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SEC COLUMNS

TOSOH BIOSCIENCE

Zettachring 6, 70567 Stuttgart, Germany


Tel: +49 (0)711 13257 0 Fax: +49 (0)711 13257 89
info.tbg@tosoh.com www.tosohbioscience.com
B11L02A
SEC COLUMNS

TOSOH BIOSCIENCE

Zettachring 6, 70567 Stuttgart, Germany


Tel: +49 (0)711 13257 0 Fax: +49 (0)711 13257 89
info.tbg@tosoh.com www.tosohbioscience.com
B11L02A
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 are
volume. It is usually applied to large molecules such as well suited for a wide range of applications in R&D, method
proteins or synthetic polymers. When an aqueous mobile development and quality control. TSKgel SW and SWXL
phase is used, SEC is also referred to as gel filtration are silica SEC phases with pore size distributions suited to
chromatography (GFC). When an organic eluent is applied, protein separations. TSKgel SW-type packings feature low
SEC is referred to as gel permeation chromatography (GPC). adsorption and well-defined pore size distribution. It is the
GPC is typically used to determine the molecular weight leading SEC column series for HPLC due to its excellent
(MW) and the MW distribution of synthetic polymers while 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 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.
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SEC
HOW IT WORKS
SEC

Size exclusion chromatography (SEC) is a method in which Smaller molecules can partially or completely enter the
components of a mixture are separated according to their porous particles. Because these smaller molecules have
molecular size, based on the flow of the sample through to flow through the interparticle space, as well as through
a porous packing. In contrast to all other modes of liquid the pore volume, they will elute from the column after the
chromatography the prerequisite for SEC is that the analyte excluded sample components. Molecules small enough to
does not interact with the surface of the stationary phases. penetrate the whole pore system of the stationary phase
Differences in elution time are ideally based solely on the will pass the entire pore and interparticle volume, and will
volume the analyte passes. elute late. Their retention volume is referred to as ‘total
permeation’ in SEC, whereas it is interpreted as ‘unretained
Large biomolecules that cannot penetrate the pores of the peak’ in conventional LC modes.
packing material elute first from the column. They are said
to be excluded from the packing; they flow with the mobile SEC is a very simple method for separating biomolecules,
phase in the interparticle space of the packed column. The because it is not necessary to change the composition of
exclusion limit characterizes the upper limit of molecular the mobile phase during elution. However, the separation
weight (or size), beyond which molecules will elute at the capacity of this method is limited. For a baseline separation
same retention volume called the exclusion or void volume it is necessary that the molecular weights of the molecules
of the column. Many SEC columns are referred to by their differ by at least 10 to 20 %.
exclusion limit.

Sample
application

buffer/
low salt
concentration

Elution

buffer/
low salt
concentration

t
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 its excellent resolution.
of proteins, peptides and other biological macro-molecules.
The complete TSKgel SW, PW, Alpha and SuperAW column TSKGEL PW SERIES
lines consist of either silica based or polymer based packings,
ranging in particle size from 4 µm to 20 µm. Columns are TSKgel PW and PWXL columns are packed with hydrophilic,
available in analytical through semi- preparative size, in rigid polymethacrylate beads. They are commonly used for
stainless steel, PEEK or glass. the separation of synthetic water soluble polymers because
they exhibit a much larger separation range, better linearity
COLUMN SELECTION of calibration curves, and less adsorption than the TSKgel
SW columns. While a TSKgel SW column is typically the first
The main criterion in choosing between the TSKgel SW, PW, column to try for biopolymers, TSKgel PW columns have
Alpha and SuperAW SEC columns is the molecular weight demonstrated good results for smaller peptides (<1,000 Da),
of the sample and its solubility. The fact that the TSKgel SW protein aggregates, DNA fragments, and viruses. TSKgel
columns are based on silica and the TSKgel PW, Alpha and PWXL-CP columns are especially suited for the separation of
SuperAW columns are derived from a hydrophilic polymer cationic polymers at low salt.
network has less impact on the separation than the particle
and pore size differences. TSKgel AW/ALPHA SERIES

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

TABLE 1

CHARACTERISTICS OF TSKgel SIZE EXCLUSION COLUMN LINES

Column line TSKgel SW / TSKgel PW / TSKgel Alpha /


SWXL /SuperSW PWXL SuperAW

Resin type Silica Polymethacrylate highly crosslinked Polymethacrylate

No. of available pore sizes 3/2 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

Max. flow 6.0 (SW) 1.2 (SWXL) 1.2 (PW) 1.0 (Alpha)
rate (mL/min) 0.4 (SuperSW) 1.0 (PWXL) 0.6 (SuperAW)

Pressure**(MPa) 1.0 - 12.0 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.
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SEC
TSKgel SEC COLUMN SELECTION
SEC

SAMPLE COLUMN SELECTION SELECTION CRITERIA

FIRST CHOICE ALTERNATIVE


Carbohydrates polysaccharides TSKgel GMPWXL TSKgel G5000PWXL large pore size, linear calibration
TSKgel SuperMultiporePW and TSKgel curve, small particles, high
G3000PWXL 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 suitable pore sizes
TSKgel BioAssist G4SWXL
TSKgel SuperSW3000 or
TSKgel G3000SWXL
TSKgel BioAssist G3SWXL
oligonucleotides TSKgel G2500PWXL small pore size, ionic interaction
Proteins normal size TSKgel SuperSW3000 TSKgel G3000PWXL small particles
small-medium TSKgel G3000SWXL or G4000PWXL small to medium range pore sizes
proteins TSKgel BioAssist G3SWXL
TSKgel G4000SWXL
TSKgel BioAssist G4SWXL
TSKgel SuperSW2000 or
TSKgel G2000SWXL
TSKgel BioAssist G2SWXL
large proteins low TSKgel G6000PWXL or large pore sizes
density TSKgel G5000PWXL
lipoprotein
gelatin TSKgel GMPWXL TSKgel G5000PWXL large pore size,
TSKgel SuperMultiporePW-M and G3000PWXL linear calibration curve
TSKgel G3000SWXL
Peptides large TSKgel SuperSW3000 TSKgel small to medium
TSKgel G3000SWXL SuperSW2000 or range pore size, versatile
TSKgel BioAssist G3SWXL or G3000PWXL
TSKgel G2000SWXL
TSKgel BioAssist G2SWXL
small TSKgel G2500PWXL TSKgel linear calibration curve,
SuperSW2000 or high resolving power
G2000SWXL
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 and G3000PWXL or linear calibration curve
TSKgel SuperMultiporePW TSKgel Alpha-5000
and Alpha-3000
cationic TSKgel G3000PWXL-CP medium to large pore size, low
TSKgel G5000PWXL-CP adsorption, linear calibration curve
TSKgel G6000PWXL-CP
Synthetic nonionic TSKgel G-Oligo-PW TSKgel G2500PW or small pore size, high resolving
oligomers TSKgel G2500PWXL or SuperAW2500 power
TSKgel Alpha-2500
TSKgel SuperOligoPW and
TSKgel SuperMultiporePW-N
anionic TSKgel G2500PWXL or TSKgel G2500PW or small pore size, ionic interaction
TSKgel Alpha-2500 SuperAW2500
TOSOH BIOSCIENCE ANALYSIS 5

SEC
TSKgel SW SERIES

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

Molecular weight (Da)


as shown by the calibration curve in Figure 1. From this data, 3
4 5
recommended separation ranges for globular proteins can 6
10 4 7
be made for each column (see Table 2). Different particle 8 9
sizes, column dimensions and column hardware materials
are available.
10 3
TSKgel G2000SWXL
The resulting differences in column characteristics allow the
TSKgel G3000SWXL 10
scientist to select the appropriate column to his individual TSKgel G4000SWXL
10 2
separation requirements.

8 6 10 12
HIGHLIGHTS Elution volume (mL)
Column: TSK-GEL SWXL columns, 5 or 8 µm ,7.8 mm ID x 30 cm L
Rigid spherical silica gel chemistry bonded with Sample:
PROTEIN 1. thyroglobulin
CALIBRATION CURVES (660,000
FORDa ); 2. IgGSWXL
TSKgel (160,000COLUMNS
Da );
hydrophilic groups 3. BSA (67,000 Da); 4. ovalbumin (43,000 Da);
Column: TSKgel 5. peroxidase
SWXL (40,2005Da);
columns, or 6.8 β-lactoglobulin
µm, 7.8 mm (18,400ID x 30 Da);cm L
Well defined pore size distribution
Sample: 1. thyroglobulin (660,000
7. myoglobin (16,900 DDa); 2.ribonuclease
a); 8. IgG (160,000 Da); Da);
A (12,600 3. BSA
Low non specific adsorption (67,000 Da); 4. ovalbumin
9. cytochrome(43,000
C (12,400Da);
Da);5. 10.peroxidase (40,200
glycine tetramer Da);
(246 Da)
Highest resolution and sensitivity 6. β-lactoglobulin
Elution: (18,400
0.3 mol/LDa);
NaCl7.inmyoglobin (16,900
0.1 mol/L sodium Da); 8. ribonuclease
phosphate buffer, pH 7.0
PEEK column hardware for SWXL packings A (12,600 Da); 9. cytochrome
Detection: UV @ 220 nmC (12,400 Da); 10. glycine tetramer (246 Da)
Mobile phase: 0.3 mol/L NaCl in 0.1 mol/L sodium phosphate buffer,
Short TSKgel QC-PAK columns for fast analysis
pH 7.0; Detection:UV @ 220 nm
Semi-preparative stainless steel columns for precise
scale up

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 (Å) THEORET. PLATES OF PROTEINS (DA)

SuperSW2000 4.6 x 30 4 125 30,000 5 x 103–1.5 x 105

G2000SWXL 7.8 x 30 5 125 20,000 5 x 103–1.5 x 105

BioAssist G2SWXL 7.8 x 30 5 125 20,000 5 x 103–1.5 x 105

QC-PAK GFC 200 7.8 x 15 5 125 10,000 5 x 103–1.5 x 105

G2000SW 7.5 x 30/60 10 125 10,000/20,000 5 x 103–1.5 x 105


21.5 x 30/60 13 125 10,000/20,000 5 x 103–1.5 x 105

SuperSW3000 4.6 x 30 4 250 30,000 1 x 104–5 x 105

G3000SWXL 7.8 x 30 5 250 20,000 1 x 104–5 x 105

BioAssist G3SWXL 7.8 x 30 5 250 20,000 1 x 104–5 x 105

QC-PAK GFC 300 7.8 x 15 5 250 10,000 1 x 104–5 x 105

G3000SW 7.5 x 30/60 10 250 10,000/20,000 1 x 104–5 x 105


21.5 x 30/60 13 250 10,000/20,000 1 x 104–5 x 105

G4000SWXL 7.8 x 30 8 450 16,000 2 x 104–7 x 106

BioAssist G4SWXL 7.8 x 30 8 450 16,000 2 x 104–7 x 106

G4000SW 7.5 x 30/60 13 450 8,000/16,000 2 x 104–7 x 106


21.5 x 30/60 17 450 8,000/16,000 2 x 104–7 x 106
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SEC
TSKgel SW SERIES APPLICATIONS
SEC

AGGREGATE ANALYSIS USE OF DETERGENTS

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

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.

