Chromatography Forum

hi,
I am working to seperation of sapoonins using methods developed previosly. All the well seperations into four major components Q7, Q18, Q21 and Q13 ( this include 90% of toatal saponins) in my sample. My sample is not purified and the same I will use for my biological studies.
I have task to seperate these four fractions using HPLC C18 or C8 column if possible. These compoents are well seperated using C4 column. I have run the methods using C18 column by changing different parameters like mobile phases (water and ACN) but not tried other solvents. Saponins are highly water soluble so according to Literature, they shows difficulties in sererations .

Please any suggestions from forum side, I shall be really thankful.

Regards

HILIC does a very good job with saponins, separating variants that coelute in reversed-phase. For an early example with QS-21 isomers, see: S. Soltysik et al., Ann. New York Acad. Sci. 690 (1993) 392. Just be sure to use an appropriate column.

Since you can consider these as amino sugars you can use cation-exchange mixed-mode. Even if you have a mixture it is hard to find compounds which have exactly the samehydrophobic and ionic properties:
http://www.sielc.com/Technology_2D_Properties.html

here are examples of amino sugars:
http://www.sielc.com/Compound-Amino-Sugar.html
http://www.sielc.com/Compound-Amikacin.html
http://www.sielc.com/Compound-Tylosin.html

If you have ability to send samples we can look at your separation in our lab free of charge.

Hi,
As per concern to separation of saponins, I started with the column with same specifications but supplied by other manufacturers. It is Vydac C4, 250,4.6, 5µ, temperature 25C. Till now i have tried various composition of mobile phase (Water ACN), from less strong to more strong mobile phase. the less strong mobile has shown a little success to separate as shown
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i have tried lower pH mobile phase with acetic acid, it work to enhance retention of saponins but unfortunately not on separation. Please any suggestion, it would be great for me.

Thanks

You cannot compare Vydac C4 column with Primesep 100 column, which is a mixed-mode reversed-phase cation-exchange column. Saponins are hydrophilic basic compounds containing basic group. There is almost no mechanism of retention on C4 column. For neutral saponin compounds you need HILIC mode on something like Primesep S or Obelisc N

Dear sir,
The method has already been developed and reported on C4 in other labs. Actually i am new in HPLC. The saponins which i am using are slightly acidic due to COOH group on glucuronic acid.

Regards
Waqas

If you have a workable separation on a C4 column, why would you want to switch to a C8 or C18?

I'm guessing the original question was about quillaia saponins, which I think are triterpene saponins. That means they're basically saturated-chicken-wire with sugars sprouting out in one or two places. The large saturated-chicken-wire section (5 interconnected alkane rings) is very hydrophobic, so of course there is a mechanism for interaction with any reverse phase column. The only question is whether the interaction is sufficiently different between the saponins you're separating, so as to allow their separation.

Many triterpene saponins contain no groups that would be ionised at neutral pH.

Andy Alpert is of course right that the sugar bits are highly suitable for Hilic, and if you don't get separation when using one mechanism of retention, using a completely different method gives you another chance.

In fact, there's a bit of column-sales psychology here: if you have a method where currently two compounds do not separate, then any column that offers different selectivity is highly likely to be an improvement (because it can't get worse...). So it's fairly easy for a new column to look "better" in this circumstance, whereas actually it's just "different". But it's always good to have a couple of totally different approaches in your tool-box ready for inconvenient coelutions.

Hello!
Ion pair agent buffered solution may turn a С18 column to some kind of С4 analogue.