CHM260

BASIC INSTRUMENTAL ANALYSIS

LABORATORY SUMMARY WRITTEN

REPORT

Name : Nurul Sabrina Binti Johar

ID No. : 2018633698
Programme : AS120
Instructor : Madam Norsakina Zurina
Zukifli
 EXPERIMENT 1:
The Visible Spectra of Soft Drinks
(Refer to Lab manual Exp. 1 and lecture notes to answer the following pre and post
laboratory questions)

A. Pre-laboratory questions

a) Define spectroscopy and state type of electromagnetic radiation used in this experiment.
- Spectroscopy is the study of interaction between electromagnetic radiation and matter or a
technique of splitting electromagnetic radiation into its constituent wavelengths (a spectrum),
in much the same way as a prism splits light into a rainbow of colours. Type of
electromagnetic radiation used in this experiment is visible light spectrum.
b) Define the terms of transmittance and absorbance.
- Transmittance, T is the fraction of incident radiation transmitted through the sample medium.
- Absorbance, A is a measurement of the amount of radiant power absorbed by the sample
defined as the -ve log of T.
c) State Beer’s law mathematically and max.
- A = abc ; A = absorbance, a = absorptivity, b = path length, c = concentration
- A = εbc ; A = absorbance, ε = molar absorptivity, b = path length, c = concentration
- max = The wavelength at which a substance has its strongest photon absorption (highest point
along the spectrum's y-axis) and the point where absorbance is greatest.
B. Post-laboratory questions

a) Based on Beer’s Law, when the concentration of an analyte increases, how will the
following be affected (increase, decrease, no change)
i. Absorbance
- Increase, as A= abc
ii. Transmittance
- Decrease, as A= - log T
b) Why essential to obtain the absorption spectrum of the soft drinks first before developing
a calibration curve?
 - The importance of obtaining the absorption spectrum before making the calibration
curve is generally we will select the wavelength of maximum absorbance for a given
sample and use it in our absorbance measurements.
c) What is the purpose of using the ‘blank’ solution?
- The purpose of using a blank is to calibrate the spectrophotometer to zero absorbance.
d) Name the colours absorbed in soft drink samples.
- Based on the lambda max, the color absorb by our soft drink is blue-green.

C. Complete the following table with wavelength and absorption given by the instructor

Wavelength (nm) Absorbance

600 0.040

580 0.214

560 0.574

540 0.770

520 0.885

500 0.796

480 0.610

460 0.383

440 0.252

420 0.213

400 0.207

380 0.194

360 0.200

λ max=520 nm
D. Complete the table of soft drinks concentration (volume %) and absorbance

Solutions Concentration (volume %) Absorbance

1 5 0.179

2 10 0.362

3 15 0.537

4 20 0.714

5 25 0.885

Unknown sample 13.54 0.245

 E. Report summary (1-2 pages)

Introduction

Spectroscopy is related to the interaction of light with matter. As light is absorbed by

matter, the result is an increase in the energy content of the atoms or molecules. When ultraviolet
radiations are absorbed, this results in the excitation of the electrons from the ground state
towards a higher energy state. Molecules containing π-electrons or non-bonding electrons (n-
electrons) can absorb energy in the form of ultraviolet light to excite these electrons to higher
anti-bonding molecular orbitals. The more easily excited the electrons, the longer the wavelength
of light it can absorb. There are four possible types of transitions (π–π*, n–π*, σ–σ*, and n–σ*),
and they can be ordered as follows: σ–σ* > n–σ* > π–π* > n–π*. The absorption of ultraviolet
light by a chemical compound will produce a distinct spectrum which aids in the identification of
the compound.

In this experiment, we will determine the concentration of an unknown solution of soft

drink. In order to accomplish this goal, we must first determine what wavelength of light the
solution best absorbs (the wavelength of maximum absorbance). This maximum wavelength will
also help us to find for the origin of the color of a soft drink sample. Then we will prepare
solutions of known concentration, measure their absorbance, and create a calibration plot. We
can then use information from that plot to determine the concentration of our unknown.

Spectrophotometer instrument that we used in this experiment is Spectronic 20 (Spec 20).

The Spectronic 20 is a single-beam spectrophotometer designed to operate in the visible
spectrum across a wavelength range of 340 nm to 950 nm, with a spectral band pass of 20 nm.
The spectrophotometer consists of a light source, a grating, slits, a sample compartment, and a
detector. The Spectronic 20 operates in the visible range only and uses a prism as its
monochromator. It is designed for quantitative absorption measurement at single wavelengths. A
spectrophotometer can be used to quantitatively establish the Beer-Lambert (Beer’s Law)
relationship for analytes that absorb in the spectrophotometer's range. Beer’s Law states that A =
εbc. Once the Beer-Lambert relationship is established, data can be collected for the
determination of the molar extinction coefficient (ε), the path length of the sample cell (b), the
concentration of an analyte in solution (c) and the predicted absorbance of a solution of known
concentration (A). The Spectronic 20 measures the absorbance of light at a pre-determined
concentration, and the concentration is calculated from the Beer-Lambert relationship.

