Lab Report on Infrared and Ultraviolet Spectroscopy

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The lab report below was submitted as part of the coursework for CM2102 Spectroscopic Applications. Please do not plagiarise from it as plagiarism might land you into trouble with your university. Do note that my report is well-circulated online and many of my juniors have received soft copies of it. Hence, please exercise prudence while referring to it and, if necessary, cite this webpage.

Abstract
This experiment aims to: 1) match the given Infrared (IR) spectrum to the respective functional groups, 2) identify 5 compounds through their IR spectrum and 3) obtain the isosbestic point of 4-methoxy-2-nitrophenol through Ultraviolet-Visible (UV) spectroscopy.

Reagents
Samples of compounds A, B, C, D and E; Solutions of pH 7, 8 and 13; One 100mL and three 10mL volumetric flasks; One 50mL beaker; Two potassium bromide (KBr) plates; IR and UV spectrophotometers
Introduction
Infrared(IR) spectroscopy involves absorption of IR radiation to elucidate structures of molecules. Radiation of this energy range corresponds to the stretching and bending of bonds. Stretching involves a displacement along the bond axis with a change in the interatomic distance while bending involves a change in bond angles between 2 bonds and an atom common to both. Only bonds that have a dipole moment that changes with time are capable of absorbing IR radiation.

Functional groups have characteristic IR absorption. The stretching and bending frequencies of a bond depends on its multiplicity and aromaticity. Stronger bonds and asymmetric stretches absorb energy of higher wavenumbers than weaker bonds and symmetric stretches. The presence of bands in regions gives direct information while their absences may be used to deduce absence of functional groups.

In ultraviolet-visible (UV) spectroscopy, transitions between electronic energy levels result in the absorption of electromagnetic radiation. The most probable transition is from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). UV spectroscopy can detect the presence of conjugated chromophores and determine concentration of solutions (according to Beer-Lambert’s law).

Experimental
Infrared Spectroscopy
A preliminary background scan was obtained by scanning dry KBr plates. The KBr plates were cleaned with chloroform, dried, before one drop of the unknown sample was added between them. It was ensured that no air bubbles were present, prior to conducting an IR scan from 400 nm to 4000 nm.

Ultraviolet Spectroscopy
To prepare 0.001M solution of 4-methoxy-2-nitrophenol, 0.0173 g was weighed in a clean, dry 50 mL beaker. 2 mL of ethanol was then added. The solution was stirred for 7 – 10 minutes before transferring into a 100 mL volumetric flask and made up to the mark with deionised water. 1.0 mL of the solution was pipetted into a 10 mL volumetric flask and it was made up to the mark with pH 7 buffer solution. Similarly, solutions of 4-methoxy-2-nitrophenol in pH 8 and pH 13 were prepared in 2 separate 10 ml volumetric flasks.  The UV spectrums for all three solutions were obtained from 300 nm to 600 nm on the same paper and their isosbestic point, determined.

Results
Exercise 1-Compounds containing oxygen
Band Region / cm-1
Vibration Mode
Functional Groups
i) 2700 – 3600
O-H Stretch
O-H (of alcohols, phenols and carboxylic acids)
ii) 1850 – 1650
C=O Stretch
C=O (of aldehydes, ketones and carboxylic acid deriatives)
iii) 1300 – 1000
C-O  Stretch
C-O (of ethers , esters, alcohols, carboxylic acids and anhydrides)

Spectrum [a]


Wavenumber/ cm-1
Peak Intensity
Vibrational Mode
About 3000
Strong, sharp
C-H stretching
About 1750
Strong, sharp
C=O stretching
About 1250
Strong, sharp
C-O stretching
Since both C-O and C=O groups are present and O-H stretching is absent, the compound is an ester, C5H11COOCH3.

Spectrum [b]
Wavenumber/ cm-1
Peak Intensity
Vibrational Mode
About 3000
Strong, sharp
C-H stretching
About 1100
Strong, sharp
C-O stretching
Since C-O stretching is present while both C=O and O-H stretching are absent, this compound is an ether, C4H9OC4H9.

