GE Healthcare

Life Sciences

Spectrophotometry
Handbook
 Handbooks from GE Healthcare Life Sciences
GE Healthcare GE Healthcare
GE Healthcare 64
7TP[cWRPaT GE Healthcare
GE, imagination at work, and GE monogram are trademarks of General Electric

Antibody Purification – Handbook

Affinity Chromatograpy Handbook – Principles and Methods

Gel filtration – Principles and Methods

Company.
ÄKTA, ÄKTAcrossflow, ÄKTAdesign, ÄKTAexplorer, ÄKTAFPLC, ÄKTApilot , ÄKTAprime,
ÄKTAprocess, ÄKTApurifier, ÄKTAxpress, Biacore, BioProcess, Capto, Cy, ECL, ECL
Advance, ECL Plex, ExcelGel, HiLoad, HiPrep, HiTrap, Hybond, Hybond LFP, LabMate,
MabSelect, MabTrap, MidiTrap, MiniTrap, MultiTrap, PhastGel, PhastSystem, PlusOne,
PrimeView, ReadyToProcess, RESOURCE, Sephadex, Sepharose, SOURCE, SpinTrap,
Superdex, Superloop, Tricorn, and UNICORN are trademarks of GE Healthcare
Companies.
Histidine-tagged protein purification: Purification and preparation of fusion proteins
and affinity peptides comprising at least two adjacent histidine residues may require
a license under US patent numbers 5,284,933 and 5,310,663 and equivalent patents
and patent applications in other countries (assignee: Hoffman La Roche, Inc).
Capto ViralQ: Separating viral particles with Capto Q products may require a license
under United States patent number 6,537,793 B2 and equivalent patents and patent
applications in other countries owned by Centelion SAS. Such a license is not included
with the purchase of Capto Q but is included with the purchase of Capto ViralQ products.
With the purchase of Capto ViralQ the customer is granted a free limited license under
US patent 6,537,793 B2 and equivalent patents and patent applications in other
countries owned by Centelion SAS to separate viral particles solely through use of the
product purchased.
ECL Advance contains Lumigen TMA-6 substrate and is sold under exclusive license
from Lumigen Inc.
ECL Plus contains Lumigen PS3 substrate and is sold under exclusive license from
Lumigen Inc.
gination at work and GE Monogram are trademarks of General
Company. Chelating Sepharose Fast Flow and Ni Sepharose 6 Fast Flow: US patent numbers
5,284,933 and 5,310,663, and equivalent patents and patent applications in other
KTAexplorer, ÄKTAFLPC, ÄKTAprime, ÄKTApurifier, Biacore, BioDirectory, countries (assignee: Hoffman La Roche, Inc) relate to the purification and preparation
ess, ECL, ECL Plus, ExcelGel, FPLC, GSTPrep, GSTrap, HisTrap, HiPrep, HiTrap, of fusion proteins and affinity peptides comprising at least two adjacent histidine
d, MAbTrap, MabSelect, MicroSpin, Microplex, Multiphor, STREAMLINE, residues (commonly known as the histidine-tag technology).
ose, Percoll, PhastSystem, PhastGel, Sephadex, Superdex, and Tricorn are Any customer that wishes to use Chelating Sepharose Fast Flow, Ni Sepharose 6 Fast
arks of GE Healthcare companies. Flow or IMAC Sepharose 6 Fast Flow for non-research/commercial applications under
these patents is requested to contact Hoffman-La Roche AG, Corporate licensing, GE, imagination at work, and GE monogram are trademarks of General Electric Company.
tion and preparation of fusion proteins and affinity peptides comprising at
attention Dr Andreas Maurer, CH-4070 Basel, Switzerland, telephone +41 61 687 2548, ÄKTA, ÄKTAexplorer, ÄKTAmicro, ÄKTAprime, ÄKTApurifier, ÄKTAxpress, Biacore, BioProcess,
wo adjacent histidine residues may require a license under US pat 5,284,933
fax +41 61 687 2113, for the purpose of obtaining a license. HiLoad, HiPrep, HiScale, HiTrap, MabSelect, MidiTrap, MiniTrap, MultiTrap, Sephacryl,
pat 5,310,663, including corresponding foreign patents (assigne: Hoffman
The Tricorn column and components are protected by US design patents USD500856, Sephadex, Sepharose, SpinTrap, Superdex, Superose, and Tricorn are trademarks of
he, Inc). GE Healthcare companies.
USD506261, USD500555, USD495060, and their equivalents in other countries.
e for commercial use of GST gene fusion vectors must be obtained from US patent numbers 5,284,933 and 5,310,663, and equivalent patents and patent
UNICORN Software: Any use of this software is subject to GE Healthcare Standard applications in other countries (assignee: Hoffman La Roche, Inc) relate to the purification
on International, Incorprated, 28820 Singel Oak Drive, Temecula, California
Software End-User License Agreement for Life Sciences Software Products. A copy of and preparation of fusion proteins and affinity peptides comprising at least two adjacent
USA. this Standard Software End User License Agreement is available on request. histidine residues (commonly known as the histidine-tag technology).
orn column and components are protected by US design patents USD500856,

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All third party trademarks are the property of their respective owners.

Affinity Chromatography
Any customer that wishes to use Chelating Sepharose Fast Flow, Ni Sepharose 6 Fast Flow
6261, USD500555, USD495060 and their equivalents in other countries. © 2002−2007 General Electric Company—All rights reserved. or IMAC Sepharose 6 Fast Flow for non-research/commercial applications under these
patents is requested to contact Hoffman-La Roche AG, Corporate licensing, attention
party trademarks are the property of their respective owners. Previously published Dec. 2002
Dr Andreas Maurer, CH-4070 Basel, Switzerland, telephone +41 61 687 2548, fax +41 61
–2007 General Electric Company – All rights reserved. All goods and services are sold subject to the terms and conditions of sale of the 687 2113, for the purpose of obtaining a license.
company within GE Healthcare that supplies them. A copy of these terms and

Antibody Purification
blished 1988. All third party trademarks are the property of their respective owners.
conditions is available on request. Contact your local GE Healthcare representative for
ds and services are sold subject to the terms and conditions of sale of the
Principles and Methods the most current information.
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© 2010 General Electric Company—All rights reserved.
First published Dec. 2000.

Gel filtration
ny within GE Healthcare that supplies them. A copy of these terms and GE Healthcare UK Ltd
ons is available on request. Contact your local GE Healthcare representative For local office contact information, Amersham Place
All goods and services are sold subject to the terms and conditions of sale of the company
within GE Healthcare which supplies them. A copy of these terms and conditions is available
most current information. Little Chalfont
please visit www.gelifesciences.com/contact Buckinghamshire, HP7 9NA, UK
on request. Contact your local GE Healthcare representative for the most current information.
lthcare Europe GmbH

Handbook
GE Healthcare UK Limited Amersham Place
ger Strasse 5 GE Healthcare Bio-Sciences Corp For local office contact information, Little Chalfont
800 Centennial Avenue Buckinghamshire, HP7 9NA
1 Freiburg, Germany GE Healthcare Bio-Sciences AB please visit www.gelifesciences.com/contact
lthcare UK Limited
am Place
Björkgatan 30
P.O. Box 1327
Piscataway, NJ 08855-1327, USA
UK
GE Healthcare Europe, GmbH
Principles and Methods
Munzinger Strasse 5
halfont 751 84 Uppsala GE Healthcare Europe GmbH
Munzinger Strasse 5,
www.gelifesciences.com/protein-purification D-79111 Freiburg
ghamshire, HP7 9NA, UK Germany
Sweden D-79111 Freiburg, Germany
GE Healthcare Bio-Sciences Corp.
lthcare Bio-Sciences Corp.
ntennial Avenue
GE Healthcare Bio-Sciences KK GE Healthcare Bio-Sciences AB 800 Centennial Avenue, P.O. Box 1327
Sanken Bldg. 3-25-1, Hyakunincho Piscataway, NJ 08855-1327
1327 www.gelifesciences.com/protein-purification Shinjuku-ku, Tokyo 169-0073, Japan Björkgatan 30 USA
way, NJ 08855-1327, USA 751 84 Uppsala GE Healthcare Bio-Sciences KK
Sanken Bldg., 3-25-1, Hyakunincho
lthcare Bio-Sciences KK
Bldg.3-25-1
Sweden Shinjuku-ku, Tokyo 169-0073
Japan
incho Shinjuku-ku
69-0073, Japan

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imagination at work
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10/15/2007 16:52:27

GE Healthcare GE Healthcare 64
7TP[cWRPaT GE, imagination at work, and GE monogram are trademarks of General Electric Company. GE Healthcare
ÄKTA, ÄKTAcrossflow, ÄKTAmicro, ÄKTAprime, ÄKTApurifier, ÄKTAxpress, AxiChrom,
Biacore, BioProcess, Capto, Cy, CyDye, DeCyder, ECL Advance, ECL Plex, Ettan, ExcelGel,
FPLC, GraviTrap, GSTPrep, GSTrap, HiLoad, HiPrep, HiScale, HiScreen, HisPrep, HisTrap,
HiTrap, LabMate, MabSelect, MabSelect SuRe, MabSelect Xtra, MAbTrap, MacroCap,
GST Gene Fusion System – Handbook

