Fundamentals of modern UV-visible spectroscopy Presentation Materials F undamentals of modern UV-visible spectroscopy 1F igure : The Electromagnetic Spectrum = c / E = h F undamentals of modern UV-visible spectroscopy 2F igure : Electronic Transitions in Formaldehyde F undamentals of modern UV-visible spectroscopy 3F igure : Electronic Transitions and Spectra of Atoms F undamentals of modern UV-visible spectroscopy 4F igure : Electronic Transitions and UV-visible Spectra in Molecules F undamentals of modern UV-visible spectroscopy 5F igure : Derivative Spectra of a Gaussian Absorbance Band 1st Derivative: 2nd Derivative: Absorbance: )(' f d dA = )(fA = )('' 2 2 f d Ad = F undamentals of modern UV-visible spectroscopy 6F igure : Resolution Enhancement * Overlay of 2 Gaussian bands with a NBW of 40 nm separated by 30 nm * Separated by 4th derivative F undamentals of modern UV-visible spectroscopy 7F igure : Transmission and Color The human eye sees the complementary color to that which is absorbed F undamentals of modern UV-visible spectroscopy 8F igure : Absorbance and Complementary Colors F undamentals of modern UV-visible spectroscopy 9F igure : Transmittance and Concentration The Bouguer-Lambert Law PathlengthConst eIIT == 0/ F undamentals of modern UV-visible spectroscopy 10F igure : Transmittance and Path Length Beer's Law ionConcentratConst eIIT == 0/ Concentration F undamentals of modern UV-visible spectroscopy 11F igure : The Beer-Bouguer-Lambert Law ( ) ( ) cbIIIITA ==-=-= /log/loglog 00 F undamentals of modern UV-visible spectroscopy 12F igure : Two-Component Mixture Example of a two-component mixture with little spectral overlap F undamentals of modern UV-visible spectroscopy 13F igure : Two-Component Mixture Example of a two-component mixture with significant spectral overlap F undamentals of modern UV-visible spectroscopy 14F igure : Influence of 10% Random Error Influence on the calculated concentrations * Little spectral overlap: 10% Error * Significant spectral overlap: Depends on similarity, can be much higher (e.g. 100%) F undamentals of modern UV-visible spectroscopy 15F igure : Absorption Spectra of Hemoglobin Derivatives F undamentals of modern UV-visible spectroscopy 16F igure : Intensity Spectrum of the Deuterium Arc Lamp * Good intensity in UV range * Useful intensity in visible range * Low noise * Intensity decreases over lifetime F undamentals of modern UV-visible spectroscopy 17F igure : Intensity Spectrum of the TungstenHalogen Lamp * Weak intensity in UV range * Good intensity in visible range * Very low noise * Low drift F undamentals of modern UV-visible spectroscopy 18F igure : Intensity Spectrum of the Xenon Lamp * High intensity in UV range * High intensity in visible range * Medium noise F undamentals of modern UV-visible spectroscopy 19F igure : Dispersion Devices * Non-linear dispersion * Temperature sensitive * Linear Dispersion * Different orders F undamentals of modern UV-visible spectroscopy 20F igure : Photomultiplier Tube Detector Anode * High sensitivity at low light levels * Cathode material determines spectral sensitivity * Good signal/noise * Shock sensitive F undamentals of modern UV-visible spectroscopy 21F igure : The Photodiode Detector * Wide dynamic range * Very good signal/noise at high light levels * Solid-state device F undamentals of modern UV-visible spectroscopy 22F igure : Schematic Diagram of a Photodiode Array * Same characteristics as photodiodes * Solid-state device * Fast read-out cycles F undamentals of modern UV-visible spectroscopy 23F igure : Conventional Spectrophotometer Schematic of a conventional single-beam spectrophotometer F undamentals of modern UV-visible spectroscopy 24F igure : Diode-Array Spectrophotometer Schematic of a diode-array spectrophotometer F undamentals of modern UV-visible spectroscopy 25F igure : Diode-Array Spectrophotometer Optical diagram of the HP 8453 diode-array spectrophotometer F undamentals of modern UV-visible spectroscopy 26F igure : Conventional Spectrophotometer Optical system of a double-beam spectrophotometer F undamentals of modern UV-visible spectroscopy 27F igure : Diode-Array Spectrophotometer Optical system of the HP 8450A diode-array spectrophotometer F