UV-VIS Spectroscopy - Chemical Analysis - Agilent Spectroscopy - Chemical Analysis Chemical Analysis Solutions Unit SiRS PhDSonia R. Sousa PhD Marketing Manager - Spectroscopy 21 January

UV-VIS Spectroscopy - Chemical Analysis

Chemical Analysis Solutions Unit

S i R S PhDSonia R. Sousa PhDMarketing Manager - Spectroscopy21 January 2009

Group/Presentation TitleAgilent Restricted

Month ##, 200XPage 1

Topics

• Basic UV-VIS Theory

UV VIS history and product offerings• UV-VIS history and product offerings

• key Instrumental parameters

• Method development

• Sample handling and measurements

• Questions and answers



THEORYTHEORY



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 dependencefollows Beer’s Law

A=εbcA = -log T

A εbcA = is absorbance (no units, since A = log(P0 / P )ε = the molar absorbtivity with units of L mol-1 cm-1

b = the path length of the sample (typically in cm)

Io = incident light intensityI = Intensity of light transmitted through solution

T= transmittance


p g p ( yp y )c = the concentration of the compound in solution mol L-1

through solution

Electronic Excitation by UV-VIS Spectroscopy

Energy Scale in UV-VIS Spectroscopy

Ref Fundamental of Molecular Spectroscopy (C N Banwell)



Ref. Fundamental of Molecular Spectroscopy (C. N. Banwell)

Table of ChromophoreN Ch h W l th [ ]Name Chromophore Wavelength [nm]

acetylide -C=C 175-180Aldehyde -CHO 210

amine -NH2 195azo -N=N- 285-400

bromide -Br 208bromide Br 208carboxyl -COOH 200-210

ester -COOR 205O 18ether -O- 185

ketone >C=O 195nitrate -ONO2 270nitrile -C=N 160nitrite -ONO 220 - 230nitro -NO 210



nitro -NO2 210

UV-VIS

Naphthalene Anthracene Pyrene

Conjugated Carbonyl Compounds

UV-VIS

HISTORY AND PRODUCTHISTORY AND PRODUCT OFFERINGS



Agilent UV-VIS Spectroscopy History



Product Offerings and Solutions

• Hardware and Software

Accessories• Accessories

• Fully test systems

• Compliance services for IQ/OQ/PV

• Supplies

G1120A Multicelltransport

89090A Tempco



Agilent UV-VIS 8453 Spectrometer

• Specifications• Wavelength range: 190 1100 nm• Wavelength range: 190 – 1100 nm• Slit width: 1 nm• Full spectrum scan: 0.1 sec

UV-VIS 8453G1103A

• Low stray light • High wavelength reproducibility and accuracy

Fully EP and USP compliant• Fully EP and USP compliant• GLP• Advanced mode: 21 CFR 11 compliant



Spectrometer Instrument DesignDual Beam Spectrometerp

Diode Array SpectrometerDiode Array Spectrometer



Conventional single beam spectrometer

Diode Array Advantages

• Simple mechanical and optical design

Open sample area• Open sample area

• Fixed arrangement of grating and detectors

• Fast acquisition of complete spectra

• Statistics



Open Sample Area

Reverse Optics

Room light cannot reach detector• Room light cannot reach detector• Narrow “acceptance angle” after sample• Geometry of spectrograph• Geometry of spectrograph

• Easy and convenient access• Better productivityBetter productivity• Less error prone

• Easy installation of acessories



ChemStation Software Mode Structure



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

Diff t i d f d t d lt• Different view modes for raw data and results• Graphical window• Table window• Table window

• New: Access to Agilent OpenLab Enterprise Content Management (ECM)



Solution for General Purpose



Solution for General Purpose



General Purpose SW - Key Features

• Graphical User Interface (GUI) with integrated status display

Includes all common application tasks• Includes all common application tasks

• Single dialog box for method setup

• Easy automation

• One page report

• Verification and diagnostics

• Execute advances method capabilityp y



Solution for Research and Development



Solution for Research and Development



Advanced SW – Key Features

• Flexible method setup

• Time based measurements• Time based measurements

• Multiple data analysis- confirmation analysis

• Single components analysis SCA and multi components analysis MCASingle components analysis SCA and multi components analysis MCA

• Automation

• Sophisticated interactive mathematical functions

• Sophisticated results statics

• Customization – macro programming

• Customer report generator



KEY INSTRUMENTALKEY INSTRUMENTAL PARAMETERS



Key Instrumental Parameters

• Spectral Resolution

Wavelength accuracy and precision• Wavelength accuracy and precision

• Photometric accuracy and precision• Stray light• Noise • DriftDrift




• Spectral Resolution – is the ability of an instrument to differentiate between two adjacent wavelengthsj g

Two wavelength are consideredresolved if the minimum betweenthe two peaks of the detector outputthe two peaks of the detector output signal is lower than 80% of maximum




• Wavelength accuracy and precision – important for the comparison of measurements made on different instrumentsp




• Photometric accuracy and precision• Stray light – is defined as detected light of any wavelength that lies

t id th b d idth f th l t d l thoutside the bandwidth of the selected wavelength• Stray light causes a negative bias in instrument response and eventually is the

limiting factor for the absorbance, and thereby concentration, that can be measuredmeasured




• Photometric accuracy and precision• Noise – affects the precision of the measurement, for a single

t i t d d i llmeasurement, may introduced error in accuracy as well.

Averaging of data pointsreduce noise

The total error at any absorbance is the Sum of the errors due to stray light and noise ( h t i d l t i i )



(photon noise and electronic noise)


• Photometric accuracy and precision• Drift normally results from variations in lamp intensity between the• Drift – normally results from variations in lamp intensity between the

measurements. Change in the instrument electronics also can cause drift.



METHOD DEVELOPMENTMETHOD DEVELOPMENT



Method Development

• Linearity

Accuracy• Accuracy

• Precision

• Sensitivity

• Range

• Selectivity



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 to the concentration of analytemathematically transformation, to the concentration of analytein sample within a given range.



Method Development

• Accuracy – is the degree of agreement between an individual test result generated by the method and the true valueg y



Method Development

• Precision – is the agreement among individual test results when the procedure is applied repeatelly to multiple samplingp pp p y p p g



Method Development

• Sensitivity – response obtained by a given analyte



Method Development

• Selectivity – the ability of a method to quantify accurately and specifically the analyte or analytes in the presence of other p y y y pcompounds



SAMPLE HANDLING ANDSAMPLE HANDLING AND MEASUREMENTS



Sample Handling and Measurement

• Cell• Material type and care• Material, type, and care

• Choice of solvent• Effect of solvent concentration pH and temperature• Effect of solvent, concentration, pH, and temperature

• Sample• Type strong or week absorbance interferenceType, strong or week absorbance, interference



Cell Material



Summary

• Advantage of diode array spectrometer• Fast spectral acquisition• Fast spectral acquisition• The full spectra can be used for error correction

• Background modeling• Derivative spectroscopy

• Simultaneous wavelength measurement• SensitivitySensitivity• Reliability



Acknowledgement

Thomas Klink Agilent Technologies• Thomas Klink – Agilent Technologies

• Tony Owens – Agilent Technologies

Fundamental of UV-VIS spectroscopy – Primer and WorkbookAgilent technologies





Documents

UV-VIS Spectroscopy - Chemical Analysis - Agilent Spectroscopy - Chemical Analysis Chemical Analysis Solutions Unit SiRS PhDSonia R. Sousa PhD Marketing Manager - Spectroscopy 21 January