1、Designation: E 685 93 (Reapproved 2005)Standard Practice forTesting Fixed-Wavelength Photometric Detectors Used inLiquid Chromatography1This standard is issued under the fixed designation E 685; the number immediately following the designation indicates the year oforiginal adoption or, in the case o
2、f 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 is intended to serve as a guide for thetesting of the performance of a photomet
3、ric detector (PD) usedas the detection component of a liquid-chromatographic (LC)system operating at one or more fixed wavelengths in the range210 to 800 nm. Measurements are made at 254 nm, if possible,and are optional at other wavelengths.1.2 This practice is intended to describe the performance o
4、fthe detector both independently of the chromatographic system(static conditions) and with flowing solvent (dynamic condi-tions).1.3 For general liquid chromatographic procedures, consultRefs (1-9).21.4 For general information concerning the principles, con-struction, operation, and evaluation of li
5、quid-chromatographydetectors, see Refs (10 and 11) in addition to the sectionsdevoted to detectors in Refs (1-7).1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 This standard does not purport to address all of thesafety
6、problems, 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 regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3E 275 Practice for Describing and M
7、easuring Performanceof Ultraviolet, Visible, and Near-Infrared Spectrophotom-etersE 682 Practice for Liquid Chromatography Terms and Re-lationships3. Terminology3.1 Definitions:3.1.1 absorbance calibration, nthe procedure that verifiesthat the absorbance scale is correct within 65%.3.1.2 drift, nthe
8、 average slope of the noise envelopeexpressed in absorbance units per hour (AU/h) as measuredover a period of 1 h.3.1.3 dynamic, nunder conditions of a flow rate of 1.0mL/min.3.1.4 linear range, nofaPD, the range of concentrationsof a test substance in a mobile phase over which the responseof the de
9、tector is constant to within 5 % as determined from thelinearity plot specified below and illustrated in Fig. 1. Thelinear range should be expressed as the ratio of the highestconcentration to the minimum detectable concentration or thelowest linear concentration, whichever is greatest.3.1.5 long-te
10、rm noise, nthe maximum amplitude in AUfor all random variations of the detector signal of frequenciesbetween 6 and 60 cycles per hour (0.1 and 1.0 cycles per min).3.1.5.1 DiscussionIt represents noise that can be mistakenfor a late-eluting peak. This noise corresponds to the observednoise only and m
11、ay not always be present.3.1.6 minimum detectability, nofaPD, that concentrationof a specific solute in a specific solvent that results in a detectorresponse corresponding to twice the static short-term noise.3.1.7 response time (speed of output), nthe detector, thetime required for the detector out
12、put to change from 10 to 90 %of the new equilibrium value when the composition of themobile phase is changed in a stepwise manner, within the linearrange of the detector.3.1.7.1 DiscussionBecause the detector volume is verysmall and the transport rate is not diffusion dependent, theresponse time is
13、generally fast enough to be unimportant. It isgenerally comparable to the response time of the recorder anddependent on the response time of the detector electrometer1This practice is under the jurisdiction of ASTM Committee E13 on MolecularSpectroscopy and is the direct responsibility of Subcommitt
14、ee E13.19 on Chroma-tography.Current edition approved Sept. 1, 2005. Published September 2005. Originallyapproved in 1979. Last previous edition approved in 2000 as E 685 93 (2000).2The boldface numbers in parentheses refer to the list of references at the end ofthis practice.3For referenced ASTM st
15、andards, 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 International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken
16、, PA 19428-2959, United States.and on the recorder amplifier. Factors that affect the observedresponse time include the true detector response time, elec-tronic filtering, and system band-broadening.3.1.8 short-term noise, nthe maximum amplitude, peak topeak, in AU for all random variations of the d
17、etector signal ofa frequency greater than one cycle per minute.3.1.8.1 DiscussionIt determines the smallest signal de-tectable by a PD, limits the precision attainable in quantitationof trace-level samples, and sets the lower limit on linearity.This noise corresponds to the observed noise only.3.1.9
18、 static, nunder conditions of no flow.4. Significance and Use4.1 Although it is possible to observe and measure each ofthe several characteristics of a detector under different andunique conditions, it is the intent of this practice that acomplete set of detector specifications should be obtainedund
