Read AN01142_2/3/03 text version

Application Note 01142

Wavelength Accuracy ­ Measurement and Effect on Performance in UV-Visible Spectrophotometry

Introduction Instrumentation

Application 1

Use of Emission Lines from a Mercury Source

Key Words · Wavelength Accuracy · Mercury Light Source · Deuterium Light Source · Emission Lines · Holmium Filter · Didymium Filter · Rare Earth Oxides · Calibration · NTRM · NIST · CRM · NPL · ISO/IEC Standard 17025 · Neutral Density Filters · Wavelength Calibration · Holmium Oxide in Perchloric Acid · Calibrating Solutions

Any quality control procedure will require that the performance of the instrument be checked on specified occasions. Verification of the wavelength accuracy will form part of any such performance check. This application note details how the wavelength accuracy of a Thermo Electron Corporation UV-Visible spectrophotometer may be checked and recalibrated if necessary. Several methods are listed below, in order of preference. Methods 1 and 2 are primary methods employing fundamental constants, and should be used when available. 1. Emission lines from a Mercury source. 2. Emission lines from a Deuterium source. 3. Calibrated traceable Holmium & Didymium glasses. 4. Rare Earth Oxides in acid solution.

Mercury sources are available as optional accessories for the following Thermo spectrophotometers:

INSTRUMENT

Nicolet® EvolutionTM 300 Nicolet Evolution 500 UV Series 300 and 500

The mercury lamp fits into the third lamp position within the optical system of the instrument, and is powered from the instrument power supply. Once installed it can be left in place and used when required.

Method: The emission spectrum of a low-pressure mercury lamp has a number of intense lines that cover a large part of the UV and visible range. The frequencies of these lines are fundamental physical properties of the source element, and therefore the wavelengths are invariant. The mercury emission spectrum is a primary wavelength standard.

nm 253.65 296.73 404.66 435.84 546.07 760.95 871.68

Factors Affecting Wavelength Accuracy

Physical Shock Accuracy may be affected if the spectrophotometer is jolted violently, dropped, or subjected to severe or prolonged vibration. Temperature Problems may be found if the operating temperature is significantly different from the temperature at which the spectrophotometer was last calibrated. All instruments are calibrated at room temperature and wavelength differences of between 0.1 and 0.3 nm per 10°C are not uncommon. It is therefore essential that instruments are warmed up to normal operating temperature before use and before performing any instrument verification test. Replacement of Key Optical Components The instrument will need recalibrating if any of the key optical components are replaced. Mechanical Wear Thermo UV-Visible spectrophotometers have been designed to minimize the effects of wear. Therefore this should not be a problem.

To obtain an emission spectrum, in addition to selecting the required wavelength range, it is important to select appropriate parameters for the scan method. Suitable values are as follows:

LOCAL CONTROL SOFTWARE VISION SOFTWARE

Scan Type Mode Bandwidth Speed Data Interval Lamp Change

Standard Scan I (Intensity Mode) 0.5 nm (if available) 30 nm/min 0.1 nm Hg

Type Data Mode Bandwidth Scan Speed Data Interval Lamp Change

Standard Intensity 0.5 nm (if available) 30 nm/min 0.1 nm Mercury

When using an instrument with a photomultiplier (PMT) detector, the FLAT EHT profile must be used for this procedure.

Requirements for Wavelength Standards

A standard method should be: · Readily available. · Easily assembled or prepared. · Easy and safe to handle. · Suited to the optical properties of the instrument e.g. spectral bandwidth, etc. · Unaffected by environmental conditions, e.g. good chemical stability, small temperature coefficients, etc.1

1.0 253.65 0.9

Sample beam intensity (l units)

250000 656.1 200000 486.0

Sample beam intensity (l)

0.8 0.7 0.6 0.5 365.02 0.4 0.3 0.2 0.1 0.0 200 250 300 350 400 450 500 550 600 404.66 546.07 576.96 435.84

150000

100000

50000 0 470

490

510

530

550

570

590

610

630

650

670

Wavelength (nm)

Figure 2: Deuterium lamp emission spectrum

Wavelength (nm)

Figure 1: A mercury emission spectrum (rescaled)

Most of the energy in the mercury spectrum is emitted in the 253.65 nm line, the intensity of which is much greater than the other lines. When the spectrum is first scanned only the 253.65 nm line will be apparent, because the vertical scale will have been expanded to accommodate it. When the scan is completed the spectrum can be rescaled as follows, in order to see all the peaks. Select MANIPULATE then rescale. Set GRAPH HIGH to 1 and GRAPH LOW to 0. The top of the line at 253.65 nm is off the scale but the lower energy lines are now clearly visible. Values for the wavelengths can be obtained from the Peak Table. The permitted tolerances for wavelength accuracy are quoted in the specifications of Thermo UV-Visible spectrophotometers. The mercury lamp accessory enables the user to demonstrate that the wavelength accuracy of the instrument is within specification quickly and conveniently, whenever necessary. If the required tolerances are not met, a wavelength calibration is necessary. Under normal circumstances this would only be expected following replacement of a major optical component. The mercury lamp calibration remeasures all seven relevant emission lines, then recalculates and replaces the software compensation equation.