One of the application of UV spectroscopy is it is one of the best methods for

determination of impurities in organic molecules. Additional peaks can be observed due to
impurities in the sample and it can be compared with that of standard raw material. By also
measuring the absorbance at specific wavelength, the impurities can be detected. In addition, it is
useful in the structure elucidation of organic molecules, such as in detecting the presence or
absence of unsaturation, the presence of hetero atoms. Moreover, UV absorption spectroscopy
can be used for the quantitative determination of compounds that absorb UV radiation. UV
absorption spectroscopy can characterize those types of compounds which absorbs UV radiation
thus used in qualitative determination of compounds. Identification is done by comparing the
absorption spectrum with the spectra of known compounds. This technique is used to detect the
presence or absence of functional group in the compound. Absence of a band at particular
wavelength regarded as an evidence for absence of particular group. Besides, kinetics of reaction
can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell
and the absorbance changes can be observed. Many drugs are either in the form of raw material
or in the form of formulation. They can be assayed by making a suitable solution of the drug in a
solvent and measuring the absorbance at specific wavelength. Molecular weights of compounds
can be measured spectrophotometrically by preparing the suitable derivatives of these
compounds.
 Methodology

A. Preparation of “Standard” Solutions of Soft Drink (Known Concentrations)

1. Soft drink is poured into a beaker and stirred to remove the carbonation.
2. 5.00 mL of the soft drink is pipetted into a 50.0 mL volumetric flask and diluted to the
mark with distilled water. The solution is covered and shaken to make a homogeneous
solution and stored it in small beaker.
3. Step 2 is repeated using 10 mL, 15 mL, 20 mL, and 25 mL of soft drink.

B. Operation of the Spectronic 20 and Determination of λ max

The Spectronic 20 is moderately expensive piece of scientific equipment and should be

treated with all due care and respect.

Instrument: Thermo Spectronic/Genesys 20.

Operating instructions
1. Spectronic 20 is turned on and waited for the instrument to warm up (minimum 15
minutes).
2. The wavelength to 600 nm is set.
3. 0% transmittance (% T) is adjusted (Adjusting dark current – nothing should be in the
sample compartment).
4. A cuvette is obtained. The cuvette may look like an ordinary test tube, but it is made of
special high quality and is much more expensive. The tube is cleaned and rinsed it with
distilled water, and then the tube is filled about ¾ full of ‘blank’ solution (the ‘blank’ is
 distilled water in this experiment). Any solution and fingerprints are carefully wiped away
from the outside of the tube using a Kimwipe.
5. 0 absorbance and 100% transmitted is adjusted with the cuvette containing the ‘blank’ in
the sample holder. The cuvette is removed and set it aside without emptying the distilled
water.
6. Another cuvette is cleaned and rinsed it with a small amount of the ‘standard’ soft drink
whose absorbance is to be measured (the most concentrated Soft Drink solutions in Part A).
Then, filled it about ¾ full with solution, wiped it with a Kimwipe and placed it in the sample
holder with hash marks aligned. The absorbance is read and recorded.
7. The cuvette is removed, the top is closed and the wavelength is changed to a setting which
is 20 nm lower.
8. 0% transmittance is reset if it has changed (sample compartment must be empty).
9. The cuvette of distilled water is inserted and the 100% T is reset. The cuvette is removed.
10. The cuvette containing the same Soft Drink solution we used in Step (6) is inserted.
11. The absorbance is read and the reading is recorded in Table 1.2.
12. Steps 8 through 11 is repeated until 360 nm, absorbance readings at each 20 nm interval
are took.
13. Using a graph paper, the absorption spectrum of our soft drink is plotted and the λ max is
determined.

C. Preparation of ‘Unknown’ Soft Drink Sample

1. Some of the soft drink is poured into a beaker and stirred to remove the carbonation.
2. The soft drink is poured without measuring the volume into a 50 mL volumetric flask and
diluted to the mark with distilled water. The flask is covered with stopper and shook to
homogenize the solution. The colour of the unknown solution prepared is not darker than the
most concentrated standard solution.
3. The ‘unknown sample’ is put into cuvette until it is about ¾ full.

D. Quantitative Analysis Of the Soft Drink Solution

 1. The Spectronic 20 is set to the wavelength maximum ( λ max) obtained from Part B.
2. 0 and 100% T is set as given in the procedure above.
3. The absorbance of each 5 ‘standard’ soft drink solution are measured and recorded.

