Eppendorf BioPhotometer User Manual

BioPhotometer

Bedienungsanleitung

Operating Manual

Mode d'emploi

Istruzioni d'impiego

Manual de Instrucciones

Bedienungsanleitung

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Operating Manual

Mode d’emploi

Istruzioni d'impiego

Manual de Instrucciones

EG-Konformitätserklärung

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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

2 Technical data

3 Safety precautions and prevention of damage

4 Installation

4.1 BioPhotometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4.2 Printer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

4.3 Cuvettes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

5 Operation

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

6.1 Programming procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

6.2 Overview of parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

6.3 Explanation of parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

6.4 Factory-set programmed values. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

7 Functions

8 Error messages, result flagging and help texts

9 Maintenance and cleaning

10 Short instructions

11 Ordering information

12 Calculation

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

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

47

48

1 Overview

1

2

4

5

8

6

3

7

1 Overview

1 Device display 5
2 9 method keys 6
3

Function

4

Cuvettes

Parameter

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.

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.

key (device functions) 7 Measuring keys

key (programming key) 8 Cuvette shaft

Dilution
Conversion

key

key

49

1 Overview

Methods

Method

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

Nucleic acids

dsDNA
ssDNA
RNA
Oligo

Double-stranded DNA
Single-stranded DNA
RNA
Oligonucleotides

Proteins

Protein
Bradford
Bradford micro
Lowry
Lowry micro
BCA
BCA micro

Direct photometric measurement
Bradford method
Bradford method, low concentration range
Lowry method
Lowry method, low concentration range
BCA method
BCA method, low concentration range

Bacteria density

OD 600

Turbidity measurement

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 program and – if necessary – adapt it to
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

Calculation

Results printout

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").

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 ( key). The
calculated mass concentrations for nucleic acids can be converted into molar

concentrations by pressing the key. This key can also be used to calculate the



Conversion

Dilution

total sample quantity ("yield") in the sample vessel.

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

2 Technical data

Photometer

Optical system: Absorption single-beam photometer with reference beam

and several fixed wavelengths

Irradiation source: Xenon flash lamp

Spectral dispersion: Holographic concave grating

Measuring wavelengths: Xe 230, 260, 280, 320, 562, 595 nm

Wavelength selection: Method-dependent, program-controlled

Spectral bandwidth: 5 nm at 230 to 320 nm

7 nm at 562 to 595 nm

Wavelength systematic error:

Wavelength random error:

Photometric measuring range: Quartz glass cuvette: 0.000 to 3.000 A

Photometric random error:

Photometric systematic error:

Accuracy of reading: 0.001 A

Stray-light proportion: < 0.05 %

Radiation detector: Silicium photo diodes

±

1 nm at 230 to 280 nm

±

2 nm at 320 to 595 nm

≤

0.1 nm

UVette® (Eppendorf): 2.5 A at 230 nm

2.6 A at 260 nm

2.8 A at 280 nm

2.9 A at 320 nm

≤

0.002 A at 0 A

≤

0.005 A at 1 A

±

1 % at 1 A

Measuring procedures

Measuring procedure: End-point against blank

Method-dependent
calculation: 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

Memory

Method memory: 12 preprogrammed, modifiable method programs

Calibration memory: For all calibration procedures

Results memory: 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: dsDNA, ssDNA, RNA, Oligo, Protein: Quartz glass or plastic

(UVette® from Eppendorf)

OD 600, Bradford, Lowry, BCA: Glass or plastic

Cuvette shaft: 12.5 mm x 12.5 mm, not temperature-controlled

Overall height of cuvettes: Min. 36 mm

Height of light beams
in the cuvette: 8.5 mm

Light bundle in the cuvette: Width: 1 mm

Height: 1.5 mm

Keypad: 19 foil keys

Display: Illuminated graphic display, 33 mm x 60 mm

User guidance: English, French, German

Results output: Via display and printer

Absorbance, concentration, ratio

General data

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

Overvoltage category:

Pollution degree: 2 (IEC 664)

Power requirement /
power output: Approx. 20 W in operation, approx. 10 W in Standby mode

Current consumption: < 0.3 A

Permitted mains interruption: Approx. 10 ms at 90 V

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

Ambient conditions: 15 to 35 °C with defined precision and accuracy

Printer connection: RS-232 C, serial, data format: 1 start bit, 8 data bits, no parity,

Standards and regulations: Complies with VDE, CE, IEC 1010-1

Dimensions: Width: 20 cm (packaged: 29 cm)

Weight: 3 kg (packaged: 4,8 kg)

II

(IEC 61010-1)

Approx. 200 ms at 220 V

–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

1 stop bit, 9600 Baud
The printer that is connected must comply with the requirements
of EN 60950 or UL 1950.

Depth: 32 cm (packaged: 43 cm)
Height: 10 cm (packaged: 20 cm)

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

4 Installation

Delivery package

– BioPhotometer
– Mains cable for BioPhotometer

– Operating manual, incl. short instructions
– Seal for cuvette shaft

4.1 BioPhotometer

Connect up
device

Space required: Width: 40 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.

Depth: 50 cm

1

2

3

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

4

54

4 Installation

4.2 Printer

Printer DPU 414

Setting the BioPhotometer
printer function

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.

– 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

4.3 Cuvettes

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.

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



Conversion



Dilution

Enter

Clear

Standard

Blank

Sample

7

dsDNA

4

Protein

1

Bradford

0

Sample No.

8

ssDNA

5

OD 600

2

Lowry

•

Function

9

RNA

6

Oligo

3

BCA

Parameter

Photometer

7

dsDNA

8

ssDNA

9

RNA

6

Oligo

4

Protein

1

Bradford

2

Lowry

5 Operation

5.1 Keypad

– 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

BCA

5

OD 600

Parameter

•

Function

0

Sample No.

Dilution

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

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

– To call up the programming level.
– To exit the programming level.

– To call up the function level.
– To exit the function level.
– To enter a point.

– To change the sample number.
– To enter figure 0.

– To enter the dilution.
– To move the cursor to the next line.

(e.g. in the parameter list or function list).



Conversion

Clear

Enter

Standard

Blank

Sample

– To calculate the molar concentration and the total amount of sample ("yield").
– To move the cursor to the previous line.

(e.g. in the parameter list or function list).

– To delete entries.

– To confirm entries.

– To measure a standard.

– To measure a blank.

– To measure a sample.

58

5 Operation

5.2 Measuring nucleic acids

This description is valid for the following methods:

– dsDNA
– ssDNA
– RNA
– Oligo

Call up method

7

dsDNA

dsDNA

PROGRAMMED FACTOR:
1 A260 = 50.0 µg/mL

Blank

Sample

or

Calculation

The factory-set factors are those which are normally used
with nucleic-acid methods for the conversion of
UV absorbance into concentration (in this example: 50). The
factors can be changed using the

Parameter

key (see
"Programming"). The number of decimal places of the result
is determined by the number of decimal places of the
programmed factor.

If a unit of concentration other than µg/mL is selected (e.g.

µg/µ

L), the BioPhotometer converts the factor internally in

order to produce the correct result.

