Dear Mucahit,

I asked this question because, even if the sample quantity injected in the column is small (ie 10 microliters) , so , according to the solvant in which you dilute your extract and according to your sample phase, it can give an "instability" in retention time, and some difficulties.

So, you can dissolve your extract in pure actetoniril if the compounds are very hydrophobic, or in the same composition of the mobile phase.

As said previously, you can use in general for hydrophobic substance, and C18 column (most often used) acetonitril + either water, or acidic solution with organic acid like formic acid (or perhaps also acetic acid) ; and a variable proportion of the two according to the "hydrophobicity" of the compound, the number of ionic groups that can be modify by acid.

First can be used a fixed composition of acetonitril and acid (ie 80/20 or 70/30) and if difficulties a gradient.

Best regards

Didier

Hi,

you could try another mobile phase. Usually A:formic acid in water (0.1% v/v) and acetonitrile gives good results using a gradient.

If you aime to quantify those analytes in fruits I suggest you use matrix sample for your method validation instead of standard samples.

Regarding phenolic extraction from fruit samples you can try methanol in different mixtures with water by ultrasound or maceration.

Dear Dr. Spinola

Thank you so much

What are you thinking about ACE 5 C18 250 x 4.6 mm id column? In your opinion, it is OK?

Dear Mucahit,

in my opinion, you can continue working with ACE column, since in theory, any C18 column can provide a good phenolic separation by reversed-phase.

Good luck!

Dear Dr. Spinola,

I want to thank you for your contributions. You suggested me the other mobil phases which are solvent A: formic acid 0.1% in water (v/v) and asetonitril. You mean that is asetonitril solvent B ? Could you send me the gradient elution program with your suggested mobil phases?

The system pressures between each run are the same? Also, a C-18 column is normally used for separation of phenolic compounds.

Try this method: M. Cioroi. Scientifical Researches. Agroalimentary Processes and

Technologies, Volume XI, No. 1 (2005), 211-216

THE IDENTIFICATION OF PHENOLIC ACIDS BY HPLC

METHOD FROM STRAWBERRIES . They used ECI detection as well!

Dear Colleagues,

What is the process for extraction of your mixture of phenolics from the fruits, and in which final solution/solvant have you your samples before a deposit on your column ?

Bst regards

Didier J

Use A=0.05%TFA, B=ACN in gradient elution mode on C-18, flow=1ml/min at 355nm.

Gudluck

:)

Dear Mucahit, try to use the pharmacopoeia method for determination of chlorogenic acid in artichok extract, I think it will works for the compounds you mentioned above.

Dear Dr. Spinola,

Thank you. I will try the gradient elution procedure that you suggested.

Dear Angelov, Dear Joshi, Dear Zimba, Thanks so much for your contributions.

Dear Mucahit,

I asked this question because, even if the sample quantity injected in the column is small (ie 10 microliters) , so , according to the solvant in which you dilute your extract and according to your sample phase, it can give an "instability" in retention time, and some difficulties.

So, you can dissolve your extract in pure actetoniril if the compounds are very hydrophobic, or in the same composition of the mobile phase.

As said previously, you can use in general for hydrophobic substance, and C18 column (most often used) acetonitril + either water, or acidic solution with organic acid like formic acid (or perhaps also acetic acid) ; and a variable proportion of the two according to the "hydrophobicity" of the compound, the number of ionic groups that can be modify by acid.

First can be used a fixed composition of acetonitril and acid (ie 80/20 or 70/30) and if difficulties a gradient.

Best regards

Didier

Dear Mucahit Pehluvan, after you have optimized your gradient elution (we have also found that acetonitrile works usually better with phenolics than methanol) you could also try to use a bit more selective wavelengths for detection (if you have an option for multiple wavelength detection); quercetin/rutin should have a clear absorption band around 370 nm and caffeic/chlorogenic acid at approx. 325 nm (check the spectra of the compounds by DAD to select the optimal detection wavelengths). You may found significant increase in sensitivity due to suppression of background noise.

You may use ACN with small amount of TFA ( Around 1%or less) , use flow rate0.5 to 0.8 in c18 colum.

Dear Mucahit,

It is possible that the sonication (10 min) can help the extraction of phenolic acids from fresh fruits. As listed above, a gradient program on your C18 with 0.1% TFA in water and 0.1% TFA in CH3CN can improve the separation. Alternatively, if TFA is not suitable for your column, you can try using 0.2% CH3COOH (phase A) and CH3CN (phase B).

Greetings

Dr. Mucahit you can try the method of Amakura et al. (2000), J. Chromat. A, 891, 183-188.

Dear Jambou

The injection volume is 20 microliters in my present study. Thaks so much your contribution and explanation about extraction and mobil phases.

Let me recommend the method described in the following article: Long-Ze and J. M. Harnly , 2007, A Screening Method for the Identification

of Glycosylated Flavonoids and Other Phenolic Compounds Using a Standard Analytical Approach for All Plant Materials. Journal of Agriculture and food chemical, 2007, Vol. 55, 4: 1084–1096, DOI: 10.1021/jf062431s, U.S.A.

To me has worked very well hydrolyze the sample by the four methods described, you will enlarge the spectrum of phenolic compounds to detect.

Best regards!

Cony

AUTHOR COPY

I am using Folin-Ciocalteu method (Piagnani et al. J of Berry Research

. 1 (2011) 1–16 DOI:10.3233/BR-2011-023). All the best

Claudia Piagnani

in the attached paper I did some work on phenolics....

http://www.tandfonline.com/doi/abs/10.1080/17429140701429228

Dear Montanaro,

Thanks for your attachment.