CHEMISTRY

Characterizing Organic
Compounds: Structural Effects
416L
EXPT 3
PAGE 19-20
in Ultra-Violet Spectroscopy
Olivia Cassandra Dela Cruz, John Francis Egenias, Krizzi Eve Garcia*
Department of Chemistry, College of Science

*Corresponding author; e-mail: [email protected]

Abstract

In ultraviolet-visible spectroscopy which is a method of quantitative analysis

and structural elucidation, the principle of UV absorption of molecules
resulting in changes in electronic states is involved. Organic compounds,
hexane, cyclohexane, anthracene, naphthalene, benzoic acid, phenol and
caffeine were characterized based on the UV spectra obtained by
determination of the λmax. The resulting spectra was analyzed with
wavelengths ranging from 200 nm to 400 nm. Factors that mainly affect the
UV absorption are structural effects and protonation effects.

Keywords: electromagnetic spectrum, spectroscopy, ultraviolet-visible light, absorbance

Introduction

In analytical chemistry, ultraviolet-visible spectroscopy is an important tool used in clinical

laboratories in the qualitative analysis and determination of chemicals. However, it is

mainly used in the quantitative determination of various organic and inorganic compounds

in solution. The electronic transitions between molecular orbitals due to the absorption of

radiation in the visible and ultraviolet regions of the electromagnetic spectrum is described

in ultraviolet spectroscopy (Porcu, 2018; Fifield and Kealey, 2000).

The method of spectroscopy is associated to the interaction of light with matter. The

increase in the energy level of atoms or molecules is due to light being absorbed by

matter. When ultraviolet radiations are absorbed, electrons jump from the ground state

towards a higher energy state which is called the excitation of the electrons (Olsen, 1976).

The theory involved states that the energy from the absorbed ultraviolet radiation is equal

to the energy difference between the higher energy state and the ground state. The

wavelength of the absorbed UV light is dependent by the electronic differences between

the orbitals in the molecule. Depending on the bond, sigma bonds are much stable than

pi bonds which are very likely to come into an excited state. While in conjugated systems,

longer chain of conjugated double bonds absorbs light at a longer wavelength thus

affecting the resulting spectra with the number of observed absorptions (Wade and

Simek, 2017). The energy changes due to absorption are relatively large that usually

corresponds to a wavelength range of 200 nm to 800 nm or a wavenumber range of

12000 to 50000 cm-1. The large energy change causes simultaneous changes in

rotational and vibrational energies. The interaction of solute and solvent molecules that

cause collisional broadening of lines in the curve formed by the spectra are not usually

observed in effect with the changes in the energy levels. While the resulting overlapping

bands coalesce to form one or more broad band-envelopes which are characterized with

the position of each maximum λmax and the corresponding intensity or molar absorptivity

ԑ. In polyatomic molecules and metal complexes, several bands from a number of

electronic transitions and their associated rotational and vibrational fine structures may

arise in the complete spectra (Pasto, 1998; Hesse et al., 2008).

In obtaining the UV-Vis spectra, the absorption plot must be obtained by the absorbance

determined through the Beer’s law which states the absorbance is directly proportional to

the concentration of the substance and the length of the path of light in the solution and

given by the equation, A = ԑcl (Fifield and Kealey, 2000; Hart et al., 2012). To measure

the UV-Vis spectrum, the sample is dissolved in a solvent that does not absorb above

200 nm and placed in a quartz cell then the spectrometer functions by comparing the

amount of light transmitted through the sample with the amount of light in the reference

beam (Wade and Simek, 2017).

Figure 1. Schematic diagram of an ultraviolet spectrometer. The monochromator

selects a wavelength which is split into two beams. The detector measures the ration of
the two beams and the printer plots the ratio as a function of wavelength (Pasto, 1998).

