PRINCIPLES OF CHROMATOGRAPHY

SEC COLUMNS The analysis, isolation, and purifi­cation of biomolecules can be

accomplished by a number of chromatographic modes. Each mode
is based on specific physical, chemical, or biological interactions
between the sample biomolecule and packing material.

Tosoh Bioscience offers a comprehensive line of TSKgel and

TOYOPEARL media and pre-packed TSKgel columns for all com-
mon modes of liquid chromatography.

The various modes of chromato­graphy involve separations that are

based on specific features of the target or sample, like size, charge,
hydropho­­
bi­
city, function or specific content of the molecule.
To find out more about general principles of liquid chromatog-
raphy and on how each of them works, visit us on our YouTube
channel [Link]

TOSOH BIOSCIENCE

Im Leuschnerpark 4 64347 Griesheim, Germany SIZE EXCLUSION CHROMATOGRAPHY

Tel: +49 6155-7043700 Fax: +49 6155-8357900
[Link]@[Link] [Link]
B15L34A
 PRINCIPLES OF CHROMATOGRAPHY

SEC COLUMNS The analysis, isolation, and purifi­cation of biomolecules can be

accomplished by a number of chromatographic modes. Each mode
is based on specific physical, chemical, or biological interactions
between the sample biomolecule and packing material.

Tosoh Bioscience offers a comprehensive line of TSKgel and

TOYOPEARL media and pre-packed TSKgel columns for all com-
mon modes of liquid chromatography.

The various modes of chromato­graphy involve separations that are

based on specific features of the target or sample, like size, charge,
hydropho­­
bi­
city, function or specific content of the molecule.
To find out more about general principles of liquid chromatog-
raphy and on how each of them works, visit us on our YouTube
channel [Link]

TOSOH BIOSCIENCE

Im Leuschnerpark 4 64347 Griesheim, Germany SIZE EXCLUSION CHROMATOGRAPHY

Tel: +49 6155-7043700 Fax: +49 6155-8357900
[Link]@[Link] [Link]
B15L34A
 TOSOH BIOSCIENCE ANALYSIS

1 2 3 SEC
OPTIMIZING SEC
TOSOH BIOSCIENCE GMBH TOSOH BIOSCIENCE LLC TOSOH CORPORATION
IM LEUSCHNERPARK 4 3604 HORIZON DRIVE, 3-8-2 SHIBA, MINATO-KU

SEC
64347 GRIESHEIM SUITE 100 TOKYO 105-8623
GERMANY KING OF PRUSSIA, PA 19406, USA JAPAN

ABOUT US
T + 49 (0) 6155 70437 00 T +1 484 805 1219 T +81 3 5427 5118 SAMPLE LOAD
F + 49 (0) 6155 83579 00 F +1 610 272 3028 F +81 3 5427 5198
[Link]@[Link] [Link]@[Link] INFO@[Link]
[Link] [Link] [Link] As SEC is a partition chromatography, sample load on the For TSKgel SW columns mobile phases a buffer concen-
column is limited. High sample loads distort peak shapes and tration between 0.1 M and 0.5 M is recommended. Under
cause an overall decrease in efficiency due to column over- low ionic strength (< 0.1 M), ionic interactions between the
load. Optimal sample load highly depends on the sample sample molecules and the silica surface may occur. Under
WITH A GLOBAL PERSPECTIVE. properties (sample matrix) and the separation task. For conditions of high ionic strength (>1.0 M), hydrophobic
analytical columns, sample concentrations of 1-20 mg/ml are interactions are more likely to occur. A neutral salt, such
Tosoh Bioscience is a leading manufacturer in the field of liquid recommended. Proteins can be loaded at higher concentra- as sodium sulphate may be added to the buffer to increase
chromatography. The portfolio of over 500 specialty products en- tions and higher total loads than synthetic macromolecules.
For preparative purposes for example, 100 mg of BSA can
buffer ionic strength. Also the ionic species of the buffer
has an effect on the separation. As a good starting point, a
compasses instruments for size exclusion/gel permeation chroma- be loaded on two 21.5 mm ID x 60 cm L TSKgel G3000SW 0.1 M sodium phosphate buffer together with 0.1 M sodium
columns, but only 20 mg of PEG 7500. sulphate has proved to be of value.
tography and a comprehensive line of media and prepacked (U)HPLC 1
Sample volume depends very much on the type of column.
On TSKgel SuperSW columns for example, a 5 µL injection As the polymeric TSKgel PW and Alpha-type resins carry
columns for all common modes of liquid chromatography. Over the 2 3
volume ensures optimal results. Standard injection volumes less residual charged groups on the surface than silica
for 7.5 and 7.8 mm ID columns are 20-100 µl, whereas for gels, salt concentration of the mobile phase can be lower.
last 40 years, TSKgel SW columns have become the worldwide in- 4
preparative purposes on 21.5 mm ID columns, injection Non-ionic, non-polar compounds such as polyethylene
volumes may be raised up to 2 ml. glycols can simply be analyzed with distilled water. For ionic
dustry standard for size exclusion chromatography of biomolecules. polymeric compounds, a neutral salt such as sodium nitrate
MOBILE PHASE is added to the aqueous eluent. Generally, a concentration
5 of 0.1 M to 0.2 M is sufficient to overcome undesirable ionic
Proper selection of the mobile phase is necessary to maximize interactions.
Tosoh manufacturing sites in Japan provide products to the sales molecular sieving mechanism and to minimize secondary
and support subsidiaries in the U.S. and Europe, ensuring full effects such as ionic and hydrophobic interaction between
the sample and the column packing material. For each sample
If hydrophobic interaction occurs between the sample and
the column matrix, a water soluble organic solvent can be
global coverage. Our technical specialists in the European Head- there will be an optimum buffer type and concentration that added to the mobile phase. The addition of acetonitrile,
results in the highest resolution and recovery. acetone, ethanol or methanol up to a concentration of 20%
quarters provide assistance in developing HPLC applications or may also prevent columns from fouling by suppressing
interaction of hydrophobic impurities of the sample. An
purification methods, in up-scaling, or packing process columns. 4
TOSOH BIOSCIENCE SHANGHAI CO. LTD.
5
TOSOH ASIA PTE. LTD.
FIGURE 24 example is shown in Figure 24 with the analysis of a pegylat-
ed protein on a TSKgel G3000SWXL column. As pegylated
We offer chromatographic workshops, on-site training, and are ROOM 301, PLAZA B,
NO. 1289 YI SHAN ROAD
63 MARKET STREET #10-03
BANK OF SINGAPORE CENTRE products are more hydrophobic, they tend to interact with
XU HUI DISTRICT SINGAPORE 048942, SINGAPORE the column matrix. Over time the pegylated product can foul
the sole sponsor of the HIC/RPC Bioseparation Conference series.
200 200
SHANGHAI, 200233, CHINA
T +86 21 3461 0856 T +65 6226 5106
the column, which is indicated by shifts of retention time
and decreasing separation performance. By adding 10% of

Intensity (mAu)

Intensity (mAu)
F +86 21 3461 0858 F +65 6226 5215
150 150
INFO@[Link] [Link]@[Link] ethanol to the elution buffer, this problem is overcome and no
[Link] [Link]
differences in performance at the first and the 150th injection
100 100
are observed (courtesy of J.J. Ratto et al. Amgen Inc., 1996).
TOSOH HISTORY
1935 TOYO SODA MANUFACTURING CO., LTD. IS FOUNDED 50 50 COLUMN PROTECTION
1936 OPERATION OF NANYO MANUFACTURING COMPLEX BEGINS
1971 SCIENTIFIC INSTRUMENTS DIVISION DEVELOPS FIRST GPC COLUMN USING TSKgel 0 0 To protect the column and increase its lifetime, the use of
1974 HPLC COLUMN PLANT IS COMPLETED 7 8 9 10 11 12 13 7 8 9 10 11 12 13 a guard column is strongly recommended. Sample purity,
1979 TOSOH DEVELOPS TOYOPEARL MEDIA Elution time (min) Elution time (min) sample load and the composition of the mobile phase have
1983 TOSOH DEVELOPS HYDROPHOBIC INTERACTION MEDIA an influence on column lifetime. For information on TSKgel
1987 TOSOHAAS US STARTS OPERATING FROM MONTGOMERYVILLE INFLUENCE OF MOBILE PHASE SEC columns for GPC analysis of organic polymers please
1989 TOSOHAAS GmbH STARTS OPERATING FROM STUTTGART refer to the TSKgel GPC column brochure.
A: No ethanol in mobile phase; B: 10% ethanol in mobile phase
1995 TOSOH NANYO GEL FACILITY RECEIVES ISO 9001 Column: TSKgel G3000SWXL columns, 5 µm, 7.8 mm ID x 30 cm L
2000/2001 FORMER TOSOHAAS US AND EUROPE OPERATIONS BECOME TOSOH BIOSEP, A 100% SUBSIDIARY OF TOSOH CORPORATION Sample: 10 mL PEG r-HuMGDF;
2002/ 2003 ALL SCIENTIFIC & DIAGNOSTIC SYSTEM RELATED COMPANIES IN EUROPE AND THE US ARE UNIFIED UNDER THE NEW NAME TOSOH BIOSCIENCE
initial injection; after 150 injections
Mobile phase: 0.1 M sodium phosphate, pH 6.9, 0.5 M NaCl,
2008 EcoSEC, THE 7 TH GENERATION GPC SYSTEM IS INTRODUCED Flow rate: 0.7 mL/min; Detection: UV @ 220 nm
2009 TOSOH BIOSCIENCE GmbH CELEBRATES ITS 20TH ANNIVERSARY
2010 TOSOH CELEBRATES ITS 75 TH YEAR IN BUSINESS AND CONTINUED RAPID EXPANSION IN CHINA
2011 TOSOH BIOSCIENCE CELEBRATES 40 YEARS OF OPERATION
2012 TOSOH RELEASES FIRST TOYOPEARL MIXED-MODE RESIN
2013 TOSOH RELEASES A HIGH CAPACITY PROTEIN A RESIN
2014 TOSOH BIOSCIENCE GmbH CELEBRATES ITS 25 TH ANNIVERSARY
2015 TOSOH BIOSCIENCE GmbH MOVES TO GRIESHEIM, GERMANY
 TOSOH BIOSCIENCE ANALYSIS

1 2 3 SEC
OPTIMIZING SEC
TOSOH BIOSCIENCE GMBH TOSOH BIOSCIENCE LLC TOSOH CORPORATION
IM LEUSCHNERPARK 4 3604 HORIZON DRIVE, 3-8-2 SHIBA, MINATO-KU

SEC
64347 GRIESHEIM SUITE 100 TOKYO 105-8623
GERMANY KING OF PRUSSIA, PA 19406, USA JAPAN

ABOUT US
T + 49 (0) 6155 70437 00 T +1 484 805 1219 T +81 3 5427 5118 SAMPLE LOAD
F + 49 (0) 6155 83579 00 F +1 610 272 3028 F +81 3 5427 5198
[Link]@[Link] [Link]@[Link] INFO@[Link]
[Link] [Link] [Link] As SEC is a partition chromatography, sample load on the For TSKgel SW columns mobile phases a buffer concen-
column is limited. High sample loads distort peak shapes and tration between 0.1 M and 0.5 M is recommended. Under
cause an overall decrease in efficiency due to column over- low ionic strength (< 0.1 M), ionic interactions between the
load. Optimal sample load highly depends on the sample sample molecules and the silica surface may occur. Under
WITH A GLOBAL PERSPECTIVE. properties (sample matrix) and the separation task. For conditions of high ionic strength (>1.0 M), hydrophobic
analytical columns, sample concentrations of 1-20 mg/ml are interactions are more likely to occur. A neutral salt, such
Tosoh Bioscience is a leading manufacturer in the field of liquid recommended. Proteins can be loaded at higher concentra- as sodium sulphate may be added to the buffer to increase
chromatography. The portfolio of over 500 specialty products en- tions and higher total loads than synthetic macromolecules.
For preparative purposes for example, 100 mg of BSA can
buffer ionic strength. Also the ionic species of the buffer
has an effect on the separation. As a good starting point, a
compasses instruments for size exclusion/gel permeation chroma- be loaded on two 21.5 mm ID x 60 cm L TSKgel G3000SW 0.1 M sodium phosphate buffer together with 0.1 M sodium
columns, but only 20 mg of PEG 7500. sulphate has proved to be of value.
tography and a comprehensive line of media and prepacked (U)HPLC 1
Sample volume depends very much on the type of column.
On TSKgel SuperSW columns for example, a 5 µL injection As the polymeric TSKgel PW and Alpha-type resins carry
columns for all common modes of liquid chromatography. Over the 2 3
volume ensures optimal results. Standard injection volumes less residual charged groups on the surface than silica
for 7.5 and 7.8 mm ID columns are 20-100 µl, whereas for gels, salt concentration of the mobile phase can be lower.
last 40 years, TSKgel SW columns have become the worldwide in- 4
preparative purposes on 21.5 mm ID columns, injection Non-ionic, non-polar compounds such as polyethylene
volumes may be raised up to 2 ml. glycols can simply be analyzed with distilled water. For ionic
dustry standard for size exclusion chromatography of biomolecules. polymeric compounds, a neutral salt such as sodium nitrate
MOBILE PHASE is added to the aqueous eluent. Generally, a concentration
5 of 0.1 M to 0.2 M is sufficient to overcome undesirable ionic
Proper selection of the mobile phase is necessary to maximize interactions.
Tosoh manufacturing sites in Japan provide products to the sales molecular sieving mechanism and to minimize secondary
and support subsidiaries in the U.S. and Europe, ensuring full effects such as ionic and hydrophobic interaction between
the sample and the column packing material. For each sample
If hydrophobic interaction occurs between the sample and
the column matrix, a water soluble organic solvent can be
global coverage. Our technical specialists in the European Head- there will be an optimum buffer type and concentration that added to the mobile phase. The addition of acetonitrile,
results in the highest resolution and recovery. acetone, ethanol or methanol up to a concentration of 20%
quarters provide assistance in developing HPLC applications or may also prevent columns from fouling by suppressing
interaction of hydrophobic impurities of the sample. An
purification methods, in up-scaling, or packing process columns. 4
TOSOH BIOSCIENCE SHANGHAI CO. LTD.
5
TOSOH ASIA PTE. LTD.
FIGURE 24 example is shown in Figure 24 with the analysis of a pegylat-
ed protein on a TSKgel G3000SWXL column. As pegylated
We offer chromatographic workshops, on-site training, and are ROOM 301, PLAZA B,
NO. 1289 YI SHAN ROAD
63 MARKET STREET #10-03
BANK OF SINGAPORE CENTRE products are more hydrophobic, they tend to interact with
XU HUI DISTRICT SINGAPORE 048942, SINGAPORE the column matrix. Over time the pegylated product can foul
the sole sponsor of the HIC/RPC Bioseparation Conference series.
200 200
SHANGHAI, 200233, CHINA
T +86 21 3461 0856 T +65 6226 5106
the column, which is indicated by shifts of retention time
and decreasing separation performance. By adding 10% of

