TSKgel Ion Exchange

Chromatography Columns

Anion Exchange columns Cation Exchange columns

TSKgel BioAssist Q TSKgel BioAssist S

TSKgel DEAE-2SW TSKgel CM-2SW
TSKgel DEAE-3SW TSKgel CM-3SW
TSKgel DEAE-5PW TSKgel CM-5PW
TSKgel DEAE-NPR TSKgel CM-STAT
TSKgel DNA-NPR TSKgel OApak-A
TSKgel DNA-STAT TSKgel SCX
TSKgel QAE-2SW TSKgel SP-2SW
TSKgel Q-STAT TSKgel SP-5PW
TSKgel SAX TSKgel SP-NPR
TSKgel Sugar AXG TSKgel SP-STAT
TSKgel Sugar AXI
TSKgel SuperQ-5PW

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 Ion Exchange Tips:
• TSKgel ion exchange columns are offered in glass, PEEK, and stainless steel hardware. Stainless steel (SS) or Pyrex
frits are embedded in the body of the column end-fittings of metal and glass columns, respectively. The nominal frit
size for SS columns is engraved in the end-fittings; Pyrex frits in the glass columns have a 10 µm nominal pore size.

• Halide salts corrode stainless steel tubing, fitting, and frits. Do not store SS columns in a mobile phase containing
NaCl and, where possible, use another salt in the operating buffer. Chlorotrifluorethylene and tetrafluorethylene are
the materials in the glass column fittings that come into contact with the mobile phase and sample.

• Good laboratory procedures demand that the analytical column be protected by a guard column. TSKgel guardgel
kits, containing column hardware and gel packing, are available to pack your own guard column. In addition, guard
cartridges and packed guard columns are available for use with TSKgel ion exchange columns.

• TSKgel ion exchange columns are supplied with an Inspection Data Sheet, which includes a QC chromatogram and
test data, and an OCS Sheet summarizing the recommended operating conditions for optimum column performance.

• A separate TSKgel Column Instruction Manual that reviews general guidelines for column installation and care, as
well as troubleshooting tips for commonly encountered problems, can be downloaded from the Tosoh Bioscience
LLC website (www.tosohbioscience.com).

80
 TSKgel Ion Exchange
Chromatography Columns

About Ion Exchange Chromatography

Ion Exchange Chromatography (IEC) is a technique based

on the difference in the strength of the interaction between
a sample ion and an oppositely charged functional group
on the support. The sample ion competes for the functional
group with a counter ion that has been added to the mobile
phase as a salt. Elution is most often accomplished by
increasing the salt concentration over time.

Ion exchange chromatography is the most common

separation mode for protein purification schemes.
Biomolecules generally have charged groups on their
surfaces, which change with the pH of the solution.

Anion Exchange Chromatography is performed with either

a strong anion exchange column, containing a quaternary
ammonium ion, or with a weak anion exchanger, having
either a tertiary or secondary amine functional group,
such as DEAE (diethylaminoethyl). A counter ion, often Cl-,
maintains electroneutrality.

Cation Exchange Chromatography is performed with either

a strong cation exchanger, containing a bonded sulfonic
acid group, such as sulfopropyl (SP), or with a weak cation
exchanger, containing a weak acid such as carboxymethyl
(CM). A counter ion, often Na +, maintains electroneutrality.
The advantage of strong vs. weak ion exchangers is that
the first are charged over a wider pH range. Weak ion
exchangers often provide slightly different selectivity from
strong exchangers.

In ion exchange chromatography, the pH of the mobile

phase buffer must be between the pI or pKa of the charged
molecule and the pKa of the charged groups on the solid
support. For example, a molecule with a pI of 8.2 is run in a
mobile phase buffer at pH 6.0 with the solid support pKa at
1.2 in cation exchange chromatography. In anion exchange
chromatography a molecule with a pI of 6.8 is run in a
mobile phase buffer at pH 8.0 with the solid support pKa at
10.3.

Figure 1: Ion Exchange Chromatography

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TSKgel Anion and Cation Exchange • TSKgel BioAssist ion exchange columns:
Chromatography Columns These columns are also based on methacrylate particle
design technology. Particles in TSKgel BioAssist Q
Tosoh Bioscience offers a broad line of high efficiency columns contain very large pores (~400 nm) that
columns for analysis and isolation of biomolecules by ion are functionalized with polyamine groups to form a
exchange chromatography. Methacrylate, silica, hydrophilic network structure. The capacity of the TSKgel BioAssist
polymer, and polystyrene are used as matrices for the Q columns is high over a wide molecular weight range
TSKgel line of anion and cation exchange columns. Tables (up to 1.0 × 106 Da). TSKgel BioAssist S columns are
1 and 2 list the available columns according to matrix and packed with particles possessing 130 nm pores
summarize the features and benefits of TSKgel ion exchange functionalized with sulfopropyl groups. TSKgel
columns. BioAssist columns are available exclusively in PEEK
housing.
• TSKgel STAT ion exchange columns:
These are nonporous polymer columns with high • TSKgel DEAE-NPR, DNA-NPR and SP-NPR
surface density of functional groups: quaternary ion exchange columns:
ammonium for anion exchange (Q- and DNA-STAT), Methacrylate is the backbone of these nonporous
carboxymethyl (CM-STAT) and sulfopropyl (SP-STAT) resin columns, which are packed with 2.5 µm particles.
for cation exchange. Particle sizes and dimensions High column efficiency coupled with low sample
of the TSKgel STAT columns are optimized either for capacity restricts the application of these columns to
highest throughput or for highest efficiency. fast analysis and micro-scale preparative isolation. Due
Applications for the TSKgel STAT columns include to the absence of large pores, protein recovery is
the separation of proteins, protein aggregates, charge generally very high on TSKgel NPR columns.
isomers of monoclonal antibodies, PEGylated proteins,
DNA fragments, nucleic acids, oligo DNA, and siRNA. • TSKgel DEAE-2SW, DEAE-3SW, QAE-2SW, SP-2SW, CM-
2SW, CM-3SW ion exchange columns:
• TSKgel DEAE-5PW, SP-5PW, CM-5PW, SuperQ-5PW Silica-based TSKgel anion and cation exchange
ion exchange columns: columns with diethylaminoethyl (DEAE), sulfopropyl
The polymethacrylate-based resin, TSKgel 5PW, is a (SP), trimethylamino (QAE), and carboxymethyl (CM)
spherical 10 µm particle with approximately 100 nm functional groups are available for analyzing smaller
pores. It is derivatized with diethylaminoethyl (DEAE), molar mass samples such as nucleotides, drug
sulfopropyl (SP) or carboxymethyl (CM) functionalities candidates, catecholamines, and small peptides or
to provide a weak anion, a strong cation, and a weak proteins. Binding capacity for small to medium size
cation exchanger, respectively. The polyamine proteins on these columns is approximately double that
chemistry employed in TSKgel SuperQ-5PW results of the TSKgel 5PW packings due to the smaller pore size
in a high capacity strong anion exchanger with a and larger surface area.
smaller effective pore size than TSKgel DEAE-5PW.
Proteins, peptides, DNA- and RNA-derived • Specialty TSKgel polystyrene-based ion
oligonucleotides, and other nucleic acid fragments exchange columns:
are typical samples that are analyzed or isolated on the These columns are available for the analysis of mono-
methacrylate-based TSKgel ion exchange columns. and disaccharides, organic acids and sugar alcohols.

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 TSKgel Ion Exchange
Chromatography Columns

Table 1: Features and benefits of TSKgel cation exchange columns

TSKgel Column Type Type/Matrix Benefit
strong(SP-STAT), Nonporous with high surface density of
CM-STAT, SP-STAT
weak (CM-STAT/polymer carboxymethyl (CM) and sulfopropyl (SP) groups
strong (SP-5PW), Polymethacrylate resin derivatized with
CM-5PW, SP-5PW
weak (CM-5PW)/polymethacrylate carboxymethyl (CM) and sulfopropyl (SP) ligands
Contain very large pores (130 nm), resulting in high
BioAssist S strong/polymethacrylate binding capacity and improved recovery of activity;
available exclusively in PEEK housing
Nonporous with 2.5 µm particles; fast analysis; high
SP-NPR strong/polymethacrylate
protein recovery
strong (SP-2SW), Silica-based with carboxymethyl (CM) and sulfopropyl
CM-2SW, CM-3SW, SP-2SW
weak (CM-2SW, CM-3SW)/silica (SP) functional groups
strong (SCX), Specialty columns for the analysis of organic acids,
SCX, OApak-A
weak (OApak-A)/ polymethacrylate saccharides and alcohols

Table 2: Features and benefits of TSKgel anion exchange columns

TSKgel Column Type Type/Matrix Benefit
strong (Q-STAT), Nonporous with high surface density of quaternary
Q-STAT, DNA-STAT
weak (DNA-STAT)/polymer ammonium groups
Polymethacrylate resin derivatized with
strong (SuperQ-5PW),
DEAE-5PW, SuperQ-5PW diethylaminoethyl (DEAE) and trimethylamino
weak (DEAE-5PW)/polymethacrylate
(SuperQ) ligands
Contain very large pores (400 nm), resulting in high
BioAssist Q strong/polymethacrylate binding capacity and improved recovery of activity;
available exclusively in PEEK housing
Nonporous with 2.5 µm particles; fast analysis; high
DEAE-NPR, DNA-NPR weak/polymethacrylate
protein recovery
DEAE-2SW, DEAE-3SW, strong ( QAE-2SW), Silica-based with diethylaminoethyl (DEAE), and
QAE-2SW weak (DEAE-2SW, DEAE-3SW)/silica trimethylamino (QAE) functional groups
Specialty columns for the analysis of mono and
Sugar AXG, Suger AXI, SAX strong/polystyrene disaccharides, as well as organic acids and sugar
alcohols

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About: TSKgel BioAssist Q Anion Exchange Columns Mouse Ascites

Especially designed for the separation of large Figure 2 compares chromatograms of mouse ascites fluid.
biomolecules, such as antibodies, the large pores of the Excellent separation between the antibody and albumin has
TSKgel BioAssist Q anion exchange column offer superior been obtained using a TSKgel BioAssist Q column versus a
capacity and resolution at a modest column back pressure. competitive Q-type product.
The anion exchange functionality of BioAssist Q columns
is introduced via a special graft polymerization technique
that results in a high density of ionic exchange groups in the 295
large particle pores that normally could only be achieved by Albumin
using particles containing a much smaller pore size. 245 Antibody

Detector response (AU)

TSKgel BioAssist Q anion exchange columns are offered in 195
B
a 4.6 mm ID × 5 cm format and a 10 mm ID × 10 cm semi-
preparative column for scale-up. The hardware for both 145
Antibody
columns is made of PEEK to reduce protein adsorption. Albumin
95

Attributes and Applications: 45

A
Table 3 lists the product attributes of TSKgel BioAssist Q -5
0 5 10 15
columns. The capacity of these columns is high over a wide
Retention time (minutes)
molecular weight range (up to 1.0 × 106 Da) and they are an
excellent choice for high throughput applications. Columns: A: TSKgel BioAssist Q, 10 µm, 4.6 mm ID × 5 cm
B: Commercial Q-type product A, 5.0 mm ID × 5 cm
Table 3: Product attributes Mobile phase: 15 min linear gradient of NaCl from 0 to 1.0 mol/L
in 20 mmol/L Tris-HCl buffer, pH 8.0
Attribute Value Flow rate: 1.0 mL/min
Detection: UV @ 280 nm
Matrix polymethacrylate Temperature: 25 ºC
Particle size (mean) 10 µm and 13 µm Injection vol.: 5 µL
Sample: mouse ascites fluid (3-fold dilution with initial eluent)
Pore size (mean) 400 nm
Functional group polyamine Figure 2: Analysis of mouse ascites fluid
Counter ion CI-
pH stability 2.0-12.0
Capacity (g BSA/L) 70
Small ion capacity 0.1 eq/L
pKa 9.4

TSKgel BioAssist Q, 4.6mm ID x 5cm

Commercial Q-type product A, 5.0mm ID x 5cm

Mobile phase: 15 min linear gradient of NaCl from 0 to 1.0mol/L

in 20mmol/L Tris-HCl buffer, pH 8.0
Flow Rate: 1.0mL/min
Detection: UV@280nm
Temperature: 25ºC
Injection vol.: 5µL
Sample: mouse ascites fluid (3-fold dilution with initial eluent)

