ASTM E334-01(2021)

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.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 31-Mar-2021
• ICS: 71.040.50 - Physicochemical methods of analysis
• Technical Committee: E13 - Molecular Spectroscopy and Separation Science
• Drafting Committee: E13.03 - Infrared and Near Infrared Spectroscopy

Relations

• Refers: ASTM E2106-00(2019) - Standard Practice for General Techniques of Liquid Chromatography-Infrared (LC/IR) and Size Exclusion Chromatography-Infrared (SEC/IR) Analyses
• Effective Date: 01-Dec-2019
• Refers: ASTM E1642-00(2016) - Standard Practice for General Techniques of Gas Chromatography Infrared (GC/IR) Analysis
• Effective Date: 01-Apr-2016
• Refers: ASTM E2106-00(2011) - Standard Practice for General Techniques of Liquid Chromatography-Infrared (LC/IR) and Size Exclusion Chromatography-Infrared (SEC/IR) Analyses
• Effective Date: 01-Nov-2011
• Refers: ASTM E131-10 - Standard Terminology Relating to Molecular Spectroscopy
• Effective Date: 01-Mar-2010
• Refers: ASTM E2105-00(2010) - Standard Practice for General Techniques of Thermogravimetric Analysis (TGA) Coupled With Infrared Analysis (TGA/IR)
• Effective Date: 01-Mar-2010
• Refers: ASTM E1642-00(2010) - Standard Practice for General Techniques of Gas Chromatography Infrared (GC/IR) Analysis
• Effective Date: 01-Mar-2010
• Refers: ASTM E1252-98(2007) - Standard Practice for General Techniques for Obtaining Infrared Spectra for Qualitative Analysis
• Effective Date: 01-Dec-2007
• Refers: ASTM E573-01(2007) - Standard Practices for Internal Reflection Spectroscopy
• Effective Date: 01-Mar-2007
• Refers: ASTM E2106-00(2006) - Standard Practice for General Techniques of Liquid Chromatography-Infrared (LC/IR) and Size Exclusion Chromatography-Infrared (SEC/IR) Analyses
• Effective Date: 01-Mar-2006
• Refers: ASTM E168-06 - Standard Practices for General Techniques of Infrared Quantitative Analysis (Withdrawn 2015)
• Effective Date: 01-Mar-2006
• Refers: ASTM E131-05 - Standard Terminology Relating to Molecular Spectroscopy
• Effective Date: 01-Sep-2005
• Refers: ASTM E2105-00(2005) - Standard Practice for General Techniques of Thermogravimetric Analysis (TGA) Coupled With Infrared Analysis (TGA/IR)
• Effective Date: 01-Sep-2005
• Refers: ASTM E1642-00(2005) - Standard Practice for General Techniques of Gas Chromatography Infrared (GC/IR) Analysis
• Effective Date: 01-Sep-2005
• Refers: ASTM E168-99(2004) - Standard Practices for General Techniques of Infrared Quantitative Analysis
• Effective Date: 01-Feb-2004
• Refers: ASTM E131-02 - Standard Terminology Relating to Molecular Spectroscopy
• Effective Date: 10-Sep-2002

Overview

ASTM E334-01(2021): Standard Practice for General Techniques of Infrared Microanalysis provides guidance on the general methods for obtaining and analyzing microgram-level samples using infrared (IR) spectrophotometric techniques, commonly referred to as infrared microanalysis. Developed by ASTM International, this standard establishes reproducible practices for labs utilizing IR microspectroscopy, analysis of chromatographic fractions, and related microanalytical approaches. By broadening standard methods, it eliminates repetitive descriptions in individual IR procedures and ensures consistency, reliability, and comparability in micro-scale infrared analysis.

Key Topics

• Infrared Microspectroscopy: Techniques for measuring IR spectra of minute samples, leveraging accessories such as beam condensers and microscopes to enhance sensitivity and minimize contamination.
• Microsample Handling: Recommendations for sample preparation, including handling liquids, solids, thin films, and evolved gases in quantities as low as a microgram.
• Contamination Control: Strategies for preventing and assessing contamination from solvents, adsorbents, chromatographic media, and sampling accessories, which is critical given the small scale of analysis.
• Coupled Chromatographic Analysis: Integration of gas chromatography (GC/IR), liquid chromatography (LC/IR), supercritical fluid chromatography (SFC/IR), and thin-layer chromatography (TLC/IR) with IR microanalysis for component separation and identification.
• Thermogravimetric Analysis (TGA/IR): Application of IR spectroscopy to analyze gases evolved from TGA, aiding in the study of composition changes upon heating.
• Use of Accessories: Guidelines for selecting microcells, microcapillary brushes, micropellet and micromull holders, and infrared-transmitting microscopes to optimize sample measurement.

Applications

The standard is highly relevant for:

• Forensic and Materials Science Laboratories: Detection and characterization of trace materials, fibers, polymers, and surface contaminants.
• Pharmaceutical and Chemical Analysis: Identification, purity assessment, and quality control through the isolation and measurement of small sample fractions separated by chromatographic means.
• Environmental Testing: Analysis of microgram-level residues or pollutants isolated via chromatography or evolved during decomposition.
• Research and Development: Investigation of material properties, thin films, and surface treatments where only minimal sample is available.
• Industrial Quality Control: Routine verification of product composition at the micro scale, especially for high-value or scarce samples.
• Academic and Institutional Laboratories: Teaching and method development for IR microanalysis.

