1、Designation: E579 04 (Reapproved 2009)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, respectively1.2 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 n
7、ot 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 ma
8、y 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 gla
9、ss 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 esta
10、blish 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, a
12、nd 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,ele
13、ctrical 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 analy
14、sisfor 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.NOTE 1To obtain the lowest reading (the best instrumental response)for the limit of detection of fluorescent material, a number of pre
15、cautionsmust be taken. The quality, condition, and position of the sample cell aremost important. The cell must be made of fused silica that does not1This test method is under the jurisdiction of ASTM Committee E13 onMolecular Spectroscopy and Separation Science and is the direct responsibility ofSu
16、bcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.Current edition approved Oct. 1, 2009. Published December 2009. Originallypublished in 1976. Last previous edition approved in 2004 as E579 04. DOI:10.1520/E0579-04R09.2Lukasiewicz, R. J., and Fitzgerald, J. M., Analytical Che
17、mistry, 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.1Copyright ASTM Internatio
18、nal, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.fluoresce 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 fluores
19、ceshould 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 intens
20、ity in the temperature range from 16 35 C is 0.62 % / Cat 450 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 photoel
21、ectric 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 rati
22、o, 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 limitof detection for the sample will be given by the instrumentreadings that give a signal equal to three times the rms value ofthe nois
23、e.NOTE 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 collect
24、ed 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 to the light beams are also important,as they affect both the desired signal and the extraneous signal that onlycontributes noise.NOTE 3Th
25、e 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 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,
26、 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 noise)and dividing by a factor that is usually taken to be 5.6, 75. Reagents5.1 Prepare a stock solution of quinine sulfate dihydrate(C20H
27、22O2N2)2H2SO42H2O 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 thesolvent. This solution contains 1 mg/mL of quinine sulfatedihydrate.N
28、OTE 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 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 i
29、n 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 0.1 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 di
30、luting aliquots of the 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 interferew
31、ith the limit of detection 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 t
32、est solution withthe blank.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 section 7.1. If Sis greater thanBby more than three times the rms noise of the empty cellsignal, then fluorescence from t
33、he solvent may be present.6. Procedure6.1 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 T
34、est Method E578. For quinine sulfatedihydrate 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 di
35、hydrate 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 signifi-cantly and independently to noise, blank, or test solution readings.6.3 Set the signal integration time to 1 s, or the instrumentale
36、quivalent.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 instrument at these settings.This readout scale is referred to as “full scale” in all sectionsthat follow. Measure the signal from the blank
37、 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 cases, removal and reinsertion of the sample cell maynot be feasible, such as, in process control (continuous flow analysis) orchromatog
38、raphic column effluent monitors. With such instrumentation,emission from 10 aliquots of solvent and 10 aliquots of test solutionshould be measured.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, 1
39、980,p. 60.6Blair, E. J., Introduction 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 (2009)26.5 Replace the
40、 pure 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 than100 % and greater than 10 % of full-scale. If the signal, s,resulting for this test solution does not fall within these limits,replac
41、e 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 difference between S, the average signalresulting
42、 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 rms noise 3 3! (3)7.2.1 Report the average LO
43、D.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 noted.9. Precision and Bias9.1 The precision of
44、 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 instrumentation, or(2), to intercompare the quantitati
45、ve performance of instru-ments of 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 mentio
46、nedin 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 by the responsible technical committee an
47、d 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 receive careful consideration at a meeting o
48、f 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 International, 100 Barr Harbor Drive, PO Box
49、 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/).E579 04 (2009)3