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 TSKgel Ion Exchange
Performance enhancement on FPLC system
Chromatography Columns

Performance Enhancement on FPLC System Dynamic Binding Capacity

Figure 3 demonstrates the performance enhancement of The dynamic binding capacity for a TSKgel BioAssist Q
a TSKgel BioAssist Q column over a competitive product column and two commercially available columns is shown
when operated side-by-side on an FPLC system. in Figure 4 as a function of protein molar mass. Dynamic
capacity is plotted against the molar mass of 4 proteins
varying in molar mass from 2.0 × 10 4 Da to 6.7 × 105 Da and
1
1,200 2 is determined by continuously loading the column with
the protein solution and calculating the amount of protein
Detector response (AU)

1,180
1,600 3 adsorbed at 10 % height of the breakthrough curve.
1,140
1,120 The binding capacity on TSKgel BioAssist Q is uniformly
i
1,100 high for all proteins, while that of Mono Q (80 nm pores)
1,080 and TSKgel SuperQ-5PW (100 nm pores) is distinctly lower
1,060 A for the larger proteins. It is evident that neither material is
1,040
optimized for the analysis of monoclonal antibodies, which
have a molar mass of 1.5 × 105 Da. Antibodies are blood
2
1,200 1 3 components and as such are most stable at pH 7.35; they
1,180 become more labile at acidic pH. Their excess positive
charge makes anion exchange chromatography the method
Detector response (AU)

1,600
1,140 of choice for their chromatographic analysis.
1,120
i
1,100
100
1,080
1,060 90
B
1,040

Dynamic binding capacity (g/L-gel)

80

0 5 10 15 70
Retention time (minutes)
60
Columns: A: TSKgel BioAssist Q, 10 µm, 4.6 mm × 5 cm
50
B: Competitor Q, 5.0 mm ID × 5 cm
1
Mobile phase: 30 min linear gradient from 0 to 1 mol/L NaCl 40
2
in 20 mmol/L sodium phosphate, pH 8.0 30
3
Flow rate: 1.0 mL/min TSKgel BioAssist Q
Detection: UV @ 280 nm 20
Mono Q
Samples: 1. conalbumin 4
10 TSKgel SuperQ-5PW
i. ovalbumin impurity
2. ovalbumin 0
3. trypsin inhibitor 104 105 106
Log molar mass

Figure 3: Performance enhancement Columns: TSKgel BioAssist Q, 10 µm, 4.6 mm ID × 1 cm

Conventional Q-type product A, 5.0 mm ID × 1 cm
TSKgel SuperQ-5PW, 4.6 mm ID × 1 cm
Mobile phase: 20 mmol/L Tris-HCI buffer, pH 8.0
Flow rate: 0.38 mL/min
Detection: UV @ 280 nm
Temperature: 25 ºC
Samples: 1. trypsin inhibitor, 10 g/L
2. human serum albumin, 10 g/L
Column: TSKgel BioAssist Q, 4.6mm x 5cm (PEEK) 3. lgG1, 2.3 g/L
Competitor Q, 5.0mm x 5cm 4. thyroglobulin, 5 g/L
Mobile phase: 30 min linear gradient from 0 to 1mol/L NaCl
in 20mmol/L sodium phosphate pH 8.0
Flow Rate: 1.0mL/min
Detection: UV@280nm Figure 4: Dynamic binding capacity as function of protein
Sample: 1) conalbumin, i) ovalbumin impurity molar mass
2) ovalbumin, 3) trypsin inhibitor

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TSKgel BioAssist Q, 4.6mm ID x 1cm
Conventional Q-type product A, 5.0mm ID x 1cm
TSKgel SuperQ-5PW, 4.60.38mL/min1.0mL/min
Detection: UV@280nm
Temperature: 25ºC
Sample solvent: 20mmol/L Tris-HCI buffer, pH 8.0
Samples: 1. trypsin inhibitor 10g/L
2. human serum albumin 10g/L
About: TSKgel DEAE-2SW, DEAE-3SW and QAE-2SW Nucleotides
Anion Exchange Columns
High performance analyses of small anionic species are
TSKgel DEAE-2SW, DEAE-3SW, and QAE-2SW columns best performed on small pore silica-based anion exchange
are packed with porous spherical silica beads which columns, such as TSKgel DEAE-2SW. This is demonstrated
are chemically modified with a weak anion exchange in Figure 5.
group. These columns are for analyzing smaller molar
mass samples such as nucleotides, drug candidates,
catecholamines, and small peptides or proteins. 4

Attributes and Applications: 5

Detector response (AU)

Table 4 lists the product attributes of the TSKgel DEAE-2SW,
DEAE-3SW, and QAE-2SW columns. These columns are 3
packed with particles composed of silica with 12.5 nm and
25 nm pores and are stable in a pH range from 2.0 – 7.5.

Table 4: Product attributes

TSKgel
DEAE-2SW DEAE-3SW QAE-2SW
column
Matrix Silica 0 12 24
Retention time (minutes)
Particle size
5 µm 10 µm 5 µm
(mean) Column: TSKgel DEAE-2SW, 5 µm, 4.6 mm ID × 25 cm
Mobile phase: A: CH 3CN in 0.1 mol/L phosphate, pH 3.0, 20/80
Pore size B: CH 3CN in 0.5 mol/L phosphate, pH 3.0, 20/80
12.5 nm 25 nm 12.5 nm
(mean) Gradient: 30 min linear gradient from buffer A to B
Functional Flow rate: 1.0 mL/min
CH2CH2N+(C2H5) 3 CH2CH2N + (C2H5) 3 trimethylamino Detection: UV @ 260 nm
group
Samples: 1. AMP 2. IMP 3. GMP 4. ADP 5. ATP
Counter ion H2PO 4 - Cl- H2PO 4 -
pH stability 2.0-7.5 Figure 5: Separation of nucleotides
Small ion
>0.3 eq/L
capacity
pKa 11.2

Column: TSKgel DEAE-2SW, 4.6mm x 25cm

Elution: 30min linear gradient from buffer A to B
Buffer A: CH3CN in 0.1mol/L phosphate, pH 3.0, 20/80
Buffer B: CH3CN in 0.5mol/L phosphate, pH 3.0, 20/80
Flow Rate: 1.0mL/min
Detection: UV@260nm

Sample: 1. AMP
2. IMP
3. GMP
4. ADP
5. ATP

86
 TSKgel Ion Exchange
Chromatography Columns

Oligonucleotides Deoxyribonucleic Acids

Backbone-modified oligonucleotides are increasingly used Figure 7 demonstrates the successful separation of
for antisense therapy. These novel oligos have the benefit of adenosine triphosphate (ATP) and deoxyribonucleic acid
longer half-lives due to resistance to endogenous nucleases. (DNA) using a TSKgel DEAE-3SW column.
One common type of backbone-modified oligonucleotides is
phosphorothioates where one of the two nonbridged oxygen
atoms in the phosphate linkage has been replaced by a
sulfur atom. The separation of several phosphorothioates on 1
a TSKgel DEAE-2SW column is shown in Figure 6.

dCsCsCsCsC 4

Detector response (AU)

10 20 30 40 50 60
Retention time (minutes)
3
dCsCsCsC
Detector response (AU)

dCsCsC 2

10 20
dCsC 1
Retention time (minutes)

Column: TSKgel DEAE-3SW, 10 µm, 6 mm ID × 15 cm

Mobile phase: ACN/0.6 mol/L ammonium formate buffer,
pH 7.0 = 20/80
20 25 30
Flow rate: 0.7 mL/min
0 5 10 15
Retention time (minutes) Detection: UV @ 260 nm
Temperature: 23 °C
Column: TSKgel DEAE-2SW, 5 µm, 4.6 mm ID × 25 cm Samples: 1. adenosine triphosphate (ATP)
Mobile phase: A: 50 mmol/L ammonium acetate 2. deoxyribonucleic acid (d-TCGAGCATAATA), DNA
B: 1.5 mol/L ammonium acetate
Gradient: linear, 0-100 % B in 60 minutes
Flow rate: 1 mL/min Figure 7: Separation of ATP and DNA
Detection: UV @ 254 nm
Temperature: 25 °C
Samples: 1. 2 base phosphorothioate oligonucleotide
2. 3 base phosphorothioate oligonucleotide
3. 4 base phosphorothioate oligonucleotide
4. 5 base phosphorothioate oligonucleotide

Figure 6: Separation of phosphorothioates

Column: TSKgel DEAE-3SW, 1um, 6mm ID x 15cm

Column: TSKgel DEAE-2SW, 4.6mm ID x 25cm Mobile phase: acetonitrile/(0.6mol/L ammonium formate buffer, pH 7.0) = 20/80
Flow rate: 1mL/min Flow Rate: 0.7mL/min
Detection: UV@260nm
Buffer A: 50mmol/L ammonium acetate
Temerature: 23°C
Buffer B: 1.5mol/L ammonium acetate
Gradient: linear, 0-100% B in 60 minutes
Detection: UV@254nm
Temperature: ambient
Sample: 1. 2 base phosphorothioate oligonucleotide
2. 3 base phosphorothioate oligonucleotide
3. 4 base phosphorothioate oligonucleotide
4. 5 base phosphorothioate oligonucleotide

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About: TSKgel DEAE-5PW and SuperQ-5PW E. coli RNA
Anion Exchange Columns
Figure 8 shows the fractionation of high molar mass E. coli
The polymethacrylate-based resin, TSKgel 5PW, is a RNA on TSKgel DEAE-5PW, effectively utilizing the large 100
spherical 10 µm particle with approximately 100 nm pores. It nm pores of the TSKgel 5PW resin.
is derivatized with a diethylaminoethyl (DEAE) functionality
to provide the weak anion exchange column, TSKgel DEAE-
5PW, and with a polyamine functionality to provide the 16S rRNA
strong anion exchange column, TSKgel SuperQ-5PW. The High MM
polyamine network chemistry employed in TSKgel SuperQ- Impurities

Detector response (AU)

5PW columns results in a much higher capacity, but also a 23S rRNA
smaller effective pore size than TSKgel DEAE-5PW columns.
4S tRNA
The TSKgel SuperQ-5PW columns are used for the 5S rRNA
separation and analysis of proteins, oligonucleotides,
and other biomolecules. These columns are offered in a
stainless steel housing in dimensions of 7.5 mm ID × 7.5 cm
and 21.5 mm ID × 15 cm and in an 8 mm ID × 7.5 cm glass 0 40 80
format. Retention time (minutes)

Column: TSKgel DEAE-5PW, 10 µm, 6 mm ID × 15 cm (custom)

TSKgel DEAE-5PW columns are also used for the Mobile phase: 300 min linear gradient from 0.3 mol/L to
separation and analysis of proteins, along with nucleotides, 1.0 mol/L NaCl in 0.1 mol/L Tris-HCl, pH 7.6
nucleosides, and other biomolecules. These columns are Flow rate: 1.0 mL/min
available in internal diameters varying from 2 mm to 21.5 Detection: UV @ 260 nm
mm and in column housings of either glass or stainless Sample: total E. coli RNA
steel.

Attributes and Applications: Figure 8: Analysis of high MM RNA

Table 5 lists the product attributes of TSKgel SuperQ-5PW

and DEAE-5PW columns. These columns are an excellent Plasmid
choice for biologically active molecules. TSKgel
SuperQ-5PW and DEAE-5PW columns are stable over Figure 9 illustrates the separation of crude pBR322 plasmid
a pH range from 2.0 – 12.0 and have a mean pore size on a TSKgel DEAE-5PW column. This chromatographic
of 100 nm. separation provides purified plasmid in one hour, as
opposed to a conventional Cs-Cl density gradient
Table 5: Product attributes ultracentrifugation, which can take up to three days.