By standardizing microanalysis methodology, ASTM E334-01(2021) enables labs to:

• Maximize data quality and consistency even with very small or delicate samples.
• Confidently couple IR detection with separation science techniques for complex mixture analysis.
• Reduce spectral interferences and contamination risks inherent to micro work.

Related Standards

ASTM E334-01(2021) is designed to supplement and align with several key ASTM standards, including:

• ASTM E168: Practices for General Techniques of Infrared Quantitative Analysis
• ASTM E573: Practices for Internal Reflection Spectroscopy
• ASTM E1252: Practice for General Techniques for Obtaining Infrared Spectra for Qualitative Analysis
• ASTM E1642: Practice for General Techniques of Gas Chromatography/Infrared (GC/IR) Analysis
• ASTM E2105: Practice for Thermogravimetric Analysis/Infrared (TGA/IR)
• ASTM E2106: Practice for General Techniques of Liquid Chromatography-Infrared (LC/IR) and Size Exclusion Chromatography-Infrared (SEC/IR) Analyses

Referencing these related standards ensures a robust theoretical and practical foundation for microanalysis using IR spectrophotometry.

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Keywords: ASTM E334, infrared microanalysis, IR microspectroscopy, microgram sample analysis, chromatographic IR analysis, sample preparation, contamination control, TGA/IR, FT-IR, laboratory standards, molecular spectroscopy.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E334 − 01 (Reapproved 2021)
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 ric Analysis (TGA) Coupled With Infrared Analysis
(TGA/IR)
1.1 Thispracticecoverstechniquesthatareofgeneralusein
E2106 Practice for General Techniques of Liquid
securing and analyzing microgram quantities of samples by
Chromatography-Infrared (LC/IR) and Size Exclusion
infrared spectrophotometric techniques. This practice makes
Chromatography-Infrared (SEC/IR) Analyses
repetition of description of specific techniques unnecessary in
individual infrared methods.
3. Terminology
1.2 These recommendations are supplementary to Practices
3.1 Definitions and Symbols—For definitions of terms and
E168,E573,andE1252,whichshouldbereferredtofortheory,
symbols, refer to Terminology E131.
general techniques of sample preparation, and calculations.
3.2 Beam Condenser—Aspecializedaccessorydesignedfor
1.3 This standard does not purport to address all of the
analysis of samples of a microgram or less, comprising an
safety concerns, if any, associated with its use. It is the
analyte area or volume of 2.0 mm diameter or less.
responsibility of the user of this standard to establish appro-
4. Contamination
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
4.1 Although the presence of contaminants is a general
1.4 This international standard was developed in accor-
problem in any type of analysis, contamination can be particu-
dance with internationally recognized principles on standard-
larly severe in micro work. For example, minor impurities in a
ization established in the Decision on Principles for the
solvent can become major components of a residue remaining
Development of International Standards, Guides and Recom-
after solvent evaporation. Materials extracted from thin-layer
mendations issued by the World Trade Organization Technical
chromatographic materials, from the paper used in paper
Barriers to Trade (TBT) Committee.
chromatography, and from solid adsorbents in general, may
include particular contaminants of concern. It should also be
2. Referenced Documents
noted that the gas-chromatographic stationary phase may lead
2.1 ASTM Standards: to significant contamination. Consideration of these and other
E131Terminology Relating to Molecular Spectroscopy
sources of contamination must always enter interpretation of
E168Practices for General Techniques of Infrared Quanti- results in microanalysis. Erroneous results can be minimized
tative Analysis
by the use of pure reagents, extreme care in sample handling,
E573Practices for Internal Reflection Spectroscopy andthefrequentuseof“blanks”inthecourseofseparationand
E1252Practice for General Techniques for Obtaining Infra-
subsequent recording of spectra.
red Spectra for Qualitative Analysis
5. General Microspectroscopic Techniques
E1642Practice for General Techniques of Gas Chromatog-
raphy Infrared (GC/IR) Analysis
5.1 Spectroscopic techniques used for the examination of
E2105Practice for General Techniques of Thermogravimet-
microsamples are usually adaptations of comparable macro
techniques, and many have been described in the literature (1,
2).
This practice is under the jurisdiction ofASTM Committee E13 on Molecular
5.2 In computerized dispersive spectrometers or Fourier
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
mittee E13.03 on Infrared and Near Infrared Spectroscopy.
transform-infrared (FT-IR) instruments, computer routines for
Current edition approved April 1, 2021. Published April 2021. Originally
multiple scanning, signal averaging, absorbance subtraction,
approved in 1990. Last previous edition approved in 2013 as E334–01 (2013).
andscaleexpansioncanbeusedveryeffectivelytoenhancethe
DOI: 10.1520/E0334-01R21.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on The boldface numbers in parentheses refer to a list of references at the end of
the ASTM website. this practice.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E334 − 01 (2021)
observed signal-to-noise ratio of weak bands and increase 5.4 Largeenergylossesbecauseofbeamattenuationmaybe
sensitivity (3, 4). Absorbance subtraction is also commonly avoided by the use of a beam-condensing accessory. This type
usedtoeliminateinterferingbandsfromthesamplematrixand ofaccessoryisdesignedtocondensethesampleradiationbeam
thus lower the limits of detection (see Practices E168). to an analyte area of 2 mm or less, accommodating the smaller
size of a microsample. A4× beam condenser is adequate for