19、er the same operating conditions. It should also be notedthat to completely specify a detectors capability, its perfor-mance should be measured at several sets of conditions withinthe useful range of the detector. The terms and tests describedin this practice are sufficiently general that they may b
20、e usedregardless of the ultimate operating parameters.4.2 Linearity and response time of the recorder or otherreadout device used should be such that they do not distort orotherwise interfere with the performance of the detector. Thisrequires adjusting the gain, damping, and calibration in accor-dan
21、ce with the manufacturers directions. If additional elec-tronic filters or amplifiers are used between the detector and thefinal readout device, their characteristics should also first beestablished.5. Noise and Drift5.1 Test ConditionsPure, degassed methanol of suitablegrade4shall be used in the sa
22、mple cell. Air or nitrogen shall beused in the reference cell if there is one. Nitrogen is preferredwhere the presence of high-voltage equipment makes it likelythat there is ozone in the air. Protect the entire system fromtemperature fluctuations because these will lead to detectabledrift.5.1.1 The
23、detector should be located at the test site andturned on at least 24 h before the start of testing. Insufficientwarm-up may result in drift in excess of the actual value for thedetector.5.2 Methods of Measurement:5.2.1 Connect a suitable device (Note 1) between the pumpand the detector to provide at
24、 least 75 kPa (500 psi) backpressure at 1.0 mL/min flow of methanol. Connect a shortlength (about 100 mm) of 0.25-mm (0.01-in.) internal-diameterstainless steel tubing to the outlet tube of the detector to retardbubble formation. Connect the recorder to the proper detectoroutput channels.NOTE 1Sugge
25、sted devices include (a)2to4mof0.1-mm (0.004-in.)internal-diameter stainless steel tubing, (b) about 250 mm of 0.25 to0.5-mm (0.01 to 0.02-in.) internal-diameter stainless steel tubing crimpedwith pliers or cutters, or (c) a constant back-pressure valve locatedbetween the pump and the injector.5.2.2
26、 Repeatedly rinse the reservoir and chromatographicsystem, including the detector, with degassed methanol toremove from the system all other solvents, any soluble mate-rial, and any entrained gasses. Fill the reservoir with methanoland pump this solvent through the system for at least 30 min tocompl
27、ete the system cleanup.5.2.3 Air or nitrogen is used in the reference cell, if any.Ensure that the cell is clean, free of dust, and completely dry.5.2.4 To perform the static test, cease pumping and allowthe chromatographic system to stabilize for at least1hatroomtemperature without flow. Set the at
28、tenuator at maximumsensitivity (lowest attenuation), that is, the setting for thesmallest value of absorbance units full-scale (AUFS). Adjustthe response time as close as possible to 2 s for a PD that hasa variable response time (Note 2). Record the response timeused.Adjust the detector output to ne
29、ar midscale on the readoutdevice. Record at least1hofdetector signal under theseconditions, during which time the ambient temperature shouldnot change by more than 2C.NOTE 2Time constant is converted to response time by multiplyingby the factor 2.2. The effect of electronic filtering on observed noi
30、se maybe studied by repeating the noise measurements for a series of response-time settings.5.2.5 Draw pairs of parallel lines, each pair correspondingto between 0.5 and 1 min in length, to form an envelope of allobserved random variations over any 15-min period (see Fig.4Distilled-in-glass or liqui
31、d-chromatography grade. Complete freedom fromparticles may require filtration, for example, through a 0.45-m membrane filter.FIG. 1 Example of a Linearity Plot for a Photometric DetectorE 685 93 (2005)22). Draw the parallel lines in such a way as to minimize thedistance between them. Measure the ver
32、tical distance, in AU,between the lines. Calculate the average value over all thesegments. Divide this value by the cell length in centimetres toobtain the static short-term noise.5.2.6 Now mark the center of each segment over the 15-minperiod of the static short-term noise measurement. Draw aseries
33、 of parallel lines encompassing these centers, each paircorresponding to 10 min in length, and choose that pair of lineswhose vertical distance apart is greatest (see Fig. 2). Dividethis distance in AU by the cell length in centimetres to obtainthe static long-term noise.5.2.7 Draw the pair of paral
34、lel lines that minimizes thevertical distance separating these lines over the1hofmea-surement (see Fig. 2). The slope of either line is the static driftexpressed in AU/h.5.2.8 Set the pump to deliver 1.0 mL/min under the sameconditions of tubing, solvent, and temperature as in 5.2.1through 5.2.3.All