TSKgel column SuperQ-5PW DEAE-5PW

Matrix polymethacrylate
1.0
Particle size (mean) 10 µm and 13 µm 10 µm, 13 µm, and 20 µm
Column: TSKgel DEAE-5PW, 6mm ID x 15cm
Detector response (AU)

Pore size (mean) 100 nm

Mobile phase: 300min linear gradient from 0.3mol/L to
Functional group trimethylamino CH2CH2N + (C2H5) 3 1.0mol/L NaCl in 0.1mol/L Tris-HCl, pH 7.6
0.5
Counter ion Cl- Flow Rate: 1.0mL/min
Detection: UV@260nm
pH stability 2.0-12.0
Sample: total E. coli RNA
Capacity (mg BSA/mL) 100 30
0 35
Small ion capacity >0.13 eq/L 0.1 eq/L Retention time (minutes)

pKa 12.2 11.5 Column: TSKgel DEAE-5PW, 10 µm, 7.5 mm ID × 7.5 cm

Mobile phase: A: 25 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0
B: 25 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0,
with 1 mol/L NaCl
Gradient: 25-60 % B in 30 minutes
Flow rate: 1 mL/min
Detection: UV @ 260 nm
Temperature: 25 °C
Sample: pBR322 plasmid
Sample load: 2.5 mg in 1 mL

Figure 9: Detection of HIV-1 PCR-Amplified 130 bp target

88
 TSKgel Ion Exchange
Chromatography Columns

Column Stability Oligonucleotides

Figures 10A & 10B demonstrate the stability of the TSKgel Figure 11 shows the analysis of a 16-mer morpholine
SuperQ-5PW columns. Ovalbumin and trypsin inhibitor oligonucleotide on a TSKgel SuperQ-5PW column using
were initially loaded onto a TSKgel SuperQ-5PW, 7.5 mm a NaCl gradient in a 10 mmol/L sodium hydroxide mobile
ID × 7.5 cm column (Figure 10A). The column was then phase.
cleaned in place (CIP) using a solution of 0.5 mol/L NaOH.
This cleaning procedure was repeated once each day for a
total of 15 days. The resolution after this cleaning protocol
was equivalent to the resolution of the initial injection of the
compounds on the column (Figure 10B).

Detector response (AU)

A B
Detector response (AU)

Detector response (AU)

%B

%B
0 10 20 30 40
Retention time (minutes)

Column: TSKgel SuperQ-5PW, 10 µm, 7.5 mm ID × 7.5 cm

Mobile phase: A: 10 mmol/L NaOH
0 30 0 30 B: 10 mmol/L NaOH with 1 mol/L NaCl
Retention time (minutes) Gradient 0 min (0 % B) 40 min (50 % B)
41 min (100 % B) 46 min (100 % B)
Column: TSKgel SuperQ-5PW, 10 µm, 7.5 mm ID × 7.5 cm Flow Rate: 1 mL/min
Mobile phase: A: 50 mmol/L Tris-HCI, pH 8.6 Detection: UV @ 254 nm
B: 0.5 mmol/L sodium cloride in 50 mmol/L Tris-HCI, Sample: 16-mer morpholine oligonucleotide,
pH 8.6 AAG AAG AAG AGG GGA G
Gradient: A-B (60min) Sample load: 0.5 O.D. (optical density)
Flow rate: 1.0 mL/min
Detection: UV @ 280 nm
Temperature: 25 °C Figure 11: Analysis of 16-mer oligonucleotide
Injection vol.: 100 µL
Sample load: each of 1 mg
Samples: 1. ovalbumin
2. trypsin inhibitor
Note: A: before CIP
B: after 15 times (15 days)

Figures 10A & 10B: Stability of TSKgel SuperQ-5PW columns

TSKgel SuperQ-5PW, 7.5mm ID x 7.5cm

Mobile phase: A: 10mmol/L NaOH

B: 10mmol/L NaOH with 1mol/L NaCl
Gradient: 0%B (0min), 50%B (40min), 100%B (41min), 100%B (46min)
Flow Rate: 1mL/min
Detection: UV@254nm
Sample: 16-mer morpholine oligonucleotide,
AAG AAG AAG AGG GGA G
Sample load: 0.5 O.D. (optical density)
TSKgel SuperQ-5PW, 10µm, 7.5mm ID x 7.5cm

Mobile phase: A: 50mmol/L Tris-HCI, pH 8.6

B: 0.5mmol/L sodium cloride in 50mmol/L Tris-HCI, pH 8.6
Gradient: A-B (60min)
Flow rate: 1.0mL/min
Detection: UV@280nm
Temperature: 25°C
Injection vol.: 100µL
Sample load: each of 1mg
Samples: 1) ovalbumin
2) trypsin inhibitor
Note: A: before CIP
B: after 15 times (15 days)

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Monoclonal Antibody Performance Data

The separation of a monoclonal antibody (IgG1) from mouse In Figure 13, a separation performed under high sample
ascites fluid using a TSKgel DEAE-5PW column is shown in load conditions was compared on various anion exchange
Figure 12. IgG1 elutes in about 15 minutes, well separated columns. When a 20 mg protein sample is loaded on a 1
from the impurities that elute before and after IgG1, such mL column volume, only the TSKgel SuperQ-5PW column
as transferrin (about 11 minutes) and albumin (about 22 shows a chromatogram with “normal” looking peaks. Other
minutes). anion exchange columns show multiple artifact peaks from
sample overloading. A 5 mm ID × 5 cm TSKgel
SuperQ-5PW column provides sufficient retention and
resolution. Thus, isolation of proteins at semi-preparative
scale is possible on TSKgel SuperQ-5PW when using an
analytical column.

STI
OVA
Detector response (AU)

300

Detector response (AU)

200

100
A
0
0 10 20 30 40

200

Detector response (AU)

0 15 30 100
Retention time (minutes)

Column: TSKgel DEAE-5PW, 10 µm, 7.5 mm ID × 7.5 cm B

0
Mobile phase: A: 20 mmol/L Tris-HCI, pH 8.5
0 10 20 30 40
B: A + 0.5 mol/L NaCl
A  B linear gradient (60 min)
Detector response (AU)

Flow rate: 1.0 mL/min 200

Detection: UV @ 280 nm
Temperature: 25 ºC
Sample: anti-human albumin (lgG1), 100
diluted solution of mouse ascites (168 µg in 40 µL)
C
0
Figure 12: Separation of monoclonal antibody 0 10 20 30 40
Retention time (minutes)

Columns: A: TSKgel SuperQ-5PW, 10 µm, 5 mm ID × 5 cm

B: Company A, Q type, 5 mm ID × 5 cm
C: Company A, perfusion Q type, 6.4 mm ID × 3 cm
All column volumes were 1.0 mL
Mobile phase: A: 50 mmol/L Tris-HCl buffer, pH 8.3
B: A + 0.5 mol/L NaCl
A  B linear gradient (60 min)
Flow rate: 0.8 mL/min
Detection: UV @ 280 nm
Temperature: 25 °C
Injection vol.: 2 mL
Samples: ovalbumin, 20 mg
trypsin inhibitor, 20 mg
Column: TSKgel DEAE-5PW, 7.5mm ID x 7.5cm
Mobile phase: A: 20mmol/L Tris-HCI (pH 8.5)
Figure 13: Comparison of various anion exchange columns under
B: A + 0.5mol/L NaCl
large sample load
A  B linear gradient (60min)
Flow rate: 1.0mL/min
Detection: UV@280nm
Temperature: 25ºC
Sample: Anti-human albumin (lgG1),
diluted solution of mouse ascites (168µg in 40µL)

90
 TSKgel Ion Exchange
Chromatography Columns

About: TSKgel DEAE-NPR and DNA-NPR DNA Digests

Anion Exchange Columns
Because of their small (2.5 µm) particle size, TSKgel
Methacrylate is the backbone of nonporous resin (NPR) DEAE-NPR nonporous columns excel in rapid separations of
columns such as TSKgel DEAE-NPR and DNA-NPR, which large polynucleotides in DNA digests. A chromatogram of a
are packed with 2.5 µm particles. High column efficiency standard Hae III digest of pBR322 plasmid DNA is shown in
coupled with low sample capacity restricts the application of Figure 14.
these columns to fast analysis and micro-scale preparative
isolation. Due to the absence of pores, protein recovery is

540
generally very high on TSKgel DEAE-NPR and DNA-NPR

504
587
columns.

Detector response (AU)

434

458
TSKgel DNA-NPR columns are packed with hydrophilic

267
polymer beads which are surface modified with a weak

234
213
184

192
anion exchange group. Because TSKgel DNA-NPR

123

12 4
104
columns contain nonporous particles, binding capacity is

89
80
64
low compared to porous columns with the same ligand

57
51
functionality. Column dimensions are optimized for the high
efficiency separation of DNA fragments, PCR products, or 2 5 10 13
Retention time (minutes)
plasmids.
Column: TSKgel DEAE-NPR, 2.5 µm, 4. 6 mm ID × 3.5 cm,
The hydrophilic polymer beads used to pack the TSKgel with guard column, 4.6 mm ID × 0.5 cm
DEAE-NPR columns are also surface modified with a weak Mobile phase: A: 0.02 mol/L Tris-HCI, pH 9.0
anion exchange group. These columns are used for the B: mobile phase A plus 1.0 mol/L NaCl
high speed separation and analysis of proteins and poly- Gradient: 15 min linear gradient from 48 % to 65 % mobile
and oligonucleotides. TSKgel DEAE-NPR columns are phase B
particularly useful for high resolution separation of DNA Flow rate: 1.5 mL/min
digests or fragments. Detection: UV @ 260 nm
Pressure: 14 MPa
Attributes and Applications: Temperature: 40 ºC
Sample: Hae III digest of pBR322 DNA,
(base pair number for each peak is indicated)
Table 6 lists the product attributes of TSKgel DNA-NPR
and DEAE-NPR columns. These columns are stable in a pH
range from 2.0 – 12.0 and are packed with spherical 2.5 µm,
Figure 14: Analysis of DNA digest
nonporous particles.

Table 6: Product attributes

Plasmid
TSKgel column DEAE-NPR DNA-NPR One of the purity checks used for plasmids in gene therapy
assays is the measure of the relative amount of open
Matrix polymethacrylate
circular plasmidColumn:
versus supercoiled
TSKgelplasmid.
DEAE-NPR, Figure 15
4.6mm x 3.5cm,
Particle size (mean) 2.5 µm with guardDNA-NPR
demonstrates the utility of the TSKgel column, 4.6mm x 0.5cm for
column
Mobile phase:
this type of analysis. 15min linear gradient from 48% to 65%
Pore size (mean) nonporous buffer B
Buffer A: 0.02mol/L Tris-HCI, pH 9.0
Functional group CH2CH2N + (C 2H5 ) 3 proprietary
Buffer B: Buffer A plus 1.0mol/L NaCl
Counter ion Cl- ClO4- supercoiled

30
Detector response (mAU)

pH stability 2.0-12.0 Flow Rate: 1.5mL/min

Capacity (mg BSA/mL) 5 20 Pressure: 2000psi
Temperature: 40ºC
Small ion capacity >0.1 eq/L 10 Detection: UV@260nm
open circular

pKa 11.2 0
Sample: Hae III digest of pBR322 DNA, (base pair
0 5 10 number15for each peak
20 is indicated)
Retention time (minutes)

Column: TSKgel DNA-NPR, 2.5 µm, 4. 6 mm ID × 7.5 cm

Mobile phase: A. 20 mmol/L Tris, pH 9.0
B. 20 mmol/L Tris, pH 9.0 with 1 mol/L NaCl
Gradient: linear gradient from 50 % to 65 %B in 10 column
volumes
Flow rate: 1 mL/min
Detection: UV @ 260 nm
Sample: PUC19 plasmid

Figure 15: Plasmid analysis

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Oligonucleotides HIV-1 PCR-amplified 130 bp Target

Figure 16 contains the chromatographic trace of the crude Figure 17 shows the detection of a 130 bp target derived
deprotected 13-mer oligonucleotide using a TSKgel DNA- from HIV using a nonporous TSKgel DEAE-NPR column.
NPR column. The early eluting peaks from 0–5 minute
exhibit a lambda max range of 220–230 nm, indicating the
presence of protecting groups used in the synthesis. The 0.066
130 bp
N-1 peak as confirmed by mass spectrometry elutes just
before the main substance peak. The PS=PO peak elutes 0.064
before N-1. Structurally, the N-1 analog is completely
thioated but is missing one nucleotide. As a result, the 0.062

