Fluorescence Spectroscopy

Measurements by fluorescence spectroscopy are subject to many more instrumental and physical variables than absorption measurements. Due to these effects, the same sample may not give identical results with different instruments even if they are of similar design. Physical effects such as quenching or self-absorption may lead to departures from expected concentration/emission relationships over quite narrow concentration ranges. For these reasons it may be impossible to produce universal “absolute” reference materials, as certified values may be instrument dependent. Provided homogeneity and stability can be established, however, reference materials can be produced that allow users to monitor the performance of a spectrofluorometer over time and can also indicate the relative performance of different instruments. Starna fluorescence references are produced in an ISO17034 accredited production facility and calibrated in an ISO/IEC 17025 accredited calibration laboratory. Several of these references are cited in USP Chapter for spectrofluorometer qualification.

Spectral Response & Linearity
Starna Quinine Sulfate References

Used to qualify the spectral response of a spectrofluorometer and can also, over a limited concentration range (up to 1 mg/l), be used as a linearity check. USP cited reference.

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Sensitivity & Signal to Noise Ratio
Starna High Purity Water Reference

This Reference Material uses Raman scattering to simulate the behaviour of fluorescent materials at very low concentrations and can be used to determine the Signal to Noise Ratio of a spectrofluorometer near its limit of detection and hence indicate its ultimate sensitivity. USP <853 > cited reference.

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Spectrofluorometer Performance and Wavelength Validation
Starna 6BF Series

Used for spectrofluorometer wavelength qualification, as a routine check of spectrofluorometer performance and for inter-instrument and inter-laboratory comparisons. Six polymer blocks containing seven organic fluorophores. Excitation range from 260 to 600 nm and emission range from 370 to 670 nm

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Phosphorescence Decay Rate and Fluorescence Lifetime
Starna SF Series Polymer Block References

Used to confirm measurement of transient fluorescentsignals. Polymer block doped with Europium III. Decay rate and phosphorescence lifetime certified for gate times of 50 μS and 2 μS and a measurement delay time of 0.1 mS using excitation and emission wavelengths of 340 nm and615 nm respectively.

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Microplate Fluorometer Qualification
Starna Fluorescence Reference Plates

For routine qualification of fluorescence-based plate readers for fluorescence intensity and well-to-well reproducibility. 96-well microplates containing inorganic fluorescent reference materials in glass matrix. The inorganic fluorophores are inherently stable, and do not suffer from photodegradation or bleaching.Formerly manufactured and supplied by Matech, USA. Available in top- and bottom-reading configurations. Range of excitation and emission wavelengths to match the user’s application.

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Starna Quantum Counter
Rhodamine B Solution Cell

Used by instrument manufacturers as a quantum counter to determine spectral response factors to calculate ‘corrected’ fluorescence spectra. Designed to provide unity quantum conversion, so that the emitted fluorescence is directly proportional to the intensityof the exciting radiation. Usable range 220 nm – 580 nm.

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Starna Quantum Yield Reference
Dilute Rhodamine 101 Solution Cell

Quantum yield of this solution is 1.0 at 25°C. Working excitation range 450 to 465 nm and emission range 550 to 650 nm. Blank-corrected absorbance of this solution is below thelevel recommended in the literature to avoid internal filter effects. Dilute Rhodamine 101 in ethanol in a sealed quartz cuvette with ethanol blank.

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Measurements by fluorescence spectroscopy are subject to many more instrumental and physical variables than absorption measurements. Due to these effects, the same sample may not give identical results with different instruments even if they are of similar design. Physical effects such as quenching or self-absorption may lead to departures from expected concentration/emission relationships over quite narrow concentration ranges. For these reasons it may be impossible to produce universal “absolute” reference materials, as certified values may be instrument dependent. Provided homogeneity and stability can be established, however, reference materials can be produced that allow users to monitor the performance of a spectrofluorometer and make inter-instrument and inter-laboratory comparisons. Starna fluorescence references are produced in an ISO17034 accredited production facility and calibrated in an ISO/IEC 17025 accredited calibration laboratory. Several of these references are cited in USP Chapter <853> for spectrofluorometer qualification.

Quinine Sulfate References -Spectral Response and Linearity

One of the first reference materials developed for fluorescence spectroscopy. Equivalent to NIST SRM 936a and cited in USP <853 >.