Measuring solutions with absorbances lower than app. 0.02
to 0.03 A

260

(corresponds to a DNA concentration of app. 1.0
to 1.5 µg/mL) should not be used. This is because with such
low absorbance, disturbances such as small particles, micro­bubbles or turbidity have a great deal of influence upon the
measuring result and often lead to unreliable results.

Measure blank

Blank

Measuring procedure

Blank measurements remain stored until the date changes.
If a blank has already been measured on the same day, the
BioPhotometer offers the following in the last line of the
display after method call-up:
– To measure a new blank

or

– To measure a sample directly and to use the stored blank.

If no blank has been measured on the same day, the
instrument will only allow blank measurement.

dsDNA BLANK

0 . 0 0 0 A

Blank

Sample

or

59

5 Operation

Enter

Measure sample

Measure
next sample

Sample

dsDNA SAMPLE 001

7 0 . 0 µg/mL

1.408 A260

1.97 260/280 0.715 A280

2.03 260/230 0.002 A320

Results display

As an indication of the purity of the nucleic acid sample which
has been measured, the absorbance at 230, 280 and 320 nm
as well as the ratios A
addition to the concentration result and the absorbance at a
wavelength of 260 nm. With pure samples, the absorbance at
320 nm should be approximately zero.

Turbid measuring solutions show increased absorbances for
all wavelengths. These can adulterate the results. In such
cases the influence on the result can be partially corrected by
switching on the "Corr. with A
"Explanation of parameters")

To measure the next sample, press the key again.

0.694 A230

260/A280

and A

260/A230

320

" parameter. (Chapter 6.3

are displayed in

Sample

Sample dilution

Conversion key



Conversion

The sample dilution in the measuring cuvette can be entered
using the key before the measurement starts and is

Dilution

included automatically in the result calculation (see
"Measuring diluted samples").

The most-recently measured concentration result can be
converted into molar concentrations and/or into nucleic acid
quantities (unit of mass or unit of mol):

CALC. AMOUNT:
TOTAL SAMPLE– – – – µ L

CALC. MOLARITY:
BASE PAIRS – – – –
MOL.MASS – – – – kDa

Entering "TOTAL SAMPLE"

The value entered is converted using the concentration
measured. The result shown is the quantity of nucleic acid
present in the sample.

Entering "BASE PAIRS" or "MOL.MASS"

It is sufficient to make an entry in only one of the two lines.
The molar concentration is calculated using the value
entered and the concentration measured.

Input fields can be skipped using the key.

60

5 Operation

Parameter

1

Bradford

Protein

Sample No.

4

0

Enter

3

BCA

0

Sample No.

0

Sample No.

Enter

Enter

Display after entry of "140 µL sample volume" and "300 base
pairs":

dsDNA SAMPLE 001

7 0 . 0 µg/mL

9.8 µ g

353.5 pmol/mL

49.5 pmol

The molar unit of concentration (here: "pmol/mL") is
preprogrammed, but can be selected and changed using the

key.

Parameter

5.3 Direct photometric measurement of protein

Call up method

4

Protein

PROTEIN

ABSORBANCE

Blank

Sample

or

Calculation

For the "protein" method, the "absorbance" calculation mode
is stored, i.e. only the absorbances which are measured
directly appear in the display. Calculations via the following
calculation procedures can be programmed using the
key (see Section 6 "Programming"):

– Factor
– Standard (One-point calibration)
– Warburg formula

The number of decimal places of the preprogrammed factor
or the preprogrammed nominal concentration of the standard
determines the number of decimal places in the result.

When programming the factor, please ensure that the factor
is adapted in line with the unit of concentration selected.

Measuring solutions with absorbances lower than app. 0.02
to 0.03 A

corresponds to a DNA concentration of app. 1.0

280

to 1.5 µg/mL should not be used. This is because with such
low absorbances, disturbances such as small particles,
microbubbles or turbidity have a great deal of influence upon
the measuring result and often lead to unreliable results.

61

5 Operation

Measuring procedure

The following example shows the measuring procedure
for the "Absorbance" calculation mode. For details of the
measuring procedure via standard (one-point calibration),
please refer to "Measuring proteins with reagent".

Blank measurements remain stored until the date changes
(For further details, please refer to "Measuring nucleic
acids").

Measure blank

Measure sample

Blank

Sample

PROTEIN BLANK

0 . 0 0 0 A

Blank

PROTEIN SAMPLE 001

Sample

or

0 . 4 3 0 A

0.245 A260

Results display

In addition to the concentration result and the absorbance at
the measuring wavelength of 280 nm, A
in the display as an indication of the purity of the sample. The
absorbance at 320 nm should be approximately zero.

Turbid measuring solutions show increased absorbances for
all wavelengths. These can adulterate the results. In such
cases the influence on the result can be partially corrected by
switching on the "Corr. with A
"Explanation of parameters")

0.430 A280

0.002 A320

260

and A

320

320

" parameter. (Chapter 6.3

appear

Measure
next sample

Sample dilution

62

To measure the next sample, press the key again.

Sample

Sample dilution in the measuring cuvette can be entered
using the key before the measurement begins and

Dilution

is then included automatically in the following calculation of
sample concentrations (see "Measuring diluted samples").

5 Operation

5.4 Measuring proteins with reagent (Bradford, BCA, Lowry)

Call up method

1

Bradford

BRADFORD

CALIBRATION RANGE
XXX – XXX µ g/mL

Blank

Standard

or

If a valid calibration (which is then stored by the device) has already
been performed, the date and time of the stored calibration appear.
In this case, the method can be recalibrated after blank
measurement or the sample measurements may begin directly and
can then be calculated using the previously stored calibration.

Micro methods

The Bradford, Lowry and BCA methods have a special feature:
Two different concentration ranges may be programmed for each
of these methods. It is possible to toggle between the two methods
(e.g. "BCA" and "BCA micro") by pressing the method key
repeatedly.

Calculation

For the Bradford, Lowry and BCA methods, the device contains a
factory-set calibration procedure via multiple-point calibration and
calculation of a calibration curve via non-linear regression. Other
calculation methods may be programmed using the key

Parameter

(see Section 6 "Programming"):

– Factor (calculation of concentration values via factor).

– Absorbance (the measured values appear as absorbance

values with no further calculation).

The following parameters may be changed for the factory-set
calculation procedure via standard (see "Programming").

– Number of standards (1 to 10).

– Number of multiple measurements per standard (1 to 3).

– Calculation procedure for multiple-point calibration (linear or

non-linear calibration).

– Nominal concentrations of the standards.

The number of decimal places of the preprogrammed factor or the
preprogrammed nominal concentration of the standard determines
the number of decimal places in the result.

In the case of calculation via factor, please ensure that the factor is
adapted in line with the unit of concentration selected.

63

5 Operation

Measuring procedure

Blank measurements remain stored until the date changes
(For further details, see "Measuring nucleic acids").

Standard measurements remain stored until they are overwritten
with new standard measurements. For the calculation of sample
measurements, the most-recently stored calibration is used.