Strategies for Protein Purification – Handbook

MBPTrap, MicroCal, MidiTrap, MiniBeads, Mini Q , Mini S, MiniTrap, MonoBeads,
Mono P, Mono Q, Mono S, MultiTrap, Polybuffer, PreDictor, PreScission, PrimeView,
ReadyToProcess, RESOURCE, Sephacryl, Sephadex, Sepharose, SOURCE, SpinTrap,
StrepTrap, Superdex, Superose, Tricorn, UNICORN, and Vivaspin are trademarks of
GE Healthcare companies.
2-D Fluorescence Difference Gel Electrophoresis: 2-D Fluorescence Difference Gel
Electrophoresis (2-D DIGE) technology is covered by US patent numbers 6,043,025,
6,127,134 and 6,426,190 and equivalent patents and patent applications in other
countries and exclusively licensed from Carnegie Mellon University. CyDye: this product
or portions thereof is manufactured under an exclusive license from Carnegie Mellon
University under US patent numbers 5,569,587, 5,627,027 and equivalent patents in
other countries. The purchase of CyDye DIGE Fluors includes a limited license to use the
CyDye DIGE Fluors for internal research and development, but not for any commercial
purposes. A license to use the CyDye DIGE Fluors for commercial purposes is subject to
a separate license agreement with GE Healthcare.
ECL Advance contains Lumigen TMA-6 substrate and is sold under exclusive license
from Lumigen Inc.
ECL Direct/Alkphos Direct labelling reagents are covered by European patent number
120376B1 and sold under license from European Molecular Biology Laboratory.
ECL Direct (RPN3000,3001,3005) is covered by US patent numbers 373017 and 513040
and foreign equivalents and is sold under license from Institut Pasteur.
ECL Plus contains Lumigen PS3 substrate and is sold under exclusive license from
Lumigen Inc.
GST Gene Fusion Vectors: A license for commercial use of GST Gene Fusion Vectors
under US patent 5,654,176 and equivalent patents and patent applications in other
countries must be obtained from Millipore Corp (formerly Chemicon International Inc).
IMAC Sepharose products and Ni Sepharose products: Purification and preparation of
agination at work, and GE monogram are trademarks of General Electric Company. fusion proteins and affinity peptides comprising at least two adjacent histidine residues
ÄKTAexplorer, ÄKTAfplc, ÄKTAprime, ÄKTApurifier, ECL, ExcelGel, FlexiPrep, GFX, may require a license under US patent numbers 5,284,933 and 5,310,663, and equivalent
ep, GSTrap, HiLoad , HiTrap, HiPrep, Hybond, MicroPlex, MicroSpin, Multiphor, patents and patent applications in other countries (assignee: Hoffman La Roche, Inc).
Gel, PreScission, Ready-To-Go, Sepharose, Superdex, and Whatman are IMAC Sepharose products, Ni Sepharose products, and Fe Sepharose products:
marks of GE Healthcare companies. These products are covered by US patent number 6 623 655 and equivalent patents
erials for research use only. Not for diagnostic or therapeutic purposes. and patent applications in other countries.
se for commercial use of GST Gene Fusion Vectors under US patent 5,654,176 and MabSelect SuRe Ligand and Cys-rProtein A Ligand: MabSelect SuRe Ligand Restricted
lent patents and patent applications in other countries must be obtained from License” and ”Cys-rProtein A Ligand Restricted License” are protected by the following

Isolation of
re Corp (formerly Chemicon International Inc). patents and equivalent patents and patent applications in other countries: US 5,151,350,
us contains Lumigen PS3 substrate and is sold under exclusive license from US 5,143,844, US 6,399,750, WO 03/00475 and EP 112338 9. A free, non-transferable
en Inc. limited license of the product from a GE Healthcare company and its licensed distributors.
Vectors are to be used for scientific investigation and research and for no other Any other use will require a separate license from a GE Healthcare company.
se whatsoever and a license for commercial use of the licensed products and StrepTrap HP and StrepTactin Sepharose High Performance: These products are covered

mononuclear cells
ocesses claimed in US patent 5,654,176 and equivalent patents and patent by US patent number 6,103,493 and equivalent patents and patent applications in other
ations in other countries must be negotiated directly with Millipore Corp (formerly countries. The purchase of StrepTrap HP and StrepTactin Sepharose High Performance
con International Inc) by the purchaser prior to such use. includes a license under such patents for non-profit and in-house research only. Please
NA Polymerase is sold under licensing arrangements with Roche Molecular contact IBA (info@iba-go.com) for further information on licenses for commercial use
ms, F Hoffmann-La Roche Ltd and the Perkin-Elmer Corporation. Purchase of of Strep-Tactin.

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oduct is accompanied by a limited license to use it in the Polymerase Chain Tricorn columns: Tricorn columns and components are protected by US design patents
on (PCR) process for research in conjunction with a thermal cycler whose use in
tomated performance of the PCR process is covered by the up-front license fee, Methodology and applications USD500856, USD506261, USD500555, USD495060 and their equivalents in other countries.
UNICORN software: Any use of UNICORN software is subject to GE Healthcare Standard
by payment to Perkin-Elmer or as purchased (i.e., an authorized thermal cycler). Software End-User License Agreement for Life Sciences Software Products. A copy of
d party trademarks are the property of their respective owners. this Standard Software End-User License Agreement is available on request.
9-2010 General Electric Company—All rights reserved. All third party trademarks are the property of their respective owners.

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ublished April 2009 © 2010 General Electric Company—All rights reserved.

GST Gene Strategies for

ds and services are sold subject to the terms and conditions of sale of the All goods and services are sold subject to the terms and conditions of sale of the
ny within GE Healthcare which supplies them. A copy of these terms and company within GE Healthcare which supplies them. A copy of these terms and
ons is available on request. Contact your local GE Healthcare representative for conditions is available on request. Contact your local GE Healthcare representative for
ost current information. the most current information.
althcare UK Limited Amersham Place GE Healthcare UK Limited Amersham Place
halfont

Fusion System Protein Purification

Little Chalfont
ghamshire, HP7 9NA For local office contact information, Buckinghamshire, HP7 9NA

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P]S
<TcW^Sb please visit www.gelifesciences.com/contact UK

althcare Europe, GmbH GE Healthcare Europe, GmbH
nger Strasse 5 Munzinger Strasse 5
11 Freiburg D-79111 Freiburg
ny www.gelifesciences.com/protein-purification Germany
althcare Bio-Sciences Corp.
ntennial Avenue, P.O. Box 1327
away, NJ 08855-1327
Handbook GE Healthcare Bio-Sciences AB
GE Healthcare Bio-Sciences Corp.
800 Centennial Avenue, P.O. Box 1327
Piscataway, NJ 08855-1327
Handbook
USA
althcare Bio-Sciences KK Björkgatan 30 GE Healthcare Bio-Sciences KK
n Bldg., 3-25-1, Hyakunincho
ku-ku, Tokyo 169-0073
751 84 Uppsala Sanken Bldg., 3-25-1, Hyakunincho
Shinjuku-ku, Tokyo 169-0073
Sweden Japan

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18-1157-58 AB 10/2010 28-9833-31 AA 09/2010

GE Healthcare GE Healthcare GE Healthcare GE Healthcare

GE, imagination at work, and GE monogram are trademarks of General Electric Company.
Hydrophobic Interaction and Reversed Phase Chromatography – Principles and Methods
Ion Exchange Chromatography & Chromatofocusing – Principles and Methods