Being the oldest spectrometric method, ultraviolet-visible spectroscopy is found to be

useful in the determination of the structure of molecules that contain conjugated systems

of double bonds always resulting in one or more intense absorption maxima at

wavelengths greater than 200 nm (Ault, 1998). The measured height of an absorption

band above the baseline of a UV-Vis spectrum is in units of absorbance while its position

is given by the wavelength of the maximum absorbance or λ max, which is measured from

the top of the band (Katritzky, 2009). The main objective of the experiment is to analyze

UV absorptions of organic compounds specifically hexane, cyclohexane, anthracene,

naphthalene, benzoic acid, phenol and caffeine in their structural effects and the effect of

adding acids and bases by UV-Vis spectroscopy.

Results and discussion

The purpose of the experiment was to characterize the compounds by their UV-Vis

spectra and analyze the effects of conjugation, type of alkyl groups attached, extent of

annulation and the addition of acids and bases using the method of UV-Vis spectrometry.

The main principles involved are the electronic transitions that arises from the absorption

of visible or ultraviolet (UV) radiation which enables the excitation of electrons thus

moving from a lower energy level to a higher energy level (“Ultraviolet-Visible

Spectroscopy”, n.d.). The table below summarizes the UV spectra obtained for hexane,

cyclohexane, naphthalene, anthracene, benzoic acid, phenol added with HCl and NaOH

separately and methanolic caffeine also added in 3% HCl and 3% NaOH.

Table 1. UV-Vis Spectroscopy Summary of Results

Samples Summary of UV-Vis Profile

Hexane λmax : 200 nm

λmin : 400 nm

Cyclohexene λmax : 200 nm

λmin : 362 nm

Anthracene λmax : 247 nm

λmin : 392 nm
 Naphthalene λmax : >200 nm; λmin : 344

nm

Benzoic Acid λmax : 200 nmλmin : 362 nm

a) With HCl λmax : 228 nmλmin : 362 nm

b) With NaOH λmax : 228 nmλmin : 344 nm

Phenol λmax : 228 nm; λmin : 362 nm

c) With HCl λmax : 244 nm; λmin : 400 nm

d) With NaOH λmax : 244 nm; λmin : 362 nm

Methanolic Caffeine λmax : 228 nm

a) With 3% HCl λmax : 228 nm; λmin : 362 nm

b) With 3% NaOH λmax : 230 nm; λmin : 388 nm

For the first compound hexane, the obtained spectrum shows that the distinct peak was

recorded at 200 nm for λmax while 400 nm for λmin. The λmax shows the wavelength at

maximum absorption in which it characterizes the electronic transition band (Němcová

et al., 1996).

Figure 2. UV Spectrum of Hexane with distinct peak at λmax=200 nm

 Figure 3. Hexane bond-line structure showing no conjugation (Pasto, 1998).

Depending on its atomic grouping (chromophore) or the capability of its electrons to

change their energy state, a substance’s absorption spectrum can be determined.

Transitions from the molecular orbitals σ or π or the nonbonding electrons n, to the

antibonding orbitals σ* or π* cause the absorptions in the UV or Visible regions. In the

compound hexane, the C-C bonds and C-H bonds have σ-σ* electronic transitions. This

transition requires greater energy at shorter wavelengths (Pasto, 1998). With the

compound having no conjugation, λmax was found at 200 nm which is also the same with

cyclohexene. The relatively low λmax shows that the spectrum is shifted on the far UV

region also called the vacuum ultraviolet because oxygen in the air absorbs the more

energetic UV photons just below 200 nm (Williamson, 2004).

Figure 4. Ultraviolet and visible regions of the spectrum and the types of absorption

bands that often occur (Olsen, 1976).

 Figure 5. UV Spectrum of Cyclohexene with distinct peak at λmax=200 nm

Cyclohexene also contained single bonds. However, the aromatic ring structure and the

presence of a double bond should have an effect on the position of the spectrum and

yield a longer wavelength at maximum absorption. Sources of error that must have

affected the resulting spectrum are stray light, inhomogeneity and noise. Stray light must

have been absorbed by the species which was detected along with the sample.

Inhomogenous distribution of absorbing species in the sample must also have been

observed in the spectrum while shot noise that arises from electrons and photons being

discrete particles which affects the precision of the analysis (Ault, 1998).