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 30 cm L 0


Sample: monoclonal antibody, Inj.volume: 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 QC ANALYSIS OF AN ANTI-TSH ANTIBODY PURIFIED FROM CELL
MALS detector: 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
TOSOH BIOSCIENCE ANALYSIS 7

SEC
TSKgel SuperSW 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 4 µm particle size featuring superior resolution and
concentrations become limited. To meet the needs of high highest sensitivity
sensitivity and high resolution protein analysis Tosoh Low non-specific adsorption
Bioscience developed TSKgel SuperSW columns packed with High reproducibility due to well-defined pore size
4 µm spherical silica particles. TSKgel SuperSW columns are distribution
available in two pore sizes, 125 Å and 250 Å, both featuring a 30,000 theoretical plates / column (4.6 mm ID)
minimum of 30,000 theoretical plates / column. Compared to Microbore columns for increased sensitivity and
the well established TSKgel SWXL (5 µm) series, SuperSW reduced buffer consumption
columns show higher resolution due to a 50 percent increase
in theoretical plate numbers (Table 3).

To further improve performance, TSKgel SuperSW media Figure 4 demonstrates the superior sensitivity reached with
are packed into columns with smaller inner diameter TSKgel SuperSW2000 compared to a TSKgel G2000SWXL
(1.0, 2.0, 4.6 mm ID). The smaller diameters are one reason column of the same length but larger inner diameter. TSKgel
for increased peak heights. In addition, the high resolution SuperSW can yield peak heights approximately 4 times that
of the 4 µm particles and accordingly smaller peak widths of TSKgel SWXL due to downsizing in column diameter and
further increase peak height provided the HPLC system is increased theoretical plates.
optimized with regard to dead volume.

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, 4.6mm ID xAND
30cm TSKgel G2000SWXL
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:UV
Detection: 5@ µL280nm
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
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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 Column: TSKgel SuperSW3000, 4.6 mm ID x 30 cm L;
inner diameter can further enhance sensitivity. TSKgel TSKgel G3000SWXL, 7.8 mm ID x 30 cm L
SuperSW3000 columns are available with 4.6; 2 and 1 mm ID. Mobile phase: 0.2 mol/L phosphate 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)
Column: TSKgel SuperSW Series, 4.6 mm ID X 30 cm L Inj.volume: 0.2 µL; Temperature.: 25 °C
Sample: Standard proteins (5 µL, 0.1 g/L each); Detection: UV @ 280 nm, cell vol. 2 µL (4.6 mm ID), 35 nL (1.0, 2.0 mm ID)
1.thyroglobulin 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
TOSOH BIOSCIENCE ANALYSIS 9

SEC
TSKgel SuperSW APPLICATIONS

SEC
QC ANALYSIS OF ANTIBODIES SEC-ESI-MS ANALYSIS OF PROTEINS

Thermally induced denaturation or aggregation of Hyphenated separation techniques like HPLC-MS or HPLC-
therapeutic antibodies can be a significant problem during ELSD allow sensitive analysis of samples with very low
different stages of its production and formulation, since analyte concentrations. Moreover MS/MS detection is a
aggregates affect the efficiency of the biotherapeutic. Thus powerful tool to provide further structural information about
the quantification of aggregates is an important parameter the compounds. These detection methods require the use
in the quality control analysis of biopharmaceuticals. Using of volatile buffer systems because the solvent must be
TSKgel SuperSW3000 columns the amounts of tri-, di- and evaporated before the sample molecules enter the detection
monomers of monoclonal antibodies can be monitored. system. For LC/MS analysis TSKgel SuperSW columns can
Quantification is facilitated by using smaller inner diameter be run with formate buffers as mobile phase, instead of the
columns since peak height is significantly increased common phosphate buffers. Figure 8 demonstrates that
(Figure 7). at least 300 mM ammonium formate is necessary to reach
separation efficiencies comparable to 100 mM phosphate
buffer.

FIGURE 7 FIGURE 8
350
70

60
0.1 mol/L
300
50
Phosphate buffer
Intensity

40
1.0 mm ID X 30 cm L
30 250
2.0 mm ID X 30 cm L Ammonium formate
20
4.6 mm ID X 30 cm L
Intensity (mV)

10 200 0.4 mol/L


0
0 5 10 15
min 150
0.3 mol/L

14 aggregates
100
0.2 mol/L
13
1.0 mm ID X 30 cm L
12
50
Intensity

2.0 mm ID X 30 cm L 0.1 mol/L


11
0
4.6 mm ID X 30 cm L 0 5 10 15
10 Time (min)

0
0 5 10 15 SEPARATION OF PROTEINS WITH AMMONIUM FORMATE
min ELUENT ON TSKgel SuperSW3000

SEPARATION OF IgG ON TSKgel SuperSW3000 Column: TSKgel SuperSW3000, 1.0, 2.0, 4.6 mm ID x 30 cm L
Sample: 1. tyroglobulin (1.0 g/L), 2. γ-globulin (2.0 g/L), 3. ovalbumin
Column: TSKgel SuperSW3000, 1.0 mm ID x 30 cm L (2.0 g/L), 4. ribonuclease A (3.0 g/L), 5. p-aminobenzoic acid (0.02 g/L)
Sample: IgG (mouse, mAb, 1.0 g/L) Mobile phase: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4
Mobile phase: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4, + 0.05% NaN3
+ 0.05 NaN3; Flow rate: 16 µL/min (1 mm ID), 65 µL/min (2 mm ID), Flow rate: 16 µL/min (1 mm ID), 65 µL/min (2 mm ID), 350 µL/min
350 µL/min (4.6 mm ID); Inj.Vol.: 0.2 µL; Temp.: 25 °C (4.6 mm ID)
Detection: UV @ 280 nm, cell vol. 2 µL (4.6 mm), 35 nL (1.0, 2.0 mm) Inj.volume: 0.2 µL; Temp.: 25 °C
Detection: UV @ 280 nm, cell vol. 2 µL (4.6 mm ID), 35 nL (1.0, 2.0
mm ID)
10 WWW.TOSOHBIOSCIENCE.COM

SEC
TSKgel SuperSW 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
columns the HPLC system should be optimized and extra of the system and might impair peak resolution. For most
column peak broadening reduced. This means reduction of separations with 4.6 mm ID TSKgel SuperSW columns a
dead volume and adjustment of sample concentration and 8-10 µL standard detector cell might be sufficient but for
injection volume. semi-micro (2 mm ID) or micro columns (1 mm ID), we
strongly recommend using semi-micro detector cells.
SYSTEM DEAD VOLUME
INJECTOR
Key components of the HPLC system with regard to dead
volume reduction are the void volume of tubings, the cell The maximum number of theoretical plates in isocratic
volume of the detector cell and the void volume of the separations can be reached when using a low diffusion
injection unit. Modern UHPLC systems designed for use with type manual injector like the Rheodyne 8125. All kinds of
sub 2 µm particles exhibit extremely small dead volumes automated HPLC injectors will deteriorate column efficiency
and can be used for SEC analysis without modification. to a certain extend but due to practical reasons, auto-samplers
are nowadays standard. All the more it is important to select
VOID VOLUME OF THE TUBING an auto-sampler capable of trace injection mode. Dead
volume of the outlet capillary should be minimized to the
The volume of tubing from injector to column, column to utmost (as short as possible, 0.1 mm ID). Figure 9 shows the
detector influences the diffusion within the tubing and the effect of injector tubings on column efficiency for a 1 mm ID
column efficiency. Column efficiency starts deteriorating column.
remarkably when the volume of the tubing exceeds 10 µL
(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 SuperSW2000 AND SuperSW3000 OPERATING CONDITIONS

For best results, it is recommended to use the following experimental conditions for TSKgel SuperSW columns:

CONNECTIONS

Tubing 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.
TOSOH BIOSCIENCE ANALYSIS 11

SEC
TSKgel SuperSW SYSTEM REQUIREMENTS

SEC
SAMPLE LOAD AND INJECTION VOLUME MOBILE PHASE

Although the efficiency of TSKgel SuperSW columns is high, The eluent plays an important role in SEC separations. When
it is obvious that it decreases at high sample loads. Figure denaturing agents are used, the exclusion limits for proteins
10 shows that sample load should not exceed 100 µg for a become smaller since they lose their compact globular
TSKgel SuperSW3000 column of 4.6 mm ID x 30 cm L. On the structure. Proper selection of eluting conditions is necessary
other hand the injection volume itself is a critical parameter. to maximize the molecular sieving mechanism and to
As for all HPLC applications injection volume should be as minimize secondary effects, such as ionic and hydrophobic
small as possible. If injection volume exceeds 20 µL on a interactions between the sample and the column packing
4.6 mm ID column, a considerable deterioration of column material. In general, the use of relatively high ionic strength
efficiency is observed for TSKgel SuperSW2000 (80 µL for buffers is recommended for most protein applications. A
TSKgel SuperSW3000). In general the sample load should be neutral salt is often added to increase ionic strength.
less than 100 µg in less than 10 µL injection volume for a 4.6
mm ID TSKgel SuperSW column. RECOVERY OF PROTEIN

FLOW RATE DEPENDENCE TSKgel SuperSW series is capable of obtaining high protein
recovery even in trace analysis with sample load of 1 µg
The effect of flow rate on column efficiency depends on or lower. Most proteins are recovered quantitatively with
particle size of packing materials, sample molecular size, TSKgel SuperSW series, but it is important to make sure
eluent viscosity, etc. The appropriate flow rate for TSKgel that samples in small concentrations are not adsorbed to
SuperSW columns is up to 0.4 mL/min for a 4.6 mm ID the sample vial or to the HPLC system itself. Similar samples
column, up to 75 µL/min for a 2 mm ID column, and up should be injected several times before measurement so
to 20 µL/min for a 1 mm ID column, respectively. If higher that adsorption points within the system are inactivated in
resolution is required the flow rate can be lowered. advance when trace analysis is performed.