Recombinant Protein Purification Handbook – Principles and Methods

2-D Electrophoresis – Principles and Methods

Cy, CyDye, DeCyder, Deep Purple, ECL, Ettan, ExcelGel, Hybond, illustra, ImageMaster,
ImageQuant, ImageScanner, Immobiline, IPGphor, Multiphor, MultiTemp, Personal Densitometer,
Pharmalyte, Rainbow, Scierra, and Typhoon are trademarks of GE Healthcare companies.
2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) technology is covered by US
patent numbers 6,043,025, 6,127,134 and 6,426,190 and equivalent patents and patent
ÄKTA, ÄKTAcrossflow, ÄKTAdesign, ÄKTAexplorer, ÄKTAFPLC, ÄKTApilot, applications in other countries and exclusively licensed from Carnegie Mellon University.
ÄKTAprime, ÄKTAprocess, ÄKTApurifier, ÄKTAxpress, AxiChrom, Biacore, Capto, CyDye: this product or portions thereof is manufactured under an exclusive license from
Deep Purple, ECL, ECL Advance, ECL Plus, ExcelGel, FPLC, GraviTrap, GSTPrep, Carnegie Mellon University under US patent numbers 5,569,587, 5,627,027 and equivalent
GSTrap, HiLoad, HiPrep, HiScreen, HisPrep, HisTrap, HiTrap, Hybond, Labmate, patents in other countries. The purchase of CyDye DIGE Fluors includes a limited license
MabSelect, MabSelect SuRe, MabSelect Xtra, MBPTrap, MidiTrap, MiniTrap, Mono Q, to use the CyDye DIGE Fluors for internal research and development, but not for any
commercial purposes. A license to use the CyDye DIGE Fluors for commercial purposes is
Multiphor, MultiTrap, PhastGel, PhastSystem, PlusOne, PreScission, PrimeView,
subject to a separate license agreement with GE Healthcare.
Rainbow, RESOURCE, Sephadex, Sephacryl, Sepharose, SpinTrap, SOURCE,
StrepTactin, StrepTrap, Superdex, Superloop, Tricorn, UNICORN, and Drop design CyDye: This product or portions thereof is manufactured under an exclusive license from
are trademarks of GE Healthcare companies. Carnegie Mellon University under US patent number 5,268,486 and equivalent patents in
the US and other countries. The purchase of CyDye products includes a limited license to
GE, imagination at work, and GE monogram are trademarks of General Electric
Company. www.gehealthcare.com/protein-purification BioProcess, BPG, Drop Design, ECL, ECL Plus, Ettan, FineLINE, FPLC, HiLoad, use the CyDye products for internal research and development but not for any commercial
purposes. A license to use the CyDye products for commercial purposes is subject to a
HiPrep, HiTrap, Hybond, MicroSpin, Mono Q, PlusOne, RESOURCE, Sephadex,
Deep Purple Total Protein Stain: Deep Purple Total Protein Stain is exclusively separate license agreement with GE Healthcare. Commercial use shall include:
licensed to GE Healthcare from Fluorotechnics Pty Ltd. Deep Purple Total www.gehealthcare.com Sepharose, SOURCE, STREAMLINE, Superdex, Tricorn, UNICORN, ÄKTA,
ÄKTAdesign, ÄKTAexplorer, ÄKTAFPLC, ÄKTApilot , ÄKTAprime, ÄKTApurifier, and 1. Sale, lease, license or other transfer of the material or any material derived or
Protein Stain may only be used for applications in life science research. Deep produced from it.
Purple is covered under a granted patent in New Zealand entitled “Fluorescent ÄKTAxpress are trademarks of GE Healthcare companies. GE, imagination at
2. Sale, lease, license or other grant of rights to use this material or any material derived
Compounds”, patent number 522291 and equivalent patents and patent work, and GE monogram are trademarks of General Electric Company. or produced from it.
applications in other countries. GE Healthcare Bio-Sciences AB, Triton is a trademark of Union Carbide Chemicals and Plastics Co. Inc. 3. Use of this material to perform services for a fee for third parties, including contract
Histidine-tagged protein purification: Purification and preparation of fusion research and drug screening.
proteins and affinity peptides comprising at least two adjacent histidine residues
a General Electric Company. Multipipette is a trademark of Eppendorf-Netheler-Hinz GmbH. If you require a commercial license to use this material and do not have one, return this
may require a license under US patent numbers 5,284,933 and 5,310,663, Coomassie is a trademark of ICI plc. material unopened to GE Healthcare Bio-Sciences AB, Bjorkgatan 30, SE-751 84 Uppsala,
and equivalent patents and patent applications in other countries (assignee: Sweden and any money paid for the material will be refunded.
Hoffman La Roche, Inc). Biacore is a trademark of Biacore AB.
IMAC Sepharose products and Ni Sepharose products: These products are
GE Healthcare Bio-Sciences AB DeCyder: This release of DeCyder (software) is provided by GE Healthcare to the customer under
Purification and preparation of fusion proteins and affinity peptides comprising a nonexclusive license and is subject to terms and conditions set out in the 2-D Differential
covered by US patent number 6,623,655 and equivalent patents and patent Gel Electrophoresis Technology Access Agreement. Customer has no rights to copy or
applications in other countries. Björkgatan 30 at least two adjacent histidine residues may require a license under US pat
duplicate or amend the Software without the prior written approval of GE Healthcare.
5,284,933 and US pat 5,310,663, including corresponding foreign patents
pGEX Vectors: pGEX Vectors are to be used for scientific investigation and
research and for no other purpose whatsoever and a license for commercial
751 84 Uppsala (assignee: Hoffman La Roche, Inc). Deep Purple Total Protein Stain is exclusively licensed to GE Healthcare from
Fluorotechnics Pty Ltd.Deep Purple Total Protein Stain may only be used for applications
use of the licensed products and the processes claimed in US patent 5,654,176 All goods and services are sold subject to the terms and conditions of sale of in life science research.Deep Purple is covered under a granted patent in New Zealand
and equivalent patents and patent applications in other countries must be Sweden the company within GE Healthcare which supplies them. GE Healthcare reserves entitled “Fluorescent Compounds”, patent number 522291 and equivalent patents and
negotiated directly with Millipore Corp (formerly Chemicon International Inc) by patent applications in other countries.
the right, subject to any regulatory and contractual approval, if required, to
the purchaser prior to such use. DIGE Gel and DIGE Buffer Kit: The buffer system in this gel and buffer kit is covered by
make changes in specifications and features shown herein, or discontinue the
StrepTrap HP and StrepTactin Sepharose High Performance: These products product described at any time without notice or obligation. Contact your local
patent application WO9616724 granted in US, EP and JP.
are covered by US patent number 6,103,493 and equivalent patents and patent Ettan CAF MALDI Sequencing Kits are protected by patents owned by Procter & Gamble
GE Healthcare representative for the most current information.
magination at work, and GE monogram are trademarks of General Electric Company. applications in other countries. The purchase of StrepTrap HP and StrepTactin Company and exclusively licensed to GE Healthcare Bio-Sciences AB and by joint patents
Sepharose High Performance includes a license under such patents for non- © 2006 General Electric Company – All rights reserved. issued to both companies. The purchase of Ettan CAF MALDI Sequencing Kits includes a
Q, Mini S, Mono Q, Mono S, Mono P, RESOURCE, SOURCE, Sepharose, BioProcess, HiTrap, profit and in-house research only. Please contact IBA (info@iba-go.com) for limited license to use the technology for internal research and development, but not for
d, HiPrep, Tricorn, FPLC, MiniBeads, MonoBeads, ÄKTA, ÄKTAexplorer, ÄKTApurifier, further information on licenses for commercial use of StrepTactin. GE Healthcare Europe GmbH any commercial purposes. No right to perform or offer commercial services or products
adex, Sephacel, Sephacryl, BPG, Pharmalyte, ÄKTAprime, ÄKTApilot, STREAMLINE, Munzinger Strasse 5 of any kind using the Sequencing Kits is hereby granted. A license to use the technology
nd, ECL, ECL Plus, Superdex, PhastGel, PlusOne, PhastSystem, BioDirectory, FineLINE Tricorn Columns: The Tricorn column and components are protected by US
D-79111 Freiburg for commercial purposes is subject to a separate license agreement with GE Healthcare
design patents USD500856, USD506261, USD500555, USD495060 and their

Hydrophobic Interaction 2-D Electrophoresis

rop Design are trademarks of GE Healthcare companies. Bio-Sciences AB. Please contact the Product Director, Mass Spectrometry and Sample
equivalents in other countries. Germany
rd party trademarks are the property of their respective owners. Handling, GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-75184, Uppsala, Sweden for
All third party trademarks are the property of their respective owners. GE Healthcare UK Ltd. details about how to obtain such a license.
04-2010 General Electric Company – All rights reserved.
Amersham Place

Recombinant Protein
© 2000–2009 General Electric Company – All rights reserved. This version of ImageMaster has been developed by the Swiss Institute of Bioinformatics in

Ion Exchange
published Apr. 2004.
First published Dec 2000. Little Chalfont collaboration with GeneBio and GE Healthcare.
ods and services are sold subject to the terms and conditions of sale of the company

and Reversed Phase

All goods and services are sold subject to the terms and conditions of sale of the Buckinghamshire, HP7 9NA All third party trademarks are the property of their respective owners.

Principles and Methods

n GE Healthcare that supplies them. A copy of these terms and conditions is available
quest. Contact your local GE Healthcare representative for the most current company within GE Healthcare that supplies them. A copy of these terms and UK © 2010 General Electric Company—All rights reserved.
mation. conditions is available on request. Contact your local GE Healthcare representative All goods and services are sold subject to the terms and conditions of sale of the company

Chromatography
GE Healthcare Bio-Sciences Corp.
for the most current information.

