Quantification of 5-HMF and fructose performed using a high-performance liquid chromatography (HPLC, Agilent 1200) equipped with a RID detector and reversed phase C-18 column at 30 °C. I used a refractive index range of 1.00 to 1.75 RIU.
The mobile phase consisted of acetonitrile and deionized water (85:15 v/v) at 0.5 mL/min flow rate and the 5-HMF will be analyzed at 280 nm.
Unfortunately, I didn't see any peak during testing my standard samples
I am not surprised. Fructose is determined by RID using a speciality amino column like Aminex (the grandfather of sugar analysis) and not C-18. HMF is also usually determined by the 'White' UV-Vis method.
Dear Noor Aljammal
I am also working on HMF and sugars in honey.
Regarding HMF,
Mobile phase : 10% Methanol
Flow rate: 1ml/min
Detection : UV 285nm
Column : Shimpack Shimadzu VP-ODS
Regarding Sugars,
Mobile phase : 80% ACN
Flow rate : 1.5ml/min
Detector: RID
Column temp: 27 degree
Column : Zorbax carbohydrate column (150 x 4.6 mm)
Regards
Sugars = HILIC conditions with NH2 column, but some separations are possible also on RP. What is the concentration of fructose?
Regards
G
In addition, HMF concentrations are in the low ppm (ug/mL) while the RID can only detect concentrations in the 0.1% range. You need a PDA/DAD/UV detector.
As I recall 1.0% is 10,000 ppm, so you will need a 10,000X increase in sensitivity which an RID cannot supply.
Bruce Neagle Many thanks for your help, I tried DAD Detector (reversed column) and it works for HMF.
I still need to figure out for fructose.
Many thanks for your helpful advice.
Regards,
Fructose can be separated from Glucose using a Biorad Aminex column or equivalent. Use 210 nm wavelength.
Ankita Sood many thanks for your help...highly appreciated. I will be taken into consideration.
Grzegorz Boczkaj Many thanks for your help.
Appreciated.
Bruce Neagle many thanks I will check it ,,,,many thanks for your help.
Regards
Make sure you fully purge the RID (w/o the column) first by pumping the the mobile phase through the reference cell.
You may have a problem with detection at 280 nm using actonitrile due to its UV cutoff, You may try going down to 210-220nm.
Donald Pennino Many thanks for your advise.
for HMF 88.5:11.5 (water: ACN), C18 and DAD detector, peaks detected at 284 nm
for fructose I am still trying to change some parameters.
Regards,
Noor
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