one question about my chromtogram

[jiang295]

this is extract from bread. why baseline has so huge bump? what are underneath in baseline. I check control, baseline is very flat. so there wont be any problem associate with detector or column. any idea?

[bisnettrj2]

Can you supply your instrument conditions? Mobile phase constituents? Injection volume? Sample diluent?

[jiang295]

column 250mm*21mm C18.
mobile phase methanol and 0,1% formic acid.
injection volumn: 2ml.
injection mass: 100mg
elution condition: 5%-90% 30min and hold 90% 8min

Can you supply your instrument conditions? Mobile phase constituents? Injection volume? Sample diluent?

[bisnettrj2]

Is the mass of sample injected equivalent to the standard that you injected that did not show the baseline hump? Was this sample injected right after the standard? How long do you re-equilibrate your column after the 8 minute hold at 90% MeOH? And what is your sample diluted in? 95:5 0.1%formic acid:methanol?

[Hollow]

I wouldn't search too far and blame something to be wrong with the system.
As you state this is an extract from bread, I would expect it to be some polymeric compounds from the sample itself.

I've seen something similiar with some plant extracts rich on polyphenolic compounds.

What was you sample prep?

[jiang295]

I went throught SPE, and using 10% methanol and 100% methanol to stepwise elute.
only collect fraction elute with 100% methanol. so polysaccharide and protein will be excluded from my sample.
I did not consider polyphenolic compounds, that is quite possible.

I wouldn't search too far and blame something to be wrong with the system.
As you state this is an extract from bread, I would expect it to be some polymeric compounds from the sample itself.

I've seen something similiar with some plant extracts rich on polyphenolic compounds.

What was you sample prep?

[jiang295]

did you prove that those are polyphenolic compounds from plant extract?

I wouldn't search too far and blame something to be wrong with the system.
As you state this is an extract from bread, I would expect it to be some polymeric compounds from the sample itself.

I've seen something similiar with some plant extracts rich on polyphenolic compounds.

What was you sample prep?

[bisnettrj2]

Can you post a chromatogram of a standard that equates to a similar concentration as your sample extract, so we can see the difference between your 'good' chromatogram and the chromatogram that you have posted?

[jiang295]

why do you think so? you think that is because of mass overload?

Can you post a chromatogram of a standard that equates to a similar concentration as your sample extract, so we can see the difference between your 'good' chromatogram and the chromatogram that you have posted?

[bisnettrj2]

Not necessarily. I just think it will be helpful to see what you consider to be a good chromatogram in this analysis, so we have a means of comparison. I suspect that Hollow is right, that it is something in your sample that isn't present in your standards, but I think it will help to rule out any instrument or inherent analysis issues.

Also, you say your SPE preparation consists of eluting the portion of interest from the SPE with 100% methanol. Do you inject 100% methanol, or do you dilute your sample with 0.1% formic acid before injection?

[jiang295]

I really appreciate your help. thanks. i just want to know reason why you want to do that.
i get rid of all methanol and redissolve in water before injection.

Not necessarily. I just think it will be helpful to see what you consider to be a good chromatogram in this analysis, so we have a means of comparison. I suspect that Hollow is right, that it is something in your sample that isn't present in your standards, but I think it will help to rule out any instrument or inherent analysis issues.

Also, you say your SPE preparation consists of eluting the portion of interest from the SPE with 100% methanol. Do you inject 100% methanol, or do you dilute your sample with 0.1% formic acid before injection?

[Hollow]

did you prove that those are polyphenolic compounds from plant extract?

proved? more or less

just considered some coindices.
- injection of a "pure" tannin sample showed also a bump like this
- the plant is known to contain such substances
- by hydrolysis with conc HCl the bump decreased
- with HCl the solutions often turns dark red -> (pro)anthocyanes
- incubating the sample with skin powder or bentonite reduces also this bump (unfortunately also other peaks)

for your bread extract:
What about Lignines and/or starch?

[Uwe Neue]

There may be ways to suppress the elution of polymers from SPE by using selective solvents for elution. If you do not know what this is, this may amount to a trial-and-error exercise. I can help you with some ideas using Oasis SPE packings.

[jiang295]

i guess it is Lignines.
does starch retain in the C18 since they are so hydrophilic?

did you prove that those are polyphenolic compounds from plant extract?

proved? more or less

just considered some coindices.
- injection of a "pure" tannin sample showed also a bump like this
- the plant is known to contain such substances
- by hydrolysis with conc HCl the bump decreased
- with HCl the solutions often turns dark red -> (pro)anthocyanes
- incubating the sample with skin powder or bentonite reduces also this bump (unfortunately also other peaks)

for your bread extract:
What about Lignines and/or starch?

[jiang295]

sure, so there is unique characteristic of this SPE material.

There may be ways to suppress the elution of polymers from SPE by using selective solvents for elution. If you do not know what this is, this may amount to a trial-and-error exercise. I can help you with some ideas using Oasis SPE packings.