1、Designation: E579 04 (Reapproved 2015)Standard Test Method forLimit of Detection of Fluorescence of Quinine Sulfate inSolution1This standard is issued under the fixed designation E579; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision
2、, 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 test method employs the signal-to-noise ratio todetermine the sensitivity of a fluorescence measuri
3、ng system intesting 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 temperatu
4、re.1.1.1 This test 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 Ramanpea
5、k intensity to 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, respectively.1.2 This test method
6、 has been applied 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
7、not be indiscriminately 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 m
8、ay undergo decomposition 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 gl
9、ass bottle.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 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 est
10、ablish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3E578 Test Method for Linearity of Fluorescence MeasuringSystems3. Summary of Test Method3.1 To measure the concentration corresponding to
11、 theLOD, the fluorescence intensity scale and gain on the detectorare adjusted such that noise observed with pure solvent in thesample cell 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,
12、and instrument settings.The concentration corresponding to the limit of detection isthat at which the noise intensity, multiplied by three, 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,el
13、ectrical pickup, and internal stray light. The sample spacemust be covered to exclude room light. The instrument shouldbe operated according to the manufacturers recommendations,or, if they are modified, the modifications must be appliedconsistently to the test for limit of detection and to the anal
14、ysisfor which the test is a requirement, so that levels of perfor-mance are comparable for both. All modifications must beincluded in the report outlined in Section 8.1This test method is under the jurisdiction of ASTM Committee E13 onMolecular Spectroscopy and Separation Science and is the direct r
15、esponsibility ofSubcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.Current edition approved May 1, 2015. Published June 2015. Originallypublished in 1976. Last previous edition approved in 2009 as E579 04 (2009).DOI: 10.1520/E0579-04R15.2Lukasiewicz, R. J., and Fitzgerald, J
16、. 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 page onthe ASTM website.Copyri
17、ght ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1NOTE 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 positio
18、n of the sample cell aremost important. The cell must be made of fused silica that 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 fluor
19、esceshould be used.4Dilute solutions of quinine sulfate dihydrate should bemade, just before use, from concentrated stock solutions.All samples usedmust be maintained at the same temperature to obviate effects due totemperature fluctuations. The average temperature coefficient for fluores-cence inte
20、nsity in the temperature range from 16 35C is 0.62 % C at450 nm for 1 g/mL quinine sulfate dihydrate in 0.1 mol/L HClO4.54. Significance and Use4.1 When determining the limiting detectable concentrationof a fluorescent substance, it is usually necessary to increasethe readout scale of a photoelectri
21、c instrument to a point wherenoise (that is, random fluctuations of the system) becomesapparent. This noise will be superimposed upon the signal fromthe sample.4.2 In molecular fluorescence spectroscopy, the limit ofdetection for the sample will be determined by the limitingsignal-to-noise ratio, S/
22、N, where the signal, S, is the differencebetween readings obtained with the sample and blanksolutions, and N is the total root-mean-square (rms) noise. Thelimit of detection for the sample will be given by theinstrument readings that give a signal equal to three times therms value of the noise.NOTE
23、2Factors other than noise affecting the sample concentrationcorresponding to the limit of detection include: the spectral bandwidths ofthe excitation and emission monochromators, the intensity of the excitinglight that can be concentrated on the sample, the fraction of thefluorescence collected by t
24、he detection system, the response time of thedetection system, and the purity of the solvent. The size and arrangementof the sample container with respect to the light beams are also important,as they affect both the desired signal and the extraneous signal that onlycontributes noise.NOTE 3The value
