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Section17:Spectrophotometry
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Spectrophotometry
Spectroscopy is the use of the absorption, emission, or scattering of electromagnetic radiation by atoms or molecules (or atomic or molecular ions) to qualitatively or quantitatively study the atoms or molecules, or to study physical processes. The interaction of radiation with matter can cause redirection of the radiation and/or transitions between the energy levels of the atoms or molecules. A transition from a lower level to a higher level with transfer of energy from the radiation field to the atom or molecule is called absorption. A transition from a higher level to a lower level is called emission if energy is transferred to the radiation field, or non-radiative decay if no radiation is emitted. Redirection of light due to its interaction with matter is called scattering, and may or may not occur with transfer of energy, i.e., the scattered radiation has a slightly different or the same wavelength.
Absorption
When atoms or molecules absorb light, the incoming energy excites a quantized structure to a higher energy level. The type of excitation depends on the wavelength of the light. Electrons are promoted to higher orbitals by ultraviolet or visible light, vibrations are excited by infrared light, and rotations are excited by microwaves. An absorption spectrum is the absorption of light as a function of wavelength. The spectrum of an atom or molecule depends on its energy level structure, and absorption spectra are useful for identification of compounds. Measuring the concentration of an absorbing species in a sample is accomplished by applying the Beer-Lambert Law.
The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and concentration of an absorbing species. The general Beer-Lambert law is usually written as:
where A is the measured absorbance, a() is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert law is written as:
where is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm1. Experimental measurements are usually made in terms of transmittance (T), which is defined as T = I / Io, where I is the light intensity after it passes through the sample and Io is the initial light intensity. The relation between A and T is:
The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of nonlinearity include:
- Deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity.
- Scattering of light due to particulates in the sample.
- Fluoresecence or phosphorescence of the sample.
- Changes in refractive index at high analyte concentration.
- Shifts in chemical equilibria as a function of concentration.
- Non-monochromatic radiation. (Deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band).
- Stray light leaking into the sample compartment.
Emission
Atoms or molecules that are excited to high energy levels can decay to lower levels by emitting radiation (emission or luminescence). For atoms excited by a high-temperature energy source this light emission is commonly called atomic or optical emission (see atomic-emission spectroscopy), and for atoms excited with light it is called atomic fluorescence (see atomic-fluorescence spectroscopy). For molecules it is called molecular fluorescence if the transition is between states of the same spin and phosphorescence if the transition occurs between states of different spin. The emission intensity of an emitting substance is linearly proportional to analyte concentration at low concentrations, and is useful for quantifying emitting species.
Scattering
When electromagnetic radiation passes through matter, most of the radiation continues in its original direction but a small fraction is scattered in other directions. Light that is scattered at the same wavelength as the incoming light is called Rayleigh scattering. Light that is scattered in transparent solids due to vibrations (phonons) is called Brillouin scattering. Brillouin scattering is typically shifted by 0.1 to 1 cm-1 from the incident light. Light that is scattered due to vibrations in molecules or optical phonons in solids is called Raman scattering. Raman scattered light is shifted by as much as 4000 cm-1 from the incident light.
Ultraviolet and Visible Absorption Spectroscopy
UV-vis spectroscopy is the measurement of the wavelength and intensity of absorption of near-ultraviolet and visible light by a sample. Ultraviolet and visible light are energetic enough to promote outer electrons to higher energy levels. UV-vis spectroscopy is usually applied to molecules and inorganic ions or complexes in solution. The uv-vis spectra have broad features that are of limited use for sample identification but are very useful for quantitative measurements. The concentration of an analyte in solution can be determined by measuring the absorbance at some wavelength and applying the Beer-Lambert Law. The light source is usually a hydrogen or deuterium lamp for uv measurements and a tungsten lamp for visible measurements. The wavelengths of these continuous light sources are selected with a wavelength separator such as a prism or grating monochromator. Spectra are obtained by scanning the wavelength separator and quantitative measurements can be made from a spectrum or at a single wavelength.
Dual-beam uv-vis spectrophotometer
In single-beam uv-vis absorption spectroscopy, obtaining a spectrum requires manually measuring the transmittance (see the Beer-Lambert Law) of the sample and solvent at each wavelength. The double-beam design greatly simplifies this process by measuring the transmittance of the sample and solvent simultaneously. The detection electronics can then manipulate the measurements to give the absorbance.
Specifications of a typical spectrophotometer:
| Wavelength range: | 200~800 nm |
| Spectral Band width: | 2 nm |
| Wavelength Accuracy: | 1 nm |
| Stray Light: | Less than 0.002%(300 nm, 630 nm) |
| Photometric Range: | Absorbance-1~3 Abs |
| Transmittance: | 0~200%T |
| Photometric Accuracy: | 0.5%T |
| Photometric Repeatability: | 0.3%T |
| Light Source: | Tungsten Lamp, Deuterium Lamp |
| Monochromator: | Diffraction grating single monochromator system |
| Cell: | Max. 4 turrets |
| Dimension: | 500mm(L) x 380 mm(W) x 230 mm(H) |
| Power Requirements: | 110/220V, 3A, 50/60 Amps. |
Fluorescence Spectroscopy
Light emission from atoms or molecules can be used to quantify the amount of the emitting substance in a sample. The relationship between fluorescence intensity and analyte concentration is:
where F is the measured fluorescence intensity, k is a geometric instrumental factor, QE is the quantum efficiency (photonsemitted/photons absorbed), Po is the radiant power of the excitation source, is the wavelength-dependent molar absorptivity coefficient, b is the path length, and c is the analyte concentration (, b, and c are the same as used in the Beer-Lambert law). Expanding the above equation in a series and dropping higher terms gives:
This relationship is valid at low concentrations (<10-5 M) and shows that fluorescence intensity is linearly proportional to analyte concentration. Determining unknown concentrations from the amount of fluorescence that a sample emits requires calibration of a fluorimeter with a standard (to determine K and QE) or by using a working curve.
Many of the limitations of the Beer-Lambert law also affect quantitative fluorimetry. Fluorescence measurements are also susceptible to inner-filter effects. These effects include excessive absorption of the excitation radiation (pre-filter effect) and self-absorption of atomic resonance fluorescence (post-filter effect).
