Implen NanoPhotometer Manual de usuario

telephone: + 49-89-726 3718 0 Fax. + 49-89-726 3718 54 Email: info@implen.de www.implen.de

NanoPhotometer® P-Class

User Manual

P 300 / P 330 / P 360

Version 2.1

P 360

P 300

P 330

NanoPhotometer® P-Class User Manual

Version 2.1 Page 2 / 70

Implen GmbH Schatzbogen 52 D-81829 Germany

Declaration of conformity for the NanoPhotometer®P-Class (P300/P330/P360)

This is to certify that the Implen NanoPhotometer®P-Class conforms to the requirements of the following Directives:

2014/35/EU Low Voltage directive (LVD)

2014/30/EU Electromagnetic Compatibility directive (EMC)

2011/65/EU Restriction on the use of certain hazardous substances in Electrical and Electronical

Equipment directive (RoHS)

2012/19/EU Waste electrical and electronic equipment directive (WEEE)

By ensuring this product is disposed of correctly, you will help prevent potential

negative consequences for the environment and human health, which could otherwise

be caused by inappropriate waste handling of this product.

2006/42/EC Machinery directive

1995/5/EC Radio and Telecommunications Terminal Equipment Directive (instruments fitted with

Bluetooth accessory only).

Standards to which conformity is declared, where relevant, are as follows:

EN 61010-1:2010 Safety requirements for electrical equipment for measurement, control and laboratory

use, General Requirements

EN 61010-2-101:2002 Safety requirements for electrical equipment for measurement, control and laboratory

use. Particular requirements for in vitro diagnostic (IVD) medical equipment

IEC 61010-1(ed. 3) Report: TRA-006858; CB Certificate: GB-TRAC 0246

UL 61010-1/CSA C22.2 No. 61010-1 Report: 25436; Listing: E113393

EN 61326-1:2013 Electrical equipment for measurement, control and laboratory use –EMC

requirements (class B)

EN ISO 12100:2010 Safety of machinery-General principles for design, risk assessment and risk reduction

For further information, including unpacking, positioning and installation of the products please refer to the user

manual.

Signed: Dated: June 21, 2016

Dr. Thomas Sahiri

Managing Director

Implen GmbH

NanoPhotometer® P-Class User Manual

Version 2.1 Page 3 / 70

TABLE OF CONTENTS

1. ESSENTIAL SAFETY NOTES...............................................................................................................................................5

Unpacking, Positioning and Installation .....................................................................................................5

2. INTRODUCTION.................................................................................................................................................................. 6

2.1 Your spectrophotometer.....................................................................................................................6

2.2 Sample handling tips ..........................................................................................................................6

2.3 Keypad and display for NanoPhotometer®P 300.............................................................................7

2.4 Keypad and display for NanoPhotometer®P 330 /P 360.................................................................8

2.5 Menu/Options.......................................................................................................................................9

3. THE NANOPHOTOMETER® P-CLASS SUBMICROLITER CELL ........................................................................................10

3.1 Technical instructions ......................................................................................................................10

3.2 Software instructions........................................................................................................................11

4. NANOVOLUME APPLICATIONS AND CUVETTE APPLICATIONS ....................................................................................13

4.1 Characterization of DNA, RNA and Oligonucleotides ...................................................................13

4.1.1 General Information ................................................................................................................13

4.1.2 Analysis of dsDNA, ssDNA and RNA .....................................................................................15

4.1.3 Analysis of Oligonucleotides...................................................................................................16

4.1.4 Dye incorporation for dsDNA, ssDNA, RNA and Oligonucleotides.........................................17

4.2 Protein Determination.......................................................................................................................19

4.2.1 General Information ................................................................................................................19

4.2.2 Protein UV Method..................................................................................................................20

4.2.3 Protein UV Dye Method ..........................................................................................................22

7.3.4 BCA Assay..............................................................................................................................24

7.3.5 Bradford Assay........................................................................................................................27

7.3.6 Lowry Assay............................................................................................................................30

7.3.7 Biuret Assay............................................................................................................................33

4.3 Bacterial Cell Culture Measurement (OD600).................................................................................36

4.3.1 General Information ................................................................................................................36

4.3.2 Analysis of Bacterial Growth...................................................................................................37

5. FUNCTIONS ......................................................................................................................................................................38

5.1 Single Wavelength –Abs and %T....................................................................................................39

5.2 Concentration ....................................................................................................................................41

5.3 Wavescan...........................................................................................................................................43

5.4 Kinetics...............................................................................................................................................46

5.5 Standard Curve..................................................................................................................................48

5.6 Multiple Wavelength..........................................................................................................................51

5.7 Absorbance Ratio..............................................................................................................................53

6. USER METHODS ..............................................................................................................................................................55