FIGURE 9 FIGURE 10

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 Column: TSKgel SuperSW series, 4.6 mm ID x 30 cm L;


Mobile phase: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4 TSKgel SWXL series, 7.8 mm ID x 30 cm L
+ 0.05 % NaN3 Sample: Bovine serum albumin
Flow rate: 16 µL/min; Inj.volume: 0.2 µL; Temp.: 25 °C Mobile phase: 0.2 mol/L phosphate buffer, pH 6.7
Detection: UV @ 280 nm Flow rate: 0.35 mL/min (SuperSW series), 1.00 mL/min (SWXL series)
Sample: p-Aminobenzoic acid (20mg/L) Temp.: 25 °C; Detection: UV @ 280 nm, micro flow cell
Tubing: ID (mm) x L (cm), Vol.
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
12 WWW.TOSOHBIOSCIENCE.COM

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)

GLASS COLUMNS
16214 QC-PAK GFC 200GL 8.0 15 5 ≥ 10,000 0.5 - 1.0 1.2 4.0
16216 QC-PAK GFC 300GL 8.0 15 5 ≥ 10,000 0.5 - 1.0 1.2 4.0
08800 G3000SW, Glass 8.0 30 10 ≥ 10,000 0.4 - 0.8 0.8 2.0
08801 G4000SW, Glass 8.0 30 13 ≥ 8,000 0.4 - 0.8 0.8 2.0

STAINLESS STEEL COLUMNS


18674 SuperSW2000 4.6 30 4 ≥ 30,000 0.1 - 0.35 0.4 12.0
21845 SuperSW3000 -NEW- 1.0 30 4 ≥ 18,000 0.016 0.02 12.0
21485 SuperSW3000 -NEW- 2.0 30 4 ≥ 25,000 0.065 0.075 12.0
18675 SuperSW3000 4.6 30 4 ≥ 30,000 0.1 - 0.35 0.4 12.0
08540 G2000SWXL 7.8 30 5 ≥ 20,000 0.5 - 1.0 1.2 7.0
08541 G3000SWXL 7.8 30 5 ≥ 20,000 0.5 - 1.0 1.2 7.0
08542 G4000SWXL 7.8 30 8 ≥ 16,000 0.5 - 1.0 1.2 3.5
16215 QC-PAK GFC 200 7.8 15 5 ≥ 10,000 0.5 - 1.0 1.2 4.0
16049 QC-PAK GFC 300 7.8 15 5 ≥ 10,000 0.5 - 1.0 1.2 4.0
05788 G2000SW 7.5 30 10 ≥ 10,000 0.5 - 1.0 1.2 2.0
05789 G3000SW 7.5 30 10 ≥ 10,000 0.5 - 1.0 1.2 2.5
05790 G4000SW 7.5 30 13 ≥ 8,000 0.5 - 1.0 1.2 1.5
05102 G2000SW 7.5 60 10 ≥ 20,000 0.5 - 1.0 1.2 4.0
05103 G3000SW 7.5 60 10 ≥ 20,000 0.5 - 1.0 1.2 5.0
05104 G4000SW 7.5 60 13 ≥ 16,000 0.5 - 1.0 1.2 3.0
06727 G2000SW 21.5 30 13 ≥ 10,000 3.0 - 6.0 8.0 1.0
06728 G3000SW 21.5 30 13 ≥ 10,000 3.0 - 6.0 8.0 1.5
06729 G4000SW 21.5 30 17 ≥ 8,000 3.0 - 6.0 8.0 1.0
05146 G2000SW 21.5 60 13 ≥ 20,000 3.0 - 6.0 8.0 2.0
05147 G3000SW 21.5 60 13 ≥ 20,000 3.0 - 6.0 8.0 3.0
05148 G4000SW 21.5 60 17 ≥ 16,000 3.0 - 6.0 8.0 2.0

PEEK COLUMNS
20027 BioAssist G2SWXL 7.8 30 5 ≥ 20,000 0.5 - 1.0 1.2 7.0
20026 BioAssist G3SWXL 7.8 30 5 ≥ 20,000 0.5 - 1.0 1.2 7.0
20025 BioAssist G4SWXL 7.8 30 8 ≥ 16,000 0.5 - 1.0 1.2 3.5
TOSOH BIOSCIENCE ANALYSIS 13

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
Seven different TSKgel PW pore sizes
The new generation of TSKgel SuperMultiporePW columns Linear SEC column line encorporating 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 (Å) MW RANGE (PEG/PEO)

G1000PW 12 <100 <1 x 103


G2000PW 12 125 <2 x 103
G2500PW 12, 17 <200 <3 x 103
G3000PW 12, 17 200 <5 x 104
G4000PW 17 500 <3 x 105
G5000PW 17 1,000 <1 x 106
G6000PW/ BioAssist G6PW 17 >1,000 <8 x 106
GMPW 17 <100-1,000 5 x 10 - 8 x 106
2

G2500PWXL 7 <200 <3 x 103


G3000PWXL 7 200 <5 x 104
G4000PWXL 10 <500 <3 x 105
G5000PWXL 10 1000 <1 x 106
G6000PWXL 13 >100 <8 x 106
G-DNA-PW 10 >1,000 <8 x 106
GMPWXL 13 100-1,000 5 x 10 - 8 x 106
2

G-Oligo-PW 7 125 <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 7 200 < 9 x 104


G5000PWXL-CP 10 1,000 < 1 x 106
G6000PWXL-CP 13 >1,000 < 2 x 107
14 WWW.TOSOHBIOSCIENCE.COM

SEC
TSKgel PW SERIES
SEC

FIGURE 13
CALIBRATION CURVES

Figure 11 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 12).
Figure 13 shows the near linear calibration curves for PEG/
PEO on TSKgel SuperMultiporePW columns.

FIGURE 12
Protein calibration curves on TSKgel PWXL columns

107

106 1 2 5 3 4
Molecular weight (Da)

a CALIBRATION CURVES FOR TSKgel SuperMultiporePW

b Sample: PEO & PEG standards;


105
c
d e Mobile phase: H2O; Flow rate: 0.6 mL/min; Detection: RI;
Temperature: 25 °C
f
104
g

103
4 6 8 10 12
Elution volume (mL)
Column: 1. G3000PWXL
2. G4000PW
PROTEIN CALIBRATION CURVES
3. G5000PW
XL
XL
ON TSKgel PWXL COLUMNS
4. G6000PWXL
5. GMPW
Column: 1. TSKgel
Sample: G3000PWXL,
XL
2. Da)
a. thyroglobulin (660,000 TSKgel G4000PWXL, 3. TSKgel
G5000PWXL, 4. TSKgel G6000PWXL,
b. γ-globulin (150,000 Da) 5. TSKgel GMPWXL
c. albumin (67,000 Da)
Sample: a. thyroglobulin (43,000 Da) Da), b. γ-globulin (150,000 Da),
(660,000
d. ovalbumin
c. albumin (67,000 Da), d. ovalbumin
e. β-lactoglobulin (43,000 Da), e. ß-lactoglobulin
(36,000 Da)
f. myoglobin (16,900 Da)
(36,000 Da), f. myoglobin (16,900
g. cytochrome Da),
C (12,400 Da) g. cytochrome C (12,400 Da)
Mobile phase: Elution:
0.2 M phosphate
0.2 M phosphatebuffer
buffer (pH(pH
6.8) 6.8); Flow rate: 1.0 mL/min;
Detection: UV Flow
@ Rate: 1.0 mL/min
280 nm
Detection: UV @ 280 nm

FIGURE 11

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 15

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 15. 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 15
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 14B.

FIGURE 14 FIGURE 15

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
16 WWW.TOSOHBIOSCIENCE.COM

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 16
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 17 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 and molecular weights were injected on the three TSKgel
the 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 16 FIGURE 17

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,
TSKgel G6000PW
Columns: TSKgel G3000PWXL-CP, 7 µm
XL
-CP, 13µm
(7.8 mm(7.8mm
ID x ID
30 xcm
30cm)
L),
10 µm; TSKgel G6000PWXL-CP, 13 µm Eluent:
TSKgel G5000PWXL-CP, 0.1mol/L
10 µmNaNO
(7.83 mm ID x 30 cm L), TSKgel
Samples: polyethylene oxides (PEO) standards; polyethylene Flow Rate: 13 µm1mL/min
G6000PWXL-CP, (7.8 mm ID x 30 cm L)
glycols (PEG) standards Mobile phase: 0.1 mol/L
Detection: RI NaNO3
Mobile phase: 0.1 mol/L NaNO3; Flow rate: 1 mL/min; FlowTemperature:
rate: 1 mL/min; Detection:
25°C RI; Temperature: 25 °C
Detection: RI; Temp: 25 °C Sample Load: 3 g/L, 100 µL
Sample Load: 3g/L, 100µL
TOSOH BIOSCIENCE ANALYSIS 17

SEC
TSKgel SuperMultipore SERIES

SEC
The new TSKgel SuperMultiporePW column line is Figure 19 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 19
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 18).

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 18

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
18 WWW.TOSOHBIOSCIENCE.COM

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


22789 SuperMultiporePW-N 6.0 15 4 >16,000 0.3 - 0.6 0.6 4.5
22790 SuperMultiporePW-M 6.0 15 5 >12,000 0.3 - 0.6 0.6 2.7
22791 SuperMultiporePW-H 6.0 15 8 (6-10) >7,000 0.3 - 0.6 0.6 0.9
22792 SuperOligoPW 6.0 15 3 >16,000 0.3 - 0.6 0.6 5.0
08031 G-Oligo-PW 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
08032 G-DNA-PW 7.8 30 10 ≥ 10,000 0.2 - 0.5 0.6 2.0
08020 G2500PWXL 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
08021 G3000PWXL 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
08022 G4000PWXL 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 2.0
08023 G5000PWXL 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 2.0
08024 G6000PWXL 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0
08025 GMPWXL 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0
21873 G3000PWXL-CP 7.8 30 7 ≥ 16,000 1.0 5.5
21874 G5000PWXL-CP 7.8 30 10 ≥ 10,000 1.0 2.5
21875 G6000PWXL-CP 7.8 30 13 ≥ 7,000 1.0 2.0
05760 G1000PW 7.5 30 12 ≥ 5,000 0.5 - 1.0 1.2 2.0
05761 G2000PW 7.5 30 12 ≥ 5,000 0.5 - 1.0 1.2 2.0
08028 G2500PW 7.5 30 12 ≥ 5,000 0.5 - 1.0 1.2 2.0
05762 G3000PW 7.5 30 12 ≥ 5,000 0.5 - 1.0 1.2 2.0
05763 G4000PW 7.5 30 17 ≥ 3,000 0.5 - 1.0 1.2 1.0
05764 G5000PW 7.5 30 17 ≥ 3,000 0.5 - 1.0 1.2 1.0
05765 G6000PW 7.5 30 17 ≥ 3,000 0.5 - 1.0 1.2 1.0
08026 GMPW 7.5 30 17 ≥ 3,000 0.5 - 1.0 1.2 1.0
05105 G2000PW 7.5 60 12 ≥ 10,000 0.5 - 1.0 1.2 4.0
08029 G2500PW 7.5 60 12 ≥ 10,000 0.5 - 1.0 1.2 4.0
05106 G3000PW 7.5 60 12 ≥ 10,000 0.5 - 1.0 1.2 4.0
05107 G4000PW 7.5 60 17 ≥ 6,000 0.5 - 1.0 1.2 2.0
05108 G5000PW 7.5 60 17 ≥ 6,000 0.5 - 1.0 1.2 2.0
05109 G6000PW 7.5 60 17 ≥ 6,000 0.5 - 1.0 1.2 2.0
08027 GMPW 7.5 60 17 ≥ 6,000 0.5 - 1.0 1.2 2.0
08030 G2500PW 21.5 60 17 ≥ 10,000 1.6 - 6.0 8.0 2.0

