Eppendorf BioPhotometer plus User Manual

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B 6132 900.017-00/1107

BioPhotometer plus

Operating manual

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Trademarks

eppendorf and UVette are registered trademarks of Eppendorf AG, Hamburg, Germany. Cy is a registered trademark of GE Healthcare UK Ltd., Buckinghamshire, UK. Alexa Fluor is a registered trademark of Molecular Probes Inc., Eugene OR, USA. LabelGuard is a trademark of Implen GmbH, München, Germany.

Registered trademarks are not marked in all cases with ™ or ® in this manual.

6132 900.017-02/032012

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345

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Table of contents

BioPhotometer plus — Operating manual

Table of contents

1 User instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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

1.2 Warning signs and hazard icons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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

1.4 Abbreviations used. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2 Product description. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.1 Main illustration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.2 Delivery package . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.3 Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.1 Intended use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.2 Warnings for intended use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.3 Application limits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

3.4 Note on product liability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

4 Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.1 Preparing installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.2 Selecting location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.3 Connect device to the main power supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.4 Cuvettes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.5 Connect printer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Operating manual

5 Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

5.1 Overview of operating controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

5.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

5.3 Summary of the measuring procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5.3.1 Prepare measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5.3.2 Select the method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5.3.3 Measure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5.3.4 Finalize the method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

5.4 Nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

5.5 Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5.5.1 Protein 280 nm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5.5.2 Protein after adding reagent (Bradford, BCA, Lowry) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5.6 Methods with evaluation via standards. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

5.7 OD 600 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.8 Dye-labeled biomolecules ("dye methods") . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.8.1 Method group "dye 550" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.8.2 Method group "dye 650" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.8.3 Frequency of incorporation "FOI" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.8.4 Correction factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.8.5 Measuring procedure and result display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.9 Methods for 340, 405 and 490 nm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

5.10 Dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

5.11 Sample number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

6 Parameter and functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

6.1 Parameter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

6.1.1 View, change and store the parameters of a method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

6.1.2 Summary and description of parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

6.1.3 Parameters preprogrammed ex factory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

6.2 Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

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Operating manual

BioPhotometer plus — Operating manual

Table of contents

7 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

7.1 Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

7.1.1 Cleaning the cuvette shaft cover. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

7.2 Disinfection / Decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

7.3 Decontaminating before shipping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

7.4 Replacing fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

7.5 Check photometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

7.5.1 Test procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

8 Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

8.1 Result flags. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

8.2 Error messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

8.3 General errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

9 Transport, storage and disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

9.1 Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

9.2 Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

9.3 Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

10 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

10.1 Power supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

10.2 Ambient conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

10.3 Weight / dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

10.4 Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

10.5 Photometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

10.6 Other technical parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

10.7 Application parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

11 Evaluation procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

11.1 Evaluation with factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

11.2 Evaluation using standards. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

11.2.1 Single point calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

11.2.2 Multi-point calibration: calibration line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

11.2.3 Multi-point calibration: Calibration curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

11.3 Dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

11.4 Special evaluation procedures for the dye methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

11.4.1 Calculating the factor for the dye from the absorbance coefficient . . . . . . . . . . . . . . . . . . . . . . . . . . 45

11.4.2 Calculation of the FOI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

11.5 Special evaluation procedures for nucleic acids and protein UV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

11.5.1 Correction A

11.5.2 Correction A

11.5.3 Conversion into molar concentrations and nucleic acid amounts . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

12 Ordering information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

340

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

550/650

Page 7

BioPhotometer plus — Operating manual

1 User instructions

1.1 Using this manual



Before using the device for the first time, please read this operating manual.

 Please view this operating manual as part of the product and keep it somewhere easily

accessible.

 If this manual is lost, please request another one. The current version can be found on our

website, www.eppendorf.com (International) or www.eppendorfna.com (North America).

1.2 Warning signs and hazard icons

Depiction Meaning

DANGER

Risk of electric shock with potential for severe injury or death as a consequence.

DANGER

Risk of explosion with potential for severe injury or death as a consequence.

Operating manual

1.3 Symbols used

Depiction Meaning



•

(example)

Text Terms used in the device display.

DANGER

Biohazard with potential for risk to health or death as a consequence.

WARNING

Warning of potential injury or health risk.

CAUTION

Refers to risk of damage to property. Refers to particularly useful information and tips.

You are requested to perform an action. Perform these actions in the sequence described.

List. Press this key to perform the action described.

1.4 Abbreviations used

DNA Deoxyribonucleic acid

dsDNA double stranded DNA

Dye methods Group of methods via the keys dye 550 and dye 650

A Absorbance

FOI Frequency of Incorporation: measure for the number of dye molecules related to the number of

nucleotides in dye-labeled biomolecules

M mol/l (molar)

OD600 Optical density for wave length 600 nm

RNA Ribonucleic acid

ssDNA Single stranded DNA

UV Ultraviolet radiation VK Coefficient of variation (standard deviation / mean), in percentages

Page 8

Operating manual

BioPhotometer plus — Operating manual

2 Product description

2.1 Main illustration

Abb. 1: Front and rear view

Fig. 1: Front and rear view

1 Device display 2 Cuvette shaft cover

Slide back or forward to open or close.

3 Cuvette shaft 4 Connection RS-232

5 ID plate 6 Mains connection socket

7 Fuse holder 8 Mains switch

9 Measuring keys 10 Keyboard

2.2 Delivery package

Number Description

1 BioPhotometer plus 1 Power cable 2 UVette

Original Eppendorf plastic cuvette, individually wrapped, for direct use in the BioPhotometer, certified RNase, DNA and protein free

1 BioPhotometer plus operating instructions, multilingual,

2.3 Features

Cuvette photometer The BioPhotometer plus is a cuvette photometer for the fast, simple and comfortable

measurement of the most important methods in the molecular biology and biochemistry research laboratory. It can also be used for the main photometric methods in cell biology.

Method programs Method programs for calculating the concentration of nucleic acids, proteins and dye-labeled

nucleic acids and proteins as well as the method "OD600" for calculating the bacteria density through measuring turbidity are already preprogrammed. However, you can modify those in many parameters. Other methods for calculating the concentration for 340, 405 and 490 nm can be freely programmed. The method "absorbance" is used for the fast absorbance measurement with any of 9 available wavelengths without further evaluation.

Method programs are combined into groups which you can open quickly via fixed keys.

Cuvettes You can use standard rectangular glass or plastic cuvettes with optical transparency for the

respective measuring wavelength. With the Eppendorf UVette you can also measure nucleic acids and proteins in the UV range using a plastic cuvette. A cover protects the cuvette shaft against dust and other contamination if the photometer is not in use. To open the cuvette shaft it is moved back, to cover it after completing the measurements it is moved forward.

Measuring keys After opening a method the device is immediately ready for measuring. A measurement is started

with one of the 3 round measuring keys.

Page 9

BioPhotometer plus — Operating manual

2 Product description

Evaluation The BioPhotometer plus converts the measured absorbance values into concentration results.

Dependent on the method the results can be calculated via fixed factors, standards, or curve calibration. In addition to the results the device also displays the absorbance values and some other important details, e.g. the common absorbance quotients for nucleic acid calculations. Sample dilutions can also be included in the evaluation. Other special evaluation procedures are provided for specific method groups. For example, when calculating the concentration of dyed nucleic acids the frequency of incorporation related to the amount of nucleic acid can also be calculated.

Output The BioPhotometer plus outputs the results via the device display and via a printer available from

Eppendorf.

Operating manual

Page 10

BioPhotometer plus — Operating manual

3 Safety

3Safety

3.1 Intended use

The intended area of use for the BioPhotometer plus is the research laboratory in molecular biology, biochemistry and cell biology. The device may only be operated by trained specialist staff.  The BioPhotometer plus is used to perform photometric measurements to quantify biomolecules as well as to perform turbidity measurements of microbiological cultures in routine laboratories. Due to the specific examination of selected parameters, the device serves to monitor laboratory processes. Use only Eppendorf accessories or accessories recommended by Eppendorf AG.

3.2 Warnings for intended use

Operating manual

DANGER!

Danger! Electric shock from damage to device/power cable.

 Only switch on the device if the device and the power cable are undamaged.  Only use devices that have been properly installed or repaired.

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

 Switch off the device and disconnect it from the power supply before starting cleaning or

disinfecting.

 Do not allow any liquids to penetrate the inside of the housing.  Do not disinfect by means of spraying.  Only reconnect the device to the power supply once it is completely dry.

Danger! Electric shock.

 Switch off the device and disconnect the power plug before opening the device to replace the

fuses. These tasks may only be performed by appropriately trained staff.

Risk of explosion!

 Do not operate the device in rooms where work is being carried out with explosive

substances.

 Do not use this device to process any explosive, radioactive or highly reactive substances.  Do not use this device to process any substances, which could create an explosive

atmosphere.

DANGER!

WARNING!

Risk when handling toxic or radioactively-marked liquids or pathogenic germs.

 Follow national regulations governing the handling of these substances.  For complete instructions regarding the handling of germs or biological material of risk group

II or higher, please refer to the "Laboratory Biosafety Manual" (Source: World Health Organization, current edition of the Laboratory Biosafety Manual).

