ASTM E2106-00

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Designation:E2106–00
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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope E1421 PracticeforDescribingandMeasuringPerformance
of Fourier Transform Infrared (FT-IR) Spectrometers:
1.1 Thispracticecoverstechniquesthatareofgeneralusein
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-
3.1 Definitions—Fordefinitionsoftermsandsymbols,refer
niques. The sample mixture is separated into fractions by the
to Terminology E131.
chromatographic separation. These fractions are subsequently
3.2 Definitions of Terms Specific to This Standard:
analyzed by an IR spectroscopic method.
3.2.1 hit quality index (HQI), n—thecomparisonofinfrared
1.2 Three different types of LC/IR techniques have been
2 spectroscopic data against a database of reference spectra of
usedtoanalyzesamples(1,2). Theseconsistofeluenttrapping
known compounds is often employed to assist in the determi-
(seePracticesE334),flowcellanddirectdeposition.Theseare
nation of the evolved gas chemical identity. Search algorithms
presented in the order that they were first used.
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.
These reference compounds are ranked with regard to a
1.4 This standard does not purport to address all of the
measurement of the comparative fit of the reference spectral
safety concerns, if any, associated with its use. It is the
data to that of the spectrum of the evolved gas.This ranking is
responsibility of the user of this standard to establish appro-
referred to as the hit quality index (HQI).
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
4. Significance and Use
4.1 This practice provides general guidelines for the prac-
2. Referenced Documents
tice of liquid chromatography or size exclusion chromatogra-
2.1 ASTM Standards:
3 phycoupledwithinfraredspectrometricdetectionandanalysis
E131 Terminology Relating to Molecular Spectroscopy
(LC/IR, SEC/IR). This practice assumes that the chromatogra-
E168 Practices for General Techniques of Infrared Quanti-
3 phy involved is adequate to resolve a sample into discrete
tative Analysis
fractions. It is not the intention of this practice to instruct the
E334 Practices for General Techniques of Infrared Mi-
3 user on how to perform liquid or size exclusion chromatogra-
croanalysis
phy (LC or SEC).
E355 Practices for Gas Chromatography Terms and Rela-
tionships
5. General LC/IR Techniques
E932 Practice for Describing and Measuring Performance
3 5.1 Three different LC/IR techniques have been used to
of Dispersive Infrared Spectrometers
analyzesamples.Theseconsistofeluenttrapping,flowcelland
E1252 Practice for General Techniques for Qualitative
3 direct deposition. These are presented in the order that they
Infrared Analysis
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
Spectroscopy and is the direct responsibility of Subcommittee E13.03 on Infrared
(FT-IR).These detectors yield either single absorption band or
Spectroscopy.
total infrared spectrum detection modes. Detection mode is
Current edition approved Sept. 10, 2000. Published November 2000.
dependent upon the type of IR detector employed and the
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard.
acquisition time required by the LC or SEC experiment.
Annual Book of ASTM Standards, Vol 03.06.
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E2106
5.2 Eluent Trapping Techniques—Eluent trapping tech- monochromators and filter infrared spectrometers permit the
niques, such as stopped flow and fraction collection, are the monitoring of a selected absorbance band, for example, 1730
−1
cm forcarbonylfunctionalgroups.Dataacquisitionforthese
simple means for obtaining LC/IR data. In these techniques,
the eluting sample is collected from the chromatograph in devices is similar to that for a typical LC detector.
discrete aliquots. These aliquots are then analyzed with the 5.3.2 The transfer line from the LC column to the flowcell
appropriate sampling accessory in an infrared spectrometer. In must be made of inert, nonporous material. This normally is
PTFE, PEEK or stainless steel tubing. The volume, internal
utilizing such techniques, it is essential that a suitable LC
detector, such as refractive index or UV/VIS, be employed to diameter, and connections of the transfer line are optimized to
reduce dead volume and mixing that can degrade the chro-
allow definition of component elution. Since the analyte of
matographic separation. When performing separations at el-
interest is trapped physically, the spectrum can be recorded
evated temperatures, the transfer line and flowcell may require
usingalongintegrationorscancoadditiontimetoimprovethe
controlled heating to maintain temperatures of the eluent.