5.3 Use of Masking Apertures—The aperture of sample
most microsample analyses.
holders used for microspectroscopic study (without the use of
5.4.1 The heat produced by the concentrated beam may be
an infrared microscope) are usually significantly smaller than
injurious to some samples, especially in the case of some
the beam at the sample position of the instrument. As a
dispersive instruments. If this difficulty is encountered, a thin
consequenceofthesesmallapertures,stepsneedtobetakento
germanium wafer between the source beam and the sample, or
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
greatly improve the results obtained (see 5.4). 5.5 Examination of Liquid Samples—Direct examination of
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 0.1mm
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-
ment background spectrum should be recorded through an 5.6 Examination of Solid Samples—The conventional tech-
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
instruments but others have general utility.
5.3.4 Some FT-IR spectrometers (especially those equipped
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 3mm by 5mm by 1 mm.The sample plate
singlebeamspectrum)theenergythroughputoftheinstrument
is mounted in a holder as near as possible to the focal point of
needs to be restricted in order to avoid detector nonlinearity theconvergingsampleradiationbeamorinabeam-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
halide that has been maintained in a drying oven at 105°C to
5.3.5 When using an infrared microscope, it is normal to
110°C. Blank samples of the stored alkali halide should be
record the reference spectrum through the same aperture as is
used to obtain frequent reference spectra, in order to guard
used for a particular sample. To accomplish this, it is most
against contamination.
convenient to use visual observation to select the aperture size
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 either 0.5mm or 1.5mm diameter.Astandard size 13mm die
single-beam background spectrum is then recorded afterwards. may be adapted for micropellet work by punching a small
The transmittance (or absorbance) spectrum of the sample is aperture in a disk of, for example, tinfoil, manila folder,
obtainedbyusingtheinstrumentsoftwaretocalculatetheratio blotting paper, or filter paper about 0.1 mm thick. About one
of the two single-beam spectra. third the usual pressure should be used for pressing the
E334 − 01 (2021)
micropellet.Thetinfoilorpaperservesasaholderforthepellet while polyethylene film is particularly useful for far-infrared
and can be positioned over the aperture of the micropellet measurements. Both materials withstand the effects of many
holder or on the beam-condenser unit. Commercially available corrosive samples.
lead micro disks are also available.
5.6.10 Another method for holding small solid samples in
the beam is to stick them on a translucent adhesive tape and
NOTE2—Stationerysupplystorescarrypaperpunchesofassortedsizes
placeanapertureoverthesample.Inthiscase,thespectrumof
and shapes that are suitable for making these apertures for micropellets.
NOTE 3—An aperture of 1mm by 4 mm is about the minimum size on the adhesive tape should be compensated for, either by placing
which some dispersive spectrometers can operate properly. If a beam
a similar aperture covered with adhesive tape in the reference
condensing accessory is used, the minimum aperture is reduced to the
beam or by computer subtraction of an adhesive tape spectrum
orderof0.5mmto1.0mmindiameter.Fouriertransforminstrumentscan
collected in a manner similar to that of the sample.
obtain spectra through a 0.5mm aperture, if necessary, without the use of
a beam condenser. 5.6.11 To avoid the need to computer-subtract the spectrum
of adhesive tape mentioned in 5.6.10, small pieces of salt
5.6.4 A very small sample may be made transferable by
window can be used to mount microsamples next to an
rubbing or abrasion, or both, using dry potassium bromide
aperture. The pieces of salt are cleaved from a used crystal by
(KBr) powder. Pellet grade KBr should be used, and subse-
using a razor blade, and can be as small as 1mm or 2 mm
quent grinding should be kept to the minimum necessary to
square.Transfer a few particles of adhesive from a (preferably
disperse the sample. This technique is also valuable for
old) piece of adhesive tape, using a probe, onto the extreme
removing a thin surface layer from a solid object.
edgesofthissaltcover.Placethesampleovertheaperture,and
5.6.5 A sample of a thin coating material may be obtained
cover with the salt plate. Pressure the salt cover onto the
by rubbing the surface with glass-paper or silicon carbide
aperture so that the adhesive holds it in place. Adhesive from
paper. The spectrum of the sample on the surface of the paper
a used piece of tape will allow the cover to be removed more
is obtained by using the diffuse reflectance technique, with a
easily after sample collection is completed.
clean piece of glass-paper or silicon carbide paper, as
5.6.12 IfusingIRSwithasmallsample,optimalresultswill
appropriate, being used as the reference.
beobtainedifthesmallsampleisplacedacrossthewidthofthe
5.6.6 Solidmaterialscanbeexaminedbyfirstdissolvingthe
internal reflection element (IRE). With very small samples,
material in a solvent (see 5.7). The resulting solution can be
optimal results will be obtained by placing the sample where
examined directly, or used to deposit the solute in a state more
thebeamenters,sothatthefirstreflectionisconcentratedatthe
advantageous for analysis, such as a thin film or in a halide
sample position (see Practices E573).
powder for the preparation of a KBr pellet or diffuse reflec-
5.6.12.1 MicroIRSaccessoriesarealsocommerciallyavail-
tance.Thesamesolventshouldbeusedtoobtainaspectrumof
able and are generally referred to as “micro-ATR” accessories.
