ASTM E334-01(2013)
(Practice)Standard Practice for General Techniques of Infrared Microanalysis
Standard Practice for General Techniques of Infrared Microanalysis
ABSTRACT
This practice establishes the standard techniques that are of general use in securing and analyzing samples in microgram quantities (microanalysis) by infrared spectrophotometry. These techniques include general microspectroscopy, analysis of gas chromatographic fractions, analysis of liquid chromatographic fractions, analysis of thin-layer chromatographic fractions, analysis of paper chromatographic fractions, analysis of gases evolved from a thermogravimetric analyzer, and infrared spectroscopy using a microscope.
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.
General Information
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E334 − 01 (Reapproved 2013)
Standard Practice for
General Techniques of Infrared Microanalysis
This standard is issued under the fixed designation E334; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
This standard has been approved for use by agencies of the U.S. Department of Defense.
1. Scope 3.2 Beam Condenser—Aspecializedaccessorydesignedfor
analysis of samples of a microgram or less, comprising an
1.1 Thispracticecoverstechniquesthatareofgeneralusein
analyte area or volume of 2.0 mm diameter or less.
securing and analyzing microgram quantities of samples by
infrared spectrophotometric techniques. This practice makes
4. Contamination
repetition of description of specific techniques unnecessary in
4.1 Although the presence of contaminants is a general
individual infrared methods.
problem in any type of analysis, contamination can be particu-
1.2 These recommendations are supplementary to Practices
larly severe in micro work. For example, minor impurities in a
E168,E573,andE1252,whichshouldbereferredtofortheory,
solvent can become major components of a residue remaining
general techniques of sample preparation, and calculations.
after solvent evaporation. Materials extracted from thin-layer
chromatographic materials, from the paper used in paper
2. Referenced Documents
chromatography, and from solid adsorbents in general, may
2.1 ASTM Standards: include particular contaminants of concern. It should also be
E131Terminology Relating to Molecular Spectroscopy
noted that the gas-chromatographic stationary phase may lead
E168Practices for General Techniques of Infrared Quanti- to significant contamination. Consideration of these and other
tative Analysis (Withdrawn 2015)
sources of contamination must always enter interpretation of
E573Practices for Internal Reflection Spectroscopy
results in microanalysis. Erroneous results can be minimized
E1252Practice for General Techniques for Obtaining Infra-
by the use of pure reagents, extreme care in sample handling,
red Spectra for Qualitative Analysis
andthefrequentuseof“blanks”inthecourseofseparationand
E1642Practice for General Techniques of Gas Chromatog-
subsequent recording of spectra.
raphy Infrared (GC/IR) Analysis
5. General Microspectroscopic Techniques
E2105Practice for General Techniques of Thermogravimet-
ric Analysis (TGA) Coupled With Infrared Analysis
5.1 Spectroscopic techniques used for the examination of
(TGA/IR)
microsamples are usually adaptations of comparable macro
E2106 Practice for General Techniques of Liquid
techniques, and many have been described in the literature (1,
Chromatography-Infrared (LC/IR) and Size Exclusion
2).
Chromatography-Infrared (SEC/IR) Analyses
5.2 In computerized dispersive spectrometers or Fourier
transform-infrared (FT-IR) instruments, computer routines for
3. Terminology
multiple scanning, signal averaging, absorbance subtraction,
3.1 Definitions and Symbols—For definitions of terms and
andscaleexpansioncanbeusedveryeffectivelytoenhancethe
symbols, refer to Terminology E131.
observed signal-to-noise ratio of weak bands and increase
sensitivity (3, 4). Absorbance subtraction is also commonly
usedtoeliminateinterferingbandsfromthesamplematrixand
This practice is under the jurisdiction ofASTM Committee E13 on Molecular
thus lower the limits of detection (see Practice E168).
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
5.3 Use of Masking Apertures—The aperture of sample
mittee E13.03 on Infrared and Near Infrared Spectroscopy.
Current edition approved Jan. 1, 2013. Published January 2013. Originally
holders used for microspectroscopic study (without the use of
approved in 1990. Last previous edition approved in 2007 as E334–01 (2007).
an infrared microscope) are usually significantly smaller than
DOI: 10.1520/E0334-01R13.
the beam at the sample position of the instrument. As a
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
consequenceofthesesmallapertures,stepsneedtobetakento
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
3 4
The last approved version of this historical standard is referenced on The boldface numbers in parentheses refer to a list of references at the end of
www.astm.org. this practice.
