ASTM E2106-00(2019)
(Practice)Standard Practice for General Techniques of Liquid Chromatography-Infrared (LC/IR) and Size Exclusion Chromatography-Infrared (SEC/IR) Analyses
Standard Practice for General Techniques of Liquid Chromatography-Infrared (LC/IR) and Size Exclusion Chromatography-Infrared (SEC/IR) Analyses
SIGNIFICANCE AND USE
4.1 This practice provides general guidelines for the practice of liquid chromatography or size exclusion chromatography coupled with infrared spectrometric detection and analysis (LC/IR, SEC/IR). This practice assumes that the chromatography involved is adequate to resolve a sample into discrete fractions. It is not the intention of this practice to instruct the user on how to perform liquid or size exclusion chromatography (LC or SEC).
SCOPE
1.1 This practice covers techniques that are of general use in qualitatively analyzing multicomponent samples by using a combination of liquid chromatography (LC) or size exclusion chromatography (SEC) with infrared (IR) spectrometric techniques. The sample mixture is separated into fractions by the chromatographic separation. These fractions are subsequently analyzed by an IR spectroscopic method.
1.2 Three different types of LC/IR techniques have been used to analyze samples (1, 2).2 These consist of eluent trapping (see Practice E334), flowcell and direct deposition. These are presented in the order that they were first used.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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.
1.5 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
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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: E2106 − 00 (Reapproved 2019)
Standard Practice for
General Techniques of Liquid Chromatography-Infrared (LC/
IR) and Size Exclusion Chromatography-Infrared (SEC/IR)
Analyses
This standard is issued under the fixed designation E2106; 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.
1. Scope E168Practices for General Techniques of Infrared Quanti-
tative Analysis
1.1 Thispracticecoverstechniquesthatareofgeneralusein
E334Practice for General Techniques of Infrared Micro-
qualitatively analyzing multicomponent samples by using a
analysis
combination of liquid chromatography (LC) or size exclusion
E1421Practice for Describing and Measuring Performance
chromatography (SEC) with infrared (IR) spectrometric tech-
of Fourier Transform Mid-Infrared (FT-MIR) Spectrom-
niques. The sample mixture is separated into fractions by the
eters: Level Zero and Level One Tests
chromatographic separation. These fractions are subsequently
analyzed by an IR spectroscopic method.
3. Terminology
1.2 Three different types of LC/IR techniques have been
3.1 Definitions—For definitions of terms and symbols, refer
used to analyze samples (1, 2). These consist of eluent
to Terminology E131.
trapping (see Practice E334), flowcell and direct deposition.
3.2 Definitions of Terms Specific to This Standard:
These are presented in the order that they were first used.
3.2.1 hit quality index (HQI), n—thecomparisonofinfrared
1.3 The values stated in SI units are to be regarded as
spectroscopic data against a database of reference spectra of
standard. No other units of measurement are included in this
known compounds is often employed to assist in the determi-
standard.
nation of the evolved gas chemical identity. Search algorithms
1.4 This standard does not purport to address all of the
generate a listing of reference compounds from the database
safety concerns, if any, associated with its use. It is the
that are spectroscopically similar to the evolved gas spectrum.
responsibility of the user of this standard to establish appro-
These reference compounds are ranked with regard to a
priate safety, health, and environmental practices and deter-
measurement of the comparative fit of the reference spectral
mine the applicability of regulatory limitations prior to use.
data to that of the spectrum of the evolved gas.This ranking is
1.5 This international standard was developed in accor-
referred to as the hit quality index (HQI).
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the 4. Significance and Use
Development of International Standards, Guides and Recom-
4.1 This practice provides general guidelines for the prac-
mendations issued by the World Trade Organization Technical
tice of liquid chromatography or size exclusion chromatogra-
Barriers to Trade (TBT) Committee.
phycoupledwithinfraredspectrometricdetectionandanalysis
(LC/IR, SEC/IR). This practice assumes that the chromatogra-
2. Referenced Documents
phy involved is adequate to resolve a sample into discrete
2.1 ASTM Standards:
fractions. It is not the intention of this practice to instruct the
E131Terminology Relating to Molecular Spectroscopy
user on how to perform liquid or size exclusion chromatogra-
phy (LC or SEC).
This practice is under the jurisdiction ofASTM Committee E13 on Molecular
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
5. General LC/IR Techniques
mittee E13.03 on Infrared and Near Infrared Spectroscopy.