In the following example, multiple-point calibration with 5 standards
in double determination and calibration calculation via non-linear
regression was programmed as a calculation procedure for the
Bradford method:

Measure blank

Measure
standards

Blank

Standard

Standard

BRADFORD BLANK

0 . 0 0 0 A

Blank

Sample

BRADFORD STD 1–1
XXXX µ g/mL

X . X X X A

NEXT: STD 1–2

The first two lines of the display contain the standard that has just
been measured. The last two lines of the display contain the next
standard which is to be measured, with the nominal concentration.

BRADFORD STD 1–2
XXXX µ g/mL

X . X X X A

Standard

or

XXXX µg/mL

Standard 1 /
first measurement

Standard 1 /
second measurement

64

NEXT: STD 2–1

XXXX µg/mL

5 Operation

Device display following all standard measurements:

BRADFORD STD 5–2
XXXX µ g/mL

X . X X X A

CV: 2.8%

CALIBRATION STORED

The CV (coefficient of variation) is a measure of the scattering of
standard values around the regression curve. If the CV is smaller
than 10 %, the calibration is stored automatically. If the CV is
greater than 10 %, the question "STORE? ENT/CLR" appears, and
you may then accept or delete the calculated calibration. Sample
measurements are calculated using the most-recent valid
calibration.

Measure sample

Measure
next sample

Sample dilution

Sample

BRADFORD SAMPLE 001

X . X X X µg/mL

X.XXX A595

Results display

In addition to the concentration result, the absorbance at the
respective wavelength (for Bradford: 595 nm) appears in the
display.

To measure the next sample, press the key again.

Sample dilution in the measuring cuvette can be entered using the

key before the measurement begins and is then included

Dilution

automatically in the result calculation (see "Measuring diluted
samples").

Sample

65

5 Operation

5.5 Measuring OD 600

Select method

Measure blank

Measure sample

5

OD 600

Blank

Sample

OD600

Blank

Sample

or

Measuring procedure

Blank measurements remain stored until the date changes
(for further details, see "Measuring nucleic acids").

OD600 BLANK

0 . 0 0 0 A

Blank

OD600 SAMPLE 001

Sample

or

0 . 4 3 0 A

Measure
next sample

Sample dilution

To measure the next sample, press the key again.

Sample

Sample dilution in the measuring cuvette can be entered
using the key before the measurement begins and

Dilution

is then included automatically in the result calculation
(see "Measuring diluted samples").

The OD 600 measurement is a stray-light measurement;
the result is therefore heavily dependent on the geometry
of the light path, which may vary between photometers
from different manufacturers.

66

5 Operation

5.6 Measuring diluted samples

Sample dilutions may be entered using the key before the measurement begins. When the result is

Dilution

calculated and displayed, the dilution factor is included automatically.

In the following example, a blank has already been measured:

dsDNA BLANK

0 . 0 0 0 A

Sample

or

Sample

or

Enter
dilution

2

Lowry

Sample No.

0

Enter

Dilution

1

Bradford

ssDNA

Sample No.

Blank

dsDNA SAMPLE 001

SAMPLE+DILUENT
– – – + – – – – µL

dsDNA SAMPLE 001

8

20+180µL

0

Blank

Enter

Measure
diluted sample

Deleting
dilution entry

Sample

dsDNA SAMPLE 001

7 0 0 . 0 µg/mL

20+180µL 0.694 A230

1.408 A260

1.97 260/280 0.715 A280

2.03 260/230 0.002 A320

The sample dilution is included in the result. The dilution
factor entered remains stored for the calculation of further
sample results until it is overwritten.

To delete the dilution factor, press the key again. The
values for "Sample" and "Diluent" are then deleted using the

Clear

key or are overwritten with "zero".

Dilution

67

5 Operation

5.7 Changing the sample number

During sample measurements, the serial number of the sample appears in the top right of the display.
The sample number is counted separately for each method and is reset to "1" when the date changes.

The sample number can be changed as desired (e.g. for repeat measurements):

dsDNA SAMPLE 005

7 0 . 0 µg/mL

2+180µL 0.694 A230

1.408 A260

1.97 260/280 0.715 A280

2.03 260/230 0.002 A320

Change
sample number

0

Sample No.

3

BCA

Enter

dsDNA SAMPLE 005

dsDNA SAMPLE 003

Blank

Sample

or

For the next sample to be measured, the sample number was
set to "3". Additional samples are counted serially from the
newly-entered number onwards.

68

6 Programming

Parameter

Enter

6 Programming

6.1 Programming procedure

For each method, parameters such as the type of calculation or the unit of concentration are stored. The
factory-set method programs can be changed using the key.

Call up method

Call up
parameter list

6

Oligo

Parameter

OLIGO

PROGRAMMED FACTOR:
1 A260 = 30.0 µg/mL

Blank

OLIGO PAGE 1–3

▲

FACTOR 30.0

CORR. WITH A320 OFF *

. . . . . . . . . . . . ON –

Sample

or

There are different parameter lists for the various different
methods, all of which can be modified (see Section 6.2 for
overview). The parameters for the "Oligo" methods extend
across three pages of the device display.

Example: Changing the factor

Any numbers that are entered are stored by pressing

:

Enter factor and
store

2

Lowry

0

Sample No.

Function

Enter

•

0

Sample No.

OLIGO PAGE 1–3

FACTOR 20.0

▲

CORR. WITH A320 OFF *

. . . . . . . . . . . . ON –

After the factor has been stored, the cursor moves to the next
parameter-selection block ("Correction with A

320

").

69

6 Programming

Example: Changing the unit

Selection parameters are selected using the cursor keys and
confirmed by pressing . The stored setting is marked

Enter

with an asterisk (*):

Select
parameter

Store
parameter

Exit
parameter level

Dilution

Enter

OLIGO PAGE 2–3

UNIT µg/mL *

. . . . . . . ng /µ L –

▲

. . . . . . . . . . µ g/µL –

M. UNIT pmol/µ L *

. . . . . . . . . . µmol/L –

OLIGO PAGE 2–3

UNIT µ g/mL –

. . . . . . . ng /µ L –

. . . . . . . . . . µg/µL *

▲

M. UNIT pmol/µ L *

. . . . . . . . . . µmol/L –

After the unit of concentration"µg/µL" has been stored, the
cursor moves to the next selection block ("molar unit").

To exit the parameter level, select the line "PARAMETER
END" and press

Enter

. Alternatively, press the key

Parameter

from any parameter line.

Parameter

OLIGO

PROGRAMMED FACTOR:
1 A

260 = 20.0 µg/mL

Blank

or

Sample

70

6 Programming

6.2 Overview of parameters

Calculation

Correction
with A

320

Unit

Molar unit

Cuvette

dsDNA

Oligo Protein Bradford
ssDNA
RNA

(Point 1) (Point 1)

Off
On

µ

g/mL

ng/µL

µg/µ

L

pmol/µL

µ

mol/L

pmol/mL

10 mm
5 mm
2 mm
1 mm

Off

On

µ

g/mL

ng/µL

µg/µ

L

pmol/µL

µ

mol/L

10 mm

5 mm

2 mm

1 mm

Absorbance
Standard
Factor
Warburg formula

Off
On

mg/mL

µ

g/mL

10 mm
5 mm
2 mm
1 mm

Brad.micro
Lowry
Low.micro
BCA
BCA micro

Absorbance
Standard
Factor

mg/mL

µ

g/mL

µ

g

10 mm
5 mm
2 mm
1 mm

OD 600

(Point 1)

(Point 2)

10 mm
5 mm
2 mm
1 mm

(For "Factor" calculation only:)

Factor

(For "Standard" calculation only:)

No. of
standards

Std.
measurement

Regression

(Point 4)

Standard

Point 1: No selection possible; "Factor" calculation is preprogrammed

Entry of
numbers

Entry of

numbers

Entry of
numbers

(Point 3)

1x
2x
3x

Entry of
numbers

Entry of
numbers

Entry of
numbers

1x
2x
3x

Linear
Non-linear

Entry of
numbers

.