Purification Handbook
ealthcare UK Ltd within GE Healthcare which supplies them. A copy of these terms and conditions is available
800 Centennial Avenue on request. Contact your local GE Healthcare representative for the most current information.
sham Place For local office contact information, visit GE Healthcare UK Ltd
P.O. Box 1327

Chromatography
Chalfont Amersham Place GE Healthcare UK Limited Amersham Place
Piscataway, NJ 08855-1327
nghamshire, HP7 9NA, UK www.gelifesciences.com/contact Little Chalfont Little Chalfont
For local office contact information,
& Chromatofocusing
Buckinghamshire, HP7 9NA, UK USA Buckinghamshire, HP7 9NA
ealthcare Bio-Sciences Corp UK
entennial Avenue
GE Healthcare Bio-Sciences AB GE Healthcare Bio-Sciences Corp GE Healthcare Bio-Sciences KK please visit www.gelifesciences.com/contact
ox 1327
away, NJ 08855-1327, USA
Björkgatan 30
800 Centennial Avenue
P.O. Box 1327 Principles and Methods Sanken Bldg.
3-25-1, Hyakunincho
GE Healthcare Europe, GmbH
Munzinger Strasse 5

Principles and Methods

D-79111 Freiburg
Piscataway, NJ 08855-1327, USA
ealthcare Europe GmbH
751 84 Uppsala
Shinjuku-ku, Tokyo 169-0073 www.gelifesciences.com/protein-purification Germany
nger Strasse 5 GE Healthcare Europe GmbH Japan
111 Freiburg, Germany
ealthcare Japan Corporation
Principles and Methods Sweden Munzinger Strasse 5
D-79111 Freiburg, Germany GE Healthcare Bio-Sciences AB
GE Healthcare Bio-Sciences Corp.
800 Centennial Avenue, P.O. Box 1327
Piscataway, NJ 08855-1327
en Bldg. 3-25-1 GE Healthcare Bio-Sciences KK USA
unincho, Shinjuku-ku, www.gelifesciences.com/protein-purification Sanken Bldg. 3-25-1 Björkgatan 30 GE Healthcare Bio-Sciences KK
169-0073, Japan
www.gelifesciences.com/purification_techsupport
Hyakunincho, Shinjuku-ku, 751 84 Uppsala Sanken Bldg., 3-25-1, Hyakunincho
Tokyo 169-0073, Japan Shinjuku-ku, Tokyo 169-0073
Sweden Japan

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Price: US$20
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11-0004-21 AB 04/2010 18-1142-75 AD 01/2009 11-0012-69 AA 02/2006 80-6429-60 AD 06/2010

2010-04-01 12:12:53

GE Healthcare GE Healthcare GE, imagination at work, and GE monogram are trademarks of General Electric Company.
GE Healthcare GE Healthcare
Life Sciences ÄKTAmicro, ÄKTA, Biacore, CyDye, Deep Purple, DeCyder, ECL Plex, Ettan, ExcelGel, Ficoll,
HiTrap, illustra, ImageQuant, Immobiline, Klari-Flex, MidiTrap, Mini Q, Mini S, MiniTrap,
Mono P, Mono Q, Mono S, MultiTrap, Percoll, Pharmalyte, RESOURCE, Sephadex,
Life Sciences
Nucleic Acid Sample Preparation for Downstream Analyses – Principles and Methods

High-throughput Process Development with PreDictor Plates – Principles and Methods

Protein Sample Preparation – Handbook

Sepharose, SOURCE, SpinTrap, Superdex, Superose, UNIFILTER, and Whatman are

trademarks of GE Healthcare companies.
2-D Fluorescence Difference Gel Electrophoresis: 2-D Fluorescence Difference Gel
Electrophoresis (2-D DIGE) technology is covered by US patent numbers 6,043,025,
6,127,134 and 6,426,190 and equivalent patents and patent applications in other
countries and exclusively licensed from Carnegie Mellon University. CyDye: this product
magination at work, and GE monogram are trademarks of General Electric Company. GE, imagination at work, and GE monogram are trademarks of General Electric Company. or portions thereof is manufactured under an exclusive license from Carnegie Mellon
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Contents

Spectrophotometry basics 3
What is spectrophotometry? 3
Definition 3
Lambert’s Law 4
Beer’s Law 4

Nucleic acid applications 6

Direct UV measurement 7
A260 /A280 Ratio 7
A260 /A230 Ratio 7
A 320 Background correction 7
Nucleic acid measurements with low volume instruments 8

Protein applications 9
Protein concentration calculations 10
Christian and Warburg 10
Colorimetric methods 10
BCA 11
Biuret 11
Bradford 11
Lowry 11
2-D Quant Kit 11
Choosing your protein assay 12
Measuring proteins in low volume instruments 14

Other applications 15
Fluorescent dyes 15
Cell culture 15
Other molecules 16

Continued overleaf

1
Spectrophotometry hints and tips 17
Choosing a cuvette 17
Beam heights 17
Choice of cuvettes 17
Buffer Compatibility 17
Low volume spectrophotometers 18
Choice of volume 18
Background correction 18

Which spectrophotometer? 19

Spectrophotometer selection guide 21

GE Healthcare spectrophotometer range 22
Datrys PC control software 22
Accessories overview 23

Glossary 24
Absorption 24
Bandwidth 24
Beam technology 25
Light source comparison 25
Methods of measurement 26
Monochromator 27
Pathlength 27
Stray Light 27
UV/Vis 27
Wavelength 27

References 28

Related products 29

2
Spectrophotometry basics
What is spectrophotometry?
Spectrophotometry is a scientific method based on the absorption of light by a substance,
and takes advantage of the two laws of light absorption.

Definition
spec·tro·pho·tom·e·ter/spektrōfōˈtämitər/

Noun: An apparatus for measuring intensity of light in a part of the spectrum,
as transmitted or emitted by particular substances.

Visible Light

Infrared Ultraviolet

Near Near

A B C

1 mm 3 µm 1400 nm 750 nm 700 nm 650 nm 600 nm 550 nm 500 nm 450 nm 400 nm 320 nm280 nm 200 nm

Fig 1. The electromagnetic spectrum. GE offers a range of spectrophotometers which operate in the UV, visible and
near infrared section of the electromagnetic spectrum.

3
Lambert’s Law (1)
The proportion of light absorbed by a medium is independent of the intensity of incident light.
A sample which absorbs 75% (25% transmittance) of the light will always absorb 75% of the
light, no matter the strength of the light source.

Lambert’s law is expressed as I/Io =T

Where I = Intensity of transmitted light

Io = Intensity of the incident light
T = Transmittance

This allows different spectrophotometers with different light sources to produce comparable
absorption readings independent of the power of the light source.

Beer’s Law (2)

The absorbance of light is directly proportional to both the concentration of the absorbing
medium and the thickness of the medium. In Spectrophotometry the thickness of the medium
is called the pathlength.

In normal cuvette-based instruments the pathlength is 10 mm. Beer’s law allows us to measure
samples of differing pathlength, and compare the results directly with each other. GE offers
a variety of instruments and accessories which allow measurement of pathlengths from 10 cm
down to 0.2 mm.
1.5

1.2
Absorbance

0.9

0.6

0.3

0.0
0.0 0.2 0.4 0.6 0.8 1.0
Path Length (cm)

Fig 2. Beer’s Law: The absorbance of light is directly proportional to both the concentration of the absorbing medium
and the thickness of the medium.

4
Short pathlength instruments are used when the sample is of limited volume, scarce and maybe
requiring recovery, or is very concentrated (e.g. > 50 µg DNA/ml), and the user wishes to avoid
the need for dilution. Many samples would traditionally require dilution for two reasons:

1. So that there is enough volume to fill a 10 mm pathlength cuvette.

2. To lower the sample concentration enough to allow accurate measurement
by the spectrophotometer.

Dilution introduces an aspect of human error and can also prevent the use of that sample in
downstream applications.

To measure concentrated samples using a 10 mm pathlength would require a very powerful
light source to give transmittance that is high enough to be detected reliably. A shorter
pathlength reduces the absorbance – increasing the transmittance – hence reducing the
incident light required to achieve a reliable result. This removes the need to dilute the sample,
or to have a larger, more powerful or more expensive instrument.

When using short pathlengths (less than 10 mm), results are generally normalised to that
of a 10 mm pathlength, e.g. In the case of a 0.2 mm pathlength, the absorbance results are
multiplied by 50. However, at the same time any error from the system of absorption by the
cuvette is also multiplied by 50, increasing the possible effect on the result.

In basic terms: Absorbance = Concentration × Pathlength

Light
Source
Adjustable
Monochromator Aperture
Sample

Photoresistor
Amplifier
Spectrum Display

of light

Light is Light is
absorbed detected
Measure is
displayed
Fig 3. General schematic of a spectrophotometer.

5
Nucleic acid applications
Spectrophotometry can be used to estimate DNA or RNA concentration and to analyse the
purity of the preparation. Typical wavelengths for measurement are 260 nm and 280 nm.
In addition measurements at 230 nm and 320 nm can provide further information.

Purines and pyrimidines in nucleic acids naturally absorb light at 260 nm. For pure samples
it is well documented that for a pathlength of 10 mm, an absorption of 1A unit is equal to a
concentration of 50 µg/ml DNA and 40 µg/ml for RNA. For oligonucleotides the concentration
is around 33 µg/ml but this may vary with length and base sequence.

So for DNA: Concentration (µg/ml) = Abs260 × 50.

These values are known as conversion factors.

A number of other substances which also absorb light at 260 nm could interfere with DNA values,
artificially increasing the result calculated from the absorption readings. To compensate for
this a selection of ratios and background corrections have been developed to help eliminate
false readings.

0.9 X: 300
Y: 0.063
0.8

0.7

0.6
Absorbance

0.5

0.4

0.3

0.2

0.1
×
0.0
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
Wavelength (nm)
Fig 4. A typical wavelength scan for a pure DNA sample.

6
There is a wide absorbance peak around 260 nm preceded by a ‘dip’ at 230 nm. Therefore
to measure the DNA absorption, the 260 nm DNA peak must be distinguishable from the
230 nm reading.

If the readings at 230 nm are too similar to those at 260 nm, DNA cannot be measured
accurately. Higher 230 nm readings can indicate contaminants in the sample. There should
also be a rapid tail-off from 260 nm down to 320 nm. For this reason, 320 nm is often used
to measure background (see background correction).

