1、Designation: E 579 04Standard Test Method forLimit of Detection of Fluorescence of Quinine Sulfate inSolution1This standard is issued under the fixed designation E 579; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of la
2、st 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 test method employs the signal-to-noise ratio todetermine the sensitivity of a fluorescence measuring system intes
3、ting for the limit of detection (LOD) of quinine sulfatedihydrate in solution. The results obtained with quinine sulfatedihydrate in solution are suitable for specifying instrumentperformance on samples having excitation and fluorescencebands wider than 10 nm at or near room temperature.1.1.1 This t
4、est method is not intended to be used as (1)arigorous test of performance of instrumentation, or (2), tointercompare the quantitative performance of instruments ofdifferent design. Intercomparison of the LOD between instru-ments is commonly expressed as the ratio of the water Ramanpeak intensity to
5、the root-mean-square (rms) noise as measuredon a fluorometer using an excitation wavelength of 350 nmThis test method uses the excitation and emission peakwavelengths for quinine sulfate dihydrate in solution, whichare approximately 350 nm and 450 nm, respectively1.2 This test method has been applie
6、d to fluorescence-measuring systems utilizing non-laser, low-energy excitationsources. There is no assurance that extremely intense illumi-nation will not cause photodecomposition2of the compoundsuggested in this test method. For this reason, it is recom-mended that this test method not be indiscrim
7、inately employedwith high intensity light sources. This test method is notintended to determine minimum detectable amounts of othermaterials. If this test method is extended to employ otherchemical substances, the user should be aware of the possibil-ity that these other substances may undergo decom
8、position oradsorption onto containers.1.3 A typical LOD for conventional fluorometers using thistest method is 1 ng of quinine sulfate per mL.1.4 The suggested shelf life of a 1 mg/mL stock solution ofquinine sulfate dihydrate is three months, when stored in thedark in a stoppered glass bottle.1.5 T
9、his 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 and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Docume
10、nts2.1 ASTM Standards:3E 578 Test Method for Linearity of Fluorescence MeasuringSystems3. Summary of Test Method3.1 To measure the concentration corresponding to theLOD, the fluorescence intensity scale and gain on the detectorare adjusted such that noise observed with pure solvent in thesample cell
11、 is large enough to measure. The test solution is thendiluted until readings on both the test solution and pure solventcan be read at the same intensity, scale, and instrument settings.The concentration corresponding to the limit of detection isthat at which the noise intensity, multiplied by three,
12、 is equal tothe signal intensity.3.2 This test for limit of detection requires an instrument tomeet the following conditions: stable, free of extraneous noise,electrical pickup, and internal stray light. The sample spacemust be covered to exclude room light. The instrument shouldbe operated accordin
13、g to the manufacturers recommendations,or, if they are modified, the modifications must be appliedconsistently to the test for limit of detection and to the analysisfor which the test is a requirement, so that levels of perfor-mance are comparable for both. All modifications must beincluded in the r
14、eport outlined in section 8.NOTE 1To obtain the lowest reading (the best instrumental response)for the limit of detection of fluorescent material, a number of precautionsmust be taken. The quality, condition, and position of the sample cell aremost important. The cell must be made of fused silica th
15、at does notfluoresce at the excitation wavelength and be free of scratches and marksthat scatter light into the fluorescence detection system. Only spectralgrade chemicals and solvents (including water) that do not fluoresce1This test method is under the jurisdiction of ASTM Committee E13 onMolecula
16、r Spectroscopy and Chromatography and is the direct responsibility ofSubcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.Current edition approved Feb. 1, 2004. Published March 2004. Originallypublished in 1976. Last previous edition approved in 1998 as E 579 84 (1998).2Lukasi
