Sample Handling Techniques

A. Infrared-Transmitting Materials

One of the features of IR spectroscopy is that solids, liquids, and gases can be run without special difficulties. Usually, some sort of IR-transmitting material is needed to support or enclose the sample. Materials such as glass and quartz are useful as windows in the near-IR but even thin windows do not transmit much below 3000 cm-1. The low wave number transmission limits of IR-transmitting materials are not sharply defined but depend on the window thickness. Four commonly used materials and their approximate low wave number limits are NaCl, 600 cm-1; KBr, 350 cm-1; CsBr, 250 cm-1; and CsI, 200 cm-1. These materials are all water-soluble. Water-insoluble materials and their low wave number limits include CaF2, 1200 cm-1; BaF2, 850 cm-1; Irtran-2, 700 cm-1; AgCl, 350 cm-1; and KRS-5 250 cm-1. Irtran-2 is made of zinc sulfide an is often used for water solutions or for making films from water solution. Silver chloride is useful but is soft, deforms easily, and darkens with exposure to light. KRS-5 is thallium bromide iodide and is often used in the internal reflection technique to be discussed later. In the far-IR, high-density polyethylene transmits to as low as 30 cm-1 but cannot be used above 600 cm-1 because of its absorption.

B. Salt Polishing

Sodium chloride can be easily polished between use. The crystal is sanded flat with a finegrade sandpaper if it is freshly cleaved, scratched, or damaged by water. Two polishing laps are prepared. There are different types, but the wet lap can be simply two thicknesses of fine nylon cloth stretched over a flat surface. The wet lap is wetted with water and sprinkled with a little fine polishing powder such as aluminum oxide or cerium oxide. This is rubbed smooth and all excess water is wiped off. The flat salt plate is rubbed about 20 strokes on the wet lap and then, without delay, is buffed about 7 strokes on the dry lap, which can be simply a layer of diaper cloth held flat. Cesium bromide can be polished the same way, but with alcohol substituted for water. The best polish comes when the lap is nearly dry.

C. Liquid Samples

The easiest samples to run on IR instrumentation are those in the liquid state. Slightly viscous samples can be simply squeezed between two polished IR-transmitting plates and run as a thin film. A typical film thickness is ~0.01 mm. If the liquid is not viscous, usually a spacer is added between the plates to keep the plates apart at the appropriate spacing. Spacer material can be metal foil or an insoluble polymeric film. Two strips roughly 10 by 2 mm can be used, for example, one on each side of the area the IR beam will pass through. These are called temporary cells and are disassembled and cleaned after each use. The thickness cannot be accurately reproduced.

Fixed cells are not disassembled after use but instead are filled, emptied, and cleaned with solvent through ports on the cell assembly. The liquid enters the leak-proof sample area between the plates through holes in the cell window. These are used for volatile liquids or when the thickness needs to be accurately known or held constant as in quantitative analysis. Many commercially available designs are used, and cells come in thickness from 0.01 to 4 mm.

If the cell windows are sufficiently flat, the cell thickness can be measured by running the IR spectrum of the empty cell and observing interference fringes in the form of percent transmission undulations. Wave number v1 (cm-1)is read at one transmission maximum, and wave number v2 is read at another transmission maximum that is 1, 2, 3, or more generally n maximum away from the first. The cell thickness t is

Interference results because part of the beam is twice reflected inside the cell and is retarded by twice the cell thickness relative to the transmitted beam with which it interferes.

D. Gas Samples

Gas cells used for IR spectroscopy come in a variety of types. The simplest is a basic cylinder 10 cm long with IR-transmitting windows on each end. These may be cemented on or clamped in place, with vacuum-tight gaskets providing the seal. Entrance and exit tubes are provided and fitted with stopcocks. The cell is filled and emptied with a gas handling system.

The sampling chamber of most IR spectrometers is not large enough to accommodate longer cell lengths directly.

However, cells of much longer path length are available that use mirrors to deflect the IR beam and to reflect it back and forth many times in the cell chamber before it leaves the cell and reenters the spectrometer. These long-path-length cells are used for detecting very small quantities of gas in pollution studies, for example.

