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i
Contents
Chapter 1 Principles and applications of UV-visible
Contents
Contents Appendix B Characteristics of diode array
- Basic principles.................................................................................................... spectroscopy
- The electromagnetic spectrum.....................................................................
- Wavelength and frequency
- Origin of UV-visible spectra
- Transmittance and absorbance
- Derivative spectra........................................................................................
- Obtaining derivative spectra
- Applications
- Signal-to-noise
- Instrumental considerations
- Qualitative analysis..............................................................................................
- Identification—spectra and structure
- Confirmation of identity
- Color............................................................................................................
- Other qualitative information
- Protein and nucleic acid melting temperature.....................................
- Enzyme activity
- Instrumental considerations.........................................................................
- Quantitative analysis............................................................................................
- Beer’s law....................................................................................................
- Sample requirements...........................................................................
- Multicomponent analysis
- Principle of additivity
- Simple simultaneous equations method..............................................
- Least squares method..........................................................................
- Other methods.....................................................................................
- Sample requirements...........................................................................
- Instrumental requirements
- Indirect quantification
- Chemical derivatization...............................................................................
- Spectrophotometric titrations
- Enzyme kinetic assays.................................................................................
- Instrumental design.............................................................................................. Chapter 2 Instrumentation
- Components.................................................................................................
- Sources................................................................................................
- Dispersion devices
- Detectors ii
- Optics
- The conventional spectrophotometer
- The diode array spectrophotometer.............................................................
- Configuration...............................................................................................
- Single-beam design.............................................................................
- Dual-beam design
- Split-beam design
- Dual-wavelength design
- Measuring a spectrum
- Key instrumental parameters
- Spectral resolution
- Wavelength accuracy and precision
- Photometric accuracy and precision............................................................
- Stray light............................................................................................
- Noise
- Linear dynamic range..................................................................................
- Drift
- Liquid samples Chapter 3 Sample handling and measurement
- Cells.............................................................................................................
- Material
- Cell types
- Sources of error...................................................................................
- Care of cells
- Choice of solvent.........................................................................................
- Effect of solvent, concentration, pH, and temperature................................
- Solid samples
- No reference
- Refractive index
- Sample geometry
- Weak absorbance
- Changing slit width
- Time averaging............................................................................................
- Wavelength averaging
- Strong absorbance................................................................................................
- Interference
- Types of interference...................................................................................
- Other absorbing compounds
- Scattering
- Correction techniques..................................................................................
- Isoabsorbance......................................................................................
- Multicomponent analysis
- Background modeling......................................................................... Contents
- Internal referencing.............................................................................
- Three-point correction
- Derivative spectroscopy......................................................................
- Photochemical problems......................................................................................
- Fluorescence................................................................................................
- Sample decomposition
- Method development Chapter 4 Method development and validation
- Linearity
- Accuracy......................................................................................................
- Precision
- Sensitivity....................................................................................................
- Range...........................................................................................................
- Selectivity....................................................................................................
- Ruggedness..................................................................................................
- Instrumental requirements
- Method validation
- Instrument performance verification.................................................................. Chapter 5 Routine operation
- Test parameters..........................................................................................
- Wavelength accuracy and precision
- Photometric accuracy and precision
- Stray light..........................................................................................
- Resolution
- Noise
- Baseline flatness
- Stability
- Linearity............................................................................................
- Standards
- Emission standards
- Solid absorption standards
- Liquid absorption standards..............................................................
- Regulatory requirements
- GLP/GMP
- European Pharmacopoeia
- United States Pharmacopoeia
- American Standard Testing Methods
- Recommendations
- Instrument self-test
- System suitability............................................................................................... iv
- Proper operation.................................................................................................
- Electronic storage
- Standard operating procedures
- Collateral data
- Confirmation wavelengths
- Full spectra
- Statistics.....................................................................................................
- Definition of terms............................................................................................. Appendix A Accuracy and precision
- Advantages of diode array spectroscopy spectrophotometers
- Fast spectral acquisition
- Simultaneous multiwavelength measurement
- Wavelength resettability............................................................................
- Sensitivity..................................................................................................
- Measurement statistics
- Ruggedness and reliability
- Open sample area
- Disadvantages of diode array spectroscopy
- Resolution..................................................................................................
- Stray light
- Sample decomposition
- Complexity
- Errors in measuring fluorescent samples...................................................
- References..................................................................
- Index...........................................................................
