T1.1 The Raman Effect and Normal Raman Scattering T1-

T1.2 Resonance-Enhanced Raman Scattering T1-

T1.3 Surface-Enhanced Raman Scattering T1-

T2.1 Holographic Optics T2-

T2.2 The Axial Transmissive Spectrograph T2-

T2.3 Low Power Lasers: HeNe and Solid State Systems T2-

T2.4 Charge Coupled Devices T2-

T3.1 Identification of Unknown Materials T3-

T3.2 Inorganic Materials Characterization T3-

T3.3 Polymer Characterization T3-

T3.4 Biochemical and Biomedical Applications T3-

T4.1 The Classical 90° Geometry T4-

T4.2 Fiber Optic Probes T4-

T4.3 The Raman Microprobe T4-

T4.4 Specialized Sampling Techniques T4-

T5.1 Fluorescence and Laser Wavelength Choice T5-

T5.2 Calibration Techniques T5-

T5.3 Correction for Instrument Response T5-

T5.4 Solution Handling Techniques T5-

T6.1 Special Data Analysis T6-

T6.2 Normal and Resonance-Enhanced Raman Spectroscopy of Foodstuffs T6-

T6.3 Thermodynamics of Hydrogen Bonding in a Benzoic Acid Solution. Raman Thermometry T6-

T6.4 Crystal Lattice Modes and Bonding Differences in Graphite and Diamond T6-

T6.5 Surface Enhanced Raman Spectroscopy of Pyridine on Silver Colloids T6-

Figure T1.1 Energy level diagram for Raman scattering; (a) Stokes Raman scattering (b) anti-Stokes Raman scattering. T1-4

Figure T2.1 Simple illustration of Bragg diffraction using tilted grating, one order evanescent. T2-4

Figure T2.2 Sketch showing how a HoloPlex grating is constructed. T2-6

Figure T2.3 Transmission curve for a Holographic 785 SuperNotch Plus filter. T2-7

Figure T2.4 Volume hologram fabrication: (a) notch filter, and (b) transmission grating. T2-9

Figure T2.5 A basic diagram of the dispersion stage of the axial transmissive spectrograph, showing locations of slit, lenses, grating and camera. T2-11

Figure T2.6 Diagram of complete Raman spectrograph, including prefilter stage. T2-13

Figure T2.7 Energy levels in the HeNe laser. T2-15

Figure T2.8 Sketch of HeNe laser, showing mirrors and cavity. T2-16

Figure T2.9 Multiple layers of a CCD device and the use of gate biasing to collect and move charge. T2-18

Figure T2.10 Three-phase gate design. To move the charge to the right, v₃ would be biased positive and v₁ switched to zero. T2-20

Figure T2.11 Quantum efficiency as a function of wavelength for a typical, front illuminated CCD. T1-21

Figure T2.12 Three designs for two-dimensional readout of a CCD. The "m"s in each diagram represents measurement amplifiers. T2-22

Figure T2.13 Multi-pin phase design incorporating negatively charged gates and doped silicon layers for the formation of well boundaries. T2-23

Figure T3.1 Comparison of Raman spectra of (a) amorphous and (b) semi-crystalline poly(ethylene terephthalate) films. The spectral features most sensitive to crystallinity are highlighted. (reprinted with permission from ref. 12). T3-5

Figure T4.1 The basic 90° illumination/collection geometry (focused laser in, collection lens to spectrograph box out). T4-3

Figure T4.2 6-around-1 probe, showing both ends of fibers. T4-5

Figure T4.3 Diagram of Mark II probe showing lenses one though five (PL1-PL5), power adjustment control (PA), grating (G), diffuse screen indicator (DS), probe head shutter, spatial filter (SF), beam combiner and notch filters, and the linear polarizers (PB). T4-6

Figure T4.4 Block diagram of microprobe. T4-7

Figure T4.5 Simple diagram of confocal geometry. T4-9

Figure T4.6 Illustration of a wave guide. T4-11

Figure T5.1 Ne lamp spectrum with wavelengths indicated for several of the lines. T5-4

Figure T6.1 The spectrum of sodium carbonate (a) before and (b) after the removal of the background Raman signal from silica. Also shown is the background silica Raman spectrum used in the subtraction, as collected from a sample of sodium chloride (c). T6-4

Figure T6.2 Raman spectra of (a) egg albumin and (b) dried egg yolk. Average of 4 exposures at 50 seconds each. T6-9

Figure T6.3 Raman spectrum from a carrot slice (a) before background subtraction and (b) after background subtraction (scaled x3). Exposure time of 20 seconds. T6-9

Figure T6.4 The temperature dependence of the Raman spectrum of 0.2 M sodium benzoate solution in the OH stretch region. The benzoic acid C-H stretch is also visible Does this/should this band have frequency or intensity temperature dependence? T6-14

Figure T6.5 Logarithm ratio of integrated band areas (weakly-hydrogen bonded to strongly hydrogen bonded) as a function of reciprocal temperature, K^-1. The best-fit line has a slope of 660 K and an intercept of -1.89. T6-14

Figure T6.6 Temperature dependence of the Raman spectrum of (a) deprotonated and (b) protonated benzoic acid in the carbonyl region. Spectra are offset vertically for clarity. T6-15

Figure T6.7 The Raman spectra of (a) diamond (5 second exposure) and (b) graphite (8 minute exposure). T6-17

Figure T6.8 The Raman spectra of (a) standard IC silicon (90 second exposure, offset for clarity) and (b) silicon from an EPROM (10 second exposure). T6-18

Figure T6.9 (a) Surface-enhanced Raman spectrum of 0.05 M pyridine on an acidified silver colloid. (b) Unenhanced 0.05 M pyridine Raman spectrum. Asterisks indicate pyridine peaks too weak to observe. Inset is expanded view of the 1000 cm^-1 peak region after background subtraction. Integration time, 30 seconds for each spectrum. T6-20

T1.1 The Raman Effect and Normal Raman Scattering T1-

T1.1.1 The Scattering Process T1-

T1.1.2 Vibrational Energies T1-

T1.1.3 Raman Selection Rules and Intensities T1-

T1.1.4 Polarization Effects T1-

T1.2 Resonance-Enhanced Raman Scattering T1-

T1.3 Surface-Enhanced Raman Scattering T1-

T1.1 The Raman Effect and Normal Raman Scattering

When light is scattered from a molecule, most photons are elastically scattered. The scattered photons, therefore, have the same energy (frequency) and wavelength as the incident photons. However, a small fraction of light (approximately 1 in 10⁷ photons) is scattered at optical frequencies different from, and usually lower than, the frequency of the incident photons. The process which leads to this inelastic scatter is termed the Raman effect. Raman scattering can occur due to a change in vibrational, rotational or electronic energy of a molecule, but chemists are concerned primarily with the vibrational Raman effect. We will use the term Raman effect to mean vibrational Raman effect only.

