ASTM E3029-15(2023) PDF
Standard Practice for Determining Relative Spectral Correction Factors for Emission Signal of Fluorescence Spectrometers
Standard Practice for Determining Relative Spectral Correction Factors for Emission Signal of Fluorescence Spectrometers
- Статус документа:
- Действующий
- Формат:
- Электронный (PDF)
- Количество страниц:
- 5
- Дата публикации:
- 1 января 2023 г.
- Издание:
- E3029
- ICS:
- 17.180.01
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
Overview
ASTM E3029-15(2023), issued by ASTM International, establishes a standard practice for determining relative spectral correction factors for the emission signal of fluorescence spectrometers. Calibration of the emission detection system as a function of emission wavelength (λEM) is essential for accurate quantification and for ensuring the true shape and peak maximum of an emission spectrum are correctly determined. The standard covers the ultraviolet-visible spectral range for grating-based fluorescence spectrometers, aiding in reliable comparison of emission intensities across different wavelengths.
This practice details three primary calibration methodologies using:
- Calibrated light sources,
- Calibrated detectors with diffuse reflectors,
- Certified reference materials.
Each method is designed to address the variable spectral responsivity of detection systems, which can significantly affect measurement accuracy within their operational wavelength range. Following standard calibration ensures greater confidence in data integrity, reproducibility, and compliance in both laboratory and industrial settings.
Key Topics
Main Areas Covered in ASTM E3029-15(2023):
- Calibration Methods: Guidance for utilizing calibrated light sources, calibrated detectors/diffuse reflectors, and certified reference materials for emission spectral correction.
- Wavelength Range: Procedures applicable from 250 nm to 830 nm, with some discussion on extending to the near-infrared range.
- Instrument Compatibility: Designed for grating-based spectrometers with a 0°/90° transmitting geometry, and use with both single channel and multichannel (CCD or diode array) detectors.
- Instrument Settings: Emphasizes maintaining consistent settings throughout calibration and measurement to ensure reliability.
- Correction Factor Calculation: Methods for determining and reporting relative spectral correction factors as a function of emission wavelength.
- Documentation: Recommendations for reporting calibration factors, settings, uncertainties, and reference materials.
Related Keywords: fluorescence calibration, emission responsivity, spectral correction factors, certified reference materials, spectrometer accuracy, laboratory calibration standards, UV-visible spectroscopy.
Applications
Practical Uses of ASTM E3029-15(2023):
- Fluorescence Spectroscopy Calibration: Enables accurate correction for detection system responsivity when measuring emission spectra, supporting quantitative results and spectrum shape verification.
- Instrument Qualification: Essential for validating the performance of new or serviced fluorescence spectrometers, fulfilling quality control and regulatory requirements.
- Analytical Chemistry: Assures accurate inter-wavelength intensity comparisons, necessary in chemical analysis, biotechnology, pharmaceuticals, and life sciences research.
- Reference Spectrum Creation: Supports the creation of accurate reference spectra for materials analysis, dye characterization, and fluorescence labeling studies.
- Educational and Regulatory Compliance: Provides laboratories with an established method to meet global standardization demands, referenced by organizations following WTO TBT principles.
Best Practices:
- Confirm wavelength accuracy and detector linearity prior to calibration.
- Use consistent instrument configurations across all calibration and measurement steps.
- Follow all safety, health, and environmental requirements specific to your laboratory.
Related Standards
- ASTM E131: Terminology Relating to Molecular Spectroscopy
- ASTM E388: Test Method for Wavelength Accuracy and Spectral Bandwidth of Fluorescence Spectrometers
- ASTM E578: Test Method for Linearity of Fluorescence Measuring Systems
- ASTM E2719: Guide for Instrument Calibration and Qualification in Fluorescence Spectroscopy
ASTM E3029-15(2023) is a key reference for laboratories aiming for precise, globally recognized fluorescence emission measurements. Adhering to its procedures facilitates greater accuracy, data quality, and international comparability in molecular spectroscopy applications.
Технические детали
- Технический комитет
- E13 - Molecular Spectroscopy and Separation Science
- SKU
- ASTM E3029-15(2023)
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