High Purity Water Reference- Sensitivity & Signal to Noise Ratio

Used to determine the Signal to Noise Ratio of a spectrofluorometer near its limit of detection and hence indicate its ultimate sensitivity. USP <853 > cited reference.

Starna SF Series Doped Polymer References - Spectral Response Qualification

Five polymer blocks containing stable organic fluorophores. Excitation range 280 to 700 nm and emission range 380 to 770 nm

Starna 6BF Series Doped Polymer References -Wavelength Validation and Performance Check

Six polymer blocks containing seven organic fluorophores. Excitation range 260 to 600 nm and emission range 370 to 670 nm.

Starna Europium III Reference - Phosphorescence Decay Rate and Fluorescence Lifetime

Polymer block doped with Europium III. Decay rate and phosphorescence lifetime certified for gate times of 50 μS and 2 μS.

Rhodamine Solution Cell - Quantum Counter

Used by instrument manufacturers to determine spectral response factors to calculate ‘corrected’ fluorescence spectra. Designed to provide unity quantum conversion,

Rhodamine in Ethanol Cell - Quantum Yield Reference

Quantum yield 1.0 at 25°C. Blank-corrected absorbance of this solution is below the level recommended in the literature to avoid internal filter effects.

Starna Rare Earth Solution Cells -Spectrofluorometer Wavelength Qualification

Cited in USP Chapter <853> for spectrofluorometer wavelength qualification.

Doped Glass Microplate References - Microplate Fluorometer Qualification

For qualification of fluorescence-based plate readers. 96-well microplates containing inorganic fluorescent reference materials in a glass matrix. Formerly supplied by Matech, USA.

Purpose

Used to determine the fluorescence quantum yield of a substance.

Description and Discussion

Solution of Rhodamine 101 in ethanol, sealed by heat fusion in to a high quality far-UV fluorometer cell, and supplied with an ethanol blank cell The fluorescence quantum yield of a sample (ΦF) is the ratio of photons absorbed to photons emitted through fluorescence. This reference has a quantum yield of 1.0 at 25°C. By measuring the fluorescence emission of a sample with similar absorbance characteristics as the reference, and under the same conditions of measurement, the quantum yield of the sample can be determined.

The excitation range for this material is approximately 460 to 600 nm; and the emission range is approximately 550 to 700 nm. For most instrumental setups, a working excitation wavelength of 535 nm is suitable.

The blank-corrected absorbance of this solution is below the level recommended in the literature to avoid internal filter effects.

Catalogue Number

	Catalogue Number
Starna Rhodamine 101 Quantum Yield reference	RM-RH101

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Data sheet

Measurements by fluorescence spectroscopy are subject to many more instrumental and physical variables than absorption measurements. Due to these effects, the same sample may not give identical results with different instruments even if they are of similar design. Physical effects such as quenching or self-absorption may lead to departures from expected concentration/emission relationships over quite narrow concentration ranges. For these reasons it may be impossible to produce universal “absolute” reference materials, as certified values may be instrument dependent. Provided homogeneity and stability can be established, however, reference materials can be produced that allow users to monitor the performance of a spectrofluorometer over time and can also indicate the relative performance of different instruments. Such references are referred to as “Reference Materials” (RMs) or Quality Control Materials (QCMs). Starna produce a range of such materials under the same tight quality control procedures as are applied to their CRMs.

For the reasons outlined above, instrument qualification may only be valid for a particular instrument and within a relatively narrow range of operating parameters – indeed it can even be method specific. The references listed below will cater for many routine situations, but Starna can also design and supply fluorescence Reference Materials tailored to individual needs.

Quinine Sulfate Solution References

used to calibrate the spectral response of a spectrofluorometer and as a linearity check.

High Purity Water (Raman) Reference

used to determine the signal-to-noise ratio of a fluorescence spectrophotometer and hence its ultimate sensitivity.

Doped Polymer Blocks (Starna SF Series)

spectral response references, traceable to NIST Fluorescence SRMs NEW!

Europium III Phosphorescence Reference

used to confirm measurements of transient fluorescentsignals.

Starna Rhodamine B Quantum Counter

used in fluorescence instrumentation for spectral correction

Starna Quantum Yield Reference

used to determine the fluorescence quantum yield of a sample

Doped Polymer Blocks (Starna 6BF Series)

for spectrofluorometer wavelength qualification andas a routine check of spectrofluorometer performance.

