Eppendorf BioSpectrometer kinetic User Manual

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Eppendorf BioSpectrometer® kinetic

Operating manual

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Trademarks

Eppendorf

and the Eppendorf logo, Eppendorf BioSpectrometer®, Eppendorf SpectraZoom® and UVette®

are registered trademarks of Eppendorf AG, Hamburg, Germany.

is a registered trademark of GE Healthcare UK Ltd., Buckinghamshire, UK.

Hellma

Trademarks are not marked in all cases with ™ or

is a registered trademark of Hellma GmbH & Co. KG, Müllheim, Germany.

in this manual.

This product is manufactured under license to issued U.S. Patent No. 6,122,052.

Protected by U.S. Patent No. 8,464,171.

6136 900.054-03/052014

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Table of contents

Eppendorf BioSpectrometer

kinetic

English (EN)

Table of contents

1 Operating instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.1 Using this manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.2 Danger symbols and danger levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.2.1 Danger symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.2.2 Danger levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.3 Symbols used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.4 Abbreviations used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2 Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.1 Main illustration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.2 Delivery package. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.3 Features. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.3.1 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.3.2 Operation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.3.3 Result output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.3.4 Device self test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3 Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.1 Intended use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.2 User profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.3 Warnings for intended use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.3.1 Personal injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.3.2 Damage to device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.4 Information on product liability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

3.5 Safety instructions located on the device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4 Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4.1 Preparing installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4.2 Selecting the location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4.3 Connecting the device to the mains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4.4 Connecting the printer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4.4.1 Thermal printer DPU-S445 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4.4.2 Thermal printer DPU-414 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4.5 Connecting PC or USB stick for data export. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5 Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

5.1 Overview of operating controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

5.1.1 Entering text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

5.2 Inserting the cuvette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

5.3 Summary of the measuring procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.3.1 Preparing the measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.3.2 Measuring procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.3.3 Important measurement instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

5.3.4 Notes on working with cuvette temperature control . . . . . . . . . . . . . . . . . . . . . . . . . . 26

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English (EN)

6 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

6.1 Selecting a method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

6.2 Photometry method description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

6.2.1 Absorbance method group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

6.2.2 Routine method group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

6.2.3 Basic method group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

6.2.4 Advanced method group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

6.3 Method parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

6.4 Method procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

6.4.1 Check parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

6.4.2 Measure standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

6.4.3 Measure samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

6.4.4 Measure samples: Results displays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

6.4.5 Process results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

6.4.6 Process results: Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

6.4.7 Print & export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

6.4.8 Finish the series of measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

7 Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

7.1 Functions of the User main group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

7.1.1 Results memory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

7.1.2 General method parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

7.1.3 Absorbance spectra library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

7.1.4 Device settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

7.1.5 Device calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

7.1.6 Info . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

8 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

8.1 Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

8.1.1 Cleaning the cuvette shaft cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

8.2 Disinfection/Decontamination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

8.3 Checking the device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

8.3.1 Checking the spectrometer unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

8.3.2 Checking the thermal module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

8.3.3 Device self-test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

8.4 Replacing fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

8.5 Decontamination before shipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

9 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

9.1 General errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

9.2 Error messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

9.3 Result flags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

10 Transport, storage and disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

10.1 Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

10.2 Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

10.3 Disposal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

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English (EN)

11 Technical data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

11.1 Power supply. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

11.2 Ambient conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

11.3 Weight/dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

11.4 Photometric properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

11.5 Incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

11.6 Further technical parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

11.7 Application parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

12 Evaluation procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

12.1 Absorbance values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

12.1.1 Blank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

12.1.2 Background correction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

12.1.3 Cuvette correction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

12.2 Evaluation with factor or standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

12.3 Evaluation with standard curve/line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

12.4 Dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

12.5 Special evaluation procedures for nucleic acids and protein UV . . . . . . . . . . . . . . . . . . . . . . . . 93

12.5.1 Correction A

and correction A

260

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

280

12.5.2 Ratios A260/A280 and A260/A230 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

12.5.3 Conversion to molar concentrations and nucleic acid quantities . . . . . . . . . . . . . . . . . 94

12.5.4 Calculating the factor for protein in "General Method Parameter" . . . . . . . . . . . . . . . 95

12.6 Special evaluation procedures for the dye methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

12.6.1 Calculating the factor for the dye from the absorbance coefficient . . . . . . . . . . . . . . . 96

12.6.2 Calculation of the FOI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

12.6.3 Conversion to amounts of dye. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

12.7 Dual wavelength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

12.8 Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

12.8.1 Measurement procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

12.8.2 Reagent blank value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

13 Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Certificates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

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Table of contents

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English (EN)

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Page 7

Operating instructions

Eppendorf BioSpectrometer

kinetic

English (EN)

1 Operating instructions

1.1 Using this manual

 Read this operating manual completely before using the device for the first time. Also observe the

instructions for use of the accessories.

 This operating manual is part of the product. Thus, it must always be easily accessible.

 Enclose this operating manual when transferring the device to third parties.

 You will find the current version of the operating manual for all available languages on our webpage

under www.eppendorf.com

1.2 Danger symbols and danger levels

The safety instructions of this operating manual indicate the following danger symbols and danger levels:

1.2.1 Danger symbols

Electric shock Explosion

Toxic substances Hazard point

Material damage

1.2.2 Danger levels

DANGER Will lead to severe injuries or death.

WARN ING May lead to severe injuries or death.

CAUTION May lead to light to moderate injuries.

NOTICE May lead to material damage.

1.3 Symbols used

Depiction Meaning

 Actions without a specified order

• List

Actions in the specified order

Press this key to perform the described action.

or sample

Page 8

Operating instructions

Eppendorf BioSpectrometer

English (EN)

Depiction Meaning

Press this softkey to perform the described action.

or [Copy]

Additional information

1.4 Abbreviations used

Absorbance

DNA

Deoxyribonucleic acid

kinetic

dsDNA

Double-stranded DNA

Dye methods Methods for dye labels group for measuring dyed biomolecules

FOI

Frequency of Incorporation: measure for the amount of dye molecules in relation to the amount of nucleotides in dyed biomolecules

mol/L (molar)

OD600

Optical density at a wavelength of 600 nm

RNA

Ribonucleic acid

ssDNA

Single-stranded DNA

Ultraviolet radiation

Vis

Visible light

Coefficient of variation (standard deviation/average value) in percent

Page 9

2 Product description

2.1 Main illustration

Abb. 2-1: Front and rear view

Product description

Eppendorf BioSpectrometer

English (EN)

kinetic

abc

def

method

ghi

jkl

mno

function

pqrs

tuv

wxyz

µ %

exit

delete

standard

enter

Fig. 2-1: Front and rear view

1Display

2Cuvette shaft

2 3

absorbance

height

8.5 mm

blank

sample

absorbance

6Fuse holder

7 Mains/power connection

3Cuvette shaft cover

4 USB port for USB stick and printer

5 Mains/power switch

8USB port for PC

9 Connection for RS-232 printer

10 Operating controls

The name plate is located at the rear left on the bottom of the device.

2.2 Delivery package

Quantity Description

1 BioSpectrometer kinetic

1 Power cord

4 4 UVettes

Original Eppendorf plastic cuvette, individually packaged, PCR clean, protein-free

1 Operating manual, in multiple languages

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Product description

Eppendorf BioSpectrometer English (EN)

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2.3 Features

The BioSpectrometer kinetic is a UV/Vis spectrophotometer for measuring liquids in cuvettes in a wavelength range of 200 nm to 830 nm. It is intended for use in development and research in the fields of molecular biology, biotechnology, biochemistry and cell biology. Glass and plastic cuvettes in a volume range of 1 μL to 3000 μL can be used.

2.3.1 Methods

Numerous methods for concentration determination of nucleic acids, proteins, and dye-marked nucleic acids and proteins, and the OD 600 method for determining bacterial density via turbidity measurement, are already preprogrammed. Furthermore, method templates for various measurement and evaluation procedures (single and multiple wavelength measurements, acquisition of spectra, kinetic methods, evaluations with factor, standard and standard curve) are preprogrammed. It is possible to create individual methods on the basis of the preprogrammed methods and templates. The templates in the Absorbance method group can be used to quickly measure absorbances or spectra without an additional evaluation.

2.3.2 Operation

The preprogrammed methods and templates are combined into clearly arranged groups from which the desired method can be quickly selected. After calling up the method, you are guided through the measuring procedure in clear steps. A help box in the display provides hints upon request. The 3 round measuring keys (standard, blank, sample) allow users to quickly start a measurement.

2.3.3 Result output

The BioSpectrometer kinetic outputs the results using the device display and a printer available from Eppendorf. With a USB connection, you can transfer result data from the device to a USB stick, a printer or directly to a PC.

2.3.4 Device self test

The device automatically checks the function of the spectrometer unit and thermal module immediately after it has been switched on. Access the Device calibration function for a more comprehensive test (see Device self-test on p. 72).

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Safety

Eppendorf BioSpectrometer

kinetic

English (EN)

3Safety

3.1 Intended use

The BioSpectrometer kinetic is to be used in molecular biology, biochemistry and cell biology research laboratories. The BioSpectrometer kinetic is exclusively intended for use indoors. All country-specific safety requirements for operating electrical equipment in the laboratory must be observed.

The BioSpectrometer kinetic is used for photometric concentration determination of analytes in liquids and recording of absorbance wavelength spectra in cuvettes.

Only use Eppendorf accessories or accessories recommended by Eppendorf.

3.2 User profile

The device and accessories may only be operated by trained and skilled personnel.

Before using the device, read the operating manual carefully and familiarize yourself with the device's mode of operation.

3.3 Warnings for intended use

3.3.1 Personal injury

DANGER! Electric shock as a result of penetration of liquid.

 Switch off the device and disconnect the power plug before starting cleaning or

disinfection work.

 Do not allow any liquids to penetrate the inside of the housing.  Do not spray clean/spray disinfect the housing.  Only plug the device back in if it is completely dry, both inside and outside.

DANGER! Risk of explosion.

 Do not operate the device in areas where work is completed with explosive substances.  Do not use this device to process any explosive or highly reactive substances.  Do not use this device for processing any substances which could generate an explosive

atmosphere.

WARNING! Electric shock due to damage to device or mains cable.

 Only switch on the device if the device and mains cable are undamaged.  Only use devices that have been properly installed or repaired.  In case of danger, disconnect the device from the mains supply by pulling the power plug

from the device or the mains socket or, by using the isolating device intended for this purpose (e.g., emergency stop switch in the laboratory).

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Safety

Eppendorf BioSpectrometer English (EN)

WARNING! Damage due to UV radiation.

Microliter cuvettes, e.g., Hellma® TrayCell (or microliter cuvettes with a similar design) divert the radiation from the light source within the cuvette so the radiation can escape upward when the lid is not closed.

 Before starting a measurement, ensure that the lid on the microliter cuvette is not open.

WARNING! Damage to health from toxic, radioactive or aggressive chemicals as well as infectious liquids and pathogenic germs.

 Observe the national regulations for handling these substances, the biological security

level of your laboratory, the material safety data sheets and the manufacturer's application notes.

 Wear personal protective equipment.  For comprehensive regulations about handling germs or biological material of risk group II

or higher, please refer to the "Laboratory Biosafety Manual" (source: World Health Organization, Laboratory Biosafety Manual, in its respectively current valid version).

kinetic

WARNING! Damage to health due to contaminated device and accessories.

 Decontaminate the device and the accessories before storage and shipping.

CAUTION! Poor safety due to incorrect accessories and spare parts.

The use of accessories and spare parts other than those recommended by Eppendorf may impair the safety, functioning and precision of the device. Eppendorf cannot be held liable or accept any liability for damage resulting from the use of incorrect or non-recommended accessories and spare parts, or from the improper use of such equipment.

 Only use accessories and original spare parts recommended by Eppendorf.

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3.3.2 Damage to device

NOTICE! Damage from the use of aggressive chemicals.

 Do not use any aggressive chemicals on the device or its accessories, such as strong and

weak bases, strong acids, acetone, formaldehyde, halogenated hydrocarbons or phenol.

 If the device has been contaminated by aggressive chemicals, immediately clean it by

means of a mild cleaning agent.

NOTICE! Damage to the device from fumigating with aggressive chemicals.

 Do not use fumigation to disinfect the device.

NOTICE! Corrosion from aggressive cleaning agents and disinfectants.

 Do not use corrosive cleaning agents, aggressive solvents or abrasive polishes.  Do not incubate the accessories in aggressive cleaning agents or disinfectants for a longer

period of time.

Eppendorf BioSpectrometer

English (EN)

Safety

kinetic

NOTICE! Damage and measurement errors due to condensation water.

With high humidity, condensation water can form on a cuvette with a temperature significantly lower than ambient temperature. The condensate may cause damage to the optics as well as incorrect measuring results.

 Do not insert any cuvettes into the cuvette shaft with a temperature that is significantly

lower than the ambient temperature.

 The temperature of the cuvette should not remain significantly below the ambient

temperature for a longer period of time.

 If required, observe the actual dew point.

NOTICE! Damage to electronic components due to condensation.

Condensate can form in the device after it has been moved from a cool environment to a warmer environment.

 After installing the device, wait at least for 3 h. Only then connect the device to the mains.

NOTICE! Function impairment due to mechanical damage.

 After mechanical damage to the device, ensure that the measuring and evaluation

functions of the device are operating correctly by completing an inspection.

NOTICE! Damage from overheating.

 Do not install the device near to any heat sources (e.g., heating, drying cabinet).  Do not expose the device to direct sunlight.  Ensure unobstructed air circulation. Keep free a clearance of at least 5 cm around all

ventilation grilles.

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Gerät nach dem Öffnen

justieren!

Adjust device after

opening!

Safety

Eppendorf BioSpectrometer English (EN)

NOTICE! Material damage from incorrect use.

 Only use the product for its intended purpose as described in the operating manual.  Ensure adequate material resistance when using chemical substances.  In case of doubt, contact the manufacturer of this product.

NOTICE! Damage as a result of incorrect packing.

Eppendorf AG is not liable for damage caused by improper packing.

 The device may only be stored and transported in its original packaging.

NOTICE! Damage due to improper cleaning of the cuvette shaft.

 Only clean the cuvette shaft using a moist cotton swab (see Cleaning on p. 65).  Do not allow any liquid to enter the cuvette shaft.  Do not reach with your fingers into the cuvette shaft.

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3.4 Information on product liability

In the following cases, the designated protection of the device may be compromised. Liability for any resulting property damage or personal injury is then transferred to the operator:

• The device is not used in accordance with the operating manual.