17、ewicz, R. J., and Fitzgerald, J. M., Analytical Chemistry, ANCHA, Vol45, 1973, p. 511.3For 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
18、 page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.should be used.4Dilute solutions of quinine sulfate dihydrate should bemade, just before use, from concentrated stock solutions.All samples usedmust be maintain
19、ed at the same temperature to obviate effects due totemperature fluctuations. The average temperature coefficient for fluores-cence intensity in the temperature range from 16 - 35 C is -0.62%/Cat 450 nm for 1 g / mLquinine sulfate dihydrate in 0.1 mol / LHClO4(2).4. Significance and Use4.1 When dete
20、rmining the limiting detectable concentrationof a fluorescent substance, it is usually necessary to increasethe readout scale of a photoelectric instrument to a point wherenoise (that is, random fluctuations of the system) becomesapparent. This noise will be superimposed upon the signal fromthe samp
21、le.4.2 In molecular fluorescence spectroscopy, the limit ofdetection for the sample will be determined by the limitingsignal-to-noise ratio, S/N, where the signal, S, is the differencebetween readings obtained with the sample and blank solu-tions, and N is the total root-mean-square (rms) noise. The
22、 limitof detection for the sample will be given by the instrumentreadings that give a signal equal to three times the rms value ofthe noise.NOTE 2Factors other than noise affecting the sample concentrationcorresponding to the limit of detection include: the spectral bandwidths ofthe excitation and e
23、mission monochromators, the intensity of the excitinglight that can be concentrated on the sample, the fraction of thefluorescence collected by the detection system, the response time of thedetection system, and the purity of the solvent. The size and arrangementof the sample container with respect
24、to the light beams are also important,as they affect both the desired signal and the extraneous signal that onlycontributes noise.NOTE 3The value of rms noise (N) can be obtained by calculating thestandard deviation of a series of readings of the signal from the sample atthe peak emission wavelength
25、 at approximately 450 nm as follows:rms 5 =(x2 x!2/n 2 1! (1)where:x = mean of the series of readings,x = value of the individual reading, andn = number of readings.Alternatively, rms noise may be estimated by noting the extreme differ-ences between the members of a series of readings (peak-to-peak
26、noise)and dividing by a factor that is usually taken to be 5.5,65. Reagents5.1 Prepare a stock solution of quinine sulfate dihydrate(C20H22O2N2)2H2SO42H2O by transferring 0.100 g of highpurity crystalline dihydrate of quinine sulfate7into a 100-mLvolumetric flask and fill the flask to volume using e
27、ither 0.1mol / L sulfuric acid or 0.1 mol / L perchloric acid as thesolvent. This solution contains 1 mg/mL of quinine sulfatedihydrate.NOTE 4Either 0.1 mol / L sulfuric acid or 0.1 mol / L perchloric acidcan be used as a solvent with quinine sulfate dihydrate, but the solvent thatis chosen must als
28、o be used as the blank. Take note that the quantum yieldof quinine sulfate dihydrate in solution has been shown to be about 13 %smaller in 0.1 mol / L sulfuric acid than in 0.1 mol / L perchloric acid,which will result in a corresponding increase in the concentration ofquinine sulfate dihydrate in 0
29、.1 mol / L sulfuric acid versus that in 0.1 mol/ L perchloric acid at the LOD for a particular instrument.5.2 Make serial dilutions by diluting aliquots of the stocksolution and successive solutions to ten times their volumewith the solvent. Repeat this process until the desired concen-tration is ob
30、tained. The sixth successive dilution will result ina concentration of 1 ng/mL.5.3 Any fluorescence from the pure solvent will interferewith the limit of detection measurement. The solvent should betested for fluorescence before being used with this method. Totest for fluorescence, follow the proced
31、ures given in sections6.1 to 6.5, but replace the blank with an empty sample cell, i.e.just air in the cell, and replace the dilute test solution with theblank.5.4 Calculate Sand B, the average signal of the blank andempty cell , respectively, and the rms noise of the signal fromthe empty cell, as d