Entry of
numbers

Point 2: No selection possible; the unit "Absorbance" is preprogrammed.
Point 3: No selection possible; the number of standards "1" is preprogrammed

.

Point 4: Selection possible only if at least "4" (or, for the single determination of the standard,

at least "5") has been entered for the "Std. number" parameter

.

71

6 Programming



Conversion

6.3 Explanation of parameters

Parameters are defined as selection parameters or as parameters for entering numbers. In the case of
selection parameters, the programmable alternatives are method-dependent (see overview in previous
section).

Parameter Entries Explanation

Calculation Selection Selection of calculation procedures: Absorbance, Factor,

Standard and Warburg formula.
In the case of calculation using the Warburg formula, the
measured value for A
and on the results printout with a " ".

is marked in the results display

260

▲

Factor Entry of numbers

(five-figure)

(Only when the calculation process "Factor" has been
selected)
Entering a factor;
the number of decimal places determines the number of
decimal places in the result.

Corr. with A

320

Selection (Only for nucleic acid methods and for the direct photometric

determination of protein)
Selection from "Corr. with A

off" and "Corr. with A

320

320

on";
"Corr. on" means: the absorbance measured at 320 nm is
subtracted from the absorbance results at 260, 280 and
230 nm. Example of application: Correction of turbidity in the
sample.
When the correction function is switched on, the measuring
value for A

is marked with a " " in the results display and

320

▲

on the results printout.

Unit Selection The selection from preprogrammed concentration units is

method-dependent.

M. unit
(molar unit)

Selection Selection is method-dependent

(for nucleic acid measurements only);
is required for the conversion of the concentration into molar
concentrations ( key).

Cuvette Selection Selection from 10 mm, 5 mm, 2 mm and 1 mm optical path

length; the result is converted for an optical path length of
10 mm (see Section 12 "Calculation").

72

6 Programming

The following parameters are offered only when the "Standard" calculation procedure has been programmed:

Parameter Entries Explanation

Std. number Entry of numbers

("1" to "10")

Std. measurement Selection Selection from "1x", "2x", "3x" repeat measurement of each

Regression Selection (Only for standard number of at least 4 (for single

Std. 1 to Std. 10 Entry of numbers

(five-figure)

Number of different standards.

standard; a mean value is formed for the further calculation
using the repeat measurements.

determination of standards: 5))
Selection from the calculation procedure linear and non-linear
regression.
For a number of standards greater than 1 and lower than
4 (or 5 respectively), calculation always takes place via linear
regression (See Section 12, "Calculation").

Entry of nominal values of standard concentrations;
the number of decimal places of the nominal concentration for
the first standard determines the number of decimal places in
the result.

73

6 Programming

OD 600

micro

BCA BCA

micro

Lowry Lowry

micro

1.000

1)

- - - - -

1)

- - - - -

1)

- - - - -

1)

- - - - -

1)

- - - - -

1)

- - - - -

Standard Standard Standard Standard Standard Standard

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

non-linear non-linear non-linear non-linear non-linear non-linear

g/mL

µ

g/mL

µ

g/mL

µ

g/mL

µ

g/mL

µ

g/mL

µ

100 1.00 100 1.00 25 0.50

dsDNA ssDNA RNA Oligo Protein Bradford Bradford

1)

Absorbance

off off off off off

320

4)

3)

2)

g/mL

µ

L

L

µ

µ

g/

µ

g/mL

µ

g/mL

µ

g/mL

µ

2) With "Standard" calculation

3) With "Standard" or "Factor" calculation

4) With "Standard" calculation: Input required from user

74

6.4 Factory-set programmed values

Calculation

Factor 50.0 37.0 40.0 30.0 - - - - -

Corr. with A

Unit

Molar unit pmol/mL pmol/mL pmol/mL pmol/

Standard 1 - - - - -

Standard 2 250 2.5 250 2.5 125 2

Standard 3 500 5 500 5 250 5

Standard 4 750 10 750 10 500 10

Standard 5 1000 15 1000 15 750 20

Standard 6 1500 25 1500 25 1000

Standard 7 1500

Standard 8 2000

Std. number 6 6 6 6 8 5

Std. measurmnt. 1x

Regression

Cuvette 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm

Notes: 1) With"Factor" calculation: Input required from user

7 Functions

Enter

•

Function

Enter

•

Function

Enter

Enter

7 Functions

Functions list

Function Entries Explanation

Display results Call up using . Display of the last 100 results

Enter

(The most-recent result appears first):



Conversion

: To select the results.

Dilution

: To print out the results that have just

been displayed.

: To return to the functions list.

Calibration report Call up using . Printout of the calibrations stored;

Enter



Conversion

: To select the method.

Dilution

: To print out the calibration report.

: To return to the functions list.

Date Entry of figures : To store.

Time Entry of figures : To store.

Stored absorbance Call up using . To print out the most-recently measured absorbances

Enter

(max. 100 measurements). Mean value, standard
deviation and CV are calculated and printed out for the
values of the most-recently measured method.

Precision measurement Call up using . To perform measurement and precision calculation of

Enter

ten consecutive measuring values of one sample. For
evaluation purposes, the method program of the most­recently selected method method is used.

Photometer test Call up using . To check the photometric accuracy and the wavelength

Enter

accuracy (see Sec. 13, "Testing the photometer").

Sprache Deutsch
Language English
Language U.S.English

Selection Selection of language version;

Please note that "English" and "U.S.English" differ due to
the format of the date.

langue française

Printer DPU 414
Printer serial

Selection DPU 414: To connect the Eppendorf thermal printer

DPU 414 (see Section 4.2, "Printer").

serial: To connect another printer

(see Section 4.2, "Printer").

Service Function is accessible to service technicians only.

75

7 Functions

Example: Changing the language version

Call up
functions list

Select
desired function

Store
function

•

Function

Dilution

Enter

FUNKTION SEITE 1–4

▲

ERGEBNISSE ANZEIGEN
KALIBRATIONS REPORT

DATUM 27.06.1998
UHRZEIT 20:44

FUNKTION SEITE 3–4

SPRACHE DEUTSCH *

▲

LANGUAGE ENGLISH –
LANGUAGE U.S.ENGL –
langue française –

FUNCTION PAGE 4–4

▲

PRINTER DPU 414 *
PRINTER SERIAL –

SERVICE – – – –

FUNCTION EXIT

Exit
function level

•

Function

To exit the function level, either select the line "FUNCTION
EXIT" and press or press the

Enter

•

key from any line

Function

of the functions list. The BioPhotometer then returns to the
last method selected.

