1、Designation: E578 07 (Reapproved 2013)Standard Test Method forLinearity of Fluorescence Measuring Systems1This standard is issued under the fixed designation E578; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last re
2、vision. 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 test method covers a procedure for evaluating thelimits of the linearity of response with fluorescence intensity offluor
3、escence-measuring systems under operating conditions.Particular attention is given to slit widths, filters, and samplecontainers. This test method can be used to test the overalllinearity under a wide variety of instrumental and samplingconditions. The results obtained apply only to the testedcombin
4、ation of slit width and filters, and the size, type andillumination of the sample cuvette, all of which must be statedin the report. The sources of nonlinearity may be the measuringelectronics, excessive absorption of either the exciting oremitted radiation, or both, and the sample handling techniqu
5、e,particularly at low concentrations.1.2 This test method has been applied to fluorescence-measuring systems utilizing continuous and low-energy exci-tation sources (for example, an excitation source of 450-Welectrical input or less). There is no assurance that extremelyintense illumination will not
6、 cause photodecomposition of thecompounds suggested in this test method.2For this reason it isrecommended that this test method not be indiscriminatelyemployed with high-intensity light sources. It is not a testmethod to determine the linearity of response of other materi-als. If this test method is
7、 extended to employ other chemicalsubstances, the principles within can be applied, but newmaterial parameters, such as the concentration range oflinearity, must be established. The user should be aware of thepossibility that these other substances may undergodecomposition, or adsorption onto contai
8、ners.1.3 This test method has been applied to fluorescence-measuring systems utilizing a single detector, that is, a photo-multiplier tube or a single photodiode. It has not been demon-strated if this method is effective for photo-array instrumentssuch as those using a CCD or a diode array detector.
9、1.4 This test method is applicable to 10-mm pathlengthcuvette formats and instruments covering a wavelength rangewithin 190 to 900 nm. The use of other sample formats has notbeen established with this test method.1.5 The values stated in SI units are to be regarded asstandard. No other units of meas
10、urement are included in thisstandard.1.6 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 regulatory lim
11、itations prior to use.2. Summary of Test Method2.1 This procedure is used for testing the linearity offluorescence-measuring systems by using solutions of quininesulfate dihydrate in sulfuric acid as standard test solutions.Other stable solutions which may be more suitable to the usercan be employed
12、 (Note 1). The standard used to determinelinearity should be stated in the report. The fluorescence of thetest solution is measured in the measuring system with thecuvettes, slits, or filters that are to be employed in projecteduse.NOTE 1Asubstitute standard should have the following properties: (1)
13、It should have a large quantum yield at very high dilution; (2) it should bestable to the exciting radiation during spectral measurements; (3) itsfluorescence and its absorption spectra overlap should be small; (4) itsquantum yield should not be strongly concentration dependent; and (5)itshould have
14、 a broad emission spectrum, so that little error is introducedwhen wide slits are used.32.2 Upper Limit of LinearityThe fluorescence intensity ofa series of standard solutions is measured, the resultantinstrument readings are plotted against concentration on alog-log graph, and a smooth curve is dra
15、wn through the datapoints. The point (concentration) at which the upper end of thecurve deviates by more than 5 % of the signal from the straightline (defined by the center region of the curve) is taken as theupper limit of linearity. The limit is expressed in microgramsper millilitre of quinine sul
16、fate dihydrate.NOTE 2Absorption of the exciting radiation at high solute concentra-tions is dependent on instrument geometry and pathlength, and can result1This test method is under the jurisdiction of ASTM Committee E13 onProtective Coating and LiningWork for Power Generation Facilities and is the
17、directresponsibility of Subcommittee D33.09 on Protective Lining for FGD Systems.Current edition approved Dec. 1, 2012. Published May 2013. Originallyapproved in 1976. Last previous edition approved in 2007 as E578 07. DOI:10.1520/E0578-07R13.2Lukasiewicz, R. J., and Fitzgerald, J. M., Analytical Ch
18、emistry, ANCHA, Vol45, 1973, p. 511.3Gill, J. E., Photochemistry and Photobiology, PHCBA, Vol 9, 1969, p. 313.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1in fluorescence signal nonlinearity.2.3 Lower Limit of LinearityThe lower li
19、mit of linearity istaken as the point (concentration) at which the lower end of thecurve deviates from the straight line defined by the centralportion of the curve by more than twice the average percentdeviation of the points that determine the straight line.3. Significance and Use3.1 The range of c
20、oncentration of a fluorescing substance insolution over which the fluorescence varies linearly with theconcentration is the range most useful for quantitative analysis.This range is affected by properties of the solution underanalysis and by features of the measuring system. This testmethod provides
21、 a means of testing the performance of afluorescence measuring system and of determining the concen-tration range over which the system is suitable for making agiven quantitative analysis.3.2 This test method is not meant for comparing theperformance of different fluorescence measuring instruments.4
22、. Apparatus4.1 Fluorescence-Measuring System, fully equipped forprojected use with a suitable UV source to cover the excitationwavelengths of quinine sulfate and a photodetector sensitive at450 nm.5. Standard Solutions5.1 Prepare a stock solution of quinine sulfate dihydrate bytransferring 0.100 g o
