RP11 Measuring glucose concentration

You can't know what an absorbance of '0.45' means unless you know what absorbance a $2\%$ solution produces.

Answer: To plot the absorbance values of known glucose concentrations, providing a reference to interpolate the concentration of the unknown sample from its absorbance.

Required practical 11. A calibration curve establishes the relationship between absorbance and known concentrations. The unknown sample's absorbance is measured, and the curve is used to find its corresponding concentration.

A colorimeter measures light passing through a clear liquid. Floating solid particles scatter light unpredictably.

Answer: They must be filtered or centrifuged to remove the brick-red precipitate, so the colorimeter measures the absorbance of the remaining blue supernatant.

Required practical 11. Benedict's test creates an insoluble $Cu_2O$ precipitate. To measure accurately, this precipitate must be removed (filtered or spun down) so the colorimeter can measure the light absorbed by the unreacted blue copper(II) sulfate in the supernatant.

Is that tube 'orange' or 'brick-red'? Two people might disagree. A machine won't.

Answer: A colorimeter provides a quantitative, objective measurement of absorbance, eliminating human error and subjective judgement of colours.

Required practical 11. Colorimetry provides objective, continuous, quantitative data (e.g., $0.45$ AU) rather than subjective, categorical data (e.g., 'greenish-yellow'), increasing the precision and reliability of the results.

You only want to measure the absorbance caused by the chemical, not the container it sits in.

Answer: To 'blank' the colorimeter, calibrating it to account for any light absorbed or scattered by the plastic cuvette and the water itself.

Required practical 11. Blanking the colorimeter sets a baseline ($0$ absorbance / $100\%$ transmission), ensuring that subsequent readings only measure the absorbance caused by the solute of interest.

Colorimeters shine a straight beam of light through a clear liquid.

Answer: The solid precipitate particles would scatter the light unpredictably, preventing an accurate measurement of the absorbance of the liquid supernatant.

Required practical 11. Suspended solids scatter light (turbidity). To measure how much copper(II) sulfate was used up, you must measure the remaining unreacted blue liquid (supernatant) clearly, which requires filtering or centrifuging out the solid $Cu_2O$.

You want to measure how much of the original blue chemical is left. You need to shine a light that the blue chemical 'eats'.

Answer: The unreacted Benedict's solution is blue. A blue solution reflects blue light but strongly absorbs red light, maximizing the sensitivity of the absorbance reading.

Required practical 11 / AT a. To measure the concentration of a blue solution, you shine its complementary colour (red) through it because the blue solution absorbs red light strongly. The higher the absorbance, the more blue $CuSO_4$ remains.

More glucose = more reaction = more blue chemical used up = paler liquid left behind.

Answer: It will have a low absorbance reading, because most of the blue copper(II) sulfate was reduced to solid precipitate, leaving a pale supernatant that absorbs very little light.

Required practical 11. A high glucose concentration reduces almost all the blue $Cu^{2+}$ ions to red $Cu^+$ solid. Once the solid is removed, the remaining liquid is very pale, so it absorbs very little red light (high transmission, low absorbance).

The curve is a translation map between the machine's light reading and actual sugar content.

Answer: They perform the exact same Benedict's test on the unknown, measure its absorbance, find that absorbance value on the y-axis of the curve, and read down to the x-axis to find the concentration.

Required practical 11. This is interpolation. By testing known standards, you graph Absorbance vs Concentration. You then test the unknown under identical conditions and use the line of best fit to translate its absorbance into a concentration.