Spectroscopy and Spectrofluorimetry

Introduction

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 nonradiative 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.

Interaction of Light with Matter

The diagram on the right illustrates the energy level structures of atoms and molecules. We need to pay our attention to how they interact with electromagnetic waves. Such interactions are at the very heart of spectroscopy.

There are lots of spectroscopic processes. The important ones are:

  • Absorption
  • Fluorescence
  • Rayleigh scattering
  • Raman scattering
  • Refraction

They appear to be different but in fact they are all closely related. We would like to examine these in much greater depth to understand them and their relationships.

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 identifying 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:

A = λ b c

where A is the measured absorbance, λ 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:A = ε b c
where ε is the wavelength-dependent molar absorptivity coefficient with a unit of M-1 cm-1.

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:A = -log T = - log (I / I o).

Modern absorption instruments can usually display the data as transmittance, % transmittance, or absorbance. An unknown concentration of an analyte can be determined by measuring the amount of light that a sample absorbs and applying Beer's law. If the absorptivity coefficient is not known, the unknown concentration can be determined using a working curve of absorbance versus concentrations derived from standards.

Limitations of the Beer-Lambert law

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 concentrations
  • shifts in chemical equilibrium as a function of concentrations
  • 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

Electromagnetic Spectrum

Type of Radiation / FrequencyRange (Hz) / WavelengthRange / Type of Transition
γ-rays / 1020-1024 / <10-12 m / nuclear
x-rays / 1017-1020 / 1 nm-1 pm / inner electron
ultraviolet / 1015-1017 / 400 nm-1 nm / outer electron
visible / 4-7.5x1014 / 750 nm-400 nm / outer electron
near-infrared / 1012-4x1014 / 2.5 um-750 nm / outer electron, molecular vibrations
infrared / 1011-1012 / 25 um-2.5 um / molecular vibrations
microwaves / 108-1012 / 1 mm-25 um / molecular rotations, electron spin flips*
radio waves / 100-108 / >1 mm / nuclear spin flips*

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, and for atoms excited with light it is called atomic fluorescence. For molecules it is called fluorescence if the transition is between states of the same spin and called phosphorescence if the transition occurs between states of different spin. In simple terms, phosphorescence is a process in which energy absorbed by a substance is released relatively slowly in the form of light.

The emission intensity of an emitting substance is linearly proportional to analyte concentrations(at low concentration range), and is useful for quantitating 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.

Raman Effect

The Raman effect occurs when light impinges upon a molecule and interacts with the electron cloud of the bonds of that molecule. The incident photon excites one of the electrons into a virtual state. For the spontaneous Raman effect, the molecule will be excited from the ground state to a virtual energy state, and relax into a vibrational excited state, which generates Stokes Raman scattering. If the molecule was already in an elevated vibrational energy state, the Raman scattering is then called anti-Stokes Raman scattering.

A molecular polarizability change, or amount of deformation of the electron cloud, with respect to the vibrational coordinate is required for the molecule to exhibit the Raman effect. The amount of the polarizability change will determine the Raman scattering intensity, whereas the Raman shift is equal to the vibrational level that is involved

Spectrofluorometer

Two general types of instruments exist:

  • Filter fluorometers use filters to isolate theincident light and fluorescentlight.
  • Spectrofluorometers use diffraction gratingmonochromators to isolate the incident light and fluorescent light.

Both types utilize the following scheme: The light from an excitation source passes through a filter or monochromator, and strikes the sample. A proportion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted in all directions. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90° to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector.

Various light sources may be used as excitation sources, including lasers, photodiodes, and lamps; xenon arcs and mercury vapor lamps in particular. A laser only emits light of high irradiance at a very narrow wavelength interval, typically under 0.01 nm, which makes an excitation monochromator or filter unnecessary. The disadvantage of this method is that the wavelength of a laser cannot be changed by much. A mercury vapor lamp is a line lamp, meaning it emits light near peak wavelengths. By contrast, a xenon arc has a continuous emission spectrum with nearly constant intensity in the range from 300-800 nm and a sufficient irradiance for measurements down to just above 200 nm.

Filters and/or monochromators may be used in fluorimeters. A monochromator transmits light of an adjustable wavelength with an adjustable tolerance. The most common type of monochromator utilizes diffraction grating, that is, collimated light enters a grating and exits with a different angle depending on the wavelength. The monochromator can then select which wavelengths to transmit. For allowing anisotropy measurements the addition of two polarization filters are necessary: One after the excitation monochromator or filter, and one before the emission monochromator or filter.

