Eppendorf BioPhotometer User Manual

BioPhotometer

Bedienungsanleitung

Operating Manual Mode d'emploi Istruzioni d'impiego Manual de Instrucciones

Bedienungsanleitung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Operating Manual. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Mode d’emploi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Istruzioni d'impiego . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Manual de Instrucciones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

EG-Konformitätserklärung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

EG Conformity Declaration Déclaration de conformité Dichiarazione di conformità CE Declaración de conformidad CEE

Nachdruck und Vervielfältigung – auch auszugsweise – nur mit Genehmigung.

No part of this publication may be reproduced without the prior permission of the copyright owner.

Toute reproduction, complète ou partielle et quel que soit le proèdè est interdiete, sauf autorisation expresse de notre part

Ristampa e riproduzione – anche di estratti – solo con autorizzazione.

Reimpresión y copia – incluso parciales – sólo con autorización.

Copyright♥ 2005 by Eppendorf AG, Hamburg

B 6131 900.102-08/0206

Contents

1	Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	49
2	Technical data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	51
3	Safety precautions and prevention of damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	53
4	Installation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	54
4.1	BioPhotometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	54
4.2	Printer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	55
4.3	Cuvettes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	56
5	Operation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	57
5.1	Keypad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	57
5.2	Measuring nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	59
5.3	Direct photometric measurement of protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	61
5.4	Measuring proteins with reagent (Bradford, BCA, Lowry) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	63
5.5	Measuring OD 600 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	66
5.6	Measuring diluted samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	67
5.7	Changing the sample number. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	68
6	Programming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	69
6.1	Programming procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	69
6.2	Overview of parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	71
6.3	Explanation of parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	72
6.4	Factory-set programmed values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	74
7	Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	75
8	Error messages, result flagging and help texts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	77
9	Maintenance and cleaning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	80
10	Short instructions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	81
11	Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	85
12	Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	86
12.1	Nucleic acids (dsDNA, ssDNA, RNA, oligo) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	86
12.2	Direct photometric determination of protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	87
12.3	Protein with addition of reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	88
12.4	OD 600 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	89
13	Testing the photometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	. .
	Conformity Declaration for BioPhotometer 6131. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	92

47

48

1 Overview

1

8

2

7

6

	3	4	5
1	Device display			5	Dilution key
2	9 method keys			6	Conversion key
3	Function key (device functions)			7	Measuring keys
4	Parameter key (programming key)			8	Cuvette shaft

The main power key, main power connection and printer connection are located on the rear of the device (see Section 4, "Installation").

	The BioPhotometer from Eppendorf is used for rapid, simple and convenient
	measurement of the most common methods in research labs in the fields of molecular
	biology and biochemistry.
Cuvettes	Standard rectangular cuvettes made of glass or plastic that transmit light at every
	measuring wavelength may be inserted into the cuvette shaft. Using the UVette→ from
	Eppendorf, it is now possible to measure nucleic acids in a plastic cuvette.
	The height of the measuring window (8.5 mm), as well as the total height (min. 36 mm)
	must be taken into consideration when the cuvettes are selected (see chap. 10 "Short
	instruction"). To ensure correct, precise results, please ensure that the cuvettes are
	clean and that the measuring solution is particle-free. A seal is included with the device
	to protect the cuvette shaft from dust when not in use.

49

1 Overview

Methods	There are twelve preprogrammed factory-set methods which can be called up at the
	push of a button:
	Nucleic acids

dsDNA

Double-stranded DNA

ssDNA

Single-stranded DNA

RNA

RNA

Oligo

Oligonucleotides

Proteins

Protein

Direct photometric measurement

Bradford

Bradford method

Bradford micro

Bradford method, low concentration range

Lowry

Lowry method

Lowry micro

Lowry method, low concentration range

BCA

BCA method

BCA micro

BCA method, low concentration range

Bacteria density

OD 600

Turbidity measurement

Method

Each method has an accompanying, factory-set program that contains different

parameters, such as units of concentration and type of calculation. The method

programs can be changed at any time using the

Parameter

key. Before using a method for

the first time, call up the corresponding method

and – if necessary – adapt it to

program

suit your requirements. For methods which are to be calculated using calibration by

standard measurements, the number and nominal concentrations of the standards

must be adapted.

