ASTM E334-96
(Practice)Standard Practice for General Techniques of Infrared Microanalysis
Standard Practice for General Techniques of Infrared Microanalysis
SCOPE
1.1 This practice covers techniques that are of general use in securing and analyzing microgram quantities of samples by infrared spectrophotometric techniques. This practice makes repetition of description of specific techniques unnecessary in individual infrared methods.
1.2 These recommendations are supplementary to Practices E168, E573, and E1252, which should be referred to for theory, general techniques of sample preparation, and calculations.
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Designation: E 334 – 96
Standard Practice for
General Techniques of Infrared Microanalysis
This standard is issued under the fixed designation E 334; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope subsequent recording of spectra.
1.1 This practice covers techniques that are of general use in
5. General Microspectroscopic Techniques
securing and analyzing microgram quantities of samples by
5.1 Spectroscopic techniques used for the examination of
infrared spectrophotometric techniques. This practice makes
microsamples are usually adaptations of comparable macro
repetition of description of specific techniques unnecessary in
techniques, and many have been described in the literature (1,
individual infrared methods.
2).
1.2 These recommendations are supplementary to Practices
5.2 In computerized dispersive spectrometers or Fourier
E 168, E 573, and E 1252, which should be referred to for
transform-infrared (FT-IR) instruments, computer routines for
theory, general techniques of sample preparation, and calcula-
multiple scanning, signal averaging, absorbance subtraction,
tions.
and scale expansion can be used very effectively to enhance the
2. Referenced Documents observed signal-to-noise ratio of weak bands and increase
sensitivity (3, 4). Absorbance subtraction is also commonly
2.1 ASTM Standards:
used to eliminate interfering bands from the sample matrix and
E 131 Terminology Relating to Molecular Spectroscopy
thus lower the limits of detection (see Practice E 168).
E 168 Practices for General Techniques of Infrared Quanti-
5.3 Use of Masking Apertures—The aperture of sample
tative Analysis
2 holders used for microspectroscopic study (without the use of
E 573 Practices for Internal Reflection Spectroscopy
an infrared microscope) are usually significantly smaller than
E 1252 Practice for General Techniques for Qualitative
2 the beam at the sample position of the instrument. As a
Infrared Analysis
consequence of these small apertures, steps need to be taken to
3. Terminology ensure that the best quality spectra be obtained, and the
techniques used will depend on the type of spectrometer being
3.1 Definitions and Symbols—For definitions of terms and
used. In general, the use of a beam condensing accessory will
symbols, refer to Terminology E 131.
greatly improve the results obtained (see 5.4).
4. Contamination
5.3.1 When a double-beam dispersive spectrometer that is
not equipped for control by minicomputer is used, the refer-
4.1 Although the presence of contaminants is a general
ence beam should be masked to a corresponding aperture. This
problem in any type of analysis, contamination can be particu-
can be accomplished by using an opaque sheet of stiff material
larly severe in micro work. For example, minor impurities in a
punched with an appropriate opening, with reference screens,
solvent can become major components of a residue remaining
or with commercially available optical attenuators. Attenuation
after solvent evaporation. Materials extracted from thin-layer
of the reference beam affects instrument performance, and
chromatographic materials, from the paper used in paper
appropriate adjustment of the instrument settings (that is, wider
chromatography, and from solid adsorbents in general, may
slits or higher gain) is necessary to produce reliable spectra at
include particular contaminants of concern. It should also be
the lower energy levels. Enhancement of sensitivity can be
noted that the gas-chromatographic stationary phase may lead
attained by the ordinate scale expansion feature available on
to significant contamination. Consideration of these and other
most spectrometers.
sources of contamination must always enter interpretation of
5.3.2 When using a single-beam spectrometer, the instru-
results in microanalysis. Erroneous results can be minimized
ment background spectrum should be recorded through an
by the use of pure reagents, extreme care in sample handling,
aperture in the sample position that has dimensions no larger
and the frequent use of “blanks” in the course of separation and
than those of the sample. Where appropriate, this can be done
by using the empty sample holder itself.
This practice is under the jurisdiction of ASTM Committee E-13 on Molecular 5.3.3 On some FT-IR spectrometers, insertion of an aperture
Spectroscopy and is the direct responsibility of Subcommittee E13.03 on Infrared
at the sample position will slightly change the observed
Spectroscopy.