Direct UV measurement
A260/A280 Ratio
The most common purity check for DNA and RNA is the A260/A280 ratio. Any protein contamination
will have maximum absorption at 280 nm. Measurements are taken at both 260 nm and 280 nm
and compared to give a ratio. For DNA the result of dividing the 260 nm absorption by the
280 nm needs to be greater or equal to 1.8 to indicate a good level of purity in the sample.
For RNA samples this reading should be 2.0 or above. Results lower than this are indicative
of impurities in the sample.

A260/A230 Ratio
An increase in absorbance at 230 nm can also indicate contamination, which may in turn affect
the 260 nm reading for DNA and RNA. A number of substances absorb at 230 nm, as this is
the region of absorbance of peptide bonds and aromatic side chains. Several buffer
components exhibit strong absorption at 260 nm and therefore can alter the results of
photometric quantification. One example of such a component is EDTA in concentrations
above 10 mM. Contaminants in a sample, such as proteins, phenol, or urea, can result in
absorption at 230 nm. Phenol contamination also increases a sample’s absorption at 280 nm
and therefore can be identified through a lower A260/A280 ratio. An A260/A230 ratio of 2 or above
is indicative of a pure sample.

A 320 Background correction

Background correction is a process whereby the absorption at a point on the spectrum unrelated
to the sample being analysed is also measured, and the reading subtracted from the peaks.
Absorption at 320 nm may be due to light scatter caused by particles, or to a precipitate in the
sample. Dirty or damaged cuvettes can cause absorption at 320 nm. Contaminations with
chaotropic salts, such as NaI, can also lead to increased light scatter.

Measuring and correcting for the reading at 320 nm therefore removes any interference from
light scatter, from the cuvette, or in cases where a blanking plate is used to target the light beam
though the sample.

Background correction is particularly useful when using small volume cells or specialist small
volume spectrophotometers.

7
Nucleic acid measurements with low volume instruments
To get meaningful results, as a rule of thumb, the following two criteria must be met:

• Abs260 > approx. twice Abs230 (A260/A230 Ratio=2)

• Abs260 = 0.1 or more (indicative of a high enough concentration, ie solution not too dilute)

Typically the only measurements checked for DNA measurement are concentration and
A260/A280 ratios. Unexpected results can often be explained by looking at the underlying A 230,
A260, A280, A320 values.

As an example:

Sample 1 Sample 3
Concentration 10.7 µg/ml Concentration 11.0 µg/ml
A230 A260 A280 A320 A230 A260 A280 A320
9.27 0.244 0.129 0.030 9.32 0.326 0.211 0.107

A260/A280 A260/A230 A260/A280 A260/A230

2.162 0.023 2.106 0.024

Sample 2 Sample 4
Concentration 11.1 µg/ml Concentration 10.5 µg/ml
A230 A260 A280 A320 A230 A260 A280 A320
9.33 0.323 0.206 0.101 9.30 0.303 0.192 0.094

A260/A280 A260/A230 A260/A280 A260/A230

2.114 0.024 2.133 0.023

In this example, the instrument is giving concentration values which are very close together
(<2% variation). So the reproducibility for concentration values is within the expected values.
However, looking at the underlying absorbance readings shows that the 230 nm reading is very
high compared to other readings making the ratio A 260 /A 230 very low. A value < 1.8 indicates
potential contamination.

Note that in this example

• The background at A 320 nm is provided for information & must be subtracted from
the A260 nm reading to arrive at the absorbance
• For information: Not all low volume instruments display the A 320 nm subtraction in
the calculation.

8
Protein applications
As with DNA, proteins absorb light at a specific wavelength, allowing direct measurement using
a spectrophotometer. The amino acids Tyrosine and Tryptophan have a very specific absorption
at 280 nm, allowing direct A280 measurement of protein concentration. Direct UV measurement
at 280 nm has many advantages, since the protein solution alone is used, without the addition
of reagents, and it is not modified or inactivated during the process. No incubation period is
required, so measurements are quick and highly reproducible.

The chemical composition of the protein will affect the absorption: the number as well as the
type of amino acids will cause variation. How much a protein absorbs at 280 nm is dependent
on the amount of the amino acids Tyrosine and especially Tryptophan: the aromatic ring of
Phenylalanine absorbs well at 260 nm, but not 280 nm. So proteins of similar molecular weight
can have quite different absorbances, since they can have completely different Tryptophan and
Tyrosine content. UV absorbance of aromatic side chains is also affected by protein structure.
Therefore conditions which affect structure, such as temperature, pH, ionic strength, or the
presence of detergents, can affect the ability of aromatic residues to absorb light at 280 nm,
and change the value of the protein’s extinction coefficient.

As with nucleic acids each protein has its own conversion factor. The common standard protein
bovine serum albumin (BSA) has a factor of 1.551.

Concentration (µg/ml) = Abs280  × Factor

The A260/A280 ratio can be used as a guide to the purity of the sample.

Some instruments also contain factors for other common proteins such as BSA or IgG which
allow users to choose the protein closest in type to their sample, if the factor for the sample
of protein is unknown.
2.2
X: 300
2.0
Y: 0.049
1.8
1.6
1.4
Absorbance

1.2
1.0
0.8
0.6
0.4
0.2 ×
0.0
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
Wavelength (nm)

Fig 5 A typical wavelength scan for a protein sample.

9
Protein concentration calculations
The protein concentration, c, in mg/ml is calculated by:

c = A280/(E280,1 mg/ml) × l)

The absorbance coefficient (E280,1 mg/ml) corresponds to the A280 of a 1 mg/ml solution of the protein
and varies between proteins.

E280,1 mg/ml can be determined:

1. by measuring the absorbance of the protein in a solution of known concentration; or

2. by the theoretical calculation

E280, 1 mg/ml = (5500nTrp + 1490nTyr + 125nS-S)/M

where nTrp, nTyr and nS-S are the number of Trp and Tyr residues, nS-S is the number of disulphide
bonds (S-S bonds) in the protein sequence, and M is the molecular weight of the protein.
Coenzymes and cofactors may also contribute. Examples of values for E280, 1 mg/ml include
0.67 for BSA, 1.37 for IgG, and 2.64 for lysozyme.

Light scattering correction of the A280 value can be made by:

A280 = A280 (measured) – 1.929 × A 330 (measured)

Christian and Warburg (3)

Nucleic acids have absorbance at 280 nm (maximum at 260 nm). If the presence of nucleic acids
is suspected, the protein concentration can be estimated (with less accuracy) according to
Christian, W. and Warburg, O. (1942):

C (mg/ml) = 1.55 × A280 - 0.76 × A260

The constants 1.55 and 0.76 refer to a specific protein used by Christian and Warburg. For best
accuracy, the factors should be determined for the target protein at hand. Refer to the
NanoVue™ Plus User Manual, 28-9574-75 from GE Healthcare.

Colorimetric methods
A wide variety of colorimetic methods for protein concentration measurements are available,
they all work in a similar way.

The reagents contain a dye which binds specifically to the proteins in a solution. The absorption of
a known set of standards is measured and a standard curve produced. Absorption measurements
of unknown samples can then be compared to the curve to establish their concentration.

It is important to check the working ranges of the commercial product being used, as these
can vary.

10
BCA (4)
This method relies on the reaction of cupric ions (Cu2+) and peptide bonds. This forms Cuprous
ions (Cu+) which are then detected with bicinchoninic acid (BCA). This gives an absorbance peak
maximum at 562 nm. Most commercially available kits state that this method is designed to
quantify 125 to 2000 μg/ml.

Biuret (5)
Like BCA, the Biuret method takes advantage of the reaction between cupric ions and peptide
bonds in alkali solution. In this case no additional development step is used. Absorption is
measured at 546 nm. Most commercially available kits state that this method is designed for
the quantification of 1000 to 15 000 μg/ml.

Bradford (6)
This method uses a dye, Coomassie Brilliant Blue, which binds to the protein. The protein
solution shows an increase in absorbance at 595 nm which is proportional to the amount of
bound dye. Bradford is very resistant to interference making it a very common method. Most
commercially available kits state that the useful range of this method is 1 to 1500 μg/ml.

Lowry (7)
Lowry takes advantage of the reaction of Folin-Ciocalteu’s phenol reagent with tyrosyl residues
of an unknown protein. The absorption at 750 nm can then be compared to a standard curve
of a known protein, normally BSA. Most commercially available kits state that this method is
useful to quantify 1 to 1500 μg/ml.

2-D Quant Kit

The 2-D Quant Kit (80-6483-56) is designed to determine protein concentration in samples
to be analyzed by high resolution electrophoresis techniques such as 2-D electrophoresis,
SDS-PAGE or IEF. Many reagents used in preparing such samples, including detergents,
reductants, chaotropes and carrier ampholytes, are incompatible with other protein assays.
The procedure quantitatively precipitates proteins, leaving interfering substances in solution.
The assay is based on the specific binding of copper ions to protein. Precipitated proteins
are resuspended in a copper-containing solution and unbound copper is measured with a
colorimetric agent. The color density is inversely related to the protein concentration. The assay
is linear in the range of 0–50 µg protein. The procedure is compatible with 2% SDS, 1% DTT,
8 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte™ and 2% IPG Buffer. Absorbance is read
at 480 nm.

See the following pages for a summary you can use for choosing your protein assay method.