Spectrum [c]
Wavenumber/ cm-1
Peak Intensity
Vibrational Mode
About 3400
Strong, broad
O-H stretching
About 3000
Strong, sharp
C-H stretching
About 1050
Strong, sharp
C-O stretching
Since there is a strong and broad peak at about 3400 cm-1 which indicates the presence of the O-H group and C-O stretching is also present, it can be deduced that the compound is an alcohol, C4H9CH(C2H5)CH2OH.

Exercise 2 – Aromatic compounds
Wavenumber/ cm-1
Functional Groups
Vibrational Mode
i) 3100-3000
Alkene
sp2 C-H stretching
ii) 2000-1700
Phenyl ring substitution overtones
C-H stretching, depends on positions of substitution
iii) 1650-1430
Aromatic
Aromatic C=C stretching
iv) 1275-1000
Aromatic
=C-H in plane bending
v) 900-690
Aromatic
=C-H out-of-plane bending

Spectrum of Compound A
        3-methylbenzonitrile
Wavenumber/ cm-1
Peak Intensity
Vibrational Mode
2062.96,3030.17
Medium, sharp
sp2 C-H stretching
2956.87,2924.09,2864.29
Medium, sharp
sp3 C-H stretching
2227.78
Strong, sharp
-CºN stretching
2000 - 1750
Weak
Aromatic  overtones
1600.92,1583.56,1485.19,1456.26
Strong
Aromatic C=C stretching
918.12,881.47,788.89
Medium, sharp
=C-H out-of-plane bending
(meta-disubstituted)
A peak is observed at 2227.78 cm-1. This peak indicates the presence of nitrile group. The pattern of the out of plane C-H bends and aromatic overtones suggest a meta-substituted aromatic ring. Therefore, sample A is 3-methylbenzonitrile.
Spectrum of Compound B
          4-bromoanisole

Wavenumber/ cm-1
Peak Intensity
Vibrational Mode
3093.82, 3070.68,3043.67
Weak
sp2 C-H stretching
3003.17,2956.87,2937.59, 2902.87,2835.36
Strong, sharp
sp3 C-H stretching
1870.95, 1845.88
Weak
Aromatic overtones (para)
1577.77,1492.90,1483.26
Strong
Aromatic C=C stretching
1288.45, 1251.80,1240.23
Strong, broad
Aromatic C-O stretching
1072.42,1031.92
Strong, sharp
Aryl C-Br stretch
823.60,804.32,790.81
Strong
=C-H out-of-plane bending (para)
The aromatic C=C stretch bands and the sp2 C-H stretch slightly above 3000 cm-1 indicate the presence of an aromatic ring. The out of plane C-H bends and aromatic overtones indicates a disubstitution in para orientation. Aromatic C-O stretches at 1288.45, 1251.80 and 1240.23 cm-1 suggests an aryl ether while The sharp peaks at 1072.42 and 1031.92 cm-1 corresponds to the aryl bromide stretch.  Therefore, sample B is 4-bromoanisole.