N-1 compound is more thioated and hydrophobic than

the PS=PO analog. The backside peak is an N+1 impurity 0.060
verified by mass spectrometry. primers
0.058

Detector response (AU)

The method conditions are designed to optimize resolution
0.056
of all impurity peaks and inhibit any aggregation, secondary
structure formation, and PS=PO conversion. Specifically,
0.054
sodium bromide acts as the eluting agent and diethylamine
provides the buffering capacity while contributing mild
0.052
chaotropic effects. The step gradient is designed to remove
all the protecting groups from the column before elution of
0.050
the impurity analogs.
0.048

0.08 0.046
13-mer oligonucleotide 0 5 10 15 20 25
0.07
Retention time (minutes)
Detector response (AU)

0.06
0.05 Column: TSKgel DEAE-NPR, 2.5 µm, 4.6 mm ID × 3.5 cm
PS=PO N-1
Mobile phase: A: 20 mmol/L Tris-HCl with 0.25 mol/L NaCl, pH 7.7
0.04
B: 20 mmol/L Tris-HCl with 1 mol/L NaCl, pH 7.7
0.03
N+1 Flow rate: 1 mL/min
0.02 Detection: UV @ 260 nm
0.01 Temperature: ambient
0.00
Sample: HIV-1 PCR-amplified 130 bp target
Sample load: 20 µL
5 10 15 20 25 30 35 40 45 50
Retention time (minutes)
Figure 17: Detection of HIV-1 PCR-Amplified 130 bp target
Column: TSKgel DNA-NPR, 2.5 µm, 4.6 mm ID × 7.5 cm
Mobile phase: A: 10 mmol/L sodium bromide, 20 mmol/L NaOH,
pH 12, 1 % diethylamine
B: 1 mol/L sodium bromide, 20 mmol/L NaOH,
pH 12, 1 % diethylamine
Gradient 3.5 min (20 %B) 12 min (20 %B) 45 min (55 %B)
Flow rate: 1.0 mL/min
Temperatures: 60 °C (column), 4 °C (sample chamber)
Sample: crude deprotected 13-mer oligonucleotide Column: TSKgel DEAE-NPR, 4.6mm ID x 3.5cm
Buffer A: 20mmol/L Tris-HCl with 0.25mol/L NaCl, pH 7.7
Buffer B: 20mmol/L Tris-HCl with 1mol/L NaCl, pH 7.7
Flow rate: 1mL/min
Figure 16: Oligonucleotide analysis
Detection: UV@260nm
Temperature: ambient
Sample: HIV-1 PCR-amplified 130bp target
Sample load: 20µL
TSKgel DNA-NPR, 2.5um, 4.6mm ID x 7.5cm

Mobile Phase: A: 10mmol/L sodium bromide, 20mmol/L NaOH, pH 12, 1% diethylamine

B: 1mol/L sodium bromide, 20mmol/L NaOH, pH 12, 1% diethylamine
Gradient 3.5min (20%B), 12min (20%B), 45min(55%B)
Flow rate: 1.0mL/min
Temperatures: 60°C (column), 40°C (sample chamber)
Sample: crude deprotected 13-mer oligonucleotide

92
 TSKgel Ion Exchange
Chromatography Columns

About: TSKgel Q-STAT and DNA-STAT Attributes and Applications:

Anion Exchange Columns
Table 7 lists the product attributes of the TSKgel Q-STAT and
TSKgel Q-STAT and DNA-STAT columns are packed with DNA-STAT columns. These columns are an excellent choice
hydrophilic nonporous resin particles of which the surface for high resolution protein and DNA separations. TSKgel
consists of an open access network of multi-layered anion Q-STAT and DNA-STAT nonporous columns are supplied in
exchange groups (see Figure 18). The innovative bonding stainless steel (SS) housing with SS fittings and PEEK frits
chemistry, combined with a relatively large particle size of and are stable in a pH range from 3.0 – 10.0.
these nonporous columns, results in a respectable loading
capacity and a low operating pressure. Table 7: Product attributes
TSKgel column Q-STAT DNA-STAT
TSKgel Q-STAT columns are packed with 7 or 10 µm
nonporous particles. Applications for these columns include Matrix hydrophilic polymer
the separation of proteins, peptides, low molar mass nucleic Particle size (mean) 7 µm and 10 µm 5 µm
acids, aggregates and charge isomers of monoclonal
antibodies, PEGylated proteins, oligo DNA, and siRNA. Pore size (mean) nonporous
Functional group quaternary ammonium
Applications for the 5 µm TSKgel DNA-STAT columns
include the separation of DNA fragments, nucleic acids and Counter ion Cl-
nucleotides.
pH stability 3.0-10.0
Static binding capacity ca. 25 (7 µm)
ca. 35 (5 µm)
Protein (mg BSA/g dry gel) ca. 20 (10 µm)
Small ion capacity 270 µeq/g dry gel
Ionic group
pKa 10.5
Hydrophilic
chain

Binding Capacities
Table 8 illustrates that despite the fact that surface area
decreases with increasing particle size, the larger TSKgel
Q-STAT and TSKgel DNA-STAT particles have higher
binding capacities than the smaller particles used in TSKgel
NPR columns. The novel bonding chemistry used in the
preparation of the TSKgel STAT resin resulted in a dramatic
increase in static binding capacity, more than compensating
for the lower external surface area of the larger particles.

Nonporous material Table 8: Binding capacities of TSKgel STAT anion exchange columns
TSKgel NPR TSKgel TSKgel
Property
Figure 18: Schematic diagram of TSKgel STAT columns column DNA-STAT Q-STAT
Particle size 2.5 µm 5 µm 7 µm 10 µm
Capacity* 9.1 38.6 27.0 20.9
*Static binding capacity in g BSA/g dry gel.

For more info visit: www.separations. asia. tosohbioscience.com 93

DNA Fragments Mouse Ascites Fluid

Mono-, di-, and tri-nucleotides were separated with Figure 20 shows the separation of mouse ascites fluid
excellent peak shape on a TSKgel DNA-STAT column packed containing a monoclonal antibody (top) and a partially
with 5 µm particles. The narrow, symmetrical peaks, as purified monoclonal antibody (bottom) on a TSKgel Q-STAT
shown in Figure 19, demonstrate the absence of micro- column. The top chromatogram clearly shows that the
pores on this new generation of nonporous resin columns. antibody and albumin components are well separated.
TSKgel DNA-STAT columns are, as the name implies, first The bottom chromatogram shows that multiple peaks are
choice for large nucleic acid fragments. present in the partially purified monoclonal sample.

MAb
C, A

95
2d-UDP
2d-ADP

85
2d-CMP

albumin

Detector response (AU)

75 Murine ascites
Detector response (mAU)

2d-CDP

ATP
TDP

2d-CTP

65
2d-AMP

2d-UTP
2d-UMP
TMP

55
2d-GMP

2d-GDP

2d-GTP
TTP

45
Purified MAb
35
U

25
T

0 5
G

15 Retention time (minutes)

5 Column: TSKgel Q-STAT, 7 µm, 4.6 mm ID × 10 cm

0 5 10 15 20 25
Retention time (minutes)
Mobile phase: A:20 mmol/L Tris-HCl buffer, pH 8.5
B:0.5 mol/L NaCl in 20 mmol/L Tris-HCl buffer, pH 8.5
Column: TSKgel DNA-STAT, 5 µm, 4.6 mm ID × 10 cm Gradient: A  B linear gradient (10 min)
Mobile phase: A: 20 mmol/L Tris-HCl, pH 8.5 Flow rate: 1.0 mL/min
B: 0.75 mol/L NaCl in buffer A Detection: UV @ 280 nm
Gradient: 0 min (50 % B) 25 min (75 % B) Temperature: 25 °C
Flow rate: 0.8 mL/min Injection vol.: 10 µL
Detection: UV @ 260 nm Sample: Top: 1/10 dilution of mouse ascites containing mAb
Bottom: purified mouse mAb
Sample diluted 10-fold with eluent A
Figure 19: Separation of large DNA fragments

Figure 20: Separation of mouse ascites fluid containing monoclonal

antibodies and purified monoclonal antibodies

Column: TSKgel Q-STAT, 4.6mm ID x 10cm

Mobile phase: A:20mmol/L Tris-HCl buffer, pH 8.5
B:0.5mol/L NaCl in 20mmol/L Tris-HCl buffer, pH 8.5
Gradient: 10min (100%B), linear
Column: TSKgel DNA-STAT, 5µm, 4.6mm ID x 10cm
Flow rate: 1.0mL/min
Mobile phase: A: 20mmol/L Tris-HCl (pH8.5)
Detection: UV@280nm
B: 0.75mol/L NaCl in buffer A
Temperature: 25°C
Gradient: 50% B (0min), 75% B (25min)
Injection vol.: 10µL
Flowrate: 0.8mL/min
Sample: Top: 1/10 dilution of mouse ascites containing mAb
Detection: UV@260nm
Bottom: purified mouse mAb
Sample diluted 10-fold with eluent A

94
 TSKgel Ion Exchange
Chromatography Columns

Immunoglobulin G (IgG)

Immunoglobulin G (IgG) is a monomeric immunoglobulin,

built of two heavy chains and two light chains. Each IgG
has two antigen binding sites. It is the most abundant
immunoglobulin and is approximately equally distributed
in blood and in tissue liquids, constituting 75 % of serum
immunoglobulins in humans. IgG was digested using
pepsin and separated on a TSKgel Q-STAT column and a
competitive nonporous WAX-10 column. As shown in Figure
21, three peaks were isolated from the TSKgel Q-STAT
column and assigned as F(ab’)2, pFc, and intact IgG by
SDS-PAGE. No correlation could be established between
the peaks obtained on the WAX-10 column and SDS-PAGE
results.
Detector response (mV)

(B)

(A)

0 2 4 6 8 10
Retention time (minutes)

F(ab’)2

pFc Intact IgG

200 kDa

116 kDa

14.4 kDa
6.5 kDa

Non-reduced SDS -PAGE

Columns: A: TSKgel Q-STAT, 7 µm, 4.6 mm ID × 10 cm

B: ProPac ® WAX-10, 10 µm, 4 mm ID × 25 cm
Mobile phase: A: 20 mmol/L Tris-HCl, pH 8.5
B: 0.5 mol/L NaCl in buffer A
Gradient: 0 min (0 % B) 10 min (100 % B)
Flow rate: 1.0 mL/min
Detection: UV @ 280 nm
Sample: pepsin digested mAb

Figure 21: Analysis of lgG

For more info visit: www.separations. asia. tosohbioscience.com 95

About: TSKgel Sugar AXG, Sugar AXI and SAX Saccharide Mixture
Anion Exchange Columns
Saccharides are retained on TSKgel Sugar AX columns
TSKgel Sugar AXG and Sugar AXI columns are specialty following the formation of negatively charged complexes
columns for the analysis of mono- and disaccharides, as with boric acid at alkaline pH. Figure 22 shows the
well as sugar alcohols. Both columns are packed with separation of twelve mono- and disaccharides using a
porous spherical polymer beads which are surface modified TSKgel Sugar AXG column.
with a strong anion exchange group.

The TSKgel Sugar AXG column contains 10 µm particles for 5

the gradient separation and analysis of monosaccharides,
disaccharides, and sugar alcohols, whereas the TSKgel
Sugar AXI column is packed with 8 µm particles for the
isocratic separation of carbohydrates where lower and 4
constant back pressures may be generated.