Warning! Damage to health from chemicals.

Hazardous chemicals cause burns and other health hazards.

 Follow the instructions for use provided by the manufacturers of reagents and other

chemicals.

Warning! Poor safety due to incorrect accessories.

The use of accessories and spare parts other than those recommended by Eppendorf may impair the safety, function and precision of the device. Eppendorf accepts no warranty or liability for damage caused by third-party parts or incorrect use.

 Use only original accessories recommended by Eppendorf.

Page 11

3 Safety

WARNING!

BioPhotometer plus — Operating manual

Warning! Risk to health from contaminated device

 Perform decontamination before storing or dispatching the device and/or its accessories.

Operating manual

CAUTION!

Caution when using aggressive chemicals.

Aggressive chemicals may damage both the device and its accessories.

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

weak alkalis, strong and weak acids, acetone, formaldehyde, chlorinated hydrocarbons or phenol.

 If the device becomes contaminated with aggressive chemicals, clean it immediately with a

neutral cleaning agent.

Caution! 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 prolonged

periods.

Caution! Damage to electronic components from condensation.

 After moving the device from a cooler environment (e.g., cool room or outdoors), wait at least

an hour before connecting it to the mains power supply.

Caution! Function may be impaired by mechanical damage.

 After a mechancial damage to the device ensure by means of an inspection that the

measuring and evaluation functions of the device function correctly.

Caution! Damage due to overheating.

 Do not place the device close to sources of heat (e.g., radiator, drying cabinet).  Do not expose the device to direct sunlight.  Allow air to circulate freely by leaving at least 5 cm to adjoining devices or to the wall and

keep the underside of the device free.

CAUTION!

Caution! 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 cases of doubt, contact the product manufacturer.

Caution! Poor safety due to missing operating manual.

 When passing on the device, always enclose the operating manual.  If you lose the operating manual, request a replacement. The current version of the

operationg manual and the safety instructions can also be found on our website www.eppendorf.com.

Caution! Damage as a result of incorrect packing.

Eppendorf accepts no warranty or liability for damage caused by incorrect packing.

 Only dispatch the device in the original packaging provided for carriage.

Page 12

BioPhotometer plus — Operating manual

3 Safety

Operating manual

CAUTION!

Caution! Damage from improper cleaning of the cuvette shaft.

 Only clean the cuvette shaft using a moist cotton swab.(see Cleaning on page 32)  Do not allow any liquid to enter the cuvette shaft.  Do not reach with your fingers into the cuvette shaft.

CAUTION!

Caution! Faulty measurement due to device confusion.

 If you use the devices Biophotometer 6131 and BioPhotometer plus 6132 in your laboratory,

note the different method designations on the keys.

3.3 Application limits

Risk of explosion!

 Do not operate the device in rooms where work is being carried out with explosive

DANGER!

substances.

 Do not use this device to process any explosive, radioactive or highly reactive substances.  Do not use this device to process any substances, which could create an explosive

atmosphere.

3.4 Note on product liability

In the following cases, the protection provided in the device may be impaired: Liability for the function of the device passes to the operator if:

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

• the device is used outside the sphere of application described here.

• the device is used with accessories and consumables (e.g. tubes and plates), which are not

recommended by Eppendorf AG.

• the device is maintained or repaired by persons not authorized by Eppendorf.

• the owner has made unauthorized modifications to the device.

Page 13

4 Installation

4.1 Preparing installation



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

 Check the completeness of delivery based on the details of the scope of delivery (see

Delivery package on page 8).

 Check all parts for any transport damage.

4.2 Selecting location

Select the location for the BioPhotometer plus in accordance with the following criteria:

• 2 power sockets with ground conductor for the BioPhotometer plus and the printer.

• Solid laboratory bench with horizontal work surface

Space requirement of the device: 40 cm (with printer: 65 cm) width, 50 cm depth.

• Temperature: 15 to 35 °C. Avoid direct sunlight.

• Humidity: 25 to 75 % relative humidity.

• Atmospheric pressure: 70 to 106 kPa.

BioPhotometer plus — Operating manual

Operating manual

4.3 Connect device to the main power supply

1. Place the BioPhotometer plus onto a suitable work surface.

2. Ensure that the mains voltage and frequency match the details for the range of mains voltages and frequencies on the device nameplate.

3. Connect the device to the power supply and switch it on from the mains power switch 8 (Fig. 1 on p. 8).

4. Remove the protective film from the device display.

4.4 Cuvettes

You can insert standard rectangular glass or plastic cuvettes into the cuvette shaft (outside diameter 12.5 mm x 12.5 mm). The optical path height must be 8.5 mm above the cuvette base and the total cuvette height must be at least 36 mm. The light beam in the cuvette is 1.0 mm wide and 1.5 mm high.

The cuvettes must be optically transparent for the respective measuring wavelength. For measurements in the UV range Eppendorf provides a plastic cuvette called UVette which is transparent from wavelengths above 220 nm and therefore also suitable for the measurement of nucleic acids.

Abb. 2: Overview of different cuvette types

Fig. 2: Overview of different cuvette types

Page 14

Operating manual

BioPhotometer plus — Operating manual

4 Installation

4.5 Connect printer

You can connect the Eppendorf thermal printer to the serial interface RS-232 C of the photometer (see Ordering information on page 48).

1. Connect the printer cable to the serial printer port 4 of the photometer and tighten the locking screws.

2. Connect the printer cable to the printer and also tighten the locking screws.

3. Connect the printer to the power supply using the plug-in power unit supplied (printer accessory) and switch it on.

4. Check the printer settings in accordance with the following table and make corrections where necessary.

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

Tab. 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.A.

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 5 (OFF) Baud 6 (ON) Rate 7 (ON) Select 8 (ON) = 9600 bps

Page 15

BioPhotometer plus — Operating manual

5 Operation

5.1 Overview of operating controls

Abb. 3: Control panel of the BioPhotometer plus.

Fig. 3: Control panel of the BioPhotometer plus.

Operating manual

Key Function Oval blue keys, e.g.:

• In the method selection: select method group.

• When entering values: enter digits.

Circular keys

Start standard measurement.

Start blank measurement.

Start sample measurement.

Oval transparent keys

• In the method selection: open parameter list.

• During the measurement procedure: enter sample dilution.

• In the method selection: open function list.

• During the measuring procedure: modify sample number.

• When entering digits: enter decimal point.

• Confirm entry or selection.

• Open selected method or function.

Cursor keys

Delete entry

Move cursor up or down in the device display.

Page 16

Operating manual

BioPhotometer plus — Operating manual

5 Operation

5.2 Methods

The following methods are available and already preprogrammed ex factory. You can modify most parameters and save them as a modified method (see Parameter on page 26).

Method group Method Explanation Wavelength

DNA dsDNA Calculating the concentration of DNA with

ssDNA OLIGO DNA

RNA RNA Analogous to method group DNA. As method group DNA.

OLIGO RNA

Protein BCA Calculating the concentration of proteins after

BCA MICRO BRADFORD 595 nm BRADFORD MICRO LOWRY 595 nm LOWRY MICRO PROTEIN 280 nm Calculating the concentration of proteins with

OD600 OD600 Turbidity measurement to determine the

dye 550 DYE 550-dsDNA For dye-labeled biomolecules: calculating the

DYE 550-ssDNA DYE 550-RNA DYE 550-OLIGO DYE 550-PROTEIN

dye 650 DYE 650-dsDNA Analogous to method group "dye 550". DNA/RNA/OLIGO: see method

DYE 650-ssDNA DYE 650-RNA DYE 650-OLIGO DYE 650-PROTEIN

assay 340 ASSAY 340/1 Calculating the concentration by measuring at

ASSAY 340/2 ASSAY 340/3

assay 405 ASSAY 405/1 Analogous to method group "assay 340". 405 nm

ASSAY 405/2 ASSAY 405/3

assay 490 ASSAY 490/1 Analogous to method group "assay 340". 490 nm

ASSAY 490/2 ASSAY 490/3

ABSORBANCE ABSORBANCE Rapid absorbance measurement after

evaluation via factor. The methods differ mainly in the preprogrammed factor.

adding reagent. The methods are preprogrammed with the evaluation procedure calibration curve. Number and target concentration of the standards can be modified.

evaluation via factor.

bacteria density.

concentration of the molecule (nucleic acid or protein) and the dye in a single measuring procedure. The frequency of incorporation of the dye in the biomolecule is also determined.

340 nm. The evaluation procedures can be freely programmed. As a sample the methods are already preprogrammed with the following evaluation procedures: 340/1: evaluation via factor 340/2: evaluation via a standard. 340/3: evaluation via calibration curve with 6 standards.

selecting the wavelength.

Measuring wavelength: 260 nm Secondary wavelengths to check for purity: 230, 280, 340 nm

550 nm

Measuring wavelength: 280 nm Secondary wavelengths to check for purity: 260, 340 nm

595 nm

DNA/RNA/OLIGO: see method groups DNA and RNA PROTEIN: see method PROTEIN 280 nm Measuring wavelength for the dye: 550 nm

groups DNA and RNA PROTEIN: see method PROTEIN 280 nm Measuring wavelength for the dye: 650 nm

340 nm

230, 260, 280, 340, 405, 490, 550, 595, 650 nm

Page 17

BioPhotometer plus — Operating manual

5 Operation

5.3 Summary of the measuring procedure

This section contains a summary of the key steps of a measuring procedure.