7. UTILITIES ..........................................................................................................................................................................56

7.1 Date and Time....................................................................................................................................57

7.2 Regional..............................................................................................................................................57

7.3 Output Options / Printer....................................................................................................................57

7.3.1 NanoPhotometer®P 300.........................................................................................................57

7.3.2 NanoPhotometer®P 330 /P 360 .............................................................................................58

7.3.3 Loading / changing the printer paper ......................................................................................59

7.4 Preferences........................................................................................................................................60

7.5 Contrast..............................................................................................................................................60

7.6 About ..................................................................................................................................................60

8. MAINTENANCE.................................................................................................................................................................61

8.1 Maintenance-free Technology..........................................................................................................61

8.2 Lamp Replacement............................................................................................................................61

8.3 Mixer replacement.............................................................................................................................61

8.4 Exchange of the gaiter......................................................................................................................61

8.5 Cleaning and general care of the instrument .................................................................................62

8.6 Error messages .................................................................................................................................63

NanoPhotometer® P-Class User Manual

Version 2.1 Page 4 / 70

8.7 Trouble shooting ...............................................................................................................................64

9. ACCESSORIES..................................................................................................................................................................64

10. SPECIFICATION AND WARRANTY ..................................................................................................................................65

11. SOLVENT COMPATIBILITY...............................................................................................................................................66

12. APPENDIX ........................................................................................................................................................................67

12.1 Nucleic acid quantification...............................................................................................................67

12.2 Nucleic acid fluorescent dye incorporation....................................................................................67

12.3 Protein quantification........................................................................................................................69

12.4 Protein fluorescent dye incorporation ............................................................................................69

NanoPhotometer® P-Class User Manual

Version 2.1 Page 5 / 70

1. ESSENTIAL SAFETY NOTES

There are a number of warning labels and symbols on your instrument. These are there to inform you where potential

danger exists or particular caution is required. Before commencing installation, please take time to familiarise yourself

with these symbols and their meaning.

Caution (refer to accompanying documents).

Background colour yellow, symbol and outline black.

Unpacking, Positioning and Installation

Check the contents of the package against the delivery note. If any shortages are discovered, inform your supplier

immediately.

Inspect the instrument for any signs of damage caused in transit. If any damage is discovered, inform your supplier

immediately.

Ensure your proposed installation site conforms to the environmental conditions for safe operation:

Indoor use only.

Temperature range 5°C to 35°C. Note that if you use the instrument in a room subjected to extremes of

temperature change during the day, it may be necessary to recalibrate (by switching off and then on again) once

thermal equilibrium has been established (2-3 hours).

Maximum relative humidity of 80% up to 31°C decreasing linearly to 50% at 40°C.

The instrument must be placed on a stable, level bench or table that can take its weight (< 4.5 kg) so that air can

circulate freely around the instrument.

The equipment should be positioned such that in the event of an emergency the mains plug can be easily located

and removed

This equipment must be connected to the power supply with the power cord supplied. It can be used on 90 –240

V, 50-60 Hz supplies.

If the instrument has just been unpacked or has been stored in a cold environment, it should be allowed to come to

thermal equilibrium for 2-3 hours in the laboratory before switching. This will prevent calibration failure as a result

of internal condensation.

Switch on the instrument via the keypad ( ) after it has been plugged in. The instrument will perform a series of

self-diagnostic checks.

Please read through this user manual prior to use.

Please contact your original supplier in the first instance if you experience technical or sample handling difficulties.

If this equipment is used in a manner not specified or in environmental conditions not appropriate for safe operation,

the protection provided by the equipment may be impaired and instrument warranty withdrawn.

NanoPhotometer® P-Class User Manual

Version 2.1 Page 6 / 70

2. INTRODUCTION

2.1 Your spectrophotometer

Your spectrophotometer is a simple-to-use UV/Visible instrument with a CCD array detector (1024 pixels). It has no

moving parts, which is the basis of the rapid scanning operating system.

The user interface is built around folders which are displayed on the main screen when the instrument is switched on.

Different folders are numbered and opened by using the associated number key on the keypad. After switching on the

NanoPhotometer®a self-calibration check is performed and the default main screen "NanoPhotometer®” is offering

the choice of:

Keypad

number

Description

Life Science methods such as nucleic acid assays

and protein assays using the NanoPhotometer® P-

Class Submicroliter Cell

Life Science methods such as nucleic acid assays,

protein assays and cell density using cuvettes

General spectroscopic methods

Contains nine folders that can store user adapted

methods (up to 81)

Instrument set up (date, time, number format),

Output Options and Baseline Compensation set up.