Detector response (AU)

TSKgel SAX columns are packed with 5 µm porous spherical
polymer beads which are surface modified with a strong 7 11
anion exchange group. They are used for the separation of
isomerized sugars, alcohols, and low molar mass organic
acids. 1
10
3
Attributes and Applications: 2 6
9 12
8

Table 9 lists the product attributes of the TSKgel Sugar AXG, Buffers: B C A
A
Sugar AXI, and SAX columns. These columns are packed 30 60
0
with silica particles and are stable in a pH range from 1.0 – Retention time (minutes)
14.0.
Column: TSKgel Sugar AXG, 10 µm, 4. 6 mm ID × 15 cm
Table 9: Product attributes Mobile phase: step gradient: 6 min buffer A, 0.6 mol/L boric acid,
pH 7.7; then 27 min buffer B, 0.7 mol/L boric acid,
TSKgel pH 7.25; then 30 min buffer C, 0.7 mol/L boric acid, pH 8.7
Sugar AXG Sugar AXI SAX
column Flow rate: 0.4 mL/min (column and post column reagent solution)
Detection: fluorescence; Ex: 331 nm, Em: 383 nm
Matrix PS-DVB polymer Pressure: 16 kg/cm2
Particle Temperature: 70 ºC (column), 100 ºC (post column reactor)
10 µm 8 µm 5 µm PC reagent: 2.5 % 2-cyanoacetamide solution
size (mean)
Samples: disaccharides, 25 nm; monosaccharides, 50 nm:
Pore size 1. cellobiose 2. maltose 3. lactose
6 nm
(mean) 4. rhamnose 5. lyxose 6. ribose
7. mannose 8. fructose 9. arabinose
Functional
trimethylamino 10. galactose 11. xylose 12. glucose
group
Counter ion HBO3- HBO3- Cl-
Figure 22: Separation of saccharide mixture
pH stability 1.0-14.0
Small ion
>1.2 eq/L >1.2 eq/L >1.0 eq/L
capacity
pKa 12.5

Column: TSKgel Sugar AXG, 4.6mm ID x 15cm

Mobile phase: step gradient: 6min buffer A, 0.6mol/L boric

acid, pH 7.7; then 27min buffer B, 0.7mol/L
boric acid, pH 7.25; then 30min buffer C,
0.7mol/L boric acid, pH 8.7
Flow Rate: 0.4mL/min (column and post column reagent solution)
Pressure: 16kg/cm2
Detection: fluorescence excitation @ 331nm,
emission @ 383nm

Temperature: 70ºC (column), 100ºC (post column reactor)

PC reagent: 2.5% 2-cyanoacetamide solution

Sample: disaccharides, 25nM; monosaccharides,

50nM: 1. cellobiose, 2. maltose, 3. lactose,
4. rhamnose, 5. lyxose, 6. ribose,
7. mannose, 8. fructose, 9. arabinose,
96 10. galactose, 11. xylose, 12. glucose
 TSKgel Ion Exchange
Chromatography Columns

Sugar Alcohol Polyphosphates

Palatinit is a sugar alcohol used as a low-calorie and The stability of the TSKgel SAX column allows a wide pH
anti-decay food additive. It can be obtained by reducing range for separations of polyphosphates. Figure 24 shows
palatinose and is composed of two isomers, 6-O-alpha-D- the monitoring of cyclooctaphosphate hydrolysis products
Glucopyranosyl-D-glucitol and 1-O-alpha-D-glucopyranosyl- over the course of 24 hours with a pH 10.2 mobile phase.
D-mannitol. As shown in Figure 23, a TSKgel Sugar AXG
column can separate the isomers.

P8m
1
Detector response (AU)

0 hr

10 20

Detector response (AU)

0 2 4 6 8 10
P1 + P2 + . . . . P8m
Retention time (minutes) 12 hr

Column: TSKgel Sugar AXG, 10 µm, 4.6 mm ID × 15 cm B

Mobile phase: 0.7 mol/L borate buffer, pH 8.6
Flow rate: 0.8 mL/min
Detection: RI
Temperature: 65 ºC 10 20
Injection vol.: 10 µL 24 hr P1 + P2 + . . . .
Samples: 1. alpha-D-glucopyranosyl-1,6-soribitol (GPS)
2. alpha-D-glucopyranosyl-1,6-mannitol (GPM)
P8m
C
Figure 23: Analysis of palatinit

0 10 20
Retention volume (mL)

Column: TSKgel SAX, 5 µm, 4 mm ID × 25 cm

Mobile phase: 0.4 mol/L KCI, 0.1 % EDTA, pH 10.2
Sample: cyclooctaphosphate hydrolysis products
A. 0 hours
B. 12 hours
C. 24 hours

Figure 24: Hydrolysis products of cyclooctaphosphate

Column: TSKgel Sugar AXG, 8640, 4.6mm ID x 15cm

Mobile phase: 0.7mol/L borate buffer (pH 8.6)
Flow rate: 0.8mL/min
Detection: RI Column: TSKgel SAX, 4mm ID x 25cm
Temperature: 65ºC Mobile phase: 0.4mol/L KCI, 0.1% EDTA, pH 10.2
Injection vol.: 10µL Sample: cyclonctaphosphate hydrolysis products
Sample: 1. alpha-D-glucopyranosyl-1,6-soribitol (GPS) 1. 0 hours
2. alpha-D-glucopyranosyl-1,6-mannitol (GPM) 2. 12 hours
3. 24 hours

For more info visit: www.separations. asia. tosohbioscience.com 97

About: TSKgel BioAssist S Cation Exchange Columns Bromelain

Specially designed for the separation of large biomolecules The application in Figure 25 shows the analysis of
such as antibodies, the large pores of the TSKgel BioAssist bromelain, a proteolytic enzyme that is used as a nutritional
S cation exchange column offer superior capacity and supplement. Bromelain is a basic glycoprotein with a molar
resolution at a low column pressure drop. Constructed mass of 33 kDa and pI of 9.55.
via a polymerization technique that allows an equivalent
density of ionic exchange groups to be incorporated into the
particle without reducing pore size, the TSKgel BioAssist
S column is unlike other ion exchange columns that use
500
graft polymerization for polymer chain introduction. The
TSKgel BioAssist S columns’ large pores are very accessible
even for high molar mass proteins. This leads to higher
chromatographic efficiency and binding capacity for 400
B
purification.

Detector response (AU)

TSKgel BioAssist S cation exchange columns are offered in 300
a 4.6 mm ID × 5 cm format and a 10 mm ID × 10 cm semi-
preparative column for scale up. Both columns are made of
PEEK to reduce protein adsorption. 200

Attributes and Applications:

100
Table 10 lists the product attributes of TSKgel BioAssist
S columns. The pore structure and bonding chemistry A
of TSKgel BioAssist S columns provide high capacity for 0
medium to large molar mass proteins. 0 5 10 15 20 25
Retention time (minutes)

Table 10: Product attributes

Columns: A: TSKgel BioAssist S, 7 µm, 4.6 mm ID × 5 cm
Attribute Value B: Competitor S, 5 mm ID × 5 cm
Mobile phase: 20 min (TSKgel) or 30 min (Competitor S)
Matrix polymethacrylate linear gradient of NaCl from 0 to 0.5 mol/L in
Particle size (mean) 7 µm and 13 µm 20 mmol/L sodium phosphate buffer, pH 7.0
Flow Rate: 0.8 mL/min for TSKgel; 1.0 mL/min for Competitor S
Pore size (mean) 130 nm Detection: UV @ 280 nm
Functional group sulfopropyl Temperature: 25 °C
Sample: crude bromelain (C4882, Sigma), 1 mg in 1 mL
Counter ion Na +
pH stability 2.0-12.0 Figure 25: Analysis of bromelain
Capacity (gamma globulin) 70
Small ion capacity 0.1 eq/L
pKa 2.4

Columns: TSKgel BioAssist S, 4.6mmID x 5cm, PEEK

Competitor S 5mmID x 5cm
Mobile phase: 20 min (TSKgel) or 30 min (Competitor S)
linear gradient of NaCl from 0 to 0.5mol/L in
20mmol/L sodium phosphate buffer, pH 7.0
Flow Rate: 0.8mL/min for TSKgel;
1.0mL/min for Competitor S
Detection: UV@280nm
Temperature: 25°C
Sample: crude bromelain (C4882, Sigma), 1mg in 100L

98
 TSKgel Ion Exchange
Chromatography Columns

Immunoglobulin M (IgM) Protein Standards

IgM is known to possess unique and beneficial Figure 27 shows a comparison of a standard protein
characteristics relative to other immunoglobulin classes; it separation on a TSKgel BioAssist S column and
is a large molecule comprised of five IgG subunits, resulting conventional ion exchange columns. It is clear that the
in a relatively unstable and difficult to purify protein. Unlike TSKgel BioAssist S column is more retentive and provides
single chain antibodies, IgM cannot be purified by Protein a higher resolution of the sample proteins compared to the
A (an affinity material commonly used for its high binding conventional products.
capacity and excellent selectivity for antibodies) due to
steric hindrance. Alternative affinity methods have been
developed with thiophillic absorbents but these methods
often result in low binding capacity. 4
2 5
3
An alternative purification method of IgM by ion exchange 1
chromatography using a TSKgel BioAssist S column was

Detector response (AU)

A
developed. As shown in Figure 26, baseline separation of
4 5
IgM from other contaminants is achieved using a 0.3 mol/L 3 2
NaCl step gradient after elution of albumin. 1
B
4 5
2 3
3.0 0.8
1
1 C
0.7
2.5
0 5 10 15 20 25
0.6
Retention time (minutes)
Concentration of NaCl (M)
Detector response (AU)

2.0
0.5
Columns: A: TSKgel BioAssist S, 7 µm, 4.6 mm ID × 5 cm
1.5 0.4
B: Conventional S type product C, 5.0 mm ID × 5 cm
0.3
C: Conventional S type product D, 4.6 mm ID × 5 cm
1.0
2 Mobile phase: A: 20 mmol/L sodium phosphate buffer, pH 6.5
0.2 B: 20 mmol/L sodium phosphate buffer
containing 1.0 mol/L NaCl, pH 6.5
0.5
0.1 Gradient: 32 min (A-B)
Flow rate: 0.8 mL/min
0.0 0 Detection: UV @ 280 nm
0 5 10 15
Retention time (minutes)
Temperature: 10 °C
Injection vol.: 20 µL
Column: TSKgel BioAssist S, 7 µm, 4.6 mm ID × 5 cm Samples: 1. myoglobin, 1 g/L
Mobile phase: 20 mmol/L sodium phosphate buffer, 2. α-chymotrypsinogen A, 2 g/L
pH 6.0 3. ribonuclease A, 4 g/L
Gradient 0 mol/L - 0.3 mol/L NaCl (5 min) 4. cytochrome C, 2 g/L
0.3 mol/L - 0.5 mol/L NaCl (10 min) 5. lysozyme, 2 g/L
Flow Rate: 1 mL/min
Detection: UV @ 280 nm
Sample: 500 µL of 9.5 g/L IgM in mouse ascites fluid; Figure 27: Analysis of protein standards
shaded peaks represent albumin and IgM TSKgel BioAssist S, 4.6mm ID x 5cm
respectively Conventional S type product C, 5.0mm ID x 5cm
Conventional S type product D, 4.6mm ID x 5cm

Mobile phase: A: 20mmol/L sodium phosphate buffer, pH 6.5

Figure 26: Separation of IgM by cation exchange chromatography B: 20mmol/L sodium phosphate buffer
containing 1.0mol/L NaCl, pH 6.5
Gradient: A-B (32min)
Flow rate: 0.8mL/min
Detection: UV@280nm
Temperature: 10°C
Injection vol.: 20µL
Samples: 1) myoglobin, 1g/L
2) α-chymotrypsinogen A, 2g/L
3) ribonuclease A, 4g/L
4) cytochrome C, 2g/L
5) lysozyme, 2g/L

TSKgel BioAssist S, 7µm, 4.6mm ID x 5cm

Mobile phase: 20mmol/L sodium phosphate buffer, pH 6.0

Gradient 0mol/L - 0.3mol/L NaCl (5min)
0.3mol/L - 0.5mol/L NaCl (10min)
Flow Rate: 1mL/min
Detection: UV@280nm
Sample: 500µL of 9.5mg/mL IgM in mouse ascites
fluid; shaded peaks represent albumin
and IgM respectively

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Peptides

Figure 28 shows chromatograms of peptides on a TSKgel

BioAssist S column. It is generally known that an accurate
quantification is difficult to obtain when peptides are
analyzed on a column with a styrene-type base material,
due to secondary interaction with the hydrophobic packing
material. However, a TSKgel BioAssist S column is capable
of analyzing such peptides as angiotensins without the need
to add an organic solvent to the mobile phase since the
acrylate packing material is hydrophilic.