OLIGO

PROGRAMMED FACTOR:
1 A260 = 20.0 µg/mL

Blank

Sample

or

76

8 Error messages, result flagging and help texts

Result flagging

Flagging Explanation

1.586 A

0.015 A

260

320

▲▲

Flagging of A
(for method "Protein direct" only):
The method was calculated using the Warburg formula.

Flagging of A
(for method "Protein direct" and for nucleic acid methods only):
The absorbances at 260, 280 and 230 nm are corrected with the absorbance
at 320 nm (see Section 6, "Programming").

in the display or on the printout

260

in the display or on the printout

320

Error texts in the results display

Error text Explanation / Cause Solution

+++++ The absorbance measured is greater

than 3.0 A.

– Dilute the sample.

– Check the cuvette

(height of light path must
be 8.5 mm).

– Clean the cuvette shaft

(see Section 9).

– Insert the cuvette correctly (the

measuring window must be facing
the light path).

! ! ! ! ! The calculated result cannot be displayed

(value too high).

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

Ratio cannot be calculated because one of
the absorbance values used for calculating
the ratio is 0 A or > 3.0 A.

– Use a cuvette made of material

that transmits light at the
measuring wavelengths used
(e.g. quartz glass or UVette® from
Eppendorf for nucleic acid
measurement).

Check the parameter
(Is the factor too high?).

Repeat the measurement
(dilute sample if necessary).

77

8 Error messages, result flagging and help texts

Error texts in measuring procedure

Error text Explanation / Cause Solution

Measure blank first No blank has been measured

for the method selected.

Measure standard first No valid calibration

for the method selected.

not within calibration (For calculation via

non-linear regression only:)
The sample result is not within
the calibration range.

Measurement module
Error 1
Measurement module
Error 2
Measurement module
Error 3

Error texts in calibration procedure

Different errors in measurement module. Contact Service.

Measure the blank.

– Measure standards.

– Program a different calculation

(fixed factor or direct absorbance
measurement).

Repeat the measurement
(dilute sample if necessary).

Error text Explanation / Cause Solution

No STD method The measuring key was pressed

Measured values
not plausible

Measured values
not monotonous

Calibration curve is not
monotonous

Standard

although "Standard" was not programmed
as a procedure for the method selected.

(For one-point calibration:)
Absorbance measured is 0 A.

(For multiple-point calibration:)
The measured values do not produce
monotonously rising or falling sequences.

(For non-linear regression:)
The calculated curve is not monotonous.

– Re-measure the methods without

standard request.

– Program the "standard"

calculation.

Re-measure standard.
(Prepare again if necessary).

Check standards and re-measure in
the correct sequence (ascending
concentration).

Check standards and re-measure in
the correct sequence (ascending
concentration).

78

8 Error messages, result flagging and help texts

Clear

Error text Explanation / Cause Solution

CV greater than 10 % (Following standard measurements:)

The scattering of the measured values
around the calculated calibration line
or curve is very large (see Section 12,
"Calculation").

Check calibration result.

Enter

– : Store calibration.

– : Abort calibration.

Recalibrate or use the calibration
stored.

Error texts in programming procedure

Error text Explanation / Cause Solution

Method parameter
incorrect. Please check

Please program
standards ascending

Method parameters incorrectly entered. Check parameters and re-enter them

if necessary.

(For multiple-point calibration:)
Standard nominal values have not been

Check programming and enter
nominal values in ascending order.

programmed in ascending order.

Other error texts

Error text Explanation / Cause Solution

Entry invalid (When a serial sample number is entered

via the key:)

0

Sample No.

Enter a number within the specified
range.

A number outside of the range 1 to 999 has
been entered.

Help texts

Error text Explanation / Cause Solution

Please
program standard

Please
program factor

(In the display after method selection:)
For the method selected, the calculation
"Standard" has been programmed; but the
nominal concentrations for the standards
have not yet been programmed.

(In the display after method selection:)
For the method selected, the calculation
"Factor" has been programmed; but the
value for the factor has not yet been
programmed.

– Program nominal concentrations

for the standards

Parameter

( key).

– Program another calculation

without standards.

– Program the value for the factor

( key).

Parameter

– Program another calculation.

79

9 Maintenance and cleaning

Photometer

Cuvette shaft

Changing the fuses

– Disconnect the device from the main power source before carrying out maintenance

work or to change the fuses.
The inside of the device is a high-voltage area. Danger!

– Wipe the entire device using a moist cloth and a mild cleaning agent.

– Disinfect the device using a lightly moistened cloth and a 70 % ethanol/water

mixture.

– Do not allow any liquid to enter the device.

– Clean the cuvette shaft using a moist cotton swab only. Do not use large quantities

of liquid (e.g. spray bottles).

– When the device is not being used, protect the cuvette shaft from dust using the

seal provided.
Dust or residue from the measuring solutions in the optical light path can cause
inaccurate measurements.

– Disconnect the device from the mains supply.

– The fuse holder is located above the mains connection (see picture in Sec. 4.1).

The holder is held in position by a small elastic stop lever on its underside.

– Push the stop lever upwards and pull out the holder.

– Change the fuses (for specifications, see Sec. 2, “Technical data“ ).

– Press the holder into the attachment until the stop lever clicks into place.

– Plug the device into the mains supply.

80

10 Short instructions

Cuvettes

Min. overall height

Min. filling level

Min. volume

36 mm

Light path
Max. height of base

10 mm

8.5 mm
7 mm

0 mm

70 µL

400 µL 1000 µL 300 µL

Semi-micro Macro

Suction

Basic area

12.5 mm x 12.5 mm

Ultra-micro

UVette

®

50 µL

Preparation

The BioPhotometer is ready to measure immediately after being switched on.

Methods

7

dsDNA

4

Protein

8

ssDNA

5

OD 600

9

RNA

6

Oligo

7

dsDNA

4

Protein

1

Bradford

8

ssDNA

5

OD 600

2

Lowry

9

RNA

6

Oligo

3

BCA

dsDNA ssDNA RNA Oligo

– Direct measurement of the nucleic acids at 260 nm.
– Ratios A

260/A280

– Optional correction of absorbance values via A

and A

260/A230

.

320.

– Measurement using quartz-glass cuvette or UVette® from Eppendorf.

OD 600

– Direct measurement of the density of bacteria suspensions
at 600 nm (turbidity measurement).
– Measurement using glass cuvette or plastic cuvette.

Protein

– Direct measurement of protein at 280 nm.
– Direct measurement of the absorbance, or calculation via factor,
standard or Warburg formula.
– Optional correction of absorbance values via A

320.

– Measurement using quartz-glass cuvette or UVette® from Eppendorf.

Bradford

1

2

Lowry

3

BCA

Bradford Lowry BCA
Bradford micro Lowry micro BCA micro

– Measurement of protein using Bradford-, Lowry- or BCA reagent.
– Direct measurement of the absorbance,
or calculation via factor or calibration
(single-point calibration, linear regression or non-linear regression).
– Number and nominal values of the calibrators are programmable.
– The protein methods are also available on a micro-scale
(Press the Method key twice).
– Measurement using glass cuvette or plastic cuvette.