thesolventblank,eitherdirectlyorasadeposit,asappropriate.
TheIREoftheseaccessoriesisonly1mmto3mmindiameter
5.6.6.1 Warning—Solvent or melt recrystallization or ap-
with an effective sampling area of 0.5mm to 2.0 mm in
plication of pressure to samples may cause changes in the
diameter, allowing analysis of smaller samples and, with a
crystalline structure of the material, and hence give changes to
diamond IRE, greater contact pressures.
the observed spectrum.
5.6.7 Some solids can be heat-softened or melted by press- 5.6.12.2 Particular cautions should be observed when using
these types of accessories.Accessory design precludes control
ingbetweentwosmallheatedKBrplatesandthenexaminedin
over the incident beam angle penetrating the IRE crystal
a demountable microcell holder (see 5.6.6.1). It is often
surface, thus, a number of incident beam angles are directed
advantageous to perform the pressing operation with the
ontothesample-crystalinterface.Theresultantspectramaynot
samplebetweentwosheetsofaluminumfoilfirst,sothatmore
be directly comparable to spectra collected from a controlled
pressurecanbeexerted.Thethinfilmisthenpeeledoffthefoil
incident angle IRE accessory or spectra collected by transmis-
and examined between the salt windows. Some solid samples
may be cut into thin wafers that may then be mounted in a sion. Additionally, if the active sampling area (0.5mm to 2.0
mm) is not completely filled by the sample, that is, the sample
micropellet holder for subsequent analysis.
issmallerthanthecrystalsurface,stray-lighteffectscandistort
5.6.8 Smallflakesofmaterialhavebeensuccessfullyexam-
the spectrum. In both cases, the “standard” ATR-correction
ined by supporting them on a salt plate and then placing an
algorithm is not sufficient to account for these effects and may
aperture over the sample. Both salt plate and aperture are
lead to even more erroneous results.
placed in the sample beam. Static forces may be used to hold
very small samples inside a pinhole aperture. Stray light may 5.6.13 For the case of intractable solid samples, the high-
be observed under both types of sample mounting, since the
pressurediamondanvilcellmaybeusedforsqueezingsamples
sample does not normally fill the aperture completely. Im- to an appropriate thickness. While the cost of a diamond anvil
proved spectral data are obtained by the use of a beam
cell is high, this is often the preferred method for reducing the
condenser (see 5.4) or, even better, an infrared transmitting thickness of samples that do not yield to simpler methods.The
microscope (see Section 11).
aperture of the cell is small, so it is necessary to use a beam
5.6.9 Samples can be held between two thin sheets of a condensing accessory, or better still, an infrared-transmitting
polymeric material that has low infrared absorbance at the microscope, to obtain the best quality spectra. Several com-
frequencies of interest, instead of being on the surface of a salt mentsshouldbemadehere,however.Diamondabsorbsenergy
–1 −1
plate as in 5.6.6 – 5.6.8. Fluorocarbon tape may be used to strongly between 1900cm and 2300 cm , which thus
obtain spectra over large portions of the mid-infrared region, renders this accessory inappropriate for the study of samples
E334 − 01 (2021)
NOTE 4—A suitable commercial version of the KBr triangle is avail-
that have significant absorptions in that region. On the other
able
hand, diamond is a good far-infrared window material and
−1
5.7.6 A microcapillary brush may be made to handle small
allows spectra to be recorded down to below 50 cm , using a
volumes of solvent (see Note 5) and can be used to cast a film
beam-condenser and suitably equipped spectrometer. Squeez-
on a remarkably small area of a salt crystal. When a micro-
ingthesampleinthecellmaychangethemorphologyandany
capillary brush containing a solution of a volatile solvent and
ordering in the structure of the sample (see 5.6.6.1).
a less volatile solute is placed on the surface of a salt plate, the
5.7 Examination of Solutions—In some instances, solutions
bristlesofthemicrobrushholdtheliquidinasmallregion.The
of liquids or solids are advantageously used for recording
non-volatilesolutemaythusbedepositedinarestrictedareaof
spectra. The preparation of solutions in microquantities has
the salt plate, ready for analysis. Working under a stereo
inherent difficulties, and solvents usually obscure some por-
microscope, deposit the solvent on the crystal, touching only
tions of the spectrum. Some of these interferences can be
theglassfiberstothecrystal (6).Makingasmallindentationin
eliminated by computer subtraction or double-beam tech-
the crystal with the point of a needle probe will help keep the
niques. Careful selection of the pathlength of the transmission
solvent localized.
cell or, with IRS, the type of IRE employed allows for dilute
NOTE 5—Following is the procedure to make a microcapillary brush.
solutions (even in water) to be examined directly using an
Insert a bundle of 20 to 30 glass wool fibers into the end of a thin-walled
FT-IR spectrometer or a computer-assisted dispersive spec-
microcapillary tube. Twirl the side of the tube near a micro burner flame
trometer. In general, solvent blank samples need to be exam- untilthefibersarefusedtothesideofthetube.(Thismaytakeafewtries
sinceitisquiteeasytosingethefibersiftheygettooneartheflame.)Once
ined in the same manner as the solutions generated, in order to
the fibers are secured to the side of the tube, snip off all but a few
identify the presence of contaminants.
millimetres of the fibers.
5.7.1 A solution may be used to prepare a micro film of
5.7.7 In practice, if there is a fair amount of residue in the
solute on a small window (approximately 8mm by 8mm by 2
solvent, it will tend to precipitate on the end of the fibers.This
mm) that has been gently scratched in order to contain the
is just as well, as the solute can then be removed, rolled onto
sample in a small area (3mm by 3 mm, or less if using an