The difference in energy between the incident photon and the Raman-scattered photon is equal to the energy of a vibration of the scattering molecule. A plot of intensity of scattered light versus energy difference, then, is a Raman spectrum.

T1.1.1 The Scattering Process

The Raman effect arises when a photon is incident on a molecule and interacts with the electric dipole of the molecule. It is a form of electronic (more accurately, vibronic) spectroscopy, although the spectrum contains vibrational frequencies. In classical terms, the interaction can be viewed as a perturbation of the molecule’s electric field. In quantum mechanics, the scattering is described as an excitation to a virtual state lower in energy than a real electronic transition with nearly coincident de-excitation and a change in vibrational energy. The scattering event occurs in 10-14 seconds or less. The virtual-state description of scattering is shown in Figure T1.1a.

The energy difference between the incident and scattered photons is represented by the arrows of different lengths in Figure T1.1a. Numerically, the energy difference between the initial and final vibrational levels, , or Raman shift in wavenumbers (cm-1), is calculated through equation 1

in which l _incident and l _scattered are the wavelengths (in cm) of the incident and Raman scattered photons, respectively. The vibrational energy is ultimately dissipated as heat. Because of the low intensity of Raman scattering, the heat dissipation does not cause a measurable temperature rise in a material.

At room temperature, the thermal population of vibrational excited states is low, although not zero. Therefore, for the majority of molecules, the initial state is the ground state, and the scattered photon will have lower energy (longer wavelength) than the exciting photon (called Stokes shift). This Stokes shifted scatter is what is usually observed in Raman spectroscopy and is depicted in Figure T1.1a.

According to the Boltzman population of states, a small fraction of the molecules are in vibrationally excited states. Raman scattering from vibrationally excited molecules leaves the molecule in the ground state. The scattered photon appears at higher energy, as shown in Figure T1.1b. This anti-Stokes-shifted Raman spectrum is always weaker than the Stokes-shifted spectrum, but at room temperature it is strong enough to be useful for vibrational frequencies less than about 1500 cm-1. The Stokes and anti-Stokes spectra contain the same frequency information. The ratio of anti-Stokes to Stokes intensity at any vibrational frequency is a measure of temperature. Anti-Stokes Raman scattering can be used for contact-less thermometry. Furthermore, the anti-Stokes spectrum can also be used when the Stokes spectrum is not directly observable, for example because of poor detector response or spectrograph efficiency.

The energy of a vibrational mode depends on molecular structure and environment. Atomic mass, bond order, molecular substituents, molecular geometry and hydrogen bonding all effect the vibrational force constant which in turn dictates the vibrational energy. For example, the stretching frequency of a phosphorus-phosphorus bond ranges from 460 to 610 to 775 cm^-1 for the single, double, and triple bonded moieties, respectively. [1] Much effort has been devoted to estimation or measurement of force constants. For small molecules, and even for some extended structures such as peptides, reasonably accurate calculations of vibrational frequencies are possible with commercially available software.

Vibrational Raman spectroscopy is not limited to intramolecular vibrations. Crystal lattice vibrations and other motions of extended solids are Raman-active. Their spectra are important in such fields as polymers and semiconductors. In the gas phase, rotational structure is resolvable on vibrational transitions. The resulting vibration/rotation spectra are widely used to study combustion and gas phase reactions generally. Vibrational Raman spectroscopy in this broad sense is an extraordinarily versatile probe into a wide range of phenomena ranging across disciplines from physical biochemistry to materials science.

T1.1.3 Raman Selection Rules and Intensities

A simple classical electromagnetic field description of Raman spectroscopy can be used to explain many of the important features of Raman band intensities. The dipole moment, P, induced in a molecule by an external electric field, E, is proportional to the field as shown in equation 2.

P = a E (2)

The proportionality constant a is the polarizability of the molecule. The polarizability measures the ease with which the electron cloud around a molecule can be distorted. The induced dipole emits or scatters light at the optical frequency of the incident light wave.

Raman scattering occurs because a molecular vibration can change the polarizability. The change is described by the polarizability derivative, , where Q is the normal coordinate of the vibration. The selection rule for a Raman-active vibration, that there be a change in polarizability during the vibration, is given in equation 3.

(3)

The Raman selection rule is analogous to the more familiar selection rule for an infrared-active vibration, which states that there must be a net change in permanent dipole moment during the vibration. From group theory it is straightforward to show that if a molecule has a center of symmetry, vibrations which are Raman-active will be inactive or forbidden in the infrared, and vice versa.

Scattering intensity is proportional to the square of the induced dipole moment, that is to the square of the polarizability derivative, .

If a vibration does not greatly change the polarizability, then the polarizability derivative will be near zero, and the intensity of the Raman band will be low. The vibrations of a highly polar moiety, such as the O-H bond, are usually weak. An external electric field can not induce a large change in the dipole moment, and stretching or bending the bond does not change this.

Typical strong Raman scatterers are moieties with distributed electron clouds, such as carbon-carbon double bonds. The pi-electron cloud of the double bond is easily distorted in an external electric field. Bending or stretching the bond changes the distribution of electron density substantially, and causes a large change in induced dipole moment. The observed strength of the Raman band is also proportional to the concentration of the species, as well as the intensity of the excitation laser.

Chemists generally prefer a quantum-mechanical approach to Raman scattering theory, which relates scattering frequencies and intensities to vibrational and electronic energy states of the molecule. The standard perturbation theory treatment assumes that the frequency of the incident light is low compared to the frequency of the first electronic excited state. The small changes in the ground state wave function are described in terms of the sum of all possible excited vibronic states of the molecule.

T1.1.4 Polarization Effects

Raman scatter is partially polarized, even for molecules in a gas or liquid, where the individual molecules are randomly oriented. The effect is most easily seen with an exciting source which is plane polarized. In isotropic media, polarization arises because the induced electric dipole has components which vary spatially with respect to the coordinates of the molecule. Raman scatter from totally symmetric vibrations will be strongly polarized parallel to the plane of polarization of the incident light. The scattered intensity from non-totally symmetric vibrations is 3/4 as strong in the plane perpendicular to the plane of polarization of the incident light as in the plane parallel to it.

The situation is more complicated in a crystalline material. In that case the orientation of the crystal is fixed in the optical system. The polarization components depend on the orientation of the crystal axes with respect to the plane of polarization of the input light, as well as on the relative polarization of the input and the observing polarizer.