PEEK
20024 BioAssist G6PW 7.8 30 17 ≥ 3,000 0.5 - 1.0 1.2 10

GUARD COLUMNS
22793 SuperMP (PW)-N Guard column 4.6 3.5 4
22794 SuperMP (PW)-M Guard column 4.6 3.5 5
22795 SuperMP (PW)-H Guard column 4.6 3.5 8
22796 SuperOligoPW Guard column 4.6 3.5 3
08034 Oligo Guard column 6.0 4.0 13 For 7.8 mm ID G-Oligo-PW columns
08033 PWXL Guard column 6.0 4.0 12 For 7.8 mm ID PWXL & G-DNA-PW (TSKgel G3000PW packing)
21876 PWXL-CP Guard column 6.0 4.0 13 For 7.8 mm ID PWXL-CP columns
06763 PW-L Guard column 7.5 7.5 13 For 7.5 mm ID G1000PW & G2000PW (TSKgel G2000PW packing)
06762 PW-H Guard column 7.5 7.5 13 For 7.5 mm ID G2500PW through GMPW columns
06758 PW-H Guard column 21.5 7.5 17 For 21.5 mm ID G2500PW through G5000PW columns
TOSOH BIOSCIENCE ANALYSIS 19

SEC
TSKgel Alpha & SuperAW SERIES

SEC
FIGURE 20
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
20). 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
the whole range.
Conditions for TP measurement: Sample: ethylene glycol
Flow rate: 1.0 mL/min; Temp.: 25 °C; Detection: RI
HIGHLIGHTS

Unique hydrophilic polyvinyl 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
20 WWW.TOSOHBIOSCIENCE.COM

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


18339 Alpha-2500 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
18340 Alpha-3000 7.8 30 7 ≥ 16,000 0.5 - 0.8 1.0 4.0
18341 Alpha-4000 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 3.0
18342 Alpha-5000 7.8 30 10 ≥ 10,000 0.3 - 0.6 1.0 3.0
18343 Alpha-6000 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0
18344 Alpha-M (mixed bed) 7.8 30 13 ≥ 7,000 0.3 - 0.6 1.0 2.0

GUARD COLUMNS
18345 Alpha Guard column 6.0 4 13 For all Alpha columns

VMPAK COLUMNS*
20011 VMpak-25 2.0 5 7 ≥ 1,000 0.1 - 0.2 0.25 2.0
20012 VMpak-25 2.0 15 7 ≥ 3,000 0.1 - 0.2 0.25 6.0

STAINLESS STEEL COLUMNS


19315 SuperAW2500 6.0 15 4 ≥ 16,000 0.3 - 0.6 6.0
19316 SuperAW3000 6.0 15 4 ≥ 16,000 0.3 - 0.6 6.0
19317 SuperAW4000 6.0 15 6 ≥ 10,000 0.3 - 0.6 4.0
19318 SuperAW5000 6.0 15 7 >10,000 0.3 - 0.6 3.0
19319 SuperAW6000 6.0 15 9 >7,000 0.3 - 0.6 2.0
19320 SuperAWM-H 6.0 15 9 >7,000 0.3 - 0.6 2.0

GUARD COLUMNS
19321 SuperAW-L Guard Column 4.6 3.5 7 For SuperAW2500-4000 columns.
19322 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 4
SEC
OPTIMIZING SEC
TOSOH BIOSCIENCE GMBH TOSOH BIOSCIENCE LLC TOSOH CORPORATION TOSOH SHANGHAI CO. LTD
ZETTACHRING 6 3604 HORIZON DRIVE, 3-8-2 SHIBA, MINATO-KU ROOM 301, PLAZA B,

SEC
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ABOUT US
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INFO.TBG@TOSOH.COM INFO.TBL@TOSOH.COM INFO@TOSOH.CO.JP PAN@TOSOH.COM.CN
WWW.TOSOHBIOSCIENCE.COM
As SEC is a partition chromatography, sample load on the For TSKgel SW columns mobile phases a buffer concen-
WWW.TOSOHBIOSCIENCE.COM WWW.TOSOHBIOSCIENCE.COM WWW.TOSOHBIOSCIENCE.COM
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 GmbH, Separations Business Unit, Stuttgart, recommended. Proteins can be loaded at higher concentra- as sodium sulphate may be added to the buffer to increase
is an acknowledged global leader in the field of bioseparations. 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
Established as TosoHaas in 1987, the original joint venture 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.
between Tosoh Corporation of Japan and the Rohm and Haas 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
Company, USA, has become synonymous with advanced products 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.
and quality support. In the year 2000, Tosoh Corporation acquired 1 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
a 100% controlling interest changing the name to TOSOH BIOSEP.
2
3
polymeric compounds, a neutral salt such as sodium nitrate
MOBILE PHASE is added to the aqueous eluent. Generally, a concentration
In the course of unifying all Tosoh affiliates, the new Brand Name
4

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 Bioscience evolved. Today, the two branches, Bioseparations molecular sieving mechanism and to minimize secondary
and Diagnostics operate with the same name Tosoh Bioscience - 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
Separations Business Unit and accordingly Diagnostics Business 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%
Unit. Tosoh manufacturing sites in Japan provide products to the may also prevent columns from fouling by suppressing
interaction of hydrophobic impurities of the sample. An
sales and support subsidiaries in the U.S. and Europe, ensuring FIGURE 22 example is shown in Figure 22 with the analysis of a pegylat-
ed protein on a TSKgel G3000SWXL column. As pegylated
full global coverage. Over the last 30 years, TSKgel SW columns products are more hydrophobic, they tend to interact with
the column matrix. Over time the pegylated product can foul
have become the worldwide industry standard for size exclusion
200 200
the column, which is indicated by shifts of retention time
and decreasing separation performance. By adding 10% of

Intensity (mAu)

Intensity (mAu)
chromatography of biomolecules. 150 150
ethanol to the elution buffer, this problem is overcome and no
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
50 50
1935 FOUNDING OF TOYO SODA MANUFACTURING CO., LTD. COLUMN PROTECTION
1936 OPERATION OF NANYO MANUFACTURING COMPLEX BEGINS

1971 SCIENTIFIC INSTRUMENTS DIVISION FORMED, FIRST GPC COLUMN USING TSK-GEL DEVELOPED BY TOSOH 0 0 To protect the column and increase its lifetime, the use of
1974 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 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,
Elution time (min) Elution time (min) sample load and the composition of the mobile phase have
1979 TOSOH DEVELOPS TOYOPEARL MEDIA

1983 TOSOH DEVELOPS HYDROPHOBIC INTERACTION MEDIA


an influence on column lifetime. For information on TSKgel
INFLUENCE OF MOBILE PHASE SEC columns for GPC analysis of organic polymers please
1987 TOSOHAAS US OPERATIONS FORMED IN MONTGOMERYVILLE
refer to the TSKgel GPC column brochure.
1989 TOSOHAAS GMBH OPERATIONS FORMED IN STUTTGART
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
Sample: 10 mL PEG r-HuMGDF;
2000 IN NOVEMBER FORMER TOSOHAAS US OPERATIONS BECOMES TOSOH BIOSEP LLC, A 100% SUBSIDIARY OF TOSOH CORPORATION
initial injection; after 150 injections
2001 IN JANUARY FORMER TOSOHAAS GMBH EUROPEAN OPERATIONS BECOMES TOSOH BIOSEP GMBH, A 100% SUBSIDIARY OF TOSOH CORPORATION Mobile phase: 0.1 M sodium phosphate, pH 6.9, 0.5 M NaCl
2002/ TOSOH CORPORATION ANNOUNCES THAT ALL TOSOH AFFILIATED SCIENTIFIC AND DIAGNOSTIC SYSTEM Flow rate: 0.7 mL/min; Detection: UV @ 220 nm
2003 RELATED COMPANIES IN EUROPE, WILL BE UNIFIED UNDER THE NEW NAME TOSOH BIOSCIENCE.

2008 ECOSEC , THE 7TH GENERATION GPC SYSTEM IS INTRODUCED GLOBALLY

2009 TOSOH BIOSCIENCE GMBH CELEBRATES ITS 20TH ANNIVERSARY IN STUTTGART

2010 TOSOH CELEBRATES ITS 75TH YEAR IN BUSINESS WITH THE OPENING OF FIVE NEW PLANTS, AND CONTINUED RAPID EXPANSION IN CHINA
ANALYSIS

TSKgel® UP-SW3000 UHPLC COLUMNS

TSKgel UP-SW3000 columns packed with 2 µm silica based HIGHLIGHTS


particles are the latest addition to the popular TSKgel
SW series, the gold standard for QC analysis of antibody Proven TSKgel SW SEC quality
therapeutics. The new silica-based UHPLC columns are Virtual absence of nonspecific interaction
based on the proven proprietary surface technology of
the renowned TSKgel SW series and facilitate the transfer Easy transfer of existing HPLC methods
of existing HPLC methods to UHPLC systems. Optimized for mAb quality control

Aqueous size exclusion chromatography (SEC) is the The new columns can be used with modern HPLC and
method of choice for the analysis of proteins fragments, UHPLC systems and are available with 15 or 30 cm length.
monomers, and aggregates under non-denaturing condi- The short one enables short analysis times; the long one
tions. Based on the flow of the sample through a porous provides higher resolution for mAb analysis. The lifetime
stationary phase SEC separates molecules according to of the columns can be improved when using the corre-
their size, or more precisely, their hydrodynamic volume. sponding guard columns. A “direct connect” (DC) guard
In aqueous elution systems SEC is also referred to as column allows minimizing extra column dead volume.
gel filtration chromatography (GFC). TSKgel G3000SWXL
columns have been the industry’s standard for quality
control of monoclonals by SEC for decades. COMPARISON OF TSKgel SW COLUMN SERIES

