I am unable to have a definite peak in the chromatogram of the five monomeric sugar and cellobiose analysis using HPLC. All five monomeric sugars, except Glucose, forms tailing peaks while Cellobiose is not detected. This tailing peaks caused overlapping when all sugar standards were run. My sugars and cellobiose were either from Merck or Sigma.
HPLC conditions are well stated. Other details are:
The brand and model of HPLC used: -HPLC Type: Fisher Scientific Thermofisher
-Brand: Shidmazu
-Model: RID-10A
Size of column: -Product name: APS-2 Hypersil
Diameter(mm): 250 x 4.6
-Particle size: (micron-N-)
Probably some column amino groups were protonated, due to some acid in mobile phase or sample.
try changing condition of detection . increase colum temperature or flow rate or both
Since you mention cellobiose + monomeric sugars I assume you want to detect wood sugars (ie. glucose, xylose, galactose, mannose, arabinose) . The amino column you use is not optimal for this kind of analysis. You should use a resin column with Pb 2+ for wood sugars separation. Ca2+ or H+ columns can also be used but not all of these sugars can be separated.
What kind of mobile phase you are using, its flow rate and temp. are very crucial. If you can share the same the problem may be resolved..
Also play around with your mobile phase and other parameters like temp and flow rate.
What mobile phase you are using. Normally for this column a isocratic solution of acetonitrile: water (70-80:30-20) is used for separation of sugars with a flow rate of 0.80 to 1.0 ml/min
Be careful NOT to use acetonitrile if Your samples contains proteines. ACN will denaturate proteins and by time block the coloumn.
@ all, I wish to thank you sincerely for your valuable contributions and suggestions. Your recommendations have been very useful.
@ Questions: I am working on lignocellulosic materials, which definitely, as said by Dr Romar, may contains some minute amount of proteins. But like I previously pointed out in my question, I had only run the pure sugars not the pre-treated sample. The mobile phase was 80%ACN.
Change of column has actually helped.
Thanks a lot
@ Dr Henrik Romar, in case of further works and a possible column blocking, which alternative mobile phase would you recommend ?
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