1、Designation: E2106 00 (Reapproved 2011)Standard Practice forGeneral Techniques of Liquid Chromatography-Infrared (LC/IR) and Size Exclusion Chromatography-Infrared (SEC/IR)Analyses1This standard is issued under the fixed designation E2106; the number immediately following the designation indicates t
2、he year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice covers techniques that are of gener
3、al use inqualitatively analyzing multicomponent samples by using acombination of liquid chromatography (LC) or size exclusionchromatography (SEC) with infrared (IR) spectrometric tech-niques. The sample mixture is separated into fractions by thechromatographic separation. These fractions are subsequ
4、entlyanalyzed by an IR spectroscopic method.1.2 Three different types of LC/IR techniques have beenused to analyze samples (1, 2).2These consist of eluenttrapping (see Practices E334), flowcell and direct deposition.These are presented in the order that they were first used.1.3 The values stated in
5、SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and healt
6、h practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3E131 Terminology Relating to Molecular SpectroscopyE168 Practices for General Techniques of Infrared Quanti-tative AnalysisE334 Practice for General Techniques of Infrared M
7、icro-analysisE1421 Practice for Describing and Measuring Performanceof Fourier Transform Mid-Infrared (FT-MIR) Spectrom-eters: Level Zero and Level One Tests3. Terminology3.1 DefinitionsFor definitions of terms and symbols, referto Terminology E131.3.2 Definitions of Terms Specific to This Standard:
8、3.2.1 hit quality index (HQI), nthe comparison of infraredspectroscopic data against a database of reference spectra ofknown compounds is often employed to assist in the determi-nation of the evolved gas chemical identity. Search algorithmsgenerate a listing of reference compounds from the databaset
9、hat are spectroscopically similar to the evolved gas spectrum.These reference compounds are ranked with regard to ameasurement of the comparative fit of the reference spectraldata to that of the spectrum of the evolved gas. This ranking isreferred to as the hit quality index (HQI).4. Significance an
10、d Use4.1 This practice provides general guidelines for the prac-tice of liquid chromatography or size exclusion chromatogra-phy coupled with infrared spectrometric detection and analysis(LC/IR, SEC/IR). This practice assumes that the chromatogra-phy involved is adequate to resolve a sample into disc
11、retefractions. It is not the intention of this practice to instruct theuser on how to perform liquid or size exclusion chromatogra-phy (LC or SEC).5. General LC/IR Techniques5.1 Three different LC/IR techniques have been used toanalyze samples. These consist of eluent trapping, flowcell anddirect de
12、position. These are presented in the order that theywere first developed. Infrared detection for any of thesetechniques can be provided by IR monochromators, IR filterspectrometers and Fourier transform infrared spectrometers(FT-IR). These detectors yield either single absorption band ortotal infrar
13、ed spectrum detection modes. Detection mode isdependent upon the type of IR detector employed and theacquisition time required by the LC or SEC experiment.1This practice is under the jurisdiction of ASTM Committee E13 on MolecularSpectroscopy and Separation Science and is the direct responsibility o
14、f Subcom-mittee E13.03 on Infrared and Near Infrared Spectroscopy.Current edition approved Nov. 1, 2011. Published December 2011. Originallyapproved in 2000. Last previous edition approved in 2006 as E2106 00 (2006).DOI: 10.1520/E2106-00R11.2The boldface numbers in parentheses refer to the list of r
15、eferences at the end ofthis standard.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM Internat
16、ional, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.2 Eluent Trapping TechniquesEluent trapping tech-niques, such as stopped flow and fraction collection, are thesimple means for obtaining LC/IR data. In these techniques,the eluting sample is collected from t
17、he chromatograph indiscrete aliquots. These aliquots are then analyzed with theappropriate sampling accessory in an infrared spectrometer. Inutilizing such techniques, it is essential that a suitable LCdetector, such as refractive index or UV/VIS, be employed toallow definition of component elution.
