DEPARTMENT OF CHEMISTRY

FACULTY OF SCIENCE OF MATHEMATICS

LAB REPORT
ANALYTICAL CHEMISTRY 2
SKA 3023

EXPERIMENT 1

DETERMINATION OF IRON USING UV-VISIBLE

(UV-VIS SPECTROHOTOMETER)
1

NAME OF GROUP MEMBERS: MATRIC NO:

AIN SUFIZA BINTI NORMAN ZAIRI D20171078202

SITI AISYAH BINTI SHA’ARI D20171078185

DAY/TIME 5 NOVEMBER 2019 ( 8- 11 AM)

LABORATORY B3-3-MK 13

LECTURER ‘S NAME DR. MAZLINA BINTI MUSA

EXPERIMENT 1

DETERMINATION OF IRON USING UV-VISIBLE (UV-VIS) SPECTROMETER

OBJECTIVES:

1. Prepare samples for UV-Visible analysis.

2. Plot calibration curve for standard solutions.
3. Perform UV-Visible analysis on aqueous solutions.

INTRODUCTION

Ultraviolet-visible (UV-Vis) spectroscopy is one of the most popular analytical

techniques because it is very versatile and able to detect nearly every molecule. With UV-Vis
spectroscopy, the UV-Vis light is passed through a sample and the transmittance of light by
a sample is measured. From the transmittance (T), the absorbance can be calculated as A=-
log (T). An absorbance spectrum is obtained that shows the absorbance of a compound at
different wavelengths. The amount of absorbance at any wavelength is due to the chemical
structure of the molecule.

UV-Vis can be used in a qualitative manner, to identify functional groups or confirm

the identity of a compound by matching the absorbance spectrum. It can also be used in a
quantitative manner, as concentration of the analyte is related to the absorbance using
Beer's Law. UV-Vis spectroscopy is used to quantify the amount of DNA or protein in a
sample, for water analysis, and as a detector for many types of chromatography. Kinetics of
chemical reactions are also measured with UV-Vis spectroscopy by taking repeated UV-Vis
measurements over time. UV-Vis measurements are generally taken with a
spectrophotometer. UV-Vis is also a very popular detector for other analytical techniques,
such as chromatography, because it can detect many compounds.

Typically, UV-Vis is not the most sensitive spectroscopy technique, because not a lot
of light is absorbed over a short path length. Other spectroscopy techniques such as
fluorescence have higher sensitivity, but they are not as generally applicable, as most
molecules are not fluorescent. UV-Vis has a similar sensitivity to other absorbance
measurements, such as infrared spectroscopy.
 A complex of iron (II) is formed with 1,10-phenantroline (Fe(C12H8N2)32+ and the
absorbance of this coloured solution is measured with a UV-Visible spectrometer. The
spectrum is plotted to determine the absorption maximum. Hydroxylamine (as the
hydrochloride salt to in increase solubility) is added to reduce any Fe3+ to Fe 2+
and to
maintain it in that state.

Equation:

4Fe 2+ + 2NH2OH ⟶ 4Fe2+ + N2O + 4H+ + H2O

MATERIALS

Chemicals

Ferrous ammonium sulphate, concentrated sulfuric acid, 1,10-phenantroline monohydrate,

hydroxylamine hydrochloride, sodium acetate, distilled water.

Apparatus

Beaker (100mL), volumetric flask (100mL and 1L), pipette (1,5,10,25 and 50 mL), measuring
cylinder (10mL).
METHODOLOGY

PART A: Preparation of solutions

1. Standard iron (II) solution was prepared by weighing 0.0700 g ferrous ammonium
sulfate Fe(NH4)2(SO4)2.6H2O. in 100mL beaker and dissolved in 30 mL distilled
water. The solution is then, quantitatively transferred to a 1L volumetric flask and
sufficient distilled water was added to dissolve the salt. 2.5 mL concentrated sulfuric
acid was added and diluted exactly to the mark with distilled water and mixed
thoroughly.
2. 1,10-phenantroline solution was prepared by dissolving 0.1 g 1,10-phenantroline
monohydrate in 100 ml distilled water in a beaker.
3. Hydroxylamine hydrochloride solution was prepared by dissolving 5g of
hydroxylamine hydrochloride in 50 mL distilled water in a beaker.
4. Sodium acetate solution was prepared by dissolving 10 g of sodium acetate in 100
mL distilled water in a beaker.

PART B: UV-Visible analysis.

