Calibration Curve

What is the purpose of a calibration curve in quantitative analysis?

A calibration curve is used to determine the concentration of a substance in an unknown sample by comparing its absorbance to a set of standard solutions of known concentration. It provides a linear relationship between absorbance and concentration.

Why is it important to prepare standard solutions when constructing a calibration curve?

Standard solutions help establish a known relationship between concentration and absorbance, ensuring accurate interpolation of unknown samples.

What is the role of the Mixed Colour Reagent (MCR) in phosphorus analysis?

The MCR reacts with orthophosphate in the sample to form a blue-colored complex, allowing for absorbance measurement at 882 nm using a spectrophotometer

How do you calculate the concentration of an unknown sample using a calibration curve?

The concentration of the unknown sample is determined using the equation of the calibration curve (y = mx + c), where absorbance (y) is used to solve for phosphorus concentration (x)

What does the coefficient of determination (R²) indicate in a calibration curve?

R2 measures how well the calibration curve fits the data. a value close to 1 suggests a strong correlation between absorbance and concentration.

Why must samples be incubated at 37°C after adding the Mixed Colour Reagent?

Incubation helps develop the blue color complex by ensuring complete reaction between orthophosphate and the reagent.

What should be done if the absorbance of an unknown sample exceeds that of the highest standard solution?

The sample should be diluted, and the dilution factor must be applied when calculating the final phosphorus concentration

What safety precautions must be followed when handling the Mixed Colour Reagent?

The MCR is corrosive. Appropriate PPE, including gloves and safety gogles must be worn.

What is the significance of the slope (m) in the calibration curve equation?

The slope represents sensitivity - how much absorbance changes per unit concentration. A steeper slope indicates higher sensitivity.

2. Absorbance Calculation using Calibration Curve

Your calibration curve equation from standard solutions is:

y=1.25x+0.03y = 1.25x + 0.03

where:

• yy = absorbance

• xx = concentration (mg P/L)

An unknown sample gives an absorbance reading of 0.78 at 882 nm.

• Calculate the phosphorus concentration in the unknown sample.

Solution: Rearranging the equation to solve for xx:

0.78=1.25x+0.030.78 = 1.25x + 0.03

0.78−0.03=1.25x0.78 - 0.03 = 1.25x

0.75=1.25x0.75 = 1.25x

x=0.751.25=0.60 mg P/Lx = \frac{0.75}{1.25} = 0.60 \text{ mg P/L}

The phosphorus concentration in the unknown sample is 0.60 mg P/L.

3. Dilution Factor Adjustment

You measured an absorbance of 1.50, but this is outside the linear range of your calibration curve. You diluted the sample 1:4 with DI water (1 part sample, 3 parts water).

• What is the original phosphorus concentration before dilution?

📌 Solution: Since the dilution factor is 4, multiply the measured concentration by 4.

Using the calibration curve equation:

y=1.25x+0.03

Solution: Since the dilution factor is 4, multiply the measured concentration by 4.

Using the calibration curve equation:

y=1.25x+0.03y = 1.25x + 0.03

Solving for xx:

1.50=1.25x+0.031.50 = 1.25x + 0.03

1.50−0.03=1.25x1.50 - 0.03 = 1.25x

1.47=1.25x1.47 = 1.25x

x=1.471.25=1.176 mg P/Lx = \frac{1.47}{1.25} = 1.176 \text{ mg P/L}

Multiplying by the dilution factor:

1.176×4=4.704 mg P/L1.176 \times 4 = 4.704 \text{ mg P/L}

So, the original concentration before dilution was 4.70 mg P/L.