4. The absorbance of the ‘unknown’ soft drink solution is measured and recorded.

E. Cleaning Up

1. The waste is poured down into the drain.

2. All cuvettes are cleaned and dried.
 Findings (figures/calibration curve/ calculation)

Graph of Absorbance against Wavelength

1
0.9
0.8
0.7
0.6
Absorbance

0.5
0.4
0.3
0.2
0.1
0
300 350 400 450 500 550 600 650
Wavelength (nm)

Graph 1: Absorbance against Wavelength

Figure 1: UV-Visible Wavelength Range

Based on the Graph 1, the readings of the absorbance are relatively increasing from
360 nm with 0.200 until it reach the peak which is at 520 nm with 0.885. After 520
nm, the absorbance readings are relatively decreasing. Based on the data obtained, the
lambda max or the wavelength of the maximum absorbance turns out to be 520 nm. Since the
lambda max falls at 520 nm, then the absorbed colour is at blue-green range based on Figure
1. Thus, the origin of the soft drink that we gain turns out to be blue and green.

Solution Concentration Absorbance

(volume %)

1 10 0.179

2 20 0.362

3 30 0.537

4 40 0.714

5 50 0.885

6 ? 0.245

Table 1: Table of Concentration and Absorbance at 520 nm

Table 1 shows the concentration and the absorbance value with the missing value of
‘Unknown’ sample concentration. By using data on Table 1, we then plot a graph to find the
‘Unknown’ sample concentration.
 Graph of Absorbance versus Concentration
1
0.9
f(x) = 0.02 x + 0.01
0.8 R² = 1
Absorbance at 520 nm

0.7
0.6
0.5
0.4
0.30.25 0.25
0.2
0.1
0
0
0 10 20 30 40 50 60
Concentration (%)

Graph 2: Graph of Absorbance versus Concentration

Thus, based on the Graph 2, the concentration of ‘Unknown’ sample is 13.54%.

Conclusion (Advantages and Limitations)

In conclusion, the origin of the soft drink is blue-green range because the wavelength
at maximum absorbance falls at 520 nm. The unknown concentration of the soft drink is
13.54% based on the graph. The Spectronic 20 is relatively simple instrument. It is capable of
measuring % transmittance and absorbance over the range of 340 to 950 nm (the range 600 to
950 nm requires a special infrared filter and a different lamp). The advantages of Spectronic
20 are it is suited for quantitative absorption measurement at a single wavelength. It also has
simplicity of instrumentation, low cost and ease of maintenance. However, two separate
readings has to be made on the light which results in some error because the fluctuations in
the intensity of the light do occur in the line voltage, the power source and in the light bulb
between measurement. In addition, the changing of wavelength is accompanied by a change
in light intensity. Thus, spectral scanning is not possible.
 References

Aryal, S. (2018, November 11). UV Spectroscopy- Principle, Instrumentation, Applications. Retrieved from
Microbe Notes: https://microbenotes.com/uv-spectroscopy-principle-instrumentation-
applications/#principle-of-uv-spectroscopy

Astronomy, S. (n.d.). Spectroscopy. Retrieved from COSMOS - The SAO Encyclopedia of Astronomy:
https://astronomy.swin.edu.au/cosmos/S/Spectroscopy

Reusch, W. (2013, 5 5). Visible and Ultraviolet Spectroscopy. Retrieved from

https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/uv-vis/spectrum.htm

1. Skoog, West, Holler and Crouch, Fundamentals of Analytical Chemistry, 8, Thomson Brooks/Cole,
2004
2. Christian, G, Analytical Chemistry, 6, John Wiley & Sons, 2006

3. Skoog, J. J. Leary, Principles of Instrumental Analysis, 4, Saunders Coll Publishing, 1992

4. Skoog, West and Holler, Analytical Chemistry: An introduction, 6, 1994

 EXPERIMENT 2
UV-Visible Determination of an unknown concentration of KmnO4 Solution

A. Post-laboratory questions

a) Why are glass materials not suitable for UV-spectroscopy cell holder?
- Glass will absorb some of the ultra violet light and will cause an inaccurate result for
the experiment as the reading will be higher than the exact one.
b) State one advantage of using the UV-Vis spectrophotometer compared with
Spectronic 20 for this analysis.
- The advantage is to record absorbance at each wavelength and rapidly scan a range
wavelength.