81

10 Short instructions

Programming

The factory-set method programs may be changed as required.

Example:

Programming of the unit "µg/mL" and of calculation via standard (500 µg/mL) for the Protein method.

PROTEIN

Select Protein method

Begin programming

Select calibration
with standard (STD)

Confirm

(After confirmation, the display
moves to the next selection block)

Dilution

4

Protein

Parameter

or

Enter



Conversion

ABSORBANCE

Blank

PROTEIN PAGE 1–3

CALCULATION STD –

▲▲

PROTEIN PAGE 1–3

CALCULATION STD –

PROTEIN PAGE 2–4

▲▲ ▲▲

CORR.WITH A

STD MEASUREMENT 1 x

ABSORBANCE

ABSORBANCE

Sample

or

FACTOR –

WARBURG –

FACTOR –

WARBURG –

320 OFF *

ON –

2 x –
3 x –

*

*

*

Select the unit µµµµg/mL

Confirm

Enter standard concen­tration and confirm

(After confirmation, the display
moves to the next selection block)

End programming

Dilution

5

OD 600

or

Enter

0

Sample No.

Enter

Parameter



Conversion

0

Sample No.

PROTEIN PAGE 3–4

UNIT mg/mL

µg/mL –

STD - - - - mg/mL

PROTEIN PAGE 3–4

UNIT mg/mL –

µg/mL

STD - - - - - µg/mL

PROTEIN PAGE 4–4

CUVETTE 10 mm

5 mm –
2 mm –
1 mm –

PARAMETER END

*

*

*

82

10 Short instructions

dsDNA

PROGRAMMED FACTOR
1 A260 = 50.0 µ g/mL

Sample

dsDNA BLANK

Blank

dsDNA SAMPLE 001

20+200 µL

0 . 0 0 0 A

CALC: AMOUNT:
TOTAL SAMPLE 140 µL

CALC: MOLARITY:
BASE PAIRS 300
MOL.MASS 198 kDa

dsDNA SAMPLE 001

20+200 µL 0.694 A

230

1.408 A260

1.97 260/280 0.715 A280

2.03 260/230 0.002 A320

5 6 3 . 2 0 µg/mL

dsDNA SAMPLE 001

20+200 µL

79 µ g

2843 pmol/mL

398 pmol

5 6 3 . 2 0 µg/mL

dsDNA SAMPLE 002

20+200 µL 0.689 A230

0.788 A260

1.85 260/280 0.623 A280

2.19 260/230 0.003 A320

2 4 9 . 7 0 µg/mL

Select dsDNA method

Measure blank

Enter

Enter

2

Lowry

Dilution

0

Sample No.

0

Sample No.

0

Sample No.

When the sample is diluted:
Example: 20 + 200 µL

2

Lowry

Measure sample

If the result of the sample
is to be converted:

Enter

Enter

Enter



Conversion

0

Sample No.

0

Sample No.

0

Sample No.

1

Bradford

4

Protein

3

BCA

Measure next sample

Sample

Sample

7

dsDNA

Blank

Blank

Sample

or

or

Sample

Blank

or

Measuring procedure for dsDNA

83

10 Short instructions

Bradford measuring procedure

BRADFORD

Select Bradford method

If "Bradford micro"
is to be selected:

Bradford

Measure blank

Measure first standard

1

1

Bradford

BRAD.micro

CALIBRATION RANGE

1.0 – 20.0 µ g/mL

Blank

Blank

Standard

CALIBRATION RANGE
100 – 2000 µ g/mL

Blank

or

Standard

BRADFORD BLANK

or

Standard

0 . 0 0 0 A

Blank

BRADFORD STD 1

or

Sample

Standard

100 µg/mL

0 . 0 4 8 A

NEXT: STD 2

200µg/mL

Measure last standard

When the sample is diluted:
Example: 20 + 200 µL

Dilution

2

Lowry

Sample No.

Measure sample

Measure next sample

2

Lowry

0

0

Sample No.

0

Sample No.

Enter

Enter

Standard

BRADFORD SAMPLE 001

20+200 µL

Blank

Sample

Sample

Sample

BRADFORD STD 6

2000 µg/mL

1 . 3 2 5 A

CV: 2.8 %
CALIBRATION STORED

or

Standard

BRADFORD SAMPLE 001

3 6 8 µg/mL

20+200 µL

BRADFORD SAMPLE 002

5 5 2 µg/mL

20+200 µL

0.525 A

0.352 A

595

595

84

11 Ordering information

11.1 Ordering information

Order no.

Photometer

6131 000.012 BioPhotometer

(230 V; 50/60 Hz; European plug)
(additional power supply variants available)

6131 810.006 BioPhotometer Software Package

for online data transfer on PC

6131 928.007 Secondary UV-VIS filter, Test Filter Set,

for checking the BioPhotometer

Printer

6131 011.006 Thermal Printer DPU 414,

incl. power supply 230 V unit and printer cable

0013 021.566 Thermal paper (5 rolls)

UVette®
(Disposable plastic cuvette for the UV / VIS range, 220 to 1,600 nm)

0030 106.300 UVette®, 80 pcs., individually packaged

4308 078.006 Cuvette stand for 16 cuvettes

11.2 Ordering information for North America

Order no.

Photometer

952 00 000-4 BioPhotometer

952 01 020-4 BioPhotometer Software Package, for online data transfer to PC

952 01 022-1 Secondary UV-VIS Filter Test Set, for verifying photometric precision and the

wavelength accuracy (NIST Traceable)

Printer

952 01 015-8 Thermal Printer DPU-414, requires power supply unit and printer cable

952 01 017-4 Power Supply Unit for Thermal Printer DPU-414, 115 V

952 01 016-6 Power Supply Unit for Thermal Printer DPU-414, 230 V

952 01 018-2 Printer Cable, for connecting serial printer

952 01 040-9 Printer Paper, 5 rolls

UVette®*

952 01 005-1 UVette®, 80 original Eppendorf disposable, individually packaged cuvettes

940 00 110-2 Cuvette Stand

* U.S. Patent No. 6,249,345

85

12 Calculation

12 Calculation

12.1 Nucleic acids (dsDNA, ssDNA, RNA, oligo)

Calculation via factor

C= A
C = Calculated concentration

260

A
F = Factor (method-specific programming using the key)

The nucleic acid methods have the following special feature: The programmed factor is always based on the
unit of concentration "µg/mL". If the unit of concentration "µg/µL" is selected, the factor is converted internally:

F’ = F / 1000
F’ = Converted factor; used for the calculation of the concentration.

Sample dilution

C

Dil, corr

C

Dil, corr

VP = Volume of the sample in the measuring solution (entered using the key)
V

Dil

260

x F

= Absorbance measured at 260 nm

Parameter

= C x (VP + V

Dil

) / V

P

= Result converted using dilution factor

Dilution

= Volume of the diluent in the measuring solution (entered using the key)

Dilution

Optical path length of the cuvette

Application: Using cuvettes with an optical path length of 1 mm, 2 mm or 5 mm.