Spectrum of Compound C

2-ethylaniline

Wavenumber/ cm-1
Peak Intensity
Vibrational Mode
3458.37 – 3215.34
Strong, broad
N-H stretching (primary amine)
3061.03,3018.60
Medium, sharp
sp2 C-H stretching
2858.51
Strong, sharp
sp3 C-H stretching
1950 – 1700
Weak
Aromatic overtones (ortho)
1616.35, 1583.56
Strong, broad
N-H bending
1494.83,1456.26
Strong, sharp
Aromatic C=C stretching
1315.45,1296.16,1274.95
Strong, broad
C-N stretching
796.60
Strong, sharp
N-H out-of-plane bending
748.38,705.95,644.22
Strong, broad
=C-H out-of-plane bending
The two N-H stretches at 3458.37 cm-1 and 3215.34 cm‑1 indicate a primary amine. The presence of sp2 C-H stretches at frequency above 3000 cm‑1 ­­and the aromatic C=C stretch peaks at 1494.83 and 1456.26 cm‑1 suggests an aromatic ring. The aromatic overtones and out of plane C-H bend at 748.38cm-1 shows that the aromatic ring is ortho substituted.  The band from 1315.45 – 1274.95 cm-1 corresponds to C-N stretch. Therefore, sample C is 2-ethylaniline.
Exercise 3 – Compounds containing methyl groups
Spectrum of Compound D               
       isobutylamine
Wavenumber/ cm-1
Peak Intensity
Vibrational Mode
3373.50,3300.20
Strong, broad
N-H stretching (primary amine)
2954.05 – 2870.08
Strong, sharp
sp3 C-H stretching
1600.92
Medium, broad
N-H bending (primary amine)
1469.76
Strong, sharp
Asymmetric deformation of the H-C-H angles of a CH3 group
1386.82, 1365.60
Medium, sharp
Isopropyl C-H  bending
1064.71
Medium, sharp
C-N stretching
786.96
Strong, broad
N-H out-of-plane bending
The presence of 2 the 2 N-H stretch peaks at 3373.50 and 3300.20 cm-1 and N-H bending at 1600.92 cm‑1 indicate that a primary amine group is present. The antisymmetric deformation of H-C-H angles of a CH3 group gives rise to very strong absorption at 1469.76 cm‑1. A doublet at 1386.82 cm-1 and 1365.60 cm-1 of equal intensity is due to the C-H bending in an isopropyl group. Hence, compound D is isobutylamine.

Spectrum of Compound E
      tert-butylamine   
Wavenumber/ cm-1
Peak Intensity
Vibrational Mode
3400 – 3200
Medium, broad
N-H stretching (primary)
2960.73,2872.01
Strong, broad
sp3 C-H stretching
1600.92
Medium, broad
N-H bending
1469.76
Strong, sharp
Asymmetric deformation of the H-C-H angles of a CH3 group
1388.75,1363.67
Strong, sharp
tert-butyl C-H bending
1244.09,1220.94
Strong, sharp
C-N stretching
The presence of broad N-H stretch peaks from 3400 – 3200 cm‑1 and N-H bending at 1600.92 cm‑1 suggests a primary amine. Two peaks of unequal intensity can be observed at 1388.75 and 1363.67 cm-1, with the lower energy peak being more intense, shows that a tert-butyl group is present. Hence, compound E is tert-butylamine.
Exercise 4 – Aromatic compounds
a)                        Aniline has an amino substituent –NH2 that carry nonbonding (n) electrons that can delocalize into the benzoic ring (as seen below) resulting in greater conjugation. This causes a decrease in energy gap for the transition of electrons and an increase in the probability of such transitions. Less energy is needed for the 1Lb transition; therefore, absorption shifts to a higher wavelength (red shift, also known as bathochromic shift). When the n electrons are more available, the shifting of the bands is greater.
Under low pH (high H+ concentration), aniline is protonated and exists mainly as the anilinium ion. The nitrogen atom in the anilinium ion has no unshared pairs of electrons for conjugation with the benzene ring. As a result, the observed bands should closely resemble that of unsubstituted benzene.  An increase in pH, on the other hand, will result in an increase in availability of n electrons and conjugation; this correspondingly leads to greater bathochromic and hyperchromic effect for the 1Lb transition.
b) The isosbestic point is defined as the wavelength at which two or more species have the same molar absorptivity during a reaction. At this point, the absorbance value is independent of the ratio of concentration of each individual absorbing species.  The presence of isosbestic point implies that only 2 dominant species, 4-methoxy-2-nitrophenol and its conjugate base 4-methoxy-2-nitrophenolate are in the solution.
The isosbestic point for 4-methoxy-2-nitrophenol occurs at 407.0 nm with absorbance = 0.257.