18、 Since the analyte ofinterest is trapped physically, the spectrum can be recordedusing a long integration or scan coaddition time to improve thesignal-to-noise ratio (SNR). Generally, the stopped flow tech-nique requires the use of a flow cell and the IR spectrumacquired contains both analyte and mo
19、bile phase spectralfeatures. The fraction collection mode permits examination ofthe eluent as a solution of analyte and mobile phase or, withproper solvent removal, the analyte alone (provided that theanalyte is nonvolatile). As such, the fraction collection modewould require either a liquid cell fo
20、r solutions or a solidsubstrate, that is, KBr window for transmission, first surfacemirror for reflection-absorption or powdered KBr for diffusereflection measurements.5.3 Flowcell DetectionWith flowcell detection, the LCeluent is monitored continuously in the timeframe of thechromatography (real-ti
21、me) by the IR spectrometer with theuse of specially designed liquid cells (3-9). Liquid cells aredesigned to minimize dead volume and analyte mixing, toconserve chromatographic resolution, and achieve maximumoptical interaction of the eluent with the infrared radiation. Asthe effluent is a condensed
22、 phase, several cell types have beendevised to accommodate most experimental approaches for IRspectrometry, that is, transmission, reflection-absorption andattenuated total reflection (7). The flowcell technique typicallyyields submicrogram detection limits for most analytes (1).Typically, flowcells
23、 are mounted within the sample compart-ment of the spectrometer and use beam condensation optics todirect the IR beam into and out of the small volume of the cell.It is important to employ a mobile phase having low orpreferably no infrared absorptions in the analytically importantspectral regions fo
24、r the analytes of interest.As such, the choiceof mobile phase may constrain the liquid chromatographicseparation. Generally, this limits the chromatographic separa-tion to a normal phase type where nonpolar solvents likechloroform and carbon tetrachloride have sufficient solventstrength to elute com
25、ponents and have low infrared absorption.In contrast, flowcell detection of reversed phase separationsinvolving aqueous mobile phases are essentially precluded asstrong absorption by water occurs across the mid-infraredspectrum. If flowcell detection of reversed phase separation isto be attenuated,
26、removal of the analytes from the aqueousmobile phase via extraction into an infrared transmissivesolvent is suggested (9).5.3.1 The rapidity with which spectra must be recordedduring a liquid chromatographic separation typically requires aFourier-transform infrared (FT-IR) spectrometer to capture th
27、ecomplete infrared spectrum. Such instruments include a com-puter that is capable of storing the large amount of spectro-scopic data generated for subsequent evaluation. Conversely,monochromators and filter infrared spectrometers permit themonitoring of a selected absorbance band, for example, 1730c
28、m1for carbonyl functional groups. Data acquisition for thesedevices is similar to that for a typical LC detector.5.3.2 The transfer line from the LC column to the flowcellmust be made of inert, nonporous material. This normally isPTFE, PEEK or stainless steel tubing. The volume, internaldiameter, an
29、d connections of the transfer line are optimized toreduce dead volume and mixing that can degrade the chro-matographic separation. When performing separations at el-evated temperatures, the transfer line and flowcell may requirecontrolled heating to maintain temperatures of the eluent.5.3.3 The flow
30、cell is made of IR transmissive windowmaterials to give maximum optical throughput to and from theeffluent chamber. Proper selection of window material isnecessary to ensure chemical inertness and IR transmissivity.The cell design and volume must maintain chromatographicresolution while maximizing o
31、ptical interaction with the eluentvia transmission, reflection-absorption or attentuated total re-flection modes. Flowcells are typically optimized so that thesampling volume accommodates the corresponding eluentvolume of a sharp chromatographic peak at the peaks fullwidth at half height (FWHH). Typ
32、ically, this volume ismatched to the scale of the liquid chromatography, that is, 10L for analytical scale and larger volume separations and lessthan 10 L for microbore separations.5.3.3.1 The optimum infrared transmission across the fullmid-infrared spectrum is obtained by using potassium bromidewi
33、ndows; however, this material is susceptible to damage bywater and cold flows under mechanical force.As the flowcell isused, small amounts of water will etch the window surfaces,and the optical throughput of the windows will drop. Eventu-ally, these windows will have to be changed. Users who expectt
34、o analyze mixtures containing water should consider usingwindows made of a water-resistant material such as zincselenide (ZnSe). IR windows of high refractive index like ZnSeand zinc sulfide (ZnS) will result in a noticeable drop ininfrared transmission due to the optical properties, that is,reflect
