1、Designation: E685 93 (Reapproved 2013)Standard Practice forTesting Fixed-Wavelength Photometric Detectors Used inLiquid Chromatography1This standard is issued under the fixed designation E685; the number immediately following the designation indicates the year oforiginal adoption or, in the case of
2、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 is intended to serve as a guide for thetesting of the performance of a photometric
3、 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 ofth
4、e 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,construction, operation, and evaluation of liquid-
5、chromatography detectors, see Refs (10 and 11) in addition tothe sections devoted to detectors in Refs (1-7).1.5 This standard does not purport to address all of thesafety problems, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety
6、 and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3E275 Practice for Describing and Measuring Performance ofUltraviolet and Visible SpectrophotometersE682 Practice for Liquid Chromatography Terms and Rela-tionships
7、3. Terminology3.1 Definitions:3.1.1 absorbance calibration, nthe procedure that verifiesthat the absorbance scale is correct within 65%.3.1.2 drift, nthe 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
8、of a flow rate of 1.0mL/min.3.1.4 linear range, n of a PD, the range of concentrationsof a test substance in a mobile phase over which the responseof the detector is constant to within 5 % as determined from thelinearity plot specified below and illustrated in Fig. 1. Thelinear range should be expre
9、ssed as the ratio of the highestconcentration to the minimum detectable concentration or thelowest linear concentration, whichever is greatest.3.1.5 long-term 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
10、.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 may not always be present.3.1.6 minimum detectability, nof a PD, that concentrationof a specific solute in a specific solvent that results in a d
11、etectorresponse corresponding to twice the static short-term noise.3.1.7 response time (speed of output), nthe detector, thetime required for the detector output 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
12、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 generally fast enough to be unimportant. It isgenerally comparable to the response time of the recorder anddependent on the response time of t
13、he detector electrometerand 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 detector
14、 signal ofa frequency greater than one cycle per minute.1This practice is under the jurisdiction of ASTM Committee E13 on MolecularSpectroscopy and Separation Science and is the direct responsibility of Subcom-mittee E13.19 on Separation Science.Current edition approved Jan. 1, 2013. Published Janua
15、ry 13. Originallyapproved in 1979. Last previous edition approved in 2005 as E685 93 (2005).DOI: 10.1520/E0685-93R13.2The boldface numbers in parentheses refer to the list of references at the end ofthis practice.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Cu
16、stomer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.8.1 DiscussionIt determines th
17、e smallest signal detect-able by a PD, limits the precision attainable in quantitation oftrace-level samples, and sets the lower limit on linearity. Thisnoise corresponds to the observed noise only.3.1.9 static, nunder conditions of no flow.4. Significance and Use4.1 Although it is possible to obser
18、ve 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 obtainedunder the same operating conditions. It should also be notedthat to completely specify a detectors c
19、apability, 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 be usedregardless of the ultimate operating parameters.4.2 Linearity and response time of the reco
20、rder 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-dance with the manufacturers directions. If additional elec-tronic filters or amplifiers are used be
21、tween 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 sample cell.Air or nitrogen shall beused in the reference cell if there is one. Nitrogen is preferr
22、edwhere 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 detector should be located at the test site andturned on at least 24 h before the start of testing
23、. 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 least 75 kPa (500 psi) backpressure at 1.0 mL/min flow of methanol. Connect a shortlength (about
24、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 1Suggested devices include (a)2to4mof0.1-mm (0.004-in.)internal-diameter stainless steel tubing, (b) abo
25、ut 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 Repeatedly rinse the reservoir and chromatographicsystem, including the detector, with degassed m
26、ethanol toremove from the system all other solvents, any solublematerial, and any entrained gasses. Fill the reservoir withmethanol and pump this solvent through the system for at least30 min to complete the system cleanup.5.2.3 Air or nitrogen is used in the reference cell, if any.Ensure that the c
27、ell 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 attenuator at maximumsensitivity (lowest attenuation), that is, the setting for thesmallest value of a
28、bsorbance 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 near midscale on the readoutdevice. Record at least1hofdetector signal under theseconditions, during w
29、hich time the ambient temperature shouldnot change by more than 2C.NOTE 2Time constant is converted to response time by multiplying bythe factor 2.2. The effect of electronic filtering on observed noise may bestudied by repeating the noise measurements for a series of response-timesettings.5.2.5 Dra
30、w 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.2). Draw the parallel lines in such a way as to minimize thedistance between them. Measure the vertical distance, in AU,between t
31、he 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 of parallel lines encompassing
32、 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.4Distilled-in-glass or liquid-chromatography grade. Complet
33、e freedom fromparticles may require filtration, for example, through a 0.45-m membrane filter.FIG. 1 Example of a Linearity Plot for a Photometric DetectorE685 93 (2013)25.2.7 Draw the pair of parallel lines that minimizes thevertical distance separating these lines over the 1 h of mea-surement (see
34、 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.Allow 15 min for the system to stabilize. Recordat least1hofsignal under these flowing conditions, du
35、ringwhich time the ambient temperature should not change bymore than 2C.5.2.9 Draw pairs of parallel 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 o
36、utlined in 5.2.6.5.2.11 Draw the pair of parallel lines as directed in 5.2.7.The slope of these 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, sinceF
37、IG. 2 Example for the Measurement of the Noise and Drift of a PD (Chart Recorder Output).E685 93 (2013)3observed noise is a function of the frequency, speed ofresponse, and bandwidth of the readout device.6. Minimum Detectability, Linear Range, andCalibration6.1 Methods of MeasurementFor the determi
38、nation 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 Dissolve in methanol a suitable compound with anultraviolet spectral absorbance that changes rapidly at
39、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 linear range, that is, down to the minimum detectableconcentration.6Rinse the sample cell with methanol
40、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 detector output. After rinsing the syringe thor-oughly with the next more concentrated solution, fill
41、 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) toconcentration (g/mL) for each solution and plot these ratiosversus log concentration (see Fig. 1). The region o
42、f 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 bedeviation in either direction. Draw horizontal lines 5 % aboveand below the line of constant response
43、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 is either theminimum detectable concentration (see 6.1.3) or the concen-tration at which the line o
44、f 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 test substance by calculating theconcentration that would correspond to twice the static short-term n
45、oise. 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 worst case situation, the linearrange may be expected to be greater for compounds having abroad spectral
46、 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 through the origin. Calcu-late the molar absorptivity, , of the test solution as follows: 5slope 3MWb(
47、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 themanufacturer.Compare the value of obtained with an experimentallydetermined value or one from the literature (Note 3). Shouldthe values differ by more
48、than 5 %, the PD may requireadjustment. Consult the manufacturers directions.NOTE 3For example, the values of molar absorptivity for uracil inmethanol are 7.7 103at 254 nm and 1.42 103at 280 nm; for potassiumdichromate in 0.01 N sulfuric acid they are 4.22 103at 254 nm and3.60 103at 280 nm.7. Respon
49、se 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 electronic filter if it is variable.7.2 Method of Measurement:7.2.1 The composition of the mobile phase is
