SPECTRONIC Standards User Guide - Cole-Parmer · SPECTRONIC Standards User Guide 5 Photometric Accuracy Standards . The photometric performance standards test photometric accuracy

SPECTRONIC StandardsUser Guide

The information in this publication is provided for reference only. All information contained in this publication is believed to be correct and complete. Thermo Fisher Scientific shall not be liable for errors contained herein nor for incidental or consequential damages in connection with the furnishing, performance or use of this material. All product specifications, as well as the information contained in this publication, are subject to change without notice. This publication may contain or reference information and products protected by copyrights or patents and does not convey any license under our patent rights, nor the rights of others. We do not assume any liability arising out of any infringements of patents or other rights of third parties. We make no warranty of any kind with regard to this material, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Customers are ultimately responsible for validation of their systems. © 2007-2010 Thermo Fisher Scientific Inc. All rights reserved. No part of this publication may be stored in a retrieval system, transmitted, or reproduced in any way, including but not limited to photocopy, photograph, magnetic or other record, without our prior written permission. For Technical Support, please contact: Thermo Fisher Scientific 5225 Verona Road Madison, WI 53711-4495 U.S.A. Telephone: 1 800 532 4752 E-mail: [email protected] World Wide Web: http://www.thermo.com/spectroscopy For International Support, please contact: Thermo Fisher Scientific Telephone: +1 608 273 5017 E-mail: [email protected] World Wide Web: http://www.thermo.com/spectroscopy Microsoft, Windows and Windows NT are either trademarks or registered trademarks of Microsoft Corporation in the United States and/or other countries. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. 269-220100, Rev. C

Contents

Introduction.........................................................................................1 Conventions used in this manual .............................................................. 2

23344555

6

888

10101011

131315

1616

Questions or concerns ............................................................................... Description ...............................................................................................

0% Transmittance Standard .................................................................. Wavelength Standard............................................................................. Stray Light Standards............................................................................. Photometric Accuracy Standards............................................................ Empty Filter Holder .............................................................................. Recertification and repair .......................................................................

Handling and Care of Standards ..........................................................

Instrument Setup.................................................................................. Operating technique ................................................................................. Adapter and accessory filters......................................................................

Recording and Interpreting Test Results ............................................ Recording test results .............................................................................. Interpreting test results............................................................................ Use of Absorbance mode.........................................................................

Test Procedures .................................................................................. 0% transmittance and stray light test....................................................... Wavelength accuracy test ........................................................................

Wavelength peak selection SPECTRONIC 20+ and SPECTRONIC 20D+ ....................................................................

Photometric accuracy/linearity test..........................................................

SPECTRONIC Standards User Guide i

Introduction

SPECTRONIC Standards are an affordable, accurate and stable set of filters for performance validation purposes.

SPECTRONIC Standards enable the spectrophotometer user to quickly and reliably evaluate the major performance parameters of Thermo Scientific's spectrophotometers. As a part of the laboratory's normal quality control program, SPECTRONIC Standards are an invaluable aid in the detection of incipient instrument problems before errors occur.

SPECTRONIC Standards User Guide 1

Conventions used in this manual This manual includes safety precautions and other important information presented in the following format:

Note Notes contain helpful supplementary information.

Notice Follow instructions labeled “Notice” to avoid damaging the system hardware or losing data.

Caution Indicates a hazardous situation which, if not avoided, could result in minor or moderate injury.

Warning Indicates a hazardous situation which, if not avoided, could result in death or serious injury.

Danger Indicates a hazardous situation which, if not avoided, will result in death or serious injury.

Questions or concerns In case of emergency, follow the procedures established by your facility. If you have questions or concerns about safety or need assistance with operation, repairs or replacement parts, you can contact our sales or service representative in your area or use the information at the beginning of this document to contact us.

2 SPECTRONIC Standards User Guide

Description SPECTRONIC Standards consists of the following items:

• 220 nm stray light filter standard • 340 nm stray light filter standard • 400 nm stray light filter standard • Wavelength accuracy standard • % Transmittance standard • Two photometric accuracy standards with stated transmittance values

near 10%T at 590 nm (working and reserve) • Two photometric performance standards with stated transmittance

values near 50%T at 590 nm (working and reserve) • Empty filter holder control standard for photometric accuracy • Carrying and storage case • Certificate of calibration

All standards have the same external dimensions as a standard 10 mm-pathlength square cuvette, so they will fit in standard spectrophotometer cell holders. An adapter is required to use the standards in a test-tube-holder sample compartments.

Your set of SPECTRONIC Standards has been individually certified and the values are traceable to NIST.