A Primer
Fundamentals of
UV-visible
spectroscopy
Tony Owen
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Preface
Preface In 1988 we published a primer entitled “ The Diode-Array
Advantage in UV/Visible Spectroscopy”. At the time, although diode array spectrophotometers had been on the market since 1979, their characteristics and their advantages compared with conventional scanning spectrophotometers were not well-understood. We sought to rectify the situation. The primer was very well-received, and many thousands of copies have been distributed. Much has changed in the years since the first primer, and we felt this was an appropriate time to produce a new primer. Computers are used increasingly to evaluate data; Good Laboratory Practice has grown in importance; and a new generation of diode array spectrophotometers is characterized by much improved performance. With this primer, our objective is to review all aspects of UV-visible spectroscopy that play a role in obtaining the best results. Microprocessor and/or computer control has taken much of the drudgery out of data processing and has improved productivity. As instrument manufacturers, we would like to believe that analytical instruments are now easier to operate. Despite these advances, a good knowledge of the basics of UV-visible spectroscopy, of the instrumental limitations, and of the pitfalls of sample handling and sample chemistry remains essential for good results. With this primer, we also want to show that the conventional “single measurement at a single wavelength” approach to obtaining results is insufficient for assuring optimum results. Multiple measurements at multiple wavelengths or (preferably) full spectra yield the best accuracy and precision of results and provide the information necessary to detect erroneous results. I would like to take this opportunity to thank my colleagues, too numerous to mention by name, at Hewlett-Packard from whom I have learned so much about UV-visible spectroscopy over the years.
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Principles and applications of UV-visible spectroscopy
This chapter outlines the basic theories and principles of
UV-visible spectroscopy. These provide valuable insight into
the uses and limitations of this technique for chemical
analysis. The primary applications of UV-visible
spectroscopy are also briefly reviewed.
Basic principles
The electromagnetic
spectrum
Ultraviolet (UV) and visible radiation comprise only a small part of the electromagnetic spectrum, which includes such other forms of radiation as radio, infrared (IR), cosmic, and X rays (see Figure 1).
Figure 1 The electromagnetic spectrum
Frequency [Hz]
Wavelength [m]
Ultraviolet Visible Infrared
Cosmic rayGamma rayX ray UltravioletvisibleInfraredMicrowaveRadarTelevisionNMRRadioUltrasonicSonic(audible) Infrasonic
Principles and applications of UV-visible spectroscopy
The energy associated with electromagnetic radiation is defined by the following equation:
where E is energy (in joules), h is Planck’s constant (6.62 × 10-34^ Js), and ν is frequency (in seconds).
Wavelength and frequency Electromagnetic radiation can be considered a combination of
alternating electric and magnetic fields that travel through space with a wave motion. Because radiation acts as a wave, it can be classified in terms of either wavelength or frequency, which are related by the following equation:
where ν is frequency (in seconds), c is the speed of light (3 × 10^8 ms-1), and λ is wavelength (in meters). In UV-visible spectroscopy, wavelength usually is expressed in nanometers (1 nm = 10-9^ m). It follows from the above equations that radiation with shorter wavelength has higher energy. In UV-visible spectroscopy, the low-wavelength UV light has the highest energy. In some cases, this energy is sufficient to cause unwanted photochemical reactions when measuring sample spectra (remember, it is the UV component of light that causes sunburn).
Origin of UV-visible
spectra
When radiation interacts with matter, a number of processes can occur, including reflection, scattering, absorbance, fluorescence/phosphorescence (absorption and reemission), and photochemical reaction (absorbance and bond breaking). In general, when measuring UV-visible spectra, we want only absorbance to occur. Because light is a form of energy, absorption of light by matter causes the energy content of the molecules (or atoms) to increase. The total potential energy of a molecule generally is represented as the sum of its electronic, vibrational, and rotational energies:
E = hν
ν =c ⁄λ
Principles and applications of UV-visible spectroscopy
Figure 3 Electronic transitions and spectra of atoms
However, for molecules, vibrational and rotational energy levels are superimposed on the electronic energy levels. Because many transitions with different energies can occur, the bands are broadened (see Figure 4). The broadening is even greater in solutions owing to solvent-solute interactions.
Figure 4 Electronic transitions and UV-visible spectra in molecules
electronic energy levels vibrational energy levels rotational energy levels electronic transition
Principles and applications of UV-visible spectroscopy
Transmittance and
absorbance
When light passes through or is reflected from a sample, the amount of light absorbed is the difference between the incident radiation ( I o ) and the transmitted radiation ( I ). The amount of light absorbed is expressed as either transmittance or absorbance. Transmittance usually is given in terms of a fraction of 1 or as a percentage and is defined as follows:
or %
Absorbance is defined as follows:
For most applications, absorbance values are used since the relationship between absorbance and both concentration and path length normally is linear.