As mentioned before, the fluorescence is most often measured at a 90° angle relative to the excitation light. This geometry is used instead of placing the sensor at the line of the excitation light at a 180° angle in order to avoid interference of the transmitted excitation light. No monochromator is perfect and it will transmit some stray light, that is, light with other wavelengths than the targeted. An ideal monochromator would only transmit light in the specified range and have a high wavelength-independent transmission. When measuring at a 90° angle, only the light scattered by the sample causes stray light. This results in a better signal-to-noise ratio, and lowers the detection limit by approximately a factor 10000 when compared to the 180° geometry. Furthermore, the fluorescence can also be measured from the front, which is often done for turbid samples.

The detector can either be single-channeled or multichanneled. The single-channeled detector can only detect the intensity of one wavelength at a time, while the multichanneled detects the intensity at all wavelengths simultaneously, making the emission monochromator or filter unnecessary. The different types of detectors have both advantages and disadvantages.

The most versatile fluorimeters with dual monochromators and a continuous excitation light source can record both an excitation spectrum and a fluorescence spectrum. When measuring fluorescence spectra, the wavelength of the excitation light is kept constant, preferably at a wavelength of high absorption (i.e., maximum absorbance, ex), and the emission monochromator scans the spectrum. For measuring excitation spectra, the wavelength passing through the emission filter or monochromator is kept constant and the excitation monochromator is scanning. The excitation spectrum generally is identical to the absorption spectrum as the fluorescence intensity is proportional to the absorption.

Excitation sources

A xenon arc lamp is an artificial light source. Powered by electricity, it uses ionizedxenon gas to produce a bright white light that closely mimics natural daylight.Xenon arc lamps can be roughly divided into three categories:

  • Continuous-output xenon short-arc lamps
  • Continuous-output xenon long-arc lamps
  • Xenon flash lamps (which are usually considered separately)

Each consists of a glass or fused quartz arc tube with tungsten metal electrodes at each end. The glass tube is first evacuated and then re-filled with xenon gas. For xenon flashtubes, a third "trigger" electrode usually surrounds the exterior of the arc tube.

Our spectrofluorometer is equipped withContinuous-output xenon short-arc lamps where the spectrum is between 200 nm and 650 nm.

Monochromator designs

A typical monochromator design is shown on the right. It consists of a diffraction grating (dispersing element), slits, and spherical mirrors. The light source emits a broad spectrum of radiation as represented by the multi-colored line from the lamp to the grating. (The yellow color of the light source represents all colors.) The diffraction grating disperses light by diffracting different wavelengths at different angles. The grating is positioned so that green light passes through the exit slit and all other colors are blocked. The particular wavelength that passes through the monochromator is selected by rotating the angle of the grating. The mirror and slit positions remain fixed. If this grating was rotated clockwise slightly, what color light would pass through the exit slit? Scanning a spectrum is accomplished by rotating the grating with a motor. The detector measures the power of the light that strikes it, converting the light power to an electrical signal.

Monochromator parameters

  • Bandpass: The wavelength range that the monochromator transmits.
  • Dispersion: The wavelength dispersing power, usually given as spectral range / slit width (nm/mm). Dispersion depends on the focal length, grating resolving power, and the grating order.
  • Resolution: The minimum bandpass of the spectrometer, usually determined by the aberrations of the optical system.
  • Acceptance angle (f/#): A measure of light collecting ability, focal length / mirror diameter
  • Blaze wavelength: The wavelength of maximum intensity in first order.

Photomultiplier Tube (PMT)

Photomultiplier tubes (PMTs) convert photons to an electrical signal. They have a high internal gain and are sensitive detectors for low-intensity applications such as fluorescence spectroscopy.

Design. A PMT consists of a photocathode and a series of dynodes in an evacuated glass enclosure. When a photon of sufficient energy strikes the photocathode, it ejects a photoelectron due to the photoelectric effect. The photocathode material is usually a mixture of alkali metals, which make the PMT sensitive to photons throughout the visible region of the electromagnetic spectrum. The photocathode is at a high negative voltage, typically -500 to -1500 volts. The photoelectron is accelerated towards a series of additional electrodes called dynodes. These electrodes are each maintained at successively less negative potentials. Additional electrons are generated at each dynode. This cascading effect creates 105 to 107 electrons for each photoelectron that is ejected from the photocathode. The amplification depends on the number of dynodes and the accelerating voltage. This amplified electrical signal is collected at an anode at ground potential, which can be measured.

Phototubes are similar to PMTs, but consist of only a photocathode and anode. Since phototubes do not have a dynode chain to provide internal amplification, they are used in less sensitive applications such as absorption spectrometers.