The optical path length of the cuvette can be programmed for each method using the key.

A

cuv, corr

A

cuv, corr

A

cuv, corr

A

cuv, corr

Correction A

= A x 2 (with an optical path length of 5 mm)
= A x 5 (with an optical path length of 2 mm)
= A x 10 (with an optical path length of 1 mm)

= Absorbance converted in accordance with an optical path length of 10 mm

320

Parameter

Application: Partial correction of incorrect absorbance caused by turbidity in the measuring solution.

The calculation procedure with or without correction A

Parameter

key.

A
A

= Ax - A

x, corr

= Absorbance at wavelength of 230, 260 and 280 nm, corrected mathematically

x, corr

320

320

can be programmed for each method using the

Ax = Absorbance measured at wavelength of 230, 260 and 280 nm
A

320

= Absorbance measured at wavelength of 320 nm

The corrected absorbance is used for further calculation of results.

Conversion key: Calculating the quantity

Application: Calculating the quantity of nucleic acid in the total sample volume.

M = C x V

P, total

M = Calculated overall quantity of nucleic acid in sample vessel
C = Calculated concentration
V

86

= Volume of the sample in the sample vessel (entered using the key)

P, total



Conversion

12 Calculation

Conversion key: Calculating the molar concentration

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

C

= C / N

mol

C

= Molar concentration (calculated)

mol

N = Relative molar mass, in kDa (entered using the key)

If, instead of the relative molar mass, the number of bases / base pairs per molecule has been entered, N is
calculated using the number of bases / base pairs:

dsDNA: N = bp x 2 x 330 x 10-3
ssDNA, RNA, Oligo: N = b x 330 x 10-3

N = Calculated relative molar mass, in kDa
bp = Number of base pairs per molecule (dsDNA)
b = Number of bases per molecule (ssDNA, RNA, Oligo)

The unit for molar concentration is programmed for each method using the key.



Conversion

Parameter

12.2 Direct photometric determination of protein

Selection for calculation of results:

– Absorbance
– Calculation of the concentration via factor
– Calculation of the concentration via one-point calibration
– Calculation of the concentration via Warburg formula

Calculation of the concentration via facto

See Section 12.1; Measuring wavelength: 280 nm

When the factor is entered using the key, the unit of concentration which has been programmed must

Parameter

be taken into consideration.

Calculation of the concentration via standard (one-point calibration)

F = CS / AS
F = Calculated factor

CS = Nominal concentration of the standard (method-specific programming using the key)
AS = Measured absorbance of the standard

If the standard multiple measurement (2x, 3x) has been programmed, calculation is based on the
absorbances measured, including the zero value, via linear regression. After the regression has been
calculated, a CV (coefficient of variation in "%") value is formed as a measure of the scattering of the
measured values. If the CV value is greater than 10 %, it appears in the display. In this case, the calibration is
not stored automatically; it must first be confirmed by the user (see Section 12.3).

r

Parameter

The calculation of the sample concentration is carried out using the calculated factor:

280

C = A

x F

Calculation of the concentration via Warburg formula

C = 1.55 x A
C = (1.55 x E

280

- 0.76 x A

280

– 0.76 x A

260

for "mg/mL" concentration unit

260

) x 1000 for "µg/mL" concentration unit

87

12 Calculation

Sample dilution, optical light path of the cuvette and correction A

320

See Section 12.1.

12.3 Protein with addition of reagent

Methods: Bradford, Bradford micro, BCA, BCA micro, Lowry, Lowry micro

Selection for the calculation of results:

– Absorbance
– Calculation of the concentration via factor
– Calculation of the concentration via standard

Selection for the calculation procedures via standard:

– One-point calibration
– Multiple-point calibration (standard line)
– Multiple-point calibration (standard curve)

Calculating the concentration via factor and calculating the concentration via standard
(one-point calibration)

See Section 12.2; Measuring wavelength: 595 nm (Bradford; Lowry) or 562 nm (BCA)

Calculating the concentration via standard (multiple-point calibration; calibration line)

A calibration line (concentration as a function of the absorbance) is calculated from 2 to 10 standards, which
are measured in single, double or triple determination. The equation of the line is calculated via linear
regression.

C = ao + a1A
a1 = Slope of the calibration line (Factor)

ao = Intersection point of the calibration lines with the concentration axis

(concentration of a sample with the absorbance "0" [Offset])

After the calibration has been calculated, the CV value (coefficient of variation in "%") is calculated (exception:
two-point calibration with single determination of the two standards). The CV value is a measure for the
scattering of the measured values around the calculated calibration line. If the value is greater than 10 %, the
calibration is not stored automatically; it must first be confirmed by the user. In the case of more than two
standards, the CV value always appears in the display (even when the value is lower than 10 %).

The calculated parameters ("ao" and "a1") of the stored calibration line can be printed out by calling up the
functions list by pressing the

•

Function

key.

Calculating the concentration via standard (multiple-point calibration; calibration curve)

A calibration curve (concentration as function of the absorbance) is calculated from 5 to 10 standards
measured in single determination or from 4 to 10 standards measured in double or triple determination. The
non-linear regression is calculated via a third-grade polynomial.

C = ao + a1A + a2A2 + a3A3 + . . .
a = Coefficients (The coefficients are determined using the least square method).

CV value: see above (linear regression).

The calculated parameters of the stored calibration line can be printed via the

•

Function

key.

88

12 Calculation

Sample dilution and optical light path of the cuvette

See Section 12.1.

12.4 OD 600

The measured values appear as absorbance values measured at a wavelength of 595 nm.

Sample dilution and optical light path of the cuvette

See Section 12.1.

89

13 Testing the photometer

13 Testing the photometer

To enable the photometric accuracy and the wavelength accuracy to be tested, a filter set (secondary UV-VIS
filter) is available from Eppendorf. This set contains three filters ("Sample A1", "Sample A2" and "Sample A3")
for testing the photometric accuracy and two filters ("Sample 260 nm" and "Sample 280 nm") for testing the
wavelength accuracy. The absorbance of the filters is measured against a blank filter ("Blank A0").

To carry out these measurements, blank filters and "sample filters" (test filters) are inserted into the cuvette
holder in the same manner as cuvettes. When doing so, please ensure that the label with the filter description
is facing the user. The absorbance values measured for the test filters are compared to those within the range
of permitted values. The limits for the permitted range are contained in a table found on the inside of the lid of
the filter box (see Figure: "X.XXX – X.XXX A").

