Introduction and Purpose

In this experiment, the mass of the iron content in a vitamin capsule is determined using

UV-Vis spectrophotometry. An indicator called ortho-phenanthroline is used to form complex

with iron, and a colour is produced for spectrometric measurement. And before the

measurements, a blank solution is used to calibrate the spectrometer. Since a blank solution

should contain everything except the analyte, the blank solution used in this lab contained

sodium citrate solution, hydroquinone, and o-phenanthroline solution. The spectrometer

measures the transmittance of the solution and gives an absorption value. The absorbance is

the log ratio of the incident light beam and the exiting light beam. And, according to Beer

Lambert Law, the absorbance is directly proportional to the concentration of the solution, the

path length, and the molar attenuation coefficient. Therefore, since they have a linear

relationship, a calibration curve can be constructed using the data points of standard solutions

with different concentrations. And the linear equation of the calibration curve can then be used

to calculate the concentration of the analyte. Besides the unknown concentration, the

calibration curve could also be used to calculate the limit of detection and limit of linear

response. 1

An example from literature that contained the utilization of calibration curve was an

experiment done in 2013, where a homemade photometer was being tested. And the

concentration of sodium in a sports drink was determined using a calibration curve. Sodium is

an electrolyte that is critical to human bodies, helping cells to function properly.2 They can be

measured by electrodes or flame photometry. 3 In this experiment, the sport drink was diluted

into seven standards: 0, 5, 10, 20, 30, 40, and 50ppm Na. 3 Then, a sample of Gatorade was
diluted into 0.05 times of its original concentration and was measured using the homemade

photometer. After that, all the points of the standard solutions were plotted on a graph, and a

calibration curve was formed. Lastly, the concentration value of the sodium content in

Gatorade was obtained by interpolation.

Calculations

[Part 1]

𝟎. 𝟎𝟔𝟓 + 𝟎. 𝟎𝟔𝟒 + 𝟎. 𝟎𝟔𝟕 + 𝟎. 𝟎𝟔𝟕 + 𝟎. 𝟎𝟕𝟎 + 𝟎. 𝟎𝟕𝟏

𝑨𝒗𝒈 𝑨𝒃𝒔 𝒐𝒇 𝟏𝒎𝑳 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 =
𝟔

≈ 𝟎. 𝟎𝟔𝟕𝟑

𝑆𝑡𝑑 𝐷𝑒𝑣

(0.065 − 0.0673)! + (0.064 − 0.0673)! + (0.067 − 0.0673)! + (0.067 − 0.0673)! + (0.070 − 0.0673)! + (0.071 − 0.0673)!
=)
6−1

= 0.00273

𝟎. 𝟏𝟐𝟗 + 𝟎. 𝟏𝟐𝟗 + 𝟎. 𝟏𝟑𝟏 + 𝟎. 𝟏𝟐𝟗 + 𝟎. 𝟏𝟐𝟗 + 𝟎. 𝟏𝟑𝟎

𝑨𝒗𝒈 𝑨𝒃𝒔 𝒐𝒇 𝟐𝒎𝑳 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 =
𝟔

≈ 𝟎. 𝟏𝟐𝟗𝟓

𝑆𝑡𝑑 𝐷𝑒𝑣

(0.129 − 0.1295)! + (0.129 − 0.1295)! + (0.131 − 0.1295)! + (0.129 − 0.1295)! + (0.129 − 0.1295)! + (0.130 − 0.1295)!
=)
6−1

= 0.000837

𝟎. 𝟑𝟑𝟒 + 𝟎. 𝟑𝟑𝟓 + 𝟎. 𝟑𝟑𝟒 + 𝟎. 𝟑𝟑𝟑 + 𝟎. 𝟑𝟑𝟒 + 𝟎. 𝟑𝟑𝟒

𝑨𝒗𝒈 𝑨𝒃𝒔 𝒐𝒇 𝟓𝒎𝑳 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 =
𝟔

≈ 𝟎. 𝟑𝟑𝟒𝟎
𝑆𝑡𝑑 𝐷𝑒𝑣

(0.334 − 0.3340)! + (0.335 − 0.3340)! + (0.334 − 0.3340)! + (0.333 − 0.3340)! + (0.334 − 0.3340)! + (0.334 − 0.3340)!
=)
6−1

= 0.000632

𝟎. 𝟔𝟒𝟑 + 𝟎. 𝟔𝟒𝟐 + 𝟎. 𝟔𝟒𝟑 + 𝟎. 𝟔𝟒𝟏 + 𝟎. 𝟔𝟒𝟏 + 𝟎. 𝟔𝟒𝟑

𝑨𝒗𝒈 𝑨𝒃𝒔 𝒐𝒇 𝟏𝟎𝒎𝑳 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 =
𝟔

≈ 𝟎. 𝟔𝟒𝟐𝟐

𝑆𝑡𝑑 𝐷𝑒𝑣

(0.643 − 0.6422)! + (0.642 − 0.6422)! + (0.643 − 0.6422)! + (0.641 − 0.6422)! + (0.641 − 0.6422)! + (0.643 − 0.6422)!
=)
6−1

