ASTM E2106-00(2006)
(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 E 334), 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.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:E2106–00(Reapproved 2006)
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 3. Terminology
1.1 Thispracticecoverstechniquesthatareofgeneralusein 3.1 Definitions—Fordefinitionsoftermsandsymbols,refer
qualitatively analyzing multicomponent samples by using a to Terminology E131.
combination of liquid chromatography (LC) or size exclusion 3.2 Definitions of Terms Specific to This Standard:
chromatography (SEC) with infrared (IR) spectrometric tech- 3.2.1 hit quality index (HQI), n—thecomparisonofinfrared
niques. The sample mixture is separated into fractions by the spectroscopic data against a database of reference spectra of
chromatographic separation. These fractions are subsequently known compounds is often employed to assist in the determi-
analyzed by an IR spectroscopic method. nation of the evolved gas chemical identity. Search algorithms
1.2 Three different types of LC/IR techniques have been generate a listing of reference compounds from the database
usedtoanalyzesamples(1,2). Theseconsistofeluenttrapping that are spectroscopically similar to the evolved gas spectrum.
(see Practices E334), flowcell and direct deposition. These are These reference compounds are ranked with regard to a
presented in the order that they were first used. measurement of the comparative fit of the reference spectral
1.3 The values stated in SI units are to be regarded as data to that of the spectrum of the evolved gas.This ranking is
standard. referred to as the hit quality index (HQI).
1.4 This standard does not purport to address all of the
4. Significance and Use
safety concerns, if any, associated with its use. It is the
4.1 This practice provides general guidelines for the prac-
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica- tice of liquid chromatography or size exclusion chromatogra-
bility of regulatory limitations prior to use. 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
E131 Terminology Relating to Molecular Spectroscopy user on how to perform liquid or size exclusion chromatogra-
E168 Practices for General Techniques of Infrared Quanti- phy (LC or SEC).
tative Analysis
5. General LC/IR Techniques
E334 Practice for General Techniques of Infrared Micro-
5.1 Three different LC/IR techniques have been used to
analysis
E1421 Practice for Describing and Measuring Performance analyzesamples.Theseconsistofeluenttrapping,flowcelland
direct deposition. These are presented in the order that they
of Fourier Transform Mid-Infrared (FT-MIR) Spectrom-
eters: Level Zero and Level One Tests were first developed. Infrared detection for any of these
techniques can be provided by IR monochromators, IR filter
spectrometers and Fourier transform infrared spectrometers
This practice is under the jurisdiction ofASTM Committee E13 on Molecular
(FT-IR).These detectors yield either single absorption band or
Spectroscopy and is the direct responsibility of Subcommittee E13.03 on Infrared
total infrared spectrum detection modes. Detection mode is
Spectroscopy.
Current edition approved March 1, 2006. Published March 2006. Originally
dependent upon the type of IR detector employed and the
approved in 2000. Last previous edition approved in 2000 as E2106–00. DOI:
acquisition time required by the LC or SEC experiment.
10.1520/E2106-00R06.
2 5.2 Eluent Trapping Techniques—Eluent trapping tech-
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. niques, such as stopped flow and fraction collection, are the
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
simple means for obtaining LC/IR data. In these techniques,
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
the eluting sample is collected from the chromatograph in
Standards volume information, refer to the standard’s Document Summary page on
discrete aliquots. These aliquots are then analyzed with the
the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2106–00 (2006)
appropriate sampling accessory in an infrared spectrometer. In 5.3.2 The transfer line from the LC column to the flowcell
utilizing such techniques, it is essential that a suitable LC must be made of inert, nonporous material. This normally is
detector, such as refractive index or UV/VIS, be employed to
PTFE, PEEK or stainless steel tubing. The volume, internal
allow definition of component elution. Since the analyte of diameter, and connections of the transfer line are optimized to
interest is trapped physically, the spectrum can be recorded
reduce dead volume and mixing that can degrade the chro-
usingalongintegrationorscancoadditiontimetoimprovethe
matographic separation. When performing separations at el-
signal-to-noise ratio (SNR). Generally, the stopped flow tech-
evated temperatures, the transfer line and flowcell may require
nique requires the use of a flow cell and the IR spectrum
controlled heating to maintain temperatures of the eluent.
acquired contains both analyte and mobile phase spectral
5.3.3 The flowcell is made of IR transmissive window
features. The fraction collection mode permits examination of
materials to give maximum optical throughput to and from the
the eluent as a solution of analyte and mobile phase or, with
effluent chamber. Proper selection of window material is
proper solvent removal, the analyte alone (provided that the
necessary to ensure chemical inertness and IR transmissivity.
analyte is nonvolatile). As such, the fraction collection mode
The cell design and volume must maintain chromatographic
would require either a liquid cell for solutions or a solid
resolutionwhilemaximizingopticalinteractionwiththeeluent
substrate, that is, KBr window for transmission, first surface
via transmission, reflection-absorption or attentuated total re-
mirror for reflection-absorption or powdered KBr for diffuse
flection modes. Flowcells are typically optimized so that the
reflection measurements.
sampling volume accommodates the corresponding eluent
5.3 Flowcell Detection—With flowcell detection, the LC
volume of a sharp chromatographic peak at the peak’s full
eluent is monitored continuously in the timeframe of the
width at half height (FWHH). Typically, this volume is
chromatography (real-time) by the IR spectrometer with the
matched to the scale of the liquid chromatography, that is, 10
use of specially designed liquid cells (3-9). Liquid cells are
µL for analytical scale and larger volume separations and less
designed to minimize dead volume and analyte mixing, to than 10 µL for microbore separations.