Emission spectra

1. Fluorescence

Characteristics of Excited States

  1. Energy
  2. Lifetime
  3. Quantum Yield
  4. Polarization

2. Phosphorescence occurs at longer wavelength than does fluorescence.

Often, the emission band is red-shifted relative to the absorption band: "Stokes shift"

3. Quantum Yield (QY) = F

FF = number of fluorescence quanta emitted divided by number of quanta absorbed to a singlet excited state;

FF = ratio of photons emitted to photons absorbed

Quantum yield is the ratio of photons emitted to photons absorbed by the system: FF = kF / kF + kISC + knr + kq + kr

4. Polarization

Molecule of interest is randomly oriented in a rigid matrix (organic solvent at low temperature or room temperature polymer). Plane polarized light is used as the excitation source.

Polarization of fluorescence of phenol in propylene glycol at -70°C shows that the transition moments of the corresponding absorption bands are mutually perpendicular.

  • Phosphorescence is usually slow (seconds), therefore, quenching by impurities including oxygen usually makes phosphorescence difficult to observe. Low temperature glasses and rigorous exclusion of oxygen are usually necessary to observe phosphorescence. Since this condition is not biological, fluorescence is the primary emission process of biological relevance.
  • Relative quantum yields are determined by using a standard such as quinine sulfate in 1 N H2SO4 (fF = 0.70), or fluorescein in 0.1 N NaOH (fF = 0.93). The areas under the emission band of the standard relative to the sample are compared. It is, of course, important that the absorption at lex are matched.
  • Excited-state decay rates can be measured by exciting the sample with a short pulse of light and monitoring the emission as a function of time.

Fluorescence decay of a pure sample follows a single exponential decay. The dark line shows the excitation pulse.Time correlated single photon counting was used to obtain this data. This technique counts the number of emitted photons hitting a detector at times, t, following excitation.

One critical difference between steady-state and kinetic measurements of fluorescence is that the value of tF is not a function of concentration of the sample while the value of FF is concentration dependent. Only at low concentration is the value of FF linearly dependent on concentration. The reason is the so-called inner filter effect.

5. The inner filter effect

At low concentration, the emission of light is uniform from the front to the back of sample cuvette. At high concentration, more light is emitted from the front than the back. Since emitted light only from the middle of the cuvette is detected the concentration must be low to assure accurate FF measurements.

Fluorescence characteristics of chromophores found in proteins and nucleic acids. Generally, quantum yields are low and lifetimes are short.

Absorption / Fluorescence
Substance / Condition / lmax (nm) / lmax(nm) / fF / tF(nsec)
Tryptophan / H2O, pH 7 / 280 / 348 / 0.20 / 2.6
Tyrosine / H2O, pH 7 / 274 / 303 / 0.14 / 3.6
Phenylalanine / H2O, pH 7 / 257 / 282 / 0.04 / 6.4
Adenine / H2O, pH 7 / 260 / 321 / 2.6 • 10-4 / <0.02
Guanine / H2O, pH 7 / 275 / 329 / 2.6 • 10-4 / <0.02
Cytosine / H2O, pH 7 / 267 / 313 / 0.8 • 10-4 / <0.02
Uracil / H2O, pH 7 / 260 / 308 / 0.4 • 10-4 / <0.02
NADH / H2O, pH 7 / 340 / 470 / 0.019 / 0.40

Define: lmax (nm): maximum wavelength of the absorption; lmax (nm):maximum wavelength of the emission; fF: Quantum yield;tF (nsec): lifetime of the excited state.

Fluorescence emission spectra of human serum albumin (solid line), tryptophan alone (dashed line), and an 18:1 molar ratio of tyrosine to tryptophan (gray line): Excitation at 245 nm.

The 18:1 sample approximates the relative occurrence of these amino acids in the protein. Note that the spectrum of the protein closely resembles that of pure tryptophan because tyrosine sensitivity is low and its emission is most likely quenched by tryptophan (via energy-transfer mechanism). (Dr. Rima: where is the figure for this paragraph?)

Commonly, fluorescent probe molecules are used to characterize protein and nucleic acids.

Sensitivity is higher.

lmax is also different from biomolecule so selective excitation is possible.

Fluorescence generally is much more sensitive to the environment of the chromophore than is light absorption. Therefore, fluorescence is an effective technique for following the binding of ligands or conformational changes.

The sensitivity of fluorescence is a consequence of the relatively long time a molecule stays in an excited singlet state before deexcitation. Absorption, or CD, is a process that is over in 10-15 sec. On this time scale, the molecule and its environment are effectively static. In contrast, during the 10-9 to 10-8 sec that a singlet remains excited, all kinds of processes can occur, including protonation or deprotonation reactions, solvent-cage relaxation, local conformational changes, and any processes coupled to translational or rotational motion.