T1.2 Resonance-Enhanced Raman Scattering

Raman spectroscopy is conventionally performed with green, red, or near-infrared lasers. The wavelengths are below the first electronic transitions of most molecules, as assumed by scattering theory. The situation changes if the wavelength of the exciting laser is within the electronic spectrum of a molecule. In that case the intensity of some Raman-active vibrations increases by a factor of 10²-10⁴. This resonance enhancement or resonance Raman effect can be quite useful.

Metalloporphyrins, carotenoids, and several other classes of biologically important molecules have strong allowed electronic transitions in the visible. The spectrum of the chromophoric moiety is resonance enhanced and that of the surrounding protein matrix is not. This allows the physical biochemist to probe the chromophoric site (often the active site) without spectral interference from the surrounding protein. Resonance Raman spectroscopy is also a major probe of the chemistry of fullerenes, polydiacetylenes, and other "exotic" molecules which strongly absorb in the visible. Although many more molecules absorb in the ultraviolet, the high cost of lasers and optics for this spectral region have limited UV resonance Raman spectroscopy to a small number of specialists.

The vibrations whose Raman bands are resonance enhanced fall into two or three general classes. The most common case is Franck-Condon enhancement, in which a component of the normal coordinate of the vibration is in a direction in which the molecule expands during an electronic excitation. The more the molecule expands along this axis when it absorbs light, the larger the enhancement factor. The easily visualized ring breathing (in-plane expansion) modes of porphyrins fall into this class. Vibrations which couple two electronic excited states are also resonance enhanced. This mechanism is called vibronic enhancement. In both cases enhancement factors roughly follow the intensities of the absorption spectrum. The theory of resonance enhancement is beyond the scope of this tutorial, and the interested reader is referred to specific reviews. [2]

Resonance enhancement does not begin at a sharply defined wavelength. In fact, enhancement of 5X-10X is commonly observed if the exciting laser is even within a few hundred wavenumbers below the electronic transition of a molecule. This pre-resonance enhancement can be experimentally useful.

T1.3 Surface-Enhanced Raman Scattering

The Raman scattering from a compound (or ion) adsorbed on or even within a few Angstroms of a structured metal surface can be 10³-10⁶ X greater than in solution. This surface-enhanced Raman scattering is strongest on silver, but is observable on gold and copper as well. At practical excitation wavelengths, enhancement on other metals is unimportant. Surface-enhanced Raman scattering (SERS) arises from two mechanisms.

The first is an enhanced electromagnetic field produced at the surface of the metal. When the wavelength of the incident light is close to the plasma wavelength of the metal, conduction electrons in the metal surface are excited into an extended surface electronic excited state called a surface plasmon resonance. Molecules adsorbed or in close proximity to the surface experience an exceptionally large electromagnetic field. Vibrational modes normal to the surface are most strongly enhanced.

The second mode of enhancement is by the formation of a charge-transfer complex between the surface and analyte molecule. The electronic transitions of many charge transfer complexes are in the visible, so that resonance enhancement occurs.

Molecules with lone-pair electrons or pi clouds show the strongest SERS. The effect was first discovered with pyridine. Other aromatic nitrogen or oxygen containing compounds, such as aromatic amines or phenols, are strongly SERS active. The effect can also been seen with other electron-rich functionalities such as carboxylic acids.

The intensity of the surface plasmon resonance is dependent on many factors including the wavelength of the incident light and the morphology of the metal surface. The wavelength should match the plasma wavelength of the metal. This is about 382 nm for a 5 m m silver particle, [3] but can be as high as 600 nm for larger ellipsoidal silver particles. The plasma wavelength is to the red of 650 nm for copper and gold, [4] the other two metals which show SERS at wavelengths in the 350-1000 nm region. The best morphology for surface plasmon resonance excitation is a small (<100 nm) particle or an atomically rough surface.

SERS is used to study monolayers of materials adsorbed on metals, including electrodes. Many formats other than electrodes can be used. The most popular include colloids, metal films on dielectric substrates and, recently, arrays of metal particles bound to metal or dielectric colloids through short linkages. Although SERS allows easy observation of Raman spectra from solution concentrations in the micromolar (1 X 10^-6) range, slow adsorption kinetics and competitive adsorption limit its application in analytical chemistry.

T2.1 Holographic Optics T2-

T2.1.1 Holographic Gratings T2-

T2.1.2 Multiplexed Gratings T2-

T2.1.3 Holographic Filters T2-

T2.1.4 Hologram Construction T2-

T2.2 The Axial Transmissive Spectrograph T2-

T2.3 Low Power Lasers: HeNe and Solid State Systems T2-

T2.3.1 The Helium-Neon Laser T2-

T2.3.2 The Diode-pumped Neodymium/YAG Laser (DPY) T2-

T2.3.3 The GaAlAs diode laser T2-

T2.4 Charge Coupled Devices T2-

T2.4.1 Charge Origin and Collection

T2.4.2 Charge Movement and Measurement T2-

T2.4.3 Two-Dimensional Readout T2-

T2.4.4 Sources of Noise T2-

T2.4.5 Imaging vs. Spectroscopic CCDs T2-

The HoloLab 1000, like all Kaiser Optical Systems instruments, uses volume-phase holograms to perform filtering and dispersion functions. As optical elements, these holograms offer unique combinations of high efficiency, controllable spectral response, and low scattering. [1] Volume-phase holograms entered spectroscopic instrumentation less than ten years ago as high performance band rejection filters. The first spectrograph to use a volume-phase holographic grating, the Kaiser Optical Systems HoloSpec f/1.8i, appeared shortly thereafter.

Volume-phase holograms are periodic refractive index gradients generated in transparent materials. Because the structures are periodic, they diffract light. Depending on the

application, the hologram can be as little as 3-4 mm thick to about 100 mm thick. They operate in the Bragg, or volume, diffraction regime. X-ray diffraction is the most familiar application of Bragg diffraction. Acousto-optic tunable filters and laser beam deflectors are also based on Bragg diffraction. [2] Most conventional reflection based diffraction gratings operate in the surface diffraction regime.

In the Bragg regime, diffraction occurs through an angle, q , given by equation 1 and illustrated in Figure T2.1.

In equation 1, m is the diffraction order, l is the wavelength and d is the fringe spacing. By convention, m is positive if diffraction is to the right, as viewed from the light source. For a sufficiently thick grating, diffraction is efficient only in +1 order and in the 0 order.

T2.1.1 Holographic Gratings

In order to obtain a large spectral bandwidth, the gratings in many Kaiser Optical Systems spectrographs use thin films, which can result in light being diffracted into orders other than the +1 and 0 orders. For some grating geometries, however, such as the geometry shown in Figure T2.1, all orders other than +1 and 0 are evanescent, and thus no light is diffracted into these orders. The gratings in many Kaiser Optical Systems spectrographs have diffraction in one order only. In a tilted grating, diffraction of s-polarized light can be close to 100% efficient. But in this configuration the electrical field vectors of incident and refracted p-polarized light are not orthogonal, and diffraction efficiency is reduced. Overall, a volume transmission holographic grating can have a diffraction efficiency of 80% or greater for unpolarized light.

