E13.01 - Ultra-Violet, Visible, and Luminescence Spectroscopy

SIGNIFICANCE AND USE
4.1 When determining the limiting detectable concentration of a fluorescent substance, it is usually necessary to increase the readout scale of a photoelectric instrument to a point where noise (that is, random fluctuations of the system) becomes apparent. This noise will be superimposed upon the signal from the sample.  
4.2 In molecular fluorescence spectroscopy, the limit of detection for the sample will be determined by the limiting signal-to-noise ratio, S/N, where the signal, S, is the difference between readings obtained with the sample and blank solutions, and N is the total root-mean-square (rms) noise. The limit of detection for the sample will be given by the instrument readings that give a signal equal to three times the rms value of the noise.
Note 2: Factors other than noise affecting the sample concentration corresponding to the limit of detection include: the spectral bandwidths of the excitation and emission monochromators, the intensity of the exciting light that can be concentrated on the sample, the fraction of the fluorescence collected by the detection system, the response time of the detection system, and the purity of the solvent. The size and arrangement of the sample container with respect to the light beams are also important, as they affect both the desired signal and the extraneous signal that only contributes noise.
Note 3: The value of rms noise (N) can be obtained by calculating the standard deviation of a series of readings of the signal from the sample at the peak emission wavelength at approximately 450 nm as follows:
   where:
     =  mean of the series of readings,   x  =  value of the individual reading, and   n  =  number of readings.   Alternatively, rms noise may be estimated by noting the extreme differences 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, 7
SCOPE
1.1 This test method employs the signal-to-noise ratio to determine the sensitivity of a fluorescence measuring system in testing for the limit of detection (LOD) of quinine sulfate dihydrate in solution. The results obtained with quinine sulfate dihydrate in solution are suitable for specifying instrument performance on samples having excitation and fluorescence bands wider than 10 nm at or near room temperature.  
1.1.1 This test method is not intended to be used as (1) a rigorous test of performance of instrumentation, or (2), to intercompare the quantitative performance of instruments of different design. Intercomparison of the LOD between instruments is commonly expressed as the ratio of the water Raman peak intensity to the root-mean-square (rms) noise as measured on a fluorometer using an excitation wavelength of 350 nm This test method uses the excitation and emission peak wavelengths for quinine sulfate dihydrate in solution, which are approximately 350 nm and 450 nm, respectively.  
1.2 This test method has been applied to fluorescence-measuring systems utilizing non-laser, low-energy excitation sources. There is no assurance that extremely intense illumination will not cause photodecomposition2 of the compound suggested in this test method. For this reason, it is recommended that this test method not be indiscriminately employed with high intensity light sources. This test method is not intended to determine minimum detectable amounts of other materials. If this test method is extended to employ other chemical substances, the user should be aware of the possibility that these other substances may undergo decomposition or adsorption onto containers.  
1.3 A typical LOD for conventional fluorometers using this test method is 1 ng of quinine sulfate per mL.  
1.4 The suggested shelf life of a 1 mg/mL stock solution of quinine sulfate dihydrate is three months, when stored in the dark in a stoppered glass bottle.  
1.5 The values stated in SI units are to be regarded as standard. No o...

SIGNIFICANCE AND USE
3.1 Calibration of the responsivity of the detection system for emission (EM) as a function of EM wavelength (λEM), also referred to as spectral correction of emission, is necessary for successful quantification when intensity ratios at different EM wavelengths are being compared or when the true shape or peak maximum position of an EM spectrum needs to be known. Such calibration methods are given here and summarized in Table 1. This type of calibration is necessary because the spectral responsivity of a detection system can change significantly over its useful wavelength range (see Fig. 1). It is highly recommended that the wavelength accuracy (see Test Method E388) and the linear range of the detection system (see Guide E2719 and Test Method E578) be determined before spectral calibration is performed and that appropriate steps are taken to insure that all measured intensities during this calibration are within the linear range. For example, when using wide slit widths in the monochromators, attenuators may be needed to attenuate the excitation beam or emission, thereby, decreasing the fluorescence intensity at the detector. Also note that when using an EM polarizer, the spectral correction for emission is dependent on the polarizer setting. (2) It is important to use the same instrument settings for all of the calibration procedures mentioned here, as well as for subsequent sample measurements.  
FIG. 1 Example of Relative Spectral Responsivity of Emission Detection System (Grating Monochromator-PMT Based), (see Test Method E578) for which a Correction Needs to be Applied to a Measured Instrument-Specific Emission Spectrum to Obtain its True Spectral Shape (Relative Intensities).  
3.2 When using CCD or diode array detectors with a spectrometer for λEM selection, the spectral correction factors are dependent on the grating position of the spectrometer. Therefore, the spectral correction profile versus λEM must be determined separately for each grating position ...
SCOPE
1.1 This practice (1)2 describes three methods for determining the relative spectral correction factors for grating-based fluorescence spectrometers in the ultraviolet-visible spectral range. These methods are intended for instruments with a 0°/90° transmitting sample geometry. Each method uses different types of transfer standards, including 1) a calibrated light source (CS), 2) a calibrated detector (CD) and a calibrated diffuse reflector (CR), and 3) certified reference materials (CRMs). The wavelength region covered by the different methods ranges from 250 nm to 830 nm with some methods having a broader range than others. Extending these methods to the near infrared (NIR) beyond 830 nm will be discussed briefly, where appropriate. These methods were designed for scanning fluorescence spectrometers with a single channel detector, but can also be used with a multichannel detector, such as a diode array or a CCD.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