25、 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 at approximately 450 nm as follows:rms 5 =(x 2 x!2/n 2 1! (1)where:x = mean of the series of readings,x = value of the individual reading, andn
26、= number of readings.Alternatively, rms noise may be estimated by noting the extremedifferences between the members of a series of readings (peak-to-peaknoise) and dividing by a factor that is usually taken to be 5.6, 75. Reagents5.1 Prepare a stock solution of quinine sulfate dihydrate(C20H22O2N2)2
27、H2SO42H2O by transferring 0.100 g of highpurity crystalline dihydrate of quinine sulfate8into a 100-mLvolumetric flask and fill the flask to volume using either 0.1mol/L sulfuric acid or 0.1 mol/L perchloric acid as the solvent.This solution contains 1 mg/mL of quinine sulfate dihydrate.NOTE 4Either
28、 0.1 mol/L sulfuric acid or 0.1 mol/L perchloric acid canbe used as a solvent with quinine sulfate dihydrate, but the solvent that ischosen must also 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 sul
29、furic acid than in 0.1 mol/L perchloric acid, whichwill result in a corresponding increase in the concentration of quininesulfate dihydrate in 0.1 mol/L sulfuric acid versus that in 0.1 mol/Lperchloric acid at the LOD for a particular instrument.5.2 Make serial dilutions by diluting aliquots of the
30、stocksolution and successive solutions to ten times their volumewith the solvent. Repeat this process until the desired concen-tration is obtained. 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 detect
31、ion measurement. The solvent should betested for fluorescence before being used with this method. Totest for fluorescence, follow the procedures given in sections6.1 to 6.5, but replace the blank with an empty sample cell, thatis, just air in the cell, and replace the dilute test solution withthe bl
32、ank.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 described in 7.1.IfSis greater than Bbymore than three times the rms noise of the empty cell signal,then fluorescence from the solvent may be present.6. Proc
33、edure6.1 Adjust the fluorescence-measuring system for normaloperating conditions. The widest excitation and emissionbandwidth available on the instrument should be used (not toexceed 40 nm).6.2 Set the excitation wavelength and emission wavelengthin accordance with Test Method E578. For quinine sulf
34、atedihydrate in solution, the peak wavelengths will be approxi-mately 350 and 450 nm, respectively.NOTE 5In some fluorescence measuring systems, it may not bepossible to adjust excitation or emission wavelengths to obtain themaximum fluorescence of quinine sulfate dihydrate in solution. However,user
35、s of such instruments should be aware of the Raman scatter phenom-enon due to solvent alone. Such Raman scatter may contribute signifi-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 solven
36、t in the sample cell and adjustinstrument settings such that the peak-to-peak noise is approxi-mately 5 % of full range of the instrument 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 read
37、ings, removing and reinserting the sample cellafter each reading. The average of these ten signals, B, is usedin 7.1.4The procedure used to recognize fluorescence in a solvent is given in 6.3 and6.4.5Velapoldi, R. A., and Mielenz, K. D., NBS Special Publication 26064, 1980,p. 60.6Blair, E. J., Intro
38、duction to Chemical Instrumentation, McGraw-Hill, NewYork, NY, 1962.7Landon, V. D., Proceedings of the I. R. E. and Waves and Electrons , PIWEB,Vol 29, 1941, p. 50.8National Institute of Standards and Technology SRM 936a, or the equivalent.E579 04 (2015)2NOTE 6In some cases, removal and reinsertion
39、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 pure solvent with a dilute test
40、solution (1ng/mL or greater) in the same cell. Note the readings of thesignal from this sample. The meter readings should be less than100 % and greater than 10 % of full-scale. If the signal, s,resulting for this test solution does not fall within these limits,replace the test solution with a soluti
41、on, 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 difference between S, the average signalresulting from the sample solution measur
42、ements 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 rms noise 33! (3)7.2.1 Report the average LOD.8. Report8.1 Report the LOD of
43、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 noted.9. Precision and Bias9.1 The precision of this test method is limited by t
44、heroot-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 instrumentation, or(2), to intercompare the quantitative performance of instru-ments of
45、 different design. No statement of bias can be made.10. Keywords10.1 fluorescence spectrometers; molecular luminescence;molecular spectroscopyASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of thi
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