The fringe spacing can be made small, resulting in a high dispersion grating. Volume-phase holographic gratings used in Raman spectrographs have dispersions as high as 5000 grooves/mm. As a consequence, it is possible to construct compact, high resolution spectrographs using these holographic transmission gratings.

Every volume-phase holographic diffraction grating is an original, not a replica. By proper choice of the thickness of the hologram, its refractive index modulation, and the spatial frequency (periodicity) of the fringes, the grating can be optimized for any desired wavelength range.

T2.1.2 Multiplexed Gratings

Transmission gratings can be stacked one after another to extend their operating range, as shown in Figure T2.2. The Kaiser Optical Systems HoloPlex grating uses this technique to allow coverage of a wider wavelength region than would be available with a single grating without a loss in spectral resolution. The grating fringes are tilted and the gratings rotated slightly so that light from one grating is deflected upwards and the from the other downwards. The effect is to increase the frequency coverage of the grating system without reducing dispersion. A 100-4400 cm-1 operating range is available with the HoloPlex grating designed for Raman spectroscopy with green (532 nm) laser excitation and for a 26 mm detector width. The range is 100-3500 cm-1 with the HoloPlex grating designed for NIR excitation.

The spectrum obtained with a HoloPlex grating appears on the CCD detector as two pieces, each on different regions of the detector. The instrument software contains routines to splice these pieces together properly, so that the user sees only one continuous high resolution Raman spectrum. The HoloLab 1000 uses a single diffraction grating, not stacked gratings. It is designed for a wide spectral window at moderate resolution.

A notch filter is usually operated at normal incidence. Tilting the filter at a small angle (<15°) shifts the rejection band to lower frequencies. This simple strategy allows use of a SuperNotch-Plus filter at frequencies as close as 40 cm^-1 from the exciting laser line, but it is not without cost. As the filter is tilted further from normal incidence, its reflection efficiency decreases and its sensitivity to polarization increases.

The greatest practical advantage of the holographic notch filter is the smoothness of its transmission curve, which makes its low wavenumber performance greatly superior to the performance of a thin film dielectric filter. [4] A dielectric filter has undulations in its transmission curve which arise from the abrupt changes in refractive index at the layers of the filter. Undulations are especially severe in the transition region between high and low transmission. They seriously degrade the low wavenumber performance of the thin film filter to the point where measurement of vibrational frequencies below about 400-500 cm^-1 becomes difficult or impossible. In addition, the holographic filter has the narrower rejection band, further adding to its low frequency performance advantage.

Other optical elements can be constructed by volume holography. Currently, the most important commercially available device for spectroscopy is the laser bandpass filter. Filters are available for many important laser wavelengths ranging from 442 nm (HeCd) to 1064 nm (Nd:YAG ) in the near infrared.

The bandpass filter is a diffraction grating which is mounted between prisms which are used to deviate the input and output beams so as to obtain an overall 90° or in-line operation. The transmission of the holographic band pass filter is greater than 90%. Dielectric band pass filters have transmissions in the 50-90% range, depending on the wavelength of operation, the width of the pass band and the fabrication technique and materials used.

T2.1.4 Hologram Construction

Volume-phase holograms are periodic refractive index distributions in an optically thick (3-100 mm) medium. They are formed by a photographic process which is illustrated in Figure T2.4. The photographic medium is exposed to two mutually coherent beams, derived from the same laser. When these beams interfere they form periodic regions of exposed/unexposed film. The latent image is developed to form the hologram.

The angle of the incident laser beam and the wavelength of the laser together determine the fringe spacing and, therefore, the reflection wavelength. It is not necessary that the laser which generates the hologram be at the center reflection wavelength of the subsequently generated notch filter.

Transmission gratings are constructed by bringing two beams into the film, usually from the same side of the film, as in Figure T2.4b. The beams are derived from a single laser with a beamsplitter. If the beams are incident at equal and opposite angles with respect to the normal to the film plane, the fringes will be perpendicular to the surface. As with the notch filter, the illuminating laser wavelength does not have to be at the design center wavelength of the grating.

Holograms can be made in many different materials, including ordinary photographic film. The preferred material for spectroscopic quality holograms is dichromated gelatin (DCG), which is prepared fresh for each batch of holograms. DCG yields holograms which have high transmission and very low scattered light, but efficient diffraction.

A uniform layer of dichromated gelatin is carefully spread on a flat glass or quartz plate, which may be 10x10 cm2 square or larger. The "film" is exposed to laser light, forming cross-links in the gelatin matrix. After exposure the hologram is washed in water, which removes the residual sensitizer and swells the film. It is then washed in alcohol to remove the water. Finally, it is rapidly dried to collapse the swollen film. The dried hologram is sealed between two layers of glass with

an optical cement. It is cut into standard sizes for final use, and the edges are sealed to prevent moisture from entering.

While the process is simple in principle, production of high quality holograms requires carefully controlled conditions and strict attention to all details of the process. To prevent particulate contamination, Kaiser Optical Systems holographic optical elements are fabricated in clean rooms similar to those used in semiconductor manufacture. Particles in a hologram scatter light and render the contaminated device useless for spectroscopy.

The holograms are exposed with stabilized argon ion lasers, using mirrors, beamsplitters and holographic media mounted on vibration-isolation optical tables. As in any interferometric process, good isolation from room vibrations is necessary to ensure formation of well-defined fringes. Development, washing, and drying are performed with close control of time and temperature.

Volume holographic optical elements are best suited for use in the visible or near infrared. Because dichromated gelatin is used as the recording medium, the window for maximum transmission is about 380 nm - 2.5 mm. Aromatic amino acids limit the ultraviolet transmission of gelatin-based devices. Because most volume holographic filters and gratings are designed for operation above 400 nm, the support medium is usually glass. Quartz can be used to extend the operating range of thin holograms into the near ultraviolet.

T2.2 The Axial Transmissive Spectrograph

The axial transmissive spectrograph uses a volume holographic transmission grating in combination with well-corrected photographic or video lenses to achieve almost aberration-free imaging performance in a compact package. Figure T2.5 illustrates the basic design of the instrument. [5]

These photographic lenses are used on-axis as collimating and focusing elements with a volume holographic transmission grating placed in the collimated region between the lenses. With this on-axis design, aberrations are inherently less than in an off-axis configuration, such as the familiar Czerny-Turner spectrograph. Residual aberrations are inherently easier to correct in an on-axis design.