Measurement

For measurement purposes, the desired method should be called up using the

appropriate measuring key. The Bradford, Lowry and BCA methods have the same

special feature: For each of these methods, two different calculation ranges may be

programmed. It is possible to toggle between the two method programs (e.g. "BCA"

and "BCA micro") by pressing the method key repeatedly.

Pressing one of the three oval measuring keys starts the measurement. The device is

ready to measure immediately after being switched on. An indication as to which of the

three measuring keys should be used for a measurement can be found in the lower part

of the device display (Details on the measuring process can be found in Section 5,

"Operation").

Calculation

It is possible to calculate the result automatically using method-specific programmed

calculation modes (factor, calibration, Warburg formula or direct absorbance output). In

addition to the calculated results, the absorbances and (for nucleic acids) the common

absorbance ratios appear in the display.

Sample dilutions can also be included in the calculation process (

Dilution

key). The

calculated mass concentrations for nucleic acids can be converted

molar

into

concentrations by pressing the

key. This key can also be used to calculate the

Conversion

total sample quantity ("yield") in

sample vessel.

the

Results printout

The results appear in the device display and can be printed out (if the printer is

connected). A data transfer program is available from Eppendorf for evaluating your

results on a computer using a calculation program (see Sec. 11, "Ordering

information").

Sample results and calibration results are stored; this data can be called up by pressing

the

key.

•

Function

50

2 Technical data

Photometer

Optical system:

Irradiation source: Spectral dispersion: Measuring wavelengths: Wavelength selection: Spectral bandwidth:

Wavelength systematic error:

Wavelength random error: Photometric measuring range:

Photometric random error:

Photometric systematic error: Accuracy of reading: Stray-light proportion: Radiation detector:

Measuring procedures

Measuring procedure:

Method-dependent calculation:

Memory

Method memory:

Calibration memory:

Results memory:

Absorption single-beam photometer with reference beam and several fixed wavelengths

Xenon flash lamp

Holographic concave grating

Xe 230, 260, 280, 320, 562, 595 nm

Method-dependent, program-controlled

5 nm at 230 to 320 nm

7 nm at 562 to 595 nm

±1 nm at 230 to 280 nm

±2 nm at 320 to 595 nm

≤ 0.1 nm

Quartz glass cuvette: 0.000 to 3.000 A

UVette→ (Eppendorf): 2.5 A at 230 nm 2.6 A at 260 nm

2.8A at 280 nm

2.9A at 320 nm

≤0.002 A at 0 A

≤0.005 A at 1 A

± 1 % at 1 A

0.001 A

< 0.05 %

Silicium photo diodes

End-point against blank

Absorbance Concentration via factor

Concentration via Warburg formula

Concentration via calibration with 1 to 10 standards One-point calibration (1 standard)

Linear regression (2 to 10 standards) Non-linear regression

(3rd degree polynomer; 4 or 5 to 10 standards; see Section 12, "Calculation")

1 x, 2 x or 3 x determination

For nucleic acids:

Ratio 260/280

Ratio 260/230

Molar concentration

Total yield

12 preprogrammed, modifiable method programs

For all calibration procedures

For 100 results with absorbance and ratio values,

sample number, sample dilution, date and time (calendar up to 2090)

51

2 Technical data

Operation

Cuvette material:

Cuvette shaft:

Overall height of cuvettes:

Height of light beams in the cuvette:

Light bundle in the cuvette:

Keypad:

Display:

User guidance:

Results output:

General data

Supply voltage:

Overvoltage category:

Pollution degree:

Power requirement / power output:

Current consumption:

Permitted mains interruption:

Fuses:

Ambient conditions:

Printer connection:

Standards and regulations:

Dimensions:

Weight:

dsDNA, ssDNA, RNA, Oligo, Protein: Quartz glass or plastic
	(UVette→ from Eppendorf)
OD 600, Bradford, Lowry, BCA:	Glass or plastic