0% Transmittance Standard

The 0% transmittance (0%T) standard is completely opaque. It is used to check the 0%T reading of the SPECTRONIC spectrophotometers by blocking the light beam from the instrument's source lamp. This standard helps determine if a light leak is present and determine the correct zero setting. The 0% transmittance standard's apparent transmittance, if any, can be used to estimate the stray light in instrument.

This standard is not traceable to NIST.

The 0% transmittance standard cannot be used with spectrophotometers that do not provide a 0% transmittance adjustment. Using this standard in absorbance mode will produce readings that are out of range. Do not use this standard in absorption mode.


Wavelength Standard

The wavelength standard produces three widely spaced, symmetrical, high transmittance peaks in the regions of 400 nm, 525 nm and 780 nm. The primary peak is the certified wavelength for your wavelength standard. This value is recorded on the certificate accompanying the SPECTRONIC Standard. Only the primary peak near 525 nm is certified and NIST traceable.

The peaks near 400 and 780 nm can be used for checking the repeatability of your instrument.

Stray Light Standards

Three stray light standards are provided with the SPECTRONIC Standards. Each standard is essentially opaque at its test wavelength and highly transmitting at longer wavelengths, as shown below.

Stray light standards are not traceable to NIST.



Photometric Accuracy Standards

The photometric performance standards test photometric accuracy and linearity. Transmittance of these standards is constant in the region of 590 nm, as shown below. Consequently, no bandwidth compensation is required.

Empty Filter Holder

The empty filter holder is identical to the holders for the other standards. The empty filter holder should be used when setting 100%T to ensure that the filter holders of the other standards do not affect the transmittance values.

Recertification and repair

We offer a recertification and repair service for the SPECTRONIC Standards. It is recommended that you return your standards for recertification every two years — or whenever you have reason to believe that a standard no longer exhibits its certified value. Before returning your standards, please contact technical support.

Handling and Care of Standards

The transmittance of the photometric accuracy standards is significantly affected by the condition of the optical surfaces. Any dirt (lint, fingerprints, oil, dust, smoke film, etc.) can cause erroneous test results. The cleaning procedure for the photometric accuracy standards involves a risk of damaging the surfaces. So, prevention of soiling is far better than cleaning.

Always keep the standards in their case when not in use. Always keep the cover closed. Never expose the standards to airborne contaminants like smoke, dust, oil vapor or chemical fumes.

Before using the photometric accuracy standards, visually inspect them. Remove any dust or lint by gentle brushing.

If it becomes necessary to clean a photometric accuracy standard with a liquid, clean only one standard at a time, and proceed as follows:

1. Read and record the standard value in percent transmittance (%T) at 590 nm before cleaning.

2. Use a nut driver to loosen the nut securing the glass filter and back the nut out almost all the way, but do not remove it.

3. Remove the filter, holding it by the edges and being careful not to scrape the glass against the filter holder — especially the metal lip that projects near the bottom of the holder.

Note You must use the appropriate liquid when cleaning the glass:

• Photometric accuracy standards - use only reagent quality isopropyl alcohol

• Wavelength or stray light standards - use only distilled or deionized water.

4. Clean the glass using only the recommended liquid and a soft,

clean, lint-free cloth.

5. Reassemble the filter and inspect it for cleanliness and scratches.



6. Read the standard value at 590 nm.

The percent transmittance (%T) reading should remain the same or increase.

7. If the value has decreased, repeat the cleaning process.

Instrument Setup

Set up the spectrophotometer as indicated in the operator's manual to make transmittance measurements, being sure to allow the recommended warm-up time.

Make all settings and readings as accurately as possible. This will minimize the borderline normal/abnormal results which give no clear indication of whether the spectrophotometer needs professional service.

The SPECTRONIC Standards have the same outside dimensions as a 10 mm square cuvette. If necessary, install an appropriate adapter (see Adapter and accessory filters) in the instrument's sample compartment.

Operating technique The test results you obtain using SPECTRONIC Standards will be influenced by your technique in operating the instrument as well as by the condition of the instrument. Refer to the instrument operating manual to make sure you use the instrument correctly and avoid introducing errors into the test results.

Adapter and accessory filters To use the SPECTRONIC Standards with the SPECTRONIC 20+ the Standard Adapter (for ½-inch square cuvettes) must first be installed. The appropriate cuvette holder is also required.

Insert standards into the cuvette holder as shown below. Orient the holder so that the window side of the standard faces to the right. Push the holder down into the sample compartment as far as possible, and close the sample compartment lid.



Note To accommodate the SPECTRONIC Standards, the ½-inch cuvette adapter and the 10 mm square cuvette holder are required.

It is highly recommended that the Accessory Filter Kit (for all SPECTRONIC 20+ series) be used to optimize the performance of the spectrophotometers — both in routine use and during tests with SPECTRONIC Standards.

