1、Designation: E 1657 98 (Reapproved 2006)Standard Practice forTesting Variable-Wavelength Photometric Detectors Used inLiquid Chromatography1This standard is issued under the fixed designation E 1657; the number immediately following the designation indicates the year oforiginal adoption or, in the c
2、ase 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 the testing of the performance of avariable-wavelength photometric
3、detector (VWPD) used as thedetection component of a liquid-chromatographic (LC) systemoperating at one or more wavelengths in the range 190 to 800nm. Many of the measurements are made at 254 nm forconsistency with Practice E 685. Measurements at other wave-lengths are optional.1.2 This practice is i
4、ntended to describe the performance ofthe 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-struc
5、tion, operation, and evaluation of liquid-chromatographydetectors, see Refs (10, 11) in addition to the sections devotedto detectors in Refs (1-7).1.5 The values stated in SI units are to be regarded asstandard.1.6 This standard does not purport to address all of thesafety concerns, if any, associat
6、ed 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 685 Practice for Testing Fixed-Wavelength PhotometricDetect
7、ors Used in Liquid Chromatography3. Terminology3.1 Definitions:3.1.1 absorbance calibrationthe procedure that verifiesthat the absorbance scale is correct within 65%.3.1.2 driftthe average slope of the noise envelope ex-pressed in absorbance units per hour (AU/h) as measured overa period of 1 h.3.1.
8、3 dynamicunder conditions of a flow rate of 1.0mL/min.3.1.4 linear rangeof a VWPD, the range of concentrationsof a test substance in a test solvent over which the ratio ofresponse of the detector versus concentration of test substanceis constant to within 5 % as determined from the linearity plotspe
9、cified in 7.1.2 and illustrated in Fig. 1. The linear rangeshould be expressed as the ratio of the upper limit of linearityobtained from the plot to either a) the lower linear concentra-tion, or b) the minimum detectable concentration, if theminimum detectable concentration is greater than the lower
10、linear concentration.3.1.5 long-term noisethe maximum amplitude in AU forall 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
11、to the observednoise only and may not always be present.3.1.6 minimum detectability of a VWPD, that concentra-tion of a specific solute in a specific solvent that results in adetector response corresponding to twice the static short-termnoise.3.1.6.1 DiscussionThe static short-term noise is a mea-su
12、rement of peak-to-peak noise. A statistical approach to noisesuggests that a value of three times the rms (root-mean-square)noise would insure that any value outside this range would notbe noise with a confidence level of greater than 99 %. Sincepeak-to-peak noise is approximately five times the rms
13、 noise(12), the minimum detectability defined in this practice is amore conservative estimate.3.1.7 response time (speed of output) the detector, thetime required for the detector output to change from 10 % to90 % of the new equilibrium value when the composition ofthe mobile phase is changed in a s
14、tepwise manner, within thelinear range 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 is1This practice is under the jurisdiction of ASTM Committee E13 on Mol
15、ecularSpectroscopy and is the direct responsibility of Subcommittee E13.19 on Chroma-tography.Current edition approved March 1, 2006. Published March 2006. Originallyapproved in 1994. Last previous edition approved in 2001 as E 1657 98 (2001).2The boldface numbers in parentheses refer to the list of
16、 references at the end ofthis practice.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 Intern
17、ational, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.generally comparable to the response time of the recorder anddependent on the response time of the detector electrometerand on the recorder amplifier. Factors that affect the observedresponse time include th
18、e true detector response time, elec-tronic filtering, and system band-broadening.3.1.8 short-term noisethe maximum amplitude, peak topeak, in AU for all random variations of the detector signal ofa frequency greater than one cycle per minute.3.1.8.1 DiscussionIt determines the smallest signal de-tec
19、table by a VWPD, limits the precision attainable in quanti-tation of trace-level samples, and sets the lower limit onlinearity. This noise corresponds to the observed noise only.3.1.9 staticunder conditions of no flow.3.1.10 wavelength accuracythe deviation of the observedwavelength maximum from the
20、 maximum of a known testsubstance.3.1.11 wavelength precisiona measure of the ability of aVWPD to return to the same spectral position as measured bythe reproducibility of absorbance values when the detector isreset to a wavelength maximum of a known test substance.4. Significance and Use4.1 Althoug
21、h 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 obtainedunder the same operating conditions. It should also be notedthat to complet
22、ely 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 be usedregardless of the ultimate operating parameters.4.2 Linearity and
23、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-dance with the manufacturers directions. If additional elec-tronic filters
24、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 methanol4shall beused in the sample cell. Air or nitrogen shall be used in thereference cell if there is one. Nitrogen is
25、 preferred where thepresence of high-voltage equipment makes it likely that there isozone in the air. Protect the entire system from temperaturefluctuations because these will lead to detectable drift.5.1.1 The detector should be located at the test site andturned on at least 24 h before the start o