Intensity (mAu)

Intensity (mAu)
F +86 21 3461 0858 F +65 6226 5215
150 150
INFO@[Link] [Link]@[Link] ethanol to the elution buffer, this problem is overcome and no
[Link] [Link]
differences in performance at the first and the 150th injection
100 100
are observed (courtesy of J.J. Ratto et al. Amgen Inc., 1996).
TOSOH HISTORY
1935 TOYO SODA MANUFACTURING CO., LTD. IS FOUNDED 50 50 COLUMN PROTECTION
1936 OPERATION OF NANYO MANUFACTURING COMPLEX BEGINS
1971 SCIENTIFIC INSTRUMENTS DIVISION DEVELOPS FIRST GPC COLUMN USING TSKgel 0 0 To protect the column and increase its lifetime, the use of
1974 HPLC COLUMN PLANT IS COMPLETED 7 8 9 10 11 12 13 7 8 9 10 11 12 13 a guard column is strongly recommended. Sample purity,
1979 TOSOH DEVELOPS TOYOPEARL MEDIA Elution time (min) Elution time (min) sample load and the composition of the mobile phase have
1983 TOSOH DEVELOPS HYDROPHOBIC INTERACTION MEDIA an influence on column lifetime. For information on TSKgel
1987 TOSOHAAS US STARTS OPERATING FROM MONTGOMERYVILLE INFLUENCE OF MOBILE PHASE SEC columns for GPC analysis of organic polymers please
1989 TOSOHAAS GmbH STARTS OPERATING FROM STUTTGART refer to the TSKgel GPC column brochure.
A: No ethanol in mobile phase; B: 10% ethanol in mobile phase
1995 TOSOH NANYO GEL FACILITY RECEIVES ISO 9001 Column: TSKgel G3000SWXL columns, 5 µm, 7.8 mm ID x 30 cm L
2000/2001 FORMER TOSOHAAS US AND EUROPE OPERATIONS BECOME TOSOH BIOSEP, A 100% SUBSIDIARY OF TOSOH CORPORATION Sample: 10 mL PEG r-HuMGDF;
2002/ 2003 ALL SCIENTIFIC & DIAGNOSTIC SYSTEM RELATED COMPANIES IN EUROPE AND THE US ARE UNIFIED UNDER THE NEW NAME TOSOH BIOSCIENCE
initial injection; after 150 injections
Mobile phase: 0.1 M sodium phosphate, pH 6.9, 0.5 M NaCl,
2008 EcoSEC, THE 7 TH GENERATION GPC SYSTEM IS INTRODUCED Flow rate: 0.7 mL/min; Detection: UV @ 220 nm
2009 TOSOH BIOSCIENCE GmbH CELEBRATES ITS 20TH ANNIVERSARY
2010 TOSOH CELEBRATES ITS 75 TH YEAR IN BUSINESS AND CONTINUED RAPID EXPANSION IN CHINA
2011 TOSOH BIOSCIENCE CELEBRATES 40 YEARS OF OPERATION
2012 TOSOH RELEASES FIRST TOYOPEARL MIXED-MODE RESIN
2013 TOSOH RELEASES A HIGH CAPACITY PROTEIN A RESIN
2014 TOSOH BIOSCIENCE GmbH CELEBRATES ITS 25 TH ANNIVERSARY
2015 TOSOH BIOSCIENCE GmbH MOVES TO GRIESHEIM, GERMANY
 TOSOH BIOSCIENCE ANALYSIS 1

SEC
SIZE EXCLUSION CHROMATOGRAPHY

SEC
Size exclusion chromatography (SEC) separates molecules Tosoh Bioscience offers a broad portfolio SEC columns
based on their size, or more precisely, their hydrodynamic packed with silica or polymer based porous beads. They
volume. It is usually applied to large molecules such as are well suited for a wide range of applications in R&D,
proteins or synthetic polymers. When an aqueous mobile method development and quality control. TSKgel SW, SWXL,
phase is used, SEC is also referred to as gel filtration SuperSW, UltraSW, and UP-SW are silica SEC phases with
chromatography (GFC). When an organic eluent is applied, pore size distributions suited to protein separations. TSKgel
SEC is referred to as gel permeation chromatography (GPC). SW-type packings feature low adsorption and well-defined
GPC is typically used to determine the molecular weight pore size distribution. It is the leading SEC column series for
(MW) and the MW distribution of synthetic polymers while (U)HPLC due to its excellent resolution.
GFC is used to separate biopolymers based on their size.
Polymeric TSKgel PW and PWXL columns are designed for
Aqueous SEC is a popular technique for the separation and GFC of water soluble organic polymers, polysaccharides,
purification of proteins because of its effectiveness and oligosaccharides, DNA and RNA. The TSKgel Alpha and
non-denaturing mobile phase conditions. It is popular for SuperAW series, based on a unique hydrophilic, polyvinyl
the isolation of proteins, removal of aggregates, desalting resin, is suited for SEC of water-soluble and polar organic-
or characterization of water-soluble polymers used in food soluble polymers. TSKgel columns for gel permeation
products, paints, pharmaceutical formulations and the like. chromatography of organic soluble polymers are described
Stationary phases for aqueous SEC range from soft packing in a separate brochure on TSKgel GPC columns.
materials, such as dextran or agarose, over hydrophilic
polymers to silica. Soft particles were employed as stationary Tosoh Corporation employs state-of-the-art manufacturing
phases for early GFC whereas today porous silica particles techniques that result in uniformly bonded packing materials
with high mechanical strength are applied for aqueous SEC with narrow pore size distributions and well-defined particle
in high performance liquid chromatography (HPLC). sizes to ensure high performance and efficiency.
2 [Link]

SEC
HOW IT WORKS
SEC

Size exclusion chromatography (SEC) is a method in which

components of a mixture are separated according to their
molecular size, based on the flow of the sample through
a porous packing. In contrast to all other modes of liquid
chromatography the prerequisite for SEC is that the analyte
does not interact with the surface of the stationary phases.
Differences in elution time are ideally based solely on the
volume the analyte passes.

Large biomolecules that cannot penetrate the pores of the

packing material elute first from the column. They are said
to be excluded from the packing; they flow with the mobile
phase in the interparticle space of the packed column. The
exclusion limit characterizes the upper limit of molecular
weight (or size), beyond which molecules will elute at the
same retention volume called the exclusion or void volume
of the column. Many SEC columns are referred to by their
exclusion limit.

Smaller molecules can partially or completely enter the

porous particles. Because these smaller molecules have
to flow through the interparticle space, as well as through
the pore volume, they will elute from the column after the
excluded sample components. Molecules small enough to
penetrate the whole pore system of the stationary phase
will pass the entire pore and interparticle volume, and will
elute late. Their retention volume is referred to as ‘total
permeation’ in SEC, whereas it is interpreted as ‘unretained
peak’ in conventional LC modes.

SEC is a very simple method for separating biomolecules,

because it is not necessary to change the composition of
the mobile phase during elution. However, the separation
capacity of this method is limited. For a baseline separation
it is necessary that the molecular weights of the molecules
differ by at least 10 to 20 %.
 TOSOH BIOSCIENCE ANALYSIS 3

SEC
TSKgel SEC COLUMNS

SEC
Tosoh Corporation has a proud history of innovation in size TSKgel SW-type packings feature low adsorption and well-
exclusion chromatography. TSKgel SEC columns are known defined pore size distribution. They are the leading SEC
worldwide for their reliability and suitability for the analysis columns in bioanalysis due to their excellent resolution.
of proteins, peptides and other biological macro-molecules. The new TSKgel UP-SW columns offer the opportunity to
The complete TSKgel SW, PW, Alpha and SuperAW column transfer existing TSKgel SW series HPLC methods to UHPLC.
lines consist of either silica based or polymer based packings,
ranging in particle size from 2 μm to 20 μm. Columns are TSKgel PW SERIES
available in analytical through semi- preparative size, in
stainless steel, PEEK or glass. TSKgel PW and PWXL columns are packed with hydrophilic,
rigid polymethacrylate beads. They are commonly used for
COLUMN SELECTION the separation of synthetic water soluble polymers because
they exhibit a much larger separation range, better linearity
The main criterion in choosing between the TSKgel SW, PW, of calibration curves, and less adsorption than the TSKgel
Alpha and SuperAW SEC columns is the molecular weight SW columns. While a TSKgel SW column is typically the first
of the sample and its solubility. The fact that the TSKgel SW column to try for biopolymers, TSKgel PW columns have
columns are based on silica and the TSKgel PW, Alpha and demonstrated good results for smaller peptides (<1,000 Da),
SuperAW columns are derived from a hydrophilic polymer protein aggregates, DNA fragments, and viruses. TSKgel
network has less impact on the separation than the particle PWXL-CP columns are especially suited for the separation of
and pore size differences. cationic polymers at low salt.

TSKgel SW SERIES TSKgel AW/ALPHA SERIES

Tosoh Bioscience TSKgel SW, SWXL, SuperSW, UltraSW, The TSKgel Alpha series columns offer a new alternative for
and UP-SW series are silica SEC phases with pore size performing SEC. Their compatibility with a wide range of
distributions suited to protein separations. A hydrophilic diol- solvents makes them useful for both GFC and GPC. TSKgel
type bonded phase shields the silica surface from interacting SuperAW columns are based on the same chemistry as
with protein samples. Due to their high resolving power, the Alpha columns but have smaller particle sizes and
TSKgel SW columns are suitable for the separation of mono- shorter, narrower column dimensions for high throughput
disperse biopolymers such as proteins and nucleic acids. applications.

TABLE 1

CHARACTERISTICS OF TSKgel SIZE EXCLUSION COLUMN LINES

Column line TSKgel SW / SWXL / TSKgel PW / TSKgel Alpha /

SuperSW/UltraSW/UP-SW PWXL SuperAW

Resin type Silica Polymethacrylate highly crosslinked Polymethacrylate

No. of available pore sizes 3/3/2/1/1 7 5

PH stability 2.5 - 7.5 2.0 - 12.0 2.0 - 12.0

Solvent compatability 100% polar 50% polar 100% polar, and nonpolar

Max. temp. 30°C 80°C* 80°C

Pressure**(MPa) 1.0 - 34.0 1.0 - 4.0 2.0 - 4.0

Application focus Proteins Water-soluble Intermediate

polymers polar polymers

* Except for the TSKgel G-DNA-PW, which can be operated up to 50°C. When operating below 10°C, reduce the flow rate to ensure that
the maximum pressure is not exceeded.

