PAG 05 - 5.2: Colorimeter to determine concentratio of glucose

Are the food tests quantitative or qualitative?

Qualitavtive - they showed presence or absense

What type of Benedict's reagent is used in PAG 5.2, and how does it differ from the standard qualitative test?

Quantitative Benedict's reagent is used. Instead of forming a brick-red precipitate, it forms a white precipitate (copper(I) thiocyanate), leaving a clear blue solution whose colour intensity is proportional to the remaining copper(II) ions.

How can a Bednedic’s tests be used quantitatively?

By measuring the absorbance or transmission (using a colorimeter) of a certain concentration of glucose

When measuring things for this PAG, do you need to be specific?

You need to be very specific with measurements as this is a quantitative testing practical

How must a calibration curve be constructed in order to calculate unknown concentrations?

1 - Measure the absorbance of known concentrations of glucose using a colorimeter

2 - Plot graph of transmission over concentration

3 - Unknown sample is then tested, using the sample procedure.

4 - The absorbance is compared to the calibration curve to find the concentration.

How do you prepare the standard glucose solutions?

Prepare a serial dilution from a stock solution (typically 1.0% glucose) using distilled water, plus a 0.0% distilled water blank.

Why must you centrifuge (or filter) the samples after heating with Benedict's reagent?

To remove the solid precipitate so it does not scatter light and give false absorbance/transmission readings in the colorimeter.

Which colour filter must be used in the colorimeter for this experiment, and why?

A red filter is used because red light is complementary to the blue solution, meaning it is absorbed most strongly by the remaining blue Cu²⁺ ions.

How do you calibrate (zero) the colorimeter before taking readings?

Place a cuvette filled with distilled water (the blank) into the colorimeter and set absorbance to 0 (or transmission to 100%).

What precautions should you take when handling cuvettes?

Handle them only by the frosted/ribbed sides to avoid leaving fingerprints on the clear optical faces, and ensure there are no air bubbles inside.

What happens chemically to the copper ions when glucose is heated with Benedict's reagent?

Glucose reduces blue copper(II) ions (Cu²⁺) into insoluble copper(I) ions

How does increasing glucose concentration affect the colour of the supernatant and light transmission?

Higher glucose concentration reduces more Cu²⁺ ions, making the supernatant paler blue. This allows more light to pass through (higher % transmission, lower absorbance).

Which variables go on the x-axis and y-axis of the calibration curve?

Glucose concentration (%) goes on the x-axis (independent variable), and absorbance or % transmission goes on the y-axis (dependent variable).

How do you use the calibration curve to find the concentration of the unknown sample?

Find the unknown's absorbance/% transmission on the y-axis, draw a horizontal line to the curve of best fit, and read vertically down to the x-axis.

What should you do if your unknown sample's reading falls outside the range of your calibration curve?

Dilute the unknown sample by a known factor (e.g., 1 in 10), re-test it, and multiply the concentration read from the graph by the dilution factor.

Name three key control variables in PAG 5.2.

1 - Volume of glucose solution used

2 - volume and concentration of quantitative Benedict's reagent added

3 - temperature and duration of heating in the water bath

What is step 1 of this PAG?

1 - Create serial dilutions of 0.00%, 0.06%, 0.13%, 0.25%, 0.50%, 1.00%

What is step 2 of this PAG?

2 - Add equal volumes of quantitative Benedict's reagent to each dilution and your unknown sample. Heat all tubes in a 90°C–100°C water bath for about 15 minutes, then let them cool.

What is step 3 of this PAG?

3 - Separate the liquid: Centrifuge (or filter) the cooled samples to remove the solid precipitate. Transfer the clear liquid (supernatant) into clean cuvettes.

What is step 4 of this PAG?

4 - Measure with colorimeter: Set the colorimeter to a red filter and zero it using distilled water. Record the absorbance (or % transmission) for every sample.

What is step 5 of this PAG?

5 - Plot and read: Plot a calibration curve of glucose concentration (x-axis) against absorbance/% transmission (y-axis). Use your unknown sample's reading to find its concentration on the graph.

What is the summary of the results shown after removing solutions from the water bath?

More glucose present = more copper ions form percipitate = less blue the solution

What is the correlation between concentration and the absorbance and why?

The higher the concentration, the lower the absorbance. Because, when light is shone through, more light is transmitted and less is absorbed.

What is a biosensor?

• A device that uses a biological component to detect a chemical substance and converts this detection into a signal.

What are the three main components of a biosensor?

A receptor, a transducer, and a display.

What biological molecules can act as the receptor in a biosensor, and how are they positioned?

An enzyme, an antibody, or single-stranded DNA, which is attached to a surface.

What happens immediately after a specific chemical binds to the receptor?

It causes a localised change, such as a change in pH.

What is the role of the transducer in a biosensor?

It detects the change caused by the chemical binding to the receptor and converts it into a response.

Give two examples of responses produced by a transducer.

A change in a coloured dye or the production of an electric current.

What is the function of the display in a biosensor?

It provides a visible signal of the response/change detected by the transducer.