BioPhotometer

Secondary / Sekundär - UV - VIS - Filter Order No. / Best.Nr.: 6131 928.007

Function: PHOTOMETERTEST

Limits

Grenzwerte

6131 914.XXX 916.XXX 917.XXX 921.XXX 922.XXX 923.XXX

Filter

Type

230 nm 0.000 X.XXX - X.XXX X.XXX - X.XXX X.XXX - X.XXX

260 nm 0.000 X.XXX - X.XXX X.XXX - X.XXX X.XXX - X.XXX X.XXX - X.XXX

280 nm 0.000 X.XXX - X.XXX X.XXX - X.XXX X.XXX - X.XXX X.XXX - X.XXX

320 nm 0.000 X.XXX - X.XXX X.XXX - X.XXX X.XXX - X.XXX

562 nm 0.000 X.XXX - X.XXX X.XXX - X.XXX X.XXX - X.XXX

595 nm 0.000 X.XXX - X.XXX X.XXX - X.XXX X.XXX - X.XXX

Blank

A

0

Reference

(A) / (E) Limiting values (A) / Grenzwerte (E)

Sample

260 nm

of wavelength / der Wellenlänge of photometer / des Photometers

Nist: SRM 2034, SN: 99 Nist: SRM 2031a, SN: 577

measured against Blank A

gemessen gegen Blank A

Sample

280 nm

Systematic error / Systematische Messabweichung

Traceable to / rückführbar auf

Random error / Zufällige Messabweichung

0 at approx. 20 °C
0 bei ca. 20 °C

Sample

A

1

Sample

A

2

Sample

A

3

empty

leer

of wavelength / der Wellenlänge of photometer / des Photometers

230 –

595 nm

Please protect against dust, heat and liquid

The limits are valid for max. 2 years.

Bitte vor Staub, Hitze und Flüssigkeiten schützen

Die Grenzwerte gelten für max. 2 Jahre.

0.000 ≤ 3.0 % ≤ 3.0 % ≤ 1.0 % ≤ 1.5 %

Limiting values CV (%) / Grenzwerte VK (%)

Fig.: Inside of the lid of the filter box

90

Date Signature

Datum Unterschrift

eppendorf

13 Testing the photometer

Test procedure

– Carry out the test at approximately 20 °C.

– Remove the filter from the filter box for a brief period only. Make sure that the surface of the filter is not

contaminated or damaged.

– Protect the filter from dust, heat, liquid and aggressive vapors.

– When inserting the filter, ensure that the label with the filter description is facing the user.

– Select the function "Photometer test".

This function is contained in devices with a software version of V 1.20 onwards. Contact Eppendorf before
using the test filter with an older software version.

– Select the test filter.

–"A1", "A2" or "A3" for the measurement of the photometric accuracy at 230, 260, 280, 320, 562

and 595 nm.

–"A

– Follow the instructions in the photometer display for the measurement of "Blank" and "Sample".

The device carries out 10 measuring cycles and then prints out the mean values for the absorbances at
the respective wavelengths.

260

" or "A

" for the measurement of the wavelength accuracy at 260 or 280 nm.

280

– Compare the absorbance values with the permitted value range.

– In addition to information on accuracy values, the printout contains details of precision as well. The mean

value and the CV are calculated from each of the ten measurements.

If the absorbances measured are not within the permitted value range, please contact the Service Depart­ment at Eppendorf. The filters should be recalibrated by Eppendorf after two years.

91

Conformity Declaration for BioPhotometer 6131

in accordance with enclosure 15 of "Eichordnung" (German standardization regulations)

Descr

iption of measurement

Device used: Single-beam filter photometer with reference beam and fixed wavelengths
Type: BioPhotometer 6131
Manufacturer / Distributor: Eppendorf AG, Hamburg
Mode of instruction: Operating manual

1. Measur

ing system

Light path: Lamp > aperture > lens > aperture > cuvette > aperture >

diffraction grating > aperture > photodiode

Light source: Xenon flash lamp

Continuum spectral range 220 to 2,000 nm
Spectral apparatus: Grating polychromator
Radiation receiver: Silicon photodiode

Spectral range 200 to 1,100 nm
Cuvette: Quartz glass, optical special glass or plastic, depending on measuring wavelength
Cuvette types: 10 mm macro min. vol. 1000 µl

10 mm semi-micro min. vol. 400 µl

10 mm suction min. vol. 300 µl

10 mm ultra-micro min. vol. 70 µl

Cuvette temperature: Not available

Results display: Illuminated, graphic LCD, 33 x 66 mm

2

Measured values displayed: Absorption, mass concentration, molar concentration

2. Measur

ing procedures
Determination of the
cuvette blank: Wavelength-dependent individual measured value of the cuvette used
Concentration determination: Lambert-Beer-Bourguer law
Reference measurement
on reference material: Check with calibrated secondary standards

3. Measur

ing range of the spectral absorption rate

0.000 to 3.000 A
The error limits listed can be exceeded outside these measuring ranges as well as
with nominal conditions of use other than those listed below.

4. Nominal conditions of use
Cuvette blank: Depending on cuvette used
Wavelengths: Xenon 230, 260, 280, 320, 562, 595 nm
Warm-up time: None
Supply voltage: 100 to 240 V ± 10 %, 50 to 60 Hz ± 5 %
Ambient temperature: 15 to 35 °C
Relative humidity: 15 to 70 %

5. Error limits and other limiting v

alues
Relative photometric uncertainty of the spectral absorption rate with all wavelengths for
an individual measurement:

±

1.5 % at 1 A

Relative photometric
short-time standard deviation:≤ 0.5 % at 1 A
Wavelength systematic error:± 1 nm at 230 to 280 nm, ± 2 nm at 320 to 595 nm

Spectral half-intensity width:
Integral fault-radiation level:

≤

5 nm at 230 to 320 nm, ≤ 7 nm at 562 and 595 nm

≤

0.03 % at 260 nm with GG 375-3 (Schott)

Date: 25.09.2000
Eppendorf AG
Quality and standards

92

EG-Konformitätserklärung

EC Conformity Declaration

Das bezeichnete Produkt entspricht den einschlägigen grundlegenden Anforderungen der

aufgeführten EG-Richtlinien und Normen. Bei einer nicht mit uns abgestimmten Änderung des

Produktes oder einer nicht bestimmungsgemäßen Anwendung verliert diese Erklärung ihre Gültigkeit.

the EC directives and standards listed. In the case of unauthorized modifications to the product

Produktbezeichnung, Product name:

The product named below fulfills the relevant fundamental requirements of

or an unintended use this declaration becomes invalid.

BioPhotometer 6131

Produkttyp, Product type:

Photometer

Einschlägige EG-Richtlinien/Normen, Relevant EC directives/standards:

73/23/EWG, EN 61010-1

89/336/EWG, EN 55011/B, EN 61000-6-1, EN 61000-3-2, EN 61000-3-3

Vorstand, Board of Management: Projektmanagement, Project Management:

07.08.2003

Hamburg, Date:

0015 033.509-02

Eppendorf AG · Barkhausenweg 1 · 22339 Hamburg · Germany

6131 900.994-01

Your local distributor:
www.eppendorf.com/worldwide

Eppendorf AG
22331 Hamburg · Germany
Tel. [phone]
Fax [phone]
E-Mail: [e-mail]

Eppendorf North America, Inc.
One Cantiague Road, P.O. Box 1019
Westbury, N.Y. 11590-0207 USA
Tel. [phone]
Toll free phone [phone]
Fax [phone]
E-Mail: [e-mail]

Application Support

Europe, International:
Tel. [phone]
E-Mail: [e-mail]

North America:
Tel. [phone] ext. 2258
E-Mail: [e-mail]

Asia, Pacific:
Tel. [phone]
E-Mail:
[e-mail]

Printed in Germany eppendorf® is a registered trademark