thesurfaceofaninfraredtransmittingwindow,andplacedover
FT-IR). It should be noted that the window must be made of a
an aperture for examination. The “drop and suck” trick can be
material that is not harmed by the solvent in use. Condensates
used with one of these brush capillaries. Use the brush to
from micro (capillary scale) pyrolysis can also be run in this
redepositthesolutiononthecrystalinasmallareatomaximize
manner.Alternatively, the deposit may be made directly onto a
sensitivity. Use an aperture of appropriate size to mask the rest
micro ATR and the spectrum obtained by IRS.
of the crystal or examine the sample using an infrared-
5.7.2 Asmall amount of a solution may be deposited onto a
transmitting microscope.
salt window using a capillary tube. In this case, the capillary
5.7.8 The technique of incorporating microgram samples
action of the tube may be used to pick up a droplet of the
intoalkalihalidesbylyophilization(freezedrying)workswell,
solution.When the end of the tube is brought into contact with
although some additional precautions are necessary. Freeze
the window, the solution should partially flow onto the surface
drying is the removal of solvent from a mixture by low-
ofthewindow.Thesolventcanthenbeevaporatedtoleavethe
temperature sublimation, normally done under vacuum condi-
residual solute as a micro film. If necessary, the capillary tube tions. Spectra of lyophilized materials often differ from those
can be fitted with a small rubber bulb to allow more sample to
of the same material that is simply ground with the alkali
be drawn into the tube, or a fine Pasteur pipette can be used. halide.Precoatingthelyophilizationtubewithafrozenlayerof
an alkali halide aqueous solution minimizes the loss of some
5.7.3 A solution can be evaporated onto a powdered solid
types of samples because of adsorption on the glass surfaces.
such as potassium chloride (KCl) for diffuse reflection tech-
Contamination frequently arises from this procedure (for
niques. The resulting powder is examined in a diffuse reflec-
example,frompumpbackstreaming)andshouldbecheckedby
tance micro-cup.
using blanks of alkali halide powder alone. It should be noted
5.7.4 Alternatively, the solution can be evaporated onto dry
that some solids have sufficient vapor pressure that a small
KBr powder which can then be used to prepare a micro KBr
sample will be reduced or even eliminated when being worked
pellet (as in 5.6.2 – 5.6.4 ).
with during lyophilization.
5.7.5 Another technique employs a porous triangle of
5.8 Micropyrolysis of Solid Samples— Pyrolysis is often
pressed KBr in a capped glass vial having a small hole in the
usedtoobtainspectrafrommaterialslikecarbon-filledrubbers
cap. The solution is allowed to evaporate at the KBr triangle
tip, leaving the solute concentrated there. This accomplishes
filtration of adsorbent and deposition of the sample on KBr in
Thesolesourceofsupplyofthisapparatusknowntothecommitteeatthistime
a single step. The tip of the triangle (after evaporation of the
is Harshaw, Cochran Rd., Solon, OH 44139. If you are aware of alternative
solvent) is used to prepare a micro KBr pellet. If preferred, the
suppliers, please provide this information to ASTM International Headquarters.
diffuse reflectance technique can be used to obtain the spec-
Your comments will receive careful consideration at a meeting of the responsible
trum of the solute in the KBr. technical committee, which you may attend.
E334 − 01 (2021)
are multicomponent until proven otherwise.
that are too opaque or heavily filled to yield spectra by other
methods. The optimum method used to pyrolyze the sample
7. Analysis of Liquid Chromatography Fractions
will depend on its size.
7.1 There are many factors that must be considered when
5.8.1 The simplest method for micropyrolysis involves the
combining liquid chromatography and FT-IR. The LC effluent
use of a disposable pipette. The sample is inserted into the
can be coupled directly to the FT-IR spectrometer using flow
pipetteandrolledtotheneckregion,andthelargeendissealed
through cells (13) or specialized IRE accessories (14).
in a small flame. When the sealed end cools, the polymer is
Alternatively, the LC effluent may be deposited onto infrared
tapped into that end. The sample is heated gently, producing
transmissive powders (15) or moving substrates (16) for
pyrolysis products that condense on the walls of the pipette.
analysis by diffuse reflection or reflection absorption spectros-
The portion of the pipette containing the ash is then removed
copy. The commercially available LC/IR systems can offer
by scoring between the ash and the condensate and breaking
detection limits at the microgram level for a broad range of
thetube.Asingledropletofsolventcanthenbeadded,washing
separations. (see Practice E2106).
the entire pyrolysate onto a salt plate for analysis.
5.8.2 Very small amounts of material can be pyrolyzed in a
7.2 Caution must be taken when interpreting LC/IR results
capillary tube instead of the pipette mentioned in 5.8.1.
as solvent interferences may obscure critical areas of the
5.8.3 A microcapillary brush (see 5.7.6) may be used to spectrum necessary for correct identification or interpretation
obtain a spectrum from a fragment that is too small to produce
of the analyte spectra. Cross contamination and peak tailing is
enough pyrolyzate by an ordinary pyrolysis analysis. Place the also more prevalent in LC/IR interfaces.
fragmentintheendofthecapillarybrushthatisawayfromthe
7.3 Supercritical fluid chromatography (SFC) can also be
fibersandworkthefragmenttowardthecenterofthetube.Seal
coupled to an FT-IR spectrometer (17). High-pressure flow-
the end of the tube. Then twirl the tube near a micro-flame in
through cells or solvent-elimination systems exist, offering
the area of the particle to pyrolyze the sample, being careful
dection limits between 10ng and 40 ng.
