UV-VIS Spectroscopy Lab · CAPE Chemistry

5.1 Origin of Absorption in UV/VIS Spectroscopy

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Electromagnetic Radiation and Energy

UV/VIS spectroscopy is based on the electronic excitation of molecules. When a molecule absorbs electromagnetic radiation in the ultraviolet (200–400 nm) or visible (400–800 nm) region, the energy supplied is used to promote an electron from a lower-energy occupied molecular orbital to a higher-energy unoccupied (antibonding) orbital.

ΔE = hν = hc/λ where h = 6.63 × 10⁻³⁴ J s, c = 3.00 × 10⁸ m s⁻¹

The wavelength absorbed corresponds exactly to the energy gap between orbitals: shorter wavelength → higher energy → larger orbital energy gap.

Molecular Orbitals Involved

When two atomic orbitals overlap, they form two molecular orbitals. In UV/VIS spectroscopy we are concerned with the following orbital types:

Bonding

σ (sigma)

formed by head-on overlap

Bonding

π (pi)

formed by side-on overlap

Non-bonding

n

lone pairs, not in bond

Anti-bonding

σ* (sigma star)

higher energy

Anti-bonding

π* (pi star)

higher energy

Energy order (lowest → highest): σ < π < n < π* < σ*

Electronic Transitions

Six possible electronic transitions occur in UV/VIS spectroscopy, but only four are relevant to the ordinary UV/VIS range (200–800 nm):

Transition	Typical λ Range	Energy Required	Compounds
σ → σ*	< 150 nm (far UV)	Very High	Saturated alkanes (not accessible)
π → π*	170–250 nm	High	Alkenes, conjugated dienes, aromatics
n → σ*	180–250 nm	Moderate–High	Alcohols, ethers, amines, halides
n → π*	250–400 nm	Moderate	Carbonyls (C=O), azo groups
d–d transitions	400–800 nm	Low–Moderate	Transition metal aquo-complexes
Charge-transfer	Varies widely	Variable	Metal–ligand complexes (intense)

Key Points

The σ → σ* transition requires so much energy it falls in the far UV (< 150 nm), outside the range of ordinary spectrophotometers. Saturated compounds like alkanes cannot therefore be analysed by routine UV spectroscopy.
The n → π* transition requires the least energy and gives absorption at longest wavelengths (least energetic photons).
When UV/VIS radiation passes through a sample, only photons whose energy exactly equals ΔE for a transition are absorbed.

5.2 Why Some Species Absorb UV/VIS Radiation and Others Do Not

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Chromophores

A chromophore is the part of a molecule responsible for absorbing UV/VIS radiation. For a molecule to absorb, it must contain a chromophore — a group that provides accessible energy levels in the UV/VIS range.

Organic Molecules: Conjugated Systems

Organic molecules with conjugated π systems (alternating double and single bonds) absorb UV/VIS radiation due to π → π* transitions. As the degree of conjugation increases, the energy gap between HOMO and LUMO decreases, shifting absorption to longer wavelengths (bathochromic / red shift).

Compound	Double Bonds	λmax (nm)	Region
Ethene (CH₂=CH₂)	1	171	Far UV
Buta-1,3-diene (conjugated)	2	217	UV
Hexa-1,3,5-triene	3	258	UV
β-Carotene (11 conjugated)	11	454	Visible (absorbs violet/blue)

Carbonyl groups (C=O) in aldehydes and ketones exhibit both π → π* (strong, ~190 nm) and n → π* (weak, ~270 nm) transitions. The n → π* band in butanone appears near 275 nm.

Transition Metal Ions: d-d Transitions

In isolated transition metal ions, the five d orbitals are degenerate (same energy). When ligands surround the metal ion, the d orbitals split into two groups of different energy. An electron can be promoted from the lower d group to the higher d group by absorbing visible light energy. The wavelength absorbed (and thus the colour observed) depends on the energy difference between the split d levels, which in turn depends on the ligand and the metal.

Did You Know?