ABSTRACT
This test method covers the testing of the spectral bandwidth and wavelength accuracy of fluorescence spectrometers that use a monochromator for emission wavelength selection and photomultiplier tube detection. The method can be applied to instruments that use multi-element detectors, such as diode arrays, but results must be interpreted carefully. Atomic lines between 250 nm and 1000 nm are used in the method. The difference between the apparent wavelength and the known wavelength for a series of atomic emission lines is used as a test for wavelength accuracy. The apparent width of some of these lines is used as a test for spectral bandwidth.
SCOPE
1.1 This test method covers the testing of the spectral bandwidth and wavelength accuracy of fluorescence spectrometers that use a monochromator for emission wavelength selection and photomultiplier tube detection. This test method can be applied to instruments that use multi-element detectors, such as diode arrays, but results must be interpreted carefully. This test method uses atomic lines between 250 nm and 1000 nm.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 These practices are a source of general information on the techniques of ultraviolet and visible quantitative analyses. They provide the user with background information that should help ensure the reliability of spectrophotometric measurements.  
4.2 These practices are not intended as a substitute for a thorough understanding of any particular analytical method. It is the responsibility of the users to familiarize themselves with the critical details of a method and the proper operation of the available instrumentation.
SCOPE
1.1 These practices are intended to provide general information on the techniques most often used in ultraviolet and visible quantitative analysis. The purpose is to render unnecessary the repetition of these descriptions of techniques in individual methods for quantitative analysis.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 By following the general guidelines (Section 5) and instrument calibration methods (Sections 6 – 16) in this guide, users should be able to more easily conform to good laboratory and manufacturing practices (GXP) and comply with regulatory and QA/QC requirements, related to fluorescence measurements.  
4.2 Each instrument parameter needing calibration (for example, wavelength, spectral responsivity) is treated in a separate section. A list of different calibration methods is given for each instrument parameter with a brief usage procedure. Precautions, achievable precision and accuracy, and other useful information are also given for each method to allow users to make a more informed decision as to which method is the best choice for their calibration needs. Additional details for each method can be found in the references given.
SCOPE
1.1 This guide (1)2 lists the available materials and methods for each type of calibration or correction for fluorescence instruments (spectral emission correction, wavelength accuracy, and so forth) with a general description, the level of quality, precision and accuracy attainable, limitations, and useful references given for each entry.  
1.2 The listed materials and methods are intended for the qualification of fluorometers as part of complying with regulatory and other quality assurance/quality control (QA/QC) requirements.  
1.3 Precision and accuracy or uncertainty are given at a 1 σ confidence level and are approximated in cases where these values have not been well established.3  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Stray radiant power can be a significant source of error in spectrophotometric measurements. SRP usually increases with the passage of time; therefore, testing should be performed periodically. Moreover, the SRPR test is an excellent indicator of the overall condition of a spectrophotometer. A control-chart record of the results of routinely performed SRPR tests can be a useful indicator of need for corrective action or, at least, of the changing reliability of critical measurements.  
5.2 This test method provides a means of determining the stray radiant power ratio of a spectrophotometer at selected wavelengths in a spectral range, as determined by the SRP filter used, thereby revealing those wavelength regions where significant photometric errors might occur. It does not provide a means of calculating corrections to indicated absorbance (or transmittance) values. The test method must be used with care and understanding, as erroneous results can occur, especially with respect to some modern grating instruments that incorporate moderately narrow bandpass SRP-blocking filters. This test method does not provide a basis for comparing the performance of different spectrophotometers.
Note 8: Kaye (3) discusses correction methods of measured transmittances (absorbances) that sometimes can be used if sufficient information on the properties and performance of the instrument can be acquired. See also A1.2.5.  
5.3 This test method describes the performance of a spectrophotometer in terms of the specific test parameters used. When an analytical sample is measured, absorption by the sample of radiation outside of the nominal bandpass at the analytical wavelength can cause a photometric error, underestimating the transmittance or overestimating the absorbance, and correspondingly underestimating the SRPR.  
5.4 The SRPR indicated by this test method using SRP filters is almost always an underestimation of the true value (see 1.3). A value cited in a manufacturer’s li...
SCOPE
1.1 Stray radiant power (SRP) can be a significant source of error in spectrophotometric measurements, and the danger that such error exists is enhanced because its presence often is not suspected (1-4).2 This test method affords an estimate of the relative radiant power, that is, the Stray Radiant Power Ratio (SRPR), at wavelengths remote from those of the nominal bandpass transmitted through the monochromator of an absorption spectrophotometer. Test-filter materials are described that discriminate between the desired wavelengths and those that contribute most to SRP for conventional commercial spectrophotometers used in the ultraviolet, the visible, the near infrared, and the mid-infrared ranges. These procedures apply to instruments of conventional design, with usual sources, detectors, including array detectors, and optical arrangements. The vacuum ultraviolet and the far infrared present special problems that are not discussed herein.
Note 1: Research (3) has shown that particular care must be exercised in testing grating spectrophotometers that use moderately narrow bandpass SRP-blocking filters. Accurate calibration of the wavelength scale is critical when testing such instruments. Refer to Practice E275.  
1.2 These procedures are neither all-inclusive nor infallible. Because of the nature of readily available filter materials, with a few exceptions, the procedures are insensitive to SRP of very short wavelengths in the ultraviolet, or of lower frequencies in the infrared. Sharp cutoff longpass filters are available for testing for shorter wavelength SRP in the visible and the near infrared, and sharp cutoff shortpass filters are available for testing at longer visible wavelengths. The procedures are not necessarily valid for “spike” SRP nor for “nearby SRP.” (See Annexes for general discussion and definitions of these terms.) However, they are adequate in most cases and for typical applications. They do cover...