The instrument is equipped with a standard USB port (back) and the P330/P360 also with a USB flash drive port (right

side). The NanoPhotometer®P-Class Software Package is necessary to connect the NanoPhotometer®P-Class to a PC

or to read the saved data on the USB flash drive. The software enables the user to “print through” the PC directly to the

printer that is connected to it. Data may be stored as Excel spreadsheet (report and/or table format), EMF graphics file,

a comma delimited (csv) data file, a tab delimited (txt) data file or in native NanoPhotometer®P-Class Software format

for later access (see also NanoPhotometer®P-Class PVC Installation and User Manual).

Alternatively, results may be saved on a SD Memory Card (P300 only) or sent to the PC via a Bluetooth (P300 only)

accessory; these can either be supplied pre-installed or are available as an optional accessory if the need for the use

arises after installation of the product.

A thermal built-in printer is available for the instrument; this may either be supplied pre-installed or is available as an

optional accessory if the need for its use arises after installation of the product.

2.2 Sample handling tips

The NanoPhotometer®P-Class includes an integrated vortexer (P 330 / P 360 only) to assure a good homogeneity

of the sample. It is recommended to mix every sample before a measurement.

Note that the light beam is directed from RIGHT to LEFT through the cell chamber; therefore please ensure the

measurement cell is inserted in the correct alignment.

Insert the measurement cell always in the same direction.

The cell holder supplied with the instrument accepts the NanoPhotometer®P-Class Submicroliter Cell and standard

10 mm pathlength quartz, glass or plastic cells.

The optical height of the NanoPhotometer®P-Class is 15 mm.

The minimum volume that can be used is 0.3 µl with the NanoPhotometer® P-Class Submicroliter Cell.

12 mm test tubes may be used (e.g. for cell cultures), however they are not recommended as higher quality data is

produced by using disposable cuvettes for the analysis. If used, align the indicator line on 12 mm test tubes in the

same direction to ensure reproducible positioning of the tube. Note that test tubes do not last forever, and that the

surface becomes scratched and blemished through repetitive use; if this is the case they should be replaced.

NanoPhotometer® P-Class User Manual

Version 2.1 Page 7 / 70

2.3 Keypad and display for NanoPhotometer®P 300

The back-lit liquid crystal display is very easy to navigate around using the alphanumeric entry and navigation arrow

keys on the hard wearing, spill proof membrane keypad.

Key

Action

On/off key

Turns the instrument on/off.

Arrow keys

Use the four arrow keys to navigate around the display and select the required

setting from the active (highlighted) option.

View Options

View options for that application mode. Some of these are common to all

applications and described on page 8. Menu unique to an application are

described in the relevant section of the NanoPhotometer®P-Class User Manual.

Alphanumeric keys

Use these to enter parameters and to write text descriptions where appropriate,

or required. Use repeated key presses to cycle through lower case, number and

upper case. Leave for 1 second before entering next character. Use C button to

backspace and 1 to enter a space.

Escape/Cancel/Back:

Escape from a selection and return to the previous folder. Cancel a selection.

Stop making measurements.

Blank/Reference

Set reference to 0.000 A or 100%T on a reference solution at the current

wavelength in the mode selected. When in scan mode, does a reference scan.

Sample/Enter selection/OK:

Enter, or confirm a selection. Take a measurement.

ON/OFF key

Arrow keys

View options

Alphanumeric keys

Escape/Cancel/Back

Blank/Reference

Sample/Enter selection/OK

LCD Display

Cellholder

NanoPhotometer® P-Class User Manual

Version 2.1 Page 8 / 70

2.4 Keypad and display for NanoPhotometer®P 330 /P 360

The back-lit liquid crystal display is very easy to navigate around using the alphanumeric entry and navigation arrow

keys on the hard wearing, spill proof membrane keypad.

Key

Action

On/Off Key

Turns the instrument on/off.

Arrow Keys

Use the four arrow keys to navigate around the display and select the required

setting from the active (highlighted) option.

View Menu

View menu for that application mode. Some of these are common to all

applications and described on page 8. Menu unique to an application are

described in the relevant section of the NanoPhotometer®P-Class User Manual.

Alphanumeric Keys

Use these to enter parameters and to write text descriptions where appropriate,

or required. Use repeated key presses to cycle through lower case, number and

upper case. Leave for 1 second before entering next character. Use C button to

backspace and 1 to enter a space.

Escape/Cancel/Back:

Escape from a selection and return to the previous folder. Cancel a selection. Stop

making measurements.

Blank/Reference

Set reference to 0.000 A or 100%T on a reference solution at the current

wavelength in the mode selected. When in scan mode, does a reference scan.

Sample/Enter Selection/OK:

Enter, or confirm a selection. Take a measurement.

(P 330 and P 360 only)

Prints the results shown on the screen on the built-in printer, if a built-in printer is

connected to the NanoPhotometer®.