not to melt the tube. Cut off the sealed end of the tube
containing the ash, draw a microdroplet of clean solvent up
8. Analysis of Thin-Layer Chromatographic Fractions
into the tube to dissolve the pyrolyzate, and then use the brush
8.1 The spots containing the components of interest, plus
to deposit the solution onto a crystal.
the associated absorbent, are generally collected by scraping
5.9 Interest in coupling chromatographic methods with
them from the plate; the components can be recovered by
FT-IR spectroscopy arises from the need to separate and
extraction with a suitable infrared solvent, and the spectrum of
identify the components of mixtures. Chromatographic meth-
the solution can be determined by the usual methods. If
ods commonly used in conjunction with FT-IR analysis of the
preferred, the spectrum of the analyte may be obtained after
elutingcomponentsaregaschromatography,highperformance
transference to a porous triangle of KBr (see 5.7.5).
liquidchromatography,supercriticalfluidchromatography,and
8.2 Extractionofthespotisusuallyrequiredbeforespectral
thin-layer paper chromatography (respectively known as GC,
determination of the component of interest because the com-
HPLC, SFC, and TLC), and paper chromatography. For GC
mon TLC absorbants (silica gel and alumina) are infrared
and SFC the identification is usually performed in real-time
absorbers. Potassium bromide (KBr) can be used as an absor-
using an FT-IR spectrometer, whereas the analysis of the
bant for some systems. When the areas of KBr containing the
compounds separated by other chromatographic techniques
components of interest have been located, the adsorbent is
may be performed in an off-line manner. For detailed guide-
recovered as before and either a KBr pellet is prepared in the
lines concerning the practice of GC/IR and LC/IR, see Prac-
conventional manner, or a spectrum is obtained by the diffuse
tices E1642 and E2106.
reflectance method.An automated extraction system for analy-
sis by diffuse reflectance has been described (18).
6. Analysis of Gas-Chromatographic Fractions (7-9)
NOTE 7—The quantity of analyte available from any one spot may be
6.1 Gas chromatographic fractions are normally examined
insufficient to produce a usable spectrum. In this case it is usually
directly as gases in a GC/FT-IR combination system in which
necessary to stripe the sample onto preparativeTLC plates and to recover
the gas chromatograph is coupled directly to the FT-IR
the total eluted band in which the sought components are located.
spectrometerandtheseparatedcomponentsanalyzedinthegas Programmed multiple development, a form of TLC in which the chroma-
tography is performed using several developments, often concentrates the
phase as they emerge from the GC column (see Practice
TLCspotsofsamplesothatsufficientquantitiesofmaterialarepresentto
E1642).
give identifiable IR spectra.
6.2 Gas chromatographic fractions can also be trapped
8.3 Quantitative or semiquantitative estimates of concentra-
separately for subsequent infrared analysis by passing the
tions may be obtained from direct comparison of values for an
streamissuingfromtheventlineofthechromatographthrough
unknown sample with those obtained for a standard sample.
a solvent, a powdered solid, or a cold trap (7). Alternative
procedures for GC/FT-IR detection involve the on-line trap-
9. Analysis of Paper Chromatographic Fractions
ping of submicrogram quantities of GC eluate at low tempera-
9.1 The areas of interest in paper chromatograms are cut
tures (10-12).Somecommerciallyavailablecryogenicsystems
from paper. These fractions may be recovered by solvent
can provide detection limits at the subnanogram level.
extractions, as in 8.2, or may be examined in-situ using
NOTE6—Itmustbeassumedthatallfractionsobtainedusingacoldtrap infraredreflectancetechniques.Withthelattermethod,spectral
E334 − 01 (2021)
subtraction is used to eliminate contributions from the paper wavelength, and thus the spectrum obtained represents an
substrate. The reference spectrum used for subtraction should increasingly larger area as the wavelength increases.
beobtainedfromapieceofthepaperthathasbeentreatedwith
NOTE 9—The energy from a point, when imaged by an optical system,
the solvent used.
does not come to a point, but rather to a central bright spot followed by a
succession of dark and bright rings (21). The bright rings are called lobes
or pods, and they contain energy from the original point. For any
10. Analysis of the Gases Evolved from a
unobscured optical imaging system, roughly 85% of the energy is in the
Thermogravimetric Analyzer
central maximum of the pattern. (The objectives used for infrared
microscopeshaveacentralobscuration,whichlowerstheapparentenergy
10.1 Asasampleisheatedunderacontrolledatmospherein
in this region, typically by some 10%.) The remainder of the infrared
a TGA experiment, gases may be evolved from the sample
energy lies in the bright rings, which will be outside of the optical image
during times of weight loss. The evolved gases can be trapped
andthusmaybeabsorbedbyunexpectedpartsofanysamplethatislarger
in the condensed phase or passed through a transfer line into a
thantheapertureused.Toillustratewhattheimplicationsoftheresolution
limit are for infrared microspectrometry, consider the longest infrared
gasanalysiscell (19, 20).TGA/FT-IRaccessoriesareavailable
−1
wavelength of interest, for example, 20 µm (a frequency of 500 cm ).
for FT-IR spectrometers, and some combined TGA/FT-IR
When this wavelength is used in the diffraction equation (21), along with
instruments are also available. With such equipment, it is
a numerical aperture of 0.5, the calculations indicate that for a point
possible to measure the evolution of some individual gases,
source the first dark ring occurs at 24 µm from the sample edge.
eventhoughtheyareevolvedaspartofamixture.Detectionof Successive dark rings occur at 44µm, 64µm, and 84µm. Roughly 5% of