• The device is used outside of its intended use.

• The device is used with accessories or consumables which are not recommended by Eppendorf.

• The device is maintained or repaired by people not authorized by Eppendorf.

• The user makes unauthorized changes to the device.

3.5 Safety instructions located on the device

Depiction Meaning Location

Hazard point

 Follow the operating manual.

The device needs to be readjusted after it has been opened.

 Do not open the device.

Rear side of the device

Bottom of the device

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Installation

Eppendorf BioSpectrometer

kinetic

English (EN)

4 Installation

4.1 Preparing installation

 Keep the transport carton and the packing material for subsequent safe transport or storage.

 Check the completeness of the delivery using the information in the delivery package (see Delivery

package on p. 9).

 Check all parts for any transport damage.

4.2 Selecting the location

Select the location for the BioSpectrometer kinetic according to the following criteria:

• 2 grounded sockets for the BioSpectrometer kinetic and for the printer.

• Solid laboratory bench with horizontal work surface Space requirement of the device: 50 cm (with printer: 75 cm) width, 50 cm depth.

• Temperature: 15°C to 35°C.

• Avoid temperature fluctuations (e.g, caused by open windows).

• Avoid direct sunlight.

• Humidity: 25% to 70% relative humidity.

Ensure that no objects (e.g., loose sheets, notebooks) that could impede the flow of air are positioned under the device.

4.3 Connecting the device to the mains

1. Place the BioSpectrometer kinetic on a suitable work surface.

2. Verify that the mains/power supply voltage and mains/power frequency match the information on the name plate.

3. Connect the device to the mains/power line and switch it on with the power switch.

4. Remove the protective film from the display.

4.4 Connecting the printer

4.4.1 Thermal printer DPU-S445

Prerequisites

Software version 3.4.4.0 or higher is installed on the device.

Connect the thermal printer DPU-S445 to the USB port for printers.

1. Connect the printer cable with the USB port for printers 4 (see Main illustration on p. 9).

2. Connect the printer cable with the printer.

3. Connect the printer to the mains/power line using the supplied mains/power adaptor and mains/power cord (printer accessory) and switch it on.

For information on the printer, refer to the operating manual of the printer.

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Installation

Eppendorf BioSpectrometer

kinetic

English (EN)

4.4.2 Thermal printer DPU-414

Connect the thermal printer DPU-414 to the serial printer connection.

1. Connect the printer cable to the serial printer connection 9 and tighten the locking screws. (see Main illustration on p. 9).

2. Connect the printer cable to the printer and tighten the locking screws as well.

3. Connect the printer to the mains/power line using the supplied mains/power adaptor and mains/power cord (printer accessory) and switch it on.

Information about modifying printer settings can be found in the operating manual for the printer.

The DIP switches are preset for the BioSpectrometer kinetic according to the following table.

Tab. 4-1: Setting the DIP SW for the thermal printer

DIP SW-1 Meaning

1 (OFF) Input = Serial

2 (ON) Printing Speed = High

3 (ON) Auto Loading = ON

4 (OFF) Auto LF = OFF

5 (ON) Setting Command = Enable

6 (OFF) Printing

7 (ON) Density

8 (ON) = 100%

DIP SW-2 Meaning

1 (ON) Printing Columns = 40

2 (ON) User Font Back-up = ON

3 (ON) Character Select = Normal

4 (ON) Zero = Normal

5 (ON) International

6 (ON) Character

7 (ON) Set

8 (OFF) = U.S.

DIP SW-3 Meaning

1 (ON) Data Length = 8 bits

2 (ON) Parity Setting = NO

3 (ON) Parity Condition = Odd

4 (OFF) Busy Control = XON/XOFF

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Installation

Eppendorf BioSpectrometer

English (EN)

DIP SW-3 Meaning

5 (OFF) Baud

6 (ON) Rate

7 (ON) Select

8 (ON) = 9600 bps

4.5 Connecting PC or USB stick for data export

You can connect a FAT 32-formatted USB stick to the USB port 4 (see Main illustration on p. 9).

Alternatively, you can connect the device for the data export directly to a PC by using a USB cable:

Prerequisites

• PC with Windows, version XP, SP2 or higher version.

• USB cable with a type A and type B plug each.

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 Connect the device to the PC by using the USB cable on the USB port 8 (see Main illustration on p. 9).

• You do not need any special PC software for the data transmission: the transferred data packets are recognized by the PC like a USB stick as a removable medium. For viewing the data, you only need to open the registered data packet.

• The transmission of data to the USB stick or to the PC is started after completing the series of measurement in the print & export (see Print & export on p. 55) method step.

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Installation

Eppendorf BioSpectrometer English (EN)

kinetic

Page 19

5 Operation

5.1 Overview of operating controls

Abb. 5-1: Control panel of the BioSpectrometer kinetic

Eppendorf BioSpectrometer

English (EN)

Operation

kinetic

Fig. 5-1: Control panel of the BioSpectrometer kinetic

Key: Function

Keypad: Enter digits and text. Keys 1 to 9 as well as 0: When entering text, next to numbers you also can enter letters and special characters by pressing the key several times. Alternatively, you can switch to a displayed keyboard with the [Keyboard] key.

Outside of entry fields: Call up method selection.

Outside of entry fields: Call up function selection.

Softkey: Select functions. The key assignment changes along with the software dialog. The current function is displayed directly above the key on the display.

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Operation

Eppendorf BioSpectrometer

kinetic

English (EN)

Key: Function

Move the cursor to the left, right, up, down.

• Navigation between input fields.

• and keys inside an entry field: Navigate within the character string.

• and keys in a result display: Navigate between the sample results of the series of measurement.

• and keys within a graph: Navigate on the x-axis of the graph, e.g. for displaying the wavelength-dependent absorbance values in a scan.

and keys in an absorbance wavelength spectrum: Change image

section (SpectraZoom procedure) (see Tab. on p. 51).

Exit the current selection for the next higher level.

Delete entry. Within a sequence of signs, the sign on the left of the cursor is deleted

•Call up selected method or function.

• Open the selection list.

• Confirm entry or selection.

Start standard measurement.

Start blank measurement.

Start sample measurement.

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Operation

Eppendorf BioSpectrometer

kinetic

English (EN)

5.1.1 Entering text

You can enter texts when assigning method names and result units. Restriction: Only digits, letters and the underscore "_" are allowed for method names.

Entry via keyboard: Use the and cursor keys to navigate within the

entry field and to change single positions in the name. Softkeys:

• [Keyboard]: Display keyboard.

• [abc]: Change between upper and lower case letters when making entries with the keypad.

• [Save]: Save entered text.

• [Cancel]: Cancel text input.

Entry via the displayed keyboard: Use the cursor keys to select the displayed signs and respectively confirm your selection with the enter key. As for a PC key pad, you can use the "Shift" resp. the "Caps Lock" key for changing the capitalization for the next entry or for all following entries. Softkeys:

• [Numbers]: Switch to entry using the keyboard.

• [Save]: Save entered text.

• [Cancel]: Cancel text input.

5.2 Inserting the cuvette

Standard rectangular glass or plastic cuvettes can be inserted in the cuvette shaft:

• External dimensions: 12.5 mm × 12.5 mm

• Height of light path: 8.5 mm higher than cuvette base

• Total height: min. 36 mm

The cuvettes must be optically transparent for the respective measuring wavelength. For measurements in the UV range, Eppendorf offers the plastic cuvette UVette which is transparent for wavelengths of 220 nm and higher and therefore also is suitable for measuring nucleic acids.

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Operation

Eppendorf BioSpectrometer English (EN)

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Cuvettes

Basic area 12.5 mm × 12.5 mm Min. overall height 36 mm

Min. filling level 10 mm Light path 8.5 mm Max. height of base 7 mm 0 mm Min. volume Photometry

Eppendorf

µCuvette G1.0

See manufacturer

information

Hellma

See manufacturer

* or similar microliter cuvette

TrayCell *

information

50 µL 70 µL 400 µL 1000 µL

Prerequisites

• The cuvette is free from contamination by dust or fingerprints and free from scratches.

• The cuvette shaft is free from particles, dust and liquid.

• The measuring volume in the cuvette is sufficient. Ensure that the minimum measuring volume has been reached.

• The measuring solution is free from particles and bubbles.

The direction of the light path is marked with an arrow on the housing.

MacroSemi-microUltra-microUVette

1. Position the cuvette so that the optical window of the cuvette is pointing towards the direction of the light path.

2. When inserting the cuvette, press it completely to the bottom against the slight resistance.

5.3 Summary of the measuring procedure

5.3.1 Preparing the measurement

1. Switch on the device and, if required, the printer.

The device performs a self test (taking approx. 1 minute) and displays the method selection.

2. Make ready the cuvettes for the measurements (see Inserting the cuvette on p. 21).

3. Prepare the measuring solutions for measuring the blank values, if required, also the standards and the samples.

4. Open the cover of the cuvette shaft. The cover can remain open during the measurements.

You should not use any measuring solution for standards and samples with a lower absorbance than 0.02 to 0.03 A (e.g. dsDNA concentration between 1.0 and 1.5 μg/mL). The detection limit of the device may be significantly lower, nevertheless, the impact of disturbances from the measuring solutions (particles, bubbles, turbidity) on the reliability of the result is very high for these low absorbance values.

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5.3.2 Measuring procedure

5.3.2.1 Selecting a method

Operation

Eppendorf BioSpectrometer

kinetic

English (EN)

 Use the cursor keys to select the desired

method and call up the method with the enter

key. For an overview and a detailed description of the methods, refer to the next chapter (see Methods on p. 29).

Wizard: The wizard at the top of the display will take you through the method procedure step-by-step. Help box: You will receive help texts in the lower right of the display during each step of the procedure. Softkeys: The [< Back] and [Next >] softkeys allow you to move between method steps in the wizard.

5.3.2.2 Checking parameters

 Check the parameter setting. The [Page dn] and

[Page up] softkeys allow you to call up the

parameter list pages. You can modify and save

parameters using [Edit].

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Operation

Eppendorf BioSpectrometer English (EN)

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5.3.2.3 Measuring the blank and standards

For evaluations without standards (e.g. DNA measurements), this method step is omitted.

1. Start by measuring a blank (blank key).

2. Then measure all standards one by one

(standard key). The display always marks the standard that is to be measured next. Use the [Graph] resp. [Table] softkey to change the result view.

5.3.2.4 Measuring samples

 Press [Next] to accept the evaluation calculated

from the standard results.

 The sample key is used for measuring your

samples consecutively. Blank results will remain saved for the duration of one series of measurement. However, a new blank measurement always is possible. (The adjacent figure shows a measuring procedure with evaluation via the standard curve and, in addition to the sample result, the graph of the standard evaluation.)

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5.3.2.5 Finalizing the method

5.3.2.6 Optional: process results

Operation

Eppendorf BioSpectrometer

1. Press [Finish], to complete the measuring series

and return to the method selection.

2. After all measurements have been completed,

switch off the device and close the cuvette shaft

cover to protect the cuvette shaft from

contamination.

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English (EN)

5.3.2.7 Printing and exporting

For some methods, you can postprocess the results in the process results method step. For example, you can use the SpectraZoom zoom function in the spectra.

 Use the and cursor keys for selecting

systematically any results of the series of

measurement for postprocessing.

1. Compose data packets for all samples or for

selected samples.

2. Print the data, save them to a USB stick or

transfer them to a PC via a USB cable.

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Operation

Eppendorf BioSpectrometer English (EN)

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5.3.3 Important measurement instructions

Check for each measurement:

• For plastic cuvettes: How many consecutive measurements can be reliably carried out in the cuvette?

• Measure the cuvette blank value before the sample or standard measurements in order to compensate the cuvette blank value in addition to the reagent blank.

• Blank results remain saved for one measuring series, but a new blank result measurement can be performed at any time, even between sample measurements.

• The displayed absorbance values always correspond to the directly measured values. The dilution or cuvette factor as well as background absorbances only will be incorporated for the following result calculation (see Absorbance values on p. 89).

• The measuring result is typically displayed 2 to 3 seconds after a measurement has been started. If a small amount of light reaches the receiver, the measuring time automatically can be extended to 9 seconds in order to increase the precision of the measurement. For kinetic measurements, the automatic extension of the measuring time is not applied in order to prevent any conflicts with the preprogrammed interval time for the measuring point recording.

• Observe that the measured absorbance values do not exceed the upper limit of the photometric measuring range. In this case, reject the measuring result. The upper limit of the photometric measuring range does not only depend on the wavelength (see Photometric properties on p. 86) but also on the cuvette blank. Ultra-micro cuvettes with a small diaphragm, such as TrayCell (Hellma), may have a cuvette blank of approx. A = 1. The available photometric measuring range is reduced by this amount. You can estimate the cuvette blank by measuring the cuvette filled with demineralized water as a sample in comparison with the empty cuvette shaft as a blank. The cuvette blank of the Eppendorf μCuvette G1.0 is negligible (approximately A = 0).

• After the measurement, remove the measuring solution completely before filling in the next measuring solution in order to minimize carry-over. If a carry-over from one sample to the next sample can be expected due to a high concentration difference, rinse the cuvette between the measurements.

• If the temperature between the lamp and the ambience differs, photometric drift may occur. Therefore a device from a colder ambience first has to be adjusted to the ambient temperature. Avoid quick changes of temperature. Carry out a new blank measurement for a long series of measurements or measurements over a long period of time.

5.3.4 Notes on working with cuvette temperature control

Temperature control is regulated using a measurement on the cuvette holder. The temperature in the measuring solution may deviate from the temperature on the cuvette holder.

The extent of the deviation is dependent on the measuring volume, cuvette material, cuvette shape and ambient temperature. The temperature control speed is also dependent on these factors. The temperature control of plastic cuvettes is slower compared to glass cuvettes. The surface of the cuvette in direct contact with the wall of the cuvette holder should be as large as possible to ensure quick temperature control. Therefore, the temperature control of plastic semi-micro cuvettes as well as, for example, the UVette is only performed slowly.

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Operation

Eppendorf BioSpectrometer

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English (EN)

Measured values typical for Eppendorf for temperature control in closed cuvettes, with closed cuvette shaft covers, are shown in the following tables. The temperature was measured in the measuring solution; the ambient temperature was 24.5°C.