** Depends on column dimensions and particle size

Note: The operating conditions and specifications for each column are listed on the Operating Conditions and Specifications sheet (OCS)
shipped with the column.
4 [Link]

SEC
TSKgel SEC COLUMN SELECTION
SEC

SAMPLE COLUMN SELECTION SELECTION CRITERIA

FIRST CHOICE ALTERNATIVE
Carbohydrates polysaccharides TSKgel GMPWXL TSKgel G5000PWXL & large pore size, small particles, linear
TSKgel SuperMultiporePW TSKgel G3000PWXL calibration curve, high resolving
power
oligosaccharides TSKgel G-Oligo-PW TSKgel G2500PWXL small particles, high resolving
TSKgel SuperOligoPW power
Nucleic DNA fragments large TSKgel G-DNA-PW or large pore size, small particles,
acids TSKgel G5000PWXL high resolving power
medium TSKgel G4000SWXL, suitable pore sizes
and small TSKgel BioAssist G4SWXL
TSKgel SuperSW3000 or
TSKgel G3000SWXL TSKgel BioAssist G3SWXL
RNA TSKgel G4000SWXL TSKgel BioAssist G4SWXL suitable pore sizes
TSKgel SuperSW3000 or
TSKgel G3000SWXL TSKgel BioAssist G3SWXL
oligonucleotides TSKgel G2500PWXL small pore size, ionic interaction
Proteins small to medium TSKgel UP-SW3000 small particles
sized proteins TSKgel SuperSW3000 TSKgel G3000/G4000PWXL small to medium range pore sizes
TSKgel G3000SWXL TSKgel BioAssist G3SWXL
TSKgel G4000SWXL TSKgel BioAssist G4SWXL
TSKgel SuperSW2000 or
TSKgel G2000SWXL TSKgel BioAssist G2SWXL
antibodies TSKgel SuperSW mAB HR/HTP fragments/monomer & dimer
TSKgel UP-SW3000 higher aggregates
TSKgel UltraSW Aggregate
large proteins low density TSKgel G6000PWXL or large pore sizes
lipoprotein TSKgel G5000PWXL
gelatin TSKgel GMPWXL TSKgel G5000PWXL & large pore size,
TSKgel SuperMultiporePW-M G3000PWXL linear calibration curve
TSKgel G3000SWXL
Peptides large TSKgel SuperSW3000 TSKgel SuperSW2000 / small to medium
TSKgel G3000SWXL TSKgel G3000PWXL range pore size, versatile
TSKgel BioAssist G3SWXL or
TSKgel G2000SWXL TSKgel BioAssist G2SWXL
small TSKgel G2500PWXL TSKgel SuperSW2000 / linear calibration curve,
TSKgel G2000SWXL high resolving power
Viruses TSKgel G6000PWXL or large pore size, high resolving
TSKgel G5000PWXL power
TSKgel SuperMultiporePW-H
Synthetic TSKgel GMPWXL or TSKgel G5000PWXL & large pore size, low adsorption,
polymers TSKgel Alpha-M G3000PWXL / TSKgel Alpha- linear calibration curve
TSKgel SuperMultiporePW 5000 & Alpha-3000
cationic TSKgel G3000PWXL-CP medium to large pore size, low
TSKgel G5000PWXL-CP adsorption, linear calibration
TSKgel G6000PWXL-CP curve
Synthetic nonionic TSKgel G-Oligo-PW TSKgel G2500PW / small pore size, high resolving
oligomers TSKgel G2500PWXL or TSKgel SuperAW2500 power
TSKgel Alpha-2500
TSKgel SuperOligoPW and
TSKgel SuperMultiporePW-N
anionic TSKgel G2500PWXL or TSKgel G2500PW / small pore size, ionic interaction
TSKgel Alpha-2500 TSKgel SuperAW2500
 TOSOH BIOSCIENCE ANALYSIS 5

SEC
TSKgel SW SERIES

SEC
FIGURE 1
TSKgel SW-type columns (SW, SWXL, SuperSW, UltraSW,
and UP-SW) are all based on spherical silica particles with
Protein calibration curves for TSK-GEL SWXL columns
very high internal pore volume. They are stable from pH 2.0
10 6
to 7.5 and have excellent solvent stability up to 100% polar 1
organic solvents.
2
Three different pore sizes of the SW and SWXL packings 10 5

Molecular weight (Da)

result in different exclusion limits for several sample types, 3
4 5
as shown by the calibration curve in Figure 1. From this data, 6
recommended separation ranges for globular proteins can 10 4 7
8 9
be made for each column (see Table 2).

Different particle sizes, column dimensions, and column

10 3
hardware materials are available. The resulting differences TSKgel G2000SWXL
in column characteristics allow the scientist to select the TSKgel G3000SWXL 10
appropriate column to his individual separation requirements. 2 TSKgel G4000SWXL
10
Latest innovations in SW technology comprise dedicated
small particle columns for antibody analysis (Super SW
mAb and UltraSW Aggregate) and UHPLC columns with 2
µm particle and 25 nm pore size (UP-SW3000). 8 6 10 12
Elution volume (mL)
HIGHLIGHTS Column: TSK-GEL SWXL columns, 5 or 8 µm ,7.8 mm ID x 30 cm L
Sample: CALIBRATION
PROTEIN 1. thyroglobulin
CURVES(660,000
FOR Da ); 2. IgG
TSKgel SW(160,000 Da );
XL COLUMNS

3. BSA (67,000 Da); 4. ovalbumin (43,000 Da);

Rigid spherical silica gel chemistry Column: TSKgel SWXL columns,
5. peroxidase (40,2005 Da);
or 86.µm, 7.8 mm ID (18,400
β-lactoglobulin x 30 cmDa);L
bonded with hydrophilic groups Sample: 1. thyroglobulin (660,000 Da); 2. IgG (160,000 Da); 3. BSA
Well defined pore size distribution (67,000 Da); [Link]
myoglobin (16,900
(43,000DDa); a); [Link]
peroxidase A (12,600 Da);
(40,200 Da);
9. (18,400
6. β-lactoglobulin cytochrome C myoglobin
Da); 7. (12,400 Da);(16,900
10. glycine
Da);tetramer (246 Da)
8. ribonuclease
Low non specific adsorption Elution:
A (12,600 Da); 9.0.3 mol/L NaClC in
cytochrome 0.1 mol/L
(12,400 Da);sodium phosphate
10. glycine tetramer buffer, pH 7.0
(246 Da)
SuperSW and UltraSW designed for IgG analysis Detection:
Mobile phase: 0.3
UV @ mol/L NaCl in 0.1 mol/L sodium phosphate buffer,
220 nm
UHPLC columns with 2 µm particles available pH 7.0; Detection:UV @ 220 nm
6 [Link]

SEC
TSKgel SW SERIES
SEC

TABLE 2

PROPERTIES AND SEPARATION RANGES FOR TSKgel SW TYPE PACKINGS

TSKgel COLUMN ID (mm) X PARTICLE PORE MIN. NO. MOLECULAR WEIGHT

LENGTH (cm L) SIZE (μm) SIZE (nm) THEORET. PLATES OF PROTEINS (Da)

SuperSW2000 4.6 x 30 4 12.5 30,000 5 x 103–1.5 x 105

G2000SWXL 7.8 x 30 5 12.5 20,000 5 x 103–1.5 x 105

BioAssist G2SWXL 7.8 x 30 5 12.5 20,000 5 x 103–1.5 x 105

QC-PAK GFC 200 7.8 x 15 5 12.5 10,000 5 x 103–1.5 x 105

G2000SW 7.5 x 30/60 10 12.5 10,000/20,000 5 x 103–1.5 x 105

21.5 x 30/60 13 12.5 10,000/20,000 5 x 103–1.5 x 105

SuperSW3000 4.6 x 30 4 25 30,000 1 x 104–5 x 105

UP-SW3000 4.6 x 15/30 2 25 25,000/45,000 1 x 104–5 x 105

SuperSW mAb HTP 4.6 x 15 4 25 15,000 1 x 104–5 x 105

SuperSW mAb HR 7.8 x 30 4 25 30,000 1 x 104–5 x 105

G3000SWXL 7.8 x 30 5 25 20,000 1 x 104–5 x 105

BioAssist G3SWXL 7.8 x 30 5 25 20,000 1 x 104–5 x 105

QC-PAK GFC 300 7.8 x 15 5 25 10,000 1 x 104–5 x 105

G3000SW 7.5 x 30/60 10 25 10,000/20,000 1 x 104–5 x 105

UltraSW mAb Aggregate 7.8 x 30 3 30 35,000 1 x 104–2 x 106

G4000SWXL 7.8 x 30 8 45 16,000 2 x 104–7 x 106

BioAssist G4SWXL 7.8 x 30 8 45 16,000 2 x 104–7 x 106

G4000SW 7.5 x 30/60 13 45 8,000/16,000 2 x 104–7 x 106

21.5 x 30/60 17 45 8,000/16,000 2 x 104–7 x 106
 TOSOH BIOSCIENCE ANALYSIS 7

SEC
TSKgel SW SERIES APPLICATIONS

SEC
AGGREGATE ANALYSIS Some SEC separations require denaturing conditions like
sodiumdodecylsulfate (SDS) containing eluents. In other
Protein aggregation is a common issue encountered cases the formulations of biopharmaceuticals contain some
during expression, purification and formulation of protein detergents (e.g. Tween 20 or Triton). TSKgel SW type columns
biotherapeutics, which needs to be characterized and can be operated under these conditions although certain
controlled during the development and production of protein amounts of the detergent will stick to the column, affecting
pharmaceuticals such as monoclonal antibodies (mAbs). column lifetime and the future use of the column. If analysis
Even small amounts of aggregates can alter the therapeutic under denaturing conditions was performed once, the
function. TSKgel G3000XL columns are the industry standard affected column should be used with detergent containing
for quality control of MAbs by SEC. Besides the traditional eluents only. Regular maintenance of the column, the use
detection of proteins using their UV absorption at 280 nm, of guard columns and monitoring of the column status by
multi angle light scattering (MALS) detection gains more and analyzing control samples are recommended as well.
more interest in protein analysis. Being a universal detection
method, MALS can deliver valuable additional information.

As it will also detect several other impurities, pure solvents

and samples are of utmost importance. This also applies to
the stationary phase, which should not generate interfering
baseline noise under the conditions used for analysis. Figure 2
shows the analysis of MAb aggregates of a commercial
monoclonal antibody with UV, refractive index (RI) and
MALS detection. Separation was performed on a TSKgel
G3000SWXL column under standard conditions. With modern
(U)HPLC instrumentation resolution can be further enhanced
by using the latest generation of SW columns, TSKgel
UltraSW Aggregate or TSKgel UP-SW3000 (see pages 10 ff)

When the analysis of proteins needs to be performed in

a metal free environment, the BioAssistSW series offers
TSKgel SWXL packings in PEEK housings, featuring the same
performance as stainless steel columns. Figure 3 shows a
typical separation performed with a BioAssist SW PEEK
column.

USE OF DETERGENTS
FIGURE 2 FIGURE 3

70

0.15
60 Fr1
Monoclonal
Multimer
detector voltage (V)

50
Dimer

0.10

Fr2

0.05
40
(mV)

30 Fr3
0.00

0.0 5.0 10.0 20 IgG

time (min)

Fr4
SEC-MALS-UV-RI ANALYSIS OF MAB AGGREGATES 10

Column: TSKgel G3000SWXL column, 5 µm, 7.8 mm ID x 0

30 cm L, Sample: monoclonal antibody, [Link]: 20 µL, 6 14
4 8 10 12
Mobile phase: phosphate buffered saline (PBS); Flow rate: 1 mL/min, (min)
Detection: MALS (red), refractive index (blue) & UV @ 280 nm (green)
HPLC System: LC-20A prominence, Shimadzu, MALS detector: QC ANALYSIS OF AN ANTI-TSH ANTIBODY PURIFIED FROM CELL
miniDAWNTM TREOS, Wyatt Techn. Corp. CULTURE SUPERNATANT

Column: TSKgel BioAssist G3SWXL, 5 µm, 7.8 mm ID x 30 cm L, Mobile

phase: 0.3 mol/L phosphate buffer, pH 7.0, Flow rate: 1.0 mL/min;
Inj. volume: 50 μL
8 [Link]

SEC
TSKgel SuperSW & SuperSW mAb SERIES
SEC

Speed and resolution is an increasing demand in liquid HIGHLIGHTS

chromatography. The need for high sensitivity applicable
to trace analysis is increasing as sample size or sample SuperSW mAb columns tailored to antibody
concentrations become limited. To meet the needs of high analysis
sensitivity and high resolution protein analysis Tosoh High troughput and high resolution columns availble
Bioscience developed TSKgel SuperSW columns packed with 4 µm particle size featuring superior resolution and
4 µm spherical silica particles. TSKgel SuperSW columns are highest sensitivity
available in two pore sizes, 125 Å and 250 Å, both featuring Low non-specific adsorption
a minimum of 30,000 theoretical plates / column. Compared High reproducibility due to well-defined pore size
to the well established TSKgel SWXL (5 µm) series, SuperSW distribution
columns show higher resolution due to a 50 percent increase 30,000 theoretical plates / column (30 cm L)
in theoretical plate numbers (Table 3).

To further improve performance, TSKgel SuperSW media A new series of TSKgel SuperSW mAb columns was
are packed into columns with smaller inner diameter developed to improve IgG analysis in QC or research
(1.0, 2.0, 4.6 mm ID). The smaller diameters are one reason applications. They feature the SuperSW3000 stationary
for increased peak heights. In addition, the high resolution phase packed in dimensions that are either ideal for either
of the 4 μm particles and accordingly smaller peak widths reaching maximum resolution or speeding up analysis
further increase peak height provided the HPLC system is without compromising resolution much.
optimized with regard to extra column dead volume.

Figure 4 demonstrates the superior sensitivity reached with

TSKgel SuperSW2000 compared to a TSKgel G2000SWXL
column of the same length but larger inner diameter. TSKgel
SuperSW can yield peak heights approximately 4 times that
of TSKgel SWXL due to downsizing in column diameter and
increased theoretical plates.

