Hi Olivia

I did not do a brief check of all the process, but maybe a simple change can help you.

The phenolic group can act as an acid, you have only water in you mobile phase.  Perhaps you dont know much, about the extraction process of your substrate or the number of ist free phenolic Groups.

Try to add 0,1% TFA to the water and look, if the peak is still visible or if you observe any Change.

As you have a "Poly"-Phenol, you have multiple groups for acidic - ionic interaction, so I would keep the acid inside the mobile phase for you future Analysis.  TFA also does not disturb f.E. further  HPLC/MS processes, because you can vaporize it.

It can also be something, that is not dissolving in pure Methanol, because it is too polar or already a salt, sometimes you remove this during the filtration because it attaches to the menbrane.

Good luck

Hi @frank hoger

thanks for replying. I realized I forgot to input 1 detail. I actually also add in 0.1% formic acid into the water mobile phase.

So yes, even with the added acid. The extra 5 mins retention time appear that does not appear in only methanol sample.

and with extra filtration of 0.45um before dilution, no retention readings at 9+minutes. 

Hi,

Do you have a PDA detector? if yes, then you have a look at the online-UV of the extra retention time. I hope it can be helpful.

Jean-Pierre Mufusama.

@jean-pierre mufusama 

Do not have.. Only connected with DAD or RID detector 

I think you can have access to the online-UV with a DAD.

Formic acid is an aldehyde, which could potentially create a methylene bridge between two polyphenols.

(+)-Catechin Disappearance: As previously described (3,

11-14), (+)-catechin reacts with aldehydes in winelike model

systems (Figure 2), and the rate of (+)-catechin disappearance

depends on the type of aldehyde used.

Dimer Evolution: The two last reaction steps, R3 + R4, of

the (+)-catechin-aldehyde condensation (Figure 5) involve RI1

protonation (R3) leading to a benzylic carbocation RI2. RI2

undergoes subsequent (+)-catechin nucleophilic attack leading

to the formation of bridged dimers. Four possible bridged dimers

would be possible: one (8-8) isomer, two stereoisomers

(6-8), and one (6-6) isomer (3, 11, 12). During the reaction,

the amount of each dimer (8-8, 6-8, and 6-6) was monitored

(Figure 6). The condensation reaction was regioselective with

regard to C-8 substitution; the 8-8-bridged dimers were always

higher than 6-8- and 6-6-bridged dimers. Overall, 6-6-bridged

dimers were formed in very small quantities. This is consistent

with previous data (11) and can be explained by the sterically

favored substitution at C-8 as opposed to substitution at C-6

(24).

(+)-Catechin−Aldehyde Condensations:  Competition between Acetaldehyde and Glyoxylic Acid

http://pubs.acs.org/doi/abs/10.1021/jf0504723

@zach wiersma

What I meant was:

1) What is this retention peak at 5minutes?  

yes I know 9.8+minutes is my sample/polyphenolic compound peak. But what is this peak at 5 minutes+?

Is definitely not water or methanol peaks (solvent peaks) as those solvent does not have any peaks at retention time of 5 minutes (Number 1 and number 2) 

2) Besides that, 

Any possibility why when filter with 0.45um, the compound/polyphenolic in water does not show any retention at 9.8+ minutes?

However, if I do not filter. there is

as your standard has > 98 % purity, any unknown peaks may be due to  presence of unknown impurity .

for more clarity ,i need your chromatogram

refer below links

https://www.ncbi.nlm.nih.gov/pubmed/12456084

https://www.researchgate.net/publication/229404742_Spectrophotometric_potentiometric_and_chromatographic_pKa_values_of_polyphenolic_acids_in_water_and_acetonitrile-water_media

Article Spectrophotometric, potentiometric and chromatographic pKa v...

Plant Polyphenols 2: Chemistry, Biology, Pharmacology, Ecology

In addition to molecular ion peaks, proanthrocyanidins can be detected as non-covalent adducts (clusters), leading to possible artifacts when interpreting ESI–MS data. In particular, formation of non-covalent adducts with formic acid may give addition signals.

Green Tea Polyphenols: Nutraceuticals of Modern Life

The A-ring tends to carry out electrophillic substitution reactions with electrophiles such as aldehydes. Aldehyde and formaldehyde readily react at the C-8 of C-6 of the A-ring, affording condensation products having two catechin moieties.

The filter could be a red-herring. 

Could the peak at ~5.5m in H₂O from elution #2 simply be the phenolic compund itself, and the peak at ~9.8m in H₂O from elution #3 be a dimer formed with formic acid (catalyzed through centrifugation + additional time) which just so happens to be very similar to the elution time of the monomer in CH₃OH (elution #1)?

It certainly is bizarre. I also read that you used different concentrations: from 10ppm in CH₃OH to 30,000ppm in water. Just this alone might raise a trace impurity to the Limit of Detection (but I'm willing to bet it was a typo based on the solubility of polyphenols), and if that trace impurity was larger than 450nm it could explain the difference between elution #2 and #3.

But that would be an extremely large trace impurity. 

Maybe you can do a run with water and acetic acid and see what happens? 

@travis J Schwochert

1) My friends ask me why would I consider 5.5m as the phenolic compound when it shows a retention peak at 9+m in methanol. I really have no idea, if you mention dimer formed. Can it really be so happen to be similar

2) Yes because as I written no matter what concentration of Phenolic in Methanol be it 10ppm to 50ppm to 200ppm to 1000ppm, there will always be a big peak at 9+minutes

Whereas, likewise no matter the concentration of Phenolic in water be in 1000ppm to 30000ppm, there is a retention time at 5minutes and at 9+minutes(if it didnt went through a 0.45um filter)

I wanted to see what is this phenolic solubility in water, thats why I mix it with water.

You proposed to inject a sample composing of water+acetic acid?

Well...if you look at formic acid, you can see that it's actually both an aldehyde and a carboxylic acid. https://www.quora.com/Does-formic-acid-give-test-reaction-for-aldehyde-as-well

And certain polyphenols reacts with aldehydes. 

So my pet theory was that a dimer was formed. Just a guess. I noticed when I was reading about these things that acetic acid or trifluoroacetic acid is often used with water during polyphenol chromatography.

If you try a different acid, then you can rule out dimer formation.

But a dimer does not explain why a filter would have any effect at all. This is why it's so hard to comprehend what is happening.