5.3.1 Prepare measurement

1. Switch on the device and, if necessary, the printer also. The device is immediately ready for measuring after being switched on.

2. Have the cuvettes for the measurements available. When selecting the cuvettes observe the respective instructions (see Cuvettes on page 13).

3. Provide the measuring solutions for measuring the blanks and, if necessary, the standards and samples.

Measuring solutions for standards and samples with lower absorbances than 0.02 to 0.03 A (this range corresponds e.g. to a dsDNA concentration of 1.0 to 1.5 μg/ml) should not be used. Whilst the detection level of the photometer is well below these values, the effect of interferences from the measuring solutions (particles, bubbles, turbidity) on the reliability of the results is very high for these low absorbances.

Operating manual

4. Slide the cover of the cuvette shaft back to open the cuvette shaft.

5.3.2 Select the method

1. Using a key select the method group.

2. Using the cursor keys select the desired method.

Before opening the method you should check the parameters of the desired method and correct

Hint!

them, if necessary (see Parameter on page 26).

3. Open the selected method using the Enter key.

The displayed factor defined in the method parameters always relates to an optical path length of 10 mm. However, for calculating the result the device automatically takes into account the optical path length defined in the method parameters. Therefore, you do not need to modify the factor in the method parameters for other optical path lengths.

In the device display you see a list of methods provided in the selected group.

The startup screen of the method is displayed:

• Top: method name and programmed optical path

length of the cuvette.

• Center: programmed evaluation (e.g. factor or

information about the evaluation with standards).

• Bottom: keys for the next measurement. The keys

are not shown if there is insufficient space to display them, but they can be made visible using the Enter key in this case.

Page 18

Operating manual

5 Operation

5.3.3 Measure

Hint!

BioPhotometer plus — Operating manual

Check for each measurement:

• Is there enough measuring solution in the cuvette? The light path height of the BioPhotometer

plus is 8.5 mm. The height of the light beam in the cuvette is 1.5 mm.

• Is the measuring solution free from particles and bubbles?

• Is the measuring surface of the cuvette free from contamination due to dust or finger prints

and free from scratches?

• When inserting the cuvette press it all the way down against a slight resistance.

• Is the cuvette positioned correctly? The optical surface of the cuvette must point towards the

direction of the light path. The direction of the light path in the BioPhotometer plus is indicated by an arrow on the blue cuvette shaft cover.

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

cuvette?

• Carry out a blank measurement for each cuvette before any sample or standard

measurement to compensate for the cuvette blank in addition to the reagent blank value.

• Check whether the measured absorbance values exceed the upper limit of the photometric

measuring range. Discard the measuring result in this case. The upper limit of the photometric measuring range not only depends on the wavelength (see Photometer on page 41) but also on the cuvette blank. Ultramicro cuvettes with a small aperture such as "TrayCell" (Hellma) or "LabelGuard" (Implen) may have a cuvette blank of up to A = 1. The available photometric measuring range is reduced by this amount. You can estimate the cuvette blank if you measure a water-filled cuvette as a sample against the empty cuvette shaft as a blank.

• Remove the measuring solution completely after measurement before filling the next

measuring solution in order to minimise carry-over. If a carry-over between samples is expected due to the high concentration differences then flush the cuvette between measurements.

• With temperature differences between the lamp and the environment photometric drift may

occur. Therefore a device being brought in from a colder environment should first reach the ambient temperature. Alternatively you can bring the lamp to the right temperature by carrying out a few measurements. In long series of measurements or in measurements over a long period of time carry out a new blank measurement.

1. Open the cuvette shaft by sliding the blue cover back.

blank 2. Fill the cuvette with blank solution and insert the filled cuvette into the cuvette shaft.

3. Press the blank key.

• Top: method name and display of the sample type

(here: "BLANK")

• Center: result (for blank: 0.000 A)

Blank results remain stored as long as the method remains open. However, we recommend to

Hint!

Standards

(optional)

Samples 5. Fill the cuvette with sample solution and press the sample key.

check the blank at regular intervals of e.g. one hour. To do so carry out a measurement with the blank solution as sample. If the measuring result differs significantly from 0 a new blank measurement must be carried out.

4. Only for methods with standard evaluation: Measure the required standards consecutively if you want to carry out a new calibration (see Methods with evaluation via standards on page 21).

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5 Operation

5.3.4 Finalize the method

1. Press one of the method group keys to return to the method selection and open the next

2. After all measurements are complete switch off the device.

3. Slide the cuvette shaft cover forward to protect the shaft against contamination.

5.4 Nucleic acids

The methods provided in the method groups "DNA" and "RNA" differ mainly in the preprogrammed factor.

Additionally, for the methods "OLIGO DNA" and "OLIGO RNA" the selection of the parameter "MOL. UNIT" (molar concentration unit) differs from that of other nucleic acid methods. This parameter is only required for special conversions described at the end of this section.

As optical path length of the cuvette the value "10 mm" has been preprogrammed. If you modify the value the modified optical path length is taken into account by the device when calculating the results. Therefore you do not need to change the factor for the evaluation.

Results display

BioPhotometer plus — Operating manual

method, if necessary.

Operating manual

Top: method name and sample number Center: result and unit Bottom left: absorbance quotient ("ratios") Bottom right: absorbance results

In addition to the concentration result and the absorbance at measuring wavelength 260 nm the absorbances at 3 additional wavelengths and the quotients 260/280 and 260/230 are displayed as an indication for the purity of the measured nucleic acid sample. The absorbance at 340 nm should be near zero for pure samples.

Turbid measuring solutions have raised absorbances at all measuring wavelengths distorting the measuring result. You can partially correct this by enabling the parameter "CORRECTION A The absorbance value for 340 nm is then highlighted in the display with the cursor to indicate that the correction is enabled.

The last measured concentration result can be converted into molar concentrations and/ or nucleic acid amounts (mass unit or molar unit) if desired:

Top: input of the total amount of the sample Bottom: input of the base pairs (for single strand nucleic acid: the bases) or molar mass. One of the two entries suffices. All entries must be confirmed using the Enter key.

Display of the calculated results for the molar concentration and the sample amounts in the total amount.

340

Page 20

Operating manual

BioPhotometer plus — Operating manual

5 Operation

5.5 Proteins

You can measure the protein concentrations directly by measuring in the UV range at 280 nm or after adding reagent in the VIS range.

5.5.1 Protein 280 nm

The measurements can be evaluated via a factor entered into the parameters or via a single point calibration (measuring a standard).

• Factor: This evaluation mode is preprogrammed ex factory. However, you still have to enter

the factor before the first measurement. If you modify the parameter "CUVETTE" (preprogrammed to 10 mm) the modified thickness of the layer will be taken into account by the device when calculating the results. This means you do not need to adjust the factor for evaluation but have to provide the input for an optical path length of 10 mm.

• Standard: Alternatively you can program the evaluation mode "Standard" ("STD") (see

Methods with evaluation via standards on page 21).

Results display

Top: method name and sample number Center: result and unit Bottom right: absorbance results

In addition to the concentration result and the absorbance at measuring wavelength 280 nm the absorbances at 2 additional wavelengths are displayed as an indication for the purity of the measured protein sample. The absorbance at 340 nm should be near zero for pure samples.

Tubid measuring solutions have raised absorbances at all measuring wavelengths distorting the measuring result. You can partially correct the distortion by enabling the parameter "CORRECTION A

5.5.2 Protein after adding reagent (Bradford, BCA, Lowry)

You can evaluate these methods via factor or calibration (standard measurement).

340

• Factor: If you evaluate via factor you must take into account when making the entry that the

factor is adjusted to the selected result unit. If you modify the parameter "CUVETTE" the modified optical path length is, however, taken into account by the device when calculating the results. This means you do not need to adjust the factor for evaluation but have to provide the input for an optical path length of 10 mm.

• Standard: For evaluation via calibration you can program up to 10 different standards for

these methods. The procedure for calibration is described in the next chapter (see Methods with evaluation via standards on page 21).

Page 21

BioPhotometer plus — Operating manual

5 Operation

5.6 Methods with evaluation via standards

For the following methods you can define an evaluation via calibration (measuring of standards) in the parameters:

• PROTEIN 280nm (here only calibration with one standard is possible)

• BRADFORD, BRADFORD micro, BCA, BCA micro, LOWRY, LOWRY micro

• Methods via the keys "assays 340 / 405 / 490"

You can program up to 10 standards each in single up to triple measurement. For evaluation with several standards you can choose between the procedures "Linear regression" (for calibration lines) and "Non-linear regression" (for calibration curves). Dependent on the number of programmed standards the following applies:

Evaluation procedure Number of standards

Calculation of a factor 1 Linear regression 2 to 10 standards Non-linear regression for single measurement: 5 to 10 standards

Operating manual

for double or triple measurement: 4 to 10 standards

Measuring procedure

Standard measurements remain stored for an unlimited period of time after a valid calibration until they are overwritten with a new calibration. Exception: If method parameters are modified the calibration is deleted.

