objectives

CXA101
Lecture 2 Spectrophotometers UV-Vis

The components of a spectrophotometer Light Sources Wavelength selectors/monochromators Sample Compartment Detectors Processes in the cuvette Types of spectrophotometers

reminder: UV-vis Spectroscopy

molecules absorb light in the UV and visible regions of the electromagnetic spectrum results in electronic transitions compounds with extended system of alternating double and single bonds will absorb UV light anything with color absorbs visible light UV-vis spectroscopy is applicable to a wide range of samples

Spectrophotometer types
Single beam Dual beam Diode array

More about these later

Principles of Spectrophotometry
A spectrophotometer consists of two parts a spectrometer for producing light of any selected color (wavelength), photometer for measuring the intensity of light.

1 Light Sources
Stable light source of good intensity and as large a range as possible UV/Vis spectrophotometers utilize two light sources:

Some spectrophotometers use xenon flash lamps, which offer intensity over both the UV and visible regions.

a deuterium arc lamp for the UV range (190 to 380 nm) tungsten- halogen lamp for the visible spectrum (380 to about 800 nm).

The reality
In practice most sources do not produce a continuous spectrum of constant intensity

2. Wavelength Selectors/ Monochromators

Ideally you would pass light of exactly the right wavelength, max, for the compound of interest through the cell Why? Which Wavelength?

Well.what do we want to do?

Abs concentration

Consider the following situation

Measure the absorbance using white light of a solution that contains a substance that absorbs blue light and is at a concentration that absorbs 40% of incident blue light
Po = 100 units P = 86 units Abs = -log(86/100) = 0.036

0.06

What if we..
Isolated the blue light somehow and only shone the 14 units of blue light through the cuvette

What if we..
Isolated the blue light somehow and only shone the 14 units of blue light through the cuvette
Abs = -log(8/14) = 0.243 Po = 14 units P = 8 units

Increased Sensitivity
We get a much larger absorbance reading even though exactly the same amount of light has been absorbed By making Po more specific to the max we have improved sensitivity

Types of monochromators
Filters
Coloured Interference

Dispersing Elements
Prism diffraction Grating reflection gratings

Coloured Filters
Coloured filters contain a compound or compounds that absorb light of specific allowing other to pass Broad range of pass through The bandwidth is large

Whats bandwidth?
Width of light peak passing through the sample
Coloured filter >50 nm Interference Filter > 10 nm Prism >2 nm Diffraction Grating > 0.1 nm

monochromators : Interference Filters

This causes some to be removed and some enhanced depending on the nature of the filter

monochromators: Prism
dispersing ability of prism can be used as a monochromator By moving the prism you can change the passing through the slit

Monochromators: Diffraction Grating

Relies on the reflectance pattern of a beam of light shone onto an extremely fine grating

Monochromators: reflection grating

Diffraction grating Combined with mirrors

Most commonly used these days

Standard Czerny-Turner configuration

3. Sample compartment
Typically plastic cuvettes with a 1 cm pathwidth are used Plastic absorbs strongly in the UV spectrum Glass or quartz cuvettes are required for < 300 nm

Plastic Absorption
UV Transparent Plastic Cuvettes Plain Plastic Cuvettes

4. Detectors
Convert light into an electric current
Phototubes Photomultiplier tubes Silicon Diodes Diode Arrays

Photomultiplier tubes

gas-filled or vacuum tube that is sensitive to light. incoming photons strike a photocathode, generating electrons, which are attracted toward the anode.

Silicon Wafer Diodes (photodiode) Diode arrays: several photodiode detectors are arranged on a silicon crystal

Diode array with up to 1024 specific elements

Blue light specific elements

Factors affecting spectrophotometric measurements

Picking the peak Making sure your absorbance measurement is made at max

Setting Po (ie setting blank)

Po

Po = P when the sample compartment contains a blank solution

Processes in the cuvette

Many processes in the cuvette can affect the spectrophotometric measurement

Types of spectrophotometers

Schematic of a wavelength-selectable, single-beam UV-Vis spectrophotometer

dual-beam uv-vis spectrophotometer

Diode array detector

Array-Detector Spectrophotometer: advantages

transmitted light simultaneously recorded at multiple wavelengths Provides sample absorption spectrum Provides max after reading more precision, more sensitivity and more reproducible results Can analyse mixtures instruments are relatively small and robust.
quick

Difference: Diode array v conventional

Review: The components of a spectrophotometer

Light source Wavelength Selectors/Monochromators Sample Compartment Detectors Factors affecting measurements Spectrophotometer types