Comparison of TSKgel SuperSW2000 and TSKgel G2000SWXL

TABLE 3 FIGURE 4
for the separation of proteins
60 60
SPECIFICATIONS OF TSKgel SuperSW SERIES COMPARED TO 4
TSKgel SWXL SERIES 50
A. G2000SWXL
50
B. SuperSW2000
40 40
TSKgel PARTICLE COLUMN SIZE GUARANTEED 3
Rs:15.6
mV

mV

COLUMN SIZE (µm) (mm ID X cm L) THEOR. PLATES 30 30 Rs:6.2

Rs:4.8
4 2
20 Rs:4.7 Rs:4.6 Rs:11.9
20 1
TSKgel 4 4.6 x 30 30,000 3
1 2
10 10
SuperSW2000
0 0
3 5 7 9 11 13 15 3 5 7 9 11 13 15
TSKgel 4 4.6 x 30 30,000 Minutes Minutes
SuperSW3000
Column: A. TSKgel G2000SWXL, 7.8mm ID x 30cm;
COMPARISON OF TSKgel
B. TSKgel SuperSW2000
SuperSW2000, AND TSKgel G2000SWXL
4.6mm ID x 30cm
TSKgel 5 7.8 x 30 20,000
FOR THE SEPARATION
Sample: 1. thyroglobulinOF PROTEINS
(0.2mg/mL); 2. albumin (1.0mg/mL);
G2000SWXL
3. ribonuclease A (1.0mg/mL); 4. p-aminobenzoic acid (0.01mg/mL)
Column:
Inj. Volume: A.
5µL TSKgel G2000SWXL, 7.8 mm ID x 30 cm L;
TSKgel 5 7.8 x 30 20,000
B. TSKgel SuperSW2000,
Elution: 0.1mol/L phosphate 4.6buffer
mm+ID x 30 cm
0.1mol/L Na2LSO4 + 0.05% NaN3 (pH 6.7)
G3000SWXL
Sample:
Flow Rate: 1.0.35mL/min
thyroglobulin (0.2 mg/mL);
for SuperSW2000; 2. albumin
1.0mL/min (1.0 mg/mL);
for G2000SW XL
3. ribonuclease
Temp: 25°C A (1.0 mg/mL); 4. p-aminobenzoic acid (0.01 mg/mL)
Inj. volume: 5 µL,
Detection: UV @ 280nm Mobile phase: 0.1 mol/L phosphate buffer +
0.1 mol/L Na2SO4 + 0.05% NaN3 (pH 6.7), Flow rate: 0.35 mL/min
(SuperSW2000), 1.0 mL/min (G2000SWXL), Temp: 25°C; Detection:
UV @ 280 nm
 TOSOH BIOSCIENCE ANALYSIS 9

SEC
TSKgel SuperSW SERIES

SEC
TABLE 4
SEPARATION RANGE OF TSKgel SuperSW
DETECTION LIMIT FOR PROTEINS (S/N=3)
The TSKgel SuperSW series has the same pore sizes as the
TSKgel SuperSW TSKgel SWXL
conventional TSKgel SWXL series with equivalent grade.
Therefore it has similar calibration curves and separation FLOW CELL STANDARD CELL STANDARD CELL
ranges as well. Method transfer from conventional SEC (LOW DEAD (LOW DEAD
VOLUME TYPE) VOLUME TYPE)
to high resolution SEC is very straight forward. TSKgel
SuperSW columns are available in two pore sizes, 125 Å Light path
(TSKgel SuperSW2000) and 250 Å (TSKgel SuperSW3000). length 10 mm 10 mm
Figure 5 shows the SEC calibration curves for standard
Thyro-
proteins. In general, TSKgel SuperSW2000 is suited to
globulin 70 ng 200 ng
separate proteins with molecular weights of 150 KDa or
smaller. TSKgel SuperSW3000 can be used for the separation γ-globulin 50 ng 100 ng
of proteins with molecular weights up to 500 KDa.
Bovine serum
albumin 70 ng 200 ng
INCREASED DETECTION LIMIT
Ovalbumin 50 ng 100 ng
Table 4 shows the detection limits for some proteins. The
Myoglobin 15 ng 30 ng
high sensitivity allows for analysis of nanogram sample
amounts. If sample amount is limited a reduction of column
inner diameter can further enhance sensitivity. TSKgel Column: TSKgel SuperSW3000, 4.6 mm ID x 30 cm L; TSKgel
SuperSW3000 columns are available with 4.6; 2 and 1 mm ID. G3000SWXL, 7.8 mm ID x 30 cm L, Mobile phase: 0.2 mol/L phos-
phate buffer, pH 6.7, Detection: UV @ 220 nm
Figure 6 shows the levels of sensitivity which can be reached
with semi-micro or micro columns. When limited sample
amount is an issue (e.g. in proteomics research) enhancing
detection limits by using a micro column can increase the
number of hits.

FIGURE 5 FIGURE 6

10.000.000 140
5
Protein
120 4
1
1.000.000
TSKgel SuperSW3000 100
2 3
2

3 80 1
Intensity

100.000
4
60
Molecular weight

5
1.0 mm ID
10.000 40
TSKgel SuperSW2000 6
2.0 mm ID
20
4.6 mm ID
1.000
0
0 5 10 15 20
7 Time (min)

100
ESTIMATION OF SENSITIVITY

Column: TSKgel SuperSW3000, 1.0, 2.0, 4.6 mm ID x 30 cm L,

10
Sample: 1. thyroglobulin (1.0 g/L), 2. γ-globulin (2.0 g/L), 3. ovalbumin
1.5 2 2.5 3 3.5 4 4.5
(2.0 g/L), 4. ribonuclease A (3.0 g/L), 5. p-aminobenzoic acid (0.02 g/L)
Elution volume (ml)
Mobile phase: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4
PROTEIN CALIBRATION CURVES FOR TSKgel SuperSW + 0.05% NaN3, Flow rate: 16 µL/min (1 mm), 65 µL/min (2 mm); 350
µL/min (4.6 mm), [Link]: 0.2 µL; Temperature.: 25 °C, Detection:
Column: TSKgel SuperSW Series, 4.6 mm ID X 30 cm L, Sample: UV @ 280 nm, cell vol. 2 µL (4.6 mm ID), 35 nL (1.0, 2.0 mm ID)
Standard proteins (5 µL, 0.1 g/L each); [Link] 2. γ-globulin 3.
bovine serum albumin, 4. ß-lactoglobulin 5. lysozyme 6. cytochrome
C 7. glycine tetramer, Mobile phase: 0.2 mol/L phosphate buffer
(pH 6.7), Flow rate: 0.35 mL/min; Detection: UV @ 280 nm
10 [Link]

SEC - TSKgel SuperSW mAb HTP/HR

FOR mAb APPLICATIONS
SEC

QC ANALYSIS OF ANTIBODIES FAST ANALYSIS OF AGGREGATION

Thermally induced denaturation or aggregation of TSKgel SuperSW mAb HTP - “HTP” indicating high
therapeutic antibodies can be a significant problem during throughput - was developed to enable an easy transfer
different stages of its production and formulation, since of established methods to fast (U)HPLC analysis. The
aggregates affect the efficiency of the biotherapeutic. Thus SuperSW3000 stationary phase is packed into column
the quantification of aggregates is an important parameter hardware with smaller (4.6 mm) inner diameter. This column
in the quality control analysis of biopharmaceuticals. Using enables doubling the throughput without compromising
TSKgel SuperSW mAb columns the amounts of tri-, di- and resolution too much. Figure 8 shows the optimization of
monomers of monoclonal antibodies can be monitored. the separation of mAb Aggregates with regard to analysis
time. Compared to the standard separation with maximum
TSKgel SuperSW mAb columns are specifically designed resolution using TSKgel SuperSW mAb HR (see Figure 7) the
to facilitate analysis of antibodies. Each of the columns is analysis time can be reduced by factor 3 when using TSKgel
tailored to a specific separation problem. TSKgel SuperSW SuperSW mAb HTP and adjusting the flow rate accordingly.
mAb HR - “HR” indicating high resolution - delivers superior Resolution between IgG monomer and dimer is still sufficient
resolution over the whole range of typical mAb SEC analysis, for quantification.
from fragments to aggregates. It has the same column
dimensions as the established TSKgel G3000SWXL column.

Figure 7 shows a comparison of the analysis oft a mouse-

human chimeric IgG using these two columns. Resolution is
improved with TSKgel SuperSW mAb HR.

FIGURE 7 FIGURE 8

A monomer
monomer

dimer

trimer
aggregates monomer Flow rate: 0.50 mL/min
dimer
Pressure: 5.0 MPa
trimer
aggregates
Rs（dimer/monomer）＝1.91

5 6 7 8 9 10 11 12 min

Flow rate: 0.35 mL/min

B monomer IgG dimer
Pressure: 3.6 MPa
fragment trimer
dimer Rs（dimer/monomer）＝2.13
aggregates

trimer
aggregates 0 1 2 3 4 5 6 7 min

FAST ANALYSIS OF mAb AGGREGATION

5 6 7 8 9 10 11 12 min
Column: TSKgel SuperSW mAb HTP (4.6 mm ID x 15 cm)
monomer
C dimer Elution: 0.2 mol/L phosphate buffer (pH 6.7) + 0.05% NaN3
COMPARISON OF AGGREGATE ANALYSIS Flow rate: 0.50 mL/min, 0.35 mL/min; Detection: UV @ 280 nm
Temp.: 25°C; Sample: monoclonal antibody (mouse-human chimeric
Columns: A. TSKgel G3000SWXL,trimer
B. TSKgel SuperSW mAb HR, IgG, Erbitux), 5 μL
aggregates
Dimension: 7.8 mm ID × 30 cm; 溶
Eluent: 0.2 mol/L phosphate buffer (pH 6.7) + 0.05% NaN3
Flow rate: 0.8 mL/min; Detection: UV @ 280 nm; Temp.: 25°C;
Sample: monoclonal antibody, (mouse-human chimeric IgG, Erbitux),10 μL
5 6 7 8 9 10 11 12 min
 TOSOH BIOSCIENCE ANALYSIS 11

SEC
TSKgel UltraSW COLUMN

SEC
TSKgel UltraSW AGGREGATE FEATURES HIGHLIGHTS

TSKgel UltraSW Aggregate provides a smaller particle size of High resolution through 3 µm particle size
3 micron and a higher exclusion limit through slightly larger Large pore size expands separation range for
pores (30 nm) than TSKgel SuperSW mAb columns. This higher aggregates
column was developed to offer a wider separation window Dimensions optimized for mAb aggregate analysis
in the molecular mass range of antibody aggregates.
ANALYSIS OF mAb AGGREGATES
Figure 9 shows the calibration curve of TSKgel UltraSW
Aggregate compared to the calibration curves of TSK- The wide separation window of TSKgel UltraSW Aggregate
gel SuperSW mAb and G3000 SWXL. The calibration curve at higher molecular weights combined with the small par-
of TSKgel SuperSW mAb HR is most similar to the one of ticle size results in a superior separation of higher antibody
TSKgel G3000SWXL, the current industrial standard for an- aggregates. Resolution of dimer, trimer, and higher aggre-
tibody analysis. The curve for TSKgel UltraSW Aggregate gates is higher than for TSKgel SuperSW mAb HR and TSK-
shows a shallower slope in the high molecular weight region gel G3000 SWXL (Figure10).
(MW > 300 000).

FIGURE 9 FIGURE 10

7
SuperSW mAb HTP
1 SuperSW mAb HR
6 UltraSW Aggregate
G3000SWXL
2
5 3
Log MW

4
4

5
2

1
0 2 4 6 8 10 12 min
CALIBRATION CURVES COMPARISON OF AGGREGATE ANALYSIS

Columns: A. TSKgel G3000SWXL, B. TSKgel SuperSW mAb HR, Columns: TSKgel UltraSW Aggregate (7.8 mm ID x 30 cm),
C. TSKgel UltraSW Aggregate; Dimension: 7.8 mm ID × 30 cm; 溶 TSKgel SuperSW mAb HR(7.8 mm ID x 30 cm), ​
Eluent: 0.2 mol/L phosphate buffer (pH 6.7) + 0.05% NaN3 TSKgel G3000SWXL (7.8 mm ID x 30 cm)
Flow rate: 0.8 mL/min; Detection: UV @ 280 nm; Temp.: 25°C; Eluent: 0.2 mol/L phosphate buffer (pH 6.7) + 0.05% NaN3;
Sample: monoclonal antibody, (mouse-human chimeric IgG, Erbitux),10 μL Flow rate: 1.0 mL/min; Temperature: 25°C; Detection: U
​ V @ 280 nm
Injection vol.: 20 μL;
Sample: IgG (human monoclonal, 2.5 g/L), 1 monomer, 2 dimer, 3 trimer,
4 tetramer, 5 multimers
12 [Link]

SEC
TSKgel UP-SW3000 SERIES
SEC

TSKgel UP-SW3000 FEATURES HIGHLIGHTS

TSKgel UP-SW3000 columns packed with 2 µm silica based Highest resolution through 2 µm particle size
particles are the latest addition to the popular TSKgel SW se- Easy transfer of existing methods
ries, the gold standard for QC analysis of antibody therapeu- Optimized for mAb quality control
tics. The new silica-based UHPLC columns are based on the Short runtimes with 15 cm column
proven proprietary surface technology of the renowned TSK-
gel SW series and were engineered to facilitate the transfer UHPLC ANALYSIS OF ANTIBODIES
of existing HPLC methods to UHPLC systems.
The separation of an antibody sample on the new 2 mikron
The new SEC columns can be used with modern, low extra UP-SW3000 column compared to the competitor UHPLC col-
column dead volume HPLC and UHPLC systems and are umn is depicted in figure 12. The two columns differ slightly
available in the lengths of 15 or 30 cm. The short one enables in pore size and particle size (25 nm for 2 µm TSKgel versus
short analysis times; the long one provides higher resolu- 20 nm for the 1.7 µm material). This difference in pore sizes
tion for mAb analysis. The lifetime of the columns can be results in a better separation in the molecular weight range
improved when using the corresponding guard columns. A of antibodies, fragments, and aggregates on TSKgel UP-SW.
“direct connect” (DC) guard column is available to minimize
extra column dead volume. Based on the wider separation window the resolution
between monomer and dimer as well as dimer and trimer
Figure 11 shows the calibration curve and the molecular is slightly higher with TSKgel UP-SW3000 although par-
weight range of the new 2 µm TSKgel UP-SW3000 compared ticle size is slightly larger than in the competitor column.
to the 5 micron TSKgel G3000SWXL and 4 micron TSKgel Moreover, also the fragment peak is more clearly separated
SuperSW3000. The calibration curves and mass ranges are from the monomer peak.
almost identical which facilitates method transfer from exist-
ing methods.

