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Week6 Lecture2

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5 views

Week6 Lecture2

Uploaded by

Zimmer Petermgbrs
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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UV-Vis

spectrophotometry
Prof. Joaquin Rodriguez-Lopez
Week 6 — Lecture 2
Qualitative versus quantitative
analysis via UV-vis absorption
• What are the objectives of
qualitative versus quantitative UV-
visible absorption spectroscopy?
• How might the application guide slit
width selection?
• Large slit width = good sensitivity but
poor resolution
• Small slit width = poor sensitivity but
good resolution

Visible region absorbance spectrum for


cytochrome c with spectral bandwidths of
(1) 20 nm, (2) 10 nm, (3) 5 nm, and (4) 1 nm.
Transmission and absorbance and
losses
• The reduction in the intensity of light
transmitted through a sample can be
used to quantitate the amount of an
unknown material.

P Psample
T 
P0 Pblank
P0 Pblank
A  log T log log
P Psample
Beer’s Law
• Quantitative relationship between
P0
absorbance and concentration of analyte A log  bc
• See derivation in text (Skoog: pages 337-338) P
• Absorption is additive for mixtures
 molar absorptivity
b pathlength
Amixture  A1  A2  ...  An c concentration
Amixture 1bc1   2 bc2  ... n bcn
Really: Al = elbc
Beer’s Law is always wavelength-specific
Limitations and
deviations from
Beer’s Law
• Real limitations
• Non-linearities due to intermolecular
interactions
• Self aggregation effects and electrolyte
effects
• Apparent
• Dynamic dissociation or association of
analyte
• Instrumental
• Polychromatic radiation
• Different molar absorptivities at different
wavelength leads to non-linearities in
Beer’s Law
• Stray radiation
https://www.ssi.shimadzu.com/products/uv-vis-spectrophotometers/faqs/resolution-and-stray-light.html
How to make a UV-vis absorption
measurement
1) Make a 0%T (dark current) measurement

2) Make a 100%T (blank) measurement

3) Measure %T of sample

4) Determine %T ratio and thus the absorbance value


Instrumental noise
• Precision of measurement is limited by instrumental noise sources
Light sources for UV-vis
• Deuterium lamp
• Most common UV source
• Arc between oxide-coated filament and metal electrode
• Low voltage and low pressure of D2
• Aperture gives 1-1.5 mm spot
• Continuum from 190-400 nm, emission lines >400nm
Light sources for UV-vis, continued
• Tungsten filament
• Most common visible and NIR source
• Blackbody radiator useful from 350-2500 nm
• Power varies as (operating voltage)4; need stable power supply!
• Tungsten-halogen sources can operate at higher temperatures and give off more UV
light.
Light sources for UV-vis, continued
• LEDs
• 375-1000 nm
• Semi-monochromatic (20-50 nm FWHM)
• “White” LEDs use phosphor to give 400-
800 nm continuum
• Keychain flashlights
• Xenon arc lamps
• Very intense source
• Continuum from 200-1000 nm, peaking
at 500 nm
Single-beam UV-vis spectrometers

Skoog, Fig. 13-13

Good light throughput, but


what if the source
power fluctuates?
Double-beam in time UV vis
spectrometers
• Beam is split in two, but measured by same detector

“in time” because


the beam appears in 2
places over one cycle in
time
- Sample
- Reference
- Sample
- Reference

What if the source


power fluctuates?

Skoog, Fig. 13-13


Double-beam in space UV-vis
spectrometers
• Beam is split into two paths and measured by matched detectors
• Difficult to find perfectly matched detectors

“in space” because Continuous


two beams are always Reference
present in space

What if the source


power fluctuates? Continuous
Sample
Cary 100 double beam spectrometer

- Sample
- Dark
- Reference
- Dark
Multichannel UV-vis spectrometers
• Dispersing optic (grating or prism)
used to separate different
wavelengths in space.
• Detection with diode array or CCD
• Fast acquisition of entire
spectrum, but much lower
resolution optical spectra
Diode array spectrophotometers

http://www.oceanoptics.com/products/usb4000.asp

Fairly inexpensive, but good


quality fiber optic models
available for ~$3000.
• Ocean Optics
• StellarNet 89 mm
3.5 inches
250 specta per sec
Attributes of UV-visible absorption for
quantitative analysis
1) Applicable to organic and inorganic species
2) Good detection limits: 10-100 mM or better
• Possible need for larger slit widths to achieve best sensitivities
3) Moderate to high selectivity
4) Accuracy: 1-3% or better
5) Ease and convenience ($$$) of data acquisition
Considerations for using UV-vis for
quantitative measurements
• Directly monitor absorbing analytes; usually non-destructive
• Can use reagents that react with colorless analyte to generate
measureable species
• Greatly increase molar absorptivity
• Thiocyanate (Fe, Co, Mo), H2O2 (Ti, V, Cr), iodide (Bi, Pd, Te)

• Monitor at wavelength of max absorption, max at lmax


• Greatest change in absorbance per unit concentration
• Absorbance least sensitive to a small change in wavelength
• Relaxes requirement on instrument to stringently achieve the exact
same wavelength
• UV-visible absorbance sensitive to environment, pH, temperature,
high electrolyte concentration, interfering species. Be careful with
standards
Calibration and mixture analysis

• Generate calibration curve (linear) using


external standards A1  M  bcM   N bc N
1 1
• Must use multiple standards
A2  M  bcM   N bc N
• Standards hopefully match sample matrix 2 2

• Matrix matching is hard—consider using


standard addition.
• Mixtures are additive
• Need to monitor at as many wavelengths as
components to be analyzed.
• Requirement of solving multiple equations
with multiple unknowns.

Isosbestic point
An isosbestic point

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