35、ivity, of such materials. Additionally, high refractiveindex materials may cause fringing, that is, create an opticalinterference pattern in the baseline of the IR spectrum.NOTE 1Fringing is due to multiple reflection optical paths createdwhen windows are placed as parallel plates separated by a dis
36、cretepathlength. These reflection optical paths permit light, which is retarded toa greater extent than light from the transmitted optical path, to reach thedetector. This reflection optical path light is out of phase with thetransmitted optical path light and yields interferences fringes in theresu
37、ltant spectrum. Fringing may be reduced by making the windowsnonparallel or by placing the cell slightly askew, that is, 515, in theoptical beam of the spectrometer. Please refer to Practices E168 foradditional information on fringing effects.5.3.3.2 The optical energy throughput of the flowcell sho
38、uldbe periodically monitored, since this is a good indicator of theoverall condition of the LC/IR interface. If a Fourier transformspectrometer is used, it is recommended that records be kept ofthe interferogram signal strength, single-beam energy re-sponse, and the ratio of two successive single-be
39、am curves (asappropriate to the instrument used). For more information onsuch tests, refer to Practice E1421. These tests will also revealE2106 00 (2011)2when a mercury cadmium telluride (MCT) detector is perform-ing poorly due to loss of the Dewar vacuum and consequentbuildup of ice on the detector
40、 face.As noted further in this text,an MCT detector is commonly used with these experiments asthey provide greater detectivity and fast data acquisition times.5.3.3.3 Care must be taken to stabilize or, preferably,remove interfering spectral features resulting from atmosphericabsorptions in the opti
41、cal beam path of the spectrometer. Bestresults will be obtained by purging the complete optical pathwith dry nitrogen gas.Alternatively, dry air can be used for thepurge gas, but has interferences in the regions of carbondioxide IR absorption (2500 to 2200 cm1and 668 cm1).Commercially-available air
42、scrubbers that remove water vaporand carbon dioxide also provide adequate purging of thespectrometer. In some instruments, the beam path is sealed inthe presence of a desiccant, but interferences from both carbondioxide and water vapor (1900 to 1400 cm1) may still befound. In all cases, the instrume
43、nt atmosphere must be stabi-lized before data collection commences. Atmospheric stabilityinside the instrument can be judged by recording the single-beam energy response and the ratio of two successive single-beam spectra.5.4 Direct Deposition LC/IRInitial attempts at direct de-position LC/IR employ
44、ed eluent deposition onto powderedKC1 (10). After evaporation of the mobile phase, the analysisof analytes was conducted by diffuse reflection. More recently,the direct deposition LC/IR technique is accomplished bydeposition of the eluent onto a flat, moving surface to allowanalysis by transmission
45、or reflection-absorption (11, 12).Inthese methods, the eluent is passed through a nebulizer toatomize the mobile phase, the aerosol is passed through aheated transfer zone to evaporate the mobile phase and theresidue is deposited onto an appropriate optical substrate. Thisallows for these methods to
46、 detect as low as subnanogramamounts of material. By capturing the eluent onto a substrate,the components of the sample are effectively trapped. It ispossible, therefore, to analyze the chromatographic distributionof analytes after the LC/IR experiment as well as to performanalyses in real-time.5.4.
47、1 For transmission spectra, the eluent is deposited di-rectly onto an infrared transmissive plate maintained at atemperature sufficient to permit further evaporation of themobile phase (11). Infrared spectra are then obtained via aninfrared transmission method.5.4.2 For reflection absorption measure
48、ments, the eluent isdeposited upon a front surface mirror. The infrared beam isthen transmitted through the analyte, reflected off the mirrorsurface and transmitted back through the analyte. A modifica-tion of this method has been introduced where the eluent isdeposited upon a thin germanium wafer.
49、The back surface ofthis wafer is vapor coated with aluminum to yield a reflectivesurface (12). As germanium is IR transmissive, the beampasses through the deposited analyte twice and, dependingupon the angle of incidence and reflection, yields an approxi-mate doubling of the pathlength. The advantage of thisapproach over that of a first surface mirror is to reduce spuriousoptical effects such as specular reflection which may occur aslight passes through the spotted analyte.5.4.3 Direct deposition techniques provide the advantage ofpost-run spectral data acquisition an
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