FIGURE 11 FIGURE 12

COMPARISON OF CALIBRATION CURVES COMPARISON OF mAb ANALYSIS

Columns : ​A: TSKgel UP-SW3000 (4.6 mm ID × 30 cm, red)​​​​

B: TSKgel SuperSW3000 ( 4.6 mm ID × 30 cm, blue) Column RS (peak 1/2) RS (peak 2/3)
C: TSKgel G3000SWXL (7.8 mm ID × 30 cm, grey)​​​​
TSKgel UP-SW3000 2 μm 1.52 3.56
Eluent: 1​ 00 mmol/L phosphate buffer (pH 6.7) + 100 mmol/L sodium sulfate +
0.05% NaN3; Flow rate: ​A & B: 0.35 mL/min; C: 1.0 mL/min Competitor UHPLC-SEC 1.7 μm 1.25 3.47
Temperature: 2 ​ 5°C; Detection: UV @ 280 nm; Injection vol.: ​10 μL
Samples: ​1. thyroglobulin (640,000 Da); (1’ thyroglobulin aggregate);
2. γ-globulin (155,000 Da); (2’ γ-globulin dimer); 3. ovalbumin (47,000 Da);
4. ribonuclease A (13,700 Da); 5. p-amino benzoic acid (137 Da)
 TOSOH BIOSCIENCE ANALYSIS 13

SEC - TSKgel SuperSW, UltraSW AND UP-SW

SYSTEM REQUIREMENTS

SEC
OPTIMIZATION OF HPLC EQUIPMENT DETECTOR CELL VOLUME

To benefit from the improved features of TSKgel SuperSW, The detector cell volume also contributes to the dead volume
UltraSW and UP-SW columns the HPLC system should be of the system and might impair peak resolution. For most
optimized and extra column peak broadening reduced. This separations with 4.6 mm ID TSKgel SuperSW columns a
means reduction of dead volume and adjustment of sample 8-10 μL standard detector cell might be sufficient but for
concentration and injection volume. semi-micro (2 mm ID) or micro columns (1 mm ID), and for
TSKgel UP-SW we strongly recommend using semi-micro/
SYSTEM DEAD VOLUME micro detector cells.

Key components of the HPLC system with regard to dead INJECTOR

volume reduction are the void volume of tubings, the cell
volume of the detector cell and the void volume of the The maximum number of theoretical plates in isocratic
injection unit. Modern UHPLC systems designed for use with separations can be reached when using a low diffusion
sub 2 μm particles exhibit extremely small dead volumes type manual injector like the Rheodyne 8125. All kinds of
and can be used for SEC analysis without modification. automated HPLC injectors will deteriorate column efficiency
to a certain extend but due to practical reasons, auto-samplers
VOID VOLUME OF THE TUBING are nowadays standard. All the more it is important to select
an auto-sampler capable of trace injection mode. Dead
The volume of tubing from injector to column, column to volume of the outlet capillary should be minimized to the
detector influences the diffusion within the tubing and the utmost (as short as possible, 0.1 mm ID). Figure 13 shows the
column efficiency. Column efficiency starts deteriorating effect of injector tubings on column efficiency for a 1 mm ID
remarkably when the volume of the tubing exceeds 10 μL column.
(e.g. 0.1 mm ID x 150 cm L). Shortening of tubings of 0.1 or
0.125 mm inner diameter is often better than using longer
capillaries with smaller inner diameters. The backpressure
increases with smaller inner diameters and the system
becomes more susceptible towards clogging.

TSKgel SuperSW, UltraSW, AND UP-SW OPERATING CONDITIONS

For best results, it is recommended to use the following experimental conditions for TSKgel SuperSW, UltraSW, and UP-SW columns:

CONNECTIONS The conventional 0.1 mm tubing may be used, but length should be kept as short as possible. Void volume
between the column and detector cell should be less than 20 µL.

INJECTOR Best results are obtained with a low diffusion type manual injector (Rheodyne 8152). Autosampler outlet void
volume should be as low as possible.

SAMPLE VOLUME Sample volume should be 10 µL or less. Sample load should be less than 100 µg (4.6 mm ID column).

GUARD COLUMN A guard column or an inline filter is highly recommended to reduce clogging and contamination.

DETECTOR

Flow Cell For best results, use a flow cell with a maximum of 2 µL. The 2 µL flow cell will give the highest efficiencies.
A 2-10 µL flow cell can be used for 4.6 mm ID columns. However, theoretical plates will be reduced.

Time Constant A small time constant (less than 0.5 sec) is needed to achieve best column performance.

PUMP A pump capable of accurately delivering a flow rate between 0.01 mL/min and 0.35 mL/min is recommended.
14 [Link]

SEC - TSKgel SuperSW, UltraSW AND UP-SW

SYSTEM REQUIREMENTS
SEC

MOBILE PHASE
SAMPLE LOAD AND INJECTION VOLUME
The eluent plays an important role in SEC separations. When
Although the efficiency of TSKgel Super/Ultra/UP-SW denaturing agents are used, the exclusion limits for proteins
columns is high, it is obvious that it decreases at high become smaller since they lose their compact globular
sample loads. Figure 14 shows that sample load should not structure. Proper selection of eluting conditions is necessary
exceed 100 μg for a TSKgel SuperSW3000 column of 4.6 mm to maximize the molecular sieving mechanism and to
ID x 30 cm L. On the other hand the injection volume itself minimize secondary effects, such as ionic and hydrophobic
is a critical parameter. As for all HPLC applications injection interactions between the sample and the column packing
volume should be as small as possible. If injection volume material. In general, the use of relatively high ionic strength
exceeds 20 μL on a 4.6 mm ID column, a considerable buffers is recommended for most protein applications. A
deterioration of column efficiency is observed for TSKgel neutral salt is often added to increase ionic strength.
SuperSW2000 (80 μL for TSKgel SuperSW3000). In general
the sample load should be less than 100 μg in less than 10 μL RECOVERY OF PROTEIN
injection volume for a 4.6 mm ID TSKgel SuperSW column.
TSKgel SuperSW series is capable of obtaining high protein
FLOW RATE DEPENDENCE recovery even in trace analysis with sample load of 1 μg
or lower. Most proteins are recovered quantitatively with
The effect of flow rate on column efficiency depends on TSKgel SuperSW series, but it is important to make sure
particle size of packing materials, sample molecular size, that samples in small concentrations are not adsorbed to
eluent viscosity, etc. The appropriate flow rate for TSKgel the sample vial or to the HPLC system itself. Similar samples
SuperSW columns is up to 0.4 mL/min for a 4.6 mm ID should be injected several times before measurement so
column, up to 75 μL/min for a 2 mm ID column, and up that adsorption points within the system are inactivated in
to 20 μL/min for a 1 mm ID column, respectively. If higher advance when trace analysis is performed.
resolution is required the flow rate can be lowered.

FIGURE 13 FIGURE 14

34.000 160.0

32.000 140.0 TSKgel SuperSW3000

120.0 TSKgel G3000SWXL
30.000
Theoretical plates

0.050 mm ID 100.0
HETP (µm)

28.000
x
0.075 mm ID 80.0
26.000
x 0.130 mm ID 60.0
24.000
x 40.0
22.000 20.0
x
20.000 0.0
0 10 20 30 40 50 60 70 0.1 1 10 100 1.000 10.000
Tubing length (cm) Sample loading (µg)

INFLUENCE OF TUBING (INJECTOR TO COLUMN) EFFECT OF SAMPLE LOAD

Column: TSKgel SuperSW3000 1.0 mm ID x 30 cm L, Mobile phase: Column: TSKgel SuperSW series, 4.6 mm ID x 30 cm L; TSKgel
0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4 + 0.05 % NaN3 SWXL series, 7.8 mm ID x 30 cm L, Sample: Bovine serum albumin,
Flow rate: 16 µL/min; [Link]: 0.2 µL; Temp.: 25 °C, Detection: Mobile phase: 0.2 mol/L phosphate buffer, pH 6.7, Flow rate: 0.35
UV @ 280 nm, Sample: p-Aminobenzoic acid (20mg/L), mL/min (SuperSW series), 1.00 mL/min (SWXL series), Temp.: 25 °C;
Tubing: ID (mm) x L (cm), Vol. Detection: UV @ 280 nm, micro flow cell
0.050 x 20, 393 nL; 0.050 x 40, 785 nL; 0.050 x 60, 1178 nL;
0.075 x 20, 883 nL; 0.075 x 40, 1766 nL; 0.075 x 60, 2469 nL;
0.130 x 20, 2653 nL; 0.130 x 40, 5307 nL; 0.130 x 60, 7960 nL
 TOSOH BIOSCIENCE ANALYSIS 15

SEC
TSKgel SW SERIES ORDERING INFORMATION

SEC
ORDERING INFORMATION

PART # DESCRIPTION ID LENGTH PARTICLE NUMBER FLOW RATE (mL/min) MAXIMUM

(mm) (cm) SIZE (µm) THEORETICAL RANGE MAX. PRESSURE
PLATES DROP (MPa)

STAINLESS STEEL COLUMNS

0023448 UP-SW3000 -NEW- 4.6 30 2 ≥ 45000 0.10–0.35 34

0023449 UP-SW3000 -NEW- 4.6 15 2 ≥ 25000 0.10–0.35 25
0022856 UltraSW Aggregate -NEW- 7.8 30 4 ≥ 35,000 0.5 - 1.0 12.0
0018674 SuperSW2000 4.6 30 4 ≥ 30,000 0.1 - 0.35 0.4 12.0
0021845 SuperSW3000 1.0 30 4 ≥ 18,000 0.016 0.02 12.0
0021485 SuperSW3000 2.0 30 4 ≥ 25,000 0.065 0.075 12.0
0018675 SuperSW3000 4.6 30 4 ≥ 30,000 0.1 - 0.35 0.4 12.0
0022855 SuperSW mAb HTP -NEW- 4.6 15 4 ≥ 15,000 0.1 - 0.35 8.0
0022854 SuperSW mAb HR -NEW- 7.8 30 4 ≥ 30,000 0.5 - 1.0 12.0
0008540 G2000SWXL 7.8 30 5 ≥ 20,000 0.5 - 1.0 1.2 7.0
0008541 G3000SWXL 7.8 30 5 ≥ 20,000 0.5 - 1.0 1.2 7.0
0008542 G4000SWXL 7.8 30 8 ≥ 16,000 0.5 - 1.0 1.2 3.5
0016215 QC-PAK GFC 200 7.8 15 5 ≥ 10,000 0.5 - 1.0 1.2 4.0
0016049 QC-PAK GFC 300 7.8 15 5 ≥ 10,000 0.5 - 1.0 1.2 4.0
0005788 G2000SW 7.5 30 10 ≥ 10,000 0.5 - 1.0 1.2 2.0
0005789 G3000SW 7.5 30 10 ≥ 10,000 0.5 - 1.0 1.2 2.5
0005790 G4000SW 7.5 30 13 ≥ 8,000 0.5 - 1.0 1.2 1.5
0005102 G2000SW 7.5 60 10 ≥ 20,000 0.5 - 1.0 1.2 4.0
0005103 G3000SW 7.5 60 10 ≥ 20,000 0.5 - 1.0 1.2 5.0
0005104 G4000SW 7.5 60 13 ≥ 16,000 0.5 - 1.0 1.2 3.0
0006727 G2000SW 21.5 30 13 ≥ 10,000 3.0 - 6.0 8.0 1.0
0006728 G3000SW 21.5 30 13 ≥ 10,000 3.0 - 6.0 8.0 1.5
0006729 G4000SW 21.5 30 17 ≥ 8,000 3.0 - 6.0 8.0 1.0
0005146 G2000SW 21.5 60 13 ≥ 20,000 3.0 - 6.0 8.0 2.0
0005147 G3000SW 21.5 60 13 ≥ 20,000 3.0 - 6.0 8.0 3.0
0005148 G4000SW 21.5 60 17 ≥ 16,000 3.0 - 6.0 8.0 2.0

PEEK COLUMNS
0020027 BioAssist G2SWXL 7.8 30 5 ≥ 20,000 0.5 - 1.0 1.2 7.0
0020026 BioAssist G3SWXL 7.8 30 5 ≥ 20,000 0.5 - 1.0 1.2 7.0
0020025 BioAssist G4SWXL 7.8 30 8 ≥ 16,000 0.5 - 1.0 1.2 3.5

GLASS COLUMNS
0008800 G3000SW, Glass 8.0 30 10 ≥ 10,000 0.4 - 0.8 0.8 2.0
0008801 G4000SW, Glass 8.0 30 13 ≥ 8,000 0.4 - 0.8 0.8 2.0
16 [Link]

SEC
TSKgel SW SERIES ORDERING INFORMATION
SEC

ORDERING INFORMATION

PART # DESCRIPTION ID LENGTH PARTICLE

(mm) (cm) SIZE (µm)