B. Complete the table of concentration and absorbance

Solution Concentration (ppm) Absorbance

Standard 1 5 0.135
Standard 2 10 0.278
Standard 3 15 0.418
Standard 4 20 0.562
Unknown 9.08 0.251

D. Report summary
 Introduction

All absorbance spectrophotometers contain a light source, a sample compartment, and a

detector. Many spectrophotometers also contain one or more monochromators, a device used to
separate light into its component wavelengths. Spectrophotometers that measure in the UV and
visible region are of two general types which are scanning and diode-array. In contrast, a
scanning UV-Vis spectrophotometer contains a monochromator, usually consisting of
holographic gratings, which allows light of individual wavelength to be sequentially imparted to
the sample. Spectroscopy involves the observations of absorption or emission of electromagnetic
radiation resulting from transitions of atoms or molecules from one energy level to another.
When a molecule at its “ground state” (the state of lowest energy) absorbs energy of some type,
the molecule is said to undergo a “transition” to a higher energy state. The higher energy state is
referred to as an “excited state”. A molecule can only absorb energy if the input energy exactly
matches a molecular transition from one energy level to another. Many organic and biological
molecules have transitions that occur between energy levels of electronic states of atoms or
molecules. Therefore, our focus will be on the visible and ultraviolet regions of the
electromagnetic spectrum in this lab. The visible and ultraviolet region of interest is found
between 170 and 800 nm, though the most useful region for experimental use is between 250 and
700 nm.

In this experiment, we want to determine the concentration of an unknown solution of

Potassium Permanganate but we need to find for the maximum wavelength first. By knowing its
maximum wavelength, we prepared different concentration of Potassium Permanganate solution
that is stated on the procedure and record the absorbance value. We can continue our experiment
to plot the calibration curve using the data of concentration and absorbance that we have
recorded. Different value of concentration will give a different value of absorbance, and based on
the Beer’s Law, the amount of energy absorbed or transmitted by a solution is proportional to the
solution's molar absorptivity and the concentration of solute. In simple terms, a more
concentrated solution absorbs more light than a more dilute solution does. Thus, the slope of the
graph absorbance against concentration is equal to the molar absorptivity coefficient, ε x l. 
Therefore, based on the graph, we can determine the concentration of an unknown solution of
Potassium Permanganate.
 Spectrophotometer that we used in this experiment is the Varian/Cary 50 UV- VIS
Spectrophotometer. The innovative design of the Cary 50, which incorporates a Xenon flash
lamp, enables it to offer many advantages over traditional UV-Vis spectrophotometers. As the
Xenon lamp is very intense, the Cary 50 can use a beam splitter without the loss in energy
causing excessive photometric noise. The beam splitter allows simultaneous reference beam
correction, so peaks will not shift as the scan speed changes. The Xenon lamp flashes only when
acquiring a data point, unlike a diode array which exposes the sample to the whole wavelength
range with each reading, causing degradation of photosensitive samples.

Methodology

A. Preparation of the KMnO4 Standard Solutions

 1. 0.01 g KMnO4 is weighed accurately to the nearest mg on a weighing paper. The
reading is recorded. The solid is transferred to a 100 mL volumetric flask by using a
funnel.

2. The solid is dissolved with a few mL of distilled water. The flask is stoppered and
shaken. The distilled water is added to the mark, and the last few drops is added by using
a medicine dropper. The flask is stoppered and shaken several times to homogenize the
solution.

3. The ‘stock’ solution is poured into a beaker. The beaker is labeled as ‘100 ppm’.

4. 5.00 mL of the ‘stock’ solution is pipetted and diluted with distilled water in a 100 mL
volumetric flask.

5. The solution is transferred into a beaker and the beaker is labeled as ‘5 ppm’.

6. Step 4 is repeated by using 10 mL, 15 mL and 20 mL stock solution and then are
transferred into small beakers.

7. The beakers are labeled as ’10 ppm’, ’15 ppm’, and ’20 ppm’ respectively.

B. Preparation of the Unknown

1. Between 5.00 to 20.00 mL of the ‘stock’ KMnO4 solution is pipetted and diluted with
distilled water in a 100 mL volumetric flask.

2. The solution is transferred into a beaker and it is labeled as ‘Unknown’.

C. Operation of the UV-Vis Spectrophotometer

Instrument: Varian/ Cary 50 UV-Vis Spectrophotometer.

Operating instructions

1. Cary Win UV icon is selected.

2. ‘Scan’ is clicked.
 3. Setup is chosen, the ‘CARY’ is clicked and then the required start and stop scan
wavelength (nm) is keyed in.

i. Y-mode (min = 0 and max = 1).

ii. X-mode = 800-200 nm

iii. Beam mode = Dual Beam

4. Cycle mode is selected if more than 1 cycle is required.

5. “Scan Control Speed” is selected to fast.

6. ‘Baseline’ icon and check ‘Baseline Correction Function’ is clicked.

7. ‘Accessories 1’ is clicked and check on the Use Cell Charger cells is selected.

8. At the peak table option, maximum peak is selected.

9. In the Auto Store icon, ‘storage on (Prompt At Start)’ is set.

10. To save the method, go to the file and the scan method is saved.

11. The ‘BLANK’ cuvette solution is filled with distilled water.

12. The ‘BLANK’ cuvette solution is put and ‘Baseline’ is clicked.

13. The ‘BLANK’ cuvette is removed and the ‘SAMPLE’ cuvette is put.

14. ‘Start’ icon is clicked to start the measurement.