Photographic and closed circuit TV lenses are ideally suited for use in this spectrograph. They are designed for projection of a large, flat field with almost complete correction of spherical aberration, coma, and astigmatism over a large wavelength range. High quality lenses are available at moderate cost because they are produced in large volumes. They can be made with transmission of 90% or higher and with ¦ /numbers as low as ¦ /1.4.

The diffracted light from the spectrograph stage is focused onto the surface of a CCD detector, which records the actual spectrum. The light path is axially symmetric through the input and output lenses, which minimizes aberrations in the system. The grating is at an angle close to 45° , so that diffracted light is forced into the +1 order for highest diffraction efficiency. The grating angle makes the overall optical path approximately L-shaped.

The axial transmissive design allows operation at very low ¦ /number. Most Kaiser Optical Systems spectrographs for Raman spectroscopy operate at ¦ /1.8. This extraordinary light-gathering power results in greater intensity delivered to the detector than is possible with a Czerny-Turner design. Coupling to optical fibers, which typically have ¦ /numbers close to ¦ /2, is nearly 100% efficient.

By contrast, spherical aberration limits the light gathering power of a Czerny-Turner spectrograph to ¦ /4. With equal laser power on a sample the Czerny-Turner may deliver to the detector a signal only 25% as intense as that produced by the axial transmissive spectrograph. The image quality of the Czerny-Turner is degraded by astigmatism and spherical aberration. Even in the best designs, which employ toroidal mirrors to correct for astigmatism, imaging is reasonably good only at the very center of the field of view. However, image quality along with resolution and the ability to view extended sources such as fiber bundles, deteriorates rapidly towards the edges of the field.

The HoloLab 1000 and other Kaiser Optical Systems spectrographs intended for Raman spectroscopy include a prefilter stage, which contains the notch filter for laser line rejection. The complete spectrograph is shown in Figure T2.6.

The first lens collimates the input signal, which then passes through the notch filter to attenuate laser light. The filtered signal, with laser light intensity reduced by at least 10⁶X using Notch-Plus or SuperNotch-Plus holographic filters, is focused on the spectrograph entrance slit with the second lens. The slit functions as a spatial filter to minimize transmission of stray light into the dispersion stage of the spectrograph.

Because of the excellent correction of the lenses used in the instrument, the image of a point can be focused to nearly the diffraction limit. Slit widths between 25, 50 or 100 mm are commonly used. The use of high transmission lenses and an efficient diffraction grating results in a Raman spectrograph with high throughput. The high quality lenses and the high dispersion gratings result in a compact instrument.

T2.3 Low Power Lasers: HeNe and Solid State Systems

Few applications on a modern single stage Raman spectrograph require the 1-5 Watt illumination common in earlier systems. Instead, lasers with a maximum output of 5-300 mW are used. The benefits include reduced risk of sample damage, greater operator safety, convenience, and lower capital and operating costs.

The laser is based on stimulated emission between atomic or molecular energy levels, which can be made to dominate over absorption, in a three-level or four-level system. The kinetic equations describing this system can be found in any introductory treatment of laser theory, such as Demtroder’s. [6] Four-level systems dominate, because it is easier to maintain a population inversion than in a three-level system.

T2.3.1 The Helium-Neon Laser

The helium-neon (HeNe) laser illustrates the basics of population inversion. The energy levels for Helium and Neon are illustrated in Figure T2.7. The laser cavity itself is a long, narrow gas discharge tube, operated at high voltage (1500-3000 V) and low current (<10 mA).

Collisions with electrons in the discharge excite He to the F₃ and F₂ levels. These states have long lifetimes, so collisional energy transfer to Ne atoms produce an efficient population in E₆ and E₄ states. The wavelengths for the allowed transitions are given in Figure T2.7. From the E₆ level one can obtain 3.39 mm or 632.8 nm emission. From the E₄ state one can obtain 1.15 mm. There are also several other transitions the 540-600 nm range suitable for laser use which are not shown in Figure T2.7. Wavelength-selective mirrors determine which transition is observed in the laser.

Stimulated emission will be the dominant process at 632.8 nm if the population of E₆ can be kept higher than the population of E₃. E₆ is replenished by collision with excited Ne atoms, while E₃ is emptied by rapid spontaneous emission to E₂. He atoms can be excited efficiently from E₂ to E₃, however. This process counteracts population inversion. A small diameter discharge tube is used to facilitate collisional de-excitation from E₂ to E₁. The narrow discharge tube limits the maximum current, so that only relatively low powers are available from the HeNe laser.

The Kaiser Optical Systems HoloLab 1000 and all other modern Raman spectrographs use charge coupled device (CCD) detectors. The CCD detector has been universally adopted because it combines large dynamic range, low noise, and high quantum efficiency with the ability to store and measure charge in two dimensions and thereby represent images. [7] The devices designed for spectroscopy and scientific imagery are constructed somewhat differently than video CCD chips. While both types work on the same premises, this latter type of chip is designed for rapid readout, not low noise.

T3.1 Identification of Unknown Materials T3-

T3.2 Inorganic Materials Characterization T3-

T3.3 Polymer Characterization T3-

T3.4 Biochemical and Biomedical Applications T3-

T3.1 Identification of Unknown Materials

As a major structure elucidation tool supporting chemical synthesis, vibrational spectroscopy has given way to nuclear magnetic resonance spectroscopy and mass spectroscopy. Identification of unknown compounds in microscopic samples or in forms such as irregular solids, which are incompatible with other spectroscopies, remains an important application area of Raman spectroscopy. For detective work, the Handbook of Characteristic Frequencies of Organic Compounds complied by Lin-Vien et al is invaluable, as are the Raman and Infrared Atlas of Organic Compounds edited by B. Schrader and the Handbook of Infrared and Raman Spectra of Inorganic Compounds and Organic Salts edited by R.A. Nyquist et al. [1-3]

T3.2 Inorganic Materials Characterization

Because the vibrational spectrum is sensitive to the local chemical environment, Raman spectroscopy is widely used to characterize the properties of materials. Crystal lattice dimensions change when dopants are added, or materials are subjected to mechanical or thermal stresses. These changes cause small (typically 0.1-10 cm^-1) changes in some bands. Band widths may also change. The changes are easily monitored and even mapped to assess the state of a material. The polarization dependence of the Raman spectrum can be used to measure crystal orientation. We discuss applications to three important materials: diamond, graphite and silicon.

Raman spectroscopy is a major tool in the study and characterization of diamond and other forms of carbon. [4,5] Diamond has a single triply-degenerate first order phonon, which is Raman-active and silent in the infrared. It appears as an intense, narrow band at 1332 cm^-1. For thin films the intensity varies linearly with the film thickness. Because their spectra are intense and characteristic, Raman spectroscopy is used to identify and quantify graphitic impurities in diamond and amorphous carbon impurities in graphites as well.