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E334 − 01 (2013)
ensure that the best quality spectra be obtained, and the a stream of cooling air directed upon the sample, will provide
techniques used will depend on the type of spectrometer being some protection for the sample.
used. In general, the use of a beam condensing accessory will
5.5 Examination of Liquid Samples—Direct examination of
greatly improve the results obtained (see 5.4).
liquidsamplescanbeaccomplishedbyusingsealedmicrocells
5.3.1 When a double-beam dispersive spectrometer that is
ormicrocavitycells,whicharecommerciallyavailableandare
not equipped for control by minicomputer is used, the refer-
characterized by small apertures and volumes of the order of a
encebeamshouldbemaskedtoacorrespondingaperture.This
few microlitres. Beam-condensing accessories are available
can be accomplished by using an opaque sheet of stiff material
that can accommodate such microcells. The volume of de-
punched with an appropriate opening, with reference screens,
mountable microcells that are suitable for liquids of low
orwithcommerciallyavailableopticalattenuators.Attenuation
volatility is about 0.5 µL when assembled with a 0.1-mm
of the reference beam affects instrument performance, and
spacer. Micro quantities of non-volatile liquids can be conve-
appropriateadjustmentoftheinstrumentsettings(thatis,wider
niently examined using micro internal reflection spectroscopy
slits or higher gain) is necessary to produce reliable spectra at
(IRS) (see Practices E573). Sometimes the most convenient
the lower energy levels. Enhancement of sensitivity can be
way to handle microquantities of a volatile liquid is to contain
attained by the ordinate scale expansion feature available on
itinagascellhavingalargelength-to-volumeratio,sothatthe
most spectrometers.
material is examined in the vapor phase.
5.3.2 When using a single-beam spectrometer, the instru-
5.6 Examination of Solid Samples—The conventional tech-
ment background spectrum should be recorded through an
niques for handling macro amounts of solids are equally
aperture in the sample position that has dimensions no larger
applicable for microgram quantities when scaled down acces-
than those of the sample. Where appropriate, this can be done
sories are used. Just as for liquids, compensation for the
by using the empty sample holder itself.
sample-beam attenuation or the use of a beam condenser is
5.3.3 OnsomeFT-IRspectrometers,insertionofanaperture
necessary for the recording of useful spectra; ordinate scale
at the sample position will slightly change the observed
expansion, multiple scans, or signal averaging may be needed
frequency positions of bands, as a result of modification of the
to enhance the sensitivity.
optical path. Hence, sample and reference aperture must be
carefully aligned at the same position, particularly if computer
NOTE1—Arangeofaccessoriessuchasmicromullholders,micropellet
differencing is to be done.
holders, etc. are commercially available. Some are designed for specific
5.3.4 Some FT-IR spectrometers (especially those equipped instruments but others have general utility.
with cooled mercury cadmium telluride (MCT) detectors) are
5.6.1 A small quantity of finely ground powder can be
so sensitive that under normal operating conditions (that is,
mulled in an agent such as mineral oil and smeared on a small
when examining macro samples or recording the reference
sample plate about 3 by 5 by 1 mm. The sample plate is
singlebeamspectrum)theenergythroughputoftheinstrument
mountedinaholderasnearaspossibletothefocalpointofthe
needs to be restricted in order to avoid detector nonlinearity
converging sample radiation beam or in a beam-condensing
(5). This is typically done by insertion of an aperture or wire
unit.
screen into the path of the beam. However, when the same
5.6.2 Alkali halide disk or pellet techniques are of consid-
instrument is employed to examine microsamples using a
erableimportanceinmicrosampling.Compromisesintheusual
sample holder, which is in itself an aperture, this throughput
recommended procedures may be required to permit analysis
restriction should be removed.
of ultra-micro samples. It is advantageous to use an alkali
5.3.5 When using an infrared microscope, it is normal to
halide that has been maintained in a drying oven at 105 to
record the reference spectrum through the same aperture as is
110°C.Blanksamplesofthestoredalkalihalideshouldbeused
used for a particular sample. To accomplish this, it is most
to obtain frequent reference spectra, in order to guard against
convenient to use visual observation to select the aperture size
contamination.
required to mask the sample area of interest. The single-beam
5.6.3 Commercial micropellet dies usually produce disks of
spectrum of this sample area is recorded, and the reference
either0.5or1.5-mmdiameter.Astandardsize13-mmdiemay
single-beambackgroundspectrumisthenrecordedafterwards.
be adapted for micropellet work by punching a small aperture
The transmittance (or absorbance) spectrum of the sample is
in a disk of, for example, tinfoil, manila folder, blotting paper,
obtainedbyusingtheinstrumentsoftwaretocalculatetheratio
or filter paper about 0.1 mm thick. About one third the usual
of the two single-beam spectra.
pressure should be used for pressing the micropellet. The
5.4 Largeenergylossesbecauseofbeamattenuationmaybe
tinfoil or paper serves as a holder for the pellet and can be
avoided by the use of a beam-condensing accessory. This type
positionedovertheapertureofthemicropelletholderoronthe
ofaccessoryisdesignedtocondensethesampleradiationbeam
beam-condenserunit.Commerciallyavailableleadmicrodisks
to an analyte area of 2 mm or less, accommodating the smaller
are also available.
size of a microsample. A4× beam condenser is adequate for
NOTE2—Stationerysupplystorescarrypaperpunchesofassortedsizes
most microsample analyses.
and shapes that are suitable for making these apertures for micropellets.