the energy from the point source is still present beyond the fourth dark
µg amounts of evolved gases can be achieved with an FT-IR
ring. In practice, of course, the source used must have significant size.
spectrometer, which represents a 0.01% weight loss from a
11.2 Microscope attachments are commercially available
10mg sample. (see Practice E2105)
that allow for spectra to be recorded in a transmittance mode,
11. Infrared Spectroscopy Using a Microscope (21-23)
where the beam passes through the specimen plane, or in a
NOTE8—Namesthathavebeenusedreferringtothistechniqueinclude
reflectance mode, where the beam reflects at the specimen
viewing infrared microspectroscopy, infrared microspectrometry, infrared
plane. Reflectance may occur at the specimen surface, from a
microspectroscopy, and micro IR. Infrared ultramicrospectrometry (or
reflective support, or sometimes at both planes.
ultramicrospectroscopy) refers to a special method in which the sample is
physically masked to below the diffraction limit (smaller than 20 µm).
11.3 Allcommerciallyavailablemicroscopeattachmentsfor
infrared microspectroscopy allow for the positioning of an
11.1 Spectra collected with infrared transmitting micro-
aperture of variable size at a specimen image plane, or planes,
scope accessories can differ from conventional spectra in
in the optical path of the microscope. The function of this
several important aspects. Therefore, care should be taken to
aperture is to limit the area of specimen being studied. The
carefully document the experimental conditions used when
image plane where the remote aperture is placed is an optical
spectra are obtained by infrared microspectroscopy. The most
conjugate of the specimen plane, related in size through the
importantdifferenceisthefactthatthespectramaybeaffected
magnification of the optical system. Thus, a relatively large
by the diffraction properties of infrared radiation. The cross
aperture can be used to mask a small dimension of the
sections of the samples being measured can be similar in size
specimen. Round and rectangular variable apertures are avail-
to the wavelength of radiation used to analyze them. Since the
abletotheuser.Theaperturegeometryshouldbeselectedsoas
sample area is defined by masking at an image plane, and
to match the shape of the desired specimen area as closely as
diffractionoftheradiationaffectsthespectrarecorded,thiscan
possible. This is particularly important for photometric accu-
show distortions in band shape or in relative intensity, or in
racy when recording spectra of small samples. Radiation
both.
reachingthedetectorthatdoesnotpassthroughthesamplewill
11.1.1 The experimental parameters to be recorded when
cause distortions in relative absorption intensities.
publishing results of an infrared study using a microscope are:
11.4 An additional remote image mask may be placed at an
(1) the area of the specimen being analyzed, (2) the size and
image plane before the specimen, so that there is an aperture
type of the detector element, (3) whether the spectra were
before and after the specimen. This aperture needs to be the
obtained using the transmittance or reflectance mode, (4) the
same size as the limiting aperture mentioned in 11.3. When
specimen geometry and method of preparation, and (5) the
usingthereflectancemodeofamicroscopeequippedwithdual
shape,location,andtypeofimageplanemasksused.Important
remote image masking, the radiation normally passes through
instrumental conditions also to be recorded are the spectral
the same aperture before hitting the sample as it does after
resolution, the data collection time, and the nature of the
reflecting off the sample surface (that is, the one aperture
reference background spectrum. It should be remembered that
serves both functions).
it is also critical to report any computer manipulation of the
spectrum, such as baseline correction or subtraction.
11.5 It is very important that the optical alignment of an
11.1.2 Thespatialdefinitionofthesamplingareaobtainable infrared-transmitting microscope be well maintained in order
with a microscope using infrared radiation is limited by to obtain good results. Both the infrared and the visible beam
diffraction effects arising from the relatively long wavelengths paths need to be co-linear and co-focal at all times; otherwise
of radiation involved. This diffraction effect is wavelength spectra can be recorded from an area different from the one
dependent and thus is particularly noticeable below a fre- visually examined. The alignment procedure for a microscope
−1
quency of about 1000 cm (10 µm).The area of the specimen operating in the transmission mode involves the use of a small
from which the radiation is collected increases with aperture, typically a 100µm pinhole, at the sample position.
E334 − 01 (2021)
With this 100µm aperture installed at the sample position, 11.6.4 Liquid and solid samples are often mounted on thin
insert one or both remote apertures having equivalent size to salt windows. It has been observed that the best spectra are
that in the sample plane, and align these apertures visually so obtained when the windows are quite thin, as this has the least
thattheyallappearcoincident.Switchtotheinfraredmodeand effectontheopticalsystemofthemicroscope.Typicallysaltor
maximize the infrared energy through these apertures, follow- KCl can be cleaved to produce windows with surface dimen-
ingthemanufacturer’sinstructions.Checkthatthevisiblelight sions of a few millimetres and a thickness of 1mm to 2 mm.
and the infrared radiation are still collinear after this These salt plates are easily cleaved to size with a single edged
adjustment, and at regular intervals. razor blade and tapping tool such as a screwdriver handle.The
11.5.1 Of particular importance is the concentration of the razor blade is placed on the surface of the salt parallel to a
