ASTM E2106-00(2011)
(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
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). These consist of eluent trapping (see Practices 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 and health practices and determine the applicability of regulatory limitations prior to use.
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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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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: E2106 − 00 (Reapproved 2011)
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 E1421Practice for Describing and Measuring Performance
of Fourier Transform Mid-Infrared (FT-MIR) Spectrom-
1.1 Thispracticecoverstechniquesthatareofgeneralusein
eters: Level Zero and Level One Tests
qualitatively analyzing multicomponent samples by using a
combination of liquid chromatography (LC) or size exclusion
3. Terminology
chromatography (SEC) with infrared (IR) spectrometric tech-
niques. The sample mixture is separated into fractions by the
3.1 Definitions—For definitions of terms and symbols, refer
chromatographic separation. These fractions are subsequently to Terminology E131.
analyzed by an IR spectroscopic method.
3.2 Definitions of Terms Specific to This Standard:
1.2 Three different types of LC/IR techniques have been 3.2.1 hit quality index (HQI), n—thecomparisonofinfrared
used to analyze samples (1, 2). These consist of eluent
spectroscopic data against a database of reference spectra of
trapping (see Practices E334), flowcell and direct deposition. known compounds is often employed to assist in the determi-
These are presented in the order that they were first used.
nation of the evolved gas chemical identity. Search algorithms
generate a listing of reference compounds from the database
1.3 The values stated in SI units are to be regarded as
that are spectroscopically similar to the evolved gas spectrum.
standard. No other units of measurement are included in this
These reference compounds are ranked with regard to a
standard.
measurement of the comparative fit of the reference spectral
1.4 This standard does not purport to address all of the
data to that of the spectrum of the evolved gas.This ranking is
safety concerns, if any, associated with its use. It is the
referred to as the hit quality index (HQI).
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
4. Significance and Use
bility of regulatory limitations prior to use.
4.1 This practice provides general guidelines for the prac-
tice of liquid chromatography or size exclusion chromatogra-
2. Referenced Documents
phycoupledwithinfraredspectrometricdetectionandanalysis
2.1 ASTM Standards:
(LC/IR, SEC/IR). This practice assumes that the chromatogra-
E131Terminology Relating to Molecular Spectroscopy
phy involved is adequate to resolve a sample into discrete
E168Practices for General Techniques of Infrared Quanti-
fractions. It is not the intention of this practice to instruct the
tative Analysis
user on how to perform liquid or size exclusion chromatogra-
E334Practice for General Techniques of Infrared Micro-
phy (LC or SEC).
analysis
5. General LC/IR Techniques
5.1 Three different LC/IR techniques have been used to
This practice is under the jurisdiction ofASTM Committee E13 on Molecular
analyzesamples.Theseconsistofeluenttrapping,flowcelland
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
mittee E13.03 on Infrared and Near Infrared Spectroscopy.
direct deposition. These are presented in the order that they
Current edition approved Nov. 1, 2011. Published December 2011. Originally
were first developed. Infrared detection for any of these
approved in 2000. Last previous edition approved in 2006 as E2106–00(2006).
techniques can be provided by IR monochromators, IR filter
DOI: 10.1520/E2106-00R11.
spectrometers and Fourier transform infrared spectrometers
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard.
(FT-IR).These detectors yield either single absorption band or
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
total infrared spectrum detection modes. Detection mode is
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
dependent upon the type of IR detector employed and the
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. acquisition time required by the LC or SEC experiment.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2106 − 00 (2011)
5.2 Eluent Trapping Techniques—Eluent trapping monochromators and filter infrared spectrometers permit the
techniques, such as stopped flow and fraction collection, are monitoring of a selected absorbance band, for example, 1730
−1
the simple means for obtaining LC/IR data. In these cm forcarbonylfunctionalgroups.Dataacquisitionforthese
techniques, the eluting sample is collected from the chromato- devices is similar to that for a typical LC detector.
graph in discrete aliquots. These aliquots are then analyzed
5.3.2 The transfer line from the LC column to the flowcell
with the appropriate sampling accessory in an infrared spec-
must be made of inert, nonporous material. This normally is
trometer. In utilizing such techniques, it is essential that a
PTFE, PEEK or stainless steel tubing. The volume, internal
suitable LC detector, such as refractive index or UV/VIS, be
diameter, and connections of the transfer line are optimized to
employed to allow definition of component elution. Since the
reduce dead volume and mixing that can degrade the chro-
analyte of interest is trapped physically, the spectrum can be matographic separation. When performing separations at el-
recorded using a long integration or scan coaddition time to
evated temperatures, the transfer line and flowcell may require
improvethesignal-to-noiseratio(SNR).Generally,thestopped controlled heating to maintain temperatures of the eluent.