26、f testing. Insufficientwarm-up may result in drift in excess of the actual value for thedetector. The detector wavelength should be set to 254 nm.5.2 Methods of Measurement:5.2.1 Connect a suitable device (see Note 1) between thepump and the detector to provide at least 75 kPa (500 psi) backpressure
27、 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 1Suggested devices include (a)2to4mof0.1-m
28、m (0.004-in.)internal-diameter stainless steel tubing, ( b) about 250 mm of 0.25 to 0.5mm (0.01 to 0.02-in.) internal-diameter stainless steel tubing crimped withpliers or cutters, or ( c) a constant back-pressure valve located between thepump and the injector.5.2.2 Repeatedly rinse the reservoir an
29、d 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 tocomplete the system cleanup.5.2.3 Air o
30、r 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 attenuator at maximumsensitivity (lo
31、west 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 VWPD thathas a variable response time (see Note 2). Record the responsetime used. Adjust the detector output to near midscale on thereadout d
32、evice. Record at least1hofdetector signal underthese conditions, during which time the ambient temperatureshould not 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 noise maybe studied by repeati
33、ng 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 all4Distilled-in-glass or liquid-chromatography grade. Complete freedom fromparticles may require filtration, for exa
34、mple, through a 0.45-m membrane filter.FIG. 1 Example of Linearity Plot for a Variable-WavelengthDetectorE 1657 98 (2006)2observed 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
35、,between the lines. Calculate the average value over all thesegments. Divide this value by the cell length in centimeters 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 en
36、compassing 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 centimeters to obtainthe static long-term noise.5.2.7 Draw the pair of parallel lines that minimi
37、zes thevertical distance separating these lines over the1hofmea-surement (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 5.2.1-5.2.3.Allow 15 min for the system to stab
38、ilize. Record at least1hofsignal under these flowing conditions, during which time theambient temperature should not change by more than 2C.FIG. 2 Example for the Measurement of the Noise and Drift of a VWD (Chart Recorder Output)E 1657 98 (2006)35.2.9 Draw pairs of parallel lines, measure the verti
39、caldistances, 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 lines is the dynamic drift.5.
40、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 device.6. Wavelength Accuracy an
41、d Precision6.1 The wavelength accuracy and precision of a VWPD areimportant parameters for the performance of chromatographicmethods. The wavelength specified in the method may becritical to the detection of different compounds having differentabsorption spectra. The stated linear range of the metho
42、d maybe compromised if the wavelength is inaccurate. Further, theprecision of adjusting the detector to the same wavelengthshould also be known. The wavelength of a VWPD isdetermined by the monochromator and the optical alignment ofthe detector. The optical alignment is performed by themanufacturer
43、and usually does not need readjustment. Somedetectors require alignment of the lamp after replacement. Thisprocedure verifies that the detector is properly aligned andmeets the manufacturers specifications for wavelength accu-racy and precision.6.2 Method of MeasurementWavelength AccuracyForthe dete
44、rmination of the wavelength accuracy of a VWPD, (13)a solution of a compound with known absorbance maxima isintroduced into the cell. The measured maxima are comparedto the known maxima for the compound. There are severalacceptable compounds and solvents.5The following procedureis recommended (Note
45、3).NOTE 3The recommended procedure is covered under U.S. Patent4,836,673. The American Society for Testing and Materials takes noposition respecting the validity of any patent rights asserted in connectionwith any item mentioned in this standard. Users of this standard areexpressly advised that dete
46、rmination of the validity of any such patentrights, and the risk of infringement of such rights, are entirely their ownresponsibility. Alternative procedures will be considered.6.2.1 Prepare the test solution. For example, dissolve2goferbium perchlorate hexahydrate6in 25 mL water.7The nominalconcent
47、ration is 0.14 M. Filter the solution with an appropriatefilter8to ensure the sample is free of particles.NOTE 4This can be conveniently done by addng water toa2gvial oferbium perchlorate hexahydrate to dissolve the solid. Transfer the contentsto a 25 mL volumetric flask and make up to volume with w
48、ater. Whilereasonable care should be observed in transferring the dissolved erbiumperchlorate into the volumetric flask, the final solution is not usedquantitatively.6.2.2 Turn on the detector and allow it to warm up accord-ing to the manufacturers recommendations. Thoroughly flushthe detector cell
49、with water preferably from the same source asthat to make up the test solution. (If using another testcompound, be sure to use the same solvent as the test solution.)Set the detector wavelength to 250 nm. Zero the absorbance ofthe detector. (Some detectors will automatically zero thedetector after changing wavelengths.) Flush the cell with atleast 1 mL of the erbium test solution. Record the absorbancereading. Increase the wavelength by 1 nm. Flush the cell withat least 1 mL of water. Zero the absorbance of the detector.Flush the cell with the erbium te