Please refer to the kit manufacturer’s product data sheets for the exact ranges for your
specific kit.

11
Choosing your protein assay

A280 Direct UV
Working Range 100 µg/ml to approx 100,000 µg/ml
This range is valid for low volume spectrophotometers such as NanoVue
(linearity in NanoVue: 100-100,000 µg/ml ± 100 µg/ml
[based on 2*SD, 20 replicates]
>10000 µg/ml ± 1% [20 replicates, %CV])
For a standard instrument working range is 0.05 to 2000 μg/ml
Advantages Simple, direct UV measure minimises need for dilution
Suited to identifying protein on column fractions
Wide working range

Disadvantages Higher order structure in the proteins will influence the absorption
Detection can be influenced by nucleic acids and other UV-absorbing
contaminants, and by light scattering from particles in the sample

Bradford
Working Range ‘Kits’ 1 to 1500 μg/ml
Advantages Dye-reagent stable for ~1 hour
More stable than A280 (2.5-25 micro assay)

Disadvantages Absorbance spectra of 2 species partially overlap (bound v. unbound)

Stains labware on contact
Susceptible to interference by high detergent concentrations in solution
Precipitates can form
High protein-to-protein signal variability

Lowry
Working Range ‘Kits’ 1 to 1500 μg/ml
Advantages Sensitive over wide working range
Commonly referenced procedure for protein determination
Reaches a stable end-point
Performed at room temperature (RT)

Disadvantages Susceptible to interference from wide range of substances

Reagent time-consuming to prepare
Reagent must be prepared fresh each time
Photosensitive assay
Amount of colour varies with different proteins
Precipitate can form in the presence of detergents
Interference from carbohydrates and some buffers
12
Micro BCA
Working Range ‘Kits’ 1 to 40 µg/ml (standard BCA range 125 to 2000 μg/ml)
Advantages Very sensitive
Not as susceptible to interference from common buffer substances
(eg. many detergents)
Low protein-to-protein variability

Disadvantages Concentrated protein samples need diluting

Sensitive to interference by strong reducing agents
Requires longer incubation (e.g. 1 hr) at higher temperatures
(e.g. 37°C-60°C) to minimise protein-protein variation effects and
improve assay sensitivity

Biuret
Working Range ‘Kits’ 1000 to 15 000 µg/ml
Advantages Wide working range
Single reagent, single incubation, performed at RT – easy to use

Disadvantages Relatively low sensitivity

Relatively large amounts of protein sample needed for accurate analysis
Amino buffer (e.g. Tris) used in pH range 8-10 can interfere with reaction
Sensitive to interference by strong reducing agents

13
Measuring proteins in low volume instruments
NanoVue Spectrophotometer can be used to determine the concentration of protein samples
by a variety of methods including Bradford, BCA, Lowry, Biuret assays, and direct UV measurement
at 280 nm. A comparative functional evaluation of the NanoVue and another low volume
spectrophotometer was performed, to measure protein samples of known and unknown
concentrations at 280 nm and also, with a commercially available Bradford colorimetric
protein assay kit.
A 595
2.5 NanoVue
Other low volume spectropohotmeter
2.0

1.5

1.0

0.5

0.0
0 500 1000 1500 2000
BSA (µg/ml)

Fig 6 Bradford protein assay curves generated from samples measured on NanoVue
and another low volume spectrophotometer (n=3 replicates; mean± 1 SD). The sample
volume used on each instrument was 4µl.

A 280
12 NanoVue
Other low volume spectropohotmeter
10

0
0 2500 5000 7500 10 000 12 500 15 000
BSA (µg/ml)

Fig 7 Protein concentration curve generated from BSA samples measured at 280 nm
using NanoVue and another low volume spectrophotometer (n=3 replicates; mean±
1 standard deviation [SD]). The sample volume used on each instrument was 2µl.

More information can be found in Use of NanoVue spectrophotometer to measure

protein concentrations Application Note 28-9468-37.

14
Other applications
Fluorescent dyes
Many spectrophotometers come with pre-programmed methods for analysis of fluorescent
dyes for DNA or protein quantification. DNA yield can be measured at 260 nm while the
incorporation of fluorescein, Cy™3, Cy5 and other dyes are measured at their respective
absorption peaks. This method is also useful for measuring the yields and brightness of
fluorescently labeled in-situ hybridization probes.

Cell culture
Specialist cell culture spectrophotometers are available for measuring cell density, which can
be used to measure the growth curve of a cultured population to allow harvesting of cells
(typically bacteria) at the optimum point.

The basic method of measurement is to analyse light scatter at 600 nm: the greater the number
of cells, the larger the amount of light scattered as it passes through the cuvette, and if more
light is scattered, less light reaches the detector.

Not all spectrophotometers can measure bacterial cell culture at 600 nm: the instruments need
to be designed to restrict the amount of scattered light that can reach the detectors. More
advanced spectrophotometers also offer background correction at 800 nm and algorithms
to give more consistent results.

Cell culture measurements are not quantitative but are used to gauge the current point of
the cell culture process and identify the peak concentration for most effective harvesting.
As the bacterial population approaches its maximum the growth rate will slow and plateau
before beginning to drop off as the cells begin to die due to lack of nutrients and increase in
toxic waste products from the growth process.

15
The numerical results from different spectrophometer designs are not directly comparable,
but the same pattern of increase, level and decrease will be seen on all instruments.

Instrument 1 Instrument 2

All scattered light hits the detector Not all scattered light hits detector therefore
a higher absorbance reading is obtained

Fig 8 Different spectrophotometers will give different readings when such readings are related to light scatter e.g. for
bacterial culture measurement. Depending on the exact set-up of an individual spectrophotometer, the light scatter,
and thus the absorbance reading, are likely to be different, as seen here.

Features of the instrument design will affect the way in which scattered light can reach the
detector and hence the numerical results e.g. The distance from the sample holder to the
detector lens.

Other molecules
As well as biomolecules such as Proteins and DNA, there are a wide range of light absorbing
substances which it is important to quantify. Spectrophotometers are used in a wide range of
industries from waste water analysis, to pharmaceutical quality control and food analysis. One
such example is the grading of olive oil.

The European regulations ECC/2568/91 and ECC/2472/97 set out the characteristics for grading
of olive oil (extra virgin, virgin etc.) One section looks at the absorption values for 232 nm and
270 nm: this is a measurement of oxidation. To be classed as extra virgin olive oil A 232 has
to be <2.5 and A270 <0.22. For virgin olive oil these values are slightly higher at 2.6 and 0.25.
A spectrophotometer is essential for the grading of olive oil in line with European regulations.

The absorbance spectrum for the molecule to be measured may vary from very narrow
(e.g. <1 nm) to broad (e.g. >3 nm). If extreme accuracy is required, for example in the quality
control of small molecules, it may therefore be necessary to alter the bandwidth of the
instrument accordingly. Instruments such as the Ultrospec 9000 have variable bandwidth to
enable more precise measurements in method development and small molecule measurements.
(For more information, see Bandwidth in glossary).

16
Spectrophotometry hints and tips
Choosing a cuvette
Beam heights
The height at which the light path of the spectrophotometer hits the cuvette is known as the beam
or Z height. Most common beam heights are 8.5 mm, 15 mm, and 20 mm. It is important to use
cuvettes which are designed for the correct beam height, firstly to ensure they are optically
clear at the point the light hits the surface, and secondly because the stated pathlength will be
at this exact point in the cuvette – and may differ at other heights on the cell. Some manufacturers
offer packing pieces to allow the use of cuvettes designed for lower beam heights on their systems.

Choice of cuvettes
The first decision when choosing a cuvette type is whether you wish to use disposable cells
or cuvettes designed to be cleaned and reused. Disposable cuvettes remove the need for
a cleaning process within the laboratory, removing the chance of carry over, but they add
an on-going cost to running the analyser, have higher native absorbencies and may not be
produced to the same precise standards as reusable items.

Reusable cuvettes are much more expensive than their disposable counterparts, and when
using a dual beam spectrophotometer the cuvettes need to be matched to ensure accurate
blank readings.

The second decision is the choice of cuvette material. Cuvettes are generally split into use for
visible only and UV/vis. Normal plastics and glass naturally absorb UV light, so a glass cuvette
would not be suitable for DNA analyses but would be fine for cell culture measurements. UV
cuvettes are more expensive than their visible alternatives as they need to be made from
specialist plastics or quartz glass.

Buffer Compatibility
There are many commonly used processes and methods in use for biological sample preparation
and purification and these often require the use or a detergent or buffer.

Many buffers and detergents absorb light themselves, interfering with the spectrophotometric
analysis of the sample. It is important to take this into account when designing the sample
preparation phase and choose a non-absorbing buffer or one which will not interfere with
the sample analysis.

Studies have found that some detergents have more of an effect on direct UV results than
others. Using detergents such at Brij 35, CHAPS and Tween 20 may give a better results than
Igepal or Triton x-100, but this will vary greatly on the concentration of detergent present in
the sample.

17
Low volume spectrophotometers
Choice of volume
For low volume spectrophotometers, the pathlength is generally much smaller than for cuvette-
based instruments (e.g. 0.1 mm to 1 mm), and may rely on the sample itself to form the
“cuvette”. Therefore contact with both the upper and lower sample plates/heads is critical.
For best results when using volumes below 2 μl with NanoVue, select the 0.2 mm pathlength.
For volumes of 2 μl or above select the 0.5 mm pathlength.