After opening a method (in the example the method "BRADFORD") you see the following device display:

The values marked "XXX" depend on the standard concentrations which you have programmed

Hint!

in the method parameters.

Because a calibration has already been stored in this example, the measuring keys provided include "sample" in addition to "standard" and "blank". Carry out a blank measurement.

Measure the first standard (in this example only as single measurement).

Measure the other standards as prompted in the bottom area of the device display.

After completing all standard measurements the calibration has been evaluated and stored. When measuring more than 2 standard samples a CV value is displayed for the calculated regression. If the calculated CV value exceeds 10% you are prompted for approval before saving. Sample measurements are evaluated using the last valid calibration.

After saving the calibration you can continue with a sample measurement. In the function list (see Functions on page 31) you can view and print the stored calibration data.

Page 22

Operating manual

BioPhotometer plus — Operating manual

5 Operation

5.7 OD 600

With method "OD 600" you can measure the bacteria density via a turbidity measurement at approx. 600 nm. Because this is a stray-light measurement the result depends on the geometry of the light path which can differ for the photometers by different manufacturers.

The exact measurement wavelength for the BioPhotometer plus is 595 nm. Measurements with suspensions of E. coli bacteria (absorbance range: approx. 0.1 to 0.3 E) showed at 600 nm about 1 to 2 % higher absorbance values than at 595 nm. You can program a corresponding correction factor in the parameters.

Results display

Top: method name and sample number Center: result. Bottom right: measured absorbance.

5.8 Dye-labeled biomolecules ("dye methods")

The preprogrammed methods for the keys dye 550 and dye 650 contain measuring procedures for dye-labeled biomolecules. During these measuring procedures both the biomolecule (nucleic acid or protein) and the dye are measured at different wavelenghts and their concentrations are determined. In addition the frequency of incorporation (concentration ratio between dye and biomolecule) is calculated.

5.8.1 Method group "dye 550"

The dye is measured at 550 nm, the nucleic acid at 260 nm and the proteins at 280 nm (see Methods on page 16). A selection of 4 dyes is available in the method parameters:

• CY 3

• ALEXA 546

• ALEXA 555

• DYE 550

The first 3 dyes are used more frequently in the laboratory, the last dye (DYE 550) is a spaceholder for other dyes.

For every dye you have to program the following corresponding data in the parameters. For the Cy and Alexa dyes values have already been preprogrammed but can be modified:

• Absorbance coefficient in the unit cm

converting the absorbance into concentration related to a cuvette optical path length of 10 mm and displays this on the start-up screen after opening the method.

• Optional: factors for the correction calculation "CORR: A

The parameters for the nucleic acid or protein component mainly match the parameters of the method group DNA and RNA and for the method Protein 280 nm.

-1·M-1

. The device uses this to calculate the factor for

" (see below for explanation).

550

5.8.2 Method group "dye 650"

The dye is measured at 650 nm. A selection of 3 dyes is available:

• CY 5

• ALEXA 647

• DYE 650

The additional explanations found under method group "dye 550" apply accordingly.

Page 23

BioPhotometer plus — Operating manual

5 Operation

5.8.3 Frequency of incorporation "FOI"

The FOI is a measure for the concentration of the dye relative to the concentration of the nucleic acid. It is not provided for protein methods. For calculating the FOI you can select between 2 units in the method parameters:

• "MOLECULE DYE / kb": dye molecules per 1000 nucleotides.

• "pmole/μg DNA": pmole dye per μg nucleic acid.

The formulae for calculating the FOI can be found separately (see Calculation of the FOI on page 45).

5.8.4 Correction factors

In addition to the options for turbidity correction described (parameter "CORRECTION A (see Nucleic acids on page 19) the affect of the dye on the measurement of the nucleic acid or the protein can also be corrected for the "DYE methods". If the degree to which the dye absorbs light is known also for the measuring wavelengths of the biomolecule (260 and 280 nm), then a correction can be made in the parameter "CORRECTION you can enter a correction factor for 260 and 280 nm. E.g. for Cy 3 the preprogrammed values are:

• 0.04 for "CORR.A

• 0.05 for "CORR.A

The use of these factors for the calculation of concentration and ratio of the biomolecule is described elsewhere (see Correction A

550 550

: F : F

260 260

Operating manual

340

550 (or 650)

" "

on page 46).

550/650

". If you enable this parameter

5.8.5 Measuring procedure and result display

After calling the method you will see the following display (example: "DYE 550 - ssDNA"):

Top: method name and cuvette selected in the parameters. Center: programmed values for the calculation of the DNA and dye concentrations: factor for ssDNA and absorbance coefficient for the dye. In addition the factor calculated from the absorbance coefficient related to a cuvette optical path length of 10 mm will be displayed. Bottom: measuring key for the next measurement.

 Carry out a blank measurement.

 Carry out a sample measurement.

Top: method name and sample number Center: result for DNA and dye. Bottom: result for the frequency of incorporation.

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BioPhotometer plus — Operating manual

5 Operation

As with the nucleic acid measurement without dye measurement (see Nucleic acids on page 19) you can use the cursor keys to access additional details of the result (absorbance value and ratio 260/280) and to carry out conversions for further results:

After entering the total volume of a sample: Total amount of the nucleic acid (mass in μg)

and the dye (in pmole).

After entering the bases or molecular mass: Molar concentration of the nucleic acid.

If the total amount of the sample has also  been entered: total amount of the nucleic acid (in pmole).

Operating manual

When displaying the absorbance values the values for 340 nm or for 550 (or 650) nm are highlighted with the cursor if "CORRECTION A enabled.

5.9 Methods for 340, 405 and 490 nm

3 method locations are provided for each wavelength. The following evaluation procedures have been preprogrammed as samples. However, you can adjust these procedures at any time:

Example: "assay 340"

• ASSAY 340/1: Evaluation via factor

• ASSAY 340/2: Evaluation via single point calibration

• ASSAY 340/3: Evaluation via curve calibration

The following applies to evaluation via factor:

• If you modify the parameter "CUVETTE" (preprogrammed to 10 mm) the modified thickness

of the layer will be taken into account by the device when calculating the results. This means you do not need to adjust the factor for evaluation but have to provide the input for an optical path length of 10 mm.

• When entering the factor ensure that it has been adjusted for the selected unit of results.

5.10 Dilution

You can enter sample dilutions before a measurement using the parameter/dilution key. The dilution factor will then be automatically taken into account when calculating the results.

For the following example the method has already been called and a blank been measured:

" or "CORRECTION A

340

550 (or 650)

" has been

1. Open the dilution input using the parameter/dilution key.

2. Enter the volumes of the sample and the dilution buffer ("diluent") and confirm each entry with enter.

This brings you back to the measuring procedure and you can start a sample measurement.

Page 25

5 Operation

BioPhotometer plus — Operating manual

3. Start a sample measurement using the sample key.

Operating manual

4. The result is calculated taking into account the sample dilution entered.

The dilution is retained for calculating all subsequent sample results until it is overwritten. To delete the dilution enter "0" for both "SAMPLE" and "DILUENT" or delete the values using the keys clear and enter.

5.11 Sample number

The device display for measuring results contains the sample number at the top right. It is counted separately continuously for every method and reset to "1" when a new method is opened.

You can modify the sample number before a measurement. For the following example 5 samples were already measured:

1. Open the sample number input using the function/·/No. key.

2. Enter the desired sample number for the next sample (here "3") and confirm the entry with Enter.

This brings you back to the measuring procedure and you can start a sample measurement.

3. Start a sample measurement using the sample key.

4. The sample result is allocated the sample number "3". All other samples are incremented continuously.

Page 26

Operating manual

BioPhotometer plus — Operating manual

6 Parameter and functions

6.1 Parameter

The methods of the BioPhotometer plus as well as the corresponding parameters have already been preprogrammed ex factory. You can modify these parameters.

6.1.1 View, change and store the parameters of a method

1. Select the desired method group using the corresponding method key.

2. Using the cursor keys select the desired method.

3. Using parameter/dilution open the parameters for this method.

4. Scroll through the parameter list with the cursor keys.

5. Modify the parameters, if necessary, and confirm the changes using Enter. There are two kinds of parameter entries:

• Selection parameters: selection via cursor keys.

• Numerical values: input via number keys.

Each parameter entered is only saved after confirmation using the Enter key.

Hint!

6. Exit the parameter list by pressing parameter/dilution again. You return to the method selection.

6.1.2 Summary and description of parameters

Parameter Entry Explanation

Cuvette Selection:

10, 5, 2, 1 or 0.2 mm

Unit Selection:

Different units dependent on the method.

MOL. Unit Selection:

Different units dependent on the method.

CALCULATION Selection:

• STD

• FACTOR

Optical optical path length of the cuvette. The factor for converting the absorbance into the concentration is corrected accordingly internally.

Result unit for the method. In some methods this selection is missing because a unit has been preprogrammed permanently.

Molar unit, only for the nucleic acid methods: for the conversion of concentration units into molar concentrations.

Evaluation procedure for calculating the sample concentration:

• with fixed factor

• with standards (calibration)

Factor Numerical input (5 digits) Factor for converting absorbance into concentration. The

number of digits after the decimal point of the factor determines the number of digits after the decimal point for the result. The factor must be entered for a cuvette optical path length of 10 mm.