Current edition approved Sept. 10, 1996. Published November 1996. Originally
published as E 334 – 90. Last previous edition E 334 – 90. The boldface numbers in parentheses refer to a list of references at the end of
Annual Book of ASTM Standards, Vol 03.06. this practice.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
E 334
frequency positions of bands, as a result of modification of the instruments but others have general utility.
optical path. Hence, sample and reference aperture must be
5.6.1 A small quantity of finely ground powder can be
carefully aligned at the same position, particularly if computer
mulled in an agent such as mineral oil and smeared on a small
differencing is to be done.
sample plate about 3 by 5 by 1 mm. The sample plate is
5.3.4 Some FT-IR spectrometers (especially those equipped
mounted in a holder as near as possible to the focal point of the
with cooled mercury cadmium telluride (MCT) detectors) are
converging sample radiation beam or in a beam-condensing
so sensitive that under normal operating conditions (that is,
unit.
when examining macro samples or recording the reference
5.6.2 Alkali halide disk or pellet techniques are of consid-
single beam spectrum) the energy throughput of the instrument
erable importance in microsampling. Compromises in the usual
needs to be restricted in order to avoid detector nonlinearity
recommended procedures may be required to permit analysis
(5). This is typically done by insertion of an aperture or wire
of ultra-micro samples. It is advantageous to use an alkali
screen into the path of the beam. However, when the same
halide that has been maintained in a drying oven at 105 to
instrument is employed to examine microsamples using a
110°C. Blank samples of the stored alkali halide should be used
sample holder, which is in itself an aperture, this throughput
to obtain frequent reference spectra, in order to guard against
restriction should be removed.
contamination.
5.3.5 When using an infrared microscope, it is normal to
5.6.3 Commercial micropellet dies usually produce disks of
record the reference spectrum through the same aperture as is
either 0.5 or 1.5-mm diameter. A standard size 13-mm die may
used for a particular sample. To accomplish this, it is most
be adapted for micropellet work by punching a small aperture
convenient to use visual observation to select the aperture size
in a disk of, for example, tinfoil, manila folder, blotting paper,
required to mask the sample area of interest. The single-beam
or filter paper about 0.1 mm thick. About one third the usual
spectrum of this sample area is recorded, and the reference
pressure should be used for pressing the micropellet. The
single-beam background spectrum is then recorded afterwards.
tinfoil or paper serves as a holder for the pellet and can be
The transmittance (or absorbance) spectrum of the sample is
positioned over the aperture of the micropellet holder or on the
obtained by using the instrument software to calculate the ratio
beam-condenser unit. Commercially available lead micro disks
of the two single-beam spectra.
are also available.
5.4 Large energy losses because of beam attenuation may be
NOTE 2—Stationery supply stores carry paper punches of assorted sizes
avoided by the use of a beam-condensing accessory. The heat
and shapes that are suitable for making these apertures for micropellets.
produced by the concentrated beam may be injurious to some
NOTE 3—An aperture of 1 by 4 mm is about the minimum size on
samples, especially in the case of some dispersive instruments.
which some dispersive spectrometers can operate properly. If a beam
If this difficulty is encountered, a thin germanium wafer
condensing accessory is used, the minimum aperture is reduced to the
order of 0.5 to 1.0 mm in diameter. Fourier transform instruments can
between the source beam and the sample, or a stream of
obtain spectra through a 0.5-mm aperture, if necessary, without the use of
cooling air directed upon the sample, will provide some
a beam condenser.
protection for the sample. A 43 beam condenser is adequate
for most microsample analyses. 5.6.4 A very small sample may be made transferable by
rubbing or abrasion, or both, using dry potassium bromide
5.5 Examination of Liquid Samples— Direct examination of
(KBr) powder. Pellet grade KBr should be used, and subse-
liquid samples can be accomplished by using sealed microcells
quent grinding should be kept to the minimum necessary to
or microcavity cells, which are commercially available and are
disperse the sample. This technique is also valuable for
characterized by small apertures and volumes of the order of a
few microlitres. Beam-condensing accessories are available removing a thin surface layer from a solid object.