Background correction
If your low volume instrument has the possibility to apply background correction, as with
NanoVue, it should always always be switched on because it can:

• Correct for drop misplacement

• Correct for sample background value of sample. eg. DNA should have zero absorbance
at 320 nm

Any reading at 320 nm can indicate a contaminant or faulty drop placement.

As a rough guide, the expected 320 nm value should be approximately ¹⁄₁₀th of the 260 nm reading.

Background correction is very important when using low volume spectrophotometers. A reading
is taken at a point in the spectrum away from the wavelength of interest and this reading is
subtracted from the main result. This removes the likelihood of any interference.

Sources of interference may include:

• Poor drop placement: This causes incomplete light transition though the sample. If the light
path intersects with the end of the sample, light is refracted, preventing it from reaching
the return optics, so it could be counted as absorption.
• Air bubbles in sample: As with poor drop placement, air drops cause refraction of the light
from its normal path, which can in turn cause some unabsorbed light not to reach the
detector and thus increasing apparent absorption.
• Particles in sample: Particle contamination in a sample causes light scatter, preventing
unabsorbed light reaching the detector. Again this will increase the apparent absorption.

18
Which spectrophotometer?
Some general features and questions to consider when choosing your next instrument are:

How does your data need to be recorded or
stored? Consider whether standard PC
software is suitable or whether
electronic records and audit
trails are required.
How many users will there be, and with what
level of experience? Pre-programmed
methods and password control of
your own methods may
be useful.

What range of sample

volumes do you need What level of
to measure? sensitivity do
you need?
Do you want to
measure multiple
samples at the
same time? What applications
are routinely run in
Do you need your laboratory?
Pharmacopoeia Could this change in
compatibility? the near future?

Consideration of system specification is a fine balancing act between bandwidth, absorbance

range, system noise and cost. Ideally, this would be a narrow bandwidth instrument with a wide
absorbance range, low noise and low cost. However, reducing the bandwidth reduces the
available light, in turn reducing the absorbance range available. To overcome this one could
use a more powerful light source and/or more sensitive detectors, which will increase system
noise, increasing the lower limit of detection. Higher specification electrical systems and
detectors will help reduce noise – but generally at a much higher cost than most laboratories
can afford.

19
GE Healthcare has a range of spectrophotometers for a broad range of scientific needs.

Ultrospec™ 10
for cells Ultrospec 9000
Novaspec™ variable band-width,
basic vis absorption/ advanced applications and
transmission/kinetics method development

GeneQuant™
flexible for DNA, RNA,
protein, cultures
Ultrospec 8000
Spectrophotometer Pharmacopoeia compatible,
dual-beam analysis
range

Ultrospec 7000
Ultrospec 2100
economical dual-beam
lab standard, DNA/RNA, protein,
functionality
kinetics - up to 8 samples

NanoVue™ micro-volume:
drop-measure-done simplicity

20
Spectrophotometer

Fluorescent probe quantitation, cDNA

probes for microarrays, PCR probes
Enzyme activity kinetics at 340 nm
selection guide

Primer quantitation and design

PCR/sequencing/hybridization

Pharma method development

(Bradford, Biuret, BCA etc)
DNA/RNA quantitation

Protein determination

Cell culture OD 600

Model Main features
Ultrospec 9000 High-performance dual-beam, variable
bandwidth UV‑Visible spectrophotometer.
Wavelength range 190‑1100 nm. European
Pharmacopeia compatible. Optional Life Science
or CFR Datrys software upgrade available.

Ultrospec 8000 High-performance dual-beam, 1 nm bandwidth

UV-Visible spectrophotometer. Wavelength
range 190-1100 nm. European Pharmacopeia
compatible. Optional Life Science or CFR
Datrys software upgrade available.

Ultrospec 7000 High-performance dual-beam, 2 nm bandwidth

UV‑Visible spectrophotometer. Wavelength
range 190‑1100 nm. Optional Life Science or
CFR Datrys software upgrade available.

Ultrospec 2100 pro 3 nm bandwidth instrument, wavelength range

190 to 900 nm with 8-position cell changer
and options for temperature control.

NanoVue Plus Novel sample plate. No cuvettes needed.

PC control now available. Low-volume
measurements (0.5 to 5 µl).

GeneQuant 1300 As GeneQuant 100, with built-in enzyme

kinetics, and CyDye™ applications plus
Bluetooth™ data output options.

GeneQuant 100 5 nm bandwidth instrument with built in

applications for nucleic acids, proteins
and cell density.

Novaspec III 7 nm bandwidth visible spectrophotometer,

330 to 800 nm.
Novaspec Plus As Novaspec III, with stored protein methods,
easy to read graphical display, and temperature
control option.

Ultrospec 10 Battery-powered, portable cell density meter.

Key: Recommended Suitable Not suitable

21
GE Healthcare spectrophotometer range
NanoVue Plus GeneQuant 1300 Novaspec III & Plus Ultrospec 10

Small and simple UV/ Good all round UV/Visible Small and simple Portable battery
Visible spectrophotometer instrument with pre- protein measurement operated cell density
for measuring small stored methods for DNA meter for bacterial cell
volumes between 0.5 and protein analysis culture, measuring OD600
and 5 μl
Pre-stored methods
for DNA and proteins,
no cuvettes required
Just Drop, Measure, Done

For most molecular For research and Ideal for teaching labs For cell culture labs
biology and teaching labs and some research labs
biochemistry labs

Versatile UV/Visible Flexible, economical Economical solution, A simple solution

spectro that quickly and solution, multivolume, perhaps multiple for measuring
accurately quantifies perhaps multiple instruments, where bacteria cell cultures,
nucleic acids and protein instruments, UV needed UV is not needed. avoiding contamination
samples, preferably for DNA/RNA measures. Note: Novaspec does not
within seconds. GeneQuant does most do UV, so if UV is needed
If a PC-piloted instrument applications and (if measuring DNA or
is needed, Datrys™ Life capillary cell can go RNA), prefer GeneQuant.
Science software down to 3 μl volumes
is available.

Datrys PC control software

An external PC with appropriate software allows the ultimate in flexibility to control and
manipulate spectrophotometry data. Whether looking for small differences in multiple spectral
overlays, or carrying out post-run manipulations on large numbers of samples, Datrys software
from GE Healthcare has the flexibility to work in the way you want. Available in four options:
Datrys Lite, Datrys Standard, Datrys Life Science and Datrys CFR and compatible with
Windows™ XP, Windows Vista™ and Windows 7 and 8 operating systems. Data export options
include Microsoft Word™ and Excel™ plus Adobe® PDF formats.

Datrys software allows PC control of Ultrospec 7000, 8000 and 9000, and also PC control
a number of other GE spectrophotometers including the NanoVue and GeneQuant.
22
 Ultrospec 2100 NEW Ultrospec 7000 NEW Ultrospec 8000 NEW Ultrospec 9000

UV/Visible 3 nm
bandwidth instrument,
wavelength range
190 to 900 nm, with
an 8-position cell
changer and options
for temperature control
Entry level dual-beam Dual beam, Deuterium Dual beam, Deuterium
instrument, with tungsten light sources, tungsten light sources,
a xenon light source, Touchscreen, USB data Touchscreen, USB
Touchscreen, USB data storage and PC control, data storage and PC
storage and PC control Pharmacopeia compatible, control, Pharmacopeia
option, 2 nm bandwidth 1 nm bandwidth compatible, Advanced
method development,
variable bandwidth
(0.5, 1, 2 and 4 nm)

For research labs, For some pharma, For pharma, some For pharma, high
core facilities some research labs, research labs specification labs
core facilities

Diverse applications Multiple applications, Multiple applications, Method development,

UV/Visible dual beam instrument extreme accuracy, extreme accuracy,
Flexibility for measuring Pharmacopeia Pharmacopeia
multiple samples, including compatibility compatibility
for temperature-controlled
enzyme kinetics

Accessories overview
Different accessories are available to expand the capabilities of GE Healthcare spectrophotometers.
These include multiple cell changers, temperature-regulated cells, sipper system for automated
sample aspiration, test tube holders etc.

A range of cuvettes are available, from capillaries (>3 µl working volume), ultra-microcells
(7 µl volume) to standard quartz cells (up to 2500 µl).

The full range of accessories can be found at www.gelifesciences.com/spectros

23
Glossary
Absorption
Absorption is the amount of light that a substance takes in and does not allow to pass through
it. Spectrophotometers actually measure transmission, the amount of light that passes
through a sample, but this is converted into absorption by comparing the bulb output to the
light that has passed through the sample.

Bandwidth
Bandwidth describes the property of the light emitted from the monochromator. When the
light is analysed, the peak may be at its maximum at the specified wavelength, but light is also
emitted to a lesser extent at the wavelengths either side of the one selected. The Spectral
Bandwidth is a measurement of the width of a peak at half-light intensity (Fig 9).
Intensity

Half Height

Spectral
Bandwidth

Wavelength
Fig 9 The Bandwidth is a measurement of the width of a peak at half-light intensity..