Derivative spectra If a spectrum is expressed as absorbance ( A ) as a function of
wavelength (λ), the derivative spectra are:
Zero order:
First order:
Second order:
Figure 5 on the next page shows the effects of derivatization on a simple Gaussian absorbance band. The derivative spectra are always more complex than the zero-order spectrum. The first derivative is the rate of change of absorbance against wavelength. It starts and finishes at zero, passing through zero at the same wavelength as λmax of the absorbance band. This
T = I ⁄ I o T = ( I ⁄ I o) × 100
A = –log T
A = f ( λ)
dA
d λ
------- = f ′ λ( )
d
2
A
d λ
2
--------- = f ″ λ( )
Principles and applications of UV-visible spectroscopy
Figure 5 Derivative spectra of a Gaussian absorbance band
Obtaining derivative spectra Optical, electronic, and mathematical methods all can be used to generate derivative spectra. Although optical and electronic techniques formed the basis of early UV-visible spectroscopy, these have been largely superseded by mathematical methods. To calculate the derivative at a particular wavelength (λ), a window of ± n data points is selected, and a polynomial
is fitted by the least squares method. The coefficients a 0 … a l at each wavelength are the derivative values, where a 1 is the first
Absorbance Absorbance
1st derivative 2nd derivative
3rd derivative 4th derivative
A λ = a 0 + a 1 λ + … + a l λ
Principles and applications of UV-visible spectroscopy
derivative, a 2 is the second derivative, and so on. Savitzky and Golay developed a highly efficient method to perform the calculations that is the basis of the derivatization algorithm in most commercial instruments. This method also smooths the data. If the polynomial order ( l ) is less than the number of data points (2n+1) in the window, the polynomial generally cannot pass through all data points. Thus the least squares fit gives a smoothed approximation to the original data points. Although transforming a UV-visible spectrum to its first or a higher derivative usually yields a more complex profile than the zero-order spectrum (see Figure 5), the intrinsic information content is not increased. In fact, it is decreased by the loss of lower-order data such as constant offset factors.
Applications Derivative spectra can be used to enhance differences among spectra, to resolve overlapping bands in qualitative analysis (see “Confirmation of identity” on page 19) and, most importantly, to reduce the effects of interference from scattering, matrix, or other absorbing compounds in quantitative analysis (see “Derivative spectroscopy” on page 81).
Signal-to-noise An unwanted effect of the derivatization process is the decrease in S/N with higher orders of derivatives. This decrease follows from the discrimination effect (see “Derivative spectroscopy” on page 81) and from the fact that noise always contains the sharpest features in the spectrum. Thus, if the spectral data used in the derivative calculation are at 2-nm intervals, the noise has a 2-nm bandwidth. If the analyte band has a bandwidth of 20 nm, the S/N of the first derivative will be 10 times worse than with the zero-order spectrum. The smoothing properties of the Savitzky-Golay polynomial technique can be used to mitigate the decrease in S/N, but care must be taken as too high a degree of smoothing will distort the derivative spectrum.
Instrumental considerations The higher resolution of derivative spectra places increased demands on the wavelength reproducibility of the spectrophotometer. Small wavelength errors can result in much
Principles and applications of UV-visible spectroscopy
The presence of an absorbance band at a particular wavelength often is a good indicator of the presence of a chromophore. However, the position of the absorbance maximum is not fixed but depends partially on the molecular environment of the chromophore and on the solvent in which the sample may be dissolved. Other parameters, such as pH and temperature, also may cause changes in both the intensity and the wavelength of the absorbance maxima. Conjugating the double bond with additional double bonds increases both the intensity and the wavelength of the absorption band. For some molecular systems, such as conjugated hydrocarbons or carotenoids, the relationship between intensity and wavelength has been systematically investigated. Transition metal ions also have electronic energy levels that cause absorption of 400–700 nm in the visible region.
Confirmation of identity Although UV-visible spectra do not enable absolute
identification of an unknown, they frequently are used to confirm the identity of a substance through comparison of the measured spectrum with a reference spectrum. Where spectra are highly similar, derivative spectra may be used. As shown in Figure 6, the number of bands increases with higher orders of derivatives. This increase in complexity of the derivative spectra can be useful in qualitative analysis, either for characterizing materials or for identification purposes. For example, the absorbance spectrum of the steroid testosterone shows a single, broad, featureless band centered at around 330 nm, whereas the second derivative shows six distinct peaks. The resolution enhancement effect may be of use as well in identifying an unknown. Figure 6 shows a computer simulation.
Nitrile RC=N Acetonitrile < 160 Nitro RNO 2 Nitromethane 271
Table 1 Selected chromophores and their absorbance maxima
Principles and applications of UV-visible spectroscopy
When two Gaussian bands with a 40-nm natural spectral bandwidth (NBW) separated by 30 nm are added in absorbance mode, a single band with a maximum midway between the two component bands results. The two components are not resolved. In the fourth derivative, these two bands are clearly visible, with maxima centered close to the λmax of the component bands.
Figure 6 Resolution enhancement
Color Color is an important property of a substance. The color of matter
is related to its absorptivity or reflectivity. The human eye sees the complementary color to that which is absorbed, as shown in Figure 7 and Figure 8.
Absorbance
4th derivative