Dilute CuSO₄ solution appears light blue because [Cu(H₂O)₆]²⁺ absorbs in the red-orange region (~810 nm). When ammonia is added, the complex [Cu(NH₃)₄]²⁺ forms, which has a larger ligand field splitting — absorbing in the yellow-red region (~620 nm) — appearing deep blue.

Species That Do NOT Absorb UV/VIS

Saturated alkanes (only σ bonds; σ → σ* needs far UV < 150 nm)
Water and most simple inorganic ions (no accessible electronic transitions)
Noble gases (no bonds, no valence electrons to promote)
Colourless simple salts (e.g., NaCl, KNO₃ — absorption only in far UV)

Summary

UV absorption: organic molecules with conjugated π systems or C=O groups (π→π*, n→π* transitions)
Visible absorption: transition metal complexes (d-d transitions) or highly conjugated organic dyes
No UV/VIS absorption: saturated compounds with only σ bonds

5.3 Basic Steps in Analysing Samples by UV/VIS Spectroscopy

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Procedure for Visible Spectrophotometry

Step 1 – Sample preparation: Dissolve the sample in an appropriate solvent (distilled water, ethanol, etc.) to form a clear solution. Dilute if necessary.
Step 2 – Select wavelength: Set the spectrophotometer to the wavelength of maximum absorption (λmax) of the analyte. This maximises sensitivity.
Step 3 – Zero the instrument: Place a blank (cuvette containing pure solvent only) in the light path. Set the absorbance to 0 (transmittance to 100%). This accounts for solvent absorption and reflection losses.
Step 4 – Measure the sample: Replace the blank with the sample cuvette. Record the absorbance (A) or %T from the digital display.
Step 5 – Repeat at other wavelengths (if scanning a full spectrum): repeat steps 3–4 for each wavelength.
Step 6 – Calibration: Measure standard solutions of known concentration to construct a calibration curve (A vs c).

Using Complexing Reagents

Many ions in solution are either colourless or only faintly coloured, making direct visible spectroscopy impractical. A complexing reagent (chromophoric reagent) is added to react with the analyte and form a more intensely coloured compound, increasing sensitivity.

Analyte	Complexing Reagent	Product Colour	λmax (nm)	ε (M⁻¹cm⁻¹)
Fe²⁺ (iron)	1,10-phenanthroline	Orange-red	510	11,100
Cu²⁺ (copper)	NH₃ (ammonia)	Deep blue	620	~50
Urea	Ehrlich's reagent + ZnSO₄	Yellow	435	—
CN⁻ (cyanide)	Br₂(aq), then p-phenylenediamine	Red	530	—

Sensitivity and Detection Limits

The sensitivity of a UV/VIS method refers to the smallest change in concentration that produces a measurable change in absorbance. This is directly proportional to the molar absorptivity (ε). Methods with high ε (like Fe–phenanthroline with ε = 11,100) are very sensitive.

The detection limit is the minimum concentration detectable above background noise. UV/VIS spectroscopy has detection limits in the range of 10⁻⁴ to 10⁻⁶ mol dm⁻³, making it suitable for trace analysis in clinical and environmental samples.

Instrument Note

Cuvettes and lenses used in UV spectroscopy must be made of quartz (silica), not ordinary glass, because glass absorbs UV radiation significantly. In visible spectroscopy, glass cuvettes are acceptable.

5.4 Beer-Lambert's Law and Calibration Curves

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Statement of Beer-Lambert's Law

Beer-Lambert's Law combines two separate observations:

Beer's Law: Absorbance is proportional to the concentration of the absorbing species.
Lambert's Law: Absorbance is proportional to the path length through which light travels.