The permitted tolerances for wavelength accuracy are quoted in the specifications of Thermo UV-Visible spectrophotometers. If the required tolerances are not met, a wavelength calibration is necessary. Under normal circumstances this would only be expected following replacement of a major optical component. The Deuterium lamp recalibration uses the Deuterium emission line at 656.1 nm to adjust the software compensation equation and is intended to remove the effects of small movements of optical components and shifts due to temperature, shock or vibration. If the errors found are linear and no more than 0.5 nm then a Deuterium calibration can be used. The process will take about five minutes and is fully described in the appropriate software manual. This option only applies where an existing calibration is present.

Application 3

Use of Rare Earth Glasses

A calibrated/traceable set of holmium & didymium glasses is available as follows:

9423 185 03111 Set of 2 calibrated/traceable wavelength filters

· Certified Reference Materials (CRM's). · Holmium filter traceable to National Physical Laboratory (NPL) and National Institute of Standards and Technology (NIST) via ISO/IEC Standard 17025 calibration processes. · Didymium filter traceable to NPL only via ISO/IEC Standard 17025 calibration process. · Supplied with calibration certificate. · UKAS accreditation number 0521. · Suitable for use with any appropriate Visible or UV-Visible spectrophotometer. · Includes wooden storage box. Further details are also available in our Standards for UV-Visible Spectrometry publication.2

NIST Traceable Reference Materials

Application 2

Use of Emission Lines from a Deuterium Source

Wavelength verification and recalibration can be performed using the deuterium lamp in UV-Visible instruments having a deuterium source (e.g. Nicolet Evolution 500, UV Series, Spectronic® UV Helios range).

Method: The emission lines produced from the deuterium lamp are again characteristic of the source element; and as a fundamental physical standard, these wavelengths are invariant. However, unlike the mercury source, only two characteristic lines are usable. These are at 486.0 and 656.1 nm. The emission spectrum (over the desired range) can be easily obtained by selecting scan method parameters as above but with lamp change set to Deuterium (D2). When using an instrument with a photomultiplier (PMT) detector, the FLAT EHT profile must be used for this procedure.

9423 CRM 9300E NIST Traceable Reference Material (NTRM) filter set 1

· Comprises 3 neutral density filters, a clear filter holder and a wavelength check filter. · Suitable for checking the photometric calibration of UV-Visible spectrophotometers in absorbance or transmittance. · Supplied with calibration certificate.

· Includes filters of nominal values 0.5, 0.7 and 1A at 546.1 nm. · Calibration values are given at 5 wavelengths. · Holmium glass wavelength filter with certified wavelengths. · Includes transport case.

9423 CRM 1930E NTRM filter set 2

Wavelength accuracy checking using a traceable holmium oxide glass filter can be conveniently carried out using one of the following performance qualification accessories:

PART NUMBER DESCRIPTION

9423 UV6 1400E 10 01 0601 9423 UV6 1460E 10 01 0701 9423 UV6 1250E 9423 UV6 1260E

· Comprises 3 neutral density filters, a clear filter holder and a wavelength check filter. · Suitable for checking the photometric calibration of UV-Visible spectrophotometers in Absorbance or transmittance. · Supplied with calibration certificate. · Includes filters of nominal values 0.3, 1.5 and 2A at 546.1 nm. · Calibration values are given at 5 wavelengths. · Holmium glass wavelength filter with certified wavelengths. · Includes transport case.

