Chromatography Forum

Dear Sir,

I am new in HPLC and I am using C4 (25cm, 4.6 ID, 300A) at 25C for seperation of saponins in Phytochemicals. the mobile phase is A (0.05% TFA in Water), B (0.05% TFA in ACN). The gradient flow is A:B (90 :10) to A:B (55 :45) in 30 mints at flow rate 0.5ml/mint. Sample solvents is the mobile phase at start. In blank, I have seen negative peak and baseline drift.
I have here some questions.
1. Is the problem unaviodable (beacuse I have run different solvent conditions suggested)
2. How to shift the solvent front to lower time scale?
3. According to some recommendations, some type of Basline drift does not effect the results. Is my chromatograph Basline is acceptable?

Your answer will be helpful for me. Please see in attachments the chromatograph for blank.
http://wikisend.com/download/193362/Bla ... pdf%5B/URL

This msy answer some of your questions:
http://chromforum.org/viewtopic.php?f=31&t=19085

Hi Tom, what an excellent and informative video, thank you.

"Is my chromatograph Basline is acceptable?"

This depends on your detector. Your detector should have baseline drift specifications that it should fall into. Of course, that specification is only applicable to a very particular solvent mixture (usually isocratic), and doesn't really mean anything when dealing with gradient drift. But, make sure your detector at least matches those specifications within the listed parameters.

A few things can be done about gradient drift due to UV absorbance/RI mismatch...

1. You can simply perform a baseline subtraction to artificially correct your baseline.
2. You can add a buffer to your aqueous phase to increase its absorbance and help it "match" the UV absorbance of your other solvent. This has worked very well for me in the past. Anyways, using buffers for HPLC is always highly recommended.
3. If possible, you can also try to set your detector to collect data at a higher wavelength, say 280nm instead of 254nm. At higher wavelengths, the UV mismatch will be become much less apparent, usually giving less drift.
4. Similar to option #3, many detectors use what is called a "reference wavelength" during data collection. The reference wavelength is usually set to a much higher wavelength than the wavelength used for detection. For instance, if using a 254nm wavelength for detection, the reference wavelength would be set to 360nm. During data collection, the computer will automatically subtract the two wavelengths, giving a very smooth baseline.

Option 4 is usually seen in Agilent systems. Most chromatographers don't like using the reference wavelength, as you have no idea if you're subtracting out important data. You can also do it by collecting a baseline at 360nm and subtracting it from your run, that way you'll have a copy of your original data and can view any discrepancies.

Thank you for reply.
I worked when I reduced the TFA concentration in ACN and increase the flow rate to 1 ml/min.
I am working to develop a HPLC method for quantification of individual saponins in a plant extract (it contains roughly more than 60 saponins types).
This time, I have seperated most of the peaks with resolution (range from 0.87 to 1.29). The recomended value of Rs should be greater than 1.
Now my HPLC condiations are,
I am using C4 (25cm, 4.6 ID, 300A) at 25C, the mobile phase is A (0.05% TFA in Water), B (0.0425% TFA in ACN). The gradient flow is A:B (90 :10) to A:B (55 :45) in 30 mints at flow rate 1 ml/mint. The sample solvents in 0.05 M Phosphate buffer solution (pH 4.5).
1. Any suggestion from you in order to enhance the resolution of peaks ?
2. Is there the same recomendations for phytochemical analysis for quantification as for pharmaceutical analysis?
Regards

It is unlikely that any reversed-phase column will separate all of your saponins. They typically have sugar residues on both ends of the molecule. Sometimes they may differ merely in the position of linkage of a sugar (e.g., 1,4- vs. 1,6-). Reversed-phase isn't sensitive to such differences. HILIC is. I suggest that you collect a few fractions from a reversed-phase column and then rerun them on a HILIC column. Using this approach, I've obtained nice separations of saponins and ginsenosides that coeluted in reversed-phase chromatography.

Thank you for your suggestions .
2. Is there the same recomendations for system suitability in phytochemical analysis/quantification as for pharmaceutical analysis??