A = ε l c

Symbol	Quantity	Typical Unit
A	Absorbance (dimensionless)	No units
ε (epsilon)	Molar absorptivity (molar extinction coefficient)	dm³ mol⁻¹ cm⁻¹ (or M⁻¹cm⁻¹)
l	Path length (width of cuvette)	cm
c	Concentration of absorbing species	mol dm⁻³

Absorbance and Transmittance

Transmittance (T) is the fraction of incident light that passes through the solution. Absorbance and transmittance are related by:

A = log₁₀(I₀/I) = −log₁₀(T) = 2 − log₁₀(%T)

If all light passes through: A = 0, %T = 100. If no light passes through: A → ∞, %T = 0.

Calibration Curves (Standard Curves)

A calibration curve is prepared by measuring the absorbance of several standard solutions of known concentration of the analyte. A graph of A (y-axis) vs c (x-axis) is plotted. By Beer-Lambert's Law, this should be a straight line through the origin with slope = εl.

To find the concentration of an unknown solution: measure its absorbance and read the corresponding concentration from the calibration curve.

Deviation from Beer-Lambert's Law

Beer-Lambert's Law is obeyed when absorbance (A) ≤ 2. At higher concentrations, deviations occur because:

At high concentrations, solute–solute interactions alter the absorbing species
The refractive index of the solution changes significantly
Stray light reaching the detector at high absorbance

For this reason, working absorbances should ideally be in the range 0.1–0.8.

Worked Example

A solution of KMnO₄ at 525 nm in a 1.0 cm cuvette has ε = 2200 M⁻¹cm⁻¹.
If c = 5.0 × 10⁻⁴ mol dm⁻³:
A = 2200 × 1.0 × 5.0 × 10⁻⁴ = 1.10
%T = 10^(2 − 1.10) × 1 = 10^0.9 ≈ 7.9%

5.5 Applications: Quantitation of Substances by UV/VIS Spectroscopy

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Iron in Iron Tablets

Iron (Fe²⁺) ions in solution are very pale green and absorb light only weakly. A complexing reagent, 1,10-phenanthroline, is added. Fe²⁺ reacts with three molecules of phenanthroline to form the deep orange-red complex [Fe(phen)₃]²⁺.

[Fe(phen)₃]²⁺: λmax = 510 nm, ε = 11,100 M⁻¹cm⁻¹

The iron tablet is dissolved in dilute HCl, Fe³⁺ is reduced to Fe²⁺ with hydroxylamine, then phenanthroline and buffer are added. The absorbance is measured at 510 nm. A calibration curve using Fe²⁺ standards is used to calculate the iron content.

Glucose in Blood

Glucose is colourless and cannot be directly analysed by visible spectroscopy. Two methods are used:

Enzymatic method: Glucose + glucose oxidase + peroxidase → coloured dye; measured at ~340 nm
Benedict's method: Glucose reduces Cu²⁺ to Cu⁺ (copper(I) oxide precipitate); the decreasing blue colour of the solution is measured

Urea in Blood

Urea is treated with zinc sulphate and Ehrlich's reagent (p-dimethylaminobenzaldehyde). The yellow product formed absorbs at 435 nm. Urea can also be determined enzymatically using urease, with the product measured at 340 nm.

Cyanide in Water

Cyanide (CN⁻) is treated with bromine water to form cyanogen bromide (CNBr). Addition of p-phenylenediamine produces a red dye that absorbs strongly at 530 nm. The method is sensitive and can detect very low concentrations of cyanide in treated water.

Industrial and Environmental Importance

UV/VIS spectroscopy is used to monitor cyanide levels in water near gold mining operations (cyanide is used in gold extraction) and industrial effluent. Iron content is routinely tested in blood samples to diagnose anaemia and in food products to verify nutritional labelling.

Substance	Matrix	Method / Reagent	λmax
Iron (Fe²⁺)	Tablets, blood, food	1,10-phenanthroline	510 nm
Glucose	Blood, urine	Glucose oxidase / Benedict's	340 nm
Urea	Blood, serum	Ehrlich's reagent + ZnSO₄	435 nm
Cyanide (CN⁻)	Water, effluent	Br₂(aq) + p-phenylenediamine	530 nm
KMnO₄	Water treatment	Direct measurement	525 nm

UV-VIS Spectroscopy — Study Notes