(Val5)-II
(Sar1, IIe6)-II (Asn1, Val5)-II
(Sar1, Thr8)-II Des-Asp1-(IIe8)-II
(Sar1, Val5, Ala8)-II
Detector response (AU)

(Val5)-II

0 0 10 15 20 25
Retention time (minutes)

Column: TSKgel BioAssist S, 7 µm, 4.6 mm ID × 5 cm

Mobile phase: A: 20 mmol/L sodium acetate buffer, pH 5.0
B: 20 mmol/L sodium acetate buffer containing
1.0 mol/L NaCl, pH 5.0
Gradient: A  B linear gradient (20 min)
Detection: UV @ 280 nm
Temperature: 25 °C

Figure 28: Analysis of peptides

Column: TSKgel BioAssist S, 4.6mm ID x 5cm, PEEK

Mobile phase: A: 20mmol/L sodium acetate buffer, pH5.0
B: 20mmol/L sodium acetate buffer containing 1.0mol/L NaCl, pH5.0
Linear gradient from eluent A to B for 20 minutes
Detection: UV@(280nm)
Temperature: 25°C

100
 TSKgel Ion Exchange
Chromatography Columns

About: TSKgel SP-2SW, CM-2SW, and CM-3SW Nucleosides

Cation Exchange Columns
Silica-based cation exchange columns are typically used
The TSKgel SP-2SW, TSKgel CM-2SW, and TSKgel in the separation of low molar mass compounds, such
CM-3SW columns are silica-based columns derivatized as pharmaceuticals, nucleotides, and small peptides. For
with sulfopropyl (SP) and carboxymethyl (CM) ligands to example, Figure 30 shows the separation of nucleosides on
provide a strong cation and weak cation exchange column, the TSKgel SP-2SW column.
respectively. They are used for the separation and analysis
of small proteins, peptides, and other biologically active
molecules. TSKgel CM-2SW has a smaller pore size than 1

TSKgel CM-3SW. 2
A. pH 3.5 B. pH 4.25

Attributes and Applications:

Table 11 shows the product attributes of the TSKgel

Detector response (AU)

SP-2SW, TSKgel CM-2SW, and TSKgel CM-3SW columns.
3
These columns are typically used for analyzing smaller 2

molar mass samples such as nucleotides, drug candidates,

catecholamines, and small peptides or proteins.
1
3
inj. inj.
Table 11: Product attributes
TSKgel column SP-2SW CM-2SW CM-3SW
Matrix silica
0 5 10 0 5 10
Particle size (mean) 5 µm 5 µm 10 µm Retention time (minutes)

Pore size (mean) 12.5 nm 12.5 nm 25 nm Column: TSKgel SP-2SW, 5 µm, 4.6 mm ID × 25 cm
Functional group sulfopropyl -CH2COO -
-CH2COO- Mobile phase: A: 0.1 mol/L sodium citrate - phosphoric
acid buffer, pH 3.5
Counter ion Na + B: 0.1 mol/L sodium citrate - acetic
acid buffer, pH 4.25
pH stability 2.0-7.5
Flow rate: 0.75 mL/min
Capacity (mg Hb/mL) ND 110 ND Detection: UV @ 260 nm
Temperature: 23 °C
Small ion capacity 0.3 eq/L >0.3 eq/L >0.3 eq/L Samples: nucleoside standards:
pKa 2.2 4.2 4.2 1. guanosine 2. cytidine 3. adenosine

Figure 30: Separation of nucleosides

Herbicides
Figure 29 shows the rapid analysis of the herbicides
paraquat and diquat in urine on the TSKgel SP-2SW column.
Detector response (AU)

Column: TSKgel SP-2SW 4.6mm x 25cm

Mobile Phase: A: 0.1 mol/L sodium citrate - phosphoric acid buffer, pH 3.5
B: 0.1 mol/L sodium citrate - acetic acid buffer, pH 4.25

Flow Rate: 0.75 mL/min

Detection: UV@260nm
0 20 40
Retention time (minutes) Temperature: 23°C

Column: TSKgel SP-2SW, 5 µm, 4.6 mm ID × 25 cm Sample: Nucleoside Standards

Mobile phase: 20 %CH 3CN in 0.2 mol/L phosphate, pH 3.0 1. Guanosine
Flow rate: 1.0 mL/min 2. Cytidine
Detection: UV @ 290 nm 3. Adenosine
Samples: 1. paraquat
2. diquat

Figure 29: Rapid analysis for the herbicides paraquat and diquat

For more info visit: www.separations. asia. tosohbioscience.com 101

Column: TSKgel SP-2SW, 4.6mm x 25cm

Elution: 20%CH CN in 0.2mol/L phosphate, pH 3.0
About: TSKgel SP-5PW and CM-5PW Differences in Selectivity
Cation Exchange Columns
Differences in selectivity between strong (TSKgel
The polymethacrylate-based resin, TSKgel 5PW, is a SP-5PW) and weak (TSKgel CM-5PW) cation exchange
spherical 10 µm particle with approximately 100 nm pores. columns are demonstrated in Figure 31, which is a
It is derivatized with sulfopropyl (SP) ligands to provide the separation of globular proteins.
strong cation exchange column, TSKgel SP-5PW, and with
carboxymethyl (CM) ligands to provide the weak cation
exchange column, TSKgel CM-5PW. 3
SP-5PW
TSKgel CM-5PW columns are used for the separation and 5
analysis of proteins, peptides, and other biologically active 1 2 4 4
molecules. These columns are offered in dimensions of 7.5

Detector response (AU)

mm ID × 7.5 cm in stainless steel housing.

TSKgel SP-5PW columns are also used for the separation

and analysis of proteins, peptides, and other biologically 3 4

active molecules. These columns are available in internal CM-5PW 5

1
diameters varying from 2 mm to 21.5 mm and in column 2

housings of either glass or stainless steel. 4

Attributes and Applications:

0 20 40
Table 12 lists the product attributes of TSKgel SP-5PW and Retention time (minutes)
CM-5PW columns. These columns are an excellent choice
for analyzing biologically active molecules. TSKgel SP-5PW Columns: TSKgel SP-5PW and TSKgel CM-5PW, 10 µm,
and CM-5PW columns are stable over the pH range of 7.5 mm ID × 7.5 cm
2.0 – 12.0 and the porous particles have a mean pore size Mobile phase: 60 min linear gradient from 0 mol/L to 0.5 mol/L NaCl
of 100 nm. in 0.02 mol/L phosphate, pH 7.0
Flow rate: 1.0 mL/min
Table 12: Product attributes Detection: UV @ 280 nm
Samples: 1. trypsinogen
TSKgel column SP-5PW CM-5PW 2. ribonuclease A
3. α-chymotrypsinogen
Matrix polymethacrylate
4. cytochrome C
Particle size (mean) 10 µm, 13 µm, and 20 µm 10 µm and 13 µm 5. lysozyme
Pore size (mean) 100 nm
Functional group -(CH2) 3SO3- -CH2COO- Figure 31: Selectivity of strong and weak TSKgel cation
exchange columns
Counter ion Na +

pH stability 2.0-12.0
Capacity (mg Hb/mL): 40 45
Small ion capacity >0.1 eq/L
pKa 2.3 4.2

Columns: TSKgel SP-5PW and TSKgel CM- 5PW,

7.5mm x 7.5cm
Elution: 60min linear gradient from 0mol/L to 0.5mol/L NaCl
in 0.02mol/L phosphate, pH7
Flow Rate: 1.0mL/min
Detection: UV@280nm

Sample: 1. trypsinogen
2. ribonuclease A
3. α-chymotrypsinogen
4. cytochrome C
5. lysozyme

102
 TSKgel Ion Exchange
Chromatography Columns

Crude Lipoxidase Peptides

The purification of 200 mg of crude lipoxidase on a 21.5 mm One of the common HPLC modes for analysis and separation
ID TSKgel SP-5PW column is illustrated in Figure 32. Scale of peptides is cation exchange. Figure 33 shows that
up is simplified as only the particle size changes from 10 separations of peptides can be efficiently separated on the
µm (7.5 mm ID) to 13 µm (21.5 mm ID) or 20 µm (55 mm ID) strong cation exchange column TSKgel SP-5PW.
columns.

3
7

Detector response (AU)

5
2 8
4
1 6
Detector response (AU)

9 10

0 10 20 30
Retention time (minutes)

Column: TSKgel SP-5PW, 10 µm, 7.5 mm ID × 7.5 cm

Mobile phase: 30 min linear gradient from 0.02 mol/L to 0.5 mol/L
0 30 60
phosphate, pH 3.0, in 30 % acetonitrile
Retention time (minutes) Flow rate: 1.0 mL/min
Detection: UV @ 220 nm
Injection vol.: 50 µL
Column: TSKgel SP-5PW, 13 µm, 21.5 mm ID × 15 cm
Samples: 2 µg each of:
Mobile phase: 120 min linear gradient from 0 mol/L to
1. oxytocin
0.5 mol/L Na2SO 4 in 0.02 mol/L acetate, pH 4.5
2. met-enkephalin
Flow rate: 4.0 mL/min
3. TRH
Detection: UV @ 280 nm
4. α-MSH
Recovery: lipoxidase activity collected between the
5. LH-RH (1 µg)
two vertical lines was 84 %
6. neurotensin
Sample: crude lipoxidase, 200 mg
7. α-MSH
8. angiotensin II
9. substance P
Figure 32: Semi-preparative purification of lipoxidase 10. β-endorphin

Figure 33: Separation of peptide mixture

Column: TSKgel SP-5PW, 21.5mm ID x 15cm

Elution: 120min linear gradient from 0mol/L to 0.5mol/L Na2SO4 in
0.02mol/L acetate, pH 4.5
Flow Rate: 4.0mL/min
Detection: UV@280nm
Recovery: Lipoxidase activity collected between the two
vertical lines was 84%
Sample: crude lipoxidase, 200mg

Column: TSKgel SP-5PW, 7.5mm ID x 7.5cm

Mobile phase: 30min linear gradient from 0.02mol/L to 0.5mol/L
phosphate, pH 3, in 30% acetonitrile
Flow rate: 1.0mL/min
Detection: UV@220nm
Injection vol.: 50µL
Samples: 2µg each of:
1. oxytocin
2. met-enkephalin
3. TRH
4. α-MSH
5. LH-RH (1µg)
6. neurotensin
7. α-MSH
8. angiotensin II
9. substance P
10. β-endorphin
For more info visit: www.separations. asia. tosohbioscience.com 103
About: TSKgel SP-STAT and CM-STAT Attributes and Applications:
Cation Exchange Columns
Table 13 lists the product attributes of TSKgel CM-STAT and
TSKgel CM-STAT and SP-STAT columns are packed with SP-STAT columns. These columns are an excellent choice
7 or 10 µm hydrophilic nonporous resin particles of which for high throughput protein separations. Nonporous TSKgel
the surface consists of an open access network of multi- CM-STAT and SP-STAT columns are supplied in stainless
layered weak cation exchange groups (see Figure 34). The steel (SS) housing with SS fittings and PEEK frits and are
innovative bonding chemistry, combined with a relatively stable in a pH range from 3.0 – 10.0.
large particle size, results in a respectable loading capacity,
low operating pressure, and rapid analysis. Table 13: Product attributes
TSKgel TSKgel
Applications for the TSKgel CM-STAT and SP-STAT columns TSKgel column
CM-STAT SP-STAT
include the separation of proteins, protein aggregates,
charge variants of monoclonal antibodies, PEGylated Particle size (mean) 7 µm and 10 µm
proteins, and peptide digests. Pore size (mean) nonporous
Functional group carboxymethyl sulfopropyl
Counter ion Na+
Protein
pH stability 3.0-10.0
Ionic group
Static binding capacity ca. 20 (7 µm) ca. 15 (7 µm)
Hydrophilic (mg lysozyme/g dry gel) ca. 15 (10 µm) ca. 10 (10 µm)
chain
Small ion capacity 100 µeq/g dry gel 23 µeq/g dry gel
pKa 4.9 2.6

Nonporous material

Figure 34: Schematic diagram of TSKgel STAT columns

104
 TSKgel Ion Exchange
Chromatography Columns

Antibody Analysis Protein Standards

The analysis profiles for five antibodies separated on a The fast separation of protein standards was investigated
TSKgel CM-STAT column (Figure 35A) were compared with using short cation exchange columns (see Figure 36). A
the profiles obtained on a competitive WCX-10 column TSKgel SP-STAT column shows superior resolution, better
(Figure 35B). Similar or higher resolution profiles were peak shape, and a shorter analysis time (<60 seconds)
obtained on the TSKgel CM-STAT column in approximately compared to a competitive monolithic SP-type column.
half the time.