Rare Earth Solution Cells

for spectrofluorometer wavelength qualification

Doped Glass Microplate References

qualification of fluorescence-based plate readers for fluorescence intensity and well-to-well reproducibility NEW!

Purpose

This Reference Material can be used to qualify the Signal to Noise Ratio of a spectrofluorometer near the limit of detection and hence indicate its ultimate sensitivity.

Description and Discussion

10mm far UV quartz fluorometer cuvette with polished faces at 90°, containing ultra-high purity water, permanently sealed by heat fusion.

Traditionally, the performance of spectrofluorometers has been tested using dilute solutions of known fluorophores such as Quinine Sulfate or Rhodamine to determine spectral response factors. Starna Certified Reference Materials are available for this purpose. To use this approach at or near the detection limit of a high-performance instrument however, is impractical because the necessary sample concentrations are so low that sample preparation becomes difficult and errorprone and there is high potential for sample contamination.

An alternative approach, employed in this Reference, is to use the phenomenon of Raman scattering. When radiation interacts with a molecule, some of the incident energy can be scattered. Most will be normal (Rayleigh) scattering, where the scattered radiation is at the same wavelength as the incident radiation. A very small proportion of the scattered photons (about 10-7) however, will exhibit Stokes Raman scattering and be emitted at a lower energy (i.e. longer wavelength) in a manner analogous to fluorescence. Because of the very small number of photons involved, the signal levels are very low and comparable to those produced by very low analyte concentrations in normal fluorescence measurements. Pure water exhibits a Raman shift of tens of nanometers when excited by UV or visible radiation, so the Raman peak can be readily identified. Uncontaminated, pure water is relatively straightforward to obtain and is therefore an ideal reference material.

The signal –to-noise ratio for a given excitation wavelength may be calculated using the instrument software.

Note that the calculated ratio will depend on the calculation protocol employed by the instrument manufacturer and therefore ‘compliance to specification’ limits will be specific to the spectrofluorometer type under test.

Typical spectra

Catalogue Number

COMPOUND	Catalogue Number
Water (Raman) Fluorescence Senitivity Reference	RM-H2O

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Data sheet

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Technique

UV-VIS References

Starna References for Fluorescence Spectroscopy

Spectral Response & Linearity Starna Quinine Sulfate References

Sensitivity & Signal to Noise Ratio Starna High Purity Water Reference

Spectrofluorometer Performance and Wavelength ValidationStarna 6BF Series

Phosphorescence Decay Rate and Fluorescence LifetimeStarna SF Series Polymer Block References

Microplate Fluorometer QualificationStarna Fluorescence Reference Plates

Starna Quantum CounterRhodamine B Solution Cell

Starna Quantum Yield ReferenceDilute Rhodamine 101 Solution Cell

Starna References for Fluorescence Spectroscopy

Quinine Sulfate References -Spectral Response and Linearity

High Purity Water Reference- Sensitivity & Signal to Noise Ratio

Starna SF Series Doped Polymer References - Spectral Response Qualification

Starna 6BF Series Doped Polymer References -Wavelength Validation and Performance Check

Starna Europium III Reference - Phosphorescence Decay Rate and Fluorescence Lifetime

Rhodamine Solution Cell - Quantum Counter

Rhodamine in Ethanol Cell - Quantum Yield Reference

Starna Rare Earth Solution Cells -Spectrofluorometer Wavelength Qualification

Doped Glass Microplate References - Microplate Fluorometer Qualification

Rhodamine 101 Solution Cell

Purpose

Starna References for Fluorescence Spectroscopy

High Purity Water Sensitivity Reference

Purpose

Spectral Response & Linearity
Starna Quinine Sulfate References

Sensitivity & Signal to Noise Ratio
Starna High Purity Water Reference

Spectrofluorometer Performance and Wavelength Validation
Starna 6BF Series

Phosphorescence Decay Rate and Fluorescence Lifetime
Starna SF Series Polymer Block References

Microplate Fluorometer Qualification
Starna Fluorescence Reference Plates

Starna Quantum Counter
Rhodamine B Solution Cell

Starna Quantum Yield Reference
Dilute Rhodamine 101 Solution Cell