D. Determination of the Unknown Concentration

1. The concentration icon is clicked.

2. Setup is chosen, Cary icon is clicked, then maximum wavelength, max is keyed in.

3. Replicate = 3 is selected.

4. ‘Standard’ icon is clicked, the ‘Calibrate During Run’ function is checked.

5. The calibration standard unit (mg/L) and the number of the standard samples is set.
 6. The Fit Type (Linear Direct) is selected.

7. Sample icon is chosen, the numbers of the samples are selected and the unknown is
keyed in.

8. Report icon is clicked, operator name and the comment is keyed in.

9. In the Auto Store icon, ‘Storage on (Prompt at start)’ is set.

10. To save the method, ‘File Save Method as Ok’ is clicked.

11. The ‘BLANK’ cuvette is put and ‘Zero’ is clicked.

12. The ‘BLANK’ cuvette is removed and the ‘SAMPLE’ cuvette is put.

13. ‘Start’ icon is clicked to start the concentration measurements.

Finding (absorbance spectrum/calculation)

Solution Concentration (ppm) Absorbance

 Standard 1 5 0.135
Standard 2 10 0.278
Standard 3 15 0.418
Standard 4 20 0.562
Unknown ? 0.251
Table 1: Table of Concentration and Absorbance at 525.6 nm

Table 1 shows the concentration and the absorbance value with the missing value of
‘Unknown’ solution of Potassium Permanganate. By using data on Table 2, we then plot a graph
to find the ‘Unknown’ solution concentration of Potassium Permanganate.

Graph of Absorbance versus Concentration of KMnO4

0.6

f(x) = 0.03 x − 0.01

0.5 R² = 1
Absorbance at 525.6 nm

0.4

0.3
0.25 0.25

0.2

0.1

0
0
0 5 10 15 20 25
Concentration (ppm)

Graph 1: Graph of Absorbance versus Concentration of KMnO4

Thus, based on the Graph 1, the concentration of an unknown solution of Potassium

Permanganate is 9.08 ppm.

Conclusion (Advantages and Limitations)

In conclusion, the maximum wavelength of Potassium Permanganate is 525.60 nm. We

also have succeeded to plot the graph of Absorbance against Concentration of KMnO 4 to
determine the unknown solution of Potassium Permanganate which is 9.08 ppm. The Cary 50
UV-Vis Spectrophotometer has making us easy to conduct this experiment because the
maximum scan rate is 24,000 nm per minute meaning we can scan the whole wavelength range
of 190-1100 nm in less than 3 seconds. In addition, the light beam is narrow, very intense and it
is unaffected by room light. We can operate with the sample compartment open or closed, we
will not notice the difference.

References

file:///C:/Users/User/Downloads/276140056-Lab-chm-261.pdf

https://dokumen.tips/documents/experiment-2-chm260.html

http://accounts.smccd.edu/batesa/chem210/labmanual/Experiments/Exp
%2005%20Photometry.pdf
 EXPERIMENT 3
Fourier Transform Infrared Spectroscopy (FTIR)

A. Post-laboratory questions

a) Explain why a background spectrum must conduct before obtaining the sample
spectrum.
- To measure the contribution of the instrument and environment to the spectrum. Also,
it can subtract the background from sample’s measurement and get the actual
sample’s spectrum.

b) Why ‘neat’ liquids, not an aqueous solution, used on salt plates?

- It is easier in preparation because no mixing is required. The aqueous solution will
dissolve the salt plates.

B. Answer the following

1. Briefly explain the observation during the preparation of the KBr pellet.
- The KBr pellet which is potassium bromide was appeared clear like a piece of glass
or transparent disk after being exposed to pressure by bench top hydraulic press.

2. Name of compound analysed

i. Benzoic Acid
ii. Polyethylene High Density (Plastic)
iii. Caffeine
iv. Acetone
v. Cyclohexane
vi. Unknown (Benzoic Acid)
3. The molecular formula of the compound is analysed.
i. C7H6O2 – Benzoic Acid
- O-H (3400-2500 cm-1),
- C=O (1760-1670 cm-1),
- C-O (1320-1210 cm-1)
- C=C (1600 -1475 cm-1)

ii. (C2H4)n – Polyethylene High Density (Plastic)

- O-H (2960-2850 cm-1)
- C-H (1470-1350 cm-1)
- C-H (1000-675 cm-1)

iii. C8H10N4O2 – Caffeine

- C=O (1680-1630 cm-1)
- C-N (1350-1000 cm-1)
- C-H (900-690 cm-1)
- C-H ( >3000 cm-1)

iv. C3H6O – Acetone

- C=O (1760-1670 cm-1)

v. C6H12 – Cyclohexane
- C-H (2960-2850 cm-1)
- C-H (1470-1350 cm-1)