Tab. 5-1: The temperature to be set for temperature control of a measuring solution

Cuvette type/measuring volume Target temperature Temperature to be set in the

method parameters

Quartz glass macrocuvette,  1 500 μL

25°C 25°C

30°C 30.4°C

37°C 37.7°C

Plastic macrocuvette,  1 000 μL

25°C 25°C

30°C 30.4°C

37°C 37.7°C

Quartz glass semimicrocuvette, 500 μL

25°C 25°C

30°C 30.4°C

37°C 37.7°C

Quartz glass ultra-micro cuvette, 60 μL

25°C 25°C

30°C 30.3 °C

37°C 37.6°C

Tab. 5-2: Duration for temperature control of a measuring solution

Cuvette type/measuring volume Initial temperature and target

temperature

Quartz glass macrocuvette,  1 500 μL

25°C - 37°C Approx. 7 min

37°C - 25°C Approx. 11 min

25°C - 30°C Approx. 7 min

Plastic macrocuvette,  1 000 μL

Quartz glass semimicrocuvette, 500 μL

Quartz glass ultra-micro cuvette, 60 μL

25°C - 37°C Approx. 13 min

37°C - 25°C Approx. 19 min

25°C - 37°C Approx. 7 min

37°C - 25°C Approx. 12 min

25°C - 37°C Approx. 7 min

37°C - 25°C Approx. 9 min

25°C - 30°C Approx. 5 min

Temperature control duration

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Operation

Eppendorf BioSpectrometer English (EN)

• For efficient temperature control, the volume of the measuring solution in the cuvette should not project beyond the edge of the cuvette holder.

• To speed up the measuring procedure during series measurements, you can pre-cool cuvettes with reagents in a thermostat outside the BioSpectrometer before inserting the cuvette in the cuvette holder and adding the sample.

• When switching from a method with temperature control to one without temperature control, please note that the temperature of the cuvette holder will slowly drift back to room temperature. The results of the method without temperature control could be affected by this.

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Methods

Eppendorf BioSpectrometer

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English (EN)

6 Methods

6.1 Selecting a method

Methods and method templates are delivered preprogrammed. The methods are organized in main groups and subgroups.

Write-protected methods The most important methods in molecular biology.

Parameters can be modified, but the modified parameters must be saved under a new method name.

Non-write-protected methods You can change parameters any number of times and start

the measurement right after saving.

New methods ("templates") Each method group contains a template which is

preprogrammed with complete parameter sets to facilitate the programming of new methods. The parameters can be changed and saved under new names any number of times.

To call a method, first use the cursor keys to select the main group, subgroup and the method. Confirm each with enter.

Tab. 6-1: Photometric methods

Absorbance Methods for fast, simple absorbance measurements without additional

evaluations

Routine Frequently used molecular biology methods. The methods are

preprogrammed. However, the parameters can be modified if saved under a new name.

Basic Methods for the evaluation of absorbance measurements with factor,

standard or standard curve/line. Method templates for measuring and evaluating kinetics.

Advanced Methods for the evaluation of two wavelength measuring methods and for

kinetics with more demanding evaluation options.

Favorites In Favorites, you can set up your own folders using <New folder>, and

copy your frequently used methods to this folder in order to quickly access them when needed.

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Methods

Eppendorf BioSpectrometer English (EN)

You can create new methods in all folders using <New Method>.

In Favorites, you can create your own folders (e.g., to allocate folders to specific people), and rename and delete the folders.

Tab. 6-2: Softkeys in method selection

[Cut] and [Paste] Cut and paste methods.

[Copy] and [Paste] Copy and paste methods.

[Delete] Delete methods.

[Rename] Rename methods.

Copied or cut methods can be added to a different folder under Favorites, or added to the original folder under a new name. Use the cursor keys to navigate to the Methods column of the desired folder and press [paste] for adding the method.

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6.2 Photometry method description

The preprogrammed methods and method templates are described in this section.

6.2.1 Absorbance method group

Single λ

• Absorbance measurement on a wavelength.

• No subsequent evaluation.

Single λ - continuous

• Repeated absorbance measurement on a wavelength.

• Parameters can be entered for temperature control between 20 and 42°C (presetting: 37°C).

• Entry of parameters for total time and interval time for the measuring points. A premature stop cannot be made during the measurement.

• Evaluation as kinetic via linear regression. A subsequent change cannot be made to the time frame for the evaluation.

• The measured values are displayed in an absorbance time graph.

• To subsequently evaluate measured values as linear regression kinetics, press the [Next >] softkey and go to the process results method step.

Multi λ

• Absorbance measurement at two to six wavelengths.

• No subsequent evaluation.

Scan

• Absorbance wavelength spectra measurement via a defined wavelength range.

• Display of wavelength and absorbance in the spectrum by navigation with a wavelength cursor.

• The spectra section can be modified using 3 different zoom variants.

• Peak detection possible.

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Methods

Eppendorf BioSpectrometer

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English (EN)

6.2.2 Routine method group

The methods for the Routine group are preprogrammed as fixed methods. Therefore, a new method name is required after the method parameters in the fixed preprogrammed methods have been modified.

Nucleic acids

• Determination of the concentration of nucleic acids through measurement at 260 nm and evaluation via factor.

• Various nucleic acid methods, such as dsDNA or RNA, are preprogrammed. The parameters vary according to the factor.

• Preprogrammed method for microliter cuvettes: Measuring DNA in sample volumes within the microliter range with 1 mm light path (with microliter cuvettes as Eppendorf μCuvette G1.0 or Hellma TrayCell).

• Additional information on the purity of the measured nucleic acid: Ratios A260/A280, ratios A260/A230, absorbance wavelength spectrum of nucleic acid, absorbance of the background wavelength (preset: 320 nm; the absorbance of the pure nucleic acid should be close to zero here).

• Partial turbidity correction can be performed via the Background parameter.

• Concentrations can be converted to molar concentrations and (after the sample volume has been entered) to nucleic acid quantities (process results method step).

Proteins direct UV

• Determination of the concentration of proteins via measurement at 280 nm and factor or standard evaluation.

• Preprogrammed methods for direct absorbance output as a result (Protein A 280) and for evaluation via albumin-specific absorbance coefficients (Albumin A 280).

• Preprogrammed method for microliter cuvettes: Measuring protein in sample volumes in the microliter range with 1 mm light path (with microliter cuvettes as Eppendorf μCuvette G1.0 or Hellma

TrayCell).

• Additional information on the purity of the measured protein: Absorbance of the background wavelength (preset: 320 nm; the absorbance of the pure protein should be close to zero here).

• Partial turbidity correction can be performed via the Background parameter.

• When programming the methods, the corresponding factor is imported through the simple selection of the protein from a predefined list. The factors are separately defined in the functions of the Gen. method param. group. Various proteins are preprogrammed in Gen. method param.; additional proteins can be added.

Proteins (with reagent)

• Concentration determination of proteins via measurement according to color reactions and evaluation using standards or factors (typical: evaluation with standard curve).

•The Bradford, Bradford micro, Lowry, Lowry micro, BCA and BCA micro methods are already preprogrammed. According to the reagent manufacturer, the "Curve fit" (standard curve type) must be changed as necessary.

Dye labels

• For dye-labeled biomolecules: Concentration determination of the biomolecule (nucleic acid or protein) via measurement at 260 or 280 nm and measurement of the dye in one measuring procedure.

• Evaluation with factor. In addition to the biomolecule, up to two dyes can be measured at the same time as two different wavelengths.

• Additional: evaluation of the frequency of incorporation (FOI) of the dye. Selection between two different FOI calculation procedures.

• Already preprogrammed methods: ssDNA, labeled with Cy 3 or Cy 5.

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• Correction of the influence of the dye spectrum on the accuracy of the biomolecule measurement is possible.

• Partial turbidity correction can be performed via the Background parameter.

• Additional information on the purity of the measured materials: Ratios A260/A280 and ratios A260/A230 (ratio values only for nucleic acids), absorbance wavelength spectrum.

• When the methods are programmed, various associated parameters, such as wavelengths and evaluation factors, are imported by simply selecting the biomolecule and the dye from predetermined lists. These parameters are separately defined in the functions of the Gen. method param. group. Various nucleic acids, proteins and dyes are preprogrammed in Gen. method param.. You may add further nucleic acids, proteins and dyes.

• Only for labeled nucleic acids: Concentrations can be converted to molar concentrations and (after the sample volume has been entered) to nucleic acid and dye quantities (process results method step).

Bacterial density

• Turbidity measurement to determine the bacteria density.

• Measurement at 600 nm is already preprogrammed.

• Additional information: absorbance wavelength spectrum.

6.2.3 Basic method group

Factor, standard

• Measurement on a wavelength and factor or standard evaluation.

• Methods for factor and standard evaluation are preprogrammed.

Calibration curve

• Measurement on a wavelength and subsequent evaluation with a series of 2 to 12 standards.

• You can select between different evaluation procedures ("Curve fit") as linear regression, non-linear regression.

• Graphical and tabular display of the standard results.

• The last saved standard evaluation can be used.

• A method for standard curve evaluation is preprogrammed.

Simple kinetics

• Measurement of a kinetics at one wavelength and subsequent evaluation by factor.

• 3 different measurement methods are available:

– "Linear regression": Evaluation in a series of measuring points recorded in intervals. – "Two point": Calculation of ΔA/min using 2 measuring points at defined times. – "Endpoint": Recording of a measuring point at a defined time.

• Parameters can be entered for temperature control between 20 and 42°C (presetting: 37°C).

• For the "linear regression" measurement method, the absorbance/time curve is depicted as a graph.

• A method for the evaluation with linear regression is preprogrammed and can be changed for other evaluation procedures ("endpoint", "two.point") via simple selection.

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6.2.4 Advanced method group

Dual wavelength

• Measurement at two wavelengths and evaluation of the measured absorbance values via two basic formulas (subtraction, division)

• Variants of the basic formulas can be defined.

• The result can be evaluated with a factor, with a standard or with a standard series.

• Methods are preprogrammed for calculation, subtraction and division, and subsequent factor evaluation.

Advanced kinetics

The following options are available via the description of the Simple kinetics method (Basic method group):

• Measurement of a reagent blanks kinetics. The blank result is subtracted from all sample results prior to the evaluation.

• An alternative to evaluation via factor is evaluation via a standard.

6.3 Method parameters

This chapter illustrates the parameters for programming the methods. The order of the parameters in the device display may slightly differ from the order in the table in order to display the parameters more clearly. The table displays all parameters available for the various methods. Only a small portion of these parameters are required for the corresponding method and will be shown in the display.

Parameter Entry Explanation

Cuvette Selection:

10 | 5 | 2 | 1 | 0.5 | 0.2 |

0.1 mm

No. of wavelengths

Wavelength Value input:

Value input: Range: 2 to 6.

Measurement wavelength in nm. Range: 200 to 830 nm.

Optical path length of the cuvette. The device always automatically converts absorbance values to the 10 mm path length of a standard cuvette (see Absorbance values on p. 89). Therefore, there is no need to change factors such as "50" for the calculation of dsDNA concentrations when modifying the Cuvette parameter.

Only for the Multi λ method group. Number of wavelengths at which the measurement is to be performed.

Measurement wavelength: The concentration is calculated based on the absorbance measured with this wavelength. For the Multi λ and Dual wavelength method groups, enter more than one wavelength. For some method groups (e.g., Nucleic acids and Proteins direct UV), the wavelengths are preprogrammed. For the Dye labels method group the measuring wavelengths are not entered into the method procedure one by one. They are automatically imported simply by selecting the biomolecule and dye from the General Method Parameters function.

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Parameter Entry Explanation

Unit Selection:

mg/mL | μg/mL |  ng/mL | pg/mL | μg/μL

Unit for the concentration result. In the preprogrammed methods of the Routine group, the

selection is restricted to units that are useful for these methods. | mg/dL | μmol/mL | nmol/mL | pmol/mL | pmol/μL | U | U/mL | U/L | % | Abs | A/min In addition, further units are freely programmable in the

General Method Parameters/Units

function. Max. 7 digits.

Formula type Selection:

division | subtraction

Only for the Dual wavelength method group.

Formula type for calculating the absorbances at the two

measurement wavelengths prior to evaluation with factor or

standard.

Formula: a Value input:

Value for a in the evaluation formula.

Only for the Dual wavelength method group.

Value for a in the formulas: 

[(a*A1) / (b*A2)] * c + d and [(a*A1) - (b*A2)] * c + d. Limit: max. of 5 digits including decimal point.

Formula: b Value input:

Value for b in the evaluation formula.

Only for the Dual wavelength method group.

Value for b in the formulas: 

[(a*A1) / (b*A2)] * c + d and [(a*A1) - (b*A2)] * c + d. Limit: max. of 5 digits including decimal point.

Formula: c Value input:

Value for c in the evaluation formula.

Only for the Dual wavelength method group.

Value for c in the formulas: 

[(a*A1) / (b*A2)] * c + d and [(a*A1) - (b*A2)] * c + d. Limit: max. of 5 digits including decimal point.

Formula: d Value input:

Value for d in the evaluation formula.

Only for the Dual wavelength method group.

Value for d in the formulas: 

[(a*A1) / (b*A2)] * c + d and [(a*A1) - (b*A2)] * c + d. Limit: max. of 5 digits including decimal point.

Calc

ulation Selection:

factor | standard

Evaluation procedure for the calculation of the sample

concentration from the measured absorbance.

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Parameter Entry Explanation

Factor Value input:

Factor. Limit: max. of 6 digits including decimal point.

Factor for converting absorbance values into the concentration.

Negative factors can also be entered for the following method

groups: Simple kinetics, Advanced kinetics, Dual

wavelength, Factor.

For the Dye labels method group the factors are not entered

into the method procedure individually. They are automatically

imported simply by selecting the biomolecule and dye from the

General Method Parameters function.

Protein Selection:

List of protein types which are stored in the

General Method Parameters/Proteins

Only for the Dye labels and Proteins direct UV method

groups.

When selecting the protein, the corresponding Factor

parameter programmed in the General Method Parameters/

Proteins function also will be imported from that function. function.

Standards Value input:

Number of standards. Range: 1 to 12.

Number of different standard concentrations for the evaluation

with standards.

For some methods the range for the number of standards is

restricted to a smaller range than 1 to 12.

Replicates Value input:

Number of replicates

Number of repeated measurements for the various standard

concentrations. per standard. Range: 1 to 3.

Std. conc. Value input:

Concentration values

Based on the number of standards, this parameter is available

for all standards (e.g.,: std. conc. 1, std. conc. 2, ...). of the standards. Limit: max. of 6 digits including decimal point.

Decimal places Value input:

Number of decimal

Number of decimal points for the calculated concentration

result. points for the result. Range: 0 to 3.

Dye 1 Selection:

List of dyes that are stored in the General

Method Parameters/ Dyes function.

Only for the Dye labels method group.

When the dye is selected, the parameters programmed in the

General Method Parameters/Dyes function, which correspond

to the dye, are also imported: factor, wavelength and, if

necessary, correction factors for the measurement at 260 or

280 nm (see the description of the following parameter).