A technique often used with FT-IR instruments is gas chromatography, or GC-FT-IR. Here the effluent from a gas chromatography column is fed through a heated light pipe with IR-transmitting windows on the ends. Source radiation passes through the cell into the FT-IR spectrometer. The gas chromatography column separates the gas-phase components and ideally sends them one by one through the light pipe, where the high speed of the FT-IR instrument is utilized to get the spectrum of each component "on the fly," so to speak.

E. Solution Spectra

The techniques for running solids in IR are quite varied. In the first case a solid can be dissolved in a suitable solvent and run as a liquid. Unfortunately, no solvent is free of absorption in the IR region and, usually, the better the solvent, the greater its absorption. This means that more than one solvent must be used to get the whole IR solution spectrum in all regions. A commonly used pair of solvents are CCU above 1330 cm-1 and CS2 below 1330 cm-1. These can be used in cells 0.1-1 mm thick, for example, with solute concentrations in the range 10-1%. In double-beam grating spectrophotometers a cell of matching thickness containing solvent only can be put into the reference beam to compensate for the solvent bands. In FT-IR instruments, a reference solvent spectrum can be subtracted from the solution spectrum to remove solvent bands. Another common solvent for solution spectra is CHCl3 often used in 0.1-mm cells with 5 to 10% solute. CHCl3 has strong bands at 1216 and 757 cm-1, where solute information is often lost or inadequately presented. Even water has been used as a solvent for some applications. Here the cell thickness must be kept small, as water is a very strong IR absorber. The internal reflection technique described in Sec. III.I has been successfully used for water solutions.

F. Films

Solid-state films of suitable thickness can be prepared from melts or solution. Such films are most suitable for amorphous materials, especially polymers. Crystalline films may scatter light and show nonreproducible orientation effects from special orientations of the crystal on the IR window surface. A sample can be heated between two salt plates until molten and allowed to solidify. Solutions can be put onto a plate and the solvent evaporated to form a film. This is a good technique for running water-soluble polymers, for example. Sometimes a film can be prepared on a substrate and stripped off and run as an unsupported film. If a film is too uniform in thickness, interference fringes similar to those from an empty cell may be seen, as discussed earlier. If a film is too irregular in thickness, a spectrum with a false percent transmittance will result from the fact that different parts of the beam go through sample areas with different thicknesses.

G. Mulls

One of the best techniques for running crystal-line solids is the use of a mineral oil or Nujol mull. Here a few milligrams of sample are finely ground with a small amount of mineral oil to make a thick paste like cold cream, for example. The paste can be prepared with a mortar and pestle and spread between two IR-transmitting windows. A well-ground sample has a brownish color like smoke when one looks through it. Most beginners do not grind the sample well enough and use too much oil. Mineral oil has only a few bands in narrow regions. The CH stretch region between 3000 and 2800 cm-1 and the CH bend region at about 1460 and 1375 cm-1 are obscured, however. If information is needed in these regions a second mull must be prepared using a halogenated oil such as Halocar-bon or Fluorolube, which contain CF2 and CFCl groups but no CH. These have no bands from 4000 to 1300 cm-1 but have strong bands below 1300 cm-1. Some people use the halogenated oil spectrum above 1300 cm-1 and the mineral oil spectrum below. In this case care must be taken to ensure that the sample thickness is the same in both preparations.

H. Potassium Bromide Disks

A very popular technique for running solids is the KBr disk technique. Here a few milligrams of sample are very finely ground and then mixed with 50 to 100 parts of dry KBr powder. The mixture is placed in a special device and compressed into a disk at high pressure. If all goes well, a transparent disk results, which is put into the spectrometer and run. Commercial KBr disk makers are available in many forms. Some are activated with wrenches or levers, while others are used with a hydraulic press. Some can be evacuated, which gives the disk transparency a longer lifetime, but this is not necessary if the disk is used promptly.