0023450 TSKgel Guardcolumn UP-SW 4.6 2.0 2

0023451 TSKgel Guardcolumn UP-SW DC 4.6 2.0 2
0022857 SuperSW mAb Guardcolumn 6.0 4.0 4
0022858 SuperSW mAb Guardcolumn 3.0 2.0 4
0022859 UltraSW Guardcolumn 6.0 4.0 3
0018762 SuperSW Guardcolumn 4.6 3.5 4 For P/N 0018674 and 0018675
0008543 SWXL Guardcolumn 6.0 4.0 7 For all SWXL and P/N 0016215 and 0016049
0005371 SW Guardcolumn 7.5 7.5 10 For all 7.5 mm ID SW columns
0005758 SW Guardcolumn 21.5 7.5 13 For all 21.5 mm ID SW columns
0008805 SW Guardcolumn, Glass 8.0 4.0 10 For all 8.0 mm ID SW and QC-PAK Glass column
0018008 BioAssist SWXL Guardcolumn 6.0 4.0 7 For all BioAssist SWXL columns
 TOSOH BIOSCIENCE ANALYSIS 17

SEC
TSKgel PW SERIES

SEC
Polymeric TSKgel PW and high resolution TSKgel PWXL The TSKgel PWXL product line also offers specialty columns
columns are designed for SEC of water soluble organic for analyzing carbohydrate oligomers (TSKgel G-Oligo-PW)
polymers, polysaccharides, DNA and RNA. They are based and DNA and RNA fragments of 500-5000 base pairs (TSKgel
on a hydrophilic polymethacrylate matrix. Stable from pH G-DNA-PW). The new SuperOligoPW semi-micro SEC
2 to 12, TSKgel PW series columns can be used in mobile column featuring a small particle size has been designed
phases of water or buffer (up to 50% polar organic solvent). to enable fast analysis of oligosaccharides and other water
A large pore G6000PW phase is available in PEEK column soluble oligomers.
hardware (TSKgel BioAssist G6PW) for ultra-low sample
adsorption during virus analysis. The properties of all TSKgel TSKgel PWXL-CP columns have the same base matrix as
PW columns are summarized in Table 5. the PWXL columns and were specifically developed for the
analysis of water-soluble cationic polymers.
When the molecular weight range of the sample is broad
or unknown, Tosoh Bioscience offers two mixed-bed HIGHLIGHTS
columns: The TSKgel GMPW column and its high resolution
counterpart, TSKgel GMPWXL, are packed with the G2500, Hydrophilic spherical polymethacrylate particles
G3000 and G6000 PW or corresponding PWXL resins. pH range of 2-12 with up to 50% polar organic solvent
Six different TSKgel PW pore sizes
The new generation of TSKgel SuperMultiporePW columns Linear SEC column line incorporating proprietary
for semi-micro SEC provide near linear calibration curves. multipore technology
They are packed with spherical, mono-disperse particles Speciality columns for challenging SEC separations
incorporating a proprietary multi-pore particle technology.
They are ideally suited to analyze water soluble polymers,
such as polyvinylpyrrolidones or dextrans.

TABLE 5
PROPERTIES AND SEPARATION RANGES OF TSKgel PW, PWXL AND PWXL-CP COLUMNS

TSKgel COLUMN PARTICLE SIZE (μm) PORE SIZE (nm) MW RANGE (PEG/PEO)

G2000PW 12 12.5 <2 x 103

G2500PW 12, 17 <20 <3 x 103
G3000PW 12, 17 20 <5 x 104
G4000PW 17 50 <3 x 105
G5000PW 17 100 <1 x 106
G6000PW/ BioAssist G6PW 17 >100 <8 x 106
GMPW 17 10-100 5 x 10 - 8 x 106
2

G2500PWXL 7 <20 <3 x 103

G3000PWXL 7 20 <5 x 104
G4000PWXL 10 <50 <3 x 105
G5000PWXL 10 100 <1 x 106
G6000PWXL 13 >100 <8 x 106
G-DNA-PW 10 >100 <8 x 106
GMPWXL 13 10-100 5 x 10 - 8 x 106
2

G-Oligo-PW 7 12.5 <5 x 103

SuperMultiporePW-N 4 n/a 3 x 102 - 5 x 104

SuperMultiporePW-M 5 n/a 5 x 102 - 1 x 106
SuperMultiporePW-H 8 (6-10) n/a 1 x 103 - 1 x 107
SuperOligoPW 3 n/a 1 x 102 - 3 x 103

G3000PWXL-CP 7 20 2 x 102 - 5 x 104

G5000PWXL-CP 10 100 4 x 102 - 5 x 105

G6000PWXL-CP 13 >100 1 x 103 - 1 x 107
18 [Link]

SEC
TSKgel PW SERIES
SEC

CALIBRATION CURVES

Figure 15 shows the calibration curves for polyethylene

glycol (PEG) and oxides (PEO) for TSKgel PW and TSKgel
PWXL columns, respectively. In general silica based SW type
columns are recommended for the analysis of proteins, but
for special applications, e.g. at basic pH or for large molecular
weight proteins, PW type columns can be applied (Figure 16).
Figure 17 shows the near linear calibration curves for PEG/
PEO on TSKgel SuperMultiporePW columns.

FIGURE 16 FIGURE 17
Protein calibration curves on TSKgel PWXL columns

107

106 1 2 5 3 4
Molecular weight (Da)

b
105
c
d e
f
104
g

103
4 6 8 10 12
Elution volume (mL)
Column: 1. G3000PWXL
2. G4000PWXL CURVES ON TSKgel PWXL COLUMNS
PROTEIN CALIBRATION CALIBRATION CURVES FOR TSKgel SuperMultiporePW
3. G5000PWXL
4. G6000PWXL
Column: 1. TSKgel G3000PWXL, 2. TSKgel G4000PWXL, 3. TSKgel Sample: PEO & PEG standards; Mobile phase: H2O; Flow rate: 0.6
5. GMPWXL
G5000PW
Sample:XL, a. thyroglobulin
4. TSKgel G6000PW
(660,000 Da) XL, 5. TSKgel GMPWXL mL/min; Detection: RI; Temperature: 25 °C
Sample: a. thyroglobulin (660,000
b. γ-globulin (150,000 Da) Da), b. γ-globulin (150,000 Da),
c. albumin (67,000 Da),
c. albumin d. ovalbumin
(67,000 Da) (43,000 Da), e. ß-lactoglobulin
(36,000 Da), f. d. ovalbumin
myoglobin (43,000 Da) Da), g. cytochrome C (12,400 Da)
(16,900
Mobile phase: β-lactoglobulin
e. 0.2 M phosphate (36,000buffer
Da) (pH 6.8); Flow rate: 1.0 mL/min;
f. myoglobin (16,900 Da)
Detection: UVg. cytochrome
@ 280 nmC (12,400 Da)
Elution: 0.2 M phosphate buffer (pH 6.8)
FIGURE
Flow 15mL/min
Rate: 1.0
Detection: UV @ 280 nm
NN
TSKgel
TSKgelPW
PW 66 TSKgel
TSKgelPW
PWXLXL 66
1010
MM
1010 GG
Columns
Columns Columns
Columns KK
(Da)
weight (Da)
(Da)
weight (Da)

55
1010 55
1010
Molecular weight
Molecular weight

DD EE FF
LL
44 44
1010 JJ
1010 CC
Molecular
Molecular

33 AA BB 33 HH
1010 1010

22 22
1010
1010

1010 1515 2020 55 1010 1515

Elution
Elutionvolume
volume(mL)
(mL) Elution
Elutionvolume
volume(mL)
(mL)

POLYETHYLENE GLYCOL AND OXIDE CALIBRATION CURVES ON TSKgel PW AND TSKgel PWXL COLUMNS

Column: TSKgel PW columns: A. G2000PW, B. G2500PW, TSKgel PWXL columns: H. G2500PWXL, J. G3000PWXL,
C. G3000PW, D. G4000PW, E. G5000PW, F. G6000PW, G. GMPW, all K. G4000PWXL, L. G5000PWXL, M. G6000PWXL, N. GMPWXL,
7.5 mm ID x 60 cm L all 7.8 mm ID x 30 cm L
Mobile phase: distilled water; Flow rate: 1.0 mL/min; Detection: RI
 TOSOH BIOSCIENCE ANALYSIS 19

SEC
TSKgel PW SERIES APPLICATIONS

SEC
LARGE DNA FRAGMENTS OLIGOMERS

For the separation of large DNA fragments greater than The influence of particle size on resolution and analysis
1,000 base pairs, a four-column system is typically required. time can be seen in Figure 19. It compares the separation
Baseline resolution of DNA fragments up to 7,000 base pairs of PEG 200 on two TSKgel G-Oligo-PW columns in series
can be achieved, provided there is a two-fold difference with 7 μm beads and two newly developed TSKgel
in the chain length of the fragments. Figure 14A shows SuperOligoPW semi-micro columns with a 3 μm material.
the elution of double stranded DNA fragments, obtained The TSKgel SuperOligoPW column is designed for high
from pBR322 DNA cleaved by both Eco RI and Bst NI, on resolution separations water soluble oligomers. Figure 19
four TSKgel G-DNA-PW columns in series. The eluted demonstrates excellent resolution of the PEG 200 obtained
peaks were collected and subjected to polyacrylamide gel by using the smaller, 3 μm particle size packing in the TSKgel
electrophoresis, which showed almost complete separation SuperOligoPW column.
of the 1060, 1857, and 4362 base pair fragments. Although
lower flow rates typically yield better separations of most
fragments, the resolution of the 1857 and 4362 base pair
fragments was slightly greater at the higher flow rate, as
shown in Figure 18B.

FIGURE 18 FIGURE 19

OLIGOMERS
Sample PEG 200

A. 0.15 mL/min B. 0.5 mL/min

c
80 B: TSKgel G-OligoPW
Intensity (mV)

a b
c

a
d b 40

d A: TSKgel SuperOligoPW
e
e
f
f

0
150 180 210 240 270 50 60 70 80 3 5 7 9 11 13 15 17 19
Minutes Minutes Elution Time (min)

SEPARATION OF LARGE DNA FRAGMENTS ON TSKgel G-DNA-PW ANALYSIS OF PEG 200. COMPARISON BETWEEN TSKgel
SuperOligoPW AND TSKgel G-OLIGO-PW

Column: TSKgel G-DNA-PW, 10 µm, 4 x 7.8 mm ID x 30 cm L Column: A. TSKgel SuperOligoPW, 6.0 mm ID x 15 cm L x 2

Sample: 60 µL of Eco RI and Bst NI cleaved pBR322 DNA, B. TSKgel G-Oligo-PW, 7.8 mm ID x 30 cm L x 2
Base pairs: a. 4362, b. 1857, c. 1060 & 928, d. 383, e. 121, f. 13 Mobile phase: H2O; Flow rate: A: 0.6 mL/min, B: 1.0 mL/min
Mobile phase: 0.3 M NaCl, 1 mM EDTA, in 0.1 M Tris-HCl, pH 7.5, Detection: RI; Temp.: 25°C; Inj. vol.: A: 20 µL, B: 100 µL
Flow rate: A. 0.15 mL/min, B. 0.5 mL/min; Detection: UV @ 260 nm
20 [Link]

SEC
TSKgel PW-CP SERIES
SEC

TSKgel PWXL-CP size exclusion columns were specifically These columns show high theoretical plate numbers, linear
developed for the analysis of water soluble cationic calibration curves and high durability. The base resin is the
polymers. Three columns are available within the TSKgel same as that used in the TSKgel PWXL columns. Figure 20
PWXL-CP series, each with a different particle size, separation shows the calibration curves for PEG/PEO calibration curves
range and exclusion limit, allowing polymers within a wide obtained with TSKgel PWXL-CP columns.
molecular mass range to be separated and characterized.
Figure 21 demonstrates that these SEC columns can be
When using conventional SEC columns the analysis of utilized for the analysis of a wide variety of cationic polymers.
cationic polymers requires a high salt concentration in the Various cationic polymers with different functional groups
mobile phase to prevent adsorption of the polymers onto the and molecular weights were injected on the three TSKgel
particles in SEC columns. The TSKgel PWXL-CP columns PWXL-CP columns (TSKgel G6000PWXL-CP, G5000PWXL-CP
eliminate ionic adsorption onto the particle by incorporating and G3000PWXL-CP, connected in series).
a cationic functionality on the particle surface. This
modification results in high recovery for cationic polymers
and enables elution under low salt conditions.