signal-to-noise ratio (SNR). Generally, the stopped flow tech-
nique requires the use of a flow cell and the IR spectrum 5.3.3 The flowcell is made of IR transmissive window
materials to give maximum optical throughput to and from the
acquired contains both analyte and mobile phase spectral
features. The fraction collection mode permits examination of effluent chamber. Proper selection of window material is
necessary to ensure chemical inertness and IR transmissivity.
the eluent as a solution of analyte and mobile phase or, with
The cell design and volume must maintain chromatographic
proper solvent removal, the analyte alone (provided that the
resolutionwhilemaximizingopticalinteractionwiththeeluent
analyte is nonvolatile). As such, the fraction collection mode
via transmission, reflection-absorption or attentuated total re-
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. Eventu-
spectrometry, that is, transmission, reflection-absorption and
ally,thesewindowswillhavetobechanged.Userswhoexpect
attenuated total reflection (7). The flowcell technique typically
to analyze mixtures containing water should consider using
yields submicrogram detection limits for most analytes (1).
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
when windows are placed as parallel plates separated by a discrete
tion to a normal phase type where nonpolar solvents like
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 re-
complete infrared spectrum. Such instruments include a com- sponse, and the ratio of two successive single-beam curves (as
puter that is capable of storing the large amount of spectro- appropriate to the instrument used). For more information on
scopic data generated for subsequent evaluation. Conversely, suchtests,refertoPracticeE1421.Thesetestswillalsoreveal
E2106
whenamercurycadmiumtelluride(MCT)detectorisperform- 5.4.3 Direct deposition techniques provide the advantage of
ing poorly due to loss of the Dewar vacuum and consequent post-run spectral data acquisition and possibly, decoupling the
buildupoficeonthedetectorface.Asnotedfurtherinthistext, chromatographic separation from the spectrometry. Through
an MCT detector is commonly used with these experiments as extended co-addition of spectra, the signal-to-noise ratio
(SNR)ofspectralresultsisimprovedoverthatobtainedduring
theyprovidegreaterdetectivityandfastdataacquisitiontimes.
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
6.1 The instrumentation used to conduct the LC/IR experi-
spectrometer. In some instruments, the beam path is sealed in
ment should be properly recorded within prescribed standard
thepresenceofadesiccant,butinterferencesfrombothcarbon
operating procedures (SOPs) or laboratory notebooks as nec-
−1
dioxide and water vapor (1900 to 1400 cm ) may still be
essarytomeetrequirementsforspecificlaboratorypractices.If
found. In all cases, the instrument atmosphere must be stabi-
the equipment is commercially available, the manufacturers’
lized before data collection commences.Atmospheric stability
names and model numbers for the complete LC/IR system, or
inside the instrument can be judged by recording the single-
the individual components, should be recorded. Additionally,
beam energy response and the ratio of two successive single-
various instrumental and software parameters are listed and
beam spectra.
discussedin6.2-6.4.5Anymodificationsmadetoacommercial
5.4 Direct Deposition LC/IR—Initial attempts at direct
instrument must be clearly noted.
deposition LC/IR employed eluent deposition onto powdered
6.2 Instrumental Parameters (IR):
KC1 (10).After evaporation of the mobile phase, the analysis
6.2.1 Detectors—Due to low optical throughput, most
of analytes was conducted by diffuse reflection. More recently,
LC/IR systems typically employ MCT narrow band photocon-
the direct deposition LC/IR technique is accomplished by
ductive detectors. It is important that the detector element be
deposition of the eluent onto a flat, moving surface to allow
properly filled with the image of the analyte spot or image of
analysis by transmission or reflection-absorption (11,12).In
the exit aperture of the interface to achieve the highest
these methods, the eluent is passed through a nebulizer to
signal-to-noise. Additionally, care must taken to ensure the
atomize the mobile phase, t
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