SIGNIFICANCE AND USE
4.1 This practice permits an analyst to compare the performance of an instrument to the manufacturer's supplied performance specifications and to verify its suitability for continued routine use. It also provides generation of calibration monitoring data on a periodic basis, forming a base from which any changes in the performance of the instrument will be evident.
SCOPE
1.1 This practice covers the parameters of spectrophotometric performance that are critical for testing the adequacy of instrumentation for most routine tests and methods2 within the wavelength range of 200 nm to 700 nm and the absorbance range of 0 to 2. The recommended tests provide a measurement of the important parameters controlling results in spectrophotometric methods, but it is specifically not to be inferred that all factors in instrument performance are measured.  
1.2 This practice may be used as a significant test of the performance of instruments for which the spectral bandwidth does not exceed 2 nm and for which the manufacturer's specifications for wavelength and absorbance accuracy do not exceed the performance tolerances employed here. This practice employs an illustrative tolerance of ±1 % relative for the error of the absorbance scale over the range of 0.2 to 2.0, and of ±1.0 nm for the error of the wavelength scale. A suggested maximum stray radiant power ratio of 4 × 10-4 yields E275 to extensively evaluate the performance of an instrument.  
1.3 This practice should be performed on a periodic basis, the frequency of which depends on the physical environment within which the instrumentation is used. Thus, units handled roughly or used under adverse conditions (exposed to dust, chemical vapors, vibrations, or combinations thereof) should be tested more frequently than those not exposed to such conditions. This practice should also be performed after any significant repairs are made on a unit, such as those involving the optics, detector, or radiant energy source.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This practice permits an analyst to compare the general performance of an instrument, as it is being used in a specific spectrophotometric method, with the performance of instruments used in developing the method.
SCOPE
1.1 This practice covers the description of requirements of spectrophotometric performance, especially for test methods, and the testing of the adequacy of available equipment for a specific method (for example, qualification for a given application). The tests give a measurement of some of the important parameters controlling results obtained in spectrophotometric methods, but it is specifically not to be concluded that all the factors in instrument performance are measured, or in fact may be required for a given application.  
1.1.1 This practice is primarily directed to dispersive spectrophotometers used for transmittance measurements rather than instruments designed for diffuse transmission and diffuse reflection.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 These practices should be used by a person who develops an analytical method to ensure that the spectral bandwidths cited in the practice are actually the ones used.
Note 2: The method developer should establish the spectral bandwidths that can be used to obtain satisfactory results.  
4.2 These practices should be used to determine whether a spectral bandwidth specified in a method can be realized with a given spectrophotometer and thus whether the instrument is suitable for use in this application. If accurate absorbance measurements are to be made on compounds with sharp absorption bands (natural half band widths of less than 15 nm) the spectral bandwidth of the spectrometer used should be better than 1/8th of the natural half band width of the compound’s absorption.  
4.3 These practices allow the user of a spectrophotometer to estimate the actual spectral bandwidth of the instrument under a given set of conditions and to compare the result to the spectral bandwidth calculated from data given in the manufacturer's literature or indicated by the instrument.
SCOPE
1.1 This practice describes procedures for estimating the spectral bandwidth of a spectrophotometer in the wavelength region of 185 nm to 820 nm.  
1.2 These practices are applicable to all modern spectrophotometer designs utilizing computer control and data handling. This includes conventional optical designs, where the sample is irradiated by monochromatic light, and ‘reverse’ optic designs coupled to photodiode arrays, where the light is separated by a polychromator after passing through the sample. For spectrophotometers that utilize servo-operated slits and maintain a constant period and a constant signal-to-noise ratio as the wavelength is automatically scanned, and/or utilize fixed slits and maintain a constant servo loop gain by automatically varying gain or dynode voltage, refer to the procedure described in Annex A1. This procedure is identical to that described in earlier versions of this practice.  
1.3 This practice does not cover the measurement of limiting spectral bandwidth, defined as the minimum spectral bandwidth achievable under optimum experimental conditions.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
3.1 The range of concentration of a fluorescing substance in solution over which the fluorescence varies linearly with the concentration is the range most useful for quantitative analysis. This range is affected by properties of the solution under analysis and by features of the measuring system. This test method provides a means of testing the performance of a fluorescence measuring system and of determining the concentration range over which the system is suitable for making a given quantitative analysis.  
3.2 This test method is not meant for comparing the performance of different fluorescence measuring instruments.
SCOPE
1.1 This test method covers a procedure for evaluating the limits of the linearity of response with fluorescence intensity of fluorescence-measuring systems under operating conditions. Particular attention is given to slit widths, filters, and sample containers. This test method can be used to test the overall linearity under a wide variety of instrumental and sampling conditions. The results obtained apply only to the tested combination of slit width and filters, and the size, type and illumination of the sample cuvette, all of which must be stated in the report. The sources of nonlinearity may be the measuring electronics, excessive absorption of either the exciting or emitted radiation, or both, and the sample handling technique, particularly at low concentrations.  
1.2 This test method has been applied to fluorescence-measuring systems utilizing continuous and low-energy excitation sources (for example, an excitation source of 450 W electrical input or less). There is no assurance that extremely intense illumination will not cause photodecomposition of the compounds suggested in this test method.2 For this reason it is recommended that this test method not be indiscriminately employed with high-intensity light sources. It is not a test method to determine the linearity of response of other materials. If this test method is extended to employ other chemical substances, the principles within can be applied, but new material parameters, such as the concentration range of linearity, must be established. The user should be aware of the possibility that these other substances may undergo decomposition, or adsorption onto containers.  
1.3 This test method has been applied to fluorescence-measuring systems utilizing a single detector, that is, a photomultiplier tube or a single photodiode. It has not been demonstrated if this method is effective for photo-array instruments such as those using a CCD or a diode array detector.  
1.4 This test method is applicable to 10 mm pathlength cuvette formats and instruments covering a wavelength range within 190 nm to 900 nm. The use of other sample formats has not been established with this test method.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 These practices are a source of general information on the techniques of ultraviolet and visible quantitative analyses. They provide the user with background information that should help ensure the reliability of spectrophotometric measurements.  
4.2 These practices are not intended as a substitute for a thorough understanding of any particular analytical method. It is the responsibility of the users to familiarize themselves with the critical details of a method and the proper operation of the available instrumentation.
SCOPE
1.1 These practices are intended to provide general information on the techniques most often used in ultraviolet and visible quantitative analysis. The purpose is to render unnecessary the repetition of these descriptions of techniques in individual methods for quantitative analysis.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
3.1 Calibration of the responsivity of the detection system for emission (EM) as a function of EM wavelength (λEM), also referred to as spectral correction of emission, is necessary for successful quantification when intensity ratios at different EM wavelengths are being compared or when the true shape or peak maximum position of an EM spectrum needs to be known. Such calibration methods are given here and summarized in Table 1. This type of calibration is necessary because the spectral responsivity of a detection system can change significantly over its useful wavelength range (see Fig. 1). It is highly recommended that the wavelength accuracy (see Test Method E388) and the linear range of the detection system (see Guide E2719 and Test Method E578) be determined before spectral calibration is performed and that appropriate steps are taken to insure that all measured intensities during this calibration are within the linear range. For example, when using wide slit widths in the monochromators, attenuators may be needed to attenuate the excitation beam or emission, thereby, decreasing the fluorescence intensity at the detector. Also note that when using an EM polarizer, the spectral correction for emission is dependent on the polarizer setting. (2) It is important to use the same instrument settings for all of the calibration procedures mentioned here, as well as for subsequent sample measurements.  
3.2 When using CCD or diode array detectors with a spectrometer for λEM selection, the spectral correction factors are dependent on the grating position of the spectrometer. Therefore, the spectral correction profile versus λEM must be determined separately for each grating position used. (3)  
3.3 Instrument manufacturers often provide an automated procedure and calculation for a spectral correction function for emission, or they may supply a correction that was determined at the factory. This correction can often be applied during spectral collection or as a post-co...
SCOPE
1.1 This practice (1)2 describes three methods for determining the relative spectral correction factors for grating-based fluorescence spectrometers in the ultraviolet-visible spectral range. These methods are intended for instruments with a 0°/90° transmitting sample geometry. Each method uses different types of transfer standards, including 1) a calibrated light source (CS), 2) a calibrated detector (CD) and a calibrated diffuse reflector (CR), and 3) certified reference materials (CRMs). The wavelength region covered by the different methods ranges from 250 to 830 nm with some methods having a broader range than others. Extending these methods to the near infrared (NIR) beyond 830 nm will be discussed briefly, where appropriate. These methods were designed for scanning fluorescence spectrometers with a single channel detector, but can also be used with a multichannel detector, such as a diode array or a CCD.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 When determining the limiting detectable concentration of a fluorescent substance, it is usually necessary to increase the readout scale of a photoelectric instrument to a point where noise (that is, random fluctuations of the system) becomes apparent. This noise will be superimposed upon the signal from the sample.  
4.2 In molecular fluorescence spectroscopy, the limit of detection for the sample will be determined by the limiting signal-to-noise ratio, S/N, where the signal, S, is the difference between readings obtained with the sample and blank solutions, and N is the total root-mean-square (rms) noise. The limit of detection for the sample will be given by the instrument readings that give a signal equal to three times the rms value of the noise.
Note 2: Factors other than noise affecting the sample concentration corresponding to the limit of detection include: the spectral bandwidths of the excitation and emission monochromators, the intensity of the exciting light that can be concentrated on the sample, the fraction of the fluorescence collected by the detection system, the response time of the detection system, and the purity of the solvent. The size and arrangement of the sample container with respect to the light beams are also important, as they affect both the desired signal and the extraneous signal that only contributes noise.
Note 3: The value of rms noise (N) can be obtained by calculating the standard deviation of a series of readings of the signal from the sample at the peak emission wavelength at approximately 450 nm as follows:
   where:  
   x  =  mean of the series of readings,   x  =  value of the individual reading, and   n  =  number of readings.   Alternatively, rms noise may be estimated by noting the extreme differences 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, 7
SCOPE
1.1 This test method employs the signal-to-noise ratio to determine the sensitivity of a fluorescence measuring system in testing for the limit of detection (LOD) of quinine sulfate dihydrate in solution. The results obtained with quinine sulfate dihydrate in solution are suitable for specifying instrument performance on samples having excitation and fluorescence bands wider than 10 nm at or near room temperature.  
1.1.1 This test method is not intended to be used as (1) a rigorous test of performance of instrumentation, or (2), to intercompare the quantitative performance of instruments of different design. Intercomparison of the LOD between instruments is commonly expressed as the ratio of the water Raman peak intensity to the root-mean-square (rms) noise as measured on a fluorometer using an excitation wavelength of 350 nm This test method uses the excitation and emission peak wavelengths for quinine sulfate dihydrate in solution, which are approximately 350 nm and 450 nm, respectively.  
1.2 This test method has been applied to fluorescence-measuring systems utilizing non-laser, low-energy excitation sources. There is no assurance that extremely intense illumination will not cause photodecomposition2 of the compound suggested in this test method. For this reason, it is recommended that this test method not be indiscriminately employed with high intensity light sources. This test method is not intended to determine minimum detectable amounts of other materials. If this test method is extended to employ other chemical substances, the user should be aware of the possibility that these other substances may undergo decomposition or adsorption onto containers.  
1.3 A typical LOD for conventional fluorometers using this test method is 1 ng of quinine sulfate per mL.  
1.4 The suggested shelf life of a 1 mg/mL stock solution of quinine sulfate dihydrate is three months, when stored in the dark in a stoppered glass bottle.  
1.5 The values stated in SI units are to be regarded as standard. No...

