1.10 Quantitative biochemical tests

How is the starch test made quantitative?

By preparing known starch concentrations, adding iodine solution, measuring absorbance using a colorimeter, and plotting a calibration curve of absorbance vs concentration.

What is the difference between qualitative and quantitative biochemical tests?

• Qualitative tests: show whether a substance is present or absent (e.g. starch, protein)

• Quantitative tests: measure how much of a substance is present, often using dilution series, colour standards, or instruments like colorimeters

What is a semi-quantitative test?

A semi-quantitative test gives an estimate of concentration rather than an exact value, often by comparing colour changes to known standards (e.g. Benedict’s test with glucose colour chart).

How is Benedict’s test made semi-quantitative?

By preparing a series of known glucose concentrations, performing Benedict’s test on each under identical conditions, and using the resulting colour changes as standards to estimate unknown concentrations.

What is the procedure for making Benedict’s colour standards?

1. Prepare stock glucose solution of known concentration

2. Make serial dilutions (e.g. 50, 20, 10, 5, 1, 0.5, 0.1 g dm⁻³)

3. Add equal volumes of Benedict’s solution

4. Heat all samples equally in a water bath

5. Cool and record colour changes as standards

How do you use Benedict’s colour standards to estimate concentration?

The test sample is treated identically, then its final colour is compared to the standards to estimate the closest concentration of reducing sugar.

Why must conditions be identical when making standards and testing samples?

To ensure valid comparison; differences in heating time, volume, or reagent concentration would affect colour intensity and make results unreliable.

What is a colorimeter used for in starch testing?

A colorimeter measures the absorbance or percentage transmission of light through a starch-iodine solution, allowing precise estimation of starch concentration.

How do you determine starch concentration using a calibration curve?

The absorbance of the unknown sample is measured, then a graph of absorbance vs concentration is used to find the corresponding starch concentration by interpolation.

Why is iodine test suitable for colorimetry?

Because the starch-iodine complex produces a measurable colour change whose intensity increases with starch concentration, allowing absorbance readings to be used quantitatively.

What is a serial dilution?

A serial dilution is a stepwise dilution of a substance to produce a range of known lower concentrations from a stock solution.

Why are serial dilutions used in biochemical tests?

They provide a range of known concentrations to create calibration curves or colour standards for estimating unknown sample concentrations.

Why is Benedict’s test only semi-quantitative?

Because the colour change is subjective and depends on observation, making it an estimate rather than an exact measurement of concentration.

Why can Benedict’s test not give exact concentration?

The colour intensity is not measured numerically, and different observers may interpret colours differently, limiting accuracy.

Why is fructose detected by Benedict’s test?

Fructose is a reducing sugar because it can isomerise under alkaline conditions to form a molecule capable of reducing Cu²⁺ ions, producing a positive result.

Estimate sugar concentration from Benedict’s results (P = orange-red, Q = green, R = blue)

• P: high concentration (around 20–50 g dm⁻³)

• Q: low concentration (around 1–5 g dm⁻³)

• R: no reducing sugar present

Limitations of Benedict’s test for concentration estimation

• Colour interpretation is subjective

• No exact numerical measurement

• Precipitate formation varies

• Cannot distinguish between different reducing sugars

Difference between qualitative and quantitative tests

• Qualitative: identifies presence/absence

• Quantitative: measures amount using instruments or calibration curves

How would you investigate starch decrease in ripening bananas?

Extract starch from bananas at different ripening stages, perform iodine test, use a colorimeter to measure absorbance, and plot concentration vs ripening stage to show decrease over time.

Why are calibration curves important in quantitative tests?

They allow unknown concentrations to be determined accurately by comparing measured absorbance or colour intensity to known standards.