UV-vis spectroscopy
- involves the spectroscopy of photons; uses light in the visible, UV and near infrared (NIR) ranges, where molecules undergo electronic transitions. - used in the quantitative determination of solutions of transition metal ions and highly conjugated organic compounds
2
X-Ray 200nm
UV 400nm
Visible 800nm
IR
Microwave
WAVELENGTH(nm)
Molecules have quantized energy levels: ex. electronic energy levels. hv energy energy
} = hv
Q: Where do these quantized energy levels come from? A: The electronic configurations associated with bonding.
Each electronic energy level (configuration) has associated with it the many vibrational energy levels.
s* p*
2p
p s
2p
s* 2s
2s s
Ethane
C C
s*
hv
s* s
s
H C C H H H H H
s*
Absorptions having max < 200 nm are difficult to observe because everything (including quartz glass and air) absorbs in this spectral region.
C C
s* p*
hv
s* p*
= hv =hc/
p s
p s
p*
C O
s* p*
n
hv
s* p*
n
p s
n
p s
p*
The n to pi* transition is at even lower wavelengths but is not as strong as pi to pi* transitions. It is said to be forbidden. Example: Acetone: ns* max = 188 nm ; = 1860 np* max = 279 nm ; = 15
Instrumentation
Instrument Parts
Source of visible light quartz halogen lamp (same to automobile) Source of ultraviolet light deuterium arc lamp (200 400 nm) Grating 1 selects a narrow band of wavelengths to enter monochromator Monochromator selects an even narrower bandto pass through the sample Photomultiplier tube detector; creates electric current proportional to P
Light Sources
UV Spectrophotometer
1. 2. 3. Hydrogen Gas Lamp Mercury Lamp Deuterium (200-400 nm)
Visible Spectrophotometer
1.
2. 1.
Tungsten Lamp
Xenon Arc lamps (200-1000 nm) Carborundum (SiC)
TUNGSTEN LAMP
Excellent source of continuous visible and near IR radiation Operates at 3000 K Useful range: 320 2500 nm
Carborundum / Globar
Source of IR radiation Useful range: 4000 200 cmMade up of silicon carbide Heated to 1500 K by electric current
Scanning Instrument
Scanning Instrument monochromator
slit
slit
slit
monochromator
Advantages/disadvantages
Scanning instrument
High spectral resolution Long data acquisition time (several minutes) Low throughput (amount of work performed by a computer within a given time)
Diode array
Fast acquisition time (a couple of seconds), compatible with on-line separations High throughput (no slits) Low resolution (2 nm)
Definition of Resolution
Spectral resolution is a measure of the ability of an instrument to differentiate between two adjacent wavelengths
UV / visible Spectroscopy
UV / visible Spectroscopy
ALWAYS
use in conjunction with NMR and infrared spectra.
HPLC-UV
HPLC Pump
Mobile phase
Sample loop
6-port valve
UV / visible Spectroscopy
1. Bathochromic shift (red shift)
lower energy, longer wavelength
CONJUGATION.
3. Hyperchromic effect
increase in intensity
4. Hypochromic effect
decrease in intensity
Every instrument has a useful range for a particular analyte. Often, you must determine that range experimentally. This is done by making a dilution series of the known solution. These dilutions are used to make a working curve.
Make a dilution series of a known quantity of analyte and measure the Absorbance. Plot concentrations v. Absorbance.
In this graph, values above A=1.0 are not linear. If we use readings above A=1.0, graph isnt accurate.
The best range of this spectrophotometer is A=0.1 to A=1.0, because of lower errors. A=0.4 is best.
Precision Accuracy
Precision + Accuracy
Precision Accuracy +
Precision + Accuracy +
MONOCHROMATOR
Disperses light into its component wavelengths and selects a narrow band of wavelengths to pass on to the sample or detector Consists of entrance and exit slits, mirrors and grating to disperse light
GRATING
A reflective or transmissive optical component with a series of closely ruled lines Different wavelengths of light are reflected or transmitted at different angles from the grating
DIFFRACTION
Bending of light rays by a grating
REFRACTION
Bending of light rays by a prism or lens
GRATING
Made up of parallel grooves with distance d Aluminum coating reflectivity SiO2 coating protection * When adjacent rays are IN PHASE, they reinforce one another and provide a source of radiation
Resolution of Grating
Resolution ability to separate two closely spaced peaks / = nN = wavelength n = diffraction order N = number of grooves that are illuminated * The greater the resolution, the smaller the difference between 2 s that can be distinguished from each other.
Dispersion
Measures the ability to separate wavelengths differing by through the difference in angle f (radians)
Dispersion Devices
Non-linear dispersion Temperature sensitive
monochromatic Ray
Red Orange Yellow Green Blue Violet
SLIT
Polychromatic Ray
PRISM
Ultraviolet
Polychromatic Ray
Monochromatic Ray
Prism - spray out the spectrum and choose the certain wavelength () that you want by moving the slit.
Stray Light
Wavelengths outside the bandwidth expected from the monochromator Caused by diffraction into unwanted orders and angles; unintended scattering; sample compartment not perfectly sealed Results to an increased measured absorbance
Filters
Remove/block wide bands of radiation from a signal to give the desired wavelength Placed in the beam Simplest types:
Colored glass Interference filters
Pass radiation in reference region; reflect other s
DETECTORS
Detector
Produces an electrical signals when struck by photons Ex: phototube emits electrons from a photosensitive, negatively charged surface when struck by visible light/UV rays Detector response is dependent on the wavelength of incident photons
Anode
Wide dynamic range Very good signal/noise at high light levels Solid-state device
Conventional Spectrophotometer
Conventional Spectrophotometer
Conventional Spectrophotometer