23、f crystalline dihydrate of quinine sulfate,(C20H24O2N2)2H2SO42H2O, National Institute of Standardsand Technology SRM 936 (or equivalent), into a 100-mLvolumetric flask and fill the flask to volume with 0.1 N sulfuricacid. This solution contains 103g/mL of quinine sulfatedihydrate.5.2 Make serial dil
24、utions by diluting successive aliquots ofthis stock solution to ten times their volume with 0.1 N sulfuricacid. Prepare, by step-wise dilution, solutions with concentra-tions of 102, 10, 100,101,102, and 103g/mL.6. Procedure6.1 Select the combination of slit widths or apertures, filters,and the size
25、, type, and illumination of cuvette for which the testis desired.6.2 Set the wavelength of the exciting radiation to 350 nmby means of filters or an excitation monochromator, whicheveris provided with the fluorescence measuring system.NOTE 3Instruments equipped with a mercury vapor lamp should beset
26、 to isolate the 365 nm mercury line.6.3 Set the central wavelength of the band pass of thefluorescence-radiation measuring system at approximately 450nm, using filters or an emission monochromator.6.4 Rinse the cuvette at least three times and fill with thereagent blank (0.1 N sulfuric acid) and rec
27、ord the reading usingthe appropriate range setting of the instrument.NOTE 4When it is necessary to change the measurement settings ofthe instrument, the reading of the reagent blank should also be determinedusing the new setting.6.5 Discard the blank solution used in 6.4, rinse the cuvetteat least t
28、hree times with the most dilute of the solutionsdescribed in Section 4, fill the cuvette with this solution, andrecord the fluorescence intensity reading.6.6 Discard the more dilute solution, rinse the cuvette atleast three times with the next most concentrated standardsolution, fill the cuvette wit
29、h this solution, and record thefluorescence intensity reading. Proceed similarly with the otherstandard solutions, ending with the 102g/mL solution.NOTE 5The 103g/mL stock solution is not a recommended testsolution due to its large absorbance, A10, for a 1cm pathlength at =450 nm, which causes extre
30、me inner filter effects and ineffective correc-tions (see Note 7).7. Calculation of Results and Data Presentation7.1 The fluorescence intensity reading minus the reading ofthe blank solution is equal to the signal, S (using the appropri-ate multiplication factors corresponding to the amplificationra
31、nges). Plot these values of S against concentration on alog-log graph and draw a smooth curve through the points.7.2 Using only the points that fall on the linear portion ofthe curve, this will include the points at concentrations of 100,101, and 102g/mL for most instruments, determine theaverage pe
32、rcent deviation of the points from the line.NOTE 6The data that falls on the linear portion of the curve should betreated by linear regression analysis, which will yield the slope of the line,the standard deviation of the slope, and the standard deviation of thepoints about the line. To determine wh
33、ich points fall in the linear range, aline connecting the points at 100,101, and 102g/mL can be drawn onthe log-log graph.7.3 Note the concentration at which the upper end of thecurve deviates by more than 5 % of the signal from the straightline defined by the center region of the curve. Report this
34、concentration, in micrograms per millilitre of quinine sulfatedihydrate, as the upper limit of linearity.NOTE 7Absorption of the excitation radiation by the sample beforereaching the detection region is usually the major inner filter effectobserved at higher concentrations. For collimated excitation
35、 radiation and90 detection region geometry, a correction for excitation radiationabsorption has been proposed:4F0F 5 (2.303Dx(X2 2 X1)(102Dx X12 102Dx X2) (1)where:F0= the corrected fluorescence intensityF = the observed fluorescence intensityDx= the optical density per centimetre of the sample at t
36、heexcitation wavelength, andX1 and X2 = the distances (in centimetres) that the detection regionboundaries are from the incident face of the sample cell.Asecondary inner filter effect, due to the absorption of emission beforeit exits the sample can also occur. For a 90 detection geometry, acorrectio
37、n for absorption of emission has also been proposed:5F0F 5 (2.303Dm(Y2 2 Y1)(102Dm Y12 102Dm Y2) (2)where:4Parker, C. A., and Barnes, W. J., Analyst, Vol 82, 1957, p. 606.5Yappert, M.C., and Ingle, Jr., J.D., Appl.Spec., Vol 43, 1989, p. 759.E578 07 (2013)2Dm= the optical density per centimetre of t
38、he sample at theemission wavelength, andY1 and Y2 = the distances (in centimetres) that the detection regionboundaries are from the exit face of the sample cell.7.4 If the plotted data for the lower concentrations deviatefrom the straight line (defined by the center region of the curve)by more than
39、twice the average percent deviation of the pointsthat determine the straight line, report the lower limit oflinearity as within this deviation down to the concentration atwhich the deviation occurs. Thus, for example, with 1 %average deviation above 103g/mL and more than 2 %deviation below this, the
40、 reports should state “linear within 2 %down to a concentration of 103g/mL.”8. Precision and Bias8.1 This test method requires a determination of the preci-sion of the test results as a part of the interpretation of theresults. The precision obtained in any application of the testwill depend on prop
41、erties of the standard test solutions used(which will vary with the chemical species involved), onsample handling technique, and on instrument performance.8.2 As this test method is not meant for comparing theperformance of different fluorescence measuring instruments,nor for comparing the performan
42、ce of any given system foranalyzing solutions of different chemical species, no statementof bias of the test method can be made.9. Keywords9.1 fluorescence spectrometers; molecular luminescence;molecular spectroscopyASTM International takes no position respecting the validity of any patent rights as
43、serted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time
44、by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will rec
45、eive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM Int
46、ernational, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).E578 07 (2013)3