FIGURE 20 FIGURE 21

1x107
TSKgel G3000PWXL-CP PAA (MW:438kDa)
TSKgel G3000PWXL-CP,
PAA 7µm
(MW:235kDa)
TSKgel G5000PWXL-CP 100 PEI (MW:266kDa)
1x106 TSKgel G5000PWXL-CP,
P(DADMACI)10µm
(MW:204kDa)
PAS (MW:7800Da)
TSKgel G6000PWXL-CP
TSKgel G6000PWXL-CP,
PAS 13µm
(MW:287kDa)
Log Molecular Weight (PEO)

Cationic Dextran (MW:11kDa)

Chitosan (MW:13.4kDa)
1x105

Mobile phase:
60 0.1mol/L NaNO3
Flow Rate: 1mL/min
mV

1x104
Detection: RI
1000 Temp: 25°C
Samples:20 polyethylene oxides (PEO) standards
polyethylene glycols (PEG) standards
100
10 15 20 25 30 35
10 -20
3 5 7 9 11 Elution Time (minutes)

Elution time (min)

Columns: TSKgel G3000PWXL-CP, 7µm (7.8mm ID x 30cm),
CALIBRATION CURVE FOR TSKgel PWXL-CP COLUMNS ANALYSIS OF CATIONIC POLYMERS
TSKgel G5000PWXL-CP, 10µm (7.8mm ID x 30cm),
Columns: TSKgel G3000PWXL-CP, 7 µm; TSKgel G5000PWXL-CP, Columns: TSKgel G3000PWTSKgel G6000PWXL-CP,mm
XL-CP, 7 µm (7.8
13µm
ID x(7.8mm
30 cm ID
L),xTSKgel
30cm)
10 µm; TSKgel G6000PWXL-CP, 13 µm; Samples: polyethylene Eluent:
G5000PW XL-CP, 10 µm0.1mol/L
(7.8 mm NaNO
ID x3 30 cm L), TSKgel G6000PWXL-
oxides (PEO) standards; polyethylene glycols (PEG) standards Flow
CP, 13 µmRate:
(7.8 mm ID1mL/min
x 30 cm L); Mobile phase: 0.1 mol/L NaNO3;
Mobile phase: 0.1 mol/L NaNO3; Flow rate: 1 mL/min; Detection: RI; FlowDetection:
rate: 1 mL/min;RIDetection: RI; Temperature: 25 °C; Sample
Temp: 25 °C Load: 3 g/L, 100 µL
Temperature: 25°C
Sample Load: 3g/L, 100µL
 TOSOH BIOSCIENCE ANALYSIS 21

SEC
TSKgel SuperMultiporePW SERIES

SEC
The new TSKgel SuperMultiporePW column line is Figure 23 shows the SEC analysis of a real sample -
incorporating Tosoh’s proprietary multi-pore particle Polyvinylpyrrolidone (PVP) K-30 - on a series of conventional
technology. These semi-micro SEC columns provide near TSKgel G3000PWXL and G5000PWXL columns compared to
linear calibration curves. They are ideally suited to analyze the one obtained with a single TSKgel SuperMultiporePW-M
the molecular weight and the MW distribution of water semi-micro linear SEC column (MW range 600,000 –
soluble polymers, such as polyvinylpyrrolidones or dextrans. 1,500,000). On a series of conventional SEC columns the
Polyvinylpyrrolidone peak shows an inflection point, which
TSKgel SuperMultiporePW columns are packed with spherical does not appear on the SuperMultiporePW-M column.
mono-disperse polymethacrylate particles, each containing Analysis is much faster and more sensitive when applying
a wide range of pore sizes. They belong to the semi-micro the new multi-pore packing.
type of SEC columns (6 mm ID, 15 cm length) providing
high theoretical plate numbers at half of the length of a FIGURE 23
conventional SEC column. The TSKgel SuperMultiporePW
series comprises of three column types covering different
molecular weight ranges (PW-N; PW-M, PW-H).

Multi-pore particle technology is the most elegant way to

achieve near linear SEC calibration curves. It solves the
known problem of peak disturbances/inflection points,
which typically occur due to a mismatch of pore sizes when
columns with different molecular weight ranges are coupled.
Particles produced by multi-pore technology contain a
broad range of pore sizes in a single polymeric bead. This
innovative approach essentially creates a linear calibration
curve within each particle (Figure 22).

Multi-pore, semi-micro SEC columns provide high resolution

and smooth peak shapes without shoulders or inflection ANALYSIS OF POLYVINYLPYRROLIDONE
points. This leads to better accuracy and reproducibility
when determining the molecular mass distribution of water Columns: TSKgel SuperMultiporePW-M, 6 mm ID x 15 cm L x 1 (red)
soluble polymers. TSKgel G3000PWXL & G5000PWXL, each 7.8 mm ID x 30 cm L
in line (blue); Sample: Polyvinylpyrrolidone (K-30); Mobile phase:
0.1 mol/L NaNO3; Flow rate: 0.6 mL/min; Detection: RI

FIGURE 22

Strategies for wide range separation using Size Exclusion Chromatography

STRATEGIES FOR WIDE RANGE SEPARATION USING SIZE EXCLUSION CHROMATOGRAPHY

Conventional Strategy New Strategy

Large Pore Medium Pore Small Pore Multiple Pore Size

Connect columns with Blend (mixed bed) packings Pure packings

different grades of packings of different grades with multi-pore size distribution
22 [Link]

SEC
TSKgel PW SERIES ORDERING INFORMATION
SEC

ORDERING INFORMATION

PART # DESCRIPTION ID LENGTH PARTICLE NUMBER FLOW RATE (mL/min) MAXIMUM

(mm) (cm) SIZE (μm) THEORETICAL RANGE MAX. PRESSURE
PLATES DROP (MPa)

STAINLESS STEEL COLUMNS

0022789 SuperMultiporePW-N 6.0 15 4 >16,000 0.3 - 0.6 0.6 4.5
0022790 SuperMultiporePW-M 6.0 15 5 >12,000 0.3 - 0.6 0.6 2.7
0022791 SuperMultiporePW-H 6.0 15 8 (6-10) >7,000 0.3 - 0.6 0.6 0.9
0022792 SuperOligoPW 6.0 15 3 >16,000 0.3 - 0.6 0.6 5.0
0008031 G-Oligo-PW 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
0008032 G-DNA-PW 7.8 30 10 ≥ 10,000 0.2 - 0.5 0.6 2.0
0008020 G2500PWXL 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
0008021 G3000PWXL 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
0008022 G4000PWXL 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 2.0
0008023 G5000PWXL 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 2.0
0008024 G6000PWXL 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0
0008025 GMPWXL 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0
0021873 G3000PWXL-CP 7.8 30 7 ≥ 16,000 1.0 5.5
0021874 G5000PWXL-CP 7.8 30 10 ≥ 10,000 1.0 2.5
0021875 G6000PWXL-CP 7.8 30 13 ≥ 7,000 1.0 2.0
0005761 G2000PW 7.5 30 12 ≥ 5,000 0.5 - 1.0 1.2 2.0
0008028 G2500PW 7.5 30 12 ≥ 5,000 0.5 - 1.0 1.2 2.0
0005762 G3000PW 7.5 30 12 ≥ 5,000 0.5 - 1.0 1.2 2.0
0005763 G4000PW 7.5 30 17 ≥ 3,000 0.5 - 1.0 1.2 1.0
0005764 G5000PW 7.5 30 17 ≥ 3,000 0.5 - 1.0 1.2 1.0
0005765 G6000PW 7.5 30 17 ≥ 3,000 0.5 - 1.0 1.2 1.0
0008026 GMPW 7.5 30 17 ≥ 3,000 0.5 - 1.0 1.2 1.0
0005105 G2000PW 7.5 60 12 ≥ 10,000 0.5 - 1.0 1.2 4.0
0008029 G2500PW 7.5 60 12 ≥ 10,000 0.5 - 1.0 1.2 4.0
0005106 G3000PW 7.5 60 12 ≥ 10,000 0.5 - 1.0 1.2 4.0
0005107 G4000PW 7.5 60 17 ≥ 6,000 0.5 - 1.0 1.2 2.0
0005108 G5000PW 7.5 60 17 ≥ 6,000 0.5 - 1.0 1.2 2.0
0005109 G6000PW 7.5 60 17 ≥ 6,000 0.5 - 1.0 1.2 2.0
0008027 GMPW 7.5 60 17 ≥ 6,000 0.5 - 1.0 1.2 2.0
0008030 G2500PW 21.5 60 17 ≥ 10,000 1.6 - 6.0 8.0 2.0

PEEK
0020024 BioAssist G6PW 7.8 30 17 ≥ 3,000 0.5 - 1.0 1.2 10

GUARD COLUMNS
0022793 SuperMP (PW)-N Guard column4.6 3.5 4
0022794 SuperMP (PW)-M Guard column4.6 3.5 5
0022795 SuperMP (PW)-H Guard column4.6 3.5 8
0022796 SuperOligoPW Guard column 4.6 3.5 3
0008034 Oligo Guard column 6.0 4.0 13 For 7.8 mm ID G-Oligo-PW columns
0008033 PWXL Guard column 6.0 4.0 12 For 7.8 mm ID PWXL & G-DNA-PW (TSKgel G3000PW packing)
0021876 PWXL-CP Guard column 6.0 4.0 13 For 7.8 mm ID PWXL-CP columns
0006763 PW-L Guard column 7.5 7.5 13 For 7.5 mm ID G1000PW & G2000PW (TSKgel G2000PW packing)
0006762 PW-H Guard column 7.5 7.5 13 For 7.5 mm ID G2500PW through GMPW columns
0006758 PW-H Guard column 21.5 7.5 17 For 21.5 mm ID G2500PW through G5000PW columns
24 [Link]

SEC TSKgel Alpha & SuperAW SERIES

ORDERING INFORMATION
SEC

ORDERING INFORMATION

PART # DESCRIPTION ID LENGTH PARTICLE NUMBER FLOW RATE (mL/min) MAXIMUM

(mm) (cm) SIZE (μm) THEORETICAL RANGE MAX. PRESSURE
PLATES DROP (MPa)

STAINLESS STEEL COLUMNS

0018339 Alpha-2500 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
0018340 Alpha-3000 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
0018341 Alpha-4000 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 3.0
0018342 Alpha-5000 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 3.0
0018343 Alpha-6000 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0
0018344 Alpha-M (mixed bed) 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0

GUARD COLUMNS
0018345 Alpha Guard column 6.0 4 13 For all Alpha columns

VMPAK COLUMNS*
0020011 VMpak-25 2.0 5 7 ≥ 1,000 0.1 - 0.2 0.25 2.0
0020012 VMpak-25 2.0 15 7 ≥ 3,000 0.1 - 0.2 0.25 6.0

STAINLESS STEEL COLUMNS

0019315 SuperAW2500 6.0 15 4 ≥ 16,000 0.3 - 0.6 6.0
0019316 SuperAW3000 6.0 15 4 ≥ 16,000 0.3 - 0.6 6.0
0019317 SuperAW4000 6.0 15 6 ≥ 10,000 0.3 - 0.6 4.0
0019318 SuperAW5000 6.0 15 7 >10,000 0.3 - 0.6 3.0
0019319 SuperAW6000 6.0 15 9 >7,000 0.3 - 0.6 2.0
0019320 SuperAWM-H 6.0 15 9 >7,000 0.3 - 0.6 2.0

GUARD COLUMNS
0019321 SuperAW-L Guard Column 4.6 3.5 7 For SuperAW2500-4000 columns.
0019322 SuperAW-H Guard Column 4.6 3.5 13 For SuperAW5000-AWM-H columns

*TSKgel VMpak-25 series contains a similar packing as TSKgel Alpha-2500. It can be used for multimodal LC and LC-MS separations.
24 [Link]

SEC TSKgel Alpha & SuperAW SERIES

ORDERING INFORMATION
SEC

ORDERING INFORMATION

PART # DESCRIPTION ID LENGTH PARTICLE NUMBER FLOW RATE (mL/min) MAXIMUM

(mm) (cm) SIZE (μm) THEORETICAL RANGE MAX. PRESSURE
PLATES DROP (MPa)

STAINLESS STEEL COLUMNS

0018339 Alpha-2500 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
0018340 Alpha-3000 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
0018341 Alpha-4000 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 3.0
0018342 Alpha-5000 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 3.0
0018343 Alpha-6000 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0
0018344 Alpha-M (mixed bed) 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0

GUARD COLUMNS
0018345 Alpha Guard column 6.0 4 13 For all Alpha columns

VMPAK COLUMNS*
0020011 VMpak-25 2.0 5 7 ≥ 1,000 0.1 - 0.2 0.25 2.0
0020012 VMpak-25 2.0 15 7 ≥ 3,000 0.1 - 0.2 0.25 6.0

STAINLESS STEEL COLUMNS

0019315 SuperAW2500 6.0 15 4 ≥ 16,000 0.3 - 0.6 6.0
0019316 SuperAW3000 6.0 15 4 ≥ 16,000 0.3 - 0.6 6.0
0019317 SuperAW4000 6.0 15 6 ≥ 10,000 0.3 - 0.6 4.0
0019318 SuperAW5000 6.0 15 7 >10,000 0.3 - 0.6 3.0
0019319 SuperAW6000 6.0 15 9 >7,000 0.3 - 0.6 2.0
0019320 SuperAWM-H 6.0 15 9 >7,000 0.3 - 0.6 2.0

GUARD COLUMNS
0019321 SuperAW-L Guard Column 4.6 3.5 7 For SuperAW2500-4000 columns.
0019322 SuperAW-H Guard Column 4.6 3.5 13 For SuperAW5000-AWM-H columns

*TSKgel VMpak-25 series contains a similar packing as TSKgel Alpha-2500. It can be used for multimodal LC and LC-MS separations.
 TOSOH BIOSCIENCE ANALYSIS 23

SEC
TSKgel Alpha & SuperAW SERIES

SEC
FIGURE 24
The TSKgel Alpha and SuperAW column series offer a new
alternative for performing SEC. The columns are packed 35,000
with a hydrophilic, highly crosslinked polymer which is
compatible to a wide range of solvents ranging from pure 30,000