Recording and Interpreting Test Results

Recording test results Test results should be recorded by date in a test log — one log for each instrument tested. (A sample of such a log is provided at the end of this manual.) By scanning the log entries, the user can detect gradual changes of test values over a period of time. Any conditions or events that might affect test results (for example, cleaning or lamp replacement) should be recorded in the Comments column.

Interpreting test results Although every effort is made to manufacture and measure the standards as accurately as possible, the wavelength and photometric performance standards have tolerances associated with them.

In interpreting test results to evaluate instrument performance, the instrument tolerance and the standard tolerance should be added to determine the acceptable total deviation from nominal. For convenience, the following table gives the maximum deviations from nominal (instrument tolerance plus standard tolerance plus allowance for indeterminate error in the test procedure).


Table 1 - Maximum Acceptable Deviations from Nominal for SPECTRONIC Standards

Test Standard

Spectrophotometer Stray Light

Wavelength Accuracy 0%T

Photometric Accuracy

SPECTRONIC 20+

SPECTRONIC 20D+ 0-0.85%T* ±3.5 nm 0.0** ±3.0%T

GENESYS™ 20 0-0.45%T ±3 nm 0.0*** ±1.2%T

SPECTRONIC standards can be used with the instruments listed below, however, other NIST traceable standards are also available for photometric and wavelength accuracy.

For increased testing confidence, please use the Wavelength/Photometric Accuracy Standards. Contact your sales or service representative for more information.

GENESYS 10 Vis ≤0.1%T ±2 nm 0.0*** ±1.2%T

GENESYS 10UVscanning ≤0.1%T ±2 nm ≤0.1%T ±1.2%T

GENESYS 6 ≤0.1%T ±2 nm ≤0.1%T ±1.2%T

BioMate 3 ≤0.1%T ±2 nm ≤0.1%T ±1.2%T

GENESYS 10S VIS <0.1%T ±2 nm 0.0*** ±1.2%T

GENESYS 10S UV-VIS <0.1%T ±2 nm ≤0.1%T ±1.2%T

GENESYS 10S Bio <0.1%T ±2 nm ≤0.1%T ±1.2%T

BioMate 3S <0.1%T ±2 nm ≤0.1%T ±1.2%T

*With Accessory Filter Kit – highly recommended accessories **An offset of ±0.2%T is acceptable ***An offset of ±0.1%T is acceptable Note All results are pictorial. All of the energy should strike the optical surface of the cell. The size and shape of the beam should be constant for the life of the instrument. .

Use of Absorbance mode The instructions in this manual assume that the spectrophotometer is used in the transmittance mode. Test results can be read in absorbance and converted mathematically to transmittance (see equations below), but the following limitations must be considered:

1. Readings near 0%T will be overrange in absorbance. Therefore, the 0%T standard cannot be used.



2. Estimates of stray light will be less accurate because they normally will be overrange in absorbance.

Because of these limitations, it is recommended that the transmittance mode be used for any spectrophotometer on which it is available.

The accuracy of wavelength tests is the same in either the transmittance or absorbance mode. A maximum in transmittance is a minimum in absorbance, so the wavelength peaks will be seen as minima in the absorbance mode. The wavelength of a peak is the same in either mode.

Equations for the conversion of absorbance readings and transmittance readings:

AAT −== 10

101 (1)

Where T = %T/100

TA 10log−= (2)

Where A = absorbance

T = transmittance

%T = percent transmittance

Test Procedures

0% transmittance and stray light test For the SPECTRONIC 20+ and SPECTRONIC 20D+ instruments follow these steps:

1. Set the wavelength to 400 nm.

2. Set the filter lever to the correct wavelength range.

3. Ensure that the sample compartment is empty.

4. Use the OFF-ON/0%T knob to set the readout to 0%T.

5. Insert the empty filter holder into the holder and then place in the sample compartment.

6. Use the front right knob or 100%T/0A knob to set the readout to 100%T.

7. Replace the empty filter holder with the 0%T standard, place in the sample compartment, and record the 0%T reading.

If the 0%T reading exceeds the Maximum Acceptable Deviations as listed in Table 1, correct it. Repeat steps 1 through 7 before proceeding.

8. Replace the 0%T standard with the SRE standard whose wavelength corresponds to the setting in step 1

9. Place the (600-900) filter from the Accessory Filter kit in the slot on the right side of the sample compartment and observe the %T reading.

10. Subtract the reading in step 7 from the reading in step 8.

11. Record the result, which is the stray light at the test wavelength.

This value should conform to the summed tolerances given in Table 1


12. To perform the SRE test at 340 nm, place the (325-405) filter from the Accessory Filter kit in the slot on the right side of the sample compartment and repeat procedure using a wavelength of 340 nm in step 1.