COMPARISON OF CALIBRATION CURVES

Figure 2

Particle
Column N (peak 4) AS (peak 4)
size
A: TSKgel UP-SW3000 2 µm 45,625 0.95
B: TSKgel SuperSW3000 4 µm 24,419 1.02

Figure 1 C: TSKgel G3000SWXL 5 µm 18,325 1.05

Columns: A: TSKgel UP-SW3000 (4.6 mm ID × 30 cm, red) Columns: A: TSKgel UP-SW3000, 2 µm, 4.6 mm ID x 30 cm
B: TSKgel SuperSW3000 ( 4.6 mm ID × 30 cm, blue) B: TSKgel SuperSW3000, 4 µm, 4.6 mm ID x 30 cm
C: TSKgel G3000SWXL (7.8 mm ID × 30 cm, grey) C: TSKgel G3000SWXL, 5 µm, 7.8 mm ID x 30 cm
Mobile phase: 100 mmol/L phosphate buffer (pH 6.7) + 100 mmol/L Mobile phase: 100 mmol/L phosphate buffer (pH 6.7) + 100 mmol/L so-
sodium sulfate + 0.05% NaN3 dium sulfate + 0.05 % NaN3
Flow rate: A & B: 0.35 mL/min; C: 1.0 mL/min Flow rate: A, B: 0.35 mL/min, C: 1.0 mL/min
Temperature: 25°C; Detection: UV @ 280 nm Temperature: 25 °C
Injection vol.: 10 µL Detect.: UV @ 280 nm (A,B: micro flow cell, C: standard flow cell)
Samples: 1. thyroglobulin (640,000 Da); (1’ thyroglobulin aggregate); Injection vol.: 10 µL
2. γ-globulin (155,000 Da); (2’ γ-globulin dimer); Sample: 1. thyroglobulin, 640,000 Da (1’ thyroglobulin dimer)
3. ovalbumin ( 47,000 Da); 2. γ-globulin, 155,000 Da (2’ γ-globulin dimer)
4. ribonuclease A (13,700 Da); 3. ovalbumin, 47,000 Da; 4. ribonuclease A, 13,700 Da
5. p-aminobenzoic acid (137 Da) 5. p-aminobenzoic acid, 137 Da
ANALYSIS

MASS RANGE REPRODUCIBLE PERFORMANCE

Figure 1 shows the calibration curve and the molecular TSKgel SW SEC columns are known for their outstanding
weight range of the new 2 µm TSKgel UP-SW3000 compared quality and reproducibility. 40 years of expertise in devel-
to those of 5 micron TSKgel G3000SWXL and 4 micron opment and production of gel filtration columns have
TSKgel SuperSW3000. Calibration curves and mass ranges paved the road to UHP-SEC. The good lot-to-lot reproduc-
are almost identical which facilitates transfer of existing ibility of TSKgel UP-SW3000 is proved in Figure 3.
methods.
APPLICATION
TSKgel UP-SW3000 has the same molecular mass separa-
tion range as the equivalent grades of conventional TSKgel TSKgel UP-SW3000 is suited for the separation of antibody
SW-type columns but much higher column efficiency: Figure dimer, monomer, and fragments in one run with ultra-high
2 shows the increase in resolution achieved by reducing the resolution (Figure 4). One TSKgel UP-SW3000 achieves
particle size from 5 (respectivly 4) micron to 2 micron. even higher resolution than two TSKgel G3000SWXL
columns connected in series.

BATCH-TO-BATCH REPRODUCIBILITY ANALYSIS OF MONOCLONAL ANTIBODIES

Figure 4

Figure 3 Rs (peak Rs (peak Rs (peak


Column
1/2) 2/3) 3/4)
Column: TSKgel UP-SW3000 4.6 mm ID x 30 cm from 4 different batches
Condition and samples: see Figure 1 A A: TSKgel G3000SWXL x2 1.60 3.63 1.77
B: TSKgel UP-SW3000 2.16 5.02 2.56

Columns: A) TSKgel G3000SWXL, 5 µm, 7.8 mm ID x 30 cm x 2;


B) TSKgel UP-SW3000, 2 µm, 4.6 mm ID x 30 cm
Mobile phase: 100 mmol/L phosphate buffer + 100 mmol/L sodium sulfate
+ 0.05% sodium azide, pH 6.7
Flow rate: A) 1.0 mL/min, B) 0.35 mL/min; Temperature: 25°C
Detection: UV @ 280 nm; Injection vol.: 10 µL
Sample: mouse-human chimeric IgG, monoclonal
1 trimer, 2 dimer, 3 monomer, 4 fragment

Ordering information
Part-No Description Matrix Housing Dimensions

0023449 TSKgel UP-SW3000, 2 µm Silica Stainless steel 4.6 mm ID x 15.0 cm L

0023448 TSKgel UP-SW3000, 2 µm Silica Stainless steel 4.6 mm ID x 30.0 cm L

0023450 TSKgel Guardcolumn UP-SW Silica Stainless steel 4.6 mm ID x 2.0 cm L

0023451 TSKgel Guardcolumn UP-SW DC Silica Stainless steel 4.6 mm ID x 2.0 cm L

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TSK-GEL PW Columns

TSK-GEL PW
Brochure

Table of Contents

I. TSK-GEL PW Columns
II. TSK-GEL PW Speciality Columns
III. Troubleshooting and Cleaning of
TSK-GEL PW Columns
TSK-GEL PW Series

HPLC columns for the size separation of biological


macromolecules

TSK-GEL PW columns are designed for aqueous, Size TSK-GEL GMPWXL is the 13 µm high resolution version
Exclusion Chromatography (SEC) of proteins, of TSK-GEL GMPW (an example is shown in Figure 9).
polysaccharides, oligosaccharides, DNA and RNA. The
column packing materials are porous, hydrophilic, rigid, TSK-GEL G-Oligo-PW is specially designed for
polymer beads with particle sizes ranging from 6 µm to oligosaccharides and other aqueous nonionic polar
22 µm. They exhibit excellent chemical and mechanical oligomers of less than 3,000 Daltons. This column is
stability, have been used from pH 2.0 to 12.0, and can based on a 6 µm version of the TSK-GEL G2000PW
be cleaned with 0.5 M NaOH.
packing material (see Figure 3, 7, 6).
They are particularly useful at pH extremes which would
adversely affect silica SEC resins (TSK-GEL SW Series).
Depending on which of the six pore size ranges is TSK-GEL G-DNA-PW is also a specially designed column
selected, these columns have 10,000 to 30,000 plates/m but for the separation of DNA and RNA molecules of less
and are available in both analytical and preparative than 7,000 base pairs and other large molecules (see
housings. Figure 4 and 8).

TSK-GEL PWXL columns are a smaller particle size Protein calibration curves on
version of the TSK-GEL PW polymer packing. These TSK-GEL PWXL columns
columns are used where the highest resolution of sample
peaks is required. There are five pore size ranges
available with chromatographic efficiencies varying 107
from 23,000 to 50,000 plates/m.

TSK-GEL PW specialty columns 106 1 2 5 3 4


Molecular weight (Da)

a
TSK-GEL GMPW is a mixed-bed SEC column comprised
b
of various pore sizes and 17 µm particles. This maximizes 105
the range of molecular weights that will elute between the c
d e
excluded volume and the permeation volume.
f
104
g
Polyethylene glycol and oxide
calibration curves for TSK-GEL PW and
TSK-GEL PWXL columns 103
4 6 8 10 12
Elution volume (mL)
TSK-GEL PW Columns TSK-GEL PWXL Columns
N
Figure 2
106 106
M Column: 1. G3000PWXL, 2. G4000PWXL, 3. G5000PWXL
G
K 4. G6000PWXL, 5. GMPWXL
Molecular weight (Da)

105 105 Sample: a. thyroglobulin, b. y-globulin, c. albumin,


D E F
L d. ovalbumin, e. ß-lactoglobulin,
104 J
104 C f. myoglobin, g. cytochrome C
103
A B 103 H Elution: 0.2 M phosphate buffer, pH 6.8
Flow rate: 1.0 ml/min
102 102 Detection: UV @ 280 nm

10 15 20 5 10 15
Elution volume (mL) Elution volume (mL)

Figure 1
Column: TSK-GEL PW columns: A. G2000PW, B. G2500PW,
C. G3000PW, D. G4000PW, E. G5000PW, F. G6000PW,
G. GMPW, all 7.5 mm L x 60 cm L
TSK-GEL PWXL columns: H. G2500PWXL, J. G3000PWXL,
K. G4000PWXL, L. G5000PWXL, M. G6000PWxL,
N. GMPWXL, all 7.8 mm ID x 30 cm L
Sample: PEO and PEG
Elution: distilled water
Flow rate: 1.0 ml/min
Detection: RI

TSKgel PW Brochure 2 www.tosohbioscience.de


TSK-GEL PW Series

Properties and molecular weight separation ranges for TSK-GEL PW packings

Molecular weight of sample (Da)


TSKgel column Particle Average Polyethylene Dextrans** Globular
Size* (µm) pore Size (Å) glycols & oxides proteins**

G2000PW 12, 17, 20 125 up to 2,000 — <5,000


G2500PWXL 6 <200 up to 3,000 — <8,000
G2500PW 12, 17, 20
G3000PWXL 6 200 up to 5 x 104 up to 6 x 104 500 – 8 x 105
G3000PW 12, 17, 20
G4000PWXL 10 500 2,000–3 x 104 1,000 – 7 x 105 1 x 104 – 1.5 x 106
G4000PW 17, 22
G5000PWXL 10 1000 4,000–1 x 106 5 x 104 – 2.5 x 106 <1 x 107
G5000PW 17, 20, 22
G6000PWXL/BioAssist G6PWXL 13 >1000 4 x 104 – 8 x 106 5 x 105 – 5 x 107 <2 x108
G6000PW 17, 25
GMPWXL 13 <100–1000 500–8 x 106 <5 x 107 <2 x 108
GMPW 17
G-Oligo-PW 6 125 up to 3,000 — <3,000
G-DNA-PW 10 >1000 4 x 104 – 8 x 106 <5 x 107 < 2 x 108

Column: TSK-GEL PW columns, 7.5 mm ID x 60 cm L; TSKgel PWXL, G-Oligo-PW & G-DNA-PW, 7.8 mm ID x 30 cm L
Elution: Polyethylene glycols and oxides: distilled water; dextrans and proteins: 0.2 M phosphate buffer, pH 6.8
Flow Rate: 1.0 mL/min
Note: * Larger particle sizes of each group are for 21.5 mm ID x 60 cm L semi-preparative and 55 mm or 108 mm ID x 60 cm L preparative
columns.
** Maximum separation range determined from estimated exclusion limits.