Theoretical Plates (TP/Column)

aqueous up to 100 % organic mobile phases (see Figure
25,000
24). Both series consist of six columns with different pore
sizes, spanning a wide MW separation range from 100 to 20,000
over 1,000,000 Da when using polyethylene glycol (PEG) as
a standard. Exclusion limits for polyethylene oxides in water 15,000

and other physical properties for the Alpha and SuperAW TSKgel Alpha-3000
10,000 TSKgel G3000PWXL
columns are listed in Table 6.
5,000
The TSKgel Alpha and SuperAW column series can be used
for separations of synthetic polymers, oligomers, additives 0

ile

2 O
SO
and detergents as well as for saccharides, nucleic acids and

to 0
SO l
l

P
F
O

F
M 2O

no

no
no

2
TH

FI
M

tr

H
/H
H
2

l/H

M
H
ha

pa
ha

ni
D

F/
D

TH
o
peptides. TSKgel SuperAW columns with reduced particle

Et

ro
et
an

ce
M
P
2-
h

A
D
Solvent

et
size and semi-micro column dimensions of 6 mm ID and 15

M
cm length provide short analysis times and higher resolution
SOLVENT COMPATABILITY OF TSKgel Alpha-3000 WITH
power. For samples with big differences in molecular ORGANIC SOLVENT
weights, the mixed bed columns TSKgel Alpha-M and
TSKgel SuperAWM-H show linear calibration curves over Conditions for solvent change: Flow rate: 1.0 mL/min; Temp.: 25 °C;
Time for purge: 8 h; Conditions for TP measurement: Sample:
the whole range.
ethylene glycol; Flow rate: 1.0 mL/min; Temp.: 25 °C; Detection: RI

HIGHLIGHTS

Unique hydrophilic polymethacrylate resin with

rigid spherical beads
Minimal swelling characteristics from 100% water to
100% non-polar solvents
Excellent mechanical and chemical stability
TSKgel SuperAW columns with reduced particle size
and shorter columns length provide short analysis
times and high resolution power

TABLE 6

PROPERTIES AND SEPARATION RANGES OF TSKgel Alpha AND SuperAW-SERIES

TSKgel COLUMN ID (mm) X PARTICLE MIN NO. EXCLUSION LIMIT

LENGTH (cm L) SIZE (μm) THEORET. PLATES (PEO/H2O)

Alpha-2500 7.8 x 30 7 16,000 5 x 103

Alpha-3000 7.8 x 30 7 16,000 9 x 104
Alpha-4000 7.8 x 30 10 10,000 4 x 105
Alpha-5000 7.8 x 30 10 10,000 1 x 106
Alpha-6000 7.8 x 30 13 7,000 >1 x 107
Alpha-M 7.8 x 30 13 7,000 >1 x 107

SuperAW2500 6.0 x 15 4 >16,000 5 x 103

SuperAW3000 6.0 x 15 4 >16,000 9 x 104
SuperAW4000 6.0 x 15 6 >10,000 1 x 106
SuperAW5000 6.0 x 15 7 >10,000 1 x 106
SuperAW6000 6.0 x 15 9 >6,000 1 x 107
SuperAWM-H 6.0 x 15 9 >6,000 1 x 107
24 [Link]

SEC TSKgel Alpha & SuperAW SERIES

ORDERING INFORMATION
SEC

ORDERING INFORMATION

PART # DESCRIPTION ID LENGTH PARTICLE NUMBER FLOW RATE (mL/min) MAXIMUM

(mm) (cm) SIZE (μm) THEORETICAL RANGE MAX. PRESSURE
PLATES DROP (MPa)

STAINLESS STEEL COLUMNS

0018339 Alpha-2500 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
0018340 Alpha-3000 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
0018341 Alpha-4000 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 3.0
0018342 Alpha-5000 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 3.0
0018343 Alpha-6000 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0
0018344 Alpha-M (mixed bed) 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0

GUARD COLUMNS
0018345 Alpha Guard column 6.0 4 13 For all Alpha columns

VMPAK COLUMNS*
0020011 VMpak-25 2.0 5 7 ≥ 1,000 0.1 - 0.2 0.25 2.0
0020012 VMpak-25 2.0 15 7 ≥ 3,000 0.1 - 0.2 0.25 6.0

STAINLESS STEEL COLUMNS

0019315 SuperAW2500 6.0 15 4 ≥ 16,000 0.3 - 0.6 6.0
0019316 SuperAW3000 6.0 15 4 ≥ 16,000 0.3 - 0.6 6.0
0019317 SuperAW4000 6.0 15 6 ≥ 10,000 0.3 - 0.6 4.0
0019318 SuperAW5000 6.0 15 7 >10,000 0.3 - 0.6 3.0
0019319 SuperAW6000 6.0 15 9 >7,000 0.3 - 0.6 2.0
0019320 SuperAWM-H 6.0 15 9 >7,000 0.3 - 0.6 2.0

GUARD COLUMNS
0019321 SuperAW-L Guard Column 4.6 3.5 7 For SuperAW2500-4000 columns.
0019322 SuperAW-H Guard Column 4.6 3.5 13 For SuperAW5000-AWM-H columns

*TSKgel VMpak-25 series contains a similar packing as TSKgel Alpha-2500. It can be used for multimodal LC and LC-MS separations.
 TOSOH BIOSCIENCE ANALYSIS

1 2 3 SEC
OPTIMIZING SEC
TOSOH BIOSCIENCE GMBH TOSOH BIOSCIENCE LLC TOSOH CORPORATION
IM LEUSCHNERPARK 4 3604 HORIZON DRIVE, 3-8-2 SHIBA, MINATO-KU

SEC
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[Link]@[Link] [Link]@[Link] INFO@[Link]
[Link] [Link] [Link] As SEC is a partition chromatography, sample load on the For TSKgel SW columns mobile phases a buffer concen-
column is limited. High sample loads distort peak shapes and tration between 0.1 M and 0.5 M is recommended. Under
cause an overall decrease in efficiency due to column over- low ionic strength (< 0.1 M), ionic interactions between the
load. Optimal sample load highly depends on the sample sample molecules and the silica surface may occur. Under
WITH A GLOBAL PERSPECTIVE. properties (sample matrix) and the separation task. For conditions of high ionic strength (>1.0 M), hydrophobic
analytical columns, sample concentrations of 1-20 mg/ml are interactions are more likely to occur. A neutral salt, such
Tosoh Bioscience is a leading manufacturer in the field of liquid recommended. Proteins can be loaded at higher concentra- as sodium sulphate may be added to the buffer to increase
chromatography. The portfolio of over 500 specialty products en- tions and higher total loads than synthetic macromolecules.
For preparative purposes for example, 100 mg of BSA can
buffer ionic strength. Also the ionic species of the buffer
has an effect on the separation. As a good starting point, a
compasses instruments for size exclusion/gel permeation chroma- be loaded on two 21.5 mm ID x 60 cm L TSKgel G3000SW 0.1 M sodium phosphate buffer together with 0.1 M sodium
columns, but only 20 mg of PEG 7500. sulphate has proved to be of value.
tography and a comprehensive line of media and prepacked (U)HPLC 1
Sample volume depends very much on the type of column.
On TSKgel SuperSW columns for example, a 5 µL injection As the polymeric TSKgel PW and Alpha-type resins carry
columns for all common modes of liquid chromatography. Over the 2 3
volume ensures optimal results. Standard injection volumes less residual charged groups on the surface than silica
for 7.5 and 7.8 mm ID columns are 20-100 µl, whereas for gels, salt concentration of the mobile phase can be lower.
last 40 years, TSKgel SW columns have become the worldwide in- 4
preparative purposes on 21.5 mm ID columns, injection Non-ionic, non-polar compounds such as polyethylene
volumes may be raised up to 2 ml. glycols can simply be analyzed with distilled water. For ionic
dustry standard for size exclusion chromatography of biomolecules. polymeric compounds, a neutral salt such as sodium nitrate
MOBILE PHASE is added to the aqueous eluent. Generally, a concentration
5 of 0.1 M to 0.2 M is sufficient to overcome undesirable ionic
Proper selection of the mobile phase is necessary to maximize interactions.
Tosoh manufacturing sites in Japan provide products to the sales molecular sieving mechanism and to minimize secondary
and support subsidiaries in the U.S. and Europe, ensuring full effects such as ionic and hydrophobic interaction between
the sample and the column packing material. For each sample
If hydrophobic interaction occurs between the sample and
the column matrix, a water soluble organic solvent can be
global coverage. Our technical specialists in the European Head- there will be an optimum buffer type and concentration that added to the mobile phase. The addition of acetonitrile,
results in the highest resolution and recovery. acetone, ethanol or methanol up to a concentration of 20%
quarters provide assistance in developing HPLC applications or may also prevent columns from fouling by suppressing
interaction of hydrophobic impurities of the sample. An
purification methods, in up-scaling, or packing process columns. 4
TOSOH BIOSCIENCE SHANGHAI CO. LTD.
5
TOSOH ASIA PTE. LTD.
FIGURE 24 example is shown in Figure 24 with the analysis of a pegylat-
ed protein on a TSKgel G3000SWXL column. As pegylated
We offer chromatographic workshops, on-site training, and are ROOM 301, PLAZA B,
NO. 1289 YI SHAN ROAD
63 MARKET STREET #10-03
BANK OF SINGAPORE CENTRE products are more hydrophobic, they tend to interact with
XU HUI DISTRICT SINGAPORE 048942, SINGAPORE the column matrix. Over time the pegylated product can foul
the sole sponsor of the HIC/RPC Bioseparation Conference series.
200 200
SHANGHAI, 200233, CHINA
T +86 21 3461 0856 T +65 6226 5106
the column, which is indicated by shifts of retention time
and decreasing separation performance. By adding 10% of

Intensity (mAu)

Intensity (mAu)
F +86 21 3461 0858 F +65 6226 5215
150 150
INFO@[Link] [Link]@[Link] ethanol to the elution buffer, this problem is overcome and no
[Link] [Link]
differences in performance at the first and the 150th injection
100 100
are observed (courtesy of J.J. Ratto et al. Amgen Inc., 1996).
TOSOH HISTORY
1935 TOYO SODA MANUFACTURING CO., LTD. IS FOUNDED 50 50 COLUMN PROTECTION
1936 OPERATION OF NANYO MANUFACTURING COMPLEX BEGINS
1971 SCIENTIFIC INSTRUMENTS DIVISION DEVELOPS FIRST GPC COLUMN USING TSKgel 0 0 To protect the column and increase its lifetime, the use of
1974 HPLC COLUMN PLANT IS COMPLETED 7 8 9 10 11 12 13 7 8 9 10 11 12 13 a guard column is strongly recommended. Sample purity,
1979 TOSOH DEVELOPS TOYOPEARL MEDIA Elution time (min) Elution time (min) sample load and the composition of the mobile phase have
1983 TOSOH DEVELOPS HYDROPHOBIC INTERACTION MEDIA an influence on column lifetime. For information on TSKgel
1987 TOSOHAAS US STARTS OPERATING FROM MONTGOMERYVILLE INFLUENCE OF MOBILE PHASE SEC columns for GPC analysis of organic polymers please
1989 TOSOHAAS GmbH STARTS OPERATING FROM STUTTGART refer to the TSKgel GPC column brochure.
A: No ethanol in mobile phase; B: 10% ethanol in mobile phase
1995 TOSOH NANYO GEL FACILITY RECEIVES ISO 9001 Column: TSKgel G3000SWXL columns, 5 µm, 7.8 mm ID x 30 cm L
2000/2001 FORMER TOSOHAAS US AND EUROPE OPERATIONS BECOME TOSOH BIOSEP, A 100% SUBSIDIARY OF TOSOH CORPORATION Sample: 10 mL PEG r-HuMGDF;
2002/ 2003 ALL SCIENTIFIC & DIAGNOSTIC SYSTEM RELATED COMPANIES IN EUROPE AND THE US ARE UNIFIED UNDER THE NEW NAME TOSOH BIOSCIENCE
initial injection; after 150 injections
Mobile phase: 0.1 M sodium phosphate, pH 6.9, 0.5 M NaCl,
2008 EcoSEC, THE 7 TH GENERATION GPC SYSTEM IS INTRODUCED Flow rate: 0.7 mL/min; Detection: UV @ 220 nm
2009 TOSOH BIOSCIENCE GmbH CELEBRATES ITS 20TH ANNIVERSARY
2010 TOSOH CELEBRATES ITS 75 TH YEAR IN BUSINESS AND CONTINUED RAPID EXPANSION IN CHINA
2011 TOSOH BIOSCIENCE CELEBRATES 40 YEARS OF OPERATION
2012 TOSOH RELEASES FIRST TOYOPEARL MIXED-MODE RESIN
2013 TOSOH RELEASES A HIGH CAPACITY PROTEIN A RESIN
2014 TOSOH BIOSCIENCE GmbH CELEBRATES ITS 25 TH ANNIVERSARY
2015 TOSOH BIOSCIENCE GmbH MOVES TO GRIESHEIM, GERMANY
 PRINCIPLES OF CHROMATOGRAPHY

SEC COLUMNS The analysis, isolation, and purifi­cation of biomolecules can be

accomplished by a number of chromatographic modes. Each mode
is based on specific physical, chemical, or biological interactions
between the sample biomolecule and packing material.

Tosoh Bioscience offers a comprehensive line of TSKgel and

TOYOPEARL media and pre-packed TSKgel columns for all com-
mon modes of liquid chromatography.

The various modes of chromato­graphy involve separations that are

based on specific features of the target or sample, like size, charge,
hydropho­­
bi­
city, function or specific content of the molecule.
To find out more about general principles of liquid chromatog-
raphy and on how each of them works, visit us on our YouTube
channel [Link]

TOSOH BIOSCIENCE

Im Leuschnerpark 4 64347 Griesheim, Germany SIZE EXCLUSION CHROMATOGRAPHY

Tel: +49 6155-7043700 Fax: +49 6155-8357900
[Link]@[Link] [Link]
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