40
A 1
Detector response (mV)

2
30

E
3
20

Detector response (mV)

D
C B
10
B
A
0
0 10 20 30 40
Retention time (minutes)

B A
15
Detector response (mV)

0 0.2 0.4 0.6 0.8 1 1.2

Retention time (minutes)
10
E
Columns: A: TSKgel SP-STAT, 10 µm, 3.0 mm ID × 3.5 cm
D
B: ProSwift ® SCX-1S Monolith, 4.6 mm ID × 5 cm
5 C
Mobile phase: A: 20 mmol/L sodium acetate, pH 5.0
B
B: 1.0 mol/L NaCl in mobile phase A, pH 5.0 for
0 A column A
0 20 40 60 80
1.5 mol/L NaCl in mobile phase A, pH 5.0 for
Retention time (minutes) column B
Gradient: 0 min (0 % B) 1 min (100 % B)
Columns: A: TSKgel CM-STAT, 7 µm, 4.6 mm ID × 10 cm Flow rate: A: 2.0 mL/min
B: ProPac WCX-10, 10 µm, 4 mm ID × 25 cm B: 4.73 mL/min
Mobile phase: A: 20 mmol/L MES, pH 6.0 Detection: UV @ 280 nm
B: 20 mmol/L MES + 0.5 mol/L NaCl, pH 6.0 Samples: 1. α-chymotrypsinogen A
Gradient: A: 0 min (10 % B) 15 min (30 % B) 15 min (100 % B) 2. cytochrome C
17 min (0 % B) 17 min (10 % B) 21 min (10 % B) 3. lysozyme
B: 0 min (10 % B) 30 min (30 % B) 30 min (100 % B)
32 min (100 % B) 32 min (10 % B) 36 min (10 % B)
Flow rate: A: 1.0 mL/min B: 2.0 mL/min Figure 36: Fast separation of protein standards
Detection: UV @ 280 nm
Temperature: 25 °C
Injection vol.: 20 µL
Samples: monoclonal antibodies (mAb A through E)

Figures 35A & 35B: Antibody analysis

A: TSKgel CM-STAT, 7µm, 4.6mm ID x 10cm
B: ProPac WCX-10, 10µm, 4mm ID x 25cm A: TSKgel Q-STAT, 10µm, 3.0mm ID x 3.5cm
B: ProSwift SCX-1S Monolith, 4.6mm ID x 5cm
Mobile phase: A: 20mmol/L MES, pH6.0 B: 20mmol/L MES + 0.5mol/L NaCl, pH 6.0
Gradient: Mobile phase:ࠉ
A: 10%B (0min), 30%B (15min), 100%B (15min), 0%B (17min), 10%B (17min), 10%B (21min) A: 20mmol/L sodium acetate, pH 5.0
B: 10%B (0min), 30%B (30min), 100%B (30min), 100%B (32min), 10%B (32min), 10%B (36min) B: 1.0mol/L NaCl in buffer A, pH 5.0 for column A
Flow rate: A: 1.0mL/min B: 2.0mL/min 1.5mol/L NaCl in buffer A, pH 5.0 for column B
Detection: UV@280nm Gradient: 0%B (0min), 100%B (1min)
Temperature: ambient Flow rate: A: 2.0mL/min
Injection vol.: 20µL B: 4.73mL/min
Sample: monoclonal antibodies (mAb A through E) Detection: UV@280nm
Samples: 1. α-chymotrypsinogen A
2. cytochrome C
3. lysozyme

For more info visit: www.separations. asia. tosohbioscience.com 105

Reaction Monitoring Charge Isomers

A sample of β-lactoglobulin (5 mg/mL) was reacted with As shown in Figure 38, the TSKgel CM-STAT column can
polyethylene glycol (5 kDa) in a pH 6.5 phosphate buffer. also be used to separate charge isomers of a purified
The formation of pegylated protein reaction products was monoclonal antibody by pH gradient.
monitored in 5 minute intervals on a 3.5 cm TSKgel
SP-STAT column. As demonstrated in Figure 37, peak
areas of mono-, di-, and tri-pegylated β-lactoglobulin 7
increased with reaction time, while the area of unreacted
β-lactoglobulin declined.

Detector response (AU)

6

50

pH
45
5
40 Native
Detector response (mAU)

35

30 4
0 10 20 30 40
25 Mono- PEG
Retention time (minutes)

20 Di- PEG

Tri -PEG
Column: TSKgel CM-STAT, 7 µm, 4.6 mm ID × 10 cm
15 Mobile phase: A: 50 mmol/L sodium acetate buffer, pH 5.0
10 B: 30 mmol/L sodium acetate buffer
(pH not adjusted)
5 Column equilibrated with mobile phase A, the
0 0.5 1.0 1.5 2.0
Retention time (minutes) sample is injected, then eluted stepwise to 100 %
mobile phase B
Flow rate: 1.0 mL/min
100
Detection: UV @ 280 nm
90
Native
Temperature: 25 °C
80 Injection vol.: 10 µL
Sample: purified mAb
70
Sample concentration: 1 g/L
60
Peak %

50
Figure 38: Separation of charge isomers
40
Mono-PEG
30

20
Di -PEG
10 Column: TSKgel CM-STAT, 4.6mm ID x 10cm
0
Tri- PEG Mobile phase: A: 50mmol/L sodium acetate buffer, pH 5.0
0 10 20 30 40 50 60 70 B: 30mmol/L sodium acetate buffer
Reaction time (minutes) (pH not adjusted)
Column equilibrated with eluent A, the sample is injected, then eluted stepwise to 100% elue
Column: TSKgel SP-STAT, 10 µm, 3 mm ID × 3.5 cm Flow rate: 1.0mL/min
Mobile phase: A: 20 mmol/L sodium acetate buffer, pH 5.0 Detection: UV@280 nm
B: 1.0 mol/L NaCl in mobile phase A, pH 5.0 Temperature: 25°C
Gradient: 0 min (0 %B) 2 min (100 %B) Injection vol.: 10µL
Flow rate: 2.0 mL/min Sample: purified mAb
Detection: UV @ 280 nm Sample concentration: 1g/L
Sample: pegylated β-lactoglobulin

Figure 37: Monitoring of reaction products

TSKgel SP-STAT, 10µm, 3mm ID x 3.5cm

Mobile phase: A: 20mmol/L sodium acetate buffer, pH 5.0 Flow rate: 2.0mL/min
B: 1.0mol/L NaCl in buffer A, pH 5.0 Detection: UV@280nm
Gradient: 0%B (0min), 100%B (2min) Sample: pegylated β-lactoglobulin

106
 TSKgel Ion Exchange
Chromatography Columns

About: TSKgel SCX and OApak-A Saccharide, Organic Acid, and Alcohol Mixture
Cation Exchange Columns
Ion exclusion chromatography can be used as an effective
The TSKgel SCX column is packed with porous polystyrene method for separating alcohols. An example of saccharide,
divinylbenzene polymer beads of which the surface has organic acid, and alcohol separation is shown in Figure 39
been modified with strong cation exchange groups that are on two TSKgel SCX (H +) columns in series.
surrounded by Na + counterions. This column is optimized
for the separation and analysis of organic acids, saccharides,
and alcohols. The TSKgel SCX column is also available in the 4

H + form for the separation of isomerized sugars, alcohols, UV 5

and lower organic acids.
8
3

Detector response (mV)

A TSKgel OApak-A column is packed with porous hydrophilic
polymer beads which have been chemically modified with
a weak cation exchange group. This column is optimized 1 23
7
for the separation and analysis of organic acids by an ion
exclusion mechanism. Applications include: organic acids in
fruit juices, wine, beer, coffee, and salt solutions. 6

RI 4 5 8
The TSKgel OApak-A column is to be used in conjunction
with the TSKgel OApak-P guard column which has a strong
0 15 30 45
cation exchange group for the removal of dissociated strong
acids under the isocratic mobile phase conditions of 0.75 Retention time (minutes)

mmol/L H2SO4. Column: TSKgel SCX (H+), 5 µm, 7.8 mm ID × 30 cm × 2

Mobile phase: 0.05 mol/L HClO 4
Attributes and Applications: Flow rate: 0.8 mL/min
Detection: UV @ 210 nm, RI
Samples: 1. maltose
Table 14 shows the product attributes of the TSKgel SCX
2. glucose
column and the TSKgel OApak-A column. Both of these
3. fructose
columns are composed of 5 µm particles and are stable in 4. lactic acid
the pH range of 2.0 – 12.0. 5. acetic acid
6. methanol
Table 14: Product attributes 7. ethanol
TSKgel column SCX OApak-A 8. butyric acid

Matrix PS-DVB hydrophilic polymer

Figure 39: Separation of saccharide, organic acid, and
Particle size (mean) 5 µm alcohol mixture
Pore size (mean) 6 nm ND
Functional group sulfonic acid proprietary
Counter ion H and Na
+ +
H+
pH stability 2.0-12.0
Small ion capacity >1.5 eq/L

Column: TSKgel SCX(H+), two 7.8mm ID x 30cm (in series)

Elution: 0.05mol/L HClO4
Flow Rate: 0.8mL/min
Detection: UV@210nm, Refractive Index

Sample: 1. maltose
2. glucose
3. fructose
4. lactic acid
5. acetic acid
6. methanol
7. ethanol
8. butyric acid

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Organic Acids in Wine and Beer Column Stability

Figure 40 demonstrates the separation of organic acids An example of the stability of the TSKgel SCX column is
commonly found in wines and beers on the TSKgel OApak-A demonstrated in Figure 41 where 1 mol/L NaOH is used as
column. the mobile phase for the separation of organic acids.

2
Detector response (mS/cm)

0.016 O.D.

3 6
4

Detector response (AU)

3
1 1
7
8

10 20 30
Retention time (minutes)

Column: TSKgel OApak-A, 5 µm, 7.8 mm ID × 30 cm

TSKgel OApak-P guard, 6 mm ID × 4 cm
Mobile phase: 0.75 mmol/L sulfuric acid
Flow rate: 0.8 mL/min 0 2 20
Detection: conductivity Retention time (minutes)
Temperature: 60 °C
Injection vol.: 20 µL Column: TSKgel SCX (Na+), 5 µm, 8 mm ID × 10 cm
Samples: 1. pyruvic acid (50 mg/L) Mobile phase: 1 mol/L NaOH
2. tartaric acid (500 mg/L) Flow rate: 0.8 mL/min
3. citric acid (500 mg/L) Detection: UV @ 210 nm
4. malic acid (500 mg/L) Samples: 1. formic acid (50 mg/L)
5. pyroglutamic acid (500 mg/L) 2. acetic acid (50 mg/L)
6. lactic acid (1,000 mg/L) 3. propionic acid (100 mg/L)
7. acetic acid (2,000 mg/L)
8. succinic acid (1,000 mg/L)
Figure 41: Separation of acids

Figure 40: Separation of organic acids commonly found in beer

and wine

Column: TSKgel OApak-P, 6mm x 4cm,

TSKgel OApak-A, 7.8mm x 30cm
Mobile phase: 0.75mmol/L Sulfuric acid
Flow Rate: 0.8mL/min Column: TSKgel SCX, 8mm ID x 10cm
Detection: Conductivity Mobile phase: 1mol/L NaOH
Temperature: 60°C Flow Rate: 0.8mL/min
Injection vol.: 20L Detection: UV@210nm
Sample: 1. pyruvic acid (50ppm); 2. tartaric acid (500ppm); Sample: 1. formic acid (50ppm)
3. citric acid (500ppm); 4. malic acid (500ppm); 2. acetic acid (50ppm)
5. pyroglutamic acid (500ppm); 3. proionic acid (100ppm)
6. lactic acid (1000ppm); 7. acetic acid (2000ppm);
8. succinic acid (1000ppm)

108
 TSKgel Ion Exchange
Chromatography Columns

About: TSKgel SP-NPR Cation Exchange Columns Hemoglobin A1c

The TSKgel SP-NPR column is packed with spherical, The analysis of hemoglobin A1c levels in blood is used to
nonporous (NPR) hydrophilic polymer beads of which the monitor glucose levels in diabetic patients. Figure 43 shows
surface has been modified with a strong cation exchange that the HbA1c fraction can be separated from other human
group. Nonporous resin columns provide fast separations Hb variants on a TSKgel SP-NPR column by running a linear
due to their small (2.5 µm) particle size. High column pH gradient in 10 minutes.
efficiency coupled with low sample capacity restricts the
application of these columns to fast analysis and micro-scale
preparative isolation.

