At the current time, you have no method and no sample retention so it is not possible to integrate or process any results. We can provide suggestions which may allow you to start the method development process, but first we need your help to provide us with the basic analysis conditions you are using. Please describe the exact HPLC column, its name, brand and type as well as its dimensions (L x ID, particle size). Please tell us the sample concentration injected (mass/volume). Please tell us which of the many saponins you are trying to retain and resolve and what they are in (if you do not know which ones yet, please make sure that you can actually "SEE" them at 230 nm because many saponins do NOT absorb at all at such high wavelengths. Many are detected using UV at 205 nm with poor sensitivity or with ELSD/CAD type detectors). *Sample prep is critical with these types of samples (very dirty). Have you performed a clean-up and filtering (0.45u) step before injecting into the HPLC?

• Please turn OFF the Reference Wavelength feature on your HPLC DAD module (!) It should always be set to 'OFF' and please share this info with other HPLC users (see link).

Many purified saponins can be retained and resolved on a good quality C18 column with a linear gradient of ACN and Water. After sample prep and filtering are complete, start with 10% ACN and ramp to 90% ACN with a proper hold time to make sure everything has been flushed off the column. BTW: MeOH is better for this, BUT ACN is used because the detection of saponins is very poor and we need a UV clear solvent for use at very low UV wavelengths to see anything at all. MeOH would absorb too much at 205 nm to be useful, but HPLC quality ACN absorbs far less so works much better (if you use an ELSD or CAD system instead of UV, then you can use MeOH w/o any problem and get better results).

Most importantly of all, please get help from your teacher/professor with this analysis as you need someone local with HPLC experience to guide you. The web is not always the best resource for students who are just starting out. It is great for providing papers and articles as examples to follow, but not very good for providing hands-on practical advice which is what you need at this stage. Your school is in the best position to help you in that regard.

http://hplctips.blogspot.com/2011/03/reference-wavelengths-as-used-in-hplc.html

http://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html

http://hplctips.blogspot.com/2015/06/k-prime-also-known-as-capacity-factor.html

What column are you using and which compounds are you analyzing?

The separation depends on your column.  I assume you are you a 250 cm C18 HPLC column.  For gradient work (which you will need a complex mixture) use a 5 cm C18 column and a composition of mobile phase ;A  95% pH 3 phosphate buffer/5% ACN AND mobile phase B; 5% phosphate buffer/95% ACN.  For a flow of 1.0 mL/min. the 'magic' occurs between 5-10 minutes (you are eluting too early!).  Adjust the ramp as required to 'tease apart' the peaks.

A long 25 cm HPLC column is capable of resolving maybe 5 peaks but cannot a complex mixture containing many molecules.

Hi Ravinder,

I agree with Bruce, you have nearly no separation accuring since all components elute right after the dead volume of the column - either your gradient is too steep (too much AcN) or the column material is not suitable. If you use C18 column (I guess), the compounds are too hydrophilic to be separated since they do not interact much with the hydrophobic stationary phase (and therefore elute after the dead volume passed the column). Try C8 or even C5 column which suit better to your compounds.

Best regards,

Marcus

At the current time, you have no method and no sample retention so it is not possible to integrate or process any results. We can provide suggestions which may allow you to start the method development process, but first we need your help to provide us with the basic analysis conditions you are using. Please describe the exact HPLC column, its name, brand and type as well as its dimensions (L x ID, particle size). Please tell us the sample concentration injected (mass/volume). Please tell us which of the many saponins you are trying to retain and resolve and what they are in (if you do not know which ones yet, please make sure that you can actually "SEE" them at 230 nm because many saponins do NOT absorb at all at such high wavelengths. Many are detected using UV at 205 nm with poor sensitivity or with ELSD/CAD type detectors). *Sample prep is critical with these types of samples (very dirty). Have you performed a clean-up and filtering (0.45u) step before injecting into the HPLC?

• Please turn OFF the Reference Wavelength feature on your HPLC DAD module (!) It should always be set to 'OFF' and please share this info with other HPLC users (see link).

Many purified saponins can be retained and resolved on a good quality C18 column with a linear gradient of ACN and Water. After sample prep and filtering are complete, start with 10% ACN and ramp to 90% ACN with a proper hold time to make sure everything has been flushed off the column. BTW: MeOH is better for this, BUT ACN is used because the detection of saponins is very poor and we need a UV clear solvent for use at very low UV wavelengths to see anything at all. MeOH would absorb too much at 205 nm to be useful, but HPLC quality ACN absorbs far less so works much better (if you use an ELSD or CAD system instead of UV, then you can use MeOH w/o any problem and get better results).

