Download UV-VIS Spectroscopy - Chemical Analysis

UV-VIS Spectroscopy - Chemical Analysis
Chemical Analysis Solutions
Unit
Sonia
S
i R
R. S
Sousa PhD
Marketing Manager - Spectroscopy
21 January 2009
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Topics
• Basic UV-VIS Theory
• UV-VIS
UV VIS history and product offerings
• key Instrumental parameters
• Method development
• Sample handling and measurements
• Questions and answers
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THEORY
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Theory – UV-VIS
The wavelength and amount of light that a compound absorbs depends on
its molecular structure and the concentration of the compound used.
Concentration dependence
follows Beer’s Law
A = -log T
A=εbc
A
εbc
A = is absorbance (no units, since A = log(P0 / P )
ε = the molar absorbtivity with units of L mol-1 cm-1
b = the p
path length
g of the sample
p (typically
( yp
y in cm))
c = the concentration of the compound in solution mol L-1
Io = incident light intensity
I = Intensity of light transmitted
through solution
T= transmittance
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Electronic Excitation by UV-VIS Spectroscopy
Energy Scale in UV-VIS Spectroscopy
Ref Fundamental of Molecular Spectroscopy (C
Ref.
(C. N
N. Banwell)
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Table of Chromophore
Page 7
N
Name
Ch
Chromophore
h
W
Wavelength
l
th [nm]
[
]
acetylide
-C=C
175-180
Aldehyde
-CHO
210
amine
-NH2
195
azo
-N=N-
285-400
bromide
-Br
Br
208
carboxyl
-COOH
200-210
ester
-COOR
205
ether
-OO
18
185
ketone
>C=O
195
nitrate
-ONO2
270
nitrile
-C=N
160
nitrite
-ONO
220 - 230
nitro
-NO2
210
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UV-VIS
Naphthalene
Anthracene
Pyrene
Conjugated Carbonyl Compounds
UV-VIS
HISTORY AND PRODUCT
OFFERINGS
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Agilent UV-VIS Spectroscopy History
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Product Offerings and Solutions
• Hardware and Software
• Accessories
• Fully test systems
• Compliance services for IQ/OQ/PV
• Supplies
G1120A Multicelltransport
89090A Tempco
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Agilent UV-VIS 8453 Spectrometer
• Specifications
• Wavelength range: 190 – 1100 nm
• Slit width: 1 nm
UV-VIS 8453
G1103A
• Full spectrum scan: 0.1 sec
• Low stray light
• High wavelength reproducibility and accuracy
• Fully EP and USP compliant
• GLP
• Advanced mode: 21 CFR 11 compliant
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Spectrometer Instrument Design
Dual Beam Spectrometer
p
Diode Array Spectrometer
Conventional single beam spectrometer
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Diode Array Advantages
• Simple mechanical and optical design
• Open sample area
• Fixed arrangement of grating and detectors
• Fast acquisition of complete spectra
• Statistics
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Open Sample Area
Reverse Optics
• Room light cannot reach detector
• Narrow “acceptance angle” after sample
• Geometry of spectrograph
• Easy and convenient access
• Better productivity
• Less error prone
• Easy installation of acessories
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ChemStation Software Mode Structure
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Common Features to all Applications
• Universal data structure
• Common basic features for
• Instrument control
• Accessory configuration and handling
• Method handling
• Raw data storage, retrieval and export
• Different
Diff
t view
i
modes
d for
f raw data
d t and
d results
lt
• Graphical window
• Table window
• New: Access to Agilent OpenLab Enterprise Content
Management (ECM)
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Solution for General Purpose
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Solution for General Purpose
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General Purpose SW - Key Features
• Graphical User Interface (GUI) with integrated status display
• Includes all common application tasks
• Single dialog box for method setup
• Easy automation
• One page report
• Verification and diagnostics
• Execute advances method capability
p
y
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Solution for Research and Development
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Solution for Research and Development
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Advanced SW – Key Features
•
Flexible method setup
•
Time based measurements
•
Multiple data analysis- confirmation analysis
•
Single components analysis SCA and multi components analysis MCA
•
Automation
•
Sophisticated interactive mathematical functions
•
Sophisticated results statics
•
Customization – macro programming
•
Customer report generator
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KEY INSTRUMENTAL
PARAMETERS
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Key Instrumental Parameters
• Spectral Resolution
• Wavelength accuracy and precision
• Photometric accuracy and precision
• Stray light
• Noise
• Drift
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Key Instrumental Parameters
• Spectral Resolution – is the ability of an instrument to
differentiate between two adjacent
j
wavelengths
g
Two wavelength are considered
resolved if the minimum between
the two peaks of the detector output
signal is lower than 80% of maximum
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Key Instrumental Parameters
• Wavelength accuracy and precision – important for the
comparison
p
of measurements made on different instruments
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Key Instrumental Parameters
• Photometric accuracy and precision
• Stray light – is defined as detected light of any wavelength that lies
outside
t id th
the b
bandwidth
d idth off the
th selected
l t d wavelength
l
th
• Stray light causes a negative bias in instrument response and eventually is the
limiting factor for the absorbance, and thereby concentration, that can be
measured
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Key Instrumental Parameters
• Photometric accuracy and precision
• Noise – affects the precision of the measurement, for a single
measurement,
t may introduced
i t d
d error iin accuracy as well.
ll
The total error at any absorbance is the
Sum of the errors due to stray light and noise
( h t noise
(photon
i and
d electronic
l t i noise)
i )
Page 31
Averaging of data points
reduce noise
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Key Instrumental Parameters
• Photometric accuracy and precision
• Drift – normally results from variations in lamp intensity between the
measurements. Change in the instrument electronics also can cause
drift.
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METHOD DEVELOPMENT
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Method Development
• Linearity
• Accuracy
• Precision
• Sensitivity
• Range
• Selectivity
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Method Development
• Linearity – is the ability of the method to produce test results
that are proportional, either directly or by a well-defined
mathematically transformation
transformation, to the concentration of analyte
in sample within a given range.
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Method Development
• Accuracy – is the degree of agreement between an individual
test result g
generated by
y the method and the true value
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Method Development
• Precision – is the agreement among individual test results
when the p
procedure is applied
pp
repeatelly
p
y to multiple
p sampling
p g
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Method Development
• Sensitivity – response obtained by a given analyte
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Method Development
• Selectivity – the ability of a method to quantify accurately and
specifically
p
y the analyte
y or analytes
y
in the p
presence of other
compounds
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SAMPLE HANDLING AND
MEASUREMENTS
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Sample Handling and Measurement
• Cell
• Material,
Material type,
type and care
• Choice of solvent
• Effect of solvent
solvent, concentration
concentration, pH
pH, and temperature
• Sample
• Type,
Type strong or week absorbance
absorbance, interference
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Cell
Material
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Summary
• Advantage of diode array spectrometer
• Fast spectral acquisition
• The full spectra can be used for error correction
• Background modeling
• Derivative spectroscopy
• Simultaneous wavelength measurement
• Sensitivity
• Reliability
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Acknowledgement
• Thomas Klink – Agilent Technologies
• Tony Owens – Agilent Technologies
Fundamental of UV-VIS spectroscopy – Primer and Workbook
Agilent technologies
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