Graph/Data

(P 330 and P 360 only)

Toggle graph on/off. The graph shows a wavescan plot across the range 220 nm

to 400 nm (for Dye methods 220 nm to 750 nm) with cursors denoting 230, 260,

280 and 320 nm (Nucleic Acid methods) and 260, 280 and 320 nm (Protein

methods).

vortexer

NanoPhotometer® P-Class User Manual

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2.5 Menu/Options

(select using key pad numbers)

Options for P 300:

After each measurement the following Options are possible:

1) Return to parameter screen.

2) Print the results via selected method.

3) Toggle graph on/off. Graph shows a wavescan plot across the

range 220 nm to 400 nm (for Dye methods 220 nm to 750 nm)

with cursors denoting 230, 260, 280 and 320 nm

4) Toggle on/off the graph in the print-out or saved file.

7) Define the sample number you wish to start from.

8) Save the parameters as a method.

9) Open printer settings, possibility to change the printer settings

within the method as described in 7.3 Output Options / Printer.

Exit Options by pressing Escape , OR wait

Menu options for P 330/P 360:

After each measurement the following options are possible in the

Menu:

1) Return to parameter screen.

2) Transfer the results via selected Output Option.

4) Toggle on/off the graph in the print-out or saved file.

7) Define the sample number you wish to start from.

8) Save the parameters as a method.

9) Open Output Options settings, possibility to change the Output

Options settings within the method as described in 7.3 Output

Options / Printer

Exit Menu by pressing Escape

, OR wait

NanoPhotometer® P-Class User Manual

Version 2.1 Page 10 / 70

3. THE NANOPHOTOMETER® P-CLASS SUBMICROLITER CELL

With its innovative optical pathway the cell is designed for optimum measurement results with submicroliter samples

ranging from 0.3 µl up to 5 µl of undiluted sample. Due to a pathlength of 0.04 mm, 0.1 mm, 0.2 mm, 1 mm and 2

mm the cell is offering an automatic dilution of 1/250, 1/100, 1/50, 1/10 and 1/5 in comparison to a standard

cuvette measurement. Because the measurements are processed with undiluted samples, the reproducibility of the

results is extremely high. If desired, samples can be retrieved after the measurement for further processing. The

NanoPhotometer®P-Class Submicroliter Cell can be used for all UV/Vis analysis utilizing the wavelength range of

190 nm to 1,100 nm.

The NanoPhotometer®P-Class Submicroliter Cell is delivered for version P 300 with one lid

with a pathlength of 0.2 mm (Lid 50), for version P 330 with two lids pathlength 0.2 mm

(Lid 50) and 1 mm (Lid 10) and for version P 360 with three lids pathlength 0.04 mm (Lid

250), 0.2 mm (Lid 50) and 0.1 mm (Lid 10). Lid 5 (2 mm pathlength), Lid 100 (0.1 mm

pathlength) and Lid 250 (0.04 mm) can be ordered optionally. The dilution factor (lid

factor) is printed on the lid. Please make sure that you use the appropriate lid for your

sample.

3.1 Technical instructions

Step 1

Insert the NanoPhotometer®P-Class Submicroliter Cell into the cell

holder with the cell windows facing the light beam. We recommend

facing the Implen logo to the front. The light beam is directed from

RIGHT to LEFT as indicated with small arrows. Insert the

NanoPhotometer®P-Class Submicroliter Cell always in the same

direction.

Step 2

Use the integrated vortexer (P 330 / P 360 only) to mix your sample well

to achieve an accurate homogeneity of the sample.

Step 3

Pipette the appropriate sample volume onto the centre of the measuring

window. Warning!! Do not overfill the well.

Lid

Sample volume

Pathlength

Dilution

5 (optional)

3.5 –5 µl

2 mm

1:5

10 (optional for P300)

1 –3 µl

1 mm

1:10

0.3 –2 µl

0.2 mm

1:50

100 (optional)

0.3 –2 µl

0.1 mm

1:100

250 (optional)

0.3 –2 µl

0.04 mm

1:250

Step 4

Make sure that for the measurements the lid fits exactly onto the

positioning supports mounted to the body of the cell. Take

measurement. Remember to consider the lid factor in your instrument

software. Please refer to the NanoPhotometer®P-Class User Manual for

detailed information.

Step 4

Take the lid off and retrieve the sample with a pipette for further

applications if desired. Remove sample residues from the measurement

window and the mirror in the lid. Clean the measurement window and

mirror in the lid well with a slightly wet fluff-free tissue. Use water, 70%

ethanol or isopropanol. Do not use aggressive solvents like strong acids

or bases or organic solvents at any time.

Important Note: Residual fluffs must be removed for optimum

performance

Your cell is ready for the next sample.

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