Current edition approved Dec. 1, 2019. Published December 2019. Originally
5.1 Three different LC/IR techniques have been used to
approved in 2000. Last previous edition approved in 2011 as E2106–00(2011).
analyzesamples.Theseconsistofeluenttrapping,flowcelland
DOI: 10.1520/E2106–00R19.
direct deposition. These are presented in the order that they
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard.
were first developed. Infrared detection for any of these
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
techniques can be provided by IR monochromators, IR filter
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
spectrometers and Fourier transform infrared spectrometers
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. (FT-IR).These detectors yield either single absorption band or
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2106 − 00 (2019)
total infrared spectrum detection modes. Detection mode is complete infrared spectrum. Such instruments include a com-
dependent upon the type of IR detector employed and the puter that is capable of storing the large amount of spectro-
acquisition time required by the LC or SEC experiment. scopic data generated for subsequent evaluation. Conversely,
monochromators and filter infrared spectrometers permit the
5.2 Eluent Trapping Techniques—Eluent trapping
monitoring of a selected absorbance band, for example, 1730
techniques, such as stopped flow and fraction collection, are
−1
cm forcarbonylfunctionalgroups.Dataacquisitionforthese
the simple means for obtaining LC/IR data. In these
devices is similar to that for a typical LC detector.
techniques, the eluting sample is collected from the chromato-
5.3.2 The transfer line from the LC column to the flowcell
graph in discrete aliquots. These aliquots are then analyzed
must be made of inert, nonporous material. This normally is
with the appropriate sampling accessory in an infrared spec-
PTFE, PEEK or stainless steel tubing. The volume, internal
trometer. In utilizing such techniques, it is essential that a
diameter, and connections of the transfer line are optimized to
suitable LC detector, such as refractive index or UV/VIS, be
reduce dead volume and mixing that can degrade the chro-
employed to allow definition of component elution. Since the
matographic separation. When performing separations at el-
analyte of interest is trapped physically, the spectrum can be
evated temperatures, the transfer line and flowcell may require
recorded using a long integration or scan coaddition time to
controlled heating to maintain temperatures of the eluent.
improvethesignal-to-noiseratio(SNR).Generally,thestopped
5.3.3 The flowcell is made of IR transmissive window
flow technique requires the use of a flow cell and the IR
materials to give maximum optical throughput to and from the
spectrum acquired contains both analyte and mobile phase
effluent chamber. Proper selection of window material is
spectral features. The fraction collection mode permits exami-
necessary to ensure chemical inertness and IR transmissivity.
nation of the eluent as a solution of analyte and mobile phase
The cell design and volume must maintain chromatographic
or, with proper solvent removal, the analyte alone (provided
resolutionwhilemaximizingopticalinteractionwiththeeluent
that the analyte is nonvolatile).As such, the fraction collection
via transmission, reflection-absorption or attentuated total re-
mode would require either a liquid cell for solutions or a solid
flection modes. Flowcells are typically optimized so that the
substrate, that is, KBr window for transmission, first surface
sampling volume accommodates the corresponding eluent
mirror for reflection-absorption or powdered KBr for diffuse
volume of a sharp chromatographic peak at the peak’s full
reflection measurements.
width at half height (FWHH). Typically, this volume is
5.3 Flowcell Detection—With flowcell detection, the LC
matched to the scale of the liquid chromatography, that is, 10
eluent is monitored continuously in the timeframe of the
µL for analytical scale and larger volume separations and less
chromatography (real-time) by the IR spectrometer with the
than 10 µL for microbore separations.
use of specially designed liquid cells (3-9). Liquid cells are
5.3.3.1 The optimum infrared transmission across the full
designed to minimize dead volume and analyte mixing, to
mid-infraredspectrumisobtainedbyusingpotassiumbromide
conserve chromatographic resolution, and achieve maximum
windows; however, this material is susceptible to damage by
optical interaction of the eluent with the infrared radiation.As
waterandcoldflowsundermechanicalforce.Astheflowcellis
the effluent is a condensed phase, several cell types have been
used, small amounts of water will etch the window surfaces,
devised to accommodate most experimental approaches for IR
and the optical throughput of the windows will drop.
spectrometry, that is, transmission, reflection-absorption and
Eventually,thesewindowswillhavetobechanged.Userswho
attenuated total reflection (7). The flowcell technique typically
expect to analyze mixtures containing water should consider
1).
yields submicrogram detection limits for most analytes (
using windows made of a water-resistant material such as zinc
Typically, flowcells are mounted within the sample compart-
selenide(ZnSe).IRwindowsofhighrefractiveindexlikeZnSe
ment of the spectrometer and use beam condensation optics to
and zinc sulfide (ZnS) will result in a noticeable drop in
directtheIRbeamintoandoutofthesmallvolumeofthecell.