ABSTRACT
This test method covers the testing of the spectral bandwidth and wavelength accuracy of fluorescence spectrometers that use a monochromator for emission wavelength selection and photomultiplier tube detection. The method can be applied to instruments that use multi-element detectors, such as diode arrays, but results must be interpreted carefully. Atomic lines between 250 nm and 1000 nm are used in the method. The difference between the apparent wavelength and the known wavelength for a series of atomic emission lines is used as a test for wavelength accuracy. The apparent width of some of these lines is used as a test for spectral bandwidth.
SCOPE
1.1 This test method covers the testing of the spectral bandwidth and wavelength accuracy of fluorescence spectrometers that use a monochromator for emission wavelength selection and photomultiplier tube detection. This test method can be applied to instruments that use multi-element detectors, such as diode arrays, but results must be interpreted carefully. This test method uses atomic lines between 250 nm and 1000 nm.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
5.1 Stray radiant power can be a significant source of error in spectrophotometric measurements. SRP usually increases with the passage of time; therefore, testing should be performed periodically. Moreover, the SRPR test is an excellent indicator of the overall condition of a spectrophotometer. A control-chart record of the results of routinely performed SRPR tests can be a useful indicator of need for corrective action or, at least, of the changing reliability of critical measurements.  
5.2 This test method provides a means of determining the stray radiant power ratio of a spectrophotometer at selected wavelengths in a spectral range, as determined by the SRP filter used, thereby revealing those wavelength regions where significant photometric errors might occur. It does not provide a means of calculating corrections to indicated absorbance (or transmittance) values. The test method must be used with care and understanding, as erroneous results can occur, especially with respect to some modern grating instruments that incorporate moderately narrow bandpass SRP-blocking filters. This test method does not provide a basis for comparing the performance of different spectrophotometers.
Note 8: Kaye (3) discusses correction methods of measured transmittances (absorbances) that sometimes can be used if sufficient information on the properties and performance of the instrument can be acquired. See also A1.2.5.  
5.3 This test method describes the performance of a spectrophotometer in terms of the specific test parameters used. When an analytical sample is measured, absorption by the sample of radiation outside of the nominal bandpass at the analytical wavelength can cause a photometric error, underestimating the transmittance or overestimating the absorbance, and correspondingly underestimating the SRPR.  
5.4 The SRPR indicated by this test method using SRP filters is almost always an underestimation of the true value (see 1.3). A value cited in a manufacturer’s li...
SCOPE
1.1 Stray radiant power (SRP) can be a significant source of error in spectrophotometric measurements, and the danger that such error exists is enhanced because its presence often is not suspected (1-4).2 This test method affords an estimate of the relative radiant power, that is, the Stray Radiant Power Ratio (SRPR), at wavelengths remote from those of the nominal bandpass transmitted through the monochromator of an absorption spectrophotometer. Test-filter materials are described that discriminate between the desired wavelengths and those that contribute most to SRP for conventional commercial spectrophotometers used in the ultraviolet, the visible, the near infrared, and the mid-infrared ranges. These procedures apply to instruments of conventional design, with usual sources, detectors, including array detectors, and optical arrangements. The vacuum ultraviolet and the far infrared present special problems that are not discussed herein.
Note 1: Research (3) has shown that particular care must be exercised in testing grating spectrophotometers that use moderately narrow bandpass SRP-blocking filters. Accurate calibration of the wavelength scale is critical when testing such instruments. Refer to Practice E275.  
1.2 These procedures are neither all-inclusive nor infallible. Because of the nature of readily available filter materials, with a few exceptions, the procedures are insensitive to SRP of very short wavelengths in the ultraviolet, or of lower frequencies in the infrared. Sharp cutoff longpass filters are available for testing for shorter wavelength SRP in the visible and the near infrared, and sharp cutoff shortpass filters are available for testing at longer visible wavelengths. The procedures are not necessarily valid for “spike” SRP nor for “nearby SRP.” (See Annexes for general discussion and definitions of these terms.) However, they are adequate in most cases and for typical applications. They do cover...

SIGNIFICANCE AND USE
4.1 These practices are a source of general information on the techniques of ultraviolet and visible quantitative analyses. They provide the user with background information that should help ensure the reliability of spectrophotometric measurements.  
4.2 These practices are not intended as a substitute for a thorough understanding of any particular analytical method. It is the responsibility of the users to familiarize themselves with the critical details of a method and the proper operation of the available instrumentation.
SCOPE
1.1 These practices are intended to provide general information on the techniques most often used in ultraviolet and visible quantitative analysis. The purpose is to render unnecessary the repetition of these descriptions of techniques in individual methods for quantitative analysis.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 By following the general guidelines (Section 5) and instrument calibration methods (Sections 6 – 16) in this guide, users should be able to more easily conform to good laboratory and manufacturing practices (GXP) and comply with regulatory and QA/QC requirements, related to fluorescence measurements.  
4.2 Each instrument parameter needing calibration (for example, wavelength, spectral responsivity) is treated in a separate section. A list of different calibration methods is given for each instrument parameter with a brief usage procedure. Precautions, achievable precision and accuracy, and other useful information are also given for each method to allow users to make a more informed decision as to which method is the best choice for their calibration needs. Additional details for each method can be found in the references given.
SCOPE
1.1 This guide (1)2 lists the available materials and methods for each type of calibration or correction for fluorescence instruments (spectral emission correction, wavelength accuracy, and so forth) with a general description, the level of quality, precision and accuracy attainable, limitations, and useful references given for each entry.  
1.2 The listed materials and methods are intended for the qualification of fluorometers as part of complying with regulatory and other quality assurance/quality control (QA/QC) requirements.  
1.3 Precision and accuracy or uncertainty are given at a 1 σ confidence level and are approximated in cases where these values have not been well established.3  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This practice permits an analyst to compare the performance of an instrument to the manufacturer's supplied performance specifications and to verify its suitability for continued routine use. It also provides generation of calibration monitoring data on a periodic basis, forming a base from which any changes in the performance of the instrument will be evident.
SCOPE
1.1 This practice covers the parameters of spectrophotometric performance that are critical for testing the adequacy of instrumentation for most routine tests and methods2 within the wavelength range of 200 to 700 nm and the absorbance range of 0 to 2. The recommended tests provide a measurement of the important parameters controlling results in spectrophotometric methods, but it is specifically not to be inferred that all factors in instrument performance are measured.  
1.2 This practice may be used as a significant test of the performance of instruments for which the spectral bandwidth does not exceed 2 nm and for which the manufacturer's specifications for wavelength and absorbance accuracy do not exceed the performance tolerances employed here. This practice employs an illustrative tolerance of ±1 % relative for the error of the absorbance scale over the range of 0.2 to 2.0, and of ±1.0 nm for the error of the wavelength scale. A suggested maximum stray radiant power ratio of 4 × 10-4 yields  
1.3 This practice should be performed on a periodic basis, the frequency of which depends on the physical environment within which the instrumentation is used. Thus, units handled roughly or used under adverse conditions (exposed to dust, chemical vapors, vibrations, or combinations thereof) should be tested more frequently than those not exposed to such conditions. This practice should also be performed after any significant repairs are made on a unit, such as those involving the optics, detector, or radiant energy source.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
3.1 The range of concentration of a fluorescing substance in solution over which the fluorescence varies linearly with the concentration is the range most useful for quantitative analysis. This range is affected by properties of the solution under analysis and by features of the measuring system. This test method provides a means of testing the performance of a fluorescence measuring system and of determining the concentration range over which the system is suitable for making a given quantitative analysis.  
3.2 This test method is not meant for comparing the performance of different fluorescence measuring instruments.
SCOPE
1.1 This test method covers a procedure for evaluating the limits of the linearity of response with fluorescence intensity of fluorescence-measuring systems under operating conditions. Particular attention is given to slit widths, filters, and sample containers. This test method can be used to test the overall linearity under a wide variety of instrumental and sampling conditions. The results obtained apply only to the tested combination of slit width and filters, and the size, type and illumination of the sample cuvette, all of which must be stated in the report. The sources of nonlinearity may be the measuring electronics, excessive absorption of either the exciting or emitted radiation, or both, and the sample handling technique, particularly at low concentrations.  
1.2 This test method has been applied to fluorescence-measuring systems utilizing continuous and low-energy excitation sources (for example, an excitation source of 450-W electrical input or less). There is no assurance that extremely intense illumination will not cause photodecomposition of the compounds suggested in this test method.2 For this reason it is recommended that this test method not be indiscriminately employed with high-intensity light sources. It is not a test method to determine the linearity of response of other materials. If this test method is extended to employ other chemical substances, the principles within can be applied, but new material parameters, such as the concentration range of linearity, must be established. The user should be aware of the possibility that these other substances may undergo decomposition, or adsorption onto containers.  
1.3 This test method has been applied to fluorescence-measuring systems utilizing a single detector, that is, a photomultiplier tube or a single photodiode. It has not been demonstrated if this method is effective for photo-array instruments such as those using a CCD or a diode array detector.  
1.4 This test method is applicable to 10-mm pathlength cuvette formats and instruments covering a wavelength range within 190 to 900 nm. The use of other sample formats has not been established with this test method.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 These practices should be used by a person who develops an analytical method to ensure that the spectral bandwidths cited in the practice are actually the ones used.Note 2—The method developer should establish the spectral bandwidths that can be used to obtain satisfactory results.  
4.2 These practices should be used to determine whether a spectral bandwidth specified in a method can be realized with a given spectrophotometer and thus whether the instrument is suitable for use in this application. If accurate absorbance measurements are to be made on compounds with sharp absorption bands (natural half band widths of less than 15 nm) the spectral bandwidth of the spectrometer used should be better than 1/8th of the natural half band width of the compound’s absorption.  
4.3 These practices allow the user of a spectrophotometer to estimate the actual spectral bandwidth of the instrument under a given set of conditions and to compare the result to the spectral bandwidth calculated from data given in the manufacturer's literature or indicated by the instrument.
SCOPE
1.1 This practice describes procedures for estimating the spectral bandwidth of a spectrophotometer in the wavelength region of 185 to 820 nm.  
1.2 These practices are applicable to all modern spectrophotometer designs utilizing computer control and data handling. This includes conventional optical designs, where the sample is irradiated by monochromatic light, and ‘reverse’ optic designs coupled to photodiode arrays, where the light is separated by a polychromator after passing through the sample. For spectrophotometers that utilize servo-operated slits and maintain a constant period and a constant signal-to-noise ratio as the wavelength is automatically scanned, and/or utilize fixed slits and maintain a constant servo loop gain by automatically varying gain or dynode voltage, refer to the procedure described in Annex A1. This procedure is identical to that described in earlier versions of this practice.  
1.3 This practice does not cover the measurement of limiting spectral bandwidth, defined as the minimum spectral bandwidth achievable under optimum experimental conditions.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This practice permits an analyst to compare the general performance of an instrument, as it is being used in a specific spectrophotometric method, with the performance of instruments used in developing the method.
SCOPE
1.1 This practice covers the description of requirements of spectrophotometric performance, especially for test methods, and the testing of the adequacy of available equipment for a specific method (for example, qualification for a given application). The tests give a measurement of some of the important parameters controlling results obtained in spectrophotometric methods, but it is specifically not to be concluded that all the factors in instrument performance are measured, or in fact may be required for a given application.  
1.1.1 This practice is primarily directed to dispersive spectrophotometers used for transmittance measurements rather than instruments designed for diffuse transmission and diffuse reflection.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
By following the general guidelines (Section 5) and instrument calibration methods (Sections 6-16) in this guide, users should be able to more easily conform to good laboratory and manufacturing practices (GXP) and comply with regulatory and QA/QC requirements, related to fluorescence measurements.
Each instrument parameter needing calibration (for example, wavelength, spectral responsivity) is treated in a separate section. A list of different calibration methods is given for each instrument parameter with a brief usage procedure. Precautions, achievable precision and accuracy, and other useful information are also given for each method to allow users to make a more informed decision as to which method is the best choice for their calibration needs. Additional details for each method can be found in the references given.
SCOPE
1.1 This guide (1) lists the available materials and methods for each type of calibration or correction for fluorescence instruments (spectral emission correction, wavelength accuracy, and so forth) with a general description, the level of quality, precision and accuracy attainable, limitations, and useful references given for each entry.
1.2 The listed materials and methods are intended for the qualification of fluorometers as part of complying with regulatory and other quality assurance/quality control (QA/QC) requirements.
1.3 Precision and accuracy or uncertainty are given at a 1 σ confidence level and are approximated in cases where these values have not been well established.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