undamentals of modern UV-visible spectroscopy 28F igure : Conventional Spectrophotometer Optical system of a split-beam spectrophotometer F undamentals of modern UV-visible spectroscopy 29F igure : Definition of Resolution Spectral resolution is a measure of the ability of an instrument to differentiate between two adjacent wavelengths F undamentals of modern UV-visible spectroscopy 30F igure : Instrumental Spectral Bandwidth The SBW is defined as the width, at half the maximum intensity, of the band of light leaving the monochromator F undamentals of modern UV-visible spectroscopy 31F igure : Natural Spectral Bandwidth The NBW is the width of the sample absorption band at half the absorption maximum F undamentals of modern UV-visible spectroscopy 32F igure : Effect of SBW on Band Shape The SBW/NBW ratio should be 0.1 or better to yield an absorbance measurement with an accuracy of 99.5% or better F undamentals of modern UV-visible spectroscopy 33F igure : Effect of Digital Sampling The sampling interval used to digitize the spectrum for computer evaluation and storage also effects resolution F undamentals of modern UV-visible spectroscopy 34F igure : Wavelength Resettability Influence of wavelength resettability on measurements at the maximum and slope of an absorption band F undamentals of modern UV-visible spectroscopy 35F igure : Effect of Stray Light Effect of various levels of stray light on measured absorbance compared with actual absorbance F undamentals of modern UV-visible spectroscopy 36F igure : Theoretical Absorbance Error The total error at any absorbance is the sum of the errors due to stray light and noise (photon noise and electronic noise) F undamentals of modern UV-visible spectroscopy 37F igure : Effect of Drift Drift is a potential cause of photometric error and results from variations between the measurement of I0 and I F undamentals of modern UV-visible spectroscopy 38F igure : Transmission Characteristics of Cell Materials Note that all materials exhibit at least approximately 10% loss in transmittance at all wavelengths F undamentals of modern UV-visible spectroscopy 39F igure : Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volume Cell Types I F undamentals of modern UV-visible spectroscopy 40F igure : Cell Types II Micro cell (a) for very small volumes and flow-through cell (b) for automated applications F undamentals of modern UV-visible spectroscopy 41F igure : Effect of Refractive Index Changes in the refractive index of reference and sample measurement can cause wrong absorbance measurements F undamentals of modern UV-visible spectroscopy 42F igure : Non-planar Sample Geometry Some sample can act as an active optical component in the system and deviate or defocus the light beam F undamentals of modern UV-visible spectroscopy 43F igure : Averaging of data points reduces noise by the square root of the number of points averaged Effect of Integration Time F undamentals of modern UV-visible spectroscopy 44F igure : * Wavelength averaging reduces also the noise (square root of data points) * Amplitude of the signal is affected Effect of Wavelength Averaging F undamentals of modern UV-visible spectroscopy 45F igure : Selection of a wavelength in the slope of a absorption band can increase the dynamic range and avoid sample preparation like dilution Increasing Dynamic Range F undamentals of modern UV-visible spectroscopy 46F igure : Scattering causes an apparent absorbance because less light reaches the detector Scattering F undamentals of modern UV-visible spectroscopy 47F igure : Scatter Spectra * Rayleigh scattering: Particles small relative to wavelength * Tyndall scattering: Particles large relative to wavelength F undamentals of modern UV-visible spectroscopy 48F igure : Absorbance at the reference wavelength must be equivalent to the interference at the analytical wavelength Isoabsorbance Corrections F undamentals of modern UV-visible spectroscopy 49F igure : Background modeling can be done if the interference is due to a physical process Background Modeling F undamentals of modern UV-visible spectroscopy 50F igure : Corrects for constant background absorbance over a range Internal Referencing F undamentals of modern UV-visible