Wednesday, October 26, 2011

IB Biology, IA, EE on glucose quantification assay using colorimeter for glucose diffusion experiment

IB Biology IA, EE, glucose assay/quantification with colorimeter using DNS
IA and EE on Glucose Quantification
1. 1% glucose std prepared (1g in 100ml)
2. 2 fold serial dil ( 1%, 0.5%, 0.25%, 0.125%, 0.0625%)
3. Add 2 ml of 1% glucose + 0.25ml of DNS (3,5 dinitrosalicylic acid) into a 50ml test tube.
4. Repeat step 1 to 3 with diff glucose concentration
5. Place all tubes in boiling water bath for 5 mins for colour formation. ( red brown)

6. Transfer 0.5 ml of sol from tube to cuvette and added 1.5ml of water to dilute it
7. Set visible spectrophotometer at 487nm.
8. Prepare a blank - 2ml water + 0.25ml DNS in test tube. Transfer 0.5ml to cuvette and add 1.5ml water to dilute it.

9. Measure absorbance at 487nm
10. Plot std calibration curve
11. Beer's Law works only for dilute solution.
12. Range for glucose ( 0.5%, 0.25%, 0.125%, 0.0625%,  0.03125%)
14. Watch video for clarification

Video, Glucose Quantification with DNS/Visible Spec

Experiment using glucose quantification
How surface area to vol ratio affect the diffusion of glucose from sweet potato strip measured with visible spec using DNS as a colour  formation?
Glucose quantification steps

  • 2 fold serial dil on 1% glucose std
  • Add 2ml glucose std + 0.25ml DNS in test tube
  • Leave in boiling water bath for 5 mins
  • Transfer 0.5ml to cuvette and add 1.5ml to dilute it
  • Measure Abs at 487nm
  • Blank done following above steps but using 2ml of water instead of glucose. 

Experiment on diffusion of glucose from sweet potato. ( Surface to volume ratio)
Sweet potato cut into 16 strips compared to 1 long uncut strip

  • Leave strips in 10 ml water over 24hrs 
  • Pipette 2ml sol and add 0.25ml DNS
  • Leave in boiling water bath for 5mins
  • Transfer 0.5ml into cuvette and add 1.5ml water to dilute it
  • Measure Abs at 487nm
  • Blank done following above steps, using 2 ml water 

Calculation of glucose from standard calibration curve
Standard Calibration Curve

  • Glucose diffusion rate = Abs change over 24hrs
  • Abs for 16 strips taken and conc is = 0.1467%
  • Abs for 1 strip taken and conc is = 0.0695%
  • Rate glucose diff = 0.1467/24 =0.00611%/hr
  • Rate glucose diff =0.0695/24 = 0.00289%/hr

Tips to make it work
* Glucose/DNS sol must be diluted with water as Beer's Law only applies for dilute sol
* Glucose sol range must be around 1% to 0.1% for colour determination.
* All item can be purchased from Carolina Biologicals or Kemtecscience
Easy Research Question for SL/HL on glucose expt
  • Starch hydrolysis with diff enzymes in producing glucose
  • Effect of pH, temp, heavy metal inhibitors, ionic salts on enzymatic hydrolysis of starch into glucose quantified using the above technique
Click HERE for different IA/EE using glucose/DNS techniques
Click HERE on glucose quantification and preparation of DNS solution.
Click HERE to purchase DNS kit
Click HERE to view MSDS toxicity of DNS.

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