Advantages of the disk over the mull include the fact that KBr, unlike mineral oil, has no bands above 400 cm-1. Also, many polymers are more easily ground in KBr. Microsamples are easier to prepare with the KBr disk. The KBr disk has disadvantages compared with the mull, however. The biggest problem is that KBr is hygroscopic, and bands from absorbed H2O appear in the spectrum in variable amounts that depend on the technique. One never knows whether the water is in the sample or the KBr preparation. Also, spectra of KBr disks are sometimes less reproducible because of changes in sample polymorphism, which result from the preparation.

I. Internal Reflectance

Internal reflectance results when a beam of radiation inside a material of relatively high index of refraction is reflected from the surface interface between this and a material of lower index of refraction. The angle of incidence a is the angle between the beam and a line perpendicular to the surface interface. If the angle of incidence is small, much of the radiation is transmitted through the surface interface and a little is internally reflected. As the angle of incidence gets larger, a certain critical angle ac, is exceeded, after which all the radiation is internally reflected from the interface and none is transmitted. The sine of the critical angle, sin ac is equal to the ratio n2/n1, where n1 is the higher index of refraction and n2 is the lower index of refraction on the two sides of the interface.

When the angle of incidence is larger than the critical angle, then the beam in the material with the higher index of refraction penetrates a little into the material with the lower index of refraction in the form of an exponentially decaying wave. It is then reflected back out. When the amplitude of the wave passing through the interface has decayed by a factor of (1/e) or about 37%, the reflective penetration (d), into the material with the lower index of refraction is given by d(1/e) = --—-, (13)

where e is the natural log base, X is the wavelength of the radiation, and a1 is the angle of incidence in the material with the higher index of refraction. If the material with the lower index of refraction should absorb part of the radiation penetrating into it, then the internally reflected beam leaving the interface will be attenuated by this absorption. Hence we have the name attenuated total reflectance (ATR) for this effect.

As used in infrared spectroscopy, one type of internal reflectance plate (Fig. 11) is made of a high index of refraction material, such as thallium bromide-iodide. The plate is usually a few millimeters thick, and the ends are beveled to allow radiation entry into one end at an angle inside the plate. The beam is multiply internally reflected and zigzags between the surfaces until it leaves at the other end. A sample with a lower index of refraction than the plate is pressed into intimate contact with the plate on one or both sides. The zigzagging beam penetrates

FIGURE 11 Plate used for internal reflection spectroscopy. The lower drawing shows the sample in contact with the plate and radiation being multiply internally reflected within the plate.

a few micrometers into the sample on the plate surface at each reflection. The sample thickness is immaterial as long as it exceeds a few micrometers. The sample contact area should go all the way across the plate so none of the beam can bypass the sample. The lengthwise coverage only affects the attenuation intensity.

When the internally reflected beam is introduced into a spectrometer, the resulting spectrum is similar to a transmission spectrum. There is one major difference. Since the radiation penetration is wavelength dependent in the penetration equation, longer wavelengths penetrate more. The internal reflectance spectrum resembles a transmission spectrum where the sample thickness gets larger in direct proportion to the radiation wavelength.

There is another effect on the penetration, and that is that the index of refraction of the sample (n2) is not constant. It changes in the region of an absorption band, becoming smaller than average on the high-wave number side of the band center and larger on the low-wave number side. From the penetration equation, the radiation penetration, and therefore the band intensity, will be decreased on the high-wave number side of the band center and increased on the low-wave number side. This distorts the band shape. To avoid this, the denominator in the penetration formula should not get too small. This means that the angle of incidence should not be too small and the index of refraction of the crystal n1 should be relatively high. In Fig. 11, the angle of incidence can be kept large enough to reduce the band distortion, but this also reduces the band intensity. This is compensated for by using multiple internal reflections.

There have been many variations in the design of internal reflection accessories. In one design, the plate in Fig. 11 is mounted horizontally at the bottom of a shell container so that a liquid can be simply spread over the top of the plate and run without further changes.

In another design, the internal reflectance crystal has a hemispherical shape. In this arrangement, the radiation enters into the curved surface, and is internally reflected off the flat surface, and then exits through the curved surface. In one version of this, the sample is in optical contact with only a small raised area of the flat surface that allows spectra to be taken of quite small areas. Since there is only one internal reflection, the crystal used, such as silicon, has a very high index of refraction.