ABSTRACT
This test method covers the testing of the spectral bandwidth and wavelength accuracy of fluorescence spectrometers that use a monochromator for emission wavelength selection and photomultiplier tube detection. The method can be applied to instruments that use multi-element detectors, such as diode arrays, but results must be interpreted carefully. Atomic lines between 250 nm and 1000 nm are used in the method. The difference between the apparent wavelength and the known wavelength for a series of atomic emission lines is used as a test for wavelength accuracy. The apparent width of some of these lines is used as a test for spectral bandwidth.
SCOPE
1.1 This test method covers the testing of the spectral bandwidth and wavelength accuracy of fluorescence spectrometers that use a monochromator for emission wavelength selection and photomultiplier tube detection. This test method can be applied to instruments that use multi-element detectors, such as diode arrays, but results must be interpreted carefully. This test method uses atomic lines between 250 nm and 1000 nm.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
These practices are a source of general information on the techniques of ultraviolet and visible quantitative analyses. They provide the user with background information that should help ensure the reliability of spectrophotometric measurements.
These practices are not intended as a substitute for a thorough understanding of any particular analytical method. It is the responsibility of the users to familiarize themselves with the critical details of a method and the proper operation of the available instrumentation.
SCOPE
1.1 These practices are intended to provide general information on the techniques most often used in ultraviolet and visible quantitative analysis. The purpose is to render unnecessary the repetition of these descriptions of techniques in individual methods for quantitative analysis.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
Stray radiant power can be a significant source of error in spectrophotometric measurements. SRP usually increases with the passage of time; therefore, testing should be performed periodically. Moreover, the SRPR test is an excellent indicator of the overall condition of a spectrophotometer. A control-chart record of the results of routinely performed SRPR tests can be a useful indicator of need for corrective action or, at least, of the changing reliability of critical measurements.
This test method provides a means of determining the stray radiant power ratio of a spectrophotometer at selected wavelengths in a spectral range, as determined by the SRP filter used, thereby revealing those wavelength regions where significant photometric errors might occur. It does not provide a means of calculating corrections to indicated absorbance (or transmittance) values. The test method must be used with care and understanding, as erroneous results can occur, especially with respect to some modern grating instruments that incorporate moderately narrow bandpass SRP-blocking filters. This test method does not provide a basis for comparing the performance of different spectrophotometers.
Note 8—Kaye (3) discusses correction methods of measured transmittances (absorbances) that sometimes can be used if sufficient information on the properties and performance of the instrument can be acquired. See also A1.2.5.
This test method describes the performance of a spectrophotometer in terms of the specific test parameters used. When an analytical sample is measured, absorption by the sample of radiation outside of the nominal bandpass at the analytical wavelength can cause a photometric error, underestimating the transmittance or overestimating the absorbance, and correspondingly underestimating the SRPR.
The SRPR indicated by this test method using SRP filters is almost always an underestimation of the true value (see 1.3). A value cited in a manufacturer’s literature represents...
SCOPE
1.1 Stray radiant power (SRP) can be a significant source of error in spectrophotometric measurements, and the danger that such error exists is enhanced because its presence often is not suspected (1-4). This test method affords an estimate of the relative radiant power, that is, the Stray Radiant Power Ratio (SRPR), at wavelengths remote from those of the nominal bandpass transmitted through the monochromator of an absorption spectrophotometer. Test-filter materials are described that discriminate between the desired wavelengths and those that contribute most to SRP for conventional commercial spectrophotometers used in the ultraviolet, the visible, the near infrared, and the mid-infrared ranges. These procedures apply to instruments of conventional design, with usual sources, detectors, including array detectors, and optical arrangements. The vacuum ultraviolet and the far infrared present special problems that are not discussed herein.
Note 1—Research (3) has shown that particular care must be exercised in testing grating spectrophotometers that use moderately narrow bandpass SRP-blocking filters. Accurate calibration of the wavelength scale is critical when testing such instruments. Refer to Practice E275.
1.2 These procedures are neither all-inclusive nor infallible. Because of the nature of readily available filter materials, with a few exceptions, the procedures are insensitive to SRP of very short wavelengths in the ultraviolet, or of lower frequencies in the infrared. Sharp cutoff longpass filters are available for testing for shorter wavelength SRP in the visible and the near infrared, and sharp cutoff shortpass filters are available for testing at longer visible wavelengths. The procedures are not necessarily valid for “spike” SRP nor for “nearby SRP.” (See Annexes for general discussion and definitions of these terms.) However, they are adequate in most cases and for typical applications. They do cover i...