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Parameter Entry Explanation

Correct A260 1 Selection:

on | off

Only for the Dye labels method group.

Correction of the influence of the dye spectrum on the

absorbance with the measuring wavelength of the biomolecule

(260 nm or 280 nm). Some of the dye spectra have a low

absorbance at 260 and 280 nm. These absorbances distort the

calculations for the nucleic acids or the proteins of these

methods. To minimize the distortion correction factors are

used, as long as these are known for the corresponding dyes.

When the parameter is switched on, the correction factor is

imported from the General Method Parameters/Dyes function.

Correct A 280 1 Selection:

on | off

Only for the Dye labels method group.

For an explanation see the description of the Correct A260 1

parameter above.

Dye 2 active Selection:

on | off

Only for the Dye labels method group.

Option to measure a second dye at the same time. Application:

Labeling a biomolecule with two dyes.

Dye 2 Selection:

List of dyes that are

Only for the Dye labels method group when measuring 2 dyes.

Selection of the second dye (see Dye 1 parameter). stored in the General

Method Parameters/ Dyes function.

Correct A260 2 Selection:

on | off

Correct A280 2 Selection:

on | off

Show scan Selection:

on | off

Start λ Value input:

Only for the Dye labels method group when measuring 2 dyes.

Analog to parameter Correct A260 1.

Only for the Dye labels method group when measuring 2 dyes.

Analog to parameter Correct A280 1.

Display of a scan (absorbance wavelength graph) in addition to

the result of the sample measurement.

Starting wavelength for recording the scan. Wavelength in nm. Range: 200 to 830 nm.

Stop λ Value input:

Stopping wavelength for recording the scan. Wavelength in nm. Range: 200 to 830 nm. Value must be higher than the value for  Start λ.

A260/A280 Selection:

on | off

Only for nucleic acids.

Display of the A260/A280 ratio in addition to the result of the

sample measurement.

A260/A230 Selection:

on | off

Only for nucleic acids.

Display of the A260/A230 ratio in addition to the result of the

sample measurement.

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Parameter Entry Explanation

FOI Selection:

none | dye/kb | pmole/ μg

Only for the Dye labels method group.

Display of the FOI in addition to the result of the sample

measurement.

The FOI (frequency of incorporation) is a measure for the

number of dye molecules per nucleic acid molecule that are

integrated into the nucleic acid. Units are "dye/kb" (dye

molecules per 1000 bases) or "pmole/μg" (pmol dye per μg

nucleic acid). "None": no FOI calculation.

Background Selection:

on | off

Prior to the calculation of the results of a sample the

absorbance of a background wavelength, during which the

analyte to be measured should exhibit the absorbance value

zero, is subtracted from the absorbance of the measuring

wavelength. Frequent application: Partial correction of

turbidity for measurement of nucleic acids (background

wavelength in this case: 320 nm or 340 nm).

Wavelength Wavelength in nm.

Range: 200 to 830 nm.

Wavelength at which the background is to be measured. The

analyte to be measured should have the absorbance value zero

in pure form here.

Background for dyes

Selection: on | off

Only for the Dye labels method group.

Application of the background correction to the measurement

of a dye (see Background parameter).

Wavelength Wavelength in nm.

Range: 200 to 830 nm.

Only for the Dye labels method group.

Wavelength at which the background for the dye is to be

measured. The pure, not contaminated dye to be measured

should have the absorbance value zero for this wavelength.

Temperature on Selection:

on | off

Temperature Value input:

Temperature in °C. Range: 20 to 42°C.

For kinetic methods only.

Use of cuvette temperature control.

For kinetic methods only.

Temperature input for the cuvette temperature control when

the Tem perature parameter is set to "on".

Notice! Damages and measurement errors due to

condensate.

At high air humidity, condensate may form on a cuvette having

a significantly lower temperature than the ambient

temperature. The condensate may cause damage to the optics

and lead to incorrect measuring results.

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 The temperature of the cuvette should not remain

significantly below the ambient temperature for a longer period of time. If required, observe the actual dew point.

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Parameter Entry Explanation

Measuring procedure

Selection: lin.regr. | endpoint | two point

For kinetic methods only.

"Linear regression": Measurement over several periods in set

time intervals within a defined period of time. Evaluation via

linear regression of the absorbance time graph within the

measuring time. Absorbance result: ΔA/min.

"Endpoint": Measurement of a measuring point after a defined

period of time. Absorbance result: A.

"Two point": Measurement of 2 measuring points at defined

times. Evaluation: Via linear interpolation between these

measuring points in the absorbance time graph. Absorbance

result: ΔA/min.

Reagent blank Selection:

on | off

For kinetic methods of the Advanced kinetics method group

and evaluation with factor only.

Measurement of a reagent blank (RB). The RB is measured

according to the same measurement method as a sample. The

absorbance result in A or ΔA/min is subtracted from the

absorbance result of the sample before the sample

concentration is calculated. Application: Correction of sample

results for kinetics with reagent drift. The reagent blank

contains the reagent and demineralized water as sample.

Delay Value input:

Time from the start to the first measuring

For kinetic methods only.

Time from the start of the measuring procedure to recording

the same measuring point. point. Range: 00:00 to 10:00 min:sec.

Measuring time Value input:

Time between first and last measuring point.

Only for kinetic methods and the "lin.regr." and "two point"

measurement methods.

Time between recording the first and the last measuring point. Range: 00:05 to 59:59 min:sec.

Total time Value input:

Time between first and

For the Single λ - cont kinetic method only.

Time between recording the first and the last measuring point. last measuring point. Range: 00:05 to 59:59 min:sec.

Interval Value input:

Time between two

Only for kinetic methods and "lin.regr." measurement methods.

Time intervals between the measuring points. measuring points. Range: 00:05 to 10:00 min:sec.

Autoprint Selection:

on | off

Printing a measuring result immediately following

measurement with the thermal printer.

Only the main result data will be printed. To output detailed

data, the required data packets can be compiled and printed in

the print & export method step at the end of a measuring

series.

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6.4 Method procedure

Wizard: the wizard at the top of the display will take you through the method procedure. The currently active method step is highlighted.

A method procedure is composed of a maximum of 5 steps. The currently active step is highlighted visually. After the last step, print & export, of a measuring series, the start of a new measuring series is offered as a next step. It once again starts with the sample measurement.

Method step Explanation

Check parameters Check method parameters. Carry out changes if required.

Measure standards Only for methods with standard evaluation:

Measure and evaluate standards. Alternatively, the last saved standard evaluation can be used.

Measure samples Measuring samples

Process results Only for some methods: postprocess results, e.g. zoom scan graphs.

Print & export Assemble data packets for printing or exporting the data.

Use the [Next >] and [< Back] softkeys to navigate between method steps. With [Abort] and [Finish] you can cancel or finish the measuring procedure. The name of this softkey changes from [Abort] to [Finish] after the first sample measurement.

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Softkeys

• [Page dn] and [Page up]: Change between 1 to 3 parameter list pages.

• [Edit]: Switch to the parameter edit mode.

Editing mode for parameters: Modified parameters are marked with a red star until the modification has been saved.

Softkeys

• [Save] and [Save as]: Save changes. When using [Save as] you have to rename the method. This is always the case when modifying the methods preprogrammed by Eppendorf in the Routine group.

• [Cancel]: Exit the edit mode without saving changes.

Saving the method under a new name: You can save the method in the same folder from which you called up the method or in any folder in the Favorites method group. The name (maximum 20 letters) can be entered using a displayed keyboard ([Keyboard] softkey) or directly using the keyboard (see Entering text on p. 21). After saving you will return to the check parameters display.

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6.4.2 Measure standards

The first standard to be measured is marked on the display. After the blank value (blank key) measure all standards (standard key) one by one. When measuring more than one replicate per standard, the average value for each standard is calculated and displayed automatically.

By using the and cursor keys, you also can select certain standards for measurement. Individual standards can be remeasured as well.

Softkeys

• [Last cal]: Call up the last saved standard evaluation for this method in order to use it for sample measurements.

• [Curve fit]: Select the procedure for the standard evaluation. If the result has not been saved, the method can also be entered later. Instructions for selecting the evaluation procedure can be found in the Evaluation procedure chapter (see Evaluation with standard curve/line on p. 91).

• [Graph]: Switch to the graphic display of the standard results.

As soon as the minimum number of results for the evaluation with the selected method (curve fit) is available, the evaluation result will be shown on the right side of the display. You can now save the evaluation and switch to sample measurements via the [Next >] key.

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Graphical view of the standard evaluation. With the and cursor keys, you navigate between

the standards to view the results. With more than one replicate per standard, you can switch between the

replicate results using and . You can also select individual standards from the graphical display and measure or remeasure them.

Softkeys

• [Table]: Switch to the table display of the standard results.

• [Next >]: Save the standard evaluation and switch to the sample measurement.

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6.4.3 Measure samples

The sample key is used for measuring your samples consecutively. Blank results remain saved for one measuring series, but a new blank result measurement can be performed at any time. With the and

keys you can navigate between the sample results that have been achieved in the measuring series up to this point.

Results display:

• The concentration result (6 digits with floating point) is clearly emphasized.

• With graphic: Result to the right of the display.

• Without graphic: Result in the middle of the display.

• In addition to the result, the basic absorbance value is shown at a smaller scale.

Additional data

• Upper right; first row: Sample number: Counted sequentially and reset to "1" for each new series of measurements. Sample dilution (if provided)

• Upper right; second row: Sample identification (ID) (if provided)

•Top left: File name with which the data in the print and export method step can be exported as Excel file (see p. 55).

Softkeys

• [Dilution]: Enter sample dilution.

• [Edit ID]: Enter sample ID

• [Data]: Display additional result data (not available with all methods).

• [Finish]: End series of measurements and return to method selection.

The displayed absorbance values always correspond to the directly measured values. The dilution or cuvette factor as well as background absorbances will be incorporated for the following result calculation (see Absorbance values on p. 89).

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Enter dilution

The [Dilution] softkey is activated after the blank (blank key) has been measured.

1. Press the [Dilution] softkey.

2. Enter the volumes for the sample (up to 3 digits) and for the dilution buffer (up to 4 digits).

The device will multiply the following sample results by the calculated dilution factor.

Softkeys

• [Clear dil.]: Delete values for sample dilution.

• [OK]: Confirm sample dilution and return to sample measurement.

• [Cancel]: Cancel entry and return to sample measurement.

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The dilution is used for all following sample results until it is changed by a new entry.

Enter sample ID

The ID will be applied to the following sample result. When an ID is being entered the last entered ID will be displayed as a default template to allow the quick entry of IDs with a consecutive structure. A single ID can only be assigned once for the same measuring series.

1. Press the [Edit ID] softkey.

2. Enter the sample ID (up to 12 digits).

Alternatives for character input:

• Keypad: If the key is pressed several times in a row, the possible entries for this key will be shown consecutively.

• Display keyboard with softkey [Keyboard]: Select character with the cursor keys and confirm with

enter.

Softkeys

• [Keyboard]: Display keyboard.

• [abc]: Change between upper and lower case letters when making entries with the keypad.

• [OK]: Confirm ID entry and return to sample measurement.

• [Cancel]: Cancel entry and return to sample measurement.

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Result image with dilution and ID

Result image with dilution and sample ID

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6.4.4 Measure samples: Results displays

This section contains a display of typical results displays for all method groups and an overview of additional results data, which can be accessed using the [Data] softkey.

Method group Results display Explanation

Absorbance main group

Single λ Results display:

• Absorbance at the measuring wavelength

• Only for dilution or with cuvettes other than 10 mm: additional display of the absorbance value before the conversion.

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Single λ continuous

Prior to measurement:

• Check parameters method step: Display temperature control. When the temperature set in the parameters has been reached and the display changes from Temperi ng to Ready, you can go to the measurement.

During the measurement:

• You can prematurely cancel the measurement using the [Stop] softkey.

Results display:

• Graph with absorbance time display and plotted regression line.

• Value for "A/min" calculated from the linear regression.

Additional data ([Data] softkey):

• Absorbance time value pairs for the first and last measuring points.

• Quality parameters for linear regression

• Navigate between the measuring points in the graph with and .

• If necessary, change the time frame for evaluation with linear regression in the process results method step.

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Method group Results display Explanation

Multi λ Results display:

• Absorbance values at the wavelengths

Additional data ([Data] softkey):

• Only for dilution or with cuvettes other than 10 mm: absorbance value before the conversion.

Scan Results display:

• Scan (graph with absorbance wavelength display)

• Navigate between the measuring points in the graph with and .

Routine main group

Nucleic acids Results display:

• Concentration result with absorbance at the measuring wavelength

• If activated in the parameters: Ratios A260/A280

• If activated in the parameters: Scan. Navigate between the measuring points on the graph which are used for

the result calculation with and .

Additional data ([Data] softkey): If the corresponding parameters have been activated:

• Absorbance value for 280 nm.

• Ratios A260/A230 and absorbance value for 230 nm.

• Absorbance value for the background wavelength.

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Method group Results display Explanation

Proteins direct UV

Results display:

• Concentration result with absorbance at the measuring wavelength

• If activated in the parameters: Scan. Navigate between the measuring points on the graph which are used for

the result calculation with and .

Additional data ([Data] softkey): If the corresponding parameters have been activated:

• Absorbance value for 260 nm.

• Absorbance value for the background wavelength.

Proteins (with reagent)

Results display:

• Concentration result with absorbance at the measuring wavelength.

• For evaluation with standard series: graph of the standard evaluation with plotted sample result.

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Dye labels Results display:

• Concentration results with absorbance at the measuring wavelength of the biomolecule.

• If activated in the parameters: Scan. Navigate between the measuring

points in the graph with and .

Additional data ([Data] softkey):. If the corresponding parameters have been activated:

• Ratios A260/A280 and A260/A230.

• Absorbance values for 280 nm and 230 nm and for the measuring wavelength of the dye.

•FOI value.

• Absorbance values for the background wavelengths.

Ratios and FOI are not displayed for the measurement of the dye-labeled proteins.

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Method group Results display Explanation

Bacterial density

Results display:

• Calculated result with absorbance at the measuring wavelength.

• If activated in the parameters: Scan. Navigate between the measuring

points in the graph with and .

Basic main group

Factor, standard

Analog to Protein direct UV (see above) Results display:

• Concentration result with absorbance at the measuring wavelength.

Calibration curve

Analog to Proteins (with reagent) (see above) Results display:

• Concentration result with absorbance at the measuring wavelength.

• Graph of the standard evaluation with plotted sample result.