= 0.000983

𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝟏𝒎𝑳 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 = 𝟕. 𝟐𝟓𝟓 × 𝟏𝟎!𝟒 𝑴 × 𝟏 𝒎𝑳 ÷ 𝟏𝟎𝟎 𝒎𝑳

= 𝟕. 𝟐𝟓𝟓 × 𝟏𝟎!𝟔 𝑴

&
3 × 10!% 𝑀 0.012 𝑚𝐿 & 0.08 𝑚𝐿 &
𝑠 = 7.255 × 10 𝑀 × CD
!$
F + H K + H K = 8.73 × 10!' 𝑀
7.255 × 10!$ 𝑀 1 𝑚𝐿 100 𝑚𝐿

𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝟐𝒎𝑳 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 = 𝟕. 𝟐𝟓𝟓 × 𝟏𝟎!𝟒 𝑴 × 𝟐 𝒎𝑳 ÷ 𝟏𝟎𝟎 𝒎𝑳

= 𝟏. 𝟒𝟓𝟏 × 𝟏𝟎!𝟓 𝑴

&
3 × 10!% 𝑀 0.012 𝑚𝐿 & 0.08 𝑚𝐿 &
𝑠 = 1.451 × 10 𝑀 × CD
!)
F + H K + H K = 8.80 × 10!' 𝑀
7.255 × 10!$ 𝑀 2 𝑚𝐿 100 𝑚𝐿

𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝟓𝒎𝑳 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 = 𝟕. 𝟐𝟓𝟓 × 𝟏𝟎!𝟒 𝑴 × 𝟓 𝒎𝑳 ÷ 𝟏𝟎𝟎 𝒎𝑳

= 𝟑. 𝟔𝟐𝟖 × 𝟏𝟎!𝟓 𝑴

&
3 × 10!% 𝑀 0.02 𝑚𝐿 & 0.08 𝑚𝐿 &
𝑠 = 3.628 × 10!) 𝑀 × CD F + H K + H K = 1.49 × 10!% 𝑀
7.255 × 10!$ 𝑀 5 𝑚𝐿 100 𝑚𝐿

𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝟏𝟎𝒎𝑳 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 = 𝟕. 𝟐𝟓𝟓 × 𝟏𝟎!𝟒 𝑴 × 𝟏𝟎 𝒎𝑳 ÷ 𝟏𝟎𝟎 𝒎𝑳

= 𝟕. 𝟐𝟓𝟓 × 𝟏𝟎!𝟓 𝑴

&
3 × 10!% 𝑀 0.04 𝑚𝐿 & 0.08 𝑚𝐿 &
𝑠 = 7.255 × 10 𝑀 × CD
!)
F +H K +H K = 2.97 × 10!% 𝑀
7.255 × 10!$ 𝑀 10 𝑚𝐿 100 𝑚𝐿
 Calibration Curve of the Iron Content in Stock Solutions
0.7000
y = 8831.3x + 0.0049
0.6000 R² = 0.9995

0.5000
Absorbance

0.4000

0.3000

0.2000

0.1000

0.0000
0.000E+00 1.000E-05 2.000E-05 3.000E-05 4.000E-05 5.000E-05 6.000E-05 7.000E-05 8.000E-05
Concentration (M)

Graph 1: The calibration curve of the iron concentrations of the stock solutions. Note that the error bars are too
small to be seen.

𝒔𝒍𝒐𝒑𝒆 = 𝟖𝟖𝟑𝟏. 𝟑 𝑴!𝟏 ± 𝟏𝟒𝟏. 𝟕 𝑴!𝟏

𝒚 − 𝒊𝒏𝒕𝒆𝒓𝒄𝒆𝒑𝒕 = 𝟎. 𝟎𝟎𝟒𝟗 ± 𝟎. 𝟎𝟎𝟓𝟗

[Part 2]

𝟎. 𝟏𝟕𝟎 + 𝟎. 𝟏𝟕𝟎 + 𝟎. 𝟏𝟕𝟎 + 𝟎. 𝟏𝟕𝟎 + 𝟎. 𝟏𝟕𝟏 + 𝟎. 𝟏𝟕𝟎

𝑨𝒗𝒈 𝑨𝒃𝒔 𝒐𝒇 𝑽𝒊𝒕𝒂𝒎𝒊𝒏 𝑺𝒕𝒐𝒄𝒌 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 − 𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝟐 =
𝟔