SIGNIFICANCE AND USE
When determining the limiting detectable concentration of a fluorescent substance, it is usually necessary to increase the readout scale of a photoelectric instrument to a point where noise (that is, random fluctuations of the system) becomes apparent. This noise will be superimposed upon the signal from the sample.
In molecular fluorescence spectroscopy, the limit of detection for the sample will be determined by the limiting signal-to-noise ratio, S/N, where the signal, S, is the difference between readings obtained with the sample and blank solutions, and N is the total root-mean-square (rms) noise. The limit of detection for the sample will be given by the instrument readings that give a signal equal to three times the rms value of the noise.
Note 2—Factors other than noise affecting the sample concentration corresponding to the limit of detection include: the spectral bandwidths of the excitation and emission monochromators, the intensity of the exciting light that can be concentrated on the sample, the fraction of the fluorescence collected by the detection system, the response time of the detection system, and the purity of the solvent. The size and arrangement of the sample container with respect to the light beams are also important, as they affect both the desired signal and the extraneous signal that only contributes noise.
SCOPE
1.1 This test method employs the signal-to-noise ratio to determine the sensitivity of a fluorescence measuring system in testing for the limit of detection (LOD) of quinine sulfate dihydrate in solution. The results obtained with quinine sulfate dihydrate in solution are suitable for specifying instrument performance on samples having excitation and fluorescence bands wider than 10 nm at or near room temperature.
1.1.1 This test method is not intended to be used as (1) a rigorous test of performance of instrumentation, or (2), to intercompare the quantitative performance of instruments of different design. Intercomparison of the LOD between instruments is commonly expressed as the ratio of the water Raman peak intensity to the root-mean-square (rms) noise as measured on a fluorometer using an excitation wavelength of 350 nm This test method uses the excitation and emission peak wavelengths for quinine sulfate dihydrate in solution, which are approximately 350 nm and 450 nm, respectively..
1.2 This test method has been applied to fluorescence-measuring systems utilizing non-laser, low-energy excitation sources. There is no assurance that extremely intense illumination will not cause photodecomposition of the compound suggested in this test method. For this reason, it is recommended that this test method not be indiscriminately employed with high intensity light sources. This test method is not intended to determine minimum detectable amounts of other materials. If this test method is extended to employ other chemical substances, the user should be aware of the possibility that these other substances may undergo decomposition or adsorption onto containers.
1.3 A typical LOD for conventional fluorometers using this test method is 1 ng of quinine sulfate per mL.
1.4 The suggested shelf life of a 1 mg/mL stock solution of quinine sulfate dihydrate is three months, when stored in the dark in a stoppered glass bottle.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
This practice permits an analyst to compare the performance of an instrument to the manufacturer's supplied performance specifications and to verify its suitability for continued routine use. It also provides generation of calibration monitoring data on a periodic basis, forming a base from which any changes in the performance of the instrument will be evident.
SCOPE
1.1 This practice covers the parameters of spectrophotometric performance that are critical for testing the adequacy of instrumentation for most routine tests and methods within the wavelength range of 200 to 700 nm and the absorbance range of 0 to 2. The recommended tests provide a measurement of the important parameters controlling results in spectrophotometric methods, but it is specifically not to be inferred that all factors in instrument performance are measured.
1.2 This practice may be used as a significant test of the performance of instruments for which the spectral bandwidth does not exceed 2 nm and for which the manufacturer's specifications for wavelength and absorbance accuracy do not exceed the performance tolerances employed here. This practice employs an illustrative tolerance of ±1 % relative for the error of the absorbance scale over the range of 0.2 to 2.0, and of ±1.0 nm for the error of the wavelength scale. A suggested maximum stray radiant power ratio of 4 × 10-4 yields 1 % absorbance bias at an absorbance of 2. These tolerances are chosen to be compatible with many chemical applications while comfortably exceeding the uncertainty of the certified values for the reference materials and typical manufacturer's specifications for error in the wavelength and absorbance scales of the instrument under test. The user is encouraged to develop and use tolerance values more appropriate to the requirements of the end use application. This procedure is designed to verify quantitative performance on an ongoing basis and to compare one instrument's performance with that of other similar units. Refer to Practice E275 to extensively evaluate the performance of an instrument.
1.3 This practice should be performed on a periodic basis, the frequency of which depends on the physical environment within which the instrumentation is used. Thus, units handled roughly or used under adverse conditions (exposed to dust, chemical vapors, vibrations, or combinations thereof) should be tested more frequently than those not exposed to such conditions. This practice should also be performed after any significant repairs are made on a unit, such as those involving the optics, detector, or radiant energy source.
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
This practice permits an analyst to compare the general performance of an instrument, as it is being used in a specific spectrophotometric method, with the performance of instruments used in developing the method.
SCOPE
1.1 This practice covers the description of requirements of spectrophotometric performance, especially for test methods, and the testing of the adequacy of available equipment for a specific method (for example, qualification for a given application). The tests give a measurement of some of the important parameters controlling results obtained in spectrophotometric methods, but it is specifically not to be concluded that all the factors in instrument performance are measured, or in fact may be required for a given application.
1.1.1 This practice is primarily directed to dispersive spectrophotometers used for transmittance measurements rather than instruments designed for diffuse transmission and diffuse reflection.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
The range of concentration of a fluorescing substance in solution over which the fluorescence varies linearly with the concentration is the range most useful for quantitative analysis. This range is affected by properties of the solution under analysis and by features of the measuring system. This test method provides a means of testing the performance of a fluorescence measuring system and of determining the concentration range over which the system is suitable for making a given quantitative analysis.
This test method is not meant for comparing the performance of different fluorescence measuring instruments.
SCOPE
1.1 This test method covers a procedure for evaluating the limits of the linearity of response with fluorescence intensity of fluorescence-measuring systems under operating conditions. Particular attention is given to slit widths, filters, and sample containers. This test method can be used to test the overall linearity under a wide variety of instrumental and sampling conditions. The results obtained apply only to the tested combination of slit width and filters, and the size, type and illumination of the sample cuvette, all of which must be stated in the report. The sources of nonlinearity may be the measuring electronics, excessive absorption of either the exciting or emitted radiation, or both, and the sample handling technique, particularly at low concentrations.
1.2 This test method has been applied to fluorescence-measuring systems utilizing continuous and low-energy excitation sources (for example, an excitation source of 450-W electrical input or less). There is no assurance that extremely intense illumination will not cause photodecomposition of the compounds suggested in this test method. For this reason it is recommended that this test method not be indiscriminately employed with high-intensity light sources. It is not a test method to determine the linearity of response of other materials. If this test method is extended to employ other chemical substances, the principles within can be applied, but new material parameters, such as the concentration range of linearity, must be established. The user should be aware of the possibility that these other substances may undergo decomposition, or adsorption onto containers.
1.3 This test method has been applied to fluorescence-measuring systems utilizing a single detector, that is, a photomultiplier tube or a single photodiode. It has not been demonstrated if this method is effective for photo-array instruments such as those using a CCD or a diode array detector.
1.4 This test method is applicable to 10-mm pathlength cuvette formats and instruments covering a wavelength range within 190 to 900 nm. The use of other sample formats has not been established with this test method.
This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
This practice should be used by a person who develops an analytical method to ensure that the spectral bandwidths cited in the practice are actually the ones used.
Note 1—The method developer should establish the spectral bandwidths that can be used to obtain satisfactory results.
This practice should be used to determine whether a spectral bandwidth specified in a method can be realized with a given spectrophotometer and thus whether the instrument is suitable for use in this application.
This practice allows the user of a spectrophotometer to determine the actual spectral bandwidth of the instrument under a given set of conditions and to compare the result to the spectral bandwidth calculated from data given in the manufacturer’literature or indicated by the instrument.
Instrument manufacturers can use this practice to measure and describe the practical spectral bandwidth of an instrument over its entire wavelength operating range. This practice is highly prefered to the general practice of stating the limiting or the theoretical spectral bandwidth at a single wavelength.
SCOPE
1.1 This practice describes a procedure for measuring the practical spectral bandwidth of a spectrophotometer in the wavelength region of 185 to 820 nm. Practical spectral bandwidth is the spectral bandwidth of an instrument operated at a given integration period and a given signal-to-noise ratio.
1.2 This practice is applicable to instruments that utilize servo-operated slits and maintain a constant period and a constant signal-to-noise ratio as the wavelength is automatically scanned. It is also applicable to instruments that utilize fixed slits and maintain a constant servo loop gain by automatically varying gain or dynode voltage. In this latter case, the signal-to-noise ratio varies with wavelength. It can also be used on instruments that utilize some combination of the two designs, as well as on those that vary the period during the scan. For digitized instruments, refer to the manufacturer's manual.
1.3 This practice does not cover the measurement of limiting spectral bandwidth, defined as the minimum spectral bandwidth achievable under optimum experimental conditions.
This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
These practices are a source of general information on the techniques of ultraviolet and visible quantitative analyses. They provide the user with background information that should help ensure the reliability of spectrophotometric measurements.
These practices are not intended as a substitute for a thorough understanding of any particular analytical method. It is the responsibility of the users to familiarize themselves with the critical details of a method and the proper operation of the available instrumentation.
SCOPE
1.1 These practices are intended to provide general information on the techniques most often used in ultraviolet and visible quantitative analysis. The purpose is to render unnecessary the repetition of these descriptions of techniques in individual methods for quantitative analysis.  
1.2 This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of whoever uses this standard to consult and establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