Simple kinetics

Prior to measurement:

Results display:

• Concentration result with absorbance or A/min with the measuring wavelength.

• Only for the "linear regression" measuring procedure: graph with absorbance time display and plotted regression line. Navigate between the measuring

points in the graph with and .

Additional data ([Data] softkey): Dependent on the measuring procedure:

• Absorbance time value pairs for the first and last measuring points ("endpoint" method: only one measuring point).

• Quality parameters for linear regression (omitted for kinetics with a slope below 0.050 E/min).

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Method group Results display Explanation

Advanced main group

Dual wavelength

Results display:

• Concentration result: calculated from

with factor or standard

calc.

evaluation.

• A

: calculated using the formula,

calc.

defined in the parameters, created from the absorbances measured on both wavelengths.

• Absorbance values that were measured at the two measuring wavelengths.

Additional data ([Data] softkey): If the corresponding parameters have been activated:

• Absorbance value for the background wavelength.

Advanced kinetics

Analog to Simple kinetics (see above). In addition to the data similar to the

Simple kinetics method group: Additional data ([Data] softkey): If the corresponding parameter was activated:

• Absorbance or A/min for the reagent blank.

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6.4.5 Process results

The sample measurement is followed by two optional steps in the method sequence: process results and print & export.

In the process results step, you can postprocess the results for some methods. Example: Changing the spectra section of a scan.

As for the result display, you can navigate between the sample results of the measurement series with the and cursor keys and select results for postprocessing.

Tab. 6-3: Options: Overview

Option Explanation Available in methods

Zoom Change the axis limit of the absorbance

wavelength graph to limit the view to enlarged sections of the graph.

More calculations Convert concentration results into molar

concentrations and (after entering the volume) into total amounts.

Peak detection Identifying peaks in absorbance wavelength

spectra.

Linear regression Modifying time windows for the evaluation of

a kinetic via linear regression.

Generally, all methods for which the Scan parameter is offered and has been activated.

• Multi λ

• Scan

• Nucleic acids

• Proteins direct UV

• Dye labels

• Bacterial density

• Dual wavelength

• Nucleic acids

• Dye labels (with nucleic acids as biomolecule)

• Scan

In principle, all kinetics methods for which the "Linear regression" measuring procedure was used.

• Single λ - continuous

• Simple kinetics

• Advanced kinetics

Options for postprocessing are presented on the two softkeys at left. In this example: [Zoom] and [More Calculations].

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After changes have been made, you can exit the current mode using the two softkeys at right:

• [Save]: Save changes and return to the process results method step.

• [Cancel]: Cancel and return to the process results method step.

After the changes have been saved you can apply them to all samples of the measuring series with [Yes].

6.4.6 Process results: Options

Zoom

Press the [Zoom] softkey and select one of the following versions.

Variant [spectra]:

• Cursor keys and : Move the wavelength cursor. It determines the zoom center above the x-axis.

• Cursor keys and : Gradually zoom in and out of the displayed section of the x-axis using the SpectraZoom procedure. The shown section of the y-axis is automatically adjusted with each step so that maximum and minimum of the data to be displayed make optimum use of the section.

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Methods

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Variant [spectra-0]: Same as the [spectra] variant, with one exception: The lower limit of the displayed section of the y-axis always equals "0 A".

Variant [free]: User-defined values for interval limits can be entered for both axes. Navigation between the entry fields by means of the

cursor keys ( , , ).

For all 3 versions, the [reset zoom] softkey will bring you back to the original display of the spectrum.

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More calculations

Press the [More calc.] softkey.

Methods

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Nucleic acids method group:

• After the molar mass has been entered (in base/ base pairs or in kDa): Convert the concentration result to the molar concentration.

• After the sample volume has been entered: Calculate the total amount in the sample.

Dye labels method group:

Nucleic acid:

• After the molar mass has been entered (in base/ base pairs or in kDa): Convert the concentration result to the molar concentration.

• After the sample volume has been entered: Calculate the total amount in the sample.

Dye:

• After entering the volume of the sample: Calculate the total amount in the sample.

•For dsDNA the calculation of the molar concentration is based on the assumption of a double-stranded nucleic acid. For the ssDNA, RNA and Oligo methods, a single-stranded nucleic acid is assumed.

• For methods which have been reprogrammed via <New Method> in the Routine main group, Nucleic acids method group, always double-stranded nucleic acids are assumed for calculating the molar concentration.

Peak detection

Press the [Peaks] softkey. For the peak detection you can alternate between two criteria:

• λ grid: Evaluation grid on the wavelength scale for the peak detection (e.g., 10 nm). 10 nm example: The spectra section from -5 nm to +5 nm is evaluated in relation to the peak to be detected.

• Min. Δ Abs: Minimum difference between the peak to be detected and the lowest absorbance in the evaluation grid. No absorbance value in the grid may be higher than the value of the peak at any given time (e.g.: 0.5).

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Methods

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Examples:

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λ grid: 100 nm, min. Δ Abs: 0.050: The peak is not detected because the λ grid is too large: The absorbance values on the left edge of the grid are higher than the absorbance of the peak.

λ grid: 20 nm, min. Δ Abs: 0.200: The peak is not detected because the predetermined value for min. Δ abs is too high. The difference of the absorbance of the peak and the lowest absorbance in the grid is less than 0.2 A.

λ grid: 20 nm, min. Δ Abs: 0.050: The peak is detected.

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Methods

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Linear regression

For kinetic methods evaluated using the "Linear regression" measurement method, you can subsequently define the start and end times for the regression evaluation in the process results method step.

Selecting measuring points

• [First pt.] softkey: Define the first measuring point for the regression evaluation. Select the

measuring point using and . Confirm with enter.

• [Last pt.] softkey: Define the last measuring point for the regression evaluation. Select the

measuring point using and . Confirm with enter.

• At the same time that the times are modified, the result is recalculated. The reagent blank is also recalculated in the newly defined time window for this purpose.

6.4.7 Print & export

In the last optional method step, you can assemble data packets for all samples of a series of measurements, or selected samples of a series of measurements, for printing to the printer, export to a USB stick or export to a PC using a USB cable.

Select data packets

• Use the cursor keys for navigating and confirm with enter.

Softkeys

• [Print]: Start printing.

• [Export]: Start export.

• [Sample]: Select individual sample results.

Select samples

• Press the [Samples] softkey to call up the sample selection.

• Use the cursor keys for navigating and confirm with enter.

Softkeys

• [Select all]: Select all samples

• [De-Sel. all]: Cancel selection.

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Methods

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Data export

The data will be transferred as Excel files (.xls) and can be read with Excel versions Excel 97 and later. For each of the selected data packets, a worksheet is created in Excel. The file name consists of the method name, the time and the date of the measuring series.

Select export version

If no USB stick is connected, the first variant cannot be selected.

Export to USB stick

1. Connect a FAT 32-formatted USB stick to the USB port 4 (see Main illustration on p. 9).

2. Start with [Export] "export to an external storage medium".

Export to PC

Requirement for the PC operating system: Windows XP, SP2 or a higher version.

1. Connect the device to the PC by using the USB cable on the USB port 8 (see Main illustration on p. 9).

2. Prior to beginning a new export make sure that any data that has been exported previously has been saved to the PC hard drive. Otherwise, the new export will overwrite the data.

3. Start with [Export] "export to PC".

4. The exported data packet will be displayed on your PC as a removable medium named "eppendorf". Open the Excel file on this drive and save it to the hard drive.

Select data packets

Results Primary result data; cannot be selected because they are always transferred.

Data Additional results data that are displayed during the measurement using the

[Data] softkey.

Graph Absorbance-wavelength-spectrum.

(Only for kinetic methods with the "linear regression" measuring procedure: absorbance time graph.)

Graph data The basic numeric data for the graph.

"export only": Only available for export, i.e., not for printing.

Parameters Method parameters

Standards/results Results data of the standard evaluation.

Standards/graph (Only for standard evaluations with several standards:) absorbance concentration

graph.

Based on the method and parameter setting, only the available data packets are presented.

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Methods

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6.4.8 Finish the series of measurements

After the print & export method step has been finished, you can start a new series of measurements using the selected method or select a new method.

Finish the series of measurements and start a new series of measurements

• [Next >] softkey: Call up new series method step

• [New] softkey: Call up measure samples method step and start a new series of measurements.

Finish the series of measurements and select a new method

• [Finish] softkey: Close the series of measurements and call up the method selection.

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Methods

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Functions

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7Functions

7.1 Functions of the User main group

With the function key or the [Function] softkey, you reach a menu containing functions like device settings or calling up saved results.

The functions are structured in 3 columns analog to the method selection. The functions in the User main group are accessible to you. As for the method selection, you navigate with the cursor keys for selecting first the desired subgroup and then the desired function in the right column. Press enter to open the function.

[Info] softkey: call up the firmware version and serial number of the BioSpectrometer kinetic.

Tab. 7-1: Overview of the functions

Subgroup Explanation

Results memory Displays saved results.

The results can be accessed structured according to methods and measuring series and can be printed and exported directly from the memory.

General method parameters

Absorbance spectra library

Device settings Editable device settings, e.g., language.

Device calibration • Option for checking the spectrophotometer; An Eppendorf filter set is

Info Open Source Licenses and information on registered trademarks.

Parameters which are used for different methods in common are stored centrally in the Functions area. Here it is possible to edit (change or create anew) these parameters. In the Check parameters method step, the comprehensive parameters can be easily selected using drop-down menus.

• Proteins, nucleic acids, dyes include parameters that are directly applied for methods of the Dye labels and Proteins direct UV groups.

• Units: Units for concentration results which can be used for many methods.

Absorbance wavelength spectra of important substances, e.g., DNA. The spectra serve as information and can be used for comparison to a spectrum of a sample result.

required for this.

• Option for checking the thermal module.

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Functions

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7.1.1 Results memory

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 In the right column, select the method for which

you would like to call up saved results.

 Confirm with enter.

 Select the desired series of measurement with

the cursor keys.

 Confirm with enter.

As in the method procedure, you can also successively switch between the display of the parameters, standards, sample results and, finally, the data packets for print and export. The assignment of the softkeys matches the assignment in the method procedure.

 If you would like to print or export results,

select the data packets. The procedure for print and export and the meaning of the function keys corresponds to the print & export method step.

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7.1.2 General method parameters

Functions

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 In the right column, select the parameter group

for which you would like to edit parameters.

 Confirm with enter.

In this example, parameter groups are summarized for various dyes (dye components for the dye methods) and stored under a name. With this name, the required parameter group can be imported into the method program during the editing of a dye method. Display:

• Left: Name of the dye. Select via and .

• Right: corresponding parameters

Softkeys

• [Edit]: Edit selected parameter group.

• [New]: Create new parameter group.

• [Delete]: Delete selected parameter group.

• [OK]: Return to the function selection.

 To edit a parameter group, use and to

select the parameter which you would like to

edit.

 Confirm with enter.

Softkeys

• [OK]: Save entry and return to the parameter group selection.

• [Cancel]: Return to the parameter group selection without making any changes.

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Functions

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When programming a method of the Dye labels or Proteins direct UV method groups, you can access the entries in General Method Parameter:

Select the name of the dye to import the corresponding parameter group into the method program. By using the "edit" selection of the "Nucleic acid" parameter, you also can get directly to the General Method Parameter function and view and edit the parameters.

Tab. 7-2: Parameter in General Method Parameter

Parameter Explanation

Proteins These parameters are loaded into the method parameters when a protein is

selected during the programming of a method in the Dye labels and Proteins direct UV groups.

• Protein name

•Factor

•A

0.1%

In order to define a factor for calculating the concentration on the basis of the absorbance, enter the following data in addition to the name and wavelength: Factor or A

or absorbance coefficient and molar mass.

0.1%

•Ext.coeff.

•Molecular mass

Nucleic acids These parameters are loaded into the method parameters when a nucleic acid

is selected during the programming of a method in the Dye labels group.

•NA name

•Factor

• Double-stranded

The factor is used to calculate the concentration on the basis of the absorbance. The double-stranded parameter affects the calculation of the molar nucleic acid concentration. (see Conversion to molar concentrations and nucleic acid

quantities on p. 94)

Dyes These parameters are loaded into the method parameters when a dye is

selected during the programming of a method in the Dye labels group.

•Dye name

• Wavelength

•Ext.coeff.

•Factor

• Corr. A260

• Corr. A280

In order to define a factor for calculating the concentration on the basis of the absorbance, enter the following data in addition to the name: Factor or absorbance coefficient. The correction factors for the absorbance values at 260 or 280 nm are used when the correction function in the method parameters is active. For more details, refer to the chapter on evaluation (see Correction A

on p. 93).

280

and correction

260

Units You can select a unit from all available units when programming method

parameters.

• Unit Entering a unit that has not yet been programmed for the concentration result.

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• Specifications for proteins which are not preset at the factory can be determined in the expasy database: http://www.expasy.org/tools/protparam.html.

• A table with A

values for many proteins can also be found in: C.N.Pace et al., Protein

Science (1995), 4: 2411–2423 (Table 5). The A return the required A

0.1%

7.1.3 Absorbance spectra library

values.

Functions

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English (EN)

values must be multiplied by 0.1 to

In the right column, you select the spectrum which you would like to call up and confirm with enter.

7.1.4 Device settings

Softkeys

• [Export] and [Print]: Export to a USB stick or print to a PC using a USB cable (see Print & export on p. 55).

• [OK]: Return to the function selection.

The following settings can be modified:

• Language: German, English, French, Spanish, Italian.

• Date and time.

• Frequency of the automatic self test of the device after switching on.

Softkeys

• [Save]: Save changes and return to the function selection.

• [Cancel]: Return to the parameter group selection without making any changes.

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Functions

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7.1.5 Device calibration

Information on checking the device is provided separately (see Checking the device on p. 66).

7.1.6 Info

The Copyright menu item contains license information on the Open Source software.

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8 Maintenance

8.1 Cleaning

DANGER! Electric shock as a result of penetration of liquid.

 Switch off the device and disconnect the power plug before starting cleaning or

disinfection work.

NOTICE! Corrosion from aggressive cleaning agents and disinfectants.

 Do not use corrosive cleaning agents, aggressive solvents or abrasive polishes.  Do not incubate the accessories in aggressive cleaning agents or disinfectants for a longer

period of time.

Maintenance

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1. Wipe down the surfaces with a cloth moistened with a mild cleaning agent.

Cleaning the cuvette shaft

2. The cuvette shaft may only be cleaned with a lint-free cotton swab that has been dampened with ethanol or isopropanol. Prevent liquid from entering the cuvette shaft. If the shaft needed to be dampened with water to remove contamination, follow this up by cleaning the shaft with a cotton swab dampened with ethanol or isopropanol to accelerate the drying process.

