you make that step change from 0 to 35% B.