Graphite has a narrow band around 1565-1585 cm^-1, which is a phonon consisting of in-plane C-C stretches. It is less intense than the diamond phonon, but still easily observed, with the exact frequency depending upon the type of graphite. The polycrystalline graphite phonon is at 1580 cm^-1, and, for highly ordered pyrolytic graphite, it is at 1576 cm^-1. In both cases the band is narrow. Amorphous sp₂ (graphitic) carbons such as glassy carbon have a pair of bands at about 1345 and 1585 cm^-1. Again, the band position varies with the type of carbon, but since these materials are disordered, the bands are much broader than for graphite or diamond.

The silicon phonon at 522 cm^-1 is a valuable diagnostic, [6] when microspectroscopy is generally employed, because of the small dimensions of semiconductor device. [7] The band is narrow (<3 cm^-1) in single crystal silicon, and the band asymmetry is indicative of microcrystalline silicon. The crystal orientation can then be deduced from polarization properties.

The most common application is probably strain mapping, because ion implantation distorts the silicon lattice, causing the phonon band to move to a lower frequency, often by as much as 10-20 cm^-1. Typically, a line through the structure is plotted, but complete 2-dimensional maps are becoming increasingly common.

Because the silicon phonon is a vibration of an extended lattice, the frequency is a function of interatomic spacings, which increases with temperature. The phonon band shifts about -0.016 cm^-1/°C. The band position, therefore, can be used to measure the temperature of a silicon wafer during processing. It can also be used to map temperature distributions in different regions of an operating device.

T3.3 Polymer Characterization

Raman spectroscopy plays multiple roles in polymer characterization. [8-10] It is particularly useful for measuring unsaturation in polymers, because of the intensity of the C=C stretching vibration. Residual unsaturation, i.e. degree of cure, is easily measured. Loss of the alkene stretch, or of some other monomer band, can be used to measure formation kinetics.

Because vibrational spectra are sensitive to the local environment, Raman spectroscopy is an important morphology diagnostic. Polymer crystallinity, for example, may result in band width and intensity changes. [11] In poly(ethylene terephthalate) films, a crystalline region has a narrower carbonyl stretch than amorphous film, but glycol bands change in intensity as well, [12] as shown in Figure T3.1. Quantitative measurements of crystallinity are best made by partial least squares, because the information is distributed in several bands. Similar changes in the Raman spectrum are used to correlate strain in fibers as well. Modern Raman spectrographs are fast and rugged enough to allow such measurements to be made on-line, to control the manufacture of the polymer film or fiber.

Physical biochemistry is a rich area of application for both normal and resonance Raman spectroscopy. Resonance Raman spectroscopy has long been used as a diagnostic for structural changes in hemes [13] and polyenes. [14] Structural changes in the polyene visual pigments can be monitored on the femtosecond time scale, as well as in the steady state. Normal Raman spectroscopy is useful for studying secondary structure of proteins and nucleic acids. [15] Using wave guide Raman spectroscopy and other techniques related to total internal reflection, it is possible to obtain Raman spectra of membranes and monolayer membrane model systems as well. [16]

The development of near-infrared Raman spectroscopy has spurred interest in the use of Raman spectroscopy in medical diagnostics. [17] Typically such applications rely on the differences in lipid/protein ratios in normal and cancerous tissue. Composition changes are reflected in the fingerprint region and in the C-H stretching region. Raman spectroscopy is attractive for these applications both because in-vivo measurements are possible with optical fiber probes and because microprobe measurements can be made on very small biopsy specimens.

T4.1 The Classical 90° Geometry T4-

T4.2 Fiber Optic Probes T4-

T4.3 The Raman Microprobe T4-

T4.4 Specialized Sampling Techniques T4-

T4.4.2 Wave Guides T4-

T4.4.3 Surface-enhanced Raman Spectroscopy T4-

T4.1 The Classical 90° Geometry

The 90° illumination/collection geometry is derived from fluorescence spectroscopy. It is shown in Figure T4.1. The laser beam is focused into the sample and scatter is observed at 90° to the laser beam. Any convenient sample container can be used. Small vials and melting point capillaries are common choices for liquids. The same containers can also be used for finely divided solids, if the laser is focused onto the front surface of the container. Bulk solids can be held in place with any convenient clamping system.

The 90° geometry minimizes direct transmission of laser light into the collection optics and so reduces the burden on the laser light rejection components of the spectrograph. Optically and mechanically this sampling system is easy to implement. Polarization ratio measurements are straightforward in this design as well. For these reasons, the standard sample compartment on most instruments designed prior to about 1990 was a 90° design. The 90° geometry is, however, difficult to align, and experience is necessary for insuring good focusing and throughput. This is particularly true for solid samples which needed to be held at a precise angle. Thus in the majority of modern instruments, the 180° geometry has been adapted.

T4.2 Fiber Optic Probes

Multimode optical fibers make excellent general purpose sample delivery and collection devices. They are small, usable in the laboratory or for remote sampling, and they provide stable, reproducible signals from solids, liquids and gases. Most common optical fibers are designed to have ¦ /number close to ¦ /2. Prior to the development of the HoloSpec ¦ /2 axial transmissive spectrograph, however, it was difficult to make full use of this extraordinary light-gathering power.

There are two different types of probes in common use in Raman spectroscopy: the concentric unfiltered fiber bundle or "n-around-1" probe and the filtered probe. [1] The fiber bundle is less expensive, while the filtered probe offers better signal-to-background in certain applications.

Figure T4.2 shows the concentric fiber bundle probe used in the HoloLab 1000. It consists of seven 100 mm core optical fibers which are cemented into a metal or plastic cylindrical holder and then polished. The central fiber delivers laser light to the sample, while the surrounding fibers collect Raman scatter. The laser beam is focused into the delivery fiber with a microscope objective or other short focal length lens. For coupling to the spectrograph entrance slit, the output ends of the collection fibers are arranged in a line, as shown in the figure. The linearized bundle is placed directly against the entrance slit of a low ¦ /number spectrograph.

Probes containing between 6 and 18 collection fibers are used because a single fiber collects only about 10-15% as much light as a multi-fiber bundle. In most designs, input and collection fibers are identical.

The fiber bundle can be inserted directly into a liquid or held a short distance from the surface of a solid. To avoid immersion in liquid, a relay lens can be used to operate the probe several centimeters or more from the sample. This is the approach taken with the unfiltered probe that is used with the HoloLab 1000.

The optical fibers themselves are a significant source of background signal. Laser light travels down a meter of fiber or more, generating an intense silica Raman spectrum. Fluorescence from the fiber cladding and from the cement holding the bundle together is also generated. These signals emerge from the delivery fiber, along with the laser light. They are reflected from the surface of any liquid or solid and collected along with the Raman spectrum of the sample. Reflected laser light generates more background as it travels through the collection fibers. In most cases, the background can be subtracted adequately, but it may obscure weak Raman signals.