STD NUMBER 1-10 Numerical input (1 to 10) Number of standards with different concentrations used for

the calibration.

STD MEASUREMENT Selection:

1x, 2x or 3x

Repeated measurements of standards.

Page 27

BioPhotometer plus — Operating manual

6 Parameter and functions

Parameter Entry Explanation

REGRESSION Selection:

Linear and non-linear regression

STD 1 to STD 10 Numerical input (numbers and

decimal point, 5 digits)

CORRECTION Selection:

• NO CORRECTION

• CORRECTION A

CY3 (or Cy5)

ALEXA .....

ABS.COEFF. Numerical input (6 digits) For "Dye methods": input of the absorbance coefficient for

CORRECTION  A

550 (650)

CORRECTION  A

550 (650)

CALCULATION FOI Selection:

: F

260

Selection For "Dye methods": selection of the dye (see Dye-labeled

Numerical input (5 digits) Only if in "Dye methods" the parameter 

340 550 (or 650)

• MOLECULE DYE / kb

• pmole/μg DNA (or RNA)

Evaluation procedure of the multi-point calibration.

• Linear regression is possible for:

2 to 10 standards

• Non-linear regression is possible for:

(single measurement:) 5 to 10 standards (double or triple measurement:) 4 to 10 standards.

Nominal concentration for each standard. Unit as entered in the parameter display 1. The number of digits after the decimal point in the nominal concentration of the first standard determines the number of digits after the decimal point in the result. The concentrations must be entered in ascending order.

CORRECTION A Can be used for the partial correction of the effect of minor

turbidity in the measuring solution: The absorbance measured at 340 nm is subtracted from the absorbance results at 230, 260 and 280 nm prior to subsequent evaluation. CORRECTION A

Can be used for the "Dye methods" to correct the effect of the dye on the measuring absorbances of the biomolecule (nucleic acid or protein): The absorbance of the dye measured at 550 (or 650) nm is multiplied with a correction factor for 260 or 280 nm. The result is subtracted from the absorbances of the biomolecule at 260 or 280 nm prior to subsequent evaluation for the biomolecule (see Dye-labeled biomolecules ("dye methods") on page 22). The correction factors are entered further down in the parameter list after selecting the dye.

biomolecules ("dye methods") on page 22).

the selected dye in the unit "cm

CORRECTION A Entry of the correction factors for taking into account the

effect of the dye on the measurement absorbances of the biomolecule (see description of the parameter CORRECTION A

For "Dye methods": selection of the procedure for calculating the frequency of incorporation (see Dye-labeled biomolecules ("dye methods") on page 22).

340

550 (or 650)

-1·M-1

has been enabled:

above).

Operating manual

Page 28

Operating manual

BioPhotometer plus — Operating manual

6 Parameter and functions

6.1.3 Parameters preprogrammed ex factory

Nucleic acids and OD 600

Parameter dsDNA ssDNA OLIGO DNA RNA OLIGO RNA OD 600

CUVETTE 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm UNIT μg/mL μg/mL μg/mL μg/mL μg/mL MOL. UNIT pmol/mL pmol/mL pmol/μL pmol/mL pmol/μL FACTOR 50.0 37.0 30.0 40.0 30.0 1.000 CORRECTION NO

CORRECTION

Proteins

NO CORRECTION

Parameter BRADFORD BRADFORD

micro

CUVETTE 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm UNIT μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL mg/ml

CORRECTION NO COR-

CALCULATION STD STD STD STD STD STD Factor FACTOR ----STD NUMBER

1-10 STD

MEASUREMENT REGRESSION NON-

STD 1 100 1.00 25 0.50 100 1.00 STD 2 250 2.5 125 2 250 2.5 STD 3 500 5 250 5 500 5 STD 4 750 10 500 10 750 10 STD 5 1000 15 750 20 1000 15 STD 6 1500 25 1000 1500 25 STD 7 1500 STD 8 2000

668566

1x 1x 1x 1x 1x 1x

NON-

LINEAR

BCA BCA micro LOWRY LOWRY

micro

NON- LINEAR

PROTEIN 280 nm

(no entry, programmed permanently)

RECTION

Page 29

BioPhotometer plus — Operating manual

6 Parameter and functions

Method group "dye 550"

Parameter DYE 550-dsDNA DYE 550-ssDNA DYE 550-RNA DYE 550-OLIGO DYE

550-PROTEIN

CUVETTE 10 mm 10 mm 10 mm 10 mm 10 mm Unit

(only for nucleic acid)

MOL. UNIT (for nucleic acid)

FACTOR (for nucleic acid or protein)

CORRECTION NO

Dye selection CY3 CY3 CY3 CY3 CY3 ABS.COEFF. (for Cy3: )

CALCULATION FOI

μg/mL μg/mL μg/mL μg/mL mg/mL 

(protein: no entry, programmed permanently)

pmol/mL pmol/mL pmol/mL pmol/μL

50.0 37.0 40.0 30.0 -----

CORRECTION

150000 MOLECULES

DYE / kb

NO CORRECTION

(for Cy3: ) 150000

MOLECULES DYE / kb

NO CORRECTION

(for Cy3: ) 150000

MOLECULES DYE / kb

NO CORRECTION

(for Cy3: ) 150000

MOLECULES DYE / kb

NO CORRECTION

(for Cy3: ) 150000

Operating manual

The unit for the dye is defined permanently with "pmol/μl" and can therefore not be programmed in the parameters.

For the various dyes the following dye-specific values have been preprogrammed as absorbance coefficients and correction factors (for the parameter CORRECTION A

Parameter CY3 ALEXA546 ALEXA555 DYE550

ABS.COEFF. 150000 ------ 150000 -----CORRECTION A

260

CORRECTION A F

280

Parameter CY5 ALEXA647 DYE650

ABS.COEFF. 250000 239000 -----CORRECTION A

CORRECTION A

0.04 0.0 0.04 0.0

550

0.05 0.0 0.04 0.0

550

The values for ALEXA546 were not known at the time of writing this manual; please contact the manufacturer (Invitrogen) with regard to these values.

Method group "dye 650"

The parameters correspond mainly to those of method group "dye 550". As preselection for the dye Cy 5 has been programmed. For the dye-specific values the following applies:

650

: F

0.0 0.0 0.0

260

0.05 0.03 0.0

280

550

Page 30

Operating manual

BioPhotometer plus — Operating manual

6 Parameter and functions

assay 340

Parameter ASSAY 340/1 ASSAY 340/2 ASSAY 340/3

CUVETTE 10 mm 10 mm 10 mm UNIT μg/mL μg/mL μg/mL CALCULATION Factor STD STD FACTOR ----STD NUMBER 1-10 1 6 STD MEASUREMENT 2x 1x REGRESSION NON-LINEAR STD 1 ----- ----STD 2 ----STD 3 ----STD 4 ----STD 5 ----STD 6 -----

assay 405 and assay 490

The parameters correspond to those of method group "assay340".

ABSORBANCE

You can define the wavelength directly in the start-up screen of the method. If you open the parameter list you are provided with the cuvette optical path length and unit for information only. However, neither parameter can be modified in this method. Unlike with other methods there is no automatic conversion to a different cuvette optical path length in this case.

Page 31

BioPhotometer plus — Operating manual

6 Parameter and functions

6.2 Functions

1. In the method selection press the key function/·/No.. A list of general device functions will open. In the method procedure or parameter mode this

key has a different function (entering the sample number or entering a decimal point when inputting values).

2. In this function list and any sub-lists (e.g. results list) select the desired function like in the parameter list (see View, change and store the parameters of a method on page 26) using the cursor keys and open it with the Enter key.

3. Press the function/·/No. key again to return from a sublevel of the functions (e.g. opening stored results in the function "RESULTS") to the next higher level in the function list and finally back to the method selection.

Tab. 2: Summary and description of functions

Function Explanation

RESULTS Display of the last 100 results (the result measured last is shown first).

For results with information about more than one device display (detailed information for nucleic acids and "dye methods") you can access the detailed displays via the cursor keys. Print the selected result using the Enter key.

STANDARDS Display of the stored calibration data for methods with evaluation via standards.

In this list you can also scroll using the cursor keys until you see the calibration data of the desired method in the device display.

PRECISION MEASUREMENT Measurement and precision calculation of 10 consecutive measured values of a

sample. The method program of the last opened method is used.

PHOTOMETER TEST NEW MEASUREMENT LAST MEASUREMENT

SPRACHE DEUTSCH LANGUAGE ENGLISH LANGUAGE U.S.ENGL LANGUE FRANCAISE

DATE Date input. Save using Enter. Time Time input. Save using Enter. Printer Printer selection:

SERVICE The function is only available to Eppendorf Service.

Checking the photometer using a filter kit from Eppendorf (see Check photometer on page 34). You can start a new check (NEW MEASUREMENT) or open the results of the last check (LAST MEASUREMENT).

Selection of the language version. "ENGLISH" and "U.S.ENGLISH" differ in their date format.

• DPU 414: Connection of the Eppendorf thermal printer.

• SERIAL: Connection of a different printer. In this case certain printer requirements

must be met. Contact your Eppendorf partner for details.