Discussion
Infrared spectroscopy
In exercise 1, IR absorption ranges were used to identify compounds with different types of oxygen bonding. We have to look for not only the presence, but also the absence of absorption bands of certain functional groups in order to deduce the unknown molecules. For example, ester has bands due to both C=O and C-O-C groups but none due to the OH group unlike in alcohol which contains O-H and C-O groups. 
For exercise 2, the positions of substituents in aromatic compounds can be determined from their C-H out-of-plane bending and aromatic overtones. The out of plane C-H bending vibrations (690-900cm‑1) are intense because of strong coupling between adjacent H atoms. The relative number and patterns of aromatic overtones, from 1650 – 2000cm-1, enables the extent of substitution and the position of substituents around the aromatic ring to be known. In addition, stretching vibrations from the substituents were also utilised to identify the unknown compounds.
Exercise 3 uses the IR spectrum to identify isopropyl and tert-butyl group based on the C-H bend in the region 1390 -1360 cm-1. Since they are structural isomers, they will have the same functional group vibration. By analysing the splitting of two peaks at around 1375 cm-1 corresponding to the -CH3 asymmetric and symmetric bending, the germinal dimethyl group may be differentiated from tert-butyl group. If isopropyl group is present, the band is split into 2 peaks of nearly equal intensity; however, a wider split of peaks with differing intensity will occur if ter-butyl group is present.
Before performing scans of samples, a preliminary background scan was conducted. This allows for the subsequent removal of ambient absorptions by any infrared active atmospheric gases. The KBr cells was thoroughly washed with chloroform first as the presence of impurities may cause inaccuracies in the spectrum. In addition, the plates should only be held by its sides. KBr disks were chosen as it does not react with sample and it does not absorb in the typical IR region of 4000 – 400 cm-1.
Ultraviolet (UV) spectroscopy
UV-Vis spectroscopy is used to study electronic transitions with compounds. The electronic transitions are basically of the HOMO-LUMO π→π* type, with an intense band at around 185 nm. The longest wavelength transition is a low-intensity system centered near 225 nm; it is known as the benezoid band.
4-methoxy-2-nitrophenol is highly conjugated and absorb strongly in the violet part of UV-visible region; hence, it appears in the complementary colour of yellow.
Both -OH and -OMe are electron donating groups that increase both the wavelength and the intensity of the benezoid band while the electron withdrawing -NO2 group have no effect on the position of the B band. When pH is raised, the compound is deprotonated to form 4-methoxy-2-nitrophenolate; it has an additional negative charge on the oxygen atom that is more available for interaction with the benzene ring, As a result, the π→π* transition shifts the spectrum to longer wavelength and increases its intensity, resulting in bathochromatic and hyperchormatic shifts in absorption. As pH increases, there is an increasing red shift and greater peak intensities.

The solutions used must be diluted to reduce the scattering of light during spectroscopy. After filling the curvettes with sample compounds, the curvettes were wiped dry and clean as any fingerprints will affect the absorbance. In addition, before a next set of solutions were to be scanned, the curvettes were rinsed several times with the new solutions to reduce contamination. All conditions such as temperature, slit, path differences must be held constant for quantitative analysis using UV spectroscopy.
Conclusion
Spectrum [a] shows an ester, C5H11COOCH3, spectrum [b] shows an ether,  C4H9OC4H9  and spectrum [c] shows an alcohol, C4H9CH(C2H5)CH2OH.

The unknown compounds A, B, C, D and E were identified by analyzing their IR spectra. Compound A is 3-methylbenzonitrile, compound B is 4-bromoanisole while compound C is ethylaniline. Compound D is isobutylamine and compound E is tert-butylamine.

The isosbestic point of 4-methoxy-2-nitrophenol was found to be at λ = 407.0 nm with an absorbance of 0.257.

References
Spectroscopy, 4th edition. Lampman, Pavia Kriz, Vyvyan.
Inorganic Chemistry 4th Edition, Shriver & Atkins, Oxford University Press.

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