Table I

Calibration curve for TSK-GEL G-Oligo-PW Calibration curve for double-stranded DNA

8000

4000

2000

104 1000

400
Base Pairs
Daltons

103
100

102

10
20 24 28 32 36
Elution volume (mL)
3 4 5 6 7 8 9 10 11
ml Figure 4
Figure 3 Column: TSKgel G-DNA-PW, four 10 µm, 7.8 mm ID X 30 cm L
Column: TSKgel G-Oligo-PW, 7.8 mm ID X 30 cm L columns in series
Sample: PEG and PEO standards Sample: double-stranded DNA fragments: EcoR I and BstN I
Flow rate: 1.0 ml/min cleaved pBR322 DNA, void volume determined with DNA
Detection: RI Elution: 0.3 M NaCl in 0.1 M Tris-HCI, pH 7.5
Flow rate: 15 ml/min
Detection: UV @ 260 nm

TSKgel PW Brochure 3 www.tosohbioscience.de


TSK-GEL PW Series

Recommended eluents for GFC of water-soluble polymers on TSK-GEL PW type columns

Type of polymer Typical sample Suitable eluent

Nonionic hydrophilic polyethylene glycol distilled water


soluble starch, methyl cellulose, pullulan 0.01 N NaOH
dextran, hydroxyethyl cellulose, 20% DMSO
polyvinyl alcohol, polyacrylamide Buffer or salt solution (e.g., 0.1– 0.5 M NaNO3)

Nonionic hydrophobic polyvinylpyrrolidone Buffer or salt solution with organic solvent


(e.g., 20% CH3CN in 0.1 M NaNO3)

Anionic hydrophilic sodium chondroitin sulfate, sodium alginate, Buffer or salt solution (e.g., 0.1 M NaNO3)
carboxymethyl cellulose, sodium polyacrylate,
sodium hyaluronate

Anionic hydrophobic sulfonated lignin sodium salt, Buffer or salt solution with organic solvent
sodium polystyrenesulfonate (e.g., 20% CH3CN in 0.1 M NaNO3)

Cationic hydrophilic glycol chitosan, DEAE-dextran, poly(ethyleneimine), 0.5 M acetic acid with 0.3 M Na2SO4,
poly(trimethylaminoethyl methacrylate) iodide salt or 0.8 M NaNO3

Cationic hydrophobic poly(4-vinylbenzyltrimethylammonium chloride), 0.5 M acetic acid with 0.3 M Na2SO4
poly(N-methyl-2-vinylpyridinium) iodide salt

Amphoteric hydrophilic peptides, proteins, poly-and oligosaccharides, Buffer or salt solution (e.g., 0.1 M NaNO3)
DNA, RNA

Amphoteric hydrophobic blue dextran, collagen, gelatin, hydrophobic Buffer or salt solution with organic solvent
proteins, hydrophobic peptides (e.g., 20% CH3CN in 0.1 M NaNO3 or
35– 45% CH3CN in 0.1% TFA)
Table II

Chaotropic Agents
PW Columns are compatible with*:
8 M Urea
Up to 50% Polar Organics 6 M Guanidine

Acetonitrile Not Compatible


Acetone
Isopropanol Toluene (not water soluble)
Methanol and other not water soluble solvents
Ethanol
DMF
DMSO

Up to 20% Nonpolar Organics

THF

*Switch to these buffers at 50% of the standard


flow rate.

Detergents

up to 0.1% SDS
up to 1% Tween, Triton

TSKgel PW Brochure 4 www.tosohbioscience.de


TSK-GEL PW Series

Elution curves for PEG standards on Separation on hydrolyzed ß-cyclodextrin


TSK-GEL G25000PWXL 1

8
6
1 PEG 7500
2 3000 2
3 2000
2
7 4 1000
5 600 3
3 7
6 400
7 200
45 4
8 ethylene glycol 6
5

5 10
15 20
Elution volume (ml)
Minutes
Figure 5
Column: TSKgel G2500PWXL, 7.8 mm ID X 30 cm L Figure 7
Sample: polyethylene glycols Column: TSKgel G-Oligo-PW, two 6 µm, 7.8 mm ID X 30 cm L
Elution: destilled water columns in series
Flow rate: 1.0 ml/min Sample: hydrolyzed ß-cyclodextrin
Detection: RI Elution: destilled water
Flow rate: 1.0 ml/min
Temperature: 60 °C
Separation of chito oligosaccharides Detection: RI

Separation of EcoR I and BstN I


cleaved pBR322 DNA
928, 1060
1857
4362

383

121

13

25 30 35
Minutes 6 9 12 15
Minutes
Figure 6
Column: TSKgel G-Oligo-PW, four 7.8 mm ID X 30 cm L in series Figure 8
Sample: chito oligosaccharides Column: TSKgel G-DNA-PW, four 10 µm, 7.8 mm ID X 30 cm L
Elution: destilled water in series
Detection: RI Sample: 1.7 µg of EcoR I cleaved pBR322 DNA and
8.0 µg of BstN I cleaved pBR322 DNA
Elution: 0.3 M NaCl in 0.1 Tris-HCI, pH 7.5 and 1 mM EDTA
Flow Rate: 0.3 ml/min
Detection: UV @ 260 nm

TSKgel PW Brochure 5 www.tosohbioscience.de


TSK-GEL PW Series

Separation of pullulan Separation of gelatin

G6000PWXL

10 15 20 G4000PWXL

Minutes 0 10 20
Minutes
Figure 9 TSKgel GMPWXL, four 7.8 mm ID x 30 cm L
umn:
Column:columns
TSKgelinGMPWXL,
series four 7.8 mm ID X 30 cm L Figure 10
mple: pullulan
in series Column: TSKgel G4000PWXL
ion: 0.1 M NaCl
wSample:
Rate: 0.1 pullulan
ml/min TSKgel G6000PWXL
ection:
Elution: RI, 0.1
LS M NaCl Sample: gelatin
Flow Rate: 1.0 ml/min Elution: 0.2 mM phosphate buffer, pH 6.9
Detection: RI, LS Flow Rate: 1.0 ml/min
Detection: RI

Troubleshooting and cleaning


TSK-GEL PW columns

Selecting mobile phase buffers

In an ideal SEC separation, the mechanism is purely Modifiers are used for proper elution of both charged and
sieving, with no chemical interaction between the column neutral hydrophobic polymers. Typical examples for a
matrix and the sample molecules. In practice, however, a variety of sample types are given in Table II. All TSK-GEL
small number of weakly charged groups on the surface of PW-type column packings are compatible with up to 50%
all TSK-GEL PW type packings can cause changes in aqueous solutions of methanol, ethanol, propanol,
elution order from that of an ideal system. Fortunately, the acetonitrile, formic acid, acetic acid, dimethyl formamide,
eluent composition can be varied greatly with TSK-GEL dimethyl sulfoxide, or acetone. Solvent exchange must be
PW columns, to be compatible with a wide range of carried out slowly.
neutral, polar, anionic, and cationic samples. Table II lists
appropriate eluents for GFC of all polymer types on TSK- Improving column performance
GEL PW type columns.
Listed below are the four most common causes for poor
For some nonionic, nonpolar polymers, such as poly- column performance and the methods you
ethylene glycols, normal chromatograms can be obtained can use to prevent these problems:
by using distilled water. Some more polar nonionic
polymers exhibit abnormal peak shapes or minor peaks 1. Void or dead volume at the column inlet, channeling,
near the void volume when eluted with distilled water, denaturation of the packing surface
due to ionic interactions between the sample and the
charged groups on the resin surface. To eliminate ionic Sudden pressure surges and higher than recommended
interactions, a neutral salt, such as sodium nitrate or flow rates can compress the column packing and cause a
sodium sulfate, is added to the aqueous eluent. Generally, void. We recommend continuous flow injection with a
a salt concentration of 0.1 M to 0.5 M is sufficient to loop injector and installation of a pulse damper to
overcome undesired ionic interactions. suppress the sudden pressure surges encountered with
quick return pumps. Bulk packing is available to refill
TSK-GEL PW-type resins are more hydrophobic than voids in the analytical and semi-preparative columns. A
polysaccharide gels such as cross-linked dextran. The guard column will protect your analytical column from
hydrophobic interaction increases as the salt concentration pressure surges and irreversibly binding contaminants.
of the eluent increases. This hydrophobic interaction Upon injection, the sample can cause a change in the
between the sample and the resin surface can be eluent pH. The guard column will allow the pH to
suppressed by the addition of a water-soluble organic equilibrate with the mobile phase before it reaches
solvents modifier (e.g. acetonitrile, isopropanol). the analytical column.

TSKgel PW Brochure 6 www.tosohbioscience.de


TSK-GEL PW Series

2. Air on the column 2. If the column is contaminated with hydrophobic


compounds, rinse with 3-5 CV of 20% organic methanol,
The column should be tightly capped when not in use. acetonitrile, THF etc.)
Mobile phases should be sparged to prevent air from 3. For precipitated protein, chaotropic reagents such as
entering the column. If air does enter the column, follow 0.1 % SDS, 8 M urea or 6 M guanidine, or proteolytic
the rehydration method. enzymes such as pepsin may be used. However, an
extended washing with buffer is required to remove
3. Column contamination or incomplete recovery of SDS and guanidine.
the sample
Unexpected elution behavior can occur if these
The cleaning directions which follow give suggestions for reagents are not completely removed.
removing ionic, hydrophobic or absorbed contaminants. Rinse with 5 CV of D. l. water after each cleaning step
Refer to “Selecting mobile phase buffers” for advice on before proceeding to the next step.
modifiers that will prevent nonspecific binding to the
media matrix. Storage

4. Frit plugging and high pressure When the column will be used the next day, allow it to
run overnight at a low flow rate in a buffer that does not
Solvents and samples should be filtered though a 0.45 µm contain a halide. When the column will not be used for
filter to prevent clogging of the column frits. If the frit more than a day, flush salt from the column and store in
becomes partially plugged, the result may be split peaks 0.05% sodium azide or 20% ethanol. Seal tightly to
or high pressure. The entire end-fitting can be removed prevent drying out.
and sonicated in 6 M nitric acid. Be careful not to disturb
the packing and rinse well after cleaning. Alternatively, we Rehydration
stock replace-ment end-fittings according to the column
ID. Furthermore, we recommend placing an in-line filter Dehydration of the column may occur after long term
before the injector, to prevent particles created by pump storage or from inadvertently pumping air over a column.
seal wear from reaching the analytical column. Rehydrate the column with the following procedure:

5. Column overload 1. Connect the column to your LC pump, but not to the
detector, in reverse flow direction.
Column overload can cause peak splitting and poor
resolution. Analytical work necessitates an injection 2. Pump a filtered mobile phase of 20% methanol in
volume less than 1% of the total column volume with distilled, deionized water over the column at half the
a sample concentration less than 10 mg/ml. recommended maximum flow rate listed in the ordering
information for your column.
Improving resolution
3. Continue this procedure for several hours until you
1. Verify that the column is not being overloaded. are confident that the column has been rehydrated.