1. A series of 100 mL volumetric flasks was added with 1, 5, 10, 25 and 50 mL of the
standard iron solution by using pipettes.
2. In another 100 mL volumetric flask, 50 mL of distilled water is placed for a blank.
3. 10 mL of solution X (unknown sample) is placed into a 100 volumetric flask by using
pipette.
4. 1 mL of the hydroxylamine hydrochloride solution, 10 mL of the 1, 10-phenantroline
solution and 8 mL of the sodium acetate solution were added to each volumetric flask
including the blank and unknown. Distilled water was added until the calibrated mark
and the solution was rested for 15 minutes and was shake once in a while before
making absorbance measurements.
5. Absorbance for the iron (II) solution is measured by using the blank solution as the
reference solution and one of the standard iron (II) solutions prepared in procedure 1
at the wavelength of 400-600 nm. Readings was taken at 20-nm intervals.
6. The graph of absorbance against wavelength was plotted and the maximum
wavelength of the absorption was selected.
7. The absorbance of each standard solutions of the maximum wavelength was
measured and a calibration curve was prepared. The unknown was measured in the
same way.
8. The graph of absorbance of the standards against concentration in ppm was plotted
and the concentration of iron in unknown was determined.
RESULTS

PART A: Preparation of Fe (II) standard solution.

Weight of ferrous ammonium sulfate: 0.07 𝑔

Concentration of Fe (II) standard solution in mg Fe 𝑚𝐿−1 : 9.97 × 10−3 𝑚𝑔 𝐹𝑒 𝑚𝐿−1

Calculation:

g
0.07 𝑔 Fe(NH4)2(SO4)2.6H2O × 1000mg × 55.845
𝑚𝑔 𝐹𝑒 𝑚𝐿 −1
= mol
1000 𝑚𝑙 × 1𝑔 × 392.14 𝑔 Fe(NH4)2(SO4)2.6H2O
= 9.97 𝑚𝑔 𝐹𝑒 𝑚𝐿−1

PART B: Curve of the absorbance of ferum-1, 10-phenantroline complex against

wavelength

Concentration of Fe (II) standard solution used: 4.99 × 10−3 𝑚𝑔 𝐹𝑒 𝑚𝐿−1

ʎ (nm) Absorbance (A)

400 0.318

420 0.356

440 0.448

460 0.514

480 0.585

500 0.614

505 0.627

510 0.634

515 0.624

520 0.591

540 0.312

560 0.103

580 0.036

600 0.009
 Absorbance (A) against ʎ (nm)
0.7

0.65

0.6

0.55

0.5

0.45
Absorbance (A)

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
ʎ (nm)

From the graph, maximum ʎ: 510 (nm)

PART C: Calibration curve for the determination of Fe (II)

Volume of Fe (II) stock Concentration, Absorbance (A) at

solution (mL) (𝒎𝒈 𝑭𝒆 𝒎𝑳−𝟏 ) maximum ʎ

1 9.97 x 10-5 0.018

5 4.99 x 10-4 0.095

10 9.97 x 10-4 0.204

25 2.49 x 10-3 0.518

50 4.99 x 10-3 0.634

Unknown 1.00 x 10-3 0.206

 Absorbance (A) against concentration (mg Fe mL-1)
0.7

0.6

0.5
Absorbance (A)

0.4

0.3

0.2

0.1

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Concentration ( × 10-3 mg Fe mL-1)

From graph, concentration of unknown: 1.0 𝑚𝑔 𝐹𝑒 𝑚𝐿−1

Calculation of the real concentration of solution x:

M1V1 = M2V2
(X)(10mL) = (100ml)(1 x 10-3 𝑚𝑔 𝐹𝑒 𝑚𝐿−1 )

X= 0.01 𝑚𝑔 𝐹𝑒 𝑚𝐿−1

Concentration of solution X: unknown: 𝟎. 𝟎𝟏 𝒎𝒈 𝑭𝒆 𝒎𝑳−𝟏

DISCUSSIONS

UV-Vis is a fast, simple and inexpensive method to determine the concentration of an

analyte in solution. It can be used for relatively simple analysis, where the type of compound
to be analyzed (‘analyte’) is known, to do a quantitative analysis to determine the
concentration of the analytes.

Standard iron (II) solution, 1,10-phenantroline solution, hydroxylamine hydrochloride solution

and sodium acetate solution was prepare before we begin our analysis. 1mL of
hydroxylamine hydrochloride solution, 8mL of sodium acetate solution and 10mL of 1,10-
phenantroline solution was added to each 1, 5, 10, 25, 50 mL of the standard iron solution
before adding distilled water to the mark. Blank sample was prepared by pouring 50 mL
distilled water into another 100 mL volumetric flask. We also pipette solution X (unknown
sample) into a 100 mL volumetric flask.