To obtain maximum sensitivity, the bandwidth of the instrument would cover the entire
bandwidth of the substance being measured. When reducing bandwidth, the amount of
available light for absorption is also reduced, so more sensitive detectors are needed to
obtain the same working range.

Increasing bandwidth reduces resolution, and measurement at too broad a bandwidth could also
possibly merge two absorption peaks into one larger one, giving an artificially high reading.

Low bandwidth instruments are very important in highly regulated environments such as
pharmaceutical and Pharmacopoeia laboratories where resolution of 1 nm is often required,
whereas a broader bandwidth is helpful for sensitive DNA quantification where absorption is
strong, and only one, broad, 5 nm peak is being measured.

24
Beam technology
Single beam
Single beam refers to the fact that the light just passes through the sample holder straight
to the detector. An initial reference is required to standardise the instrument before analysis
can begin. Single beam instruments are generally very simple and economical to purchase.
Examples of such instruments are Novaspec III & Plus.

Split Beam
The light from the source is split into two paths, with approximately 30% of the energy
being diverted from the main path into a feedback detector. 70% is then passed through a
monochromator, through the sample compartment to a detector. The feedback detector is
used to correct variations in the energy emitted by the lamp. A reference sample is required
at the start of each run to correct for cuvette and solvent absorption.

Dual Beam
Sometimes known as double beam technology. The light is split into two paths, each of which
passes through a cell holder onto its own detector. One cell holder is for the sample and the
second is for a reference. The reference should contain the same cuvette type and solvent as
the sample. The absorption of the reference path is subtracted from that of the sample path.
Every measurement has its own reference, giving more reliable results. GE Healthcare
Ultrospec 7000, 8000 and 9000 are all Dual beam instruments.

Light source
Deuterium
Deuterum arc lamps provide powerful and stable light in the UV region, 190-370 nm (Fig 10).
In higher specification spectrophotometers a special design of deuterium lamp is used to
maximise life without reducing output, called “press to read” (Pulse) technology. This allows the
lamp to go into a low power mode, where the lamp is kept warm but is not actually on. This can
increase the lamp life up to 3 times that of a conventional deuterium lamp. The deuterium
lamps of the GE Healthcare Ultrospec 8000 and 9000 can be changed by the researcher in
the lab. Deuterium lamps are paired with tungsten lamps to allow coverage of the full UV and
visible spectrum.

Tungsten
Tungsten lamps give stable light in the visible and near infra-red areas of the spectrum,
covering 320-1100 nm (Fig 10). GE tungsten lamps also employ press to read (Pulse)
technology to extend lamp life, and the lamps in the Ultrospec 8000 and 9000 can also be
changed by the researcher in the lab.

25
 Deuterium
arc lamp
Energy ➔

Tungsten
halogen lamp

200 400 600 800

λ nm
Fig 10 Both Deuterium and Tungsten lamps are required in order to cover the complete wavelength range from 190 nm
to 1100 nm.

Xenon
Xenon flash lamps are a high energy light source emitting light across the UV, Visible and near
IR spectrum. They are known as flash lamps as they are not on constantly but flash, up to 80
times a second. Xenon lamps have a very long life span, but don’t provide the optical stability
of the deuterium/tungsten set up in the higher specification systems. Xenon lamps must be
changed by a service engineer. The entry-level dual beam Ultrospec 7000 instrument has
a xenon lamp.

LED
Used for single wavelength applications, such as cell culture measurement in the Ultrospec 10.
LEDs are stable, low cost and offer a long life.

Methods of measurement
Single wavelength
A simple method which allows measurement of transmittance (t%) or absorbance of a
substance at a single fixed wavelength. E.g. 260 nm for DNA.

Wavelength scan
Measurements for a range of wavelengths are taken, allowing the system to plot absorbance
against wavelength and produce a graph. Particularly useful for analysing an unknown
sample. The start and finish wavelengths can be set to target the scan to a region or the
available spectrum.

Kinetics
A single wavelength measurement is repeated at set intervals for a set number of times.
The absorption is then plotted against time. This can be used to measure reaction progress,
or for enzyme activity studies when combined with a temperature-controlled cell holder.

26
Quantitative analysis
A single wavelength measurement of a number of known standards can be taken, from which
the instrument creates a standard curve, to which future measurements of unknown samples
can be compared to establish their concentration.

Cell Density
Light scatter from bacterial cell cultures can be used as an indication of cell concentration and
when plotted over time can be used to see if the culture is ready to be harvested. Measurements
are taken at 600 nm.

Monochromator
In a UV/Vis spectrophotometer, a monochromator is the optical device that transmits a
mechanically selected band of wavelengths of light from a wider range of wavelengths
available from the light source. From the Greek mono (single) and chroma (colour).

Pathlength
Pathlength is the distance through the sample, through which the light has to pass, in order to
reach the detector. Standard cuvettes have a pathlength of 10 mm (1 cm). GE offers instruments
covering pathlengths from 0.2 mm all the way to 100 mm.

Stray Light
Stray light is light that reaches the detector that is of a different wavelength to that the
monochromator is selecting. This can be caused by the diffraction pattern produced by the
monochromator or from light leaks from the outside environment. GE systems are designed
to reduce all possible sources of stray light.

UV/Vis
The electromagnetic spectrum is split into a number of different regions by wavelength. These
include X-ray (0.01-10 nm) Visible light (380-760 nm) and Microwaves (1-10cm). GE UV/Vis
spectrophotometers all measure in the UV (10-400 nm) visible (380-760 nm) and near infra-red
(750-2250 nm) regions covering a range of 190-1100 nm.

Wavelength
Represented by the lower case Greek letter lambda λ. Wavelength is a measurement of the
distance between successive peaks in a waveform.

Wavelength
λ

Distance
27
References
1. J.H. Lambert, (1760) Photometria sive de mensura et gradibus luminis, colorum et umbrae [Photometry, or, On the
measure and gradations of light, colors, and shade] (Augsburg (“Augusta Vindelicorum”), Germany: Eberhardt Klett).
2. Beer (1852) Bestimmung der Absorption des rothen Lichts in farbigen Flüssigkeiten (Determination of the absorption
of red light in colored liquids), Annalen der Physik und Chemie, vol. 86, pp. 78–88.
3. Warburg, O. and Christian, W. (1942) Isolierung und Kristallisation des Garungsferments Enolase.
Biochem. Z. 310:384-421.
4. Smith, P.K., et al. (1985). “Measurement of protein using bicinchoninic acid”. Anal. Biochem. 150 (1): 76–85. 3843705.
5. Layne, E. (1957) Spectrophotometric and Turbidimetric Methods for Measuring Proteins.
Methods in Enzymology 10: 447-455.
6. Bradford, M.M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal. Biochem. 72:248-254.
7. Lowry, O.H., Rosebrough, J.J., Farr, A.L., and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent.
J.Biol. Chem. 193:265-275.

28
Related products
GE Healthcare’s spectrophotometer range and life sciences consumables are now available
from your preferred supplier. Contact your local GE Healthcare representative for more details.

illustra™ Nucleic Acid Preparation

Purification (plasmid DNA, genomic DNA, RNA, sequencing
clean-up), GenomiPhi™ amplification, ExoStar™, GFX ™
(PCR clean-up), Ready-To-Go™ technology

Protein Sample Preparation

Mag Sepharose™, MiniTrap™, MultiTrap™, SpinTrap™, GraviTrap™ for
simple protein sample prep, including for recombinant proteins
and antibodies

Chromatography media and protein purification

TALON™, HisTrap™, GSTrap™, HiTrap™, Tricorn™,
MonoBeads™ complete range of media and
columns for protein purification

Amersham™ DNA and Protein Labeling, Blotting

and Detection
Cy dyes, HyPer™5 microarray dyes for array CGH and gene
expression, ECL™ Labeling and Detection range ECL Gel Box,
Rainbow™ Markers, Hybond™ Membranes Hyperfilm™

Whatman™ filtration and

sample prep range
FTA™, e-Core™, Whatman™ sterile syringe
filters, Puradisc FP30 for buffer and sample
filtration, Whatman GD/X ™ for filtration of
particulate loaded samples

29

For local office contact information, visit:
www.gelifesciences.com/contact
www.gelifesciences.com/spectros
GE Healthcare UK Limited
Amersham Place
Little Chalfont,
Buckinghamshire HP7 9NA, UK
GE, imagination at work and GE monogram are trademarks
of General Electric Company.
Cy,CyDye, Datrys, e-Core, ECL, ExoStar, FTA, GeneQuant,
GenomiPhi, GFX, GraviTrap, GSTrap, HisTrap, HiTrap, Hybond,
Hyperfilm, illustra, Mag Sepharose, MiniTrap, MonoBeads,
MultiTrap, NanoVue, Novaspec, Pharmalyte, Rainbow,
Ready-To-Go, SpinTrap, TALON, Tricorn, Whatman and
Whatman GD/X are trademarks of GE Healthcare companies.
Ultrospec is a trademark of Biochrom Ltd. Adobe is a
registered trademark of Adobe Systems Incorporated
in the United States. Microsoft, Windows, and Vista are
trademarks of Microsoft Corporation. Bluetooth is a
trademark of Bluetooth Special Interest Group (SIG).
© 2012 General Electric Company—All rights reserved.
First published October 2012.
29-0331-82 AA 10/2012