There are many applications for internal reflectance spectroscopy, and only a few will be mentioned here. Internal reflectance spectroscopy can be used to obtain the spectra of rubbery materials that are hard to grind. The rubbery material is simply pressed against the internal reflectance plate, and it is ready to run. Carbon-filled rubber or other polymers may be run using a high index of refraction germanium as the internal reflectance element. Internal reflectance is used to obtain selectively the top few micrometers of a sample surface where the composition may be different than that further down. It is also good for water solutions because the controlled penetration keeps the effective sample thickness small.

J. Diffuse Reflectance

Diffuse reflectance is a technique usually used with FT-IR instruments. A powdered sample is placed in a small container, where source radiation strikes it and is diffusely reflected in various directions. This radiation is collected and measured by the spectrometer. Usually in the mid-IR region the finely powdered sample is diluted to 5 to 10% with finely powdered KBr or KCl. The spectrum is ra-tioed against a reference spectrum of pure powdered KBr or KCl. The ratioed spectrum is processed by a computer using a function f (Rm) derived by Kubelka and Munk, which changes the reflectance spectrum into one resembling a linear absorbance spectrum:

Here (Rœ) is the reflectance of a thick scattering layer, k the molar extinction coefficient, and s a scattering coefficient, which is a function of particle size. The spectrum is quite sensitive to particle size, which affects the radiation scattering. Spectral distortion (compared with a transmission spectrum) may occur if the particle size is not uniformly fine. Black, strongly scattering materials such as coal can be run by this technique.

K. Infrared Microspectroscopy

In this method of sampling, a special type of microscope is used to select very small sample areas for examination by the infrared spectrometer. As in the macroscopic case, samples can be prepared with thicknesses on the order of 0.01 mm. Since infrared radiation must pass through the microscope, all the optics are front surface mirrors.

The sampling region can be viewed visually through the microscope, and selected areas can be isolated by masking off the unwanted parts of the field. Variable aperture masks are located in remote image planes of the sample area, located above and below the sample to reduce diffraction effects. Then, the optical path is changed so that the source radiation goes through the unmasked areas and the infrared spectrum of the sample is recorded. This can be divided by the spectrum of a similarly masked blank for example, to give a percent transmission spectrum of the sample, or from this, an absorbance-type spectrum of the sample.

The types of applications are basically similar to those handled by transmission spectroscopy, but with a significant difference. The microscope can yield good spectra on much smaller sample areas. This means that many new types of problems can be handled that were difficult or impossible to solve with standard instruments. Sample areas that are heterogeneous are now easily measured in the chemical industry, forensic work, and biological studies.

In the chemical industry for example, some polymer products may show some very small impurity areas whose chemical composition may be characterized with little difficulty by the spectra. Polymer films weathered by outdoor exposure may show chemical modification as a function of the layer depth below the surface. This may be characterized by the infrared spectra of the various layers.

In forensic work, heterogeneous fields can be examined selectively for hair strands, polymer fibers, or lint particles, for example, and their chemical composition can be characterized by their infrared spectra. Cross-sections of paint chips can be examined and the various layers can be characterized, which may be distinctive.

In the biological field, heterogeneous areas of various biological specimens can be examined, and the infrared spectrum of different microareas of the field can be taken. The spectrum of a single red blood corpuscle can be easily measured. One difference that can be observed in these samples is the ratio of protein to fat. Proteins have bands near 1650 and 1550 cm-1 for the ^C-NH group, and fats have an ester C=O band near 1740 cm-1 and an alkane chain doublet near 2925 and 2855 cm-1, with an internal unsaturation band near 3015 cm-1 for the =CH group. Some studies may reveal chemical differences between adjacent tissues of various types. The spectral characteristics of two-dimensional areas may be mapped by running a series of spectra of a grid pattern. An array consisting of a number of detectors can be used, each one of which generates the infrared spectrum of its own small area of the grid pattern.

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