1、Designation: E 578 07Standard Test Method forLinearity of Fluorescence Measuring Systems1This standard is issued under the fixed designation E 578; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number
2、 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 test method covers a procedure for evaluating thelimits of the linearity of response with fluorescence intensity offluorescence-measuri
3、ng 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 testedcombination of slit w
4、idth 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 technique,particularly
5、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 cause photodec
6、omposition 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 extended to em
7、ploy other chemicalsubstances, the principles within can be applied, but newmaterial parameters, such as the concentration range of linear-ity, must be established. The user should be aware of thepossibility that these other substances may undergo decompo-sition, or adsorption onto containers.1.3 Th
8、is 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.1.4 This te
9、st 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 This standard does not purport to address all of thesafety concerns, if any, associated with i
10、ts 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. Summary of Test Method2.1 This procedure is used for testing the linearity offluorescence-measuring systems by
11、 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 (Note 1). The standard used to determinelinearity should be stated in the report. The fluorescence of thetest solution is measured in
12、the measuring system with thecuvettes, slits, or filters that are to be employed in projecteduse.NOTE 1A substitute standard should have the following properties:(1) It should have a large quantum yield at very high dilution; (2) it shouldbe stable to the exciting radiation during spectral measureme
13、nts; (3) itsfluorescence and its absorption spectra overlap should be small; (4) itsquantum yield should not be strongly concentration dependent; and (5)itshould have a broad emission spectrum, so that little error is introducedwhen wide slits are used.32.2 Upper Limit of LinearityThe fluorescence i
14、ntensity ofa series of standard solutions is measured, the resultantinstrument readings are plotted against concentration on alog-log graph, and a smooth curve is drawn through the datapoints. The point (concentration) at which the upper end of thecurve deviates by more than 5 % of the signal from t
15、he 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 sulfate dihydrate.NOTE 2Absorption of the exciting radiation at high solute concentra-tions is dependent on instrument geometry and pathl
16、ength, and can resultin fluorescence signal nonlinearity.1This test method is under the jurisdiction of ASTM Committee E13 onMolecular Spectroscopy and Separation Science and is the direct responsibility ofSubcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.Current edition ap
17、proved March 1, 2007. Published March 2007. Originallyapproved in 1976. Last previous edition approved in 2001 as E 578 01.2Lukasiewicz, R. J., and Fitzgerald, J. M., Analytical Chemistry, ANCHA, Vol45, 1973, p. 511.3Gill, J. E., Photochemistry and Photobiology, PHCBA, Vol 9, 1969, p. 313.1Copyright
18、 ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.2.3 Lower Limit of LinearityThe lower limit of linearity istaken as the point (concentration) at which the lower end of thecurve deviates from the straight line defined by the centralportion of t
19、he curve by more than twice the average percentdeviation of the points that determine the straight line.3. Significance and Use3.1 The range of concentration of a fluorescing substance insolution over which the fluorescence varies linearly with theconcentration is the range most useful for quantitat
20、ive analysis.This range is affected by properties of the solution underanalysis and by features of the measuring system. This testmethod provides a means of testing the performance of afluorescence measuring system and of determining the concen-tration range over which the system is suitable for mak
21、ing agiven quantitative analysis.3.2 This test method is not meant for comparing theperformance of different fluorescence measuring instruments.4. Apparatus4.1 Fluorescence-Measuring System, fully equipped forprojected use with a suitable UV source to cover the excitationwavelengths of quinine sulfa
22、te and a photodetector sensitive at450 nm.5. Standard Solutions5.1 Prepare a stock solution of quinine sulfate dihydrate bytransferring 0.100 g of crystalline dihydrate of quinine sulfate,(C20H24O2N2)2H2SO42H2O, National Institute of Standardsand Technology SRM 936 (or equivalent), into a 100-mLvolu
23、metric flask and fill the flask to volume with 0.1 N sulfuricacid. This solution contains 103g/mL of quinine sulfatedihydrate.5.2 Make serial dilutions by diluting successive aliquots ofthis stock solution to ten times their volume with 0.1 N sulfuricacid. Prepare, by step-wise dilution, solutions w
24、ith 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, 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 excita
25、tion monochromator, whicheveris provided with the fluorescence measuring system.NOTE 3Instruments equipped with a mercury vapor lamp should beset 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, u
26、sing 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 record the reading usingthe appropriate range setting of the instrument.NOTE 4When it is necessary to change the measurement settings ofthe instrument, the re
27、ading 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 three times with the most dilute of the solutionsdescribed in Section 4, fill the cuvette with this solution, andrecord the fluorescence intensity reading.6
28、.6 Discard the more dilute solution, rinse the cuvette atleast three times with the next most concentrated standardsolution, fill the cuvette with this solution, and record thefluorescence intensity reading. Proceed similarly with the otherstandard solutions, ending with the 102g/mL solution.NOTE 5T
29、he 103g/mL stock solution is not a recommended testsolution due to its large absorbance, A10, for a 1cm pathlength at l =450 nm, which causes extreme inner filter effects and ineffective correc-tions (see Note 7).7. Calculation of Results and Data Presentation7.1 The fluorescence intensity reading m
30、inus the reading ofthe blank solution is equal to the signal, S (using the appropri-ate multiplication factors corresponding to the amplificationranges). 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 t
31、he linear portion ofthe curve, this will include the points at concentrations of 100,101, and 102g/mL for most instruments, determine theaverage percent 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,
32、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 which 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 w
33、hich 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 thisconcentration, in micrograms per millilitre of quinine sulfatedihydrate, as the upper limit of linearity.NOTE 7Absorption of the excitation radiation by
34、the sample beforereaching the detection region is usually the major inner filter effectobserved at higher concentrations. For collimated excitation radiation and90 detection region geometry, a correction for excitation radiationabsorption has been proposed4:F0/F = (2.303Dx(X2X1)/(10Dx X110Dx X2),whe
35、re F0is the corrected fluorescence intensity, F is the observedfluorescence intensity, Dxis the optical density per cm of the sample at theexcitation wavelength, and X1 and X2 are the distances (in centimetres)that the detection region boundaries are from the incident face of thesample cell. A secon
36、dary inner filter effect, due to the absorption ofemission before it exits the sample can also occur. For a 90 detectiongeometry, a correction for absorption of emission has also been proposed5:F0/F = (2.303Dm(Y2Y1)/(10Dm Y110Dm Y2),where Dmis the optical density per cm of the sample at the emission
37、wavelength, and Y1 and Y2 are the distances (in cm) that the detectionregion boundaries 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 twice the average percent devia
38、tion 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 %4Parker, C. A., and Barnes, W. J., Analyst, Vol 82, 1957, p. 606.5Yappert, M.C., and Ingle, JR., J.D., Ap
39、pl.Spec., Vol 43, 1989, p. 759.E578072average deviation above 103g/mL and more than 2 % devia-tion below this, the 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
40、part of the interpretation of theresults. The precision obtained in any application of the testwill depend on properties 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
41、meant for comparing theperformance of different fluorescence measuring instruments,nor for comparing the performance 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 lumin
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45、iews known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 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).E578073