These problems are eliminated in the filtered fiber optic probe, but at a substantial increase in manufacturing complexity and cost. Figure T4.3 shows the Kaiser Optical Systems Mark II filtered fiber optic probe. It uses a lens to focus filtered laser light from a delivery fiber onto the sample and to collect and collimate Raman scattered light. The collected light is focused onto a single collection fiber.

The light emerging from the delivery fiber is collimated and presented to a holographic transmission grating which diffracts the laser light to a spatial filter. Only the laser wavelength passes through this spatial filter. The collected Raman scatter, as well as Rayleigh scatter and reflected laser radiation, is passed to a pair of notch filters. These filters remove reflected laser light and avoid any fiber background generation as the collected Raman light travels to the spectrograph. The use of lenses allows efficient delivery and collection with single fibers.

Fiber coupling lends many advantages in terms of sampling flexibility. Multimode optical fibers scramble the polarization of light transmitted through them. This reduces spectral artifacts induced by the polarization dependencies of the optical elements. Polarization measurements may still be made using fiber coupling, as shown in Figure T4.3. A polarization block is inserted into the Mark II probe which determines the polarization of the laser excitation and collected Raman signal.

T4.3 The Raman Microprobe

The Raman microprobe allows acquisition of Raman spectra with spatial resolution of 0.5-2 mm from small objects ranging from semiconductors to bacteria. The microprobe is a research quality epi-fluorescence microscope coupled to a Raman spectrograph. A major advantage of Raman spectroscopy is the high spatial resolution that can be obtained, typically on the order of 1 micron (compared to 10 microns with FTIR). Single mode optical fibers are used for delivery of the laser excitation to the Mark II probe. These small core diameter fibers (2-10 microns) allow for the high spatial resolution achieved by the HoloLab 5000 research Raman microscope, shown schematically in Figure T4.4.

Light from the laser is focused onto the object, which is the material or organism under observation, and the back-scattered light is collected through the objective. The collected light is presented to a standard Raman spectrograph.

The optical configuration is essentially that of an epi-fluorescence microscope. Green (532 nm) and red (633 nm) lasers are some of the most common sources. With either laser, the entire Stokes and the useful anti-Stokes spectrum will be in the 400-800 nm region, allowing the use of microscope objectives and other optical elements designed for visible light microscopy. Low laser power (5-50 mW, typically) is used to avoid burning delicate objects with the tightly focused laser beam.

Almost all commercially available Raman microprobes include provisions for a small video camera which allows focusing and positioning the object without the use of conventional eye pieces. With the addition of a frame grabber, video provides a convenient means of image documentation as well.

The Kaiser Optical Systems microprobe uses a unique optical fiber illumination system, instead of the direct illumination scheme employed in older instruments. [2] Optical fiber delivery simplifies and stabilizes the alignment of the instrument, eliminating the need for mounting a research Raman microprobe on an optical table.

Most Raman microprobes use confocal optics. A confocal optical system blocks much of the light coming from above and below the plane on which the laser is focused. The major benefits are improved depth resolution and, consequently, an increased signal/background ratio in the Raman spectrum. The lateral spatial resolution is also improved somewhat. The principle is illustrated in Figure T4.5.

The collected light is brought to a focus at the pinhole. If the pinhole diameter is smaller than the magnified diameter of the focused laser, most of the light from above or below the focus plane of the laser will be rejected. An adjustable iris is a common form of spatial filter. If the image is magnified to the proper size, then an optical fiber can be used equally well.

Because a confocal optical system attenuates the signal coming from out of the region of best focus of the microscope, it can provide improved signal/background ratios in Raman microspectroscopy. But by blocking about 90% of the light gathered by the microscope objective, the spatial filter also reduces the intensity by an order of magnitude.

Visible light operation provides a major advantage over the infrared microprobe. Rayleigh’s criterion for the diffraction-limited resolution of a microscope is given by equation 1.

In equation 1, Dx is the minimum separation between resolved points, l is the wavelength and N.A. is the numerical aperture of the objective. The microscope objective focuses the input laser to a spot whose diameter is also given by equation 1. It is the radius of the focused beam which defines the diameter of the sampled region for microspectroscopy.

Because visible lasers are used, Raman microprobes can acquire spectra with resolution about 0.5-2 mm. The spatial resolution is much better than the 10-50 mm obtainable with an infrared microscope.

The use of visible lasers also allows use of specialized objectives with unique properties. Oil and water immersion objectives, which can have numerical aperture as high as 1.4, are the most important examples. They provide bright, high resolution images. Immersion objectives are unavailable for the infrared and are prohibitively expensive for the near-infrared.

T4.4 Specialized Sampling Techniques

T5.1 Fluorescence and Laser Wavelength Choice T5-

T5.2 Calibration Techniques T5-

T5.3 Correction for Instrument Response T5-

T5.3.1 Intensity Calibration T5-

T5.3.2 Sampling Geometry T5-

T5.4 Solution Handling Techniques T5-

Any wavelength in the ultra-violet (UV), visible, or near infra-red (near-IR) range will excite Raman scattering. Wavelength selection is based on several practical considerations. Short excitation wavelengths (blue or UV) provide higher Raman scattering intensity. Resonance enhancement usually requires blue or UV excitation, however luminescence is also efficiently excited. Luminescence backgrounds are particularly severe with excitation in the 300-550 nm range. These problems are less severe with excitation at wavelengths below 300 nm, but UV lasers and spectrographs remain expensive.

Luminescence can be circumvented with deep red or near-infrared excitation. Of course, scattering efficiency declines rapidly with increasing excitation wavelength, and thermal background problems can be severe in the near-infrared. CCDs do not respond to wavelengths much longer than 1 mm. Near-infrared array detectors are available, but these are noisy and expensive. For 1 mm excitation, anti-Stokes detection is satisfactory, with array detectors, to Raman shifts as high as about 1500 cm^-1.

Several "compromise" excitation wavelengths are commonly used in Raman scattering. The 532 nm frequency-doubled diode pumped Nd-YAG laser (DPY) is the often the best general purpose choice. Scattering intensity is strong, background problems are not too severe in many cases, and spectrographs and detectors function well in the 530-650 nm Stokes shift wavelength range. The 532 nm DPY is available at power levels ranging from about 5 mW to 10 W. Where fluorescence minimization is the most important criterion, 780-785 nm diode lasers are used. The Stokes shift region is close to the red end of the CCD response, where scattering intensity is weaker than with green excitation, and instrumentation is generally more expensive.