spectroscopy 51F igure : Three-Point Correction * Uses two reference wavelengths * Corrects for sloped linear background absorbance F undamentals of modern UV-visible spectroscopy 52F igure : Discrimination of Broad Bands * Derivatives can eliminate background absorption * Derivatives discriminate against broad absorbance bands F undamentals of modern UV-visible spectroscopy 53F igure : Scatter Correction by Derivative Spectroscopy Scatter is discriminated like a broad-band absorbance band F undamentals of modern UV-visible spectroscopy 54F igure : Effect of Fluorescence The emitted light of a fluorescing sample causes an error in the absorbance measurement F undamentals of modern UV-visible spectroscopy 55F igure : Acceptance Angles and Magnitude of Fluorescence Error * Forward optics: Absorbance at the excitation wavelengths are too low * Reversed optics: Absorbance at the emission wavelengths are too low F undamentals of modern UV-visible spectroscopy 56F igure : Inadequate Calibration * Theoretically only one standard is required to calibrate * In practice, deviations from Beer's law can cause wrong results F undamentals of modern UV-visible spectroscopy 57F igure : Calibration Data Sets * Forward optics: Absorbance at the excitation wavelengths are too low * Reversed optics: Absorbance at the emission wavelengths are too low F undamentals of modern UV-visible spectroscopy 58F igure : Wavelength(s) for Best Linearity * A linear calibration curve is calculated at each wavelength * The correlation coefficient gives an estimate on the linearity F undamentals of modern UV-visible spectroscopy 59F igure : Wavelength(s) for Best Accuracy * The quantification results are calculated at each wavelength * The calculated concentration are giving an estimate of the accuracy F undamentals of modern UV-visible spectroscopy 60F igure : Precision of an Analysis Precision of a method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings F undamentals of modern UV-visible spectroscopy 61F igure : Wavelength(s) for Best Sensitivity * Calculation of relative standard deviation of the measured values at each wavelength * The wavelength with lowest %RSD likely will yield the best sensitivity F undamentals of modern UV-visible spectroscopy 62F igure : Wavelength(s) for Best Selectivity Selectivity is the ability of a method to quantify accurately and specifically the analyte or analytes in the presence of other compounds F undamentals of modern UV-visible spectroscopy 63F igure : Ideal Absorbance and Wavelength Standards * An ideal absorbance standard would have a constant absorbance at all wavelengths * An ideal wavelength standard would have very narrow, well-defined peaks F undamentals of modern UV-visible spectroscopy 64F igure : Ideal Stray Light Filter An ideal stray light filter would transmit all wavelengths except the wavelength used to measure the stray light F undamentals of modern UV-visible spectroscopy 65F igure : Holmium Perchlorate Solution The most common wavelength accuracy standard is a holmium perchlorate solution F undamentals of modern UV-visible spectroscopy 66F igure : Potassium Dichromate Solution The photometric accuracy standard required by several pharmacopoeias is a potassium dichromate solution F undamentals of modern UV-visible spectroscopy 67F igure : Stray Light Standard Solutions The most common stray light standard and the respectively used wavelengths F undamentals of modern UV-visible spectroscopy 68F igure : Toluene in Hexane (0.02% v/v) The resolution is estimated by taking the ratio of the absorbance of the maximum near 269 nm and minimum near 266 nm F undamentals of modern UV-visible spectroscopy 69F igure : Confirmation Analysis In confirmation analysis, the absorbance at one or more additional wavelengths are used to quantify a sample F undamentals of modern UV-visible spectroscopy 70F igure : Spectral Similarity Comparative plots of similar and dissimilar spectra F undamentals of modern UV-visible spectroscopy 71F igure : Precision and Accuracy Precision ­ Precision + Precision ­ Precision + Accuracy ­ Accuracy ­ Accuracy + Accuracy + F undamentals of modern UV-visible spectroscopy 72F igure : Hydrolysis of Sultone Wavelength [nm] Absorbance[AU]