1、Designation: E 2106 00 (Reapproved 2006)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 E 2106; the number immediately following the designation indicates
2、 the 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 (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice covers techniques that are of ge
3、neral 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 subs
4、equentlyanalyzed by an IR spectroscopic method.1.2 Three different types of LC/IR techniques have beenused to analyze samples (1,2).2These consist of eluent trapping(see Practices E 334), flowcell and direct deposition. These arepresented in the order that they were first used.1.3 The values stated
5、in SI units are to be regarded asstandard.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 health practices and determine the applica-bility of regulator
6、y limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3E 131 Terminology Relating to Molecular SpectroscopyE 168 Practices for General Techniques of Infrared Quanti-tative AnalysisE 334 Practice for General Techniques of Infrared Mi-croanalysisE 1421 Practice for Describing and Measur
7、ing 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 E 131.3.2 Definitions of Terms Specific to This Standard:3.2.1 hit quality index (HQI), nthe comparison of in
8、fraredspectroscopic 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 databasethat are spectroscopically similar to the evolved gas
9、 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 and Use4.1 This practice provides general guidelines f
10、or 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 discretefractions. It is not the intention of this pract
11、ice 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 deposition. These are presented in the order that they
12、were 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 infrared spectrum detection modes. Detection mode isdepend
13、ent upon the type of IR detector employed and theacquisition time required by the LC or SEC experiment.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 coll
14、ected from the chromatograph indiscrete aliquots. These aliquots are then analyzed with the1This practice is under the jurisdiction of ASTM Committee E13 on MolecularSpectroscopy and is the direct responsibility of Subcommittee E13.03 on InfraredSpectroscopy.Current edition approved March 1, 2006. P
15、ublished March 2006. Originallyapproved in 2000. Last previous edition approved in 2000 as E 2106 00.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service a
16、t serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.appropriate sampling accessory in an infrared spe
17、ctrometer. Inutilizing such techniques, it is essential that a suitable LCdetector, such as refractive index or UV/VIS, be employed toallow definition of component elution. Since the analyte ofinterest is trapped physically, the spectrum can be recordedusing a long integration or scan coaddition tim
18、e 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 mobile phase spectralfeatures. The fraction collection mode permits examination ofthe eluent as a solution of analyte and mobile p
19、hase or, withproper solvent removal, the analyte alone (provided that theanalyte is nonvolatile). As such, the fraction collection modewould require either a liquid cell for solutions or a solidsubstrate, that is, KBr window for transmission, first surfacemirror for reflection-absorption or powdered
20、 KBr for diffusereflection measurements.5.3 Flowcell DetectionWith flowcell detection, the LCeluent is monitored continuously in the timeframe of thechromatography (real-time) by the IR spectrometer with theuse of specially designed liquid cells (3-9). Liquid cells aredesigned to minimize dead volum
21、e and analyte mixing, toconserve chromatographic resolution, and achieve maximumoptical interaction of the eluent with the infrared radiation. Asthe effluent is a condensed phase, several cell types have beendevised to accommodate most experimental approaches for IRspectrometry, that is, transmissio
22、n, reflection-absorption andattenuated total reflection (7). The flowcell technique typicallyyields submicrogram detection limits for most analytes (1).Typically, flowcells are mounted within the sample compart-ment of the spectrometer and use beam condensation optics todirect the IR beam into and o
23、ut 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 for the analytes of interest.As such, the choiceof mobile phase may constrain the liquid chromatographicseparation. Generally, thi
24、s limits the chromatographic separa-tion to a normal phase type where nonpolar solvents likechloroform and carbon tetrachloride have sufficient solventstrength to elute components and have low infrared absorption.In contrast, flowcell detection of reversed phase separationsinvolving aqueous mobile p
25、hases are essentially precluded asstrong absorption by water occurs across the mid-infraredspectrum. If flowcell detection of reversed phase separation isto be attenuated, removal of the analytes from the aqueousmobile phase via extraction into an infrared transmissivesolvent is suggested (9).5.3.1
26、The rapidity with which spectra must be recordedduring a liquid chromatographic separation typically requires aFourier-transform infrared (FT-IR) spectrometer to capture thecomplete infrared spectrum. Such instruments include a com-puter that is capable of storing the large amount of spectro-scopic
27、data generated for subsequent evaluation. Conversely,monochromators and filter infrared spectrometers permit themonitoring of a selected absorbance band, for example, 1730cm1for carbonyl functional groups. Data acquisition for thesedevices is similar to that for a typical LC detector.5.3.2 The trans
28、fer 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, and connections of the transfer line are optimized toreduce dead volume and mixing that can degrade the chro-matographic separatio
29、n. 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 flowcell is made of IR transmissive windowmaterials to give maximum optical throughput to and from theeffluent chamber. Proper selec
30、tion of window material isnecessary to ensure chemical inertness and IR transmissivity.The cell design and volume must maintain chromatographicresolution while maximizing optical interaction with the eluentvia transmission, reflection-absorption or attentuated total re-flection modes. Flowcells are
31、typically optimized so that thesampling volume accommodates the corresponding eluentvolume of a sharp chromatographic peak at the peaks fullwidth at half height (FWHH). Typically, this volume ismatched to the scale of the liquid chromatography, that is, 10L for analytical scale and larger volume sep
32、arations 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 bromidewindows; however, this material is susceptible to damage bywater and cold flows under mechanical force.As the flowcell isused, sma
33、ll 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 expectto analyze mixtures containing water should consider usingwindows made of a water-resistant material such as zincselenide (ZnSe).
