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Determination of Glucans and Mannans in YCW
  Principle
Material
Method
Results
1. PrincipleRetour en haut de page
  The YCW are submitted to a pre-solubilization with concentrated H2SO4 prior to hydrolysis with H2SO4 at 128°c in an oven This total hydrolysis of the glucans and mannans generates proportional quantities of glucose and mannose that are determined by Ionic Chromatography.
 
! This method measures the total glucans which may include residual glycogen and is not specific of ß 1-6 linked ß 1-3 glucans. Another method involving a previous alkaline treatment is available for determination of the ß-1-6 linked ß 1-3 glucans.
2. MaterialRetour en haut de page
 
  • 100 ml capped flask (Duran or Schott Glass)
  • Tube
  • Filter Acrodisc IC Pall
  • Oven
  • H2SO4 72 %
  • Ionic chromatography system with pulsed amperometry detector containing a gold electrode
  • Vortex mixer
  • NaOH 32%
  • 100 ml and 50 ml volumetric flasks
  • Distilled water
  • HPLC grade Water
  • Metrosepcarb1 ionic chromatography column (Metrohm)
3. MethodRetour en haut de page
 
  • 3.1. Standard preparation
    •  
    • Weigh 50 mg of glucose (note the exact weight Wglu) and 50 mg of mannose (note the exact weight Wman)
    • Go to step 3.3.
     
  • 3.2. Sample preparation
    •  
    • Weigh 50 mg of YCW (note the exact weight Wy)
    • Go to step 3.3.
     
  • 3.3. Pre solubilization
    •  
    • Add 3,3 ml of H2SO4 72%
    • Mix the sealed tube with a Vortex mixer
    • Leave for one hour at ambient temperature with stirring every 10 min
     
  • 3.4. Acid Hydrolysis
    •  
    • Pour the contents of the tube into a 100 ml flask
    • Add 40 ml of distilled water
    • Close the flask
    • Put the capped flask in an oven at 128°c and incubate for 3 hours
    • Take out the flask and cool it
    • Neutralize with 8,112 ml of NaOH 32%
    • Decant the contents of the flask into a 100 ml volumetric flask
    • Adjust to 100 ml with distilled water
    • Filter the solution through an Acrodisc IC filter
     
  • 3.5. Chromatography
    •  
    • 3.5.1. Preparation of Standards
      •  
      • Take 2,5 ml of the hydrolysed solutions of glucose and mannose obtained in 3.4.
      • Transfer to a 50 ml volumetric flask
      • Adjust with distilled water
      • Put in a chromatography vial for the autosampler
       
    • 3.5.2. Sample preparation
      •  
      • Take 7,5 ml of hydrolysed material obtained in 3.4.
      • Transfer to a 50 ml volumetric flask
      • Adjust with distilled water
      • Put in a chromatography vial for the autosampler
       
    • 3.5.3. Preparation of the mobile phase
      •  
      • Measure one liter of HPLC grade water.
      • Filter using a 0,45 µm membrane
      • Degas under vacuum for 1 h 30 min
      • Measure 7,57 ml of NaOH 51% into the flask intended for the mobile phase
        ! Be careful to use only a polypropylene flask for the mobile phase
      • Add the 1 liter of degassed water
      • Stir using a magnetic stirrer
       
    • 3.5.4. Calibration solution for chromatography
      •  
      • Prepare, using HPLC grade water, solutions of glucose and mannose at 10 mg/l, 30 mg/l and 40 mg/l
      • Use them for calibrating the chromatography.
       
    • 3.5.5. Chromatographic Condtions
      •  
      • Condition the Metrosep Carb1 column from Metrohm using the mobile phase at a flow rate of 1 ml/min for 2 hours.
      • Inject 20 µl of :
        • The three calibration solutions (§3.5.4.)
        • The standard solution
        • The standard solution
      Calibrate the system with the calibration solution. Trace the calibration curves
      Area = f (concentration)
4. ResultsRetour en haut de page
  The built-in software of the chromatography equipment will give the concentration in mg/l for :
 
The standard solution :
Concentration of Mannose in mg/l: CmanSt (mg:l)
Concentration of Glucose in mg/l: CgluSt (mg:l)
 
The sample solution :
Concentration of Mannose in mg/l: CmanY (mg:l)
Concentration of Glucose in mg/l: CgluY (mg:l)
  • 4.1. Yield calculation
    •  
    Calculate the recovery yield for the standard mannose and glucose solutions as follows:
    Yman = CmanSt (mg/l) / Wman (mg) x 10 x (2,5/50)
    Yglu = CgluSt (mg/l) / Wglu (mg) x 10 x (2,5/50)
     
    Wman and Wglu are the measured weights of mannose and glucose in mg (See § 3.1.)
     
  • 4.2. Concentration of mannans and glucans in YCW
    •  
    Concentration of mannans in g%g:
     
    Cmannans = 0,9* [(CmanY x (50 / 7,5) ) / (Wy (mg) x 10)] * (1 / Yman)
     
     
    Concentration of glucans in g%g:
    Cglucans = 0,9* [(CgluY x (50 / 7,5) ) / (Wy (mg) x 10)] * (1 / Yglu)
    Wy: weight of YCW (see § 3.2.)
    Yman and Yglu: yields of mannose and glucose (see § 4.1.)
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