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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.

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IB Chemistry Biology on Vitamin C quantification using UV spectrophotometer at 266nm

1. Sodium oxalate (0.0056M) - dissolving 0.075g sodium oxalate in 100ml pH 5 buffer
2. Vit C (0.001M ), prepared by dissolving 0.018g of Vit C in 100ml sodium oxalate solution
3. Perform 2 fold dilution on 0.001M Vit C.
4. Add 1 ml Vit C into a quartz cuvette and 2ml of sodium oxalate ( as a stabiliser )
5. Leave for 10 mins
6. Prepare a blank with 1ml water and 2 ml ammonium oxalate
7. Set up UV spectrophotometer at 266nm and quantify.

Result shown below

Latest journal published in April, accurate way to quantify Vit C.
Click HERE to view

* Oxalate solution used as stabiliser and pH around 5 as both affect the stability of Vitamin C

Short video on Vitamin C quantification using UV spectrophotometer

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Vitamin C quantification using iodometric titration

Potassium iodate as oxidising agent in burette
Potassium iodide as reducing agent , starch and vitamin C (reducing agent) in conical flask
End point is blue black colouration
Chemical equation below
KIO₃ + 5KI + 6H⁺ --> 3I₂ + 6K⁺ + 3H₂O
3C₆H₈O₆ + 3I₂ --> 3C₆H₆O₆ + 6I^- + 6H⁺
Mole ratio KIO3:Vitamin C = 1 :3

Video on Iodometric Titration for Vitamin C quantification

IA Research question for Chemistry/ Biology on Degradation/decomposition of Vit C

Effect of heavy metals salts like Pb2+or Cu2+
Effect of pH and temperature
Duration and exposure to light or UV light
Aerobic or anaerobic degradation
Effect/presence of different sugar and oxidising agent

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IB Biology on effect of ethanol/SDS on diffusion of beetroot pigment.
Effects of ethanol and SDS on the diffusion of beetroot pigment measured using visible spec

Steps to follow

Cut beetroot into blocks, place in water to remove red pigment due to cutting
Prepare 2 fold dilution of SDS/Ethanol and place 2 ml into well
Find max wavelength for red pigment by pipetting 1.5ml into cuvette

Max Wavelength determined using visible spectrophotometer

Pipette 1.5ml red pigment (diluted) into cuvette to determine the max wavelength
Beer Lambert Law works only for diluted beetroot solution
583nm was taken

Procedure/Steps