5.6.5 A sample of a thin coating material may be obtained
that can accommodate such microcells. The volume of de-
mountable microcells that are suitable for liquids of low by rubbing the surface with glass-paper or silicon carbide
paper. The spectrum of the sample on the surface of the paper
volatility is about 0.5 μL when assembled with a 0.1-mm
spacer. Micro quantities of non-volatile liquids can be conve- is obtained by using the diffuse reflectance technique, with a
clean piece of glass-paper or silicon carbide paper, as appro-
niently examined using micro internal reflection spectroscopy
(IRS), (see Practices E 573). Sometimes the most convenient priate, being used as the reference.
5.6.6 Solid materials can be examined by first dissolving the
way to handle microquantities of a volatile liquid is to contain
material in a solvent (see 5.7). The resulting solution can be
it in a gas cell having a large length-to-volume ratio, so that the
examined directly, or used to deposit the solute in a state more
material is examined in the vapor phase.
advantageous for analysis, such as a thin film or in a halide
5.6 Examination of Solid Samples—The conventional tech-
powder for the preparation of a KBr pellet or diffuse reflec-
niques for handling macro amounts of solids are equally
tance. The same solvent should be used to obtain a spectrum of
applicable for microgram quantities when scaled down acces-
the solvent blank, either directly or as a deposit, as appropriate.
sories are used. Just as for liquids, compensation for the
sample-beam attenuation or the use of a beam condenser is
NOTE 4—Caution: Solvent or melt recrystallization or application of
necessary for the recording of useful spectra; ordinate scale
pressure to samples may cause changes in the crystalline structure of the
expansion, multiple scans, or signal averaging may be needed material, and hence give changes to the observed spectrum.
to enhance the sensitivity.
5.6.7 Some solids can be heat-softened or melted by press-
ing between two small heated KBr plates and then examined in
NOTE 1—A range of accessories such as micromull holders, micropellet
holders, etc. are commercially available. Some are designed for specific a demountable microcell holder (see Note 4). It is often
E 334
advantageous to perform the pressing operation with the accessory inappropriate for the study of samples that have
sample between two sheets of aluminum foil first, so that more significant absorptions in that region. On the other hand,
pressure can be exerted. The thin film is then peeled off the foil diamond is a good far-infrared window material and allows
−1
and examined between the salt windows. Some solid samples spectra to be recorded down to below 50 cm , using a
may be cut into thin wafers that may then be mounted in a beam-condenser and suitably equipped spectrometer. Squeez-
micropellet holder for subsequent analysis. ing the sample in the cell may change the morphology and any
ordering in the structure of the sample (see Note 4).
5.6.8 Small flakes of material have been successfully exam-
5.7 Examination of Solutions—In some instances, solutions
ined by supporting them on a salt plate and then placing an
of liquids or solids are advantageously used for recording
aperture over the sample. Both salt plate and aperture are
spectra. The preparation of solutions in microquantities has
placed in the sample beam. Static forces may be used to hold
inherent difficulties, and solvents usually obscure some por-
very small samples inside a pinhole aperture. Stray light may
tions of the spectrum. Some of these interferences can be
be observed under both types of sample mounting, since the
eliminated by computer subtraction or double-beam tech-
sample does not normally fill the aperture completely. Im-
niques. Careful selection of the pathlength of the transmission
proved spectral data are obtained by the use of a beam
cell or, with IRS, the type of IRE employed allows for dilute
condenser (see 5.4) or, even better, an infrared transmitting
solutions (even in water) to be examined directly using an
microscope (see Section 11).
FT-IR spectrometer or a computer-assisted dispersive spec-
5.6.9 Samples can be held between two thin sheets of a
trometer. In general, solvent blank samples need to be exam-
polymeric material that has low infrared absorbance at the
ined in the same manner as the solutions generated, in order to
frequencies of interest, instead of being on the surface of a salt
identify the presence of contaminants.
plate as in 5.6.6-5.6.8. Fluorocarbon tape may be used to obtain
5.7.1 A solution may be used to prepare a micro film of
spectra over large portions of the mid-infrared region, while
solute on a small window (approximately 8 by 8 by 2 mm) that
polyethylene film is particularly useful for far-infrared mea-
has been gently scratched in order to contain the sample in a
surements. Both materials withstand the effects of many
small area (3 by 3 mm, or less if using an FT-IR). It should be
corrosive samples.
noted that the window must be made of a material that is not
5.6.10 Another method for holding small solid samples in
harmed by the so
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