≈ 𝟎. 𝟏𝟕𝟎𝟐

𝑆𝑡𝑑 𝐷𝑒𝑣

(0.170 − 0.1702)! + (0.170 − 0.1702)! + (0.170 − 0.1702)! + (0.170 − 0.1702)! + (0.171 − 0.1702)! + (0.170 − 0.1702)!
=)
6−1

= 0.000408

𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝑽𝒊𝒕𝒂𝒎𝒊𝒏 𝑺𝒕𝒐𝒄𝒌 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 − 𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 #𝟐: 𝒚 = 𝟎. 𝟏𝟕𝟎𝟐 = 𝟖𝟖𝟑𝟏. 𝟑𝒙 + 𝟎. 𝟎𝟎𝟒𝟗

𝟎. 𝟏𝟔𝟓𝟐 = 𝟖𝟖𝟑𝟏. 𝟑𝒙

𝒙 ≈ 𝟏. 𝟖𝟕 × 𝟏𝟎!𝟓 𝑴
 &
√0.000408& + 0.00586& 141.7 &
𝑠 = 1.87 × 10 𝑀 × CD
!)
F +H K = 7.30 × 10!% 𝑀
0.1652 8831.3

𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝑽𝒊𝒕𝒂𝒎𝒊𝒏 𝑺𝒕𝒐𝒄𝒌 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 − 𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 #𝟏 = 𝟏. 𝟖𝟕 × 𝟏𝟎!𝟓 × 𝟏𝟎𝟎 𝒎𝑳 ÷ 𝟏𝟎 𝒎𝑳

≈ 𝟎. 𝟎𝟎𝟎𝟏𝟖𝟕 𝑴

&
7.30 × 10!% 0.04 & 0.08 &
𝑠 = 0.000187 𝑀 × CD F + H K + H K = 7.34 × 10!$ 𝑀
1.87 × 10!) 10 100

𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝑽𝒊𝒕𝒂𝒎𝒊𝒏 𝑺𝒕𝒐𝒄𝒌 𝑺𝒐𝒍𝒖𝒕𝒊𝒐𝒏 = 𝟎. 𝟎𝟎𝟎𝟏𝟖𝟕𝑴 × 𝟏𝟎𝟎 𝒎𝑳 ÷ 𝟓 𝒎𝑳 ≈ 𝟎. 𝟎𝟎𝟑𝟕𝟒 𝑴

&
7.34 × 10!$ 0.02 & 0.08 &
𝑠 = 0.00374 𝑀 × CD F +H K +H K = 0.000148 𝑀
0.000187 5 100

𝒎𝒂𝒔𝒔 𝑭𝒆 = 𝟎. 𝟎𝟎𝟑𝟕𝟒 𝑴 × 𝟎. 𝟏 𝑳 × 𝟓𝟓. 𝟖 𝒈/𝒎𝒐𝒍 = 𝟎. 𝟎𝟐𝟎𝟗 𝒈

0.000148 & 0.08 &

𝑠 = 0.0209 𝑔 × CH K +H K = 0.000823 𝑔
0.00374 100

Results Sheets

Dilute Standard Iron Solution [FeSO4(NH4)2SO4 ⋅ 6H2O] = 7.255× 10-4 M

Calibration Curve (λ used = 530nm)

Volume of [Fe2+] Absorbance Total Mass of Fe in the
Standard Prepared Standard Solution

10mL 7.255 × 10-5 M ± 3.0 × 10-7 M 0.6422 ± 0.0010 4.048 × 10-4 g ± 1.7 × 10-6 g

5mL 3.628 × 10-5 M ± 1.5 × 10-7 M 0.3340 ± 0.0006 2.024 × 10-4 g ± 8.5 × 10-7 g

2mL 1.451 × 10-5 M ± 8.8 × 10-8 M 0.1295 ± 0.0008 8.097 × 10-5 g ± 5.0 × 10-7 g

1mL 7.255 × 10-6 M ± 8.7 × 10-8 M 0.0673 ± 0.0027 4.048 × 10-5 g ± 4.9 × 10-7 g

Vitamin Capsule Sample

Absorbance of Vitamin Stock Overall Dilution Factor Total Fe (mg) in the Capsule
Dilution #2 Provided
0.1702 1:200 0.0209g ± 0.0008g
References

1. Calibration Curves | Protocol. https://www.jove.com/v/10188/calibration-curves (accessed

Nov 4, 2021)

2. Sodium. https://labtestsonline.org/tests/sodium (accessed Nov 4, 2021).

3. LaFratta, C. N.; Jain, S.; Pelse, I.; Simoska, O.; Elvy, K. Using a Homemade Flame Photometer
to Measure Sodium Concentration in a Sports Drink. J. Chem Educ [Online] 2013, 90 (3),
372–375. https:// dx.doi.org/10.1021/ed3004758 (accessed Nov 4, 2021)