The TSKgel SP-NPR column is used for the separation and

analysis of proteins and peptides. This column is particularly
useful for adeno-associated viruses and other large
biopolymers. Hemoglobin
A1c

Detector response (AU)

Attributes and Applications
Table 15 shows the product attributes of the TSKgel SP-
NPR column. Due to the absence of all but very small pores,
protein recovery is generally high on TSKgel NPR columns.

Table 15: Product attributes

TSKgel column SP-NPR
Matrix hydroxylated methacrylic polymer
Particle size (mean) 2.5 µm
0 2 4 6 8 10
Pore size (mean) nonporous Retention time (minutes)

Functional group sulfopropyl Column: TSKgel SP-NPR, 2.5 µm, 4. 6 mm ID × 3.5 cm

Mobile phase: A: 0.02 mol/L MES, and 0.02 mol/L HEPES-NaOH,
Counter ion Na +
pH 6.0
pH stability 2.0-12.0 B: 0.02 mol/L MES, and 0.02 mol/L HEPES- NaOH,
pH 8.0
Capacity (mg Hb/mL): 5 Gradient: 10 min linear gradient from 32 % to 75 % buffer B
Small ion capacity >0.1 eq/L (pH 6.66 to pH 7.43)
Flow rate: 1.5 mL/min
pKa 2.3 Detection: VIS @ 415 nm
Sample: hemoglobin standard
Purified Adeno-Associated Virus

A purity check of adeno-associated virus (AAV), commonly Figure 43: pH gradient analysis of hemoglobin A1c
used in gene therapy research, on a TSKgel SP-NPR column
is shown in Figure 42. This 10 minute HPLC method replaces
an existing assay that took two days to perform.
Detector response (mAU)

20
15 Column: TSKgel SP-NPR, 4.6mm ID x 3.5cm
10 Elution: 10min linear gradient from 32% to 75% buffer B
(pH 6.66 to pH 7.43)
5
Buffer A: 0.02mol/L MES, and 0.02mol/L HEPES-NaOH, pH 6.0
0 Buffer B: 0.02mol/L MES, and 0.02mol/L HEPES- NaOH, pH 8.0
-5 Flow Rate: 1.5mL/min
0 2.5 5 7.5 10 Detection: VIS@415nm
Retention time (minutes) Sample: hemoglobin standard

Column: TSKgel SP-NPR, 2.5 µm, 4.6 mm ID × 3.5 cm

Mobile phase: A. 50 mmol/L HEPES, 1 mmol/L EDTA, 5 mmol/L MgCl, pH 7.5
B. 50 mmol/L HEPES, 1 mmol/L EDTA, 5 mmol/L MgCl,
pH 7.5 with 0.5 mol/L NaCl; linear gradient from 20 % to
100 % B in 10 column volumes
Gradient: 0 min (0 % B) 2 min (100 % B)
Flow rate: 1 mL/min
Detection: UV @ 280 nm
Sample: purified adeno-associated virus

Figure 42: Analysis of purified AAV

For more info visit: www.separations. asia. tosohbioscience.com 109

Ordering Information - TSKgel Anion Exchange columns

Part # Description Matrix Housing ID (mm) Length (cm)

0019685 TSKgel BioAssist Q, 10 µm, 400 nm polymer PEEK 4.6 5
0021410 TSKgel BioAssist Q, 13 µm, 400 nm polymer PEEK 10 10

0018761 TSKgel DEAE-2SW, 5 µm 12.5 nm silica Stainless Steel 2 25

0007168 TSKgel DEAE-2SW, 5 µm, 12.5 nm silica Stainless Steel 4.6 25

TSKgel Guard Cartridge for 2 mm ID TSKgel

0042154 silica Stainless Steel 2 1
DEAE-2SW column, 3 pk, 5 µm
TSKgel Guard Cartridge Holder for 2 mm ID
0019308 Stainless Steel 2 1
cartridges
TSKgel Guardgel Kit for 4.6 mm ID TSKgel
0007648 silica Stainless Steel
DEAE-2SW column, 10 µm

0007163 TSKgel DEAE-3SW, 10 µm, 25 nm silica Stainless Steel 7.5 7.5

TSKgel Guardgel Kit for 7.5 mm ID TSKgel

0007648 silica Stainless Steel
DEAE-3SW column, 10 µm

0013061 TSKgel DEAE-5PW Glass, 10 µm, 100 nm polymer Glass 5 5

0008802 TSKgel DEAE-5PW Glass, 10 µm, 100 nm polymer Glass 8 7.5
0014016 TSKgel DEAE-5PW Glass, 13 µm, 100 nm polymer Glass 20 15
0018757 TSKgel DEAE-5PW, 10 µm, 100 nm polymer Stainless Steel 2 7.5
0007164 TSKgel DEAE-5PW, 10 µm, 100 nm polymer Stainless Steel 7.5 7.5
0007574 TSKgel DEAE-5PW, 13 µm, 100 nm polymer Stainless Steel 21.5 15

TSKgel Glass Guardgel Kit for 5 mm ID and 8

0008806 polymer Glass
mm ID TSKgel DEAE-5PW columns, 20 µm
TSKgel Glass Guard Column for 20 mm ID
0014466 polymer Glass 20 2
TSKgel DEAE-5PW Glass column, 13 µm
TSKgel Guard Cartridge for 2 mm ID TSKgel
0042152 polymer Stainless Steel 2 1
DEAE-5PW column, 3 pk, 10 µm
TSKgel Guard Cartridge Holder for 2 mm ID
0019308 Stainless Steel 2 1
cartridges
TSKgel Guardgel Kit for 7.5 mm ID TSKgel
0007210 polymer Stainless Steel
DEAE-5PW column, 20 µm
TSKgel Guardgel Kit for 21.5 mm ID TSKgel
0016092 polymer Stainless Steel
DEAE-5PW column, 20 µm

0013075 TSKgel DEAE-NPR, 2.5 µm, nonporous polymer Stainless Steel 4.6 3.5

TSKgel Guard Column for 4.6 mm ID TSKgel

0017088 polymer Stainless Steel 4.6 0.5
DEAE-NPR column, 5 µm

0018249 TSKgel DNA-NPR, 2.5 µm, nonporous polymer Stainless Steel 4.6 7.5

TSKgel Guard Column for 4.6 mm ID TSKgel

0018253 polymer Stainless Steel 4.6 0.5
DNA-NPR column, 5 µm

0021962 TSKgel DNA-STAT, 5 µm, nonporous polymer Stainless Steel 4.6 10

0007166 TSKgel QAE-2SW, 5 µm, 12.5 nm silica Stainless Steel 4.6 25

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 TSKgel Ion Exchange
Chromatography Columns

Part # Description Matrix Housing ID (mm) Length (cm)

TSKgel Guardgel Kit for 4.6 mm ID TSKgel
0007646 silica Stainless Steel
QAE-2SW column, 5 µm

0021960 TSKgel Q-STAT, 10 µm, nonporous polymer Stainless Steel 3 3.5

0021961 TSKgel Q-STAT, 7 µm, nonporous polymer Stainless Steel 4.6 10

0007157 TSKgel SAX, 5 µm, 6 nm polymer Stainless Steel 6 15

0008640 TSKgel Sugar AXG, 10 µm, 6 nm polymer Stainless Steel 4.6 15

0008639 TSKgel Sugar AXI, 8 µm, 6 nm polymer Stainless Steel 4.6 15

0018386 TSKgel SuperQ-5PW Glass, 10 µm, 100 nm polymer Glass 8 7.5

0018257 TSKgel SuperQ-5PW, 10 µm, 100 nm polymer Stainless Steel 7.5 7.5
0018387 TSKgel SuperQ-5PW, 13 µm, 100 nm polymer Stainless Steel 21.5 15

TSKgel Guardgel Kit for 7.5 mm ID TSKgel

0018388 polymer Stainless Steel
SuperQ-5PW column, 20 µm

Ordering Information - TSKgel Cation Exchange columns

Part # Description Matrix Housing ID (mm) Length (cm)

0019686 TSKgel BioAssist S, 7 µm, 130 nm polymer PEEK 4.6 5
0021411 TSKgel BioAssist S, 13 µm, 130 nm polymer PEEK 10 10

0007167 TSKgel CM-2SW, 5 µm, 12.5 nm silica Stainless Steel 4.6 25

TSKgel Guardgel Kit for TSKgel CM-2SW

0007650 silica Stainless Steel
column, 10 µm

0007162 TSKgel CM-3SW, 10 µm, 25 nm silica Stainless Steel 7.5 7.5

TSKgel Guardgel Kit for TSKgel CM-2SW &

0007650 silica Stainless Steel
CM-3SW columns, 10 µm

0013068 TSKgel CM-5PW, 10 µm, 100 nm polymer Stainless Steel 7.5 7.5

TSKgel Guardgel Kit for 7.5 mm ID TSKgel

0013069 polymer Stainless Steel
CM-5PW column, 20 µm

0021965 TSKgel CM-STAT, 10 µm, nonporous polymer Stainless Steel 3 3.5

0021966 TSKgel CM-STAT, 7 µm, nonporous polymer Stainless Steel 4.6 10

0016653 TSKgel OApak-A, 5 µm, 100 nm polymer Stainless Steel 7.8 30

TSKgel Guard Column for TSKgel OApak-A,

0016654 polymer Stainless Steel 6 4
10 µm

TSKgel SCX, Strong Cation Exchange, 5 µm

0007156 polymer Stainless Steel 6 15
(Na +), 6 nm
TSKgel SCX, Strong Cation Exchange, 5 µm
0007158 polymer Stainless Steel 7.8 30
(H +), 6 nm

For more info visit: www.separations. asia. tosohbioscience.com 111

 Part # Description Matrix Housing ID (mm) Length (cm)
0007165 TSKgel SP-2SW, 5 µm, 12.5 nm silica Stainless Steel 4.6 25

TSKgel Guardgel Kit for 4.6 mm ID TSKgel

0007644 silica Stainless Steel
SP-2SW column, 5 µm

0013062 TSKgel SP-5PW Glass, 10 µm, 100 nm polymer Glass 5 5

0008803 TSKgel SP-5PW Glass, 10 µm, 100 nm polymer Glass 8 7.5
0014017 TSKgel SP-5PW Glass, 13 µm, 100 nm polymer Glass 20 15
0018758 TSKgel SP-5PW, 10 µm, 100 nm polymer Stainless Steel 2 7.5
0007161 TSKgel SP-5PW, 10 µm, 100 nm polymer Stainless Steel 7.5 7.5
0007575 TSKgel SP-5PW, 13 µm, 100 nm polymer Stainless Steel 21.5 15

TSKgel Glass Guardgel Kit for 5 mm ID &

0008807 polymer Glass
8 mm ID TSKgel SP-5PW columns, 20 µm
TSKgel Guard Cartridge for 2 mm ID TSKgel
0042153 polymer Stainless Steel 2 1
SP-5PW column, 3 pk, 10 µm
TSKgel Guard Cartridge Holder for 2 mm ID
0019308 Stainless Steel 2 1
cartridges
TSKgel Guardgel Kit for 7.5 mm ID TSKgel
0007211 polymer Stainless Steel
SP-5PW column, 20 µm
TSKgel Guardgel Kit for 21.5 mm ID TSKgel
0016093 polymer Stainless Steel
SP-5PW column, 20 µm

0013076 TSKgel SP-NPR, 2.5 µm, nonporous polymer Stainless Steel 4.6 3.5

0021963 TSKgel SP-STAT, 10 µm, nonporous polymer Stainless Steel 3 3.5

0021964 TSKgel SP-STAT, 7 µm, nonporous polymer Stainless Steel 4.6 10

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