4. The molecular formula of an unknown compound

- C7H6O2
 C. Report summary

Introduction

Fourier Transform Infrared Spectroscopy which is known also as FTIR Analysis or FTIR
Spectroscopy is an analytical used to identify organic, polymeric, and, in some cases is inorganic
materials. The FTIR Spectroscopy uses infrared light because it can scan test samples and observe

chemical properties. The FTIR instrument sends infrared radiation for about 10,000 to 100 cm -1
through a sample, which is some radiation can be absorbed and some passed through. The absorbance
radiation is converted into rotational and vibrational energy by the sample molecule. A solution was
developed which employed a very simple optical device called an interferometer. The interferometer
produces a unique type of signal which has all of the infrared frequencies ‘encoded’ into it. The signal
can be measured very quickly, usually on the order of one second or so. Thus, the time element per
sample is reduced to a matter of a few second rather than several time.

FTIR offers quantitative and qualitative analysis for organic and inorganic samples. Fourier
Transform Infrared Spectroscopy (FTIR) identifies chemical bonds in a molecule by producing an
infrared absorption spectrum. The spectra produce a profile of a sample, a distinctive molecular
fingerprint that can be used to screen and scan sample for many different component. FTIR is an
effective analytical instrument for detecting functional group and characterizing covalent bonding
information. Potassium bromide (KBr) is use on this experiment because it is as a carrier for the sample

in IR spectrum also it has a transmittance 100% in the range of wave number 4000-400 cm -1 The
objective of this experiment is to prepare the KBr pellet of an organic compound such as benzoic acid,
to carry out a qualitative analysis of an organic compound such as benzoic acid using FTIR and to
identify IR absorption peaks and the corresponding functional groups of an unknown solid or liquid or
powder.

In this experiment, we want to prepare the KBr pellet of an organic compound and carry out a
qualitative analysis of an organic compound. We also want to identify IR absorption peaks and the
corresponding functional group of a solid/liquid/powder. As a result, we can predict the molecular
formula of an unknown compound.
 Methodology

SAMPLE PREPARATION FOR IR ANALYSIS

A. (i) The KBr method

1. The solid sample (i.e benzoic acid) and KBr solid (IR grade) is obtained from the dessicator. About 1
gram of each is weighed and dried in the oven for about 2-3 hours at 110°C.

2. The agate mortar and pestle is removed for use to grind the benzoic acid and KBr.

3. 1 mg of Benzoic acid is taken and grinded by using agate mortar and pestle until the benzoic acid
become very small and shiny for about 1 minute or more.

4. 80 mg of KBr is taken and grinded it using agate mortar and pestle until it becomes powdered.

5. The benzoic acid is mixed and powdered KBr in agate mortar for ratio 1 : 80 and the mixture is grinded
until become homogeneous about 30 seconds.

6. Using a spatula, the mixture is scraped into the middle and the mixture is grinded again for about
seconds to mix the benzoic acid thoroughly with the KBr.

7. The benzoic acid and KBr is grounded finely and the mixture is scattered the infrared radiation
excessively.

8. The mixture is heap in the center of the agate mortar using a spatula.
 ii) Making the KBr pellet

1. The die set is removed from the box or storage container.

2. Die set is cleaned with ethanol.

3. The mixture of benzoic acid and KBr is put into the die set. The mixture is filled on the surface of the die
set.

4. The die set is tightly closed and put it into Hydraulic Press gauge. The Hydraulic Press gauge is
tightened.

5. The Hydraulic Press gauge is pressed until the pressure goes up to 7000 psi.

6. The air is released and let it rested for 2 minutes.

7. The Hydraulic Press gauge is pressed again until the pressure goes up to 8500 psi and let it rested for
about 1 minute. The pressure is released.

8. The die set is slowly removed from Hydraulic Press gauge.

9. The die set is opened and the KBr pellet was appeared clear like a piece of glass or transparent disk.

10. The KBr pellet is put into a pellet holder for analysis.
B. Preparing Liquid Samples (‘neat liquids’) using a Salt Plate

1. A ‘neat liquid’ sample is used.

2. Some of the liquid sample about 1-2 drops is put on the plate, and then it is covered with another plate.
The entire plate is covered by the liquid that should spread out. If it does not cover well, the top is turned to
spread the sample or added a bit more sample.

3. The sandwich is placed in the IR salt plate holder and covered with a hold-down plate.

4. At least two nuts are put on the posts of the holder and spin it down gently to hold the plates with an even
pressure. It is not being forced because it will crack the plates.

5. The holder and plate is slide into the bracket on the instrument in the sample beam.

6. The spectrum is run.

7. The salt plates from the instrument are removed and cleaned them. A little acetone is followed often by
drying with Kim Wipes. The salt plates are kept in a clean and dry place.