ABSTRACT
This test method covers the testing of the spectral bandwidth and wavelength accuracy of fluorescence spectrometers that use a monochromator for emission wavelength selection and photomultiplier tube detection. The method can be applied to instruments that use multi-element detectors, such as diode arrays, but results must be interpreted carefully. Atomic lines between 250 nm and 1000 nm are used in the method. The difference between the apparent wavelength and the known wavelength for a series of atomic emission lines is used as a test for wavelength accuracy. The apparent width of some of these lines is used as a test for spectral bandwidth.
SCOPE
1.1 This test method covers the testing of the spectral bandwidth and wavelength accuracy of fluorescence spectrometers that use a monochromator for emission wavelength selection and photomultiplier tube detection. This test method can be applied to instruments that use multi-element detectors, such as diode arrays, but results must be interpreted carefully. This test method uses atomic lines between 250 nm and 1000 nm.
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SCOPE
1.1 Stray radiant power (SRP) can be a significant source of error in spectrophotometric measurements, and the danger that such error exists is enhanced because its presence often is not suspected (). This test method affords an estimate of the relative radiant power, that is, the Stray Radiant Power Ratio (SRPR), at wavelengths remote from those of the nominal bandpass transmitted through the monochromator of an absorption spectrophotometer. Test-filter materials are described that discriminate between the desired wavelengths and those that contribute most to SRP for conventional commercial spectrophotometers used in the ultraviolet, the visible, the near infrared, and the mid-infrared ranges. These procedures apply to instruments of conventional design, with usual sources, detectors, including array detectors, and optical arrangements. The vacuum ultraviolet and the far infrared present special problems that are not discussed herein.Note 1
Research () has shown that particular care must be exercised in testing grating spectrophotometers that use moderately narrow bandpass SRP-blocking filters. Accurate calibration of the wavelength scale is critical when testing such instruments. Refer to Practice E 275.
1.2 These procedures are neither all-inclusive nor infallible. Because of the nature of readily available filter materials, with a few exceptions, the procedures are insensitive to SRP of very short wavelengths in the ultraviolet, or of lower frequencies in the infrared. Sharp cutoff longpass filters are available for testing for shorter wavelength SRP in the visible and the near infrared, and sharp cutoff shortpass filters are available for testing at longer visible wavelengths. The procedures are not necessarily valid for "spike" SRP nor for "nearby SRP." (See Annexes for general discussion and definitions of these terms.) However, they are adequate in most cases and for typical applications. They do cover instruments using prisms or gratings in either single or double monochromators, and with single and double beam instruments.Note 2
Instruments with array detectors are inherently prone to having higher levels of SRP. See Annexes for the use of filters to reduce SRP.
1.3 The proportion of SRP (that is, SRPR) encountered with a well-designed monochromator, used in a favorable spectral region, typically is 0.1 % transmittance or better, and with a double monochromator it can be less than 110-6, even with a broadband continuum source. Under these conditions, it may be difficult to do more than determine that it falls below a certain level. Because SRP test filters always absorb some of the SRP, and may absorb an appreciable amount if the specified measurement wavelength is not very close to the cutoff wavelength of the SRP filter, this test method underestimates the true SRPR. However, actual measurement sometimes requires special techniques and instrument operating conditions that are not typical of those occurring during use. When absorption measurements with continuum sources are being made, it can be that, owing to the effect of slit width on SRP in a double monochromator, these test procedures may offset in some degree the effect of absorption by the SRP filter; that is, because larger slit widths than normal might be used to admit enough energy to the monochromator to permit evaluation of the SRP, the stray proportion indicated could be greater than would normally be encountered in use (but the net effect is still more likely to be an underestimation of the true SRPR). Whether the indicated SRPR equals or differs from the normal-use value depends on how much the SRP is increased with the wider slits and on how much of the SRP is absorbed by the SRP filter. What must be accepted is that the numerical value obtained for the SRPR is a characteristic of the particular test conditions as well as of the performance of the instrument in normal use. It is an indication of whether high absorbance measuremen...

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1.1 This test method employs the signal-to-noise ratio to determine the sensitivity of a fluorescence measuring system in testing for the limit of detection (LOD) of quinine sulfate dihydrate in solution. The results obtained with quinine sulfate dihydrate in solution are suitable for specifying instrument performance on samples having excitation and fluorescence bands wider than 10 nm at or near room temperature.
1.1.1 This test method is not intended to be used as (1) a rigorous test of performance of instrumentation, or (2), to intercompare the quantitative performance of instruments of different design.Intercomparison of the LOD between instruments is commonly expressed as the ratio of the water Raman peak intensity to the root-mean-square (rms) noise as measured on a fluorometer using an excitation wavelength of 350 nm This test method uses the excitation and emission peak wavelengths for quinine sulfate dihydrate in solution, which are approximately 350 nm and 450 nm, respectively..
1.2 This test method has been applied to fluorescence-measuring systems utilizing non-laser, low-energy excitation sources. There is no assurance that extremely intense illumination will not cause photodecomposition of the compound suggested in this test method. For this reason, it is recommended that this test method not be indiscriminately employed with high intensity light sources. This test method is not intended to determine minimum detectable amounts of other materials. If this test method is extended to employ other chemical substances, the user should be aware of the possibility that these other substances may undergo decomposition or adsorption onto containers.
1.3 A typical LOD for conventional fluorometers using this test method is 1 ng of quinine sulfate per mL.
1.4 The suggested shelf life of a 1 mg/mL stock solution of quinine sulfate dihydrate is three months, when stored in the dark in a stoppered glass bottle.
1.5 This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

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1.1 This practice covers the parameters of spectrophotometric performance that are critical for testing the adequacy of instrumentation for most routine tests and methods within the wavelength range of 200 to 700 nm and the absorbance range of 0 to 2. The recommended tests provide a measurement of the important parameters controlling results in spectrophotometric methods, but it is specifically not to be inferred that all factors in instrument performance are measured.
1.2 This practice may be used as a significant test of the performance of instruments for which the spectral slit width does not exceed 2 nm and for which the manufacturer's specifications for wavelength and absorbance accuracy do not exceed the performance tolerances employed here. This practice employs an illustrative tolerance of ± 1 % relative for the error of the absorbance scale over the range of 0.2 to 2.0, and of 1.0 nm for the error of the wavelength scale. A suggested maximum stray radiant power ratio of 4 x 10-4 yields
1.3 This practice should be performed on a periodic basis, the frequency of which depends on the physical environment within which the instrumentation is used. Thus, units handled roughly or used under adverse conditions (exposed to dust, chemical vapors, vibrations, or combinations thereof) should be tested more frequently than those not exposed to such conditions. This practice should also be performed after any significant repairs are made on a unit, such as those involving the optics, detector, or radiant energy source.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
This practice permits an analyst to compare the performance of an instrument to the manufacturer’supplied performance specifications and to verify its suitability for continued routine use. It also provides generation of calibration monitoring data on a periodic basis, forming a base from which any changes in the performance of the instrument will be evident.
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1.1 This practice covers the parameters of spectrophotometric performance that are critical for testing the adequacy of instrumentation for most routine tests and methods within the wavelength range of 200 to 700 nm and the absorbance range of 0 to 2. The recommended tests provide a measurement of the important parameters controlling results in spectrophotometric methods, but it is specifically not to be inferred that all factors in instrument performance are measured.
1.2 This practice may be used as a significant test of the performance of instruments for which the spectral slit width does not exceed 2 nm and for which the manufacturer's specifications for wavelength and absorbance accuracy do not exceed the performance tolerances employed here. This practice employs an illustrative tolerance of ± 1 % relative for the error of the absorbance scale over the range of 0.2 to 2.0, and of 1.0 nm for the error of the wavelength scale. A suggested maximum stray radiant power ratio of 4 x 10-4 yields
1.3 This practice should be performed on a periodic basis, the frequency of which depends on the physical environment within which the instrumentation is used. Thus, units handled roughly or used under adverse conditions (exposed to dust, chemical vapors, vibrations, or combinations thereof) should be tested more frequently than those not exposed to such conditions. This practice should also be performed after any significant repairs are made on a unit, such as those involving the optics, detector, or radiant energy source.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