Operating manual

Page 32

7 Maintenance

7.1 Cleaning

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

 Switch off the device and disconnect it from the power supply before starting cleaning or

DANGER!

 Do not allow any liquids to penetrate the inside of the housing.  Do not disinfect by means of spraying.  Only reconnect the device to the power supply once it is completely dry.

BioPhotometer plus — Operating manual

disinfecting.

Operating manual

CAUTION!

Caution! 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 prolonged

periods.

1. Switch off the device and disconnect the power plug.

2. Wipe down the surfaces with a cloth you have moistened with a mild cleaning agent.

3. Only clean the cuvette shaft using a moist lint-free cotton swab. Prevent liquid from entering the cuvette shaft.

7.1.1 Cleaning the cuvette shaft cover

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

1. Slide the cover fully forward.

2. Gently pull the cover upwards near the front and then slowly push it to the back. After a few millimeters you can lift the cover completely.

3. Clean the cover and the cover holder with a cloth or a lint-free cotton swab wetted with a mild detergent.

4. Replace the cover on the cover holder as shown here. The button of the cover holder fits exactly into the circular enlarged recess at the bottom of the

cover.

If the photometer is not in use slide the blue cover over the cuvette shaft to protect it against dust

Hint!

and other contamination.

Page 33

BioPhotometer plus — Operating manual

7 Maintenance

7.2 Disinfection / Decontamination

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

 Switch off the device and disconnect it from the power supply before starting cleaning or

DANGER!

disinfecting.

 Do not allow any liquids to penetrate the inside of the housing.  Do not disinfect by means of spraying.  Only reconnect the device to the power supply once it is completely dry.

1. Switch off the device and disconnect the power plug.

2. Prior to disinfection clean the device using a mild detergent as described above  (see Cleaning on page 32).

3. Select a disinfecting method which meets the legal regulations and guidelines applicable to your area of application.

4. For example, use alcohol (ethanol, isopropanol) or disinfectants containing alcohol.

5. Wipe down the surfaces with a cloth you have moistened in disinfectant.

6. If the cuvette shaft needs to be removed for disinfection proceed for removal and assembly as described in the section on Cleaning (see Cleaning the cuvette shaft cover on page 32).

7. The removed cuvette shaft cover can be disinfected by spray disinfection.

7.3 Decontaminating before shipping

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

Operating manual

Warning! Risk to health from contaminated device

1. Follow the instructions in the decontamination certificate. It is available in PDF format on our

WARNING!

2. Decontaminate all the parts you want to dispatch.

3. Enclose the fully-completed decontamination certificate for returned goods (incl. the serial

7.4 Replacing fuses

Danger! Electric shock.

 Switch off the device and disconnect the power plug before opening the device to replace the

DANGER!

1. Switch off the device and disconnect the power plug.

2. The fuse holder is above the mains connection 7 (see Fig. 1 on page 8). Press the small

3. Replace the fuses (see order information in the operating manual).

4. Push the fuse holder back into its retainer until the notch lever engages.

5. Reconnect the power plug.

homepage (www.eppendorf.com/decontamination)

number of the device) with the shipment.

fuses. These tasks may only be performed by appropriately trained staff.

spring-loaded notch lever at the underside of the fuse holder and pull out the holder.

Page 34

Operating manual

BioPhotometer plus — Operating manual

7 Maintenance

7.5 Check photometer

To check the photometric accuracy and the accuracy of the wavelength Eppendorf offers a filter kit (secondary UV-VIS filter). The kit contains three filters ("sample A1", "sample A2" and "sample A3") to check the photometric acccuracy and two filters ("sample 260 nm" and "sample 280 nm") to check the accuracy of the wavelength. The absorbances of the filters are measured against a blank filter ("blank A0"). In addition to information about the accuracy you are also provided with information about the precision: from the 10 measurements each per wavelength the CV value is calculated in addition to the mean value.

To perform a blank measurement, firstly blank filters are inserted like cuvettes into the cuvette shaft followed by the test filters. The absorbance values measured for the test filters are compared to the permitted value range. The limit values for the permitted range for the individual filters are printed in a table in the lid of the filter box (see in table: "X.XXX – X.XXX E").

Tab. 3: Inside lid of the filter box (sample)

Page 35

7 Maintenance

7.5.1 Test procedure

BioPhotometer plus — Operating manual

• Carry out test at approx. 20°C.

Hint!

• Remove filter only briefly from the filter box and protect against contamination or damage to

the filter surface.

Operating manual

• Protect filter against dust, heat, liquids and aggressive vapors.

• Always insert the filter with the sticker containing the filter designation facing the user

(towards the recipient).

1. Open the function PHOTOMETER TEST / NEW MEASUREMENT.

2. Select the test filter and confirm the selection with "Enter".

3. "SAMPLE 260" or "SAMPLE 280" for measuring the accuracy of the wavelength for 260 or 280 nm.

4. "SAMPLE A1", "SAMPLE A2" or "SAMPLE A3" for measuring the photometric accuracy at 230, 260, 280, 340, 405, 490, 550, 595 and 650 nm.

5. Follow the instructions on the device display for measuring the blank ("A0") and the test filter. The device carries out 10 measurements each and then displays the mean value and the CV value for the measured absorbance at the respective wavelengths.

6. Press "Enter" in accordance with the instructions on the device display to have the values displayed and then printed - provided a printer is connected.

7. Compare the mean values and the CV values to the permitted ranges in the table supplied.

If the measured values do not match the permitted value range please contact Eppendorf Service. The filter should be recalibrated by Eppendorf after 2 years.

Page 36

Operating manual

BioPhotometer plus — Operating manual

8 Troubleshooting

8.1 Result flags

Result flags Possible cause Remedy

0.015 A

0.015 A or

0.015 A

++++++ The measured absorbance is above A = 3.

------ (Instead of a value for the ratio:)

¶ Only for nucleic acid methods and 

340

550

650

"PROTEIN 280 nm": The marking '¶' of the A

the absorbance values for 230, 260 and 280 nm are corrected with the absorbance for 340 nm (parameter "CORRECTION A

enabled). Only for method groups "Dye 550" and 

"Dye 650": The marking '¶' of the A

value indicates that the absorbance values for 260 and 280 nm are corrected with the absorbance for 550 or 650 nm (parameter "CORRECTION

Ratio cannot be calculated because one of the absorbance values used for the calculation is A = 0 or A > 3.

550 (or 650)

value indicates that

340

value or the A

550

" is enabled).

340

" is

None. The flag is only for information.

650

 Dilute sample.  Check cuvette volume (light path

height is 8.5 mm).

 Clean cuvette shaft (see Cleaning

on page 32) and insert cuvette the right way round: measuring surface towards the light path. The direction of the light path is indicated on the blue cuvette shaft cover by an arrow.

 Use cuvette from a material which

is optically transparent for light of the measuring wavelength (for measurements in UV e.g.: Eppendorf UVette).

Repeat measurement, if necessary with a diluted sample.

Page 37

BioPhotometer plus — Operating manual

8 Troubleshooting

8.2 Error messages

Device displays with error messages can be exited using Enter. If certain parameters are programmed incompletely or incorrectly the icon for the parameter key

is shown in the display. In these cases press the parameter/dilution key instead. This brings you directly to the parameter list to correct the error.

Error text Explanation / cause Remedy

(Parameter) PLEASE PROGRAM e.g.: FACTOR PLEASE

PROGRAM

MEASURE BLANK FIRST No blank has been stored for the method

MEASURE STANDARD FIRST No calibration has been stored for the method

NO STD METHOD The key standard has been pressed although

OUTSIDE OF CALIBRATION Only for non-linear calibration evaluation:

MEASURED VALUES NOT MONOTONIC

CALIBRATION CURVE IS NOT MONOTONIC

PROGRAM STANDARDS INCREASING

CV IS GREATER THAN 10% When calculating the calibration the "CV" is

CALIBRATION NOT OK! The measured standard absorbances do not

MEASURED VALUES NOT PLAUSIBLE

INVALID INPUT When entering a sample number:

MEASURING MODULE FAULT 1 (or 2 or 3)

Method parameter has not been programmed correctly or is incomplete.

opened.

no calibration evaluation has been programmed for the method.

The measured sample absorbance is outside the absorbance range of the standard.

For multi-point calibration: The absorbance values of the standard do not result in a monotonic increasing or decreasing order.

The calibration curve is not monotonic increasing or decreasing or does have a reversal point.

For multi-point calibration the target concentration of the standards has not been programmed in increasing order.

greater than 10%.

result in a valid calibration evaluation.

Value cannot be displayed; potential causes when measuring with standards:

• Standard for single point calibration has the

absorbance "0"

• Calculated factor for single point calibration

is too great to be displayed.

• Polynome for multi-point calibration cannot

be displayed with the measured absorbance values.

The number "0" was entered

Technical error messages.

 Check and correct parameter (see

Parameter on page 26).

 Measure blank.

 Measure standards to save a valid

calibration.

 Program evaluation with standards

in the measuring parameters or measure the method again without standards.

 Only measure samples which are

within the calibration range. For too highly concentrated samples: dilute samples.

 Check standards and measure

again in the correct order (increasing concentration).

 Check standards, if necessary

prepare again and re-measure.

 Program target concentration of

the standards in increasing order.

 Check standards, if necessary

prepare again and re-measure.

 Check standards and measure

again.

 For measurements with standards:

check measured absorbances; if necessary, prepare standards again and re-measure.

 Enter sample number between 1

and 999.

 Contact Eppendorf Service.

Operating manual

Page 38

Operating manual

BioPhotometer plus — Operating manual

8 Troubleshooting

8.3 General errors

Fault Possible cause Remedy

Measuring results are imprecise.

Measuring results incorrect.

• Shelf life of reagent solution

exceeded.

• Reagent not prepared

correctly.

 Ensure that the reagent is still within its shelf life

and properly prepared.

 Use clean demineralized water of adequate

quality for preparation if required.

• Pipetting incorrect.  Ensure that the pipette is calibrated and that

pipetting is being performed correctly.

• Incubation procedure before

measurement is incorrect.

 If the method procedure requires incubation

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

• Cuvette dirty.  Clean and rinse the cuvette. When changing

cuvette, ensure that the optical surface of the cuvette remains clean and has not come into contact with fingers.

 If the optical surface of the cuvette is soiled with

fingerprints, clean it by wiping with a lint-free laboratory cloth soaked in alcohol.

• Cuvette not completely full of

measuring solution without bubbles.

 Ensure that the necessary minimum volume of

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

• Measuring solution turbid.  Centrifuge turbid measuring solutions containing

particles and use the clear supernatant.

• Photometer drifting.  Contact Eppendorf Service.

• Method wrongly programmed.  Ensure that the method parameters are entered

correctly.

• Standard solution not

prepared correctly.

 Ensure that the correct standard is used and that

the measuring solution for the standard is prepared correctly.

• Reagent absorbance drifting.  (With unstable reagent absorbance and

end-point methods): when measuring a long series of samples, do not measure the reagent blank only at the beginning of the series, but also during the series of samples. If the reagent blank drifts significantly, the reagent is unsuitable for fault-free measurements and must be replaced with new reagent.

Other possible causes can be found under "Measuring results imprecise".

Other possible remedies can be found under "Measuring results imprecise".

Page 39

BioPhotometer plus — Operating manual

9 Transport, storage and disposal

9.1 Transport



Only transport the device in the original packaging.

9.2 Storage

9.3 Disposal

Air temperature Rel. humidity Air pressure

General transportation -25 to 60°C 10 to 95% 30 to 106 kPa Air freight -40 to 55 °C 10 to 95% 30 to 106kPa

Air temperature Rel. humidity Air pressure

in transport packaging -25 to 55°C 25 to 75% 70 to 106 kPa without transport

packaging

In the event of disposing of the product, please observe the applicable legal regulations.

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

The disposal of electrical devices is regulated within the European Community by national regulations based on EU Directive 2002/96/EC pertaining to waste electrical and electronic equipment (WEEE).

In accordance with this, any devices delivered after 13/08/2005 on a business-to-business basis, which includes this product, may no longer be disposed of in household waste. To document this they have been marked with the following identification:

-5 to 45°C 25 to 75% 70 to 106 kPa

Operating manual

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

Page 40

Operating manual

BioPhotometer plus — Operating manual

10 Technical data

10.1 Power supply

Power supply 100 to 240 V ± 10 % / 50 to 60 Hz ± 5 % Overvoltage category IEC 61010-1 category II Degree of contamination: IEC 61010-1 category II Power consumption approx. 20 W during operation, 

approx. 10 W in standby mode

Permitted mains interruption approx. 10 ms at 90 V

approx. 20 ms at 220 V

Fuses T 1A/250 V, 5 mm x 20 mm (2 off)

10.2 Ambient conditions

Operation Ambient temperature: 15 to 35 °C

Rel. humidity: 15 to 70% Atmospheric pressure: 86 to 106 kPa

Storage Ambient temperature: -25 to 70 °C

Rel. humidity: 15 to 70% Atmospheric pressure: 30 to 106 kPa

Not tropicalized. Protect against direct sunlight.

10.3 Weight / dimensions

Weight 3 kg (packaged: 4.8 kg) Dimensions Width: 200 mm (packaged: 290 mm)

Space required Width: 400 mm (or 650 mm including printer)

10.4 Interfaces

Interface for printer and PC serial RS-232

The printer to be connected must meet the requirements of EN 60950 or UL 1950.

Depth: 320 mm (packaged: 430 mm) Height: 100 mm (packaged: 200 mm)

Depth: 500 mm

Page 41

10 Technical data

10.5 Photometer

Measuring principle Absorption single beam photometer with reference

Light source Xenon flash light Monochromator Holographic concave grating Beam receiver Silicon photo diodes Wavelengths 230, 260, 280, 340, 405, 490, 550, 595, 650 nm Wavelength selection Dependent on method, controlled by program Spectral bandwidth 5 nm at 230 to 340 nm

Systematic wavelength error ± 1 nm at 230 to 280 nm

Photometric measuring range Quartz glass cuvette:

Reading accuracy ΔA = 0.001 Random photometric error  0.002 at A = 0

Systematic photometric error ± 1% at A = 1 Stray light component < 0.05 %

BioPhotometer plus — Operating manual

beam and several fixed wavelenghts

7 nm at 405 to 650 nm

± 2 nm at 340 to 650 nm

• A = 0 to 3, except:

• A = 0 to 2 at 340 nm

• Only for dye methods: 

A = 0 to 2 at 550/650 nm

UVette (Eppendorf):

• A = 0 to 2.5 at 230 nm

• A = 0 to 2.6 at 260 nm

• A = 0 to 2.8 at 280 nm

• Other values see quartz glass cuvette

 0.005 at A = 1

Operating manual

10.6 Other technical parameters

Cuvette material For DNA, RNA, Oligo, Protein UV, Assay 340:

Cuvette shaft 12.5 mm x 12,5 mm, not temperature-controlled Overall cuvette height Min. 36 mm Height of the light beam in the cuvette 8.5mm Light beam in the cuvette Width: 1 mm

Keyboard 19 foil keys Display Illuminated graphic display, 33 mm x 60 mm Operator guidance language English, French, German Result output Via display and printer:

Standards and regulations According to VDE, CE, IEC 1010-1

Quartz glass or UV transparent plastic  (UVette by Eppendorf) For OD600, Bradford, Lowry, BCA, Assay 405, Assay 490: Glass or plastic

Height: 1.5 mm

Absorbance, concentration, ratio, FOI

Page 42

Operating manual

BioPhotometer plus — Operating manual

10 Technical data

10.7 Application parameters

Method memory 32 preprogrammed, modifiable method

programs

Measuring methods End point against blank

Dye methods: parallel measurement of biomolecule and dye label

Method-dependent evaluation absorbance

concentration via factor concentration via calibration with 1 to 10 standards:

• single point calibration (1 standard)

• linear regression (2 to 10 standards)

• non-linear regression (polynome of 3rd

degree; 4 or 5 to 10 standards  (see Evaluation procedure on page 43)).

• 1x, 2x or 3x measurement

for nucleic acids:

• ratio 260/280

• ratio 260/230

• molar concentration

• total yield

for dye methods:

• FOI (frequency of incorporation; 

marking density) Calibration memory For all calibration methods Measured value memory For 100 results with abosrbance and ratio

values, sample number, sample dilution, date and time

Page 43

BioPhotometer plus — Operating manual

11 Evaluation procedure

Hint!

This chapter describes the available evaluation procedures in the method programs (see Parameter on page 26) and the calculation of a dilution by the device software.

When comparing the measuring results to those of other photometers / spectrometers note that the values may depend on the bandwidths 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.

You should therefore check the accuracy of the method by measuring standards.

11.1 Evaluation with factor

C = calculated concentration A = measured absorbance F = factor The factor is programmed in the parameter list and can be modified. For the dye methods the

factor for calculating the dye component is calculated from the absorbance coefficient of the device.

The factor always relates to the cuvette optical path length of 10 mm. If you change the parameter CUVETTE the modification is taken into account by the device 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.

Operating manual

11.2 Evaluation using standards

11.2.1 Single point calibration

F = calculated factor C

= nominal concentration of the standard

= measured absorbance of the standard

The nominal concentration is programmed in the parameter list and can be modified. If multiple measurement (2x, 3x) has been programmed for the standard, then the evaluation

from the measured absorbances takes place via linear regression taking into account the zero value. After calculating the regression a CV value (coefficient of variation with a unit of %) is formed as measure for the scattering of the measured values. If the CV is greater than 10% it will be displayed. In this case the calibration will only be stored after confirmation.

The calculation of the sample concentration takes place using the calculated factor:

Page 44

Operating manual

BioPhotometer plus — Operating manual

11 Evaluation procedure

11.2.2 Multi-point calibration: calibration line

For the evaluation via a calibration line the selection "LINEAR" is programmed in the parameter "REGRESSION".

From 2 to 10 standards which can be measured once, twice or three times, a linear equation is calculated via linear regression (concentration as function of absorbance):

= Interface of the calibration line with the concentration axis ("offset": concentration of a

sample with absorbance 0) a1 = slope of the calibration line ("factor") After calculating the regression the device forms a CV value (see above) as measure for the

scattering of the measured values around the calibration line (exception: two point calibration with single measurement of both standards). If the CV is greater than 10% it will be displayed. In this case the calibration will only be stored after confirmation. For more than 2 standards the CV value is always displayed (even for a value below 10%).

In the function list the calibration data can be printed.

11.2.3 Multi-point calibration: Calibration curve

For the evaluation via a calibration curve the selection "NON-LINEAR" is programmed in the parameter "REGRESSION".

From 5 to 10 standards measured once, or 4 to 10 standards measured twice or three times, an equation for the third degree polynome (concentration as function of absorbance) is calculated via a non-linear regression.

a = coefficients (the coefficients are defined using the method of smallest squares) After calculating the regression the device forms a CV value as measure for the scattering of the

measured values around the calibration line (see above). If the CV exceeds 10% the calibration is only stored after confirmation.

The details above about the CV value and printing the calibration data apply accordingly.

11.3 Dilution

Dilutions entered into the method procedure are taken into account when calculating the results:

C V V

= 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

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BioPhotometer plus — Operating manual

11 Evaluation procedure

11.4 Special evaluation procedures for the dye methods

11.4.1 Calculating the factor for the dye from the absorbance coefficient

In the dye methods a factor is calculated for the dye from the absorbance coefficient entered in the parameters and is displayed in the startup screen of the method procedure.

The factor is calculated as follows:

= factor for the dye with automatic allowance for the cuvette optical path length

d, Dye

= absorbance coefficient for the dye programmed in the method parameters; unit: cm-1M

Dye

d = cuvette optical path length, unit: cm

11.4.2 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:

Unit DYE MOLECULE DYE / kb

Unit pmole/μg DNA (or RNA)

Operating manual

-1

= measured absorbance of the dye at 550 or 650 nm

X50

= absorbance coefficient for the dye, unit: cm-1M

Dye

MMnt = average molecular mass of the nucleotides; unit: g/mol; for dsDNA/ssDNA/Oligo DNA: 325; for RNA / Oligo RNA: 337

= measured absorbance of the nucleic acid

260

F = factor for calculating the nucleic acid programmed in the method parameters. The factor relates to the cuvette optical path length of 10 mm and does not need modifying if the parameter CUVETTE is modified.

-1

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BioPhotometer plus — Operating manual

11 Evaluation procedure

11.5 Special evaluation procedures for nucleic acids and protein UV

This section relates to the evaluation of nucleic acids or proteins in the methods dsDNA, ssDNA, Oligo DNA, RNA, Oligo RNA, Protein UV and the corresponding biomolecule components in the dye methods.

Operating manual

11.5.1 Correction A

11.5.2 Correction A

340

Application: Partial correction of distortions of absorbance due to turbidity in the measuring solution.

The application of the evaluation procedure can be activated in the parameter list of the method.

= calculated corrected absorbance at a wavelength of 230, 260 and 280 nm

X, corr

= measured absorbance at a wavelength of 230, 260 and 280 nm

= measured absorbance at a wavelength of 340 nm

340

550/650

Application: Correction of the effect of the dye absorbance on the nucleic acid or protein absorbance at 260 and 280 nm.

The application of the evaluation procedure can be activated in the parameter list of the method.

= calculated corrected absorbance at a wavelength of 260 and 280 nm

X, corr

= measured absorbance at a wavelength of 260 and 280 nm

= correction factor for the wavelength 260 or 280 nm (the two correction factors for 260 and

for 280 nm are programmed in the parameter list of the method) A

= measured absorbance at a wavelength of 550 (or 650) nm

X50

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

Hint!

absorbance values.

11.5.3 Conversion into molar concentrations and nucleic acid amounts

The conversion can only be applied to nucleic acids and dye methods with nucleic acids as biomolecule component.

Calculation of amount

Application: Calculation of the amount (mass) of nucleic acid or nucleic acid dye conjugate in the whole sample volume.

M = calculated total amount (mass) of the nucleic acid or the conjugate in the sample tube C = calculated concentration of the nucleic acid or the conjugate V

= volume of the sample in the sample tube; entered by the user in the third screen of the

P, t o t al

result display (see Nucleic acids on page 19).

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BioPhotometer plus — Operating manual

11 Evaluation procedure

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 figure of the bases or base pairs per nucleic acid molecule.

= calculated molar concentration of the nucleic acid; the unit for the molar concentration is

Mol

programmed in the parameter list of the method; dependent on the programmed unit the formula above is adjusted automatically.

C = mass concentration of the nucleic acid in ng/μl or μg/ml, from the measured absorbance the device calculates the concentration result and displays it on the results screen

MM = relative molar mass in kDa If in the third results screen the number of bases or base pairs per nucleic acid molecule has

been entered instead of the relative molar mass, then the molar mass is calculated from the number of bases (or pairs):

For dsDNA:

Operating manual

For ssDNA, RNA, Oligo:

MM = calculated relative molar mass in kDa bp = entered number of base pairs per molecule b = entered number of bases per molecule

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BioPhotometer plus — Operating manual

12 Ordering information

Operating manual

12 Ordering information

Order No. (International)

6132 000.008 6132 000.016

6131 928.007 952010221

6131 011.006 6131 010.000

0013 021.566 952010409

0030 106.300 952010051

0030 106.318 952010069

4308 078.006 940001102

Order No. (North America)

952000006

952010140

Description

BioPhotometer plus

230 V / 50-60 Hz, power plug Europe, more types of mains connection available 120 V / 50-60 Hz, power plug North America

Secondary UV-VIS-Filter

Test filter set for checking photometric accuracy und wavelength accuracy (according to NIST)

Thermal Printer DPU 414

incl. power supply and printer cable 230 V 115 V

Thermo paper

5 rolls

UVette

individually packaged single cuvettes, directly usable with BioPhotometer, certified RNase-, DNA- and protein-free 80 cuvettes

UVette® routine pack

Eppendorf Quality purity level, reclosable box 200 cuvettes

Cuvette stand

for 16 cuvettes

Page 49

Page 50

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In touch with life

Your local distributor: www .eppendorf.com/worldwide

Eppendorf AG · 22331 Hamburg · Germany · Tel: +49 40 53801-0 · Fax: +49 40 538 01-556 · E-mail: eppendorf@eppendorf.com

Eppendorf North America, Inc. · 102 Motor Parkway · Hauppauge, N.Y. 11788-5178 · USA

Tel: +1 516 334 7500 · Toll free phone: +1 800-645-3050 · Fax: +1 516 334 7506 · E-mail: info@eppendorf.com

Application Support Europe: Tel: +49 1803 666 789 (Preis je nach Tarif im Ausland; 9 ct/min aus dem dt. Festnetz; Mobilfunkhöchstpreis 42 ct/min)

support@eppendorf.com

North America: Tel: +1 800 645 3050 · E-mail: techserv@eppendorf.com

Asia Pacific: Tel: +60 3 8023 6869 · E-mail: support_asiapacific@eppendorf.com

Eppendorf BioPhotometer plus User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

1 User instructions

1.1 Using this manual

1.2 Warning signs and hazard icons

1.3 Symbols used

1.4 Abbreviations used

2 Product description

2.1 Main illustration

2.2 Delivery package

2.3 Features

3.1 Intended use

3.2 Warnings for intended use

3.3 Application limits

3.4 Note on product liability

4 Installation

4.1 Preparing installation

4.2 Selecting location

4.3 Connect device to the main power supply

4.4 Cuvettes

4.5 Connect printer

5 Operation

5.1 Overview of operating controls

5.2 Methods

5.3 Summary of the measuring procedure

5.3.1 Prepare measurement

5.3.2 Select the method

5.3.3 Measure

5.3.4 Finalize the method

5.4 Nucleic acids

5.5 Proteins

5.5.1 Protein 280 nm

5.5.2 Protein after adding reagent (Bradford, BCA, Lowry)

5.6 Methods with evaluation via standards

5.7 OD 600

5.8 Dye-labeled biomolecules ("dye methods")

5.8.1 Method group "dye 550"

5.8.2 Method group "dye 650"

5.8.3 Frequency of incorporation "FOI"

5.8.4 Correction factors

5.8.5 Measuring procedure and result display

5.9 Methods for 340, 405 and 490 nm

5.10 Dilution

5.11 Sample number

6 Parameter and functions

6.1 Parameter

6.1.1 View, change and store the parameters of a method

6.1.2 Summary and description of parameters

6.1.3 Parameters preprogrammed ex factory

6.2 Functions

7 Maintenance

7.1 Cleaning

7.1.1 Cleaning the cuvette shaft cover

7.2 Disinfection / Decontamination

7.3 Decontaminating before shipping

7.4 Replacing fuses

7.5 Check photometer

7.5.1 Test procedure

8 Troubleshooting

8.1 Result flags

8.2 Error messages

8.3 General errors

9 Transport, storage and disposal

9.1 Transport

9.2 Storage

9.3 Disposal

10 Technical data

10.1 Power supply

10.2 Ambient conditions

10.3 Weight / dimensions

10.4 Interfaces

10.5 Photometer

10.6 Other technical parameters

10.7 Application parameters

11 Evaluation procedure

11.1 Evaluation with factor

11.2 Evaluation using standards

11.2.1 Single point calibration