12.5 mm x 12.5 mm, not temperature-controlled

Min. 36 mm

8.5 mm

Width: 1 mm

Height: 1.5 mm

19 foil keys

Illuminated graphic display, 33 mm x 60 mm

English, French, German

Via display and printer

Absorbance, concentration, ratio

100 to 240 V ± 10 %; 50 to 60 Hz ± 5 %

II (IEC 61010-1)

2 (IEC 664)

Approx. 20 W in operation, approx. 10 W in Standby mode

< 0.3 A

Approx. 10 ms at 90 V

Approx. 200 ms at 220 V

T 1 A / 250 V, 5 mm x 20 mm (2 pcs.)

15 to 35 °C with defined precision and accuracy –25 to 70 °C when not in operation or when stored 15 to 70 % relative humidity

Cannot be used in tropical climate Keep out of direct sunlight

RS-232 C, serial, data format: 1 start bit, 8 data bits, no parity, 1 stop bit, 9600 Baud

The printer that is connected must comply with the requirements of EN 60950 or UL 1950.

Complies with VDE, CE, IEC 1010-1

Width:	20 cm	(packaged:	29 cm)
Depth:	32 cm	(packaged:	43 cm)
Height:	10 cm	(packaged:	20 cm)
3 kg		(packaged:	4,8 kg)

Technical specifications subject to change.

52

3 Safety precautions and prevention of damage

Before using the Biophotometer please familiarize yourself completely with the operating instructions.The following points must be followed exactly to enable safe work with the device:

Technical safety

–Do not open the device.

–Do not allow any liquid to enter into the device.

–Disconnect the device from the mains supply before carrying out maintenance work or changing the fuses.

The inside of the device is a high-voltage area. Danger!

–Do not operate the device in a hazardous location or potentially explosive environment.

–Do not use the device if it is damaged, especially if the main power cable is in any way damaged or defective.

–Repairs may only be carried out by the service technicians from Eppendorf AG and by authorized contractual partners.

–The device must be connected to a power outlet that has a protective ground connection.

–If the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired.

Handling biological and chemical material

–Reagents and dilution buffers can cause cauterization and other damage to health.

–Samples (nucleic acids, proteins, bacteria cultures) can be infectious and cause serious damage to health.

–During sample preparation, measuring procedures and maintenance and cleaning work, observe all local laboratory safety precautions (e.g. wear protective clothing and gloves, use of disinfectant) regarding the handling of sample material.

–Dispose of measuring solutions and cleaning and disinfectant materials in accordance with the relevant local laboratory regulations.

Transfer

– If the device is passed on to someone else, please include the instruction manual.

Disposal

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

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

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

–According to these regulations, any devices supplied after 13.08.05 in the business- to-business sphere, to which this product is assigned, may no longer be disposed of in municipal or domestic waste. They are marked with the following symbol to indicate this.

–				As disposal regulations within the EU may vary from country to country,
				please contact your supplier if necessary.

53

4 Installation

Delivery package

– BioPhotometer

–Mains cable for BioPhotometer

–Operating manual, incl. short instructions

–Seal for cuvette shaft

4.1BioPhotometer

Connect up	Space required:	Width:	40 cm
device		Depth:	50 cm
	Power connection:	Safety socket

–Insert the mains plug of the device into the safety socket.

It is not necessary to set voltage of the device within the voltage range specified in "Technical data" because the voltage is set automatically within this range.

Ambient conditions:

see "Technical data".

– Remove the protective foil from the device display.

1

2

	3	4
1	Mains switch	4 Printer connection, serial
2	Fuse holder	(RS-232 C)
3	Mains connection

54

4 Installation

4.2 Printer

Printer DPU 414 The Eppendorf Thermal Printer DPU 414 can be connected to the serial interface RS-232 C of the BioPhotometer (see Section 11, "Ordering information").

–Insert the printer cable into the printer connection socket of the BioPhotometer (see photo) and tighten the safety screws on the plug to secure.

–Connect the printer cable to the printer and tighten the safety screws on the plug to secure.

–Connect up to the power supply using a 115 V or 230 V mains cable.