Most importantly of all, please get help from your teacher/professor with this analysis as you need someone local with HPLC experience to guide you. The web is not always the best resource for students who are just starting out. It is great for providing papers and articles as examples to follow, but not very good for providing hands-on practical advice which is what you need at this stage. Your school is in the best position to help you in that regard.

http://hplctips.blogspot.com/2011/03/reference-wavelengths-as-used-in-hplc.html

http://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html

http://hplctips.blogspot.com/2015/06/k-prime-also-known-as-capacity-factor.html

To echo what Bill is so nicely saying, ResearchGate is a resource for solving problems that cannot be easily resolved elsewhere. It is most definitely NOT a training forum, which you are clearly in need of. I am sure you probably have training resources available to you; you should take advantage of them.

That's right, I fully agree with Mark - as usual Bill always provides the best and most detailed answers on HPLC questions. There is nothing to add.

Thank you all for your early responses.

Column: Zorbax Eclipse Plus C-18, analytical, 4.6x250mm, 5-micron, make- Agilent. 

Injected filtered sample conc is 500 ppm.

I do not recommend Zorbax at all, instead I have previously conveniently used Nucleosil 10C18 (Macherey & Nagel, Düren, Germany) and/or Develosil ODS (Nomura, Seto, Aichi, Japan) (please see file; PTH Roma RP-HPLC).

In the article of chromatography, the choice of the column is essential and we describe the column name used in the research (please see articles accepted in the Journal of Chromatography).   I have been having difficulty in handling with Zorbax during my young days employed to Shimadzu Co. (Kyoto, Japan).   Shimadzu Co. has introduced an HPLC technology from the E. I. du Pont de Nemours and Company (Wilmington, DE, USA), and I have had a good chance to compare Zorbax of du Pont to other several columns.

I may be an odd Biochemist who has an HPLC technology.  I do not threaten at all, but I am only saying about my odd experience.  

Ravinder, the fact that you are using a 4.6 x 250 mm column confirms all earlier comments. You have no retention at all and no method (chromatogram shows samples coming out in the void volume). Please get some local help in learning about HPLC and you will soon have this sorted out.

Regarding Kou's odd comment above. Please ignore it. Column brand choice not only has nothing to do with your question, but also makes little difference at all when you are starting out. Zorbax brand columns are excellent in general (I recommend them all) as are the older Nucleosil brand too (I also recommend them as well). Repeated posting of non-relevant information attached to self promotional content in members threads like this one is not something that we desire to see on this or any other group.

Bill is right, try some less hydrophobic columns like C8 or C5, your compounds will interact much stronger with the stationary phase then. A binary pump would be also an excellent option for your since you can run a solvent gradient.

Thanks, Bill Letter for you valuable suggestions

I agree with Dr. Bill.

Mr Kumar,

Via UPLC. 

I'm interested in this one, because I've got a similar problem. We use saponin (usually that derived from Quillaia Saponaria, though we have also looked at saponins from Gypsophila spp and other sources) and we know that the material we buy contains a fair quantity of lactose - the powder X-ray diffraction pattern clearly identifies this, while the saponin is amorphous. QC testing is rather limited - the most useful is probably TLC, to check that we've got (approximately?) the correct saponin. This works reasonably well. Our material always has a UV absorption at 280 - 281 nm - I have to wonder if this is another impurity! Other, much stronger, UV absorption is at lower wavelengths - I've never had the time or need to investigate this, and I haven't got a believable peak wavelength because the absorbance is too high at the concentration we use to look at the 280 nm area. The business need for doing anything about this is slight, so not much will happen... but I would like to know at least if the lactose concentration is reasonably consistent batch-to-batch.

Hi David,

regarding the wavelength absorption - if using a DAD for HPLC analysis, the detector can be set up to measure a spectrum (e.g. 210nm - 600 nm) and not only single wavelengths. With that, you obtain an UV/VIS spectrum of the eluting compound (plus absorption of the current solvent mixture, e.g. AcN/H2O) right during HPLC analysis.

Marcus

At first you should see the structures of compounds under study, whether the given compound has any Ionizable groups, if it contains Ionizable groups then you can use buffer to separate peaks. You can change the column to get high resolution peaks. In this case you can use C 18 column.

You can use longer column packed with smaller particles.

Hi Ravinder, maybe this would be helpful to you: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3263049/

Greetings Markus

Dear Ravinder,

The best to get your result try followings

1) Try phosphate buffer 10 to 50 mM with various pH

2) Try 0.1% TFA or 0.1% Formic acid in water instead of plain water

3) Try Methanol instead of ACN

Gradiebnt proportion optimaization is important for better separation. The test samples should elute within runn time with out interfering with next runn so make the mobile phase more organic by changing method developent

Melese: Please refer to the original post for context. *Adding MORE organic phase will NOT improve a RP method where the sample elutes at the void volume.

Hi,

The following link may be of interest. Best of luck.

Article High-performance liquid chromatography analysis of plant sap...

With best regards,

JPM