primary and secondary mirrors of the Cassegrain objective or prominent fracture edge.The handle of the screwdriver is then
the condenser, or both. Unless absolutely necessary, adjust- gentlytappedontothebackedgeoftherazorblade,causingthe
ments to the optical system of an infrared microscope should razorbladetocleavethecrystal.Itisveryconvenienttomount
not be made without specific instructions available to achieve these small crystals over a hole in a piece of cardboard or
proper alignment. manilafolderusingasmallamountofnitrocellulosecement,or
over a hole in a thin aluminum plate with a small amount of
11.6 Sample Handling Considerations:
rubber cement.
11.6.1 While the use of a microscope for IR sampling
11.6.5 Solidparticlesareeasilyplacedonthesaltplatewith
simplifies the analysis of many samples, sample preparation is
afinepointedtungstenneedle(see11.6.10).Iftheparticlesare
critical to obtaining the desired spectral measurement.
quite thin, they need only be placed on the surface of the salt
11.6.2 The collection, handling, and mounting of micro-
plate.Itisquiteusefultoscorethesurfaceofthesaltplatewith
scopic samples must be considered in terms of the sample
the needle to produce a simple map such that the particle can
geometryneededforIRspectralmeasurements.ForIRspectral
easily be found unequivocally under the microscope. If the
measurement of organic materials in the transmission mode, a
small sample is quite thick, a variety of different techniques
sample thickness from 5µm to 20 µm is most desirable. Thick
can be used to reduce it in thickness.
samples cause both loss of detail in regions of strong absorp-
11.6.6 Micropipettes are useful for applying small amounts
tion and distortions of absorption ratios. While sample areas
of reagents, adhesive, or solvents to salt plates for particle
10µm by 10 µm can be analyzed, larger areas provide spectral
manipulation. Glass micropipettes can be purchased from
data with higher signal-to-noise ratios.To minimize diffraction
several suppliers or produced by reducing the diameter of a
effects, the smallest sample dimension should be approxi-
glass tubing, using typical glass-blowing techniques. Micropi-
mately five times the wavelength of interest when using a
pettes are also useful for applying small amounts of liquid to
single mask and two times the wavelength when dual remote
the surface of the salt plate for analysis. An alternative
image masks are used (see Note 10). When possible, a thin,
technique for placing a liquid on a salt plate is to evaporate a
uniform sample with as large an area as is practical should be
solution of the liquid on the surface of the salt plate from a
selected. The maximum sample area is determined by the
microbrush (see 5.7.6).
microscope/spectrometer optics and the detector element size.
In most current IR microscopy systems the maximum sample
11.6.7 When small amounts of liquids are to be analyzed,
area is 250µm by 250 µm, even though the specimens may be they have to be kept in a restricted area of the salt plate, or the
many times this size.
analysis is extremely difficult. This can be accomplished in
several ways. One procedure is to rupture the surface of a salt
NOTE 10—The actual values of sample dimensions for minimizing the
plate in a small area with repeated jabbing of that region with
diffraction contributions are dependent on the numerical aperture of the
the end of a small microprobe. The small salt crystals that are
microscopeobjectiveused.Forexample,withanumericalapertureof0.5,
asamplesizeoffivetimesthewavelengthforasingleaperturesystemand
produced are allowed to remain in the small well, and the
2.5 times the wavelength for a microscope with dual remote image masks
liquid is added to that well. The capillary spaces between the
will lead to approximately 95% of the incident radiance passing through
particles retain the liquid in situ, minimizing spreading.
the defined sample area. With a 0.25 numerical aperture objective, the
same conditions will be met when the sample dimensions are approxi- 11.6.8 Another useful procedure for analyzing small
mately ten and five times the wavelength, respectively.
amounts of liquid is to place a micro flat of salt on the surface
of the somewhat larger salt plate.The liquid is then allowed to
11.6.3 Collecting, mounting, and thinning of samples for
flow between these salt plates by capillary action, the liquid
microspectral measurement require special techniques and
being applied to the edge of the junction of the two salt plates
tools.SincethelargestsampleareasthatcanbeanalyzedbyIR
with a micropipette.
microscopy are just resolved by the unaided human eye,
sample preparation is aided greatly by using a low-power 11.6.9 Spreading of droplets of liquid can be minimized by
stereo binocular microscope. Magnifications of 7× to 20× are placing the salt plate on the gently heated surface of a small
most useful for locating and mounting samples for analysis. metal washer. This applies additional heat at the outside of the
salt plate and somewhat less heat near the center of the salt
Fine needles, tweezers, spear pointed probes, razor blades, and
scalpels are valuable tools to extract and manipulate samples. plate. The result is that the droplet is forced toward the center.
Rollers, presses, compression stages, and microtomes are used This technique can often minimize spreading sufficiently so
to reduce sample thickness. These tools and techniques are that good infrared spectra can be produced from very small
described in detail in the following sections. amounts of liquid.
E334 − 01 (2021)
11.6.10 Fine-Pointed Tungsten Needles: under a stereo microscope using a microprobe. If the sample is
tacky,thisshouldbedoneonasmallsaltwindow.Ifthesample
11.6.10.1 Fine-point tungsten wire needles are very useful
is hard, the rolling should be carried out on a hard surface and
for the extracting and handling of microscopic samples for IR
then transferred to a suitable mount. Met
...