HeNe 633 nm provides an excellent compromise among cost, scattering intensity and fluorescence minimization. Fluorescence backgrounds are at manageable levels in many systems, and scattering intensities are reasonably strong. HeNe lasers are inexpensive and reliable, and standard visible region optical components and CCDs can be used. For these reasons, the HeNe laser is used in the Kaiser HoloLab 1000.

T5.2 Calibration Techniques

A CCD detector simply records light intensity at each pixel, yet does so through a window over 3000 cm^-1 long. Consequently, dispersion of the spectrum is not quite linear with wavelength over the whole observation range. The spectrograph must be calibrated. There are two common methods: calibration against a spectral discharge lamp; and calibration against a reference material, such as indene, whose Raman spectrum has been carefully measured. Neon and argon lamps are the most common choices. Their spectra are accurately known, and the lamps are inexpensive and readily available. Figure T5.1 shows the neon spectrum in the HeNe Stokes shift range (633-800 nm).

The empirically-determined constants, k₀...k₃, will be more accurate if a large number of reference lines, distributed across the window, are used. It is good practice to calibrate an instrument frequently, even with an instrument which has excellent wavelength stability, such as the HoloLab 1000. Stored calibrations can be used for non-critical measurements.

Recalibration may be necessary if the optical fiber probe has been removed from the spectrograph and replaced. It will definitely be necessary if the slit has been moved. Periodic recalibration is recommended even if there have been no changes in probe or slit position.

T5.3 Correction for Instrument Response T5.3.1 Intensity Calibration

Solution Raman spectroscopy is possible with almost all solvents. Laboratory distilled or de-ionized water is satisfactory. ACS reagent or other high purity organic solvents can be used. Of course, it is desirable to choose a solvent whose own Raman spectrum contains as few bands as possible near the bands of the analyte. Even where this is impractical, solvent background subtraction is quite satisfactory. With a stable modern instrument such as the HoloLab 1000, subtraction of 10X-100X backgrounds is feasible.

While it is possible to obtain spectra from turbid solutions or even slurries, probe-generated background will be smallest from clear solutions. Filtration through a 0.5 mm membrane filter is usually adequate to remove particulates.

T6.1 Special Data Analysis T6-

T6.1.1 Silica Background Subtraction T6-

T6.1.2 Fluorescence Removal T6-

T6.2 Normal and Resonance-Enhanced Raman Spectroscopy of Foodstuffs T6-

T6.2.1 Introduction T6-

T6.2.2 Experimental and Analysis T6-

T6.3 Thermodynamics of Hydrogen Bonding in a Benzoic Acid Solution. Raman Thermometry T6-

T6.3.1 Introduction T6-

T6.3.2 Experimental Procedure and Discussion T6-

T6.4 Crystal Lattice Modes and Bonding Differences in Graphite and Diamond T6-

T6.4.1 Introduction T6-

T6.4.2 Experimental and Discussion T6-

T6.5 Surface Enhanced Raman Spectroscopy of Pyridine on Silver Colloids T6-

T6.5.1 Introduction T6-

T6.5.2 Experimental and Discussion T6-

T6.1 Special Data Analysis

Most of the experiments described here require some level of mathematical data analysis. While the experiments and data analysis discussion below assume the use of GRAMS/32 analysis routines, similar procedures can be carried out using many other spectral or mathematical analysis packages. If a different package is to be used, the manual for that package should be consulted. If the data needs to be exported to a program other than those that are explicitly supported by HoloGRAMS, the File : Save As Text option can be used to save the data to a text file which can be read by many packages.

There are many important spectral manipulations that can be performed in GRAMS/32. The user is directed to the GRAMS/32 manual for descriptions of several of the standard procedures including, for example, spectral smoothing and peak integration. Here we describe several important and non-standard manipulations that the user may find useful.

T6.2.1 Introduction

Raman spectroscopy can be a unique and informative tool for the study of many biological systems because of the large number of active vibrational modes and the relative lack of spectral interference from water. In this experiment, the utility of Raman spectroscopy will be demonstrated using a carrot and both the albumin (white) and yolk from a chicken egg.

The egg albumin consists of water (88%), proteins (11%) and a small amount of lipids, glucose and inorganic ions. [1,2] Two proteins, ovalbumin and conalbumin, make up 70% of the protein content. The strongest of the protein peaks is the amide I band which falls between 1640 and 1660 cm^-1. The egg yolk contains 47% water, 33% lipids, and 17% proteins. The yolk also contains yellow-orange carotenoids. Even with red excitation, pre-resonant enhancement (i.e. a laser wavelength slightly to the red of the electronic transition) of the Raman spectrum is observed. The C=C stretches in the 1500-1600 cm^-1 region and the C-C stretch in the 1100-1200 cm^-1 region are strongly enhanced. Carotenoids are also responsible for the color and strong Raman bands of carrots.

T6.2.2 Experimental and Analysis

• Crack open the egg and separate the yolk from the albumin

The most notable difference between the spectra of the yolk and the albumin is the presence of the 1161 and 1526 cm-1 bands in the yolk spectrum. These bands, from egg carotenoids, are not present in the albumin. The amide I protein band near 1650 cm-1 is present in both spectra but is slightly shifted between the two.



 

T6.3 Thermodynamics of Hydrogen Bonding in a Benzoic Acid Solution. Raman Thermometry

T6.3.1 Introduction

T6.4 Crystal Lattice Modes and Bonding Differences in Graphite and Diamond

T6.4.1 Introduction

In extended networks such as in crystals and polymers, vibrational modes of the extended lattice are observed. These vibrations do not follow quite the same rules as the more familiar intra-molecular vibrations. This experiment utilizes graphite and diamond to demonstrate the effect of lattice structure on vibrational frequency and intensity. The disordered carbon network present in graphite leads to relatively low vibrational energies and poor scattering efficiency relative to the highly ordered and cross-linked network present in diamond. Silicon from integrated circuit chips is used to further illustrate lattice modes.



 

T6.4.2 Experimental and Discussion

• A graphite atomic absorption furnace is a source of relatively pure graphite. Scrapings from a used furnace provide adequate sample for measurement of the graphite Raman spectrum.

T6.5 Surface Enhanced Raman Spectroscopy of Pyridine on Silver Colloids

T6.5.1 Introduction

1. P.R. Carey, Biological Applications of Raman and Resonance Raman Spectroscopies, Academic Press, New York, p. 71, 1982.

2. P.C. Painter and J.L. Koenig, Biopolymers 15, 2155 (1976).

3. K.L. Davis, K.L. Liu, M. Lanan, and M.D. Morris, Anal. Chem. 65, 293 (1993).

4. P.C. Lee and D. Meisel, J. Phys. Chem. 86, 3391 (1982).

5. R.S. Sheng, L. Zhu, and M.D. Morris, Anal. Chem. 58, 1116 (1986).