Figure 6. Cyclohexene bond-line structure showing conjugation (Pasto, 1998).

For the compound anthracene, the obtained spectrum exhibited a distinct peak at 247 nm

for λmax while 392 nm for λmin.

Figure 7. UV Spectrum of Anthracene with distinct peak at λmax=247 nm

The λmax for this compound is recorded to be higher compared with the first two

compounds, hexane and cyclohexane which have no conjugation. The promotion of π to

π* affected the resulting absorption wavelength yielding a higher value of wavelength. An

isolated chromophore C=C when conjugated with another C=C, will result to a maximum

absorption band shifting to a longer wavelength along with an increase in its intensity.

Energy changes are lower for substances with a system of conjugated double bonds

(Němcová et al., 1996).

Figure 8. Anthracene bond-line structure showing conjugation (Pasto, 1998).

The fourth compound, naphthalene, the obtained spectra had an absorption maxima of

greater than 200 nm. The added conjugation shifted the spectra towards a longer

wavelength. The π-π* transition due to the change in energy state, was due to the

presence of nonbonding electrons.

Figure 9. UV Spectrum of Naphthalene with distinct peak at λmax=247 nm

Figure 10. Naphthalene bond-line structure showing conjugation (Pasto, 1998).

For the compound benzoic acid, the resulting spectra showed that the λmax was at 200

nm. When HCl and NaOH was added however, the λmax shifted to a longer wavelength.

The presence of double bonds and an atom conjugated with a lone electron pair causes

less energetically demanding n- π* transition and have a higher λmax (Lehman, 2010).

Also, the addition of the acid or base contributed to the increase in λmax (“Ultraviolet-

Visible Spectroscopy”, n.d.).

 (a) (b)

(c)

Figure 11. UV Spectrum of Benzoic Acid a) untreated b) with HCl c) with NaOH

Figure 11. Benzoic Acid bond-line structure showing conjugation (Pasto, 1998)

The compound phenol had obtained a spectrum with a λmax of 228 nm and a λmin of 362

nm. Comparing the structure of phenol with benzoic acid, both compounds have an

aromatic ring and double bonds the only difference is the presence of the functional group

ketone in benzoic acid. Carbonyl groups causes shift in the absorption spectrum due to
the non-bonding electrons in an orbital intermediate in energy between the bonding π

orbital and the antibonding π* orbital. With higher energy, the electronic transitions

between the non-bonding orbital to the π* orbital gives smaller change in energy thus

absorbance occurs at longer wavelength (Hart et al., 2012).

(a) (b)

(c)

Figure 12. UV Spectrum of Phenol a) untreated b)with HCl c) with NaOH

Figure 13. Phenol bond-line structure showing conjugation (Pasto, 1998)

When phenol was added with 5% HCl and 5% NaOH, the resulting spectra had shifted

towards a longer wavelength, that is both compounds obtained a λmax of 244 nm. This
shift is called the bathochromic effect or red shift wherein the absorption maximum shifts

to a longer wavelength. While a shift to a shorter wavelength is called hypsochromic effect

or the blue shift (Olsen, 1976). The shift can be explained by the protonation effects

brought by the addition of an acid and base.

(a) (b)

(c)

Figure 14. UV Spectrum of Caffeine a) untreated b)with HCl c) with NaOH

Figure 15. Caffeine bond-line structure showing conjugation

For the methanolic caffeine, the obtained spectrum showed a λ max of 228 nm. When it is

added to 3% HCl and 3% NaOH, the obtained spectrum shifted to a longer wavelength

for HCl while it stayed the same for 3% NaOH.

Scheme 1. Reaction of Caffeine with the addition of HCl results in the protonation of the

compound, shifting the equilibrium to the left (Williamson, 2004).

Scheme 2. Reaction of Caffeine with the addition of NaOH results in the deprotonation

of the compound, shifting the equilibrium to the right (Williamson, 2004).