infrared transmission due to the optical properties, that is,
It is important to employ a mobile phase having low or
reflectivity, of such materials. Additionally, high refractive
preferablynoinfraredabsorptionsintheanalyticallyimportant
index materials may cause fringing, that is, create an optical
spectralregionsfortheanalytesofinterest.Assuch,thechoice
interference pattern in the baseline of the IR spectrum.
of mobile phase may constrain the liquid chromatographic
separation. Generally, this limits the chromatographic separa-
NOTE 1—Fringing is due to multiple reflection optical paths created
tion to a normal phase type where nonpolar solvents like when windows are placed as parallel plates separated by a discrete
pathlength.Thesereflectionopticalpathspermitlight,whichisretardedto
chloroform and carbon tetrachloride have sufficient solvent
a greater extent than light from the transmitted optical path, to reach the
strengthtoelutecomponentsandhavelowinfraredabsorption.
detector. This reflection optical path light is out of phase with the
In contrast, flowcell detection of reversed phase separations
transmitted optical path light and yields interferences fringes in the
involving aqueous mobile phases are essentially precluded as
resultant spectrum. Fringing may be reduced by making the windows
strong absorption by water occurs across the mid-infrared nonparallel or by placing the cell slightly askew, that is, 5–15°, in the
optical beam of the spectrometer. Please refer to Practices E168 for
spectrum. If flowcell detection of reversed phase separation is
additional information on fringing effects.
to be attenuated, removal of the analytes from the aqueous
mobile phase via extraction into an infrared transmissive 5.3.3.2 Theopticalenergythroughputoftheflowcellshould
solvent is suggested (9).
be periodically monitored, since this is a good indicator of the
5.3.1 The rapidity with which spectra must be recorded overallconditionoftheLC/IRinterface.IfaFouriertransform
duringaliquidchromatographicseparationtypicallyrequiresa spectrometerisused,itisrecommendedthatrecordsbekeptof
Fourier-transform infrared (FT-IR) spectrometer to capture the the interferogram signal strength, single-beam energy
E2106 − 00 (2019)
response, and the ratio of two successive single-beam curves mate doubling of the pathlength. The advantage of this
(as appropriate to the instrument used). For more information approachoverthatofafirstsurfacemirroristoreducespurious
on such tests, refer to Practice E1421. These tests will also optical effects such as specular reflection which may occur as
reveal when a mercury cadmium telluride (MCT) detector is light passes through the spotted analyte.
performing poorly due to loss of the Dewar vacuum and 5.4.3 Direct deposition techniques provide the advantage of
consequentbuildupoficeonthedetectorface.Asnotedfurther post-run spectral data acquisition and possibly, decoupling the
in this text, an MCT detector is commonly used with these chromatographic separation from the spectrometry. Through
experiments as they provide greater detectivity and fast data extended co-addition of spectra, the signal-to-noise ratio
acquisition times. (SNR)ofspectralresultsisimprovedoverthatobtainedduring
real-time data acquisition. It must be noted that slow sublima-
5.3.3.3 Care must be taken to stabilize or, preferably,
tion of the analyte and recrystallization may occur with direct
removeinterferingspectralfeaturesresultingfromatmospheric
deposition techniques. It is prudent to initially obtain the
absorptions in the optical beam path of the spectrometer. Best
spectral data with a short co-addition time to create reference
results will be obtained by purging the complete optical path
data to ensure the integrity of spectra obtained with longer
withdrynitrogengas.Alternatively,dryaircanbeusedforthe
co-addition times after the chromatographic separation is
purge gas, but has interferences in the regions of carbon
−1 −1
complete.
dioxide IR absorption (2500 to 2200 cm and 668 cm ).
Commercially-available air scrubbers that remove water vapor
6. Significant Parameters for LC/IR
and carbon dioxide also provide adequate purging of the
spectrometer. In some instruments, the beam path is sealed in
6.1 The instrumentation used to conduct the LC/IR experi-
thepresenceofadesiccant,butinterferencesfrombothcarbon ment should be properly recorded within prescribed standard
−1
dioxide and water vapor (1900 to 1400 cm ) may still be
operating procedures (SOPs) or laboratory notebooks as nec-
found. In all cases, the instrument atmosphere must be stabi- essarytomeetrequirementsforspecificlaboratorypractices.If
lized before data collection commences.Atmospheric stability the equipment is commercially available, the manufacturers’
inside the instrument can be judged by recording the single- names and model numbers for the complete LC/IR system, or
beam energy response and the ratio of two successive single- the individual components, should be recorded. Additionally,
beam spectra. various instrumental and software paramete
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