flow technique requires the use of a flow cell and the IR
5.3.3 The flowcell is made of IR transmissive window
spectrum acquired contains both analyte and mobile phase
materials to give maximum optical throughput to and from the
spectral features. The fraction collection mode permits exami-
effluent chamber. Proper selection of window material is
nation of the eluent as a solution of analyte and mobile phase
necessary to ensure chemical inertness and IR transmissivity.
or, with proper solvent removal, the analyte alone (provided
The cell design and volume must maintain chromatographic
that the analyte is nonvolatile).As such, the fraction collection
resolutionwhilemaximizingopticalinteractionwiththeeluent
mode would require either a liquid cell for solutions or a solid
via transmission, reflection-absorption or attentuated total re-
substrate, that is, KBr window for transmission, first surface
flection modes. Flowcells are typically optimized so that the
mirror for reflection-absorption or powdered KBr for diffuse
sampling volume accommodates the corresponding eluent
reflection measurements.
volume of a sharp chromatographic peak at the peak’s full
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
yields submicrogram detection limits for most analytes (1).
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
chloroform and carbon tetrachloride have sufficient solvent pathlength.Thesereflectionopticalpathspermitlight,whichisretardedto
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
complete infrared spectrum. Such instruments include a com- response, and the ratio of two successive single-beam curves
puter that is capable of storing the large amount of spectro- (as appropriate to the instrument used). For more information
scopic data generated for subsequent evaluation. Conversely, on such tests, refer to Practice E1421. These tests will also
E2106 − 00 (2011)
reveal when a mercury cadmium telluride (MCT) detector is optical effects such as specular reflection which may occur as
performing poorly due to loss of the Dewar vacuum and light passes through the spotted analyte.
consequentbuildupoficeonthedetectorface.Asnotedfurther
5.4.3 Direct deposition techniques provide the advantage of
in this text, an MCT detector is commonly used with these
post-run spectral data acquisition and possibly, decoupling the
experiments as they provide greater detectivity and fast data
chromatographic separation from the spectrometry. Through
acquisition times.
extended co-addition of spectra, the signal-to-noise ratio
5.3.3.3 Care must be taken to stabilize or, preferably, (SNR)ofspectralresultsisimprovedoverthatobtainedduring
removeinterferingspectralfeaturesresultingfromatmospheric real-time data acquisition. It must be noted that slow sublima-
absorptions in the optical beam path of the spectrometer. Best tion of the analyte and recrystallization may occur with direct
results will be obtained by purging the complete optical path deposition techniques. It is prudent to initially obtain the
withdrynitrogengas.Alternatively,dryaircanbeusedforthe spectral data with a short co-addition time to create reference
purge gas, but has interferences in the regions of carbon data to ensure the integrity of spectra obtained with longer
−1 −1
dioxide IR absorption (2500 to 2200 cm and 668 cm ). co-addition times after the chromatographic separation is
Commercially-available air scrubbers that remove water vapor complete.
and carbon dioxide also provide adequate purging of the
spectrometer. In some instruments, the beam path is sealed in 6. Significant Parameters for LC/IR
thepresenceofadesiccant,butinterferencesfrombothcarbon
6.1 The instrumentation used to conduct the LC/IR experi-
−1
dioxide and water vapor (1900 to 1400 cm ) may still be
ment should be properly recorded within prescribed standard
found. In all cases, the instrument atmosphere must be stabi-
operating procedures (SOPs) or laboratory notebooks as nec-
lized before data collection commences.Atmospheric stability
essarytomeetrequirementsforspecificlaboratorypractices.If
inside the instrument can be judged by recording the single-
the equipment is commercially available, the manufacturers’
beam energy response and the ratio of two successive single-
names and model numbers for the complete LC/IR system, or
beam spectra.
the individual components, should be recorded. Additionally,
various instrumental and software parameters are listed and
5.4 Direct Deposition LC/IR—Initial attempts at direct de-
discussedin6.2-6.4.5Anymodificationsmadetoacommercial
position LC/IR employed eluent deposition onto powdered
instrument must be clearly noted.
KC1 (10).After evaporation of the mobile phase, the analysis
of analytes was conducted by diffuse reflection. More recently,
6.2 Instrumental Parameters (IR):
the direct deposition LC/IR technique is accomplished by
6.2.1 Detectors—Due to low optical throughput, most
deposition of the eluent onto a flat, moving surface to allow
LC/IR systems typically employ MCT narrow band photocon-
analysi
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