Analytical Instrumentation Lab: Ultraviolet Spectroscopy

What does Ultraviolet (UV) Spectroscopy study?

It studies how compounds absorb ultraviolet light to analyze electronic transitions from ground to excited states.

What course topic does this relate to?

Analytical Instrumentation Lab (Fall 2025) — focusing on molecular spectrometry using UV radiation.

What energy state are most compounds in at room temperature?

The ground state (low energy).

What happens when molecules interact with electromagnetic radiation (EMR)?

Electronic, vibrational, and rotational transitions occur depending on the wavelength.

What does UV/Visible radiation specifically cause?

Electrons move to higher orbitals (excited state), representing ground → excited state transitions.

What does fluorescence measure?

Excited → ground state transitions, the emission of light as electrons relax.

What wavelength range does UV spectroscopy cover?

200–400 nm

Why is UV spectroscopy considered a useful quantitative technique?

It has high sensitivity (10⁻⁶ M) and can analyze colorless solutions accurately.

What physical principle does UV spectroscopy follow?

Beer’s Law, which relates absorbance to concentration.

How do shorter wavelengths affect energy?

Shorter wavelengths have higher energy, allowing detection of low-concentration analytes

What lamp is used for UV radiation (200–400 nm)?

A deuterium lamp.

What lamp is used for visible radiation (400–800 nm)?

A tungsten lamp.

What spectral range do both lamps combined cover?

200–800 nm, the UV–VIS region

What is the purpose of a monochromator?

To separate wavelengths of polychromatic light into individual components.

What device type is used in modern UV instruments?

A holographic diffraction grating, which provides high-resolution wavelength selection.

What material are UV cuvettes made from?

Quartz, because glass absorbs UV radiation.

What is the typical path length of a cuvette used in UV spectrometry?

1 mm (though many visible spectrophotometers use 1 cm).

What type of detector is used in diode-array UV spectrometers?

A diode-array detector containing hundreds of photodiodes.

How does the diode-array system function?

• Source light passes through a lens focusing polychromatic light onto the flow cell.

• Transmitted light hits a grating, dispersing it across the photodiode array.

• Each photodiode detects a different wavelength (≈ 2 nm bandwidth per diode).

What is the advantage of diode-array detectors?

They allow simultaneous measurement of multiple wavelengths for rapid spectral acquisition.

What type of instrument is the Agilent 8453?

A UV–VIS Diode Array Spectrophotometer.

What is its spectral range?

190–1100 nm total range.

What are its light sources and ranges?

• Deuterium lamp: 190–800 nm (UV region)

• Tungsten lamp: 370–1100 nm (visible + SWNIR)

What is the sample container made of?

Quartz cuvettes, required for UV transparency.

Why is UV detection often more sensitive than visible detection?

UV light enables detection of lower-concentration analytes due to higher photon energy and stronger absorption.

What is the concentration of the caffeine stock solution provided?

0.00100 M Caffeine

What standards must be prepared from the stock?

0.00010 M, 0.00007 M, and 0.00003 M standards.

• One group also prepares a 0.00005 M standard.

Which standard is used to generate the spectral scan?

The 0.00005 M standard, and this data is shared across groups.

At what wavelength should absorbance readings be taken?

At the secondary maximum (λ₂ max) of the spectral scan.

How is the standard curve constructed?

Plot the absorbance values of all four standards versus concentration to form a Beer’s Law graph.

What is provided to each group for analysis?

An unknown caffeine sample.

How is the unknown concentration determined?

By extrapolation from the standard curve obtained from known standards.

What additional calculation is required?

Determination of the molar absorptivity (ε) value for caffeine using Beer’s Law (A = εbc).