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1.1 This test method covers a procedure for evaluating the limits of the linearity of response with fluorescence intensity of fluorescence-measuring systems under operating conditions. Particular attention is given to slit widths, filters, and sample containers. This test method can be used to test the overall linearity under a wide variety of instrumental and sampling conditions. The results obtained apply only to the tested combination of slit width and filters, and the size, type and illumination of the sample cuvette, all of which must be stated in the report. The sources of nonlinearity may be the measuring electronics, excessive absorption of either the exciting or emitted radiation, or both, and the sample handling technique, particularly at low concentrations.
1.2 This test method has been applied to fluorescence-measuring systems utilizing continuous and low-energy excitation sources (for example, an excitation source of 450-W electrical input or less). There is no assurance that extremely intense illumination will not cause photodecomposition of the compounds suggested in this test method. For this reason it is recommended that this test method not be indiscriminately employed with high-intensity light sources. It is not a test method to determine the linearity of response of other materials. If this test method is extended to employ other chemical substances, the principles within can be applied, but new material parameters, such as the concentration range of linearity, must be established. The user should be aware of the possibility that these other substances may undergo decomposition, or adsorption onto containers.
1.3 This test method is applicable to 10-mm pathlength cuvette formats and instruments covering a wavelength range within 190 to 900 nm. The use of other sample formats has not been established with this test method.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
This practice permits an analyst to compare the general performance of his instrument, as he is using it in a specific spectrophotometric method, with the performance of instruments used in developing the method.
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1.1 This practice covers the description of requirements of spectrophotometric performance especially for ASTM methods, and the testing of the adequacy of available equipment for a specific method. The tests give a measurement of some of the important parameters controlling results obtained in spectrophotometric methods, but it is specifically not to be concluded that all the factors in instrument performance are measured.
1.1.1 This practice is not to be used (1) as a rigorous test of performance of instrumentation, or (2) to intercompare the quantitative performance of instruments of different design.
1.1.2 This practice is primarily directed to dispersive spectrophotometers used for transmittance measurements rather than instruments designed for diffuse transmission and diffuse reflection.

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1.1 This practice covers the description of requirements of spectrophotometric performance especially for ASTM methods, and the testing of the adequacy of available equipment for a specific method. The tests give a measurement of some of the important parameters controlling results obtained in spectrophotometric methods, but it is specifically not to be concluded that all the factors in instrument performance are measured.
1.1.1 This practice is not to be used (1) as a rigorous test of performance of instrumentation, or (2) to intercompare the quantitative performance of instruments of different design.
1.1.2 This practice is primarily directed to dispersive spectrophotometers used for transmittance measurements rather than instruments designed for diffuse transmission and diffuse reflection.

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1.1 This practice describes a procedure for measuring the practical spectral bandwidth of a spectrophotometer in the wavelength region of 185 to 820 nm. Practical spectral bandwidth is the spectral bandwidth of an instrument operated at a given integration period and a given signal-to-noise ratio.  
1.2 This practice is applicable to instruments that utilize servo-operated slits and maintain a constant period and a constant signal-to-noise ratio as the wavelength is automatically scanned. It is also applicable to instruments that utilize fixed slits and maintain a constant servo loop gain by automatically varying gain or dynode voltage. In this latter case, the signal-to-noise ratio varies with wavelength. It can also be used on instruments that utilize some combination of the two designs, as well as on those that vary the period during the scan. For digitized instruments, refer to the manufacturer's manual.  
1.3 This practice does not cover the measurement of limiting spectral bandwidth, defined as the minimum spectral bandwidth achievable under optimum experimental conditions.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

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1.1 These practices are intended to provide general information on the techniques most often used in ultraviolet and visible quantitative analysis. The purpose is to render unnecessary the repetition of these descriptions of techniques in individual methods for quantitative analysis.  
1.2 This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of whoever uses this standard to consult and establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

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1.1 This method affords an estimate of the relative radiant power (that is, the Stray Radiant Power Ratio (SRPR) at wavelengths remote from those of the nominal pass band transmitted through the monochromator of an absorption spectrophotometer. Test-filter materials are described that discriminate between the desired wavelengths and those which contribute most to SRP for conventional commercial spectrophotometers used in the ultraviolet, the visible, the near infrared, and the alkali halide infrared ranges. These procedures apply to instruments of conventional design, with usual sources, detectors, and optical designs. The vacuum ultraviolet and the far infrared present special problems that are not discussed herein.  Note 1-An equivalent test method (1,2)  for use in the ultraviolet that is easier and faster to perform might be preferred, especially when frequent testing is indicated. Note 2-Recent research (3) has shown that particular care must be exercised in testing grating spectrophotometers that use moderately narrow bandpass blocking filters. See 4.2.
1.2 These procedures are neither all-inclusive nor infallible.  Because of the nature of readily available filter materials, with a few exceptions the procedures are insensitive to SRP of shorter wavelengths in the ultraviolet or visible, or of lower frequencies in the infrared, and they are not necessarily valid for "spike" SRP nor for "nearby SRP." (See Annexes for general discussion and definitions of these terms.) However, they are adequate in most cases and for typical applications. They do cover instruments using prisms or gratings, in either single or double monochromators.  
1.3 The proportion of SRP (that is, SRPR) encountered with a well-designed monochromator, used in a favorable spectral region, is a small fraction of 1%. With a double monochromator it may easily be less than 1 ppm even with a broad-band continuum source. Under these conditions, it may be difficult to do more than determine that it falls below a certain level. Actual measurement often requires special techniques and instrument operating conditions that are not typical of those occurring during use. When absorption measurements with continuum sources are being made, it is frequently true that, owing to the effect of slit width on SRP in a double monochromator, these test procedures tend to give "conservative" results; that is, because larger slit widths than normal must be used to admit enough energy to the monochromator to permit evaluation of the SRP, the stray proportion indicated is greater than would normally be encountered in use.  
1.4 The principal reason for a test procedure that is not exactly representative of normal operation is that the effects of SRP are "magnified" in sample measurements at high absorbance. It is usually necessary to increase sensitivity in some way during the test in order to evaluate the SRP adequately. This is usually accomplished by increasing slit width and so obtaining sufficient energy to allow meaningful measurement of the SRP after the monochromatic energy has been removed by the SRP (test) filter. A further reason for increasing energy or sensitivity can be that many instruments have only absorbance scales, which obviously do not extend to zero transmittance. Even a SRP proportion as large as 1% may fall outside the measurement range.  
1.5 Many instruments that use optical attenuators to balance sample absorption make relatively inaccurate measurements below 10% transmittance, because of poor attenuator linearity. All instruments should be carefully and frequently checked if used below about 1% transmittance because of the possibility of zero shift due to electrical pickup or other causes. For these reasons, the test procedure specifies that the measurements be made within 100% and 10% on the scale. Obviously this is not required if reliable photometric linearity and zero checks are made under the conditions prevailing during the ...