2. Decrease the dead volume in your HPLC system by 4. Reconnect the column to your LC in the proper flow
using the shortest tubing lengths and the smallest tubing direction.
ID possible without exceeding the maximum pressure for
the column. 5. Rinse well with distilled, deionized water then
equilibrate with your normal mobile phase.
3. Decrease the flow rate, but not lower than 0.3 ml/min
because diffusion will increase. Perform the recommernded QC test to ensure that
the column is performing properly.
4. If using a 30 cm PW column, add an additional 30 cm
column or switch to an XL-type column which has a
smaller particle size and will improve resolution more
than two fold.

Cleaning

If the column becomes contaminated, the following


cleaning procedures are suggested with the column in
reverse flow at half the recommended maximum flow
rate (see also ODS-sheet that comes with the column
or the TSK-GEL Instruction Manual):

1. If the column is contaminated with basic com-pounds,


rinse with 3-5 column volumes (CV) of 0.5-1.0 M neutral
salt or a buffered solution at low pH (pH 2-3) or high pH
(0.1-0.2 N NaOH,pH 13.0).

TSKgel PW Brochure 7 www.tosohbioscience.de


TSK-GEL PW Series

Ordering Information
Analytical and preparative TSK-GEL Size Exclusion polymer-based column products: typical properties

Part # Description ID Length Particle Min. Number Flow Rate (mL/min) Maximum
(mm) (cm) Size (µm) Theoretical Range Max. Pressure
Plates Drop (kg/cm2)
Stainless steel columns
08031 G-Oligo-PW, 125 Å 7.8 30 6 14,000 0.5 – 0.8 1.0 40
08032 G-DNA-PW, >1.000 Å 7.8 30 10 10,000 0.2 – 0.5 0.6 20

08020 G2500PWXL, <200 Å 7.8 30 6 14,000 0.5 – 0.8 1.0 40


08021 G3000PWXL, 200 Å 7.8 30 6 14,000 0.5 – 0.8 1.0 40
08022 G4000PWXL, 500 Å 7.8 30 10 10,000 0.3 – 0.8 1.0 20
08023 G5000PWXL, 1.000 Å 7.8 30 10 10,000 0.3 – 0.8 1.0 20
08024 G6000PWXL, >1.000 Å 7.8 30 13 7,000 0.3 – 0.8 1.0 20
08025 GMPWXL, 100-1.000 Å 7.8 30 13 7,000 0.3 – 0.8 1.0 20

05761 G2000PW, 125 Å 7.5 30 12 5,000 0.5 – 1.0 1.2 20


08028 G2500PW, <200 Å 7.5 30 12 5,000 0.5 – 1.0 1.2 20
05762 G3000PW, 200 Å 7.5 30 12 5,000 0.5 – 1.0 1.2 20
05763 G4000PW, 500 Å 7.5 30 17 3,000 0.5 – 1.0 1.2 10
05764 G5000PW, 1.000 Å 7.5 30 17 3,000 0.5 – 1.0 1.2 10
05765 G6000PW, >1.000 Å 7.5 30 17 3,000 0.5 – 1.0 1.2 10
08026 GMPW, 100-1.000 Å 7.5 30 17 3,000 0.5 – 1.0 1.2 10

05105 G2000PW, 125 Å 7.5 60 12 10,000 0.5 – 1.0 1.2 40


08029 G2500PW, <200 Å 7.5 60 12 10,000 0.5 – 1.0 1.2 40
05106 G3000PW, 200 Å 7.5 60 12 10,000 0.5 – 1.0 1.2 40
05107 G4000PW, 500 Å 7.5 60 17 6,000 0.5 – 1.0 1.2 20
05108 G5000PW, 1.000 Å 7.5 60 17 6,000 0.5 – 1.0 1.2 20
05109 G6000PW, >1.000 Å 7.5 60 17 6,000 0.5 – 1.0 1.2 20
08027 GMPW, 100-1.000 Å 7.5 60 17 6,000 0.5 – 1.0 1.2 20

05150 G2000PW, 125 Å 21.5 60 17 10,000 1.0 – 6.0 8.0 20


08030 G2500PW, <200 Å 21.5 60 17 10,000 1.0 – 6.0 8.0 20
05151 G3000PW, 200 Å 21.5 60 17 10,000 1.0 – 6.0 8.0 20
05152 G4000PW, 500 Å 21.5 60 22 6,000 1.0 – 6.0 8.0 20
05153 G5000PW, 1.000 Å 21.5 60 22 6,000 1.0 – 6.0 8.0 20
05154 G6000PW, >1.000 Å 21.5 60 25 6,000 1.0 – 6.0 8.0 20

07926 G3000PW, 200 Å 55.0 60 20 4,500 15.0 – 25.0 30.0 15


07927 G5000PW , 1.000 Å 55.0 60 20 4,500 15.0 – 25.0 30.0 15

Guard columns
08034 Oligo Guard column 6.0 4.0 13 For G-Oligo-PW
08033 PWXL Guard column 6.0 4.0 12 For 7.8 mm ID PWXL and G-DNA-PW
(contains 3000PW packing)
06763 PW-L Guard column 7.5 7.5 13 For 7.5 mm ID G2000PW, (contains 2000PW packing)
06762 PW-H Guard column 7.5 7.5 13 For 7.5 mm ID G2500PW-G6000PW + GMPW
(contains 3000PW packing)
06757 PW-L Guard column 21.5 7.5 17 For 21.5 mm ID G2000PW
06758 PW-H Guard colum 21.5 7.5 17 For 21.5 mm ID G2500PW through G6000PW
07924 PW Guard colum 45.0 5.0 20 For 55 mm ID G3000PW + G5000PW

Bulk packing
08035 PWXL Top-Off, 1g wet resin 10 For all PWXL and G-DNA-PW

TSKgel PW Brochure 8 www.tosohbioscience.de


B06L10A
ProductFlyer_PWXL-CP Kopie 2 18.03.2008 11:28 Uhr Seite 1

TSK-GEL® PWXL-CP
Size Exclusion Chromatography Columns for Cationic Column Recovery
Polymer Analysis G3000 PWXL-CP 100,2%

PRODUCT HIGHLIGHTS G5000 PWXL-CP 98,8%

G6000 PWXL-CP 97,4%


High recoveries for cationic polymers (see
Table 2) TABLE 2
High reproducibility over time without adsorp-
tion (see Figure 1) These columns show high theoretical plate values, linear
Column offering allows separation of wide calibration curves and high durability because the base
MW range of polymers resin is the same as that used in the TSK-GEL PWXL
Elution under low salt conditions series columns.

INTRODUCTION PAA was injected onto a TSKgel G5000PWXL-CP column.


Each chromatogram, from the first injection (red) to the
TSK-GEL PWXL-CP size exclusion columns were specifi- fifth injection (black), showed similar elution profiles wit-
cally developed for the analysis of water soluble cationic hout any adsorption of the polymer.
polymers. Three columns are available within the TSK-
180
GEL PWXL-CP series, each with a different particle size,
separation range and exclusion limit, allowing polymers
within a wide molecular mass range to be separated and 140
characterized.
100
The analysis of cationic polymers requires a high salt
mV

concentration in the mobile phase to prevent adsorption


60
of the polymers onto the particles in SEC columns. As a
result, many polymer researchers encounter low reco-
very when analyzing cationic polymers, as well as poor 20
reproducibility from run to run.
-20
The TSK-GEL PWXL-CP columns eliminate ionic 3 5 7 9 11 13
adsorption onto the particle by incorporating a cationic Elution time (min)
functionality on the particle surface. This modification FIGURE 1
results in high recovery for cationic polymers and enab- Column: TSKgel G5000PWXL-CP
Eluent: 0.1 mol/l NaNO3
les elution under low salt conditions.
Flow rate: 1.0 ml/min
Detection: RI
PROPERTIES OF TSK-GEL PWXL-CP COLUMNS Temperature: 25°C
Sample: polyallyamine-HCI (PAA)
Sample load: 3 g/l, 100 µl
G3000 PWXL-CP G5000 PWXL-CP G6000 PWXL-CP

Base material Poly Poly Poly


methacrylate methacrylate methacrylate

Particle size 7 µm 10 µm 13 µm

Exclusion limit 100 000 1 000000 20 000 000


(Da)

Separation range 200 - 50 000 400 - 500 000 1 000 - 10 000 000
(Da), (PEO, PEG)

Theoretical plates 16 000 10 000 7 000

TABLE 1

ZETTACHRING 6 70567 STUTTGART GERMANY T: +49 (0)711 13257-0 F: +49 (0)711 13257-89 INFO.SEP.EU@TOSOH.COM WWW.TOSOHBIOSCIENCE.DE
WWW.TSKGEL.COM MEMBER OF THE TOSOH GROUP
ProductFlyer_PWXL-CP Kopie 2 18.03.2008 11:28 Uhr Seite 2

Application

Various cationic polymers with different functional


groups and molecular weights were injected on the TSK-
GEL PWXL-CP columns (TSKgel G6000PWXL-CP,
G5000PWXL-CP and G3000PWXL-CP, connected in
series). Figure 2 demonstrates that these new SEC
columns can be utilized for the analysis of a wide variety
of cationic polymers.

FIGURE 2

Column: TSKgel G3000PWXL-CP, 7 µm,


7.8 mm ID X 30 cm L
TSKgel G5000PWXL-CP, 10 µm,
7.8 mm ID X 30 cm L
TSKgel G6000PWXL-CP, 13 µm,
7.8 mm ID X
30 cm L
Eluent: 0.1 mol/l NaNO3
Flow rate: 1.0 ml/min
Detection: RI
Temp.: 25°C
Sample load: 3 g/l, 100 µl

For further details of choice and selection of the TSK-GEL® column


that best suits your particular process purification needs,
please contact us:

Tel. + 49 (0) 711 13257 0


or
info.sep.eu@tosoh.com
or
www.tskgel.com

Ordering information
TSKgel PWXL-CP

Part-No Description Matrix Housing Dimensions


21873 TSKgel G3000PWXL-CP, 7 µm, 200 Å Polymer Stainless steel 7.8 mm ID x 30 cm L
21874 TSKgel G5000PWXL-CP, 10 µm, 200 Å Polymer Stainless steel 7.8 mm ID x 30 cm L
21875 TSKgel G6000PWXL-CP, 13 µm, 200 Å Polymer Stainless steel 7.8 mm ID x 30 cm L
21876 TSKgel PWXL-CP Guardcolumn Polymer Stainless steel 6.0 mm ID x 4.0 cm L

F08L05A
Column Flyer
NEW universal steric exclusion columns

New high performance NEW SEC columns and capillaries


polymeric analytical
NEW High resolution columns
columns for SEC and GPC

Take advantage of the versatile and rigid


TSKgel SuperAW columns. Speed up your
method development and let the column do
what YOU want!
Introduction The different pore sizes of the resins allow for a wide
separation range of 100-1.000.000 Da using Polyethylene
Size exclusion chromatography (SEC) as a method to oxides (PEO), and, in addition, each column type shows
separate compounds by their molecular sizes is widely a high degree of linearity considering the specific cali-
used for the separation, purification and determination bration range for the respective column.
of molecular weight distributions of hydrophilic as well
as of hydrophobic substances. Figure 1 Calibration curves of PEO and PEG in water

Usually hydrophobic compounds are analyzed with


organic solvents on a polystyrene-divinylbenzene
matrix (represented by the H-type column series),
whereas SEC of polar compounds needs aqueous sol-
vents and a hydrophilic matrix, based either on silica
(the SW and SWXL series) or on polymethacrylate
(represented by the PW and PWXL series).
Analysis of polar, non water soluble macromolecules
always was difficult on both resin types, due to their
limited solvent compatibility.