C. Sample Preparation for FTIR (Reflectance) Solid sample was grounded finely first before
use.

1. The spectrum of benzoic acid is run using the Reflectance method.

2. An unknown is obtained from the instructor (solid/liquid/ powder) and the spectrum is run.
D. Operation of the FTIR

(i) Instrument: Thermo/ Nicolet 380 FTIR

Operating Instructions

2.Program ‘EZOMNIC’ is selected.

3.‘COLLECT’ icon was selected and ‘EXPERIMENT SET UP’ is chosen to analyze a pellet.
4.The data is checked which appear in the window,
I. No of scan: 32
II. Resolution: 4
III. Final format: Transmittance, Absorbance
IV. File handling: Interferograms was saved.
V. Background handling: Background before each sample was collected.
5.‘SAVE’ and ‘OK’ is clicked.
6.‘COLLECT’ is selected and ‘COLLECT SAMPLE’ is clicked.
7.The title of the experiment is typed then ‘OK’ is clicked.
8.‘OK’ is clicked to collect the background spectrum.
9.The pellet is placed into the sample compartment after scanning is completed.
10. ‘OK’ is clicked to obtain sample spectrum.
11. “Find peaks” and ‘replace’ is clicked after scan was completed.
12. “Process” and ‘Advanced ATR correction’ is chosen, ‘GE’ is clicked and ‘OK’.
13. It is saved and printed.
(ii) Instrument: Perkin-Elmer Spectrum-RX

Operation instrument

1. Shift, scan.
2. The silica gel is removed from the FT-IR.
3. The energy is checked. The energy must be > 70%.
4. Scan is pressed again.
5. Background button is pressed. 3 times scanning is chosen.
6. Execute is pressed and scan is pressed.
7. Background button is pressed and on the sample holder is put.
8. The axis either X, Y or Z was chosen. The screen was shown like the background spectrum.
9. Arrows to modify the graph is used.
10. The peak is marked. The shift V cursor is used or Shift H cursor. Shift is marked.
11. Lastly, the plot button is pressed to printout the results.
Finding (spectrum/ functional group)

i) Benzoic Acid

Figure 1 FTIR Spectrum for Benzoic Acid

Figure 2 Benzoic Acid Structure

Wavenumber, cm-1 Bond Functional Group Peak, cm-1

1320-1210 (m) C-O (stretching) Carboxylic Acids 1296.7
1760-1670 (s) C=O (stretching) Carboxylic Acids 1685.8
3400-2500 (b) O-H (stretching) Carboxylic Acids 2682.6, 2994
1600,1500 (w) C=C (stretching) Aromatic rings 1585.8

Based on the Figure 1, it is a benzoic acid which has a benzene ring that carrying carboxylic acid.
The molecular formula for benzoic acid is C 7 H 6 O 7. The functional group that has on spectrum graph
of benzoic acid is carboxylic acid which is the bond is C=O. The peak on spectrum graph for carboxylic
acid is 1685.8cm−1. Next, the second of the functional group has on the spectrum graph is aromatic
rings which are C=C bond. The peak that has located on spectrum graph is 1585.8 cm−1. Other than that,
the carboxylic acid is the functional group that have O-H bond on the spectrum graph and the peak
present on the spectrum are 2682.6cm−1 and 2994cm−1.
 ii) Plastic

Figure 3 FTIR Spectrum for Plastic

Figure 4 Plastic Structure

Wavenumber, cm-1 Bond Functional Group Peak, cm-1

2960-2850(s) C-H (stretching) Alkanes 2850.48, 2915.13
1470-1350(v) C-H (scissoring and Alkanes 1463.70
bending)
1000-675 (s) C-H (bending) Alkenes 721.26

The next spectrum graph on the experiment is Figure 3 which is plastic or polyethylene. It is
consist of a C-H bond. The molecular formula for ethylene is ¿. The functional groups consist of alkanes
and the peak present on the spectrum are 2850.48 cm−1, 2915.13cm−1and 1463.70cm−1. Also, the

functional group that has on spectrum graph is alkenes and the peak is 721.26 cm−1.

iii) Caffeine
 Figure 5 FTIR Spectrum for Caffeine

Figure 6 Caffeine Structure

Wavenumber, cm-1 Bond Functional Group Peak, cm-1

900-690 =C-H (bending) Aromatic Rings 605.30
1350-1000 C-N (stretching) Amine 1229.83
1680-1630 C=O (stretching) Amide 1650.38
> 3000 =C-H (stretching) Aromatic Rings 3106.7

Furthermore, for the next Figure 5 which is caffeine that has molecular formula of C 8 H 10 N 4.
The functional group is amines, amides and aromatic rings which are amines have C-N bond, amide
have C=O and aromatic rings C-H. Based on Figure 5, the peak for amines is 1229.83 cm−1, for amide is

1650.38cm−1and aromatic rings are 605.30 cm-1 and 3106.7 cm-1.