5.4.1 The heat produced by the concentrated beam may be
NOTE3—Anapertureof1by4mmisabouttheminimumsizeonwhich
injurious to some samples, especially in the case of some
somedispersivespectrometerscanoperateproperly.Ifabeamcondensing
dispersive instruments. If this difficulty is encountered, a thin
accessory is used, the minimum aperture is reduced to the order of 0.5 to
germanium wafer between the source beam and the sample, or 1.0 mm in diameter. Fourier transform instruments can obtain spectra
E334 − 01 (2013)
through a 0.5-mm aperture, if necessary, without the use of a beam
beam or by computer subtraction of an adhesive tape spectrum
condenser.
collected in a manner similar to that of the sample.
5.6.11 To avoid the need to computer-subtract the spectrum
5.6.4 A very small sample may be made transferable by
of adhesive tape mentioned in 5.6.10, small pieces of salt
rubbing or abrasion, or both, using dry potassium bromide
window can be used to mount microsamples next to an
(KBr) powder. Pellet grade KBr should be used, and subse-
aperture. The pieces of salt are cleaved from a used crystal by
quent grinding should be kept to the minimum necessary to
using a razor blade, and can be as small as 1 or 2 mm square.
disperse the sample. This technique is also valuable for
Transfer a few particles of adhesive from a (preferably old)
removing a thin surface layer from a solid object.
piece of adhesive tape, using a probe, onto the extreme edges
5.6.5 A sample of a thin coating material may be obtained
ofthissaltcover.Placethesampleovertheaperture,andcover
by rubbing the surface with glass-paper or silicon carbide
with the salt plate. Pressure the salt cover onto the aperture so
paper. The spectrum of the sample on the surface of the paper
that the adhesive holds it in place.Adhesive from a used piece
is obtained by using the diffuse reflectance technique, with a
of tape will allow the cover to be removed more easily after
clean piece of glass-paper or silicon carbide paper, as
sample collection is completed.
appropriate, being used as the reference.
5.6.12 IfusingIRSwithasmallsample,optimalresultswill
5.6.6 Solidmaterialscanbeexaminedbyfirstdissolvingthe
beobtainedifthesmallsampleisplacedacrossthewidthofthe
material in a solvent (see 5.7). The resulting solution can be
internal reflection element (IRE). With very small samples,
examined directly, or used to deposit the solute in a state more
optimal results will be obtained by placing the sample where
advantageous for analysis, such as a thin film or in a halide
thebeamenters,sothatthefirstreflectionisconcentratedatthe
powder for the preparation of a KBr pellet or diffuse reflec-
sample position (see Practices E573).
tance.Thesamesolventshouldbeusedtoobtainaspectrumof
5.6.12.1 MicroIRSaccessoriesarealsocommerciallyavail-
thesolventblank,eitherdirectlyorasadeposit,asappropriate.
able and are generally referred to as “micro-ATR” accessories.
5.6.6.1 Warning—Solvent or melt recrystallization or ap-
The IRE of these accessories is only 1 to 3 mm in diameter
plication of pressure to samples may cause changes in the
with an effective sampling area of 0.5 to 2.0 mm in diameter,
crystalline structure of the material, and hence give changes to
allowinganalysisofsmallersamplesand,withadiamondIRE,
the observed spectrum.
greater contact pressures.
5.6.7 Some solids can be heat-softened or melted by press-
5.6.12.2 Particular cautions should be observed when using
ingbetweentwosmallheatedKBrplatesandthenexaminedin
these types of accessories.Accessory design precludes control
a demountable microcell holder (see 5.6.6.1). It is often
over the incident beam angle penetrating the IRE crystal
advantageous to perform the pressing operation with the
surface, thus, a number of incident beam angles are directed
samplebetweentwosheetsofaluminumfoilfirst,sothatmore
ontothesample-crystalinterface.Theresultantspectramaynot
pressurecanbeexerted.Thethinfilmisthenpeeledoffthefoil
be directly comparable to spectra collected from a controlled
and examined betwee
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