Table 1 Solvent Compatibilities for TSK-GEL SEC Matrices

Water/aqueous solution polar organic solvent non-polar rganic solvent


Aqueous buffer systems Methanol/Ethanol/ THF/Acetone/Methylene
Isopropanol chloride* Chloroform*/
Acetonitrile/DMSO/DMF Toluene*/Hexane*

PW, PWXL

SW, SWXL, Super SW


Column: 6.0 mm ID x 15 cm L
Eluent: Water
H and HXL Flow rate: 0.6 ml/min
Temp.: ambient
HHR Detect.: RI
Sample: Standard polyethylene oxide, polyethylene
glycol, ethylene glycol
Sample conc.: 0.04-0.1% each
Alpha, SuperAW

*for H-type columns only Figure 2 Calibration curves of PEO and PEG in DMF

7
SuperAWM-H
With the introduction of Alpha-Series, and now the SuperAW6000
6
TSKgel SuperAW column series, this restriction was SuperAW5000
overcome due to a resin based on a hydrophilic, highly SuperAW4000
crosslinked vinyl polymer. This resin, which is solvent 5
SuperAW3000
Log MW

compatible from pure aqueous up to 100 % organic


SuperAW2500
mobile phases*1), enables SEC of various polymers 4
soluble either in aqueous solutions or in organic solvents.
3
Table 2 Features of TSKgel Super AW columns

1
Grade Exclusion limit Particle Theoretical Dimension
(PEO/DMF) size (µm) Plates (mm ID x cm L)
1 2 3 4
Elution volume (ml)
TSKgel SuperAW2500 2 x 103 4 > 16,000 6.0 x 15
Column: 6.0 mm ID x 15 cm L
TSKgel SuperAW3000 6 x 104 4 > 16,000 6.0 x 15
Eluent: 10mM LiBr in DMF
Flow rate: 0.6 ml/min
TSKgel Super AW4000 4 x 105 6 > 10,000 6.0 x 15 Temp.: 40 °C
Detect.: RI
TSKgel SuperAW5000 4 x 10 6 7 > 10,000 6.0 x 15 Sample: Standard polyethylene oxid, polyethylene glycol,
ethylene glycol
TSKgel SuperAW6000 > 4x 107 9 > 6,000 6.0 x 15 Sample conc.: 0.04-0.1% each
Inj. Vol.: 10 µl
TSKgel SuperAWM-H > 4 x 107 9 > 6,000 6.0 x 15

The combination of small particle sized resins and short


column dimensions results in a superior separation per-
formance of TSK-GEL SuperAW columns compared to
New TSKgel SuperAW column series conventional SEC-columns.

The TSKgel SuperAW column series consists of six As demonstrated in the plot in figure 3, showing column
SEC-column types. The analytical columns are packed performance in relation to flow rates, HETP values are
with a micro-particle sized, mechanically stable smaller and less dependent on flow rate than with
hydrophilic polymer with different pore sizes. conventional columns.
*1) Exception: Chloroform, Toluene, Hexane
Figure 3 Plots of HETP vs. linear velocity Figure 5 Analysis of Polyacrylonitrile in DMF

mV

15

-5
4 8 12 min
Column: TSKgel SuperAWM-H (6.0 mm ID x 15 cm L,
two in series)
Columns: TSKgel SuperAW2500 (6.0 mm ID x 15 cm L) Eluent: DMF containing 10mmol/L LiBr
TSKgel G2500PWXL (7.8 mm ID x 30 cm L) Flow rate: 0.6mL/min
Eluent: Water Temperature: 40 °C
Temp.: ambient Detection: Refractive index detector
Detect.: RI Sample load: 20µL (0.5g/L)
Sample: Ethylene glycol, 2.5g/l
Inj. Vol.: 5 µl (Super AW2500)
20 µl (G2500PWXL) Separation by Non-SEC mode

This, in practice means that the 15 cm TSKgel SuperAW The excellent solvent compatibility of the TSKgel
column provides almost the same theoretical plate SuperAW also enables analysis of small or complex
number than conventional SEC-columns with 30 cm compounds in different Non-SEC-modes. As shown
length, enabling shorter analysis times followed by in figure 7 different chromatograms can be obtained
reduced solvent consumption. from a surfactant by changing the eluent from 35% of
acetonitrile to 100% of acetonitrile. The sample is
Figure 4 Performance Comparison of TSKgel SuperAW2500 vs. separated based on molecular size with 60% acetoni-
TSKgel G2500PWXL
trile and it is retained on the column in other eluent
30 compositions based on either hydrophobic or ionic
SuperAW2500
interactions.
25
G2500PWXL Thus it is possible to set up different elution conditions
to suit the purpose of measurement in one column.
20
RI (mV)

15
Figure 6 Separation of Triton X-100 by non-SEC mode

10 (mV)

35 % Acetonitrile
5 109
60 % Acetonitrile
100 % Acetonitrile
0
2 4 6 8 10 12

Elution time (min)


Column: TSKgel SuperAW2500 (6.0 mm ID x 15 cm L) 79
Eluent: Water
Sample: hydrolysate of dextran T-40
Flow rate: 0.6 ml/min
Sample load: 5 µl
Column: TSKgel G2500PWXL (7.8 mm ID x 30 cm L)
Flow rate: 1.0 ml/min
Sample load: 10 µl
49
4 14 24 34
Applications (min)

Column: TSKgel SuperAW2500 (6.0 mm ID x 15cm L,


Various applications of the TSKgel SuperAW columns two in series )
are available on request. As an example the analysis Sample: Triton X-100
Eluent: dif. Acetonitrile / Water solutions
of Polyacrylonitrile in DMF containing 10 mmol Lithium
Flow rate: 0.6 ml/min
is shown (Figure 5). Temperature: 40°C
Detection: UV 280nm
Injection volume: 20 µl
Sample concentr.: 1.0 g/l
Conclusions

For further details of choice


■ TSKgel SuperAW columns are allround SEC
and selection of the
columns for initial analysis of unknown samples
suitable TSK-GEL column
■ Hydrophilic resin with little swelling and
that best meets
shrinkage is compatible to wide range of
solvent (water to organic solvent) your particular needs.
please contact us:
■ Rigid resin with excellent mechanical stability
leads to pressure stablility up to 600psi = 4.0Mpa
Tel.: +49 (0)711 132570
■ Micro-particle gel packed into analytical col-
umns allows short analysis times and high or
resolution power
info.sep.eu@tosoh.com
■ Five pore sizes and a mixed-bed enable a wide or
separation range
www.tosohbioscience.com
■ Separations possible by SEC, non-SEC or mixed
mode allow to optimize the chromatographic
method to analytical needs

Photocopy this page and fax to + 49 (0) 711 132 5789


Laboratory Catalogue
Name

Company Brochure
Title

Price List Company / Institution

Department
Column Selection Guide
on CD-ROM NEW! Address / Building

TSKgel SuperAW
City / Post Code / Country
Separation Report
Tel.

Fax

E-mail

Tosoh Bioscience GmbH


Tosoh Bioscience GmbH, Zettachring 6
70567 Stuttgart, Germany
Tel. + 49 (0) 7 11 1 32 57-0, Fax + 49 (0) 7 11 1 32 57-89
info.sep.eu@tosoh.com
www.tosohbioscience.com

Member of the TOSOH Group

F06L02A
SW cover.qxd 05.10.2007 17:21 Uhr Seite 1

TOSOH BIOSCIENCE
Separations Business Unit

Size Exclusion with TSKgel ® SuperSW

TOSOH BIOSCIENCE
TOSOH BIOSCIENCE GmbH
Zettachring 6
70567 Stuttgart, Germany
Phone: +49 (0) 711 13257-0, Fax: +49 (0) 711 13257-89
info.sep.eu@tosoh.com, www.tosohbioscience.de
www.tskgel.com

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Size Exclusion
Chromatography!
SW cover.qxd 05.10.2007 17:21 Uhr Seite 2

Why we are different Notes to be made in using TSKgel SuperSW series

Tosoh Bioscience is a global leader in the field of bioseparations


Tosoh Bioscience provides comprehensive technical and regulatory support
■ Reduce peak broadening in tubing, detector, etc.
Tosoh Bioscience offers the broadest range of columns for aqueous and organic SEC
■ Take care of sample overloading
Tosoh Bioscience is the leading manufacturer of silica based SEC columns
■ Take care of flow rate of pumping system since the required flow rate is low

Size Exclusion Chromatography


Tubing:
Sample typical ■ Use 0.1 mm ID tubing. It is recommended that the total tubing length is 100 cm or shorter
application chromatogram
■ Connection pipe set type L (product no. 018186; 0.1 mm ID x 40 cm L, 2 pieces) available,
buffer/low salt
concentration connection surface (both ends) with fine-cut finishing

■ Sections requiring 0.1 mm ID tubing

a) Between injection valve/column inlet, or auto-sampler/column inlet

b) Between column outlet/detector inlet (tubing on inlet side of the detector)

Pumping system:
■ Pumping system should be applicable to semi-micro HPLC
Elution
Size Exclusion Chromatography
■ Flow rate should be 0.01 - 0.35 ml/min
buffer/low salt (SEC)
concentration
is the general name for the chromato-
graphic mode also referred to as gel Injector:
permeation chromatography (GPC)
for non-aqueous elution systems or ■ Low diffusion type injector (Reodyne 8125) is recommended
gel filtration chromatography (GFC)
for aqueous systems. SEC is a
method in which components of a
mixture are separated according to Guard column:
their molecular size, based on the
flow of the sample through a