Changes in pH brought by the protonation of compounds due to the addition of acid,

equilibrium lies to the left based on Le Chatelier’s principle. The protonation of phenol

then results in a blue sift or the shifting to a longer wavelength. On the other hand, the

deprotonation of phenol due to the addition of a base, results in an electron lone pair that

is conjugated with π electrons which causes the shift to a longer wavelength (Katritzky,

2009). This concept also applies with the compound phenol and benzoic acid.
In addition, the presence of auxochromes or saturated groups containing heteroatoms

also modify the absorption due to a chromophore. Auxochromic substitution of

chromophores leads to a bathochromic shift for π-π* transition and hypsochromic shift for

n-π* transitions. The shift is due to resonance effects caused by the interaction of lone

pair electrons in the auxochromes with the π system of the chromophore (Fifield and

Kealey, 2000). While the annulation or the formation of rings also affects absorption. This

is due to the conjugate system being elongated. The determination of both the shape of

the spectra and the position of the visible maxima are governed by the site of annulation.

It causes strong bathochromic shifts at odd ring positions and hypsochromic shifts at even

ring positions (Katritzky, 2009).

The UV absorption of molecules can characterize the compound under study since there

are differences in the promotion of electronic states that is the wavelength at which it

absorbs light can be known. Several factors that affect the UV spectra are level of

conjugation, the presence of alkyl groups, extent of annulation and the changes in pH

due to protonation. For conjugation, the longer chain of conjugated double bonds, the

longer the wavelength at which it absorbs. Likewise, alkyl groups increase the value of

λmax by 5 nm per group. The annulation of aromatic rings also shifts the spectrum to a

longer wavelength. Lastly, protonation effects leads to a shift to a shorter wavelength.

Experimental methodology

UV-Vis Profile of Assigned Sample

A milligram of the hexane and cyclohexene was weighed using an analytical balance and

dissolved in a 10 mL spectroscopy grade hexane. The solution was stirred and mixed in

a beaker then transferred into a quartz cuvette. The cuvette was wiped with a soft tissue

to avoid any residues or fingerprints interfering with the sample’s UV spectrum. It was

then placed into the chamber of the spectrometer, taking note of the clear glass portion

of the cuvette is inserted in parallel into the sample holder.

Figure 16. Bio-Rad SmartSpec 3000 UV/Vis Spectrometer (Bio-Rad SmartSpec 3000

UV/Vis Spectrophotometer, n.d.)

The spectrophotometer was set up using a software in the computer. For the setup of

experimental parameters, the wavelength range settings were changed to 200 nm to 400

nm. The data plot was printed, that is the wavelength where maximum absorption and
minimum absorption occurs was determined. The maximum absorption and wavelength

for each band observed was noted and compared with the resulting spectra.

UV-Vis Profile of Aromatic Systems: Naphthalene and Anthracene

In obtaining the spectra for the aromatic systems, naphthalene and anthracene, the same

procedure was adapted. With the maximum absorption and wavelength noted, the

obtained spectra for the aromatic system was compared and contrasted with the sample

pair assigned to the group.

Protonation Effects: Benzoic Acid, Phenol and Caffeine

A milligram of the sample, benzoic acid, phenol and caffeine was weighed and dissolved

on a 20 mL spectroscopy grade methanol. Three samples for each compound was

prepared and for each sample 10 mL of the solution was obtained. The first sample was

untreated, the second sample was added with 3 drops of 5% HCl and the third sample

was added with 3 drops of 5% NaOH. Following the same procedure as the first and

second part, the solution was stirred and mixed then transferred on a quartz cuvette. The

UV absorbance was measured ranging from 200 to 400 nm with the use of a UV-Vis

spectrophotometer. Also, the maximum absorption and wavelength for each band

observed was noted. After obtaining the first spectra, the sample was returned to the

original beaker and 3 drops of 5% HCl will be added then stirred and transferred into a

quartz cuvette. The UV absorbance will be measured again with the same range then the

obtained spectra will be compared and contrasted with the first spectra obtained.
References

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