34、 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,reflectivity, of such materials. Additionally, high refractiveindex materials may cause fringing, that is, create an opticalinterferenc
35、e 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 discretepathlength. These reflection optical paths permit light, which is retarded toa greater extent than light from the transmitt
36、ed optical path, to reach thedetector. This reflection optical path light is out of phase with thetransmitted optical path light and yields interferences fringes in theresultant spectrum. Fringing may be reduced by making the windowsnonparallel or by placing the cell slightly askew, that is, 515, in
37、 theoptical beam of the spectrometer. Please refer to Practices E 168 foradditional information on fringing effects.5.3.3.2 The optical energy throughput of the flowcell shouldbe periodically monitored, since this is a good indicator of theoverall condition of the LC/IR interface. If a Fourier trans
38、formspectrometer 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-beam curves (asappropriate to the instrument used). For more information onsuch tests, refer to Practice E 1421. These tests will
39、 also revealwhen a mercury cadmium telluride (MCT) detector is perform-ing poorly due to loss of the Dewar vacuum and consequentbuildup of ice on the detector face.As noted further in this text,E 2106 00 (2006)2an MCT detector is commonly used with these experiments asthey provide greater detectivit
40、y 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 optical beam path of the spectrometer. Bestresults will be obtained by purging the complete optical pathwith dry nitrogen gas.Alt
41、ernatively, 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 scrubbers that remove water vaporand carbon dioxide also provide adequate purging of thespectrometer. In some instruments, th
42、e 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 instrument atmosphere must be stabi-lized before data collection commences. Atmospheric stabilityinside the instrument can be judged
43、by recording the single-beam energy response and the ratio of two successive single-beam spectra.5.4 Direct Deposition LC/IRInitial attempts at directdeposition LC/IR employed eluent deposition onto powderedKC1 (10). After evaporation of the mobile phase, the analysisof analytes was conducted by dif
44、fuse reflection. More recently,the direct deposition LC/IR technique is accomplished bydeposition of the eluent onto a flat, moving surface to allowanalysis by transmission or reflection-absorption (11,12).Inthese methods, the eluent is passed through a nebulizer toatomize the mobile phase, the aero
45、sol 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 detect as low as subnanogramamounts of material. By capturing the eluent onto a substrate,the components of the sample are effe
46、ctively trapped. It ispossible, therefore, to analyze the chromatographic distributionof analytes after the LC/IR experiment as well as to performanalyses in real-time.5.4.1 For transmission spectra, the eluent is deposited di-rectly onto an infrared transmissive plate maintained at atemperature suf
47、ficient to permit further evaporation of themobile phase (11). Infrared spectra are then obtained via aninfrared transmission method.5.4.2 For reflection absorption measurements, the eluent isdeposited upon a front surface mirror. The infrared beam isthen transmitted through the analyte, reflected o
48、ff 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. The back surface ofthis wafer is vapor coated with aluminum to yield a reflectivesurface (12). As germanium is IR transmissive,
49、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 and possibly, decoupling thechromatographic separation from the spectrometry. Throughextended co-addition of spectra, the signal-to
