Chromatography Forum

Hello, I´m Juan Manuel.
At this moment, I´m working on a new HPLC method for analysis of Silybum Marianum (flavonolignands). I used two mobile phases, 1ml/min, column Kromasil C-18 250x4,5, 20 microlitres.

1- AcN/ Water with buffer ph 2,7 (62%/38%)
Sample solvent with methanol.

2- MeOH/ Water with buffer ph 2,7 (50%/50%)
Sample solvent with methanol and water.

With the first mobile phase I only had one peak, my calibration was good.
But with other mobile phase and eluent for sample, I had two peaks because I have best resolution.
My problem is Why is area peak for a standard is different, is normally that for 50 ppm area peak is 1000 mAU and for 25 ppm area peak is 100 mAU or 50 mAU or 20 mAU. I haven´t reproducibility.

I think that problem is The water in sample, and sample preparation, Why?
Thank you, very much.

Have you checked your autosampler precision?

Have you checked your autosampler precision?

When I solvent my standar with methanol, I had a calibration of r=0,9995, but with phase movil with methanol and water buffer ph 2,8 I must solvent with methanol-water my signal is very different of 50 ppm (5 ml Don 100 ppm with only methanol + rest with water- total 10 ml) signal area 1000, with 25 ppm (5 ml of 50 ppm, above + rest with water- total 10ml) no signal.

Thanks

Hi

If I understand you correctly then you are saying that when you make up your standard solutions with methanol, you calibration plot is fine (as evidence by your correlation coefficient). However, when you make up your standards with a mixture of methanol and water, the calibration plot fails.

My response;

1) Why can you not continue to use methanol as a diluent for your samples? Your diluent does not (usually) have to be exactly the same as your mobile phase (sure you may get a bigger solvent front if the solutions are very different but this should not matter if your peaks are well resolved from the solvent front?).

2) With the MeOH/H20 diluent, your problems may be due to the way you are making up your samples. If you add 5mL MeOH to 5mL water, you don't get 10mL final volume (it 'shrinks', try it in a measuring cylinder and you will see). If you are adding 5mL MeOH to a flask and then making it up to volume with water, your volumes may not be accurate. I suggest that you make up a volume of 50% (v/w) MeOH:H20, mix it throughly and then use this to make up your samples.

Good luck

Ann

Hi

If I understand you correctly then you are saying that when you make up your standard solutions with methanol, you calibration plot is fine (as evidence by your correlation coefficient). However, when you make up your standards with a mixture of methanol and water, the calibration plot fails.

My response;

1) Why can you not continue to use methanol as a diluent for your samples? Your diluent does not (usually) have to be exactly the same as your mobile phase (sure you may get a bigger solvent front if the solutions are very different but this should not matter if your peaks are well resolved from the solvent front?).

2) With the MeOH/H20 diluent, your problems may be due to the way you are making up your samples. If you add 5mL MeOH to 5mL water, you don't get 10mL final volume (it 'shrinks', try it in a measuring cylinder and you will see). If you are adding 5mL MeOH to a flask and then making it up to volume with water, your volumes may not be accurate. I suggest that you make up a volume of 50% (v/w) MeOH:H20, mix it throughly and then use this to make up your samples.

Good luck

Ann

Hello Ann, Now I find my problem. My standard is a little solubility in water, so I don´t have signal with my detector. But I can´t solvent with only methanol, now I solvent with 25 % more or less of water.

Thank you very much

I'm glad you got it sorted

Best wishes

Ann

were the two peaks produced in the MeOH/H2O mobile phase (conditions #2) two real components?

according to Google, Silybum Marianum is a plant, the active is silymarin - this is made up of 3 flavonoids.. aren't you therefore separating and expecting 3 peaks?

Anyway, I don't understand how come your signal disappears at 25 ppm unless you're already down close to the LOD. I don't see anything inheriently wrong with making a dilution of 5 ml of methanolic sample to 10 ml total with water.. that reduces the conc to 1/2, regardless of the solvent imo.

It all sounds like a case of a sample diluent being too strong compared to the mobile phase conditions. When you weakened the diluent, it removed the "problem"..

were the two peaks produced in the MeOH/H2O mobile phase (conditions #2) two real components?

according to Google, Silybum Marianum is a plant, the active is silymarin - this is made up of 3 flavonoids.. aren't you therefore separating and expecting 3 peaks?

Anyway, I don't understand how come your signal disappears at 25 ppm unless you're already down close to the LOD. I don't see anything inheriently wrong with making a dilution of 5 ml of methanolic sample to 10 ml total with water.. that reduces the conc to 1/2, regardless of the solvent imo.

It all sounds like a case of a sample diluent being too strong compared to the mobile phase conditions. When you weakened the diluent, it removed the "problem"..

Hello JA,
I only analyze the silybin, that´s my standard. The silybin is a diastereoisomer. There´s method that don´t separate the diastereoisomer, other yes. In my case I want separate them. With option I have two problems:
a) I solvent my standard with methanol 100%, I have split (double) or the distortion peaks. Four peaks or two very distortion.
b) So I solvent with some water. But there´s other problem the silybin isn´t solubility in water 100%.

SOLUTION: My injection solvent is 75% methanol and 25 water more or less.

thanks