32、escribed in section 7.1. If Sis greater thanBby more than three times the rms noise of the empty cellsignal, then fluorescence from the solvent may be present.6. Procedure6.1 Adjust the fluorescence-measuring system for normaloperating conditions. The widest excitation and emissionbandwidth availabl
33、e on the instrument should be used (not toexceed 40 nm).6.2 Set the excitation wavelength and emission wavelengthin accordance with Test Method E 578. For quinine sulfatedihydrate in solution, the peak wavelengths will be approxi-mately 350 and 450 nm, respectively.NOTE 5In some fluorescence measuri
34、ng systems, it may not bepossible to adjust excitation or emission wavelengths to obtain themaximum fluorescence of quinine sulfate dihydrate in solution. However,users of such instruments should be aware of the Raman scatter phenom-enon due to solvent alone. Such Raman scatter may contribute signif
35、i-cantly and independently to noise, blank, or test solution readings.6.3 Set the signal integration time to 1 s, or the instrumentalequivalent.6.4 Put the pure solvent in the sample cell and adjustinstrument settings such that the peak-to-peak noise is approxi-mately 5 % of full range of the instru
36、ment at these settings.This readout scale is referred to as “full scale” in all sectionsthat follow. Measure the signal from the blank for at least tenindependent readings, removing and reinserting the sample cellafter each reading. The average of these ten signals, B, is usedin 7.1.NOTE 6In some ca
37、ses, removal and reinsertion of the sample cell maynot be feasible, such as, in process control (continuous flow analysis) orchromatographic column effluent monitors. With such instrumentation,emission from 10 aliquots of solvent and 10 aliquots of test solutionshould be measured.6.5 Replace the pur
38、e solvent with a dilute test solution (1ng/mL or greater) in the same cell. Note the readings of thesignal from this sample. The meter readings should be less than4The procedure used to recognize fluorescence in a solvent is given in 6.3 and6.4.5Blair, E. J., Introduction to Chemical Instrumentation
39、, McGraw-Hill, NewYork, NY, 1962.6Landon, V. D. Proceedings of the I. R. E. and Waves and Electrons, PIWEB,Vol 29, 1941, p. 50.7National Institute of Standards and Technology SRM 936a, or the equivalent.E579042100 % and greater than 10 % of full-scale. If the signal, s,resulting for this test soluti
40、on does not fall within these limits,replace the test solution with a solution, if applicable. Repeatthe measurement of (s) ten times as in step 6.4, removing andreinserting the sample cell after each reading. Average the 10measurements of s to obtain the average, S.7. Calculation7.1 Take the differ
41、ence between S, the average signalresulting from the sample solution measurements and B, thatresulting from the average of the ten readings of the blanksolution. This is S the net signal due to the substance in thesolvent.S 5 S2 B(2)7.2 Calculate the LOD as follows:LOD 5 sample concentration/S! 3 rm
42、s noise 3 3! (3)7.2.1 Report the average LOD.8. Report8.1 Report the LOD of quinine sulfate dihydrate in solutionin nanograms per millilitre.8.2 If the manufacturers recommendations for the opera-tion of the instrument were modified for the performance ofthis test, these modifications should be note
43、d.9. Precision and Bias9.1 The precision of this test method is limited by theroot-mean-square noise in the fluorescence measuring systemwhen the peak-to-peak noise from the blank is amplified.9.2 This test method is not intended to be used as (1)arigorous test of absolute performance of instrumenta
44、tion, or(2), to intercompare the quantitative performance of instru-ments of different design. No statement of bias can be made.10. Keywords10.1 fluorescence spectrometers; molecular luminescence;molecular spectroscopyREFERENCES(1) Lukasiewicz, R.J., and Fitzgerald, J.M., Analytical Chemistry, AN-CH
45、A, Vol. 45, 1973, p. 511.(2) Velapoldi, R.A., and Mielenz, K.D., NBS Special Publication 26064,1980, p. 60.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised th
46、at 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 by the responsible technical committee and must be reviewed every five years andif not revised, either reappr
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48、eel 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 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).E579043