For other spectrophotometer models, use the following procedures:

1. Set the wavelength to 400nm.

2. Set 100%T.

3. Insert the 0%T standard and record the 0%T reading.

If the 0%T reading exceeds the Maximum Acceptable Deviations as listed in Table 1, correct it and repeat steps 1 through 3 before proceeding.

4. Replace the 0%T standard with the SRE standard whose wavelength corresponds to the setting in step 1 and observe the %T reading.

5. Subtract the reading in step 3 from the reading in 4.

6. Record the result, which is the stray light at the test wavelength.

This value should conform to the summed tolerances given in Table 1.

7. Repeat the procedure using the wavelengths specified for your instrument in Table 2.


Table 2 – Wavelengths to use when testing 0% transmittance and stray light

Spectrophotometer Wavelengths

SPECTRONIC 20+ and SPECTRONIC 20D+ 400 nm, 340 nm

GENESYS 20 400 nm, 340 nm

GENESYS 10 Vis 400 nm, 340 nm

GENESYS 10 UV 400 nm, 340 nm, 220 nm

GENESYS 10 UVscanning 400 nm, 340 nm, 220 nm

GENESYS 6 400 nm, 340 nm, 220 nm

BioMate 3 400 nm, 340 nm, 220 nm

GENESYS 10S VIS 400 nm, 340 nm

GENESYS 10S UV-Vis 400 nm, 340 nm, 220 nm

GENESYS 10S Bio 400 nm, 340 nm, 220 nm

BioMate 3S 400 nm, 340 nm, 220 nm

Wavelength accuracy test 1. If the instrument has more than one source lamp, turn on the

tungsten lamp.

2. Refer to the certificate provided with your set of SPECTRONIC Standards and set the wavelength display to read approximately 10 nm lower than the certified wavelength of the Primary Wavelength Test Peak indicated on the certificate.

3. With no samples in your sample compartment, adjust 100%T, set auto zero or Measure Blank (depending on your instrument).

4. Place the wavelength standard in the sample compartment and close the lid. While watching the %T display, slowly change the wavelength toward the certified wavelength as the %T reading increases.

5. Continue to increase the wavelength until the %T value starts to decrease and record the wavelength that gives the maximum %T value.


This wavelength should match the certified wavelength within summed tolerances given in Table 1.

The GENESYS model spectrophotometers (excluding the GENESYS 20) wavelength accuracy tests are performed differently. The GENESYS 10 series and BioMate 3 have a Performance Validation procedure under the Test menu, where the wavelength accuracy test can be performed using the SPECTRONIC Standards.

Wavelength peak selection SPECTRONIC 20+ and SPECTRONIC 20D+

Ensure that the filter lever is positioned to the correct wavelength range.

Note For greater accuracy on non-scanning instruments, a point-by-point scan can be used. Read and plot the filter transmittance at a series of several discrete, closely-spaced wavelength settings around the expected location of a transmission peak. Carefully reset 100%T at each wavelength with the filter removed from the light beam. The advantage of this procedure is that any slope in the instrument's spectral response curve will have less effect on where the peak is found than it would in the method of direct, continuous scanning without resetting 100%T. The disadvantage is that it is time-consuming and tedious; many settings and readings must be carefully made and plotted to obtain one result. Our recommendation is to use the method given in the General Procedure rather than try to minimize error through this more time-consuming method.

Photometric accuracy/linearity test 1. Set the wavelength to 590 nm.

2. Set 0%T with the sample compartment empty.

Note There are a few 20D+ models with auto 0%T capability. If you have one of these, skip this step.

3. Insert the empty filter holder into the square cuvette holder and then place in the sample compartment. Adjust the instrument to read exactly 100%T.

4. Remove the empty filter holder from the square cuvette holder and reinsert the empty holder to open the built-in occluder.



When the empty holder is inserted, the 100%T reading should remain essentially unchanged.

5. Insert the photometric performance standard whose labeled value is near 50%T and record the instrument reading.

The reading should be within the summed tolerance given in Table 1.

6. Replace the 50%T standard with the standard whose labeled value is near 10%T and record the instrument reading.

The reading should be within the summed tolerance given in Table 1.


SPECTRONIC Standards Test Log

STRAY LIGHT WAVELENGTH ACCURACY PHOTOMETRIC ACCURACY

DATE 0%T 220 nm 340 nm 400 nm ____nm

PEAK ____nmPEAK

____nmPEAK

10%T 50%T COMMENTS

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SPECTRONIC Standards User Guide - Cole-Parmer · SPECTRONIC Standards User Guide 5 Photometric Accuracy Standards . The photometric performance standards test photometric accuracy