8.1.1 Cleaning the cuvette shaft cover

If you would like not only to clean the directly accessible surface of the cuvette shaft cover, you can remove the cover.

 Do not soak the cuvette shaft cover in cleaning agent.  Clean the cuvette shaft cover as described.

1. Lift the cuvette shaft cover with one hand.

2. With the other hand, hold the cover at the height of the locking pin and pull the cover to the right until the locking pin has been removed.

• Pull the cover to the right at a 90 degree angle.

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Maintenance

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3. Clean the cover with a cloth or lint-free cotton swab dampened with a mild cleaning agent.

4. Slide the locking pin back into the housing as far as it will go.

The locking pin has completely disappeared in the housing.

When the photometer is not being used, close the cuvette shaft using the blue cuvette shaft cover to protect it from dust and other contamination.

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8.2 Disinfection/Decontamination

DANGER! Electric shock as a result of penetration of liquid.

 Switch off the device and disconnect the power plug before starting cleaning or

disinfection work.

1. Clean the device with a mild cleaning agent before the disinfection (see Cleaning on p. 65).

2. Choose a disinfection method which corresponds to the legal regulations and guidelines in place for your range of application.

3. For example use alcohol (ethanol, isopropanol) or other alcoholic disinfectants.

4. Wipe the surfaces with a cloth which you have moisturized with a disinfectant.

5. If the cuvette shaft cover needs to be removed for the disinfection, proceed as follows for the disassembly and assembly (see Cleaning the cuvette shaft cover on p. 65).

6. You can use spray disinfection to disinfect the disassembled cuvette shaft cover.

8.3 Checking the device

Requirements

• Observe the ambient conditions (see Ambient conditions on p. 85).

• Carry out an inspection at approx. 20 °C. Avoid temperature variations (e.g, due to opened windows).

• The filter may only be removed from the filter box for a short period of time, and the filter surfaces must be protected from contamination or damage.

• Protect the filter from dust, heat, liquids and aggressive vapors.

• The filter is inserted so that the label, with the name of the filter, points toward the detector. When the spectrometer unit is checked: label points forward.

• The cuvette shaft is free from contamination.

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Maintenance

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8.3.1 Checking the spectrometer unit

Eppendorf offers a filter kit (BioSpectrometer reference filter kit) for checking the photometric accuracy and wavelength systematic error. The kit contains one blank filter (A0) and three filters (A1, A2 and A3) for checking the photometric accuracy, and 3 filters for checking the wavelength systematic error in the range of 260 nm to 800 nm. The filter absorbances are measured against blank filter A0. In addition to information on accuracy, you also obtain information on precision: average value and variation coefficient (CV value) are calculated from the sets of 15 measurements per wavelength.

For the measurement, place the blank filters (for blank measurement) and sample filters into the cuvette shaft, using the same procedure as with cuvettes. The absorbance values measured for the test filters will be compared to the permitted range of values. For the individual filters, the limit values for the permitted area are printed in a table in the lid of the filter box.

Please connect the Eppendorf thermal printer if you would like to document the values.

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Maintenance

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Abb. 8-1: Inside of the filter box lid (sample)

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Fig. 8-1: Inside of the filter box lid (sample)

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8.3.1.1 Checking photometric accuracy

Maintenance

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English (EN)

1. Select the Spectrometer unit function in the Device calibration group and confirm with enter.

2. Select whether you want to check the wavelength systematic error or photometric accuracy, and confirm with enter. Press [Next >] to switch to the measurement.

3. Follow the instructions on the device display and start by measuring the A0 blank filter, and then the first test filter A1. The device measures the test filter 15 times at 9 wavelengths, and then shows the average value and the VC values for the series of measurements for all 9 wavelengths.

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Maintenance

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4. Results display after measuring a test filter for testing photometric accuracy. Measure the other two test filters A2 and A3.

5. Results display after measuring all 3 test filters for testing the photometric accuracy. With the and keys, you can view the results for the different test filters again. Press [Finish] to complete the test.

6. Compare the average values and CV values to the supplied table. If the measured values do not agree with the permitted range of values, contact Eppendorf Service.

Have the filter recertified by Eppendorf Service after 2 years.

8.3.1.2 Checking wavelength systematic error

 To check the wavelength systematic error, proceed as follows: Measure the 3 test filters at the

corresponding wavelength.

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8.3.2 Checking the thermal module

• Carry out an inspection at approx. 20 °C.

• Make sure that the cuvette shaft is empty for the check.

Maintenance

Eppendorf BioSpectrometer

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1. Select the Temperature unit function in the Device calibration group and confirm with enter.

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2. Make sure the cuvette holder is empty, and start the test procedure with [Next >]. The following temperature control test at 5 temperatures takes approx. 30 minutes. The estimated remaining time is displayed during the test.

3. The results are displayed after the test has been completed. A positive test result is indicated by "OK" behind the temperature value. If the result "failed" is displayed for at least one temperature please contact Eppendorf Service.

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Maintenance

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8.3.3 Device self-test

The frequency of the automatic self-test (approx. 1 minute) can be set using the Device settings function (see Device settings on p. 63). The factory setting for Self-test interval is "Weekly".

The self-test checks the following:

• Verification of the detector – Determination of the random error across the entire available spectrum

• Verification of the light source – Verification of the maximum available energy of the light source and quality of the light transmission

through the device – Determination of the signal random error at the reference sensor – Determination of the signal level at the reference sensor – Separate determination of light intensity in UV range

• Determination of the systematic and random error of the wavelength – Position of an intensity peak in the UV range of the spectrum – Precision of the position of an intensity peak in the UV range of the spectrum

• Random error of the temperature sensor

• Verification of the temperature control rate

 Select the Perform self-test function in the Device calibration group and confirm with enter.

At the end of the self-test, the display shows the message PASSED. If the display shows the message FAILED, the self-test has failed. If this error cannot be corrected (see Error messages on p. 76), contact Eppendorf Service.

8.4 Replacing fuses

DANGER! Electric shock.

 Switch off the device and disconnect the power plug before starting maintenance or

cleaning work.

The fuse holder is located between the mains connection socket and the mains power switch.

1. Disconnect the power plug.

2. Press the upper and lower end of the plastic springs 1 together and pull the fuse holder 2 fully out.

3. Replace faulty fuses and reinsert the fuse holder. Make sure that the guiding rail 3 is positioned correctly.

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Maintenance

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English (EN)

8.5 Decontamination before shipment

If you are shipping the device to the authorized Technical Service for repairs or to your authorized dealer for disposal please note the following:

WARNING! Risk to health from contaminated device

1. Follow the instructions in the decontamination certificate. You find it as a PDF file on our website (www.eppendorf.com/decontamination

2. Decontaminate all the parts you would like to dispatch.

3. Include the fully completed decontamination certificate in the package.

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Maintenance

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9 Troubleshooting

9.1 General errors

Error Possible cause Remedy

Measuring results are imprecise.

• Reagent is past its shelf life.

• Reagent has not been prepared properly.

• Pipetting is not correct.

• Incubation procedure before measurement is incorrect.

• The cuvette is contaminated.

• The cuvette is not filled completely with measuring solution, and it contains bubbles.

• Turbidity of the measuring solution.

• Spectrophotometer is drifting.

• Cuvette shaft is dirty.

 Ensure that the reagent is still within its

shelf life and properly prepared.

 Use clean demineralized water of adequate

quality for preparation if required.

 Ensure that the pipette is calibrated and

that pipetting is being performed correctly.

 If the method procedure requires

incubation before the measurement, ensure that the temperature and time for incubation are correctly observed.

 Clean and rinse the cuvette. When

replacing a cuvette, pay attention that the optical window of the cuvette remains clean and that you do not touch it with your fingers.

 If the cuvette window has become soiled

from fingerprints, wipe it clean using a lint-free lab cloth soaked in ethanol or isopropanol.

 Ensure that the required minimum volume

of the cuvette for a measurement is reached and that no bubbles are in the measuring solution.

 Centrifuge the turbid measuring solutions

containing particles and use the clear supernatant.

 Contact Eppendorf Service.  Observe the ambient conditions.  Prevent temperature changes.

 Clean the cuvette shaft.

Troubleshooting

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Trou bleshooting

Eppendorf BioSpectrometer English (EN)

Error Possible cause Remedy

The measuring results are not correct.

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• The method has not been programmed correctly.

• The standard solution has not been prepared correctly.

• The absorbance of the reagent is drifting.

• The cuvette is not positioned correctly.

 Ensure that the method parameters are

entered correctly.

 Ensure that the correct standard is used

and that the measuring solution for the standard is prepared correctly.

 For instable reagent absorbance and end

point methods: When measuring a long series of samples, measure the reagent blank value not only at the beginning but also during the sample series. If the reagent blank value drifts strongly, the reagent is not appropriate for error-free measurements and has to be replaced by a new reagent.

 Position the cuvette in the cuvette shaft so

that the optical window points towards the direction of the light path.

 Photometry light path: from back to front

9.2 Error messages

You can exit device displays with error messages using the [OK] softkey.

System errors require an evaluation by the Technical Service. These errors are shown in English (System error …). Please contact Technical Service in these cases. Other error messages, for which you can carry out troubleshooting measures, are illustrated in the table below.

Problem Cause Solution

Self test failed. • Cuvette shaft cover was open during

self test.

• The cuvette shaft was not empty during the self test.

• Device is faulty.

File export failed. During data export:

• USB stick improperly formatted or faulty.

• USB stick removed from the device too early (during the export).

Failed to initialize printer.

• Printer not connected or switched off.

• Printer not configured correctly.

 Repeat the self test with empty

cuvette shaft and the cuvette shaft cover closed.

 Contact Eppendorf Service.

 Reformat or replace the USB stick.  Reconnect the USB stick and repeat

the export.

 Connect the printer and switch it on.  Reconfigure the printer.

For a correct configuration of the printer settings refer to the installation description (see Connecting the printer on p. 15).

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Eppendorf BioSpectrometer

Problem Cause Solution

Blank measurement: An intensity on a pixel that influences the main, auxiliary or scan wavelength is too low.

The entered name is not valid.

A method (or folder, dye, protein, nucleic acid, or unit) with this name already exists.

The following parameter values are not defined in

General Method Parameter:

The value of the parameter marked with * is not defined in the Gen. Param. Please correct the parameter.

Invalid zoom interval.

The entered standard concentrations are not monotonically increasing resp. monotonically falling. Correct the standard concentrations.

• The absorbance of the blank solution used for the blank measurement is too high.

• Incorrect or turbid blank solution.

• For scans: Wavelength range is too large, because the sample is very strongly absorbed in part of the wavelength range.

• Error when entering the name. Different causes are possible. For the precise cause please see the information in the help box.

• The name under which the method was saved has already been used for a different method in the same folder.

• The message also appears after editing names already given to a folder or to a nucleic acid (dye, protein, concentration unit) (under General Method Parameter).

• When opening a method with parameters which access General Method Parameter, the system determined that at least one parameter (dye, nucleic acid, protein, unit) does not exist there anymore, so probably has been deleted.

This error message appears when editing method parameters.

• Parameter in General Method Parameter is not defined.

During the Zoom process with free entry of limits ([Free]) softkey):

• The zoom area is below the lower limit.

• See the error text.

 Check the blank solution and

remeasure the blank if required.

 For scans: Match the wavelength

range to the sample spectrum.

 See information in the help box.

 Assign a different name.

 Select a different parameter from the

existing list. If necessary, program a new list entry in General Method Parameter in order to be able to use it when programming a method.

 Select a different parameter from the

existing list. If necessary, program a new list entry in General Method Parameter in order to be able to use it when programming a method.

 Enter the values so that the interval

does not fall below the range limits of

0.02 A and 10 nm.

 Enter the standard concentrations so

that the first standard receives the lowest concentration and the other standard concentrations form an increasing sequence.

Troubleshooting

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English (EN)

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Trou bleshooting

Eppendorf BioSpectrometer English (EN)

Problem Cause Solution

At least two of the entered standard concentrations are identical. Correct the standard concentrations.

The measured values are not strictly monotonous!

The ID cannot be set.

The dilution cannot be set.

Calculation not possible because of division by zero. Absorbance result or Formula "b" parameter is zero.

There is only one measurement left to be performed in this series of measurement. The maximum number of measurements within one series of measurements has been reached.

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• See the error text.

• Error when measuring a standard series: The measured absorbance values of the standard series are not continuously increasing or decreasing.

• Error when entering the sample ID. Different causes are possible. For the precise cause please see the information in the help box.

• Error when entering the dilution. Different causes are possible. For the precise cause please see the information in the help box.

• An absorbance result was divided by a "zero" value during the evaluation of a Division type method (Dual wavelength method group). This is not mathematically permissible.

• The number of measurements in one measuring series is limited to 99.

 Enter the standard concentrations so

that the first standard receives the lowest concentration and the other standard concentrations form an increasing sequence.

 Repeat the standard measurements or

delete the single, incorrectly measured standard result.

 See information in the help box.

 Check the reagents and samples used

and repeat the measurement.

 Do not enter "zero" as a value for the

Formula b parameter.

 Start a new series of measurement

after maximally 99 measurements.

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Eppendorf BioSpectrometer

Problem Cause Solution

Invalid zoom interval!

Device configuration was changed from … to … .

The temperature control system is faulty. Please program the method without temperature control or cancel the method.

The ambient temperature is too high.

Linear regression could not be applied to all measurements.

Error in the process results method step in the Zoom mode. Permissible zoom range for the wavelength scale:

• Wavelength interval at least 10 nm

• Entries for wavelengths only within the range programmed in the parameters for the method.

Permissible zoom range for the absorbance scale:

• Absorbance interval at least 0.02 A

• Upper and lower limit for absorbance interval +3 A or –3 A

• A BioSpectrometer kinetics is not detected as the kinetic variant but as a BioSpectrometer basic. The kinetic methods are therefore not displayed.

• The temperature control of the device is faulty.

For kinetic methods with temperature control:

• The ambient temperature measured by the device is above the specified range.

• For kinetic methods, the time frame for evaluation with linear regression was changed in the process results method step, and the change should be extended to all measuring results. The required number of measuring points, however, was not available for at least one sample result.

 Please observe the stated limits in the

zoom procedure.

 Switch the device off and back on

again. If the error occurs again: Contact Technical Service.

 Contact Eppendorf Service. Until the

temperature control has been repaired only use methods for which no temperature control has been programmed.

 Make sure that the ambient

temperature is within the range specified for the operation of the device.

 Only change the time frame for

evaluation with linear regression in the process results method step for samples with sufficient measuring points.