conserve chromatographic resolution, and achieve maximum
5.3.3.1 The optimum infrared transmission across the full
optical interaction of the eluent with the infrared radiation.As
mid-infraredspectrumisobtainedbyusingpotassiumbromide
the effluent is a condensed phase, several cell types have been
windows; however, this material is susceptible to damage by
devised to accommodate most experimental approaches for IR
waterandcoldflowsundermechanicalforce.Astheflowcellis
spectrometry, that is, transmission, reflection-absorption and used, small amounts of water will etch the window surfaces,
attenuated total reflection (7). The flowcell technique typically and the optical throughput of the windows will drop. Eventu-
ally,thesewindowswillhavetobechanged.Userswhoexpect
yields submicrogram detection limits for most analytes (1).
to analyze mixtures containing water should consider using
Typically, flowcells are mounted within the sample compart-
ment of the spectrometer and use beam condensation optics to windows made of a water-resistant material such as zinc
selenide(ZnSe).IRwindowsofhighrefractiveindexlikeZnSe
directtheIRbeamintoandoutofthesmallvolumeofthecell.
and zinc sulfide (ZnS) will result in a noticeable drop in
It is important to employ a mobile phase having low or
infrared transmission due to the optical properties, that is,
preferablynoinfraredabsorptionsintheanalyticallyimportant
reflectivity, of such materials. Additionally, high refractive
spectralregionsfortheanalytesofinterest.Assuch,thechoice
index materials may cause fringing, that is, create an optical
of mobile phase may constrain the liquid chromatographic
interference pattern in the baseline of the IR spectrum.
separation. Generally, this limits the chromatographic separa-
tion to a normal phase type where nonpolar solvents like
NOTE 1—Fringing is due to multiple reflection optical paths created
chloroform and carbon tetrachloride have sufficient solvent
when windows are placed as parallel plates separated by a discrete
strengthtoelutecomponentsandhavelowinfraredabsorption.
pathlength.Thesereflectionopticalpathspermitlight,whichisretardedto
a greater extent than light from the transmitted optical path, to reach the
In contrast, flowcell detection of reversed phase separations
detector. This reflection optical path light is out of phase with the
involving aqueous mobile phases are essentially precluded as
transmitted optical path light and yields interferences fringes in the
strong absorption by water occurs across the mid-infrared
resultant spectrum. Fringing may be reduced by making the windows
spectrum. If flowcell detection of reversed phase separation is
nonparallel or by placing the cell slightly askew, that is, 5–15°, in the
to be attenuated, removal of the analytes from the aqueous
optical beam of the spectrometer. Please refer to Practices E168 for
mobile phase via extraction into an infrared transmissive additional information on fringing effects.
solvent is suggested (9).
5.3.3.2 Theopticalenergythroughputoftheflowcellshould
5.3.1 The rapidity with which spectra must be recorded
be periodically monitored, since this is a good indicator of the
duringaliquidchromatographicseparationtypicallyrequiresa
overallconditionoftheLC/IRinterface.IfaFouriertransform
Fourier-transform infrared (FT-IR) spectrometer to capture the
spectrometerisused,itisrecommendedthatrecordsbekeptof
complete infrared spectrum. Such instruments include a com-
the interferogram signal strength, single-beam energy re-
puter that is capable of storing the large amount of spectro-
sponse, and the ratio of two successive single-beam curves (as
scopic data generated for subsequent evaluation. Conversely,
appropriate to the instrument used). For more information on
monochromators and filter infrared spectrometers permit the
such tests, refer to Practice E1421. These tests will also reveal
monitoring of a selected absorbance band, for example, 1730
whenamercurycadmiumtelluride(MCT)detectorisperform-
−1
cm forcarbonylfunctionalgroups.Dataacquisitionforthese
ing poorly due to loss of the Dewar vacuum and consequent
devices is similar to that for a typical LC detector. buildupoficeonthedetectorface.Asnotedfurtherinthistext,
E2106–00 (2006)
an MCT detector is commonly used with these experiments as extended co-addition of spectra, the signal-to-noise ratio
theyprovidegreaterdetectivityandfastdataacquisitiontimes. (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 parameters are listed and
5.4 Direct Deposition LC/IR—Initial attempts at direct
discussedin6.2-6.4.5Anymodificationsmadetoacommercial
deposition LC/IR employed eluent deposition onto powdered instrument must be clearly noted.
KC1 (10).After evaporation of the mobile phase, the analysis
6.2 Instrumental Parameters (IR):
of analytes was conducted by diffuse reflection. More recently,
6.2.1 Detectors—Due to low optical throughput, most
the direct deposition LC/IR technique is accomplished by
LC/IR systems typically employ MCT narrow band photocon-
deposition of the eluent onto a flat, moving surface to allow
ductive detectors. It is important that the detector element be
analysis by transmission or reflection-absorption (11,12).In
properly filled with the image of the analyte spot or image of
these methods, the eluent is passed through a nebulizer to
the exit aperture of the interface to achieve the highest
atomize the mobile phase, the aerosol is passed through a
signal-to-noise. Additionally, care must taken to ensure the
heated transfer zone
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