35、ow 15 min for the system to stabilize. RecordFIG. 2 Example for the Measurement of the Noise and Drift of a PD (Chart Recorder Output).E 685 93 (2005)3at least1hofsignal under these flowing conditions, duringwhich time the ambient temperature should not change bymore than 2C.5.2.9 Draw pairs of para
36、llel lines, measure the verticaldistances, and calculate the dynamic short-term noise follow-ing the procedure of 5.2.5.5.2.10 Make the measurement for the dynamic long-termnoise following the procedure outlined in 5.2.6.5.2.11 Draw the pair of parallel lines as directed in 5.2.7.The slope of these
37、lines is the dynamic drift.5.2.12 The actual noise of the system may be larger orsmaller than the observed values, depending upon the methodof data collection, or signal monitoring of the detector, sinceobserved noise is a function of the frequency, speed ofresponse, and bandwidth of the readout dev
38、ice.6. Minimum Detectability, Linear Range, andCalibration6.1 Methods of MeasurementFor the determination of thelinear range of a PD, (12) for a specific substance, the responseto that test substance must be determined. The followingprocedure is designed to provide a worst-case procedure.6.1.1 Disso
39、lve in methanol a suitable compound with anultraviolet spectral absorbance that changes rapidly at thewavelength of interest.5Choose a concentration that is ex-pected to exceed the linear range, typically to give an absor-bance above 2 AU. Dilute the solution accurately in a series tocover the linea
40、r range, that is, down to the minimum detectableconcentration.6Rinse the sample cell with methanol and zerothe detector with methanol in the cell. Rinse the cell with thesolution of lowest concentration until a stable reading isobtained; usually rinsing the cell with 1 mL is sufficient.Record the de
41、tector output. After rinsing the syringe thor-oughly with the next more concentrated solution, fill the cellwith the solution from each dilution in turn. Obtain a minimumof five on-scale measurements. Measure under static condi-tions.6.1.2 Calculate the ratio of detector response (AU) toconcentratio
42、n (g/mL) for each solution and plot these ratiosversus log concentration (see Fig. 1). The region of linearitywill define a horizontal line of constant response ratio. Athigher concentrations, there will typically be a negative devia-tion from linearity, while at lower concentrations there may bedev
43、iation in either direction. Draw horizontal lines 5 % aboveand below the line of constant response ratio. The upper limitof linearity is the concentration at which the line of measuredresponse ratio intersects one of the 5 % bracketing lines at thehigh concentration end. The lower limit of linearity
44、 is either theminimum detectable concentration (see 6.1.3) or the concen-tration at which the line of measured response ratio intersectsone of the bracketing lines at the low concentration end,whichever is greater.6.1.3 Determine the minimum detectability (minimum de-tectable concentration) of the t
45、est substance by calculating theconcentration that would correspond to twice the static short-term noise. Specify the solute and solvent.6.1.4 Calculate the ratio of the upper limit of linearity to thelower limit of linearity to give the linear range expressed as anumber. As this procedure is a wors
46、t case situation, the linearrange may be expected to be greater for compounds having abroad spectral band in the region of the chosen wavelength.6.1.5 Plot or calculate the detector response (AU) versusconcentrations (g/mL) for a test substance of known molarabsorptivity to find the best-fit line th
47、rough the origin. Calcu-late the molar absorptivity, e, of the test solution as follows:e5slope 3 MWb(1)where:slope = the slope of the linear portion of the plot,AUl/g,MW = molecular weight, g/mole, andb = nominal cell length, cm, as specified by the manu-facturer.Compare the value of e obtained wit
48、h an experimentallydetermined value or one from the literature (Note 3). Shouldthe values differ by more than 5 %, the PD may requireadjustment. Consult the manufacturers directions.NOTE 3For example, the values of molar absorptivity for uracil inmethanol are 7.7 3 103at 254 nm and 1.42 3 103at 280
49、nm; for potassiumdichromate in 0.01 N sulfuric acid they are 4.22 3 103at 254 nm and3.60 3 103at 280 nm.7. Response Time7.1 The response time of the detector may become signifi-cant when a short micro-particle column and a high-speedrecorder are used. Also, it is possible, by using an intentionallyslow response time, to reduce the observed noise and henceincrease the apparent linear range. Although this would havelittle effect on broad peaks, the signal from narrow peakswould be significantly degraded. Measure at the highest andlowest values of the elect