Troubleshooting

kinetic

English (EN)

Page 80

Trou bleshooting

Eppendorf BioSpectrometer English (EN)

kinetic

9.3 Result flags

Warnings and error messages for results are displayed in the bottom right of the help box. The header bar of the Help box is highlighted yellow for warnings and red for error messages.

Warnings: Decide whether the result is useful for you while taking the displayed warning into consideration.

Error messages: No result is displayed; the reason is shown in the error message.

Problem Cause Solution

The standard curve is not monotone. Please select another Curve Fit.

Some absorbance values for secondary wavelengths are too high or are not displayed.

The result is outside the range of the standard concentrations.

The coefficient of determination is <0.8.

• No usable result was returned during the evaluation of a standard curve using the "spline interpolation", "quadratic regression" or "cubic regression" Curve Fit procedures.

• For at least one secondary wavelength, the absorbance exceeded the measuring range.

• Secondary wavelengths are not needed for calculating the concentration result. They are used for different purposes. For example, dsDNA method: absorbance at 280 nm for the calculation of ratios 260/

280.

• Turbidity of the measuring solution

• Measurements at the limits of the photometric measuring range.

• For methods with evaluation via standard curves (nonlinear evaluation method): The sample result is up to 5 % outside of the standard concentration range.

• For methods with evaluation of standard series via the regression procedure: The coefficient of determination for the regression evaluation indicates a significant deviation of the measuring points from the regression line.

• Turbidity of the measuring solution.

• Measurements at the limits of the photometric measuring range.

 Select a different Curve Fit

procedure.

 If the absorbance values of the

secondary wavelengths are relevant: Dilute the sample or remove the turbidity via centrifugation and repeat the measurement.

 Accept the measurement result, or

remeasure the sample under conditions under which the result is within the range of the standard concentrations (dilute sample or modify standard concentrations and remeasure).

 Accept the result of the standard

evaluation or remeasure the standards.

 Make sure the measuring

solutions are clear.

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Eppendorf BioSpectrometer

Problem Cause Solution

The coefficient of determination for the regression evaluation of the standard series is < 0.8.

Scan: Some of the measured absorbances are too high and are not displayed.

The measurement is not complete.

The kinetics are nonlinear: The coefficient of determination is < 0.95.

The kinetics are nonlinear for at least one standard: The coefficient of determination is <0.95.

• For methods with evaluation of standard series via the regression procedure: If the regression evaluation for the standard series was nonlinear, but the standard evaluation was accepted by the user, a warning appears after samples have been measured.

• For at least one scan wavelength, the absorbance exceeded the measuring range.

• Turbidity of the measuring solution.

• Measurements at the limits of the photometric measuring range.

• Kinetic procedure: You have prematurely canceled the measurement using the [Stop] softkey. If at least 2 measuring points are available, a result is calculated and displayed.

• Kinetic procedure with "linear regression" measuring procedure: The coefficient of determination for the regression evaluation indicates a significant deviation of the measuring points from the regression line.

• Turbidity of the measuring solution.

• Measurements at the limits of the photometric measuring range.

• Activity concentration of the enzyme too high.

• Kinetic procedure with "linear regression" measuring procedure and evaluation procedure via standards: The coefficient of determination for the regression evaluation of at least one standard measurement indicates a significant deviation of the measuring points from the regression line.

• Turbidity of the measuring solution.

• Measurements at the limits of the photometric measuring range.

• Activity concentration of the enzyme too high.

 Use the sample results with the

reservation mentioned or repeat the measurement of the standard series and samples.

 If the non-displayed areas of the

scan are relevant: Dilute the sample or remove the dilution via centrifugation and repeat the measurement.

 Accept the measuring result with

the reduced measuring time or measure again with a longer measuring time.

 Accept measuring result or

remeasure the sample. Before repeating the measurement, evaluate the reason for the non-linearity and act accordingly (e.g., clear the measuring solution by centrifugation or dilute the sample).

 Accept measuring result or

remeasure the standard. Before repeating the measurement, evaluate the reason for the non-linearity and act accordingly (e.g., clear the measuring solution via centrifugation or use the standard with low concentration).

Troubleshooting

kinetic

English (EN)

Page 82

Trou bleshooting

Eppendorf BioSpectrometer English (EN)

Problem Cause Solution

During the kinetics measurement, the temperature was outside of the allowable range.

Absorbance at the measuring wavelength is too high.

The calculated result is negative.

At least one of the results is negative.

The result has more than 6 pre-decimal places.

The result is more than 5 % outside of the standard concentration range.

• Calculation not possible because of division by zero. Absorbance result is zero.

•Calculation error. Division by zero.

Calculation not possible because of division by zero. Absorbance result or parameter formula b is zero.

kinetic

• Ambient temperature outside the specified range.

• The temperature control is faulty.

• Turbidity of the measuring solution.

• Optical surfaces of the cuvette are soiled.

• Cuvette has been inserted into the cuvette shaft facing the wrong direction.

• Too high absorbance of measuring solution.

• Measuring solution not prepared correctly.

• The incorrect factor has been entered (wrong algebraic sign).

• For methods with several results (e.g., Dye labels).

• Measuring solution not prepared correctly.

• The incorrect factor has been entered (wrong algebraic sign).

•Very high sample concentration.

• Concentration unit does not match the expected range of the sample concentrations.

• For methods with evaluation via standard curves (nonlinear evaluation method): The sample result is more than 5 % outside of the standard concentration range.

• The evaluation required dividing by an absorbance result with the value of "zero". This is not mathematically permissible. Examples: Calculation of a factor at one-point calibration; calculation of a 260/280 ratio with nucleic acid measurements.

• An absorbance result was divided by a "zero" value during the evaluation of a Division type method (Dual wavelength method group). This is not mathematically permissible.

 Measure the sample at ambient

temperature within the specified range (15°C to 35°C). If the warning nevertheless appears, contact Eppendorf Service.

 Measure again considering the

possible causes.

 Measure again considering the

possible causes.

 Measure again considering the

possible causes.

 Dilute sample and measure again.  Change the concentration unit

(Parameter Unit) and measure again.

 Remeasure the sample under

conditions under which the result is within the range of the standard concentrations (dilute sample, modify standard concentrations and remeasure).

 Check the reagents and samples

used and repeat the measurement.

 Check the reagents and samples

used and repeat the measurement.

 Do not enter "zero" as a value for

the Formula b parameter.

Page 83

Transport, storage and disposal

Eppendorf BioSpectrometer

English (EN)

10 Transport, storage and disposal

10.1 Transport

 Use the original packaging for transport.

Air temperature Relative humidity Atmospheric pressure

General transport -25 °C – 60 °C 10 % – 95 % 30 kPa – 106 kPa

Air freight -40 °C – 55 °C 10 % – 95 % 30 kPa – 106 kPa

10.2 Storage

Air temperature Relative humidity Atmospheric pressure

In transport packaging -25 °C – 55 °C 25 % – 75 % 70 kPa – 106 kPa

Without transport packaging -5 °C – 45 °C 25 % – 75 % 70 kPa – 106 kPa

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10.3 Disposal

In case the product is to be disposed of, the relevant legal regulations are to be observed.

Information on the disposal of electrical and electronic devices in the European Community:

Within the European Community, the disposal of electrical devices is regulated by national regulations based on EU Directive 2012/19/EU pertaining to waste electrical and electronic equipment (WEEE).

According to these regulations, any devices supplied after August 13, 2005, in the business-to-business sphere, to which this product is assigned, may no longer be disposed of in municipal or domestic waste. To document this, they have been marked with the following identification:

Because disposal regulations may differ from one country to another within the EU, please contact your supplier if necessary.

In Germany, this is mandatory from March 23, 2006. From this date, the manufacturer has to offer a suitable method of return for all devices supplied after August 13, 2005. For all devices supplied before August 13, 2005, the last user is responsible for the correct disposal.

Page 84

Transport, storage and disposal

Eppendorf BioSpectrometer

kinetic

English (EN)

Page 85

Technical data

Eppendorf BioSpectrometer

English (EN)

11 Technical data

11.1 Power supply

Power supply 100 V to 240 V ±10 %, 50 Hz to 60 Hz

Overvoltage category II

Degree of pollution 2

Power consumption Maximum power consumption according to name plate: 50 W

Approx. 30 W during operation Approx. 5 W with the display dimmed and temperature control switched off

Permitted mains interruption Approx. 10 ms at 90 V

Approx. 20 ms at 230 V

Protection class I

Fuses T 2.5 A/250 V, 5 mm × 20 mm (2 pcs.)

kinetic

11.2 Ambient conditions

Operation Ambient temperature: 15°C to 35°C

Rel. humidity: 25% to 70% Air pressure: 86 kPa to 106 kPa

Air pressure Use up to an altitude of 2000 m above MSL

Do not expose to direct sunlight.

11.3 Weight/dimensions

Weight 5.3 kg

Dimensions Width: 295 mm

Depth: 400 mm Height:150 mm

Space required Width: 500 mm (with thermal printer: 750 mm)

Depth: 500 mm

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Technical data

Eppendorf BioSpectrometer English (EN)

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11.4 Photometric properties

Measuring principle Single beam absorption spectrophotometer with reference beam

Light source Xenon flash lamp

Monochromator Holographic aberration-corrected concave grating

Beam receiver CMOS photodiode array

Wavelengths 200 nm to 830 nm

Wavelength selection Method-dependent, freely selectable

Spectral bandwidth ≤ 4 nm

Smallest step size 1 nm

Systematic wavelength error ±1 nm

Random wavelength error ≤ 0.5 nm

Photometric measuring range 0 A to 3.0 A at 260 nm

Reading accuracy ΔA = 0.001

Random photometric error ≤ 0.002 at A = 0

≤ 0.005 (0.5 %) at A = 1

Systematic photometric error ±1 % at A = 1

Stray light component < 0.05 %

11.5 Incubation

Adjustable temperature range 20°C to 42°C

Smallest step size 0.1°C

Maximum temperature uncertainty (in relation to the temperature in the sample)

Systematic temperature error ±0.2 °C at 25 °C to 37 °C

Random temperature error ±0.15 °C at 25 °C to 37 °C

(see Tab. on p. 27)

Page 87

Technical data

Eppendorf BioSpectrometer

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English (EN)

11.6 Further technical parameters

Cuvette material For measurements in the UV:

Quartz glass or UV transparent plastic (Eppendorf UVette, 220 nm to 1600 nm) For measurements in the visible range: Glass or plastic material

Cuvette shaft 12.5 mm × 12.5 mm, tempered

Overall cuvette height Min. 36 mm

Height of the light beam in the cuvette

Key pad 22 foil keys

Result output Absorbance, concentration, scan (absorbance wavelength spectrum)

Display VGA TFT display 5.7”

Operator guidance language English, French, Spanish, Italian, German

Interfaces USB master: for USB stick and thermal printer DPU-S445

8.5 mm

6 foil keys as softkeys

Additional, method-dependent data (ratio, FOI, background absorbances)

USB slave for connecting to a PC Serial interface RS 232: for thermal printer DPU-414 Connected devices must meet the safety requirements specified in IEC 60950-1.

Page 88

Technical data

Eppendorf BioSpectrometer English (EN)

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11.7 Application parameters

Methods Preprogrammed and freely programmable methods for all measuring

and evaluation procedures:

• Absorbance measurements with one or more wavelengths, scans

• Nucleic acids and proteins, OD600, dye methods (parallel measurement of biomolecule and dye marking)

• Methods with evaluation via factor, standard and standard series

• Dual wavelength procedure with subtraction and division evaluation

• Kinetic method: End point, two-point, linear regression

Method-dependent evaluation Absorbance, concentration via factor and standard.

Concentration via standard series:

• Linear regression

• Nonlinear regression (2nd and 3rd degree polynoms)

• Spline evaluation

• Linear interpolation (point-to-point evaluation)

Absorbance calculations via subtraction and division Additional data for nucleic acids: Ratios 260/280 and 260/230; molar concentration, total yield Additional data for dye methods: FOI (frequency of incorporation, labeling density) Scans: zoom, peak evaluation Kinetics: Subsequent modification of the time window for the regression evaluation

Method memory >100 method programs

Measured value memory and calibration memory

Memory for >1 000 results with all data of the results evaluation and standard evaluation, sample number, sample name, date and used parameter set of the method program. (The number of saved results depends on the number of saved methods.)

Page 89

Evaluation procedure

Eppendorf BioSpectrometer

English (EN)

12 Evaluation procedure

This chapter describes the evaluation procedures available in the method programs as well as the calculation of a dilution using the device software.

When comparing the measuring results to the results of other photometers/ spectrophotometers, note that the values may be dependent on the bandwidth of the devices. In the following cases the differences may be significant:

• The absorbance spectrum shows a narrow peak in the measurement wavelength.

• The measurement is carried out not at the maximum but at the edge of a peak.

Therefore, check the accuracy of the methods by measuring standards.

12.1 Absorbance values

kinetic

Absorbance values are displayed as A

(XXX represents the wavelength). These displays always match

XXX

the directly measured values, i.e., without corrections, which are incorporated in the final evaluation, e.g., corrections for optical path lengths of the cuvette, or background corrections.

12.1.1 Blank

All absorbance values are always related to the last measured blank (blank). Therefore, a blank measurement is compulsory at the start of every series of measurements and can be completed at any time during a series of measurements. Ideally, the blank measurement should be able to compensate for any influences on the absorbance value of the measuring solution. The blank should therefore be measured with the same buffer that was used for the sample measurement and the same cuvette that was used to measure the sample value – unless the cuvettes used for the blank and sample measurements are optically aligned and thus have the same absorbance value at the measuring wavelength.

12.1.2 Background correction

Main application: Partial correction of distortions of the absorbance for nucleic acid measurements due to turbidity in the measuring solution. For example, the absorbance at 320 nm, which should be approx. 0 A with pure nucleic acids, is subtracted from the absorbance at 260 nm, (the measuring wavelength for nucleic acids).

AAA 

BkgrXXXcorrBkgrXXX

XXX, corrBkgr

= measured absorbance at a wavelength of XXX nm.

XXX

= measured absorbance at the background wavelength.

Bkgr

= calculated corrected absorbance at a wavelength of XXX nm.

Page 90

FAC u

Evaluation procedure

Eppendorf BioSpectrometer English (EN)

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12.1.3 Cuvette correction

All absorbance values which are used for result calculation are standardized to the cuvette layer thickness of 10 mm. If a cuvette with a different path length is used, this path length must be defined in the cuvette parameter. In this case, the measured absorbances are corrected to match measuring results with a cuvette layer thickness of 10 mm before converting them to sample results.

