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Concentration vs. Absorbance: 1. Standard Curve

The document describes a procedure to determine the concentration of an unknown iron sample using UV-Vis spectrophotometry. A standard curve is constructed by measuring the absorbance of iron solutions with known concentrations at 510 nm. The relationship between absorbance and concentration is linear with an R2 value of 0.9997. This standard curve is used to determine the concentration of three trials of a diluted unknown sample. The mean concentration is calculated to be 0.07042 mM with a standard deviation of 0.02 mM. Conditions for applying Beer's law and actions to take if a sample's absorbance is outside the linear range are also discussed.

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Hee Min
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188 views2 pages

Concentration vs. Absorbance: 1. Standard Curve

The document describes a procedure to determine the concentration of an unknown iron sample using UV-Vis spectrophotometry. A standard curve is constructed by measuring the absorbance of iron solutions with known concentrations at 510 nm. The relationship between absorbance and concentration is linear with an R2 value of 0.9997. This standard curve is used to determine the concentration of three trials of a diluted unknown sample. The mean concentration is calculated to be 0.07042 mM with a standard deviation of 0.02 mM. Conditions for applying Beer's law and actions to take if a sample's absorbance is outside the linear range are also discussed.

Uploaded by

Hee Min
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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1.

Standard curve:
Wavelength at absorbance maximum = 510 nm
The concentration of stock solution:
𝑚 0.07
nMorh’s salt = = = 0.1785 x 10-3 (mol/L) = 0.1785 (mmol/L or mM)
𝑀 392.13

Tube # 1 2 3 4 5
Dilution 1:100 1:50 1:20 1:10 1:5
Concentration of
0.1785 x 10-2 0.3570 x 10-2 0.8925 x 10-2 0.01785 0.03570
Fe (mM)
Absorbance 0.025 0.038 0.103 0.210 0.415

Fit the data points to a linear function in which absorbance is y-axis and concentration is x-axis.
(y= ax, b=0 as the absorbance = 0 when [Fe2+] = 0).

Concentration vs. Absorbance


0.45
0.4 y = 11.644x
R² = 0.9997
0.35
0.3
Absorbance

0.25
0.2
0.15
0.1
0.05
0
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04
Concentration (mM)

From the linear function above:


 Equation of the relationship between concentration and absorbance:
𝑦 = 11.644𝑥
 R = 0.9997
2

2. Determination of concentration of unknown sample:


Using the equation above, the concentration of the unknown sample can be calculate by
𝑦
formula: 𝑥 =
11.644
Trial # Absorbance Concentration (mM) in Concentration
the tube (dilute from (mM) of the
sample 10 times) sample
1 0.080 0.006870 0.06870
2 0.082 0.007042 0.07042
3 0.084 0.007214 0.07214
𝑥1 +𝑥2 +𝑥3 0.06870+0.07042+0.07214
Mean: 𝑥 = = = 0.07042 ≈ 0.07 (mM)
𝑛 3
∑𝑛
𝑖=1(𝑋𝑖 −𝑋)
2
Standard deviation: 𝑠 = √ = 0.172 × 10−2 ≈ 0.02
𝑛−1

3. Question:
a. Under what conditions that the Beer’s law can be used to determine the concentration of
sample?
 The condition for using Beer’s law valid:
- The sample is at a low concentration and must be the dilute solution. The reason is that,
at the high concentration, the distances between ions or molecules in solution are small
to make the absorbing species dependent to each other.
- The analyst must not have the reaction, dissociation or association with the solvent.
- Absorbance must be in the linear range.
- The light which passes through the sample must be monochromatic.
b. What should be done if the absorbance of a sample is above the linear range of the standard
curve?
 If the absorbance of a sample is above the linear range, that mean the sample is diluted
and the concentration of the sample and diluent blank are measured from the calibration
curve. So the concentration of the sample less the dilution blank is multiplied by the dilution
factor then from this is subtracted the concentration of the sample blank.
4. Conclusion:
 Discussion:
- The increasing in the dark of solution’s color related to the concentration of the
solution.
- As the concentration of standard solutions is known, we can use the highest
concentration solution (tube #5) to determine the wavelength where the absorbance is
maximum.
- Base on the relationship of the absorbance and the concentration of the standard
solution, we can construct a calibration curve and determine the unknown
concentration.
- The final results can be affect by the contamination during the experiment.
 Conclusion:
- The concentration of iron ion in the sample is…
- Spectrophotometric method is one of the most important technique in chemistry
analysis. When doing the experiment with this method, we should pay attention to
some factor that can affect the result.
- To apply the Beer’s law, we should make sure that the solutions are diluted and their
concentrations are not high and the light must be monochromatic.
- When using the spectrophotometer, we must make sure the machine and the cuvettes
are clean for the light passing through.

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