In this experiment the concentration of iron in unknown solution can be know from
calibration curve. We must know the maximum wavelength of absorbance to prepare the
calibration curve. Hence, we perform absorbance for the iron solution chosen at the
wavelength of 400-600 nm by using the concentration of fe(II) standard solution of 4.985 x
103 mg Fe mL-1. From the curve of absorbance of ferum-1,10-phenantroline complex against
wavelength we obtained the highest absorbance which is 0.634 A at 510 nm. Now we are
able to perform calibration curve to determine the concentration Fe(II) of the unknown by
using maximum wavelength obtained which is 510 nm. In this experiment, we used blank
solution as reference solution. The calibration curve is absorbance of the standard against
the concentration of Fe (II) in ppm. From this plot and the absorbance of unknown we will
find the concentration of iron of the stock solution by the calculation,

M1V1 = M2V2

Where,

M1 = The concentration of Fe (II) standard solution in mg Fe mL-1

V1 =Volume of pipetted Fe (II) stock solution (mL)

M2 = The concentration of Fe (II)

V2 = 100 mL
The concentration iron in unknown solution is determined by the calibration curve in which
from y-axis of absorbance (A) at 0.206 A down to the x-axis to get the concentration iron of
the unknown at 1.00 x 10-3 mg Fe mL-1.

CONCLUSIONS

 Samples for UV-Visible analysis are prepared.

 Calibration curved for standard solutions is plotted.
 UV-Visible analysis on aqueous solution is performed.

REFERENCES

 Schwartz, L.M. “Advances in Acid-Base Gran Plot Technology,” J. Chem. Educ., 64,
947-950 (1987).

 Christian, G.D., Analytical Chemistry, 6th edition. New Jersey. John Wiley, 2004.

 Filby, G. “Spreadsheets for Chemists,” VCH Publishers, New York, 1995, p. 210.

QUESTIONS

1. What types of electrons in a molecule are generally involved in the absorption

of UV or visible radiation?
 Closed-shell electrons
 Paired nonbonding outershell electrons
 Covalent single bond electrons

2. Identify factors that cause the Beer’s Law relationship to depart from linearlity?
 Gravimetric errors – eg. mood
 Incomplete spectral resolution – due to wrong slit width selection
 Turbidity – must filter the sample first to avoid cloudy
 Aggregation at high concentration – the particles stick together
 Contamination of cuvettes – must clean the cuvettes immediately after using
3. Calculate the frequency in hertz and the energy in joules of an X-ray photon
with a wavelength of 2.70Å (1eV = 1.062 J)
Frequency:
𝑐
𝑣=
ʎ
3 × 108 𝑚𝑠 −1
𝑣=
2.7 × 10−10 𝑚
𝑣 = 1.11 × 1018 𝑠 −1
Energy:
𝐸 = ℎ𝑣
𝐸 = 6.62 × 10−34 𝐽𝑠 × 1.11 × 1018 𝑠 −1
𝐸 = 7.35 × 10−16 𝐽

4. Describe the absorption phenomena taking place in the far-infrared, mid-

infrared, and visible-ultraviolet regions of the spectrum.

There are three main processes by which a molecule can absorb radiation.
and each of these routes involves an increase of energy that is proportional to the
light absorbed. The first route occurs when absorption of radiation leads to a higher
rotational energy level in a rotational transition. The second route is a vibrational
transition which occurs on absorption of quantized energy. This leads to an increased
vibrational energy level. The third route involves electrons of molecules being raised
to a higher electron energy, which is the electronic transition. It’s important to state
that the energy is quantized and absorption of radiation causes a molecule to move
to a higher internal energy level. This is achieved by the alternating electric field of
the radiation interacting with the molecule and causing a change in the movement of
the molecule. There are multiple possibilities for the different possible energy levels
for the various types of transitions.

The energy levels can be rated in the following order: electronic > vibrational
> rotational. Each of these transitions differs by an order of magnitude. Rotational
transitions occur at lower energies (longer wavelengths) and this energy is
insufficient and cannot cause vibrational and electronic transitions but vibrational
(near infra-red) and electronic transitions (ultraviolet region of the electromagnetic
spectrum) require higher energies.
 The energy of IR radiation is weaker than that of visible and ultraviolet
radiation, and so the type of radiation produced is different. Absorption of IR radiation
is typical of molecular species that have a small energy difference between the
rotational and vibrational states. A criterion for IR absorption is a net change in dipole
moment in a molecule as it vibrates or rotates.

5. You had prepared a blank solution in step 2. Discuss the characteristics of the
blank solution in this experiment.
 Contain no analyte of interest
 Used to calibrate the spectrophotometer readings