This correction is applied to:

• Methods with evaluation by factor.

•Methods of the Absorbance group, for which only absorbance values are output.

The correction is not applied to:

• Methods with evaluation by standards, as we presume that standards and samples are measured in cuvettes of the same layer thickness.

• Calculations with division: Division method (Dual wavelength method group) and calculation of ratios such as A

260/A280

(for nucleic acid measurements).

XXXcorrCuvXXX10,

Cuv

XXX, corrCuv

= measured absorbance at a wavelength of XXX nm.

XXX

Cuv = path length of the cuvette.

= calculated corrected absorbance at a wavelength of XXX nm.

12.2 Evaluation with factor or standard

C = calculated concentration.

A = absorbance.

F = factor.

The factor is programmed in the parameter list and can be modified. It always relates to an optical path length of the cuvette of 10 mm. If you change the Cuvette parameter the device will take the modification into account when calculating the results. Therefore you do not need to change the factor for the evaluation.

If, on the other hand, you modify the concentration unit, you have to ensure that the factor is adjusted for the selected unit.

Page 91

Evaluation procedure

Eppendorf BioSpectrometer

kinetic

English (EN)

The factor is either entered directly as a parameter during the "Factor" evaluation procedure or calculated during the "Standard" evaluation procedure (evaluation with a standard concentration):

F = calculated factor.

= concentration of the standard (enter as parameter).

= measured absorbance of the standard.

If multiple measurement (2 or 3 replicates) has been programmed for the standard, the average value is calculated from the measured absorbance values and inserted as A

12.3 Evaluation with standard curve/line

If evaluations are made with more than one standard, the following evaluation procedures for the standard curve/line can be selected with the [Curve fit] in the measure standards/new method step:

Evaluation procedure Description Minimum required number of

standard points

Linear interpolation Linear point-to-point connection in the

absorbance concentration graph of the standard evaluation.

Linear regression Polynome regression for first degree

polynomial.

Quadratical regression Polynome regression for second degree

polynomial.

Cubical regression Polynome regression for third degree

polynomial.

Spline interpolation Interpolation via natural cubic splines. 3 standards minimum.

For the regression procedure, one can select that the regression line (regression curve) goes through the zero point.

2 standards minimum.

3 standards minimum.

4 standards minimum.

5 standards minimum.

Page 92

Evaluation procedure

Eppendorf BioSpectrometer English (EN)

• Use the "linear regression" procedure for calibration lines.

• With curvilinear gradients, test which evaluation procedure (quadratic regression, cubic regression, spline interpolation) produces the function that is most suitable to the standard evaluation. Spline interpolation connects the measuring points by cubic polynomials, whereas the regression methods position a quadratic or cubic function between the measuring points in such a way, that the smallest possible deviation from the function results for the measuring points.

• Aside from the calculated regression equation, the regression method also displays the coefficient of determination as a measure for the scattering of the measuring points around the calculated function. At a value of < 0.8 for the coefficient of determination the result is issued with a warning.

• If the first standard hat a concentration of "0", select the setting in which the regression line (regression curve) goes through the zero point.

• If none of the procedures recommended for curvilinear gradients produce satisfactory results, select the "linear interpolation" procedure.

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12.4 Dilution

In the measure samples method step. entered dilutions are considered in the result calculation:

= result converted using the dilution factor

Dil, corr

= volume of the sample in the measuring solution

= volume of the diluent in the measuring solution

Dil

Page 93

Evaluation procedure

Eppendorf BioSpectrometer

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English (EN)

12.5 Special evaluation procedures for nucleic acids and protein UV

This section covers the evaluation of nucleic acids or proteins in the Nucleic acids and Proteins direct UV method groups, as well as the corresponding biomolecular components in the Dye labels method group.

12.5.1 Correction A

and correction A

260

280

Application: correction of the influence of dye absorbance on the nucleic acid or protein absorbance at 260 and 280 nm for the methods of the Dye labels group.

The application of the evaluation procedure can be activated in the parameters Correct A260 or Correct A280.

ACFAA u

XXX, corr

XXX

= calculated corrected absorbance for a wavelength of 260 nm or 280 nm

= measured absorbance for a wavelength of 260 nm or 280 nm

YYYXXXcorrXXX

CF = correction factor for a wavelength of 260 nm or 280 nm (the correction factors for 260 nm and 280 nm are both dye-specific and are programmed in General Method Parameter: Dyes in the Functions area).

= measured absorbance at the dye wavelength.

YYY

The absorbance values displayed in the results are the directly measured, not the corrected absorbance values.

12.5.2 Ratios A260/A280 and A260/A230

Application: Information on the purity of the measured nucleic acid. Application of the evaluation procedure can be activated in the A260/280 or A260/A230 parameters.

"Ratio" refers to the quotients of the measured absorbances at the listed wavelengths.

Literature values for ratio values with pure nucleic acids:

A260/A280

• DNA: 1.8 to 1.9

• RNA: 1.9 to 2.0 (Current Protocols in Molecular Biology, 1994)

A260/A230

For the ratios A260/A230, different information can be found in the literature for pure nucleic acids:

• DNA: 2.3 to 2.5 (The Nucleic Acids, 1955)

•DNA: 1.9 (Current Protocols in Molecular Biology, 1994)

The values are highly dependent on the pH value. Therefore, nucleic acids should not be measured in water, but in a buffer with a pH of 7 to 7.2 (e.g., TE buffer).

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Evaluation procedure

Eppendorf BioSpectrometer English (EN)

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12.5.3 Conversion to molar concentrations and nucleic acid quantities

The conversion only can be applied to nucleic acids and dye methods with nucleic acids as biomolecule components. It is realized in the process results/More calculations method step.

12.5.3.1 Calculation of amount

Application: calculating the amount (mass) of nucleic acid in the total sample volume.

M = calculated total amount (mass) of nucleic acid in the sample tube. Unit: μg.

C = nucleic acid concentration calculated from the measurement. Unit: μg/mL or ng/μL.

= total volume of the sample in the sample tube. Enter this value in More calculations. Unit: μL.

S, total

12.5.3.2 Calculation of the molar concentration

Application: calculation of the molar concentration of the nucleic acid from the mass concentration and relative molar mass. The molar mass is either entered directly or calculated by the device from the entered number of bases or base pairs per nucleic acid molecule.

Mol

= calculated molar concentration of the nucleic acid. Unit: pmol/mL.

Mol

C = nucleic acid concentration calculated from the measurement. Unit: μg/mL or ng/μL.

MM = relative molar mass. Unit: kDa

If the number of bases or base pairs per nucleic acid molecule are entered in More calculations instead of the relative molar mass, the MM is calculated from the number of the bases or base pairs:

For dsDNA:

For ssDNA, RNA, Oligo:

10u

MM = calculated relative molar mass; unit: kDa

bp = entered number of base pairs per molecule

b = entered number of bases per molecule

Page 95

Evaluation procedure

%1.0

Eppendorf BioSpectrometer

kinetic

English (EN)

•For dsDNA the calculation of the molar concentration is based on the assumption of a double-stranded nucleic acid. For the ssDNA, RNA and Oligo methods, a single-stranded nucleic acid is assumed.

12.5.4 Calculating the factor for protein in "General Method Parameter"

This section only covers the calculation of the protein components in the Dye labels and Proteins direct UV method groups. For these method groups, the protein component is selected in the parameters (see Method parameters on p. 33). The protein component is assigned a factor that will be entered in the General Method Parameter/Proteins function for each protein. Alternatively, A

coefficient plus the molar mass of the protein can be entered instead of the factor. In this case, the factor is calculated as follows:

or the absorbance

0.1%

F = factor for the protein; unit: g/L.

= absorbance of the protein at a concentration of 0.1 % (1 g/L).

0.1%

When entering the molar absorbance coefficient and the relative molar mass of the protein A calculated on this basis:

= molar absorbance coefficient of the protein; unit: cm-1M-1.

= relative molar mass of the protein; unit: Da (entry in General Method Parameter in kDa).

0.1%

can be

Page 96

NAXXX

Dye

YYY

MMA

FOI

NAXXXDye

YYY

FOI

Evaluation procedure

Eppendorf BioSpectrometer English (EN)

kinetic

12.6 Special evaluation procedures for the dye methods

12.6.1 Calculating the factor for the dye from the absorbance coefficient

For the dye methods the concentration of the dye is calculated using a factor from the measured absorbance (see Evaluation with factor or standard on p. 90). The factor is entered for each dye in the General Method Parameter/Dyes function. Alternatively, you can enter the absorbance coefficient. In this case, the factor is calculated as follows:

Dye

F = factor for the dye; unit: pmol/μL.

ε = absorbance coefficient for the dye, unit: cm

-1M-1

12.6.2 Calculation of the FOI

As a value for the ratio of dye molecules to the number of nucleotides in the nucleic acid the frequency of incorporation (FOI) is calculated and displayed for the dye methods. The calculation can be selected for two different result units:

MOLECULE dye/kb unit

pmole/μg DNA (or RNA) unit

= absorbance of the dye.

YYY

= absorbance of the nucleic acid.

XXX

= average molar mass of the nucleotides: 330 g/mol.

= factor for calculating the nucleic acid

= absorbance coefficient for the dye, unit: cm-1M

dye

-1

Page 97

Evaluation procedure

totalP

VCM

calc

u

Eppendorf BioSpectrometer

kinetic

English (EN)

12.6.3 Conversion to amounts of dye

The quantity (dimensions) of dye in the entire sample volume is calculated in the process results/More calculations method step.

M = calculated total amount (mass) of dye in the sample tube. Unit: pmol.

C = dye concentration calculated from the measurement. Unit: pmol/μL.

= total volume of the sample in the sample tube; entered by the user under More calculations. 

S, total

Unit: μL.

12.7 Dual wavelength

For methods of the Dual Wavelength group absorbances that were measured at two wavelengths can be calculated with each other before the calculated absorbance is evaluated further with the factor or standard.

To determine the calculated absorbance a division or subtraction evaluation can be defined in the parameters:



calc

, A2 = measured absorbance.

a, b, c, d = factors that are entered in the parameters. Negative figures may also be entered.

dcAbAaA

uuu

Page 98

RBcorrRB

AAA 

Evaluation procedure

Eppendorf BioSpectrometer English (EN)

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12.8 Kinetics

An absorbance value A or an absorbance difference standardized to a minute ΔA/min is determined using the measurement procedures available for selection. This determined absorbance value is entered in the concentration calculation using a factor or (only for Advanced kinetics) with an end-point standard.

12.8.1 Measurement procedures

Endpoint

At the end of the waiting period (incubation period, Delay parameter), an absorbance value is measured and used for the concentration calculation.

Two point

After the delay the measuring point is taken and after the measuring time has expired a second one is taken. ΔA/min is calculated from the absorbance and the time difference.

AAA

min tt

, t1 = absorbance and time for the first measuring point.

, t2 = absorbance and time for the second measuring point.

Linear regression

After the delay has expired measuring points are recorded at fixed time intervals from the start to the end of the measuring time. A linear regression is carried out via the measuring points in the absorbance/time diagram. Result: absorbance value in ΔA/min.



12.8.2 Reagent blank value

For the methods of the Advanced kinetics group, the measurement of a reagent blank can be preprogrammed in the parameters. Primary application: compensation of a reagent drift in the "Linear regression" measurement procedure. Instead of a sample, the reagent blank contains the reagent and demineralized water, and is measured according to the same measurement procedure as the sample. The measurement is made at the start of the sample series; the absorbance result is then subtracted from the sample absorbance result before the evaluation:

A = measured absorbance result (in A or ΔA/min) of the sample.

= corrected absorbance result (in A or ΔA/min) of the sample.

RB, corr

= absorbance result (in A or ΔA/min) of the reagent blank.

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Evaluation procedure

Eppendorf BioSpectrometer

kinetic

English (EN)

If the time frame for the kinetic evaluation with linear regression is reduced in the process results method step, the reduced measuring window is automatically used for calculating the reagent blank which was incorporated in the calculation of this sample result. That means that the reagent blank is recalculated (only) for the calculation of this sample result. For the calculation of the other sample results the reagent blank result that was calculated on the basis of the originally used time window is used.

When the kinetic method is evaluated with standards the measurement of a reagent blank is not available. Here, the reagent blank can alternatively be defined as the first standard ("zero" concentration).

Page 100

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Evaluation procedure

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Eppendorf BioSpectrometer kinetic User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

1 Operating instructions

1.1 Using this manual

1.2 Danger symbols and danger levels

1.2.1 Danger symbols

1.2.2 Danger levels

1.3 Symbols used

1.4 Abbreviations used

2 Product description

2.1 Main illustration

2.2 Delivery package

2.3 Features

2.3.1 Methods

2.3.2 Operation

2.3.3 Result output

2.3.4 Device self test

3Safety

3.1 Intended use

3.2 User profile

3.3 Warnings for intended use

3.3.1 Personal injury

3.3.2 Damage to device

3.4 Information on product liability

3.5 Safety instructions located on the device

4 Installation

4.1 Preparing installation

4.2 Selecting the location

4.3 Connecting the device to the mains

4.4 Connecting the printer

4.4.1 Thermal printer DPU-S445

4.4.2 Thermal printer DPU-414

4.5 Connecting PC or USB stick for data export

5 Operation

5.1 Overview of operating controls

5.1.1 Entering text

5.2 Inserting the cuvette

5.3 Summary of the measuring procedure

5.3.1 Preparing the measurement

5.3.2 Measuring procedure

5.3.3 Important measurement instructions

5.3.4 Notes on working with cuvette temperature control

6 Methods

6.1 Selecting a method

6.2 Photometry method description

6.2.1 Absorbance method group

6.2.2 Routine method group

6.2.3 Basic method group

6.2.4 Advanced method group

6.3 Method parameters

6.4 Method procedure

6.4.1 Check parameters

6.4.2 Measure standards

6.4.3 Measure samples

6.4.4 Measure samples: Results displays

6.4.5 Process results

6.4.6 Process results: Options

6.4.7 Print & export

6.4.8 Finish the series of measurements

7Functions

7.1 Functions of the User main group

7.1.1 Results memory

7.1.2 General method parameters

7.1.3 Absorbance spectra library

7.1.4 Device settings

7.1.5 Device calibration

7.1.6 Info

8 Maintenance

8.1 Cleaning

8.1.1 Cleaning the cuvette shaft cover

8.2 Disinfection/Decontamination

8.3 Checking the device

8.3.1 Checking the spectrometer unit

8.3.2 Checking the thermal module

8.3.3 Device self-test

8.4 Replacing fuses

8.5 Decontamination before shipment

9 Troubleshooting