9423 CRM 9400E NTRM filter sets 1 and 2

Calibration Validation Carousel (NPL) for Spectronic Helios Alpha/Beta Intelligent Calibration Validation Carousel (NPL) for Nicolet Evolution 300 Calibration Validation Carousel (NIST) for Spectronic Helios Alpha/Beta Intelligent Calibration Validation Carousel (NIST) for Nicolet Evolution 300 Calibration Validation Unit (NPL) for Nicolet Evolution 500/UV Series Calibration Validation Unit (NIST) for Nicolet Evolution 500/UV Series

Application 4

Use of Rare Earth Oxide Solutions

· Comprises 6 neutral density filters, clear filter holders and a wavelength check filter. · Suitable for checking the photometric calibration of UV-Visible spectrophotometers in absorbance or transmittance. · Supplied with calibration certificate. · Includes filters of nominal value 0.3, 0.5, 0.7, 1.0, 1.5, and 2.0A at 546.1 nm. · Calibration values are given at 5 wavelengths. · Holmium glass wavelength filter with certified wavelengths. · Includes transport case.

Method: This technique uses glasses manufactured by fusing the appropriate rare earth oxide in silica. The two most frequently used are holmium and didymium (a mixture of neodymium and praseodymium). Manufacturing can cause batch-to-batch variation in these standards. This is overcome by calibrating each standard glass against certified reference glasses from the NPL and NIST (Holmium) or NPL only (Didymium) via an ISO/IEC standard 17025 procedure. With the appropriate filter available the spectrum (over the desired range) can be quickly obtained by selecting the normal scan mode. This procedure does not allow for recalibration of the instrument, but its speed of implementation compared to Applications 1 & 2 makes it ideal for routine checking of the wavelength calibration. NB: On a variable bandwidth instrument, 0.5 nm should be used as the spectral bandwidth.

Method: This technique uses solutions of rare earth oxide, prepared by dissolution in acid media. The most frequently used is holmium oxide in perchloric acid. The reference wavelengths have been well-defined3, and a suitable solution is available from various sources. Once the solution has been obtained, the spectrum (over the desired range) can be quickly obtained by selecting the normal scan mode. This procedure does not allow for recalibration of the instrument, but its speed of implementation compared to Applications 1 & 2 makes it ideal for routine checking of the wavelength calibration. NB: The peaks produced when the holmium compound is in solution are much sharper than those produced by the glasses. They are therefore much more sensitive to spectral bandwidth. On a variable bandwidth instrument, 0.5 nm should be used. Compensation tables are available for use at other known bandwidths.3 Results:

1.4 1.2 1.0 640.3 536.4

Absorbance

0.8 0.6 0.4 0.2 0 245

278.1

361.2 485.1

249.7

385.3

295

345

395

445

495

545

595

645

Wavelength (nm)

Figure 3: Holmium oxide solution in perchloric acid

Far UV Wavelength Standard This standard consists of a solution of a rare earth oxide dissolved in dilute sulfuric acid, supplied in a permanently heat-sealed Far UV quartz cell. The absorbance spectrum of this rare earth contains a series of characteristic peaks extending into the Far UV region. Specification Material

Solution of rare earth oxide in dilute sulfuric acid. Consists of one sealed cell with certified peaks at spectral bandwidth values of 0.1, 0.2, 0.5, 1.0, 1.5, 2.0 and 3.0 nm. 200 ­ 300 nm, instruments with spectral bandpass less than 5 nm UV Wavelength filter

Usable Range 9423 UV9 5540E

Thermo Electron Corporation has direct subsidiary offices in North America, Europe and Japan. To complement these direct subsidiaries, we maintain a network of representative organizations throughout the world. Use this reference list or visit our Web site to locate the representative nearest you.

References

1. C. Burgess & A. Knowles, UV Spectrometry Group, Standards in Absorption Spectrometry, Chapman & Hall, ISBN 0-412-22470-4. 2. Data Sheet 18, "Standards for UV-Visible Spectrometry" (Thermo Electron Corporation). 3. Holmium Oxide Solution Wavelength Standard From 240 to 640 nm, SRM 2034 NBS Special Publication, 260-102.

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Thermo ARL: Tel: +86 10 6833 6715 Thermo Elemental: Tel: +86 10 6592 0232 Thermo Nicolet: Tel: +86 10 6597 3388 ext 2912

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Tel: +33 (0)1 39 30 53 00

Germany

Tel: +49 (0)6102 3671 0

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Tel: +39 02 6601 6351

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Thermo Elemental: Tel: +81 774 201245 Thermo Nicolet: Tel: +81 45 450 1112

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Tel: +31 76 5724840

Nordic

Thermo Nicolet: Tel: +358 9 3291 00 Thermo ARL: Tel: +46 (0)8 556 468 31

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www.thermo.com/spectronic

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©2002 Thermo Electron Corp. All rights reserved worldwide. We make no warranties, expressed or implied, in this product summary, and information is subject to change without notice. All product and company names are property of their respective owners. AN_S0368

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AN01142_2/3/03

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