iv) Cyclohexane
 Figure 7 FTIR Spectrum for Cyclohexane

Figure 8 Cyclohexane Structure

Wavenumber, cm-1 Bond Functional Group Peak, cm-1

2960-2850(s) C-H (stretching) Alkanes 2920.37, 2852.87
1470-1350(v) C-H (scissoring and bending) Alkanes 1449.25

Besides, in Figure 7 which is cyclohexane has molecular formula is C 6 H 12. The

functional group present in the structure is alkanes which has peaks number of 2920.37 cm−1,
2852.87 cm−1and 1449.25cm−1 based on the Figure 7.

v) Acetone
 Figure 9 FTIR Spectrum for Acetone

Figure 10 Acetone Structure

Wavenumber, cm-1 Bond Functional Group Peak, cm-1

1760-1670 (s) C=O (stretching) Ketones 1709.01

For Figure 9 which is Acetone has molecular formula of C 3 H 6O. The functional groups for acetone

are ketones. Hence, the peak number that present on the spectrum for ketones is 1709.01 cm−1.

vi) Unknown
 Figure 11 FTIR Spectrum for Unknown

Wavenumber, cm-1 Bond Functional Group Peak, cm-1

3100-3000 (m) C-H Aromatic Rings 3003.54
1760-1670 (s) C=O Carboxylic acids 1710.10
1260-1000 (s) C-O Carboxylic acid 1219.38

For the last spectrum which is graph in Figure 11 that shows for Benzoic Acid spectrum graph so
for the molecular formula is C 7 H 6 O 7. The bond that has in the graph is C-H which is the functional
group of aromatic rings, C-O which is carboxylic acids and also C=O refers to the functional group
carboxylic acids. For carboxylic acids, the peak numbers that appear on the Figure 11 are 1710.10 cm -1
and 1219.38 cm-1 while for the aromatic rings is 3003.54cm−1.

Conclusion (Advantages and Limitations)

 In conclusion, we are able to prepare the KBr pellet of an organic compound, carry out a
qualitative analysis of an organic using FTIR and identify IR absorption peaks and the
corresponding functional group of an unknown solid or liquid or powder. By using FTIR
spectrum we also can determine the functional group of a compound with the graph that has a
different wavelength. Also, the result will be occurred with detail qualitative and quantitative
chemical information by using FTIR spectroscopy without damage the sample. We managed to
determine the molecule present in the Unknown molecule which is Benzoic Acid.

The advantages of FTIR spectroscopy is low maintenance which is the system consists of
one moving part only, that is, the moving mirror. This results in greater system reliability and
less of maintenance due to reduced wear and tear. Other than that, the advantages FTIR
spectroscopy is freedom from spectral discontinuities as there are no gratings or filter changes
there are no discontinuities in the resulting spectrum.

References

Amiel, C., Mariey, L., Curk-Daubié, M. C., Pichon, P., & Travert, J. (2000). Potentiality of
Fourier transform infrared spectroscopy (FTIR) for discrimination and identification of
dairy lactic acid bacteria. Lait, 80(4), 445–459. https://doi.org/10.1051/lait:2000137
Chen, Y. (2015). Applications of Micro-Fourier Transform Infrared Spectroscopy (FTIR).
16(12). https://www.mdpi.com/1422-0067/16/12/26227/htm
Retzko, I. (2001). Chemical analysis of plasma-polymerized films: The application of X-ray
photoelectron spectroscopy (XPS), X-ray absorption spectroscopy (NEXAFS) and fourier
transform infrared spectroscopy (FTIR). Journal of Electron Spectroscopy and Related
Phenomena, 121(1–3), 111–129. https://doi.org/10.1016/S0368-2048(01)00330-9
Zarnowiec. (2015). Fourier transform infrared spectroscopy as a tool to study.pdf. 22, 1710-
1718(9).
https://www.ingentaconnect.com/content/ben/cmc/2015/00000022/00000014/art00008
 EXPERIMENT 4:
FLAME ATOMIC ABSORPTION SPECTROSCOPY (AAS)

B. Post-laboratory questions
a) Using any available software, plot a standard calibration curve of absorbance versus
concentration of Ca standard solution.

Solutions Concentration (ppm) Absorbance

Standard 1 1.161 0.024
Standard 2 2.051 0.042
Standard 3 3.144 0.064
Standard 4 4.031 0.082
Standard 5 4.820 0.098

b) In a standard addition method, you will prepare a series of solutions to add different
increments of standard solutions to fixed aliquots of unknown sample X (10.00 mL). All
solutions were prepared using a 50 mL volumetric flask. Suppose that the analysis of the
standard solution of X gave the following results:

Solutions Concentration (ppm) Absorbance

Standard 1 0.00 0.201
Standard 2 2.44 0.292
Standard 3 4.88 0.378
Standard 4 7.32 0.467
Standard 5 9.76 0.554