Setting the	BioPhotometer
printer function	– Select the function "Printer DPU 414" in the function list, and confirm.

Printer DPU 414

–Check the printer settings. If necessary, set the printer for use with the BioPhotometer, as described in the printer supplement.

Printer settings for working with the BioPhotometer:

Dip SW-1
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

Settings made by the user are not relevant for the group "Dip SW-2" because the BioPhotometer assumes these settings automatically in accordance with the language version selected.

Dip SW-3
1	(ON)	: Data Length = 8 bits
2	(ON)	: Parity Settings = No
3	(ON)	: Parity Conditions = Odd
4	(OFF)	: Busy Control = XON/XOFF
5	(OFF)	: Baud
6	(ON)	:	Rate
7	(ON)	:	Select
8	(ON)	:	=9600 bps

55

4 Installation

Other printers In addition to the DPU 414, it is also possible to connect other serial printers to the serial interface of the BioPhotometer. With the aid of an adapter cable, parallel printers can also be connected.

BioPhotometer

– Select the function "Printer serial" in the functions list, and confirm.

Printer

Requirements for the serial printer:

Busy Control	:	XON/XOFF
Baud Rate(ON)	:	9600 bps
Data Bit Length	:	8 bits
Parity Permission	:	Without
Parity Conditions	:	Odd

Parallel printers can be connected using an adapter cable which fulfills the above requirements.

4.3 Cuvettes

Commercially available rectangular cuvettes may be used in the cuvette shaft. When the height of the measuring window is 8.5 mm above the cuvette base and the overall height of the cuvette is at least 36 mm (see the graphics in "Short instructions").

The light bundle in the cuvette is 1.0 mm wide and 1.5 mm high.

For measurements, cuvettes made of glass or plastic may be used on condition that they are transparent at the respective measuring wavelength. The UVette→ from Eppendorf is a plastic cuvette which is transparent at wavelengths as low as 220 nm, which means that it is also suitable for nucleic acid measurement.

56

5 Operation

5.1 Keypad

Photometer

				Standard
	7	8	9	Blank
	dsDNA	ssDNA	RNA
	4	5	6	Sample
	Protein	OD 600	Oligo
	1	2	3	Clear
	Bradford	Lowry	BCA
				Conversion

0•

Sample No.	Parameter	Enter
	Function	Dilution

7

dsDNA

8

ssDNA

9

RNA

6

Oligo

4

Protein

1 Bradford

2

Lowry

–To call up the "Double-stranded DNA" method.

–To enter figure 7.

–To call up the "Single-stranded DNA" method.

–To enter figure 8.

–To call up the "RNA" method.

–To enter figure 9.

–To call up the "Oligonucleotide" method.

–To enter figure 6.

–To call up the "Protein (direct photometric measurement)" method.

–To enter figure 4.

–To call up the "Bradford" and "Bradford micro" methods.

–To switch between the "Bradford" and "Bradford micro" methods.

–To enter figure 1.

–To call up the "Lowry" and "Lowry micro" methods.

–To switch between the "Lowry" and "Lowry micro" methods.

–To enter figure 2.

57

5 Operation

3– To call up the "BCA" and "BCA micro" methods.

BCA	–	To switch between the "BCA" and "BCA micro" methods.
	–	To enter figure 3.

5– To call up the "OD 600 (measuring the bacteria density)" method.

			OD 600			– To enter figure 5.
						– To call up the programming level.
Parameter
						– To exit the programming level.
			•			–	To call up the function level.
Function						–	To exit the function level.
						–	To enter a point.
		0				– To change the sample number.
Sample No.						– To enter figure 0.
						–	To enter the dilution.
			Dilution			– To move the cursor to the next line.
							(e.g. in the parameter list or function list).
						– To calculate the molar concentration and the total amount of sample ("yield").
Conversion						– To move the cursor to the previous line.
							(e.g. in the parameter list or function list).
						–	To delete entries.
			Clear

Enter		– To confirm entries.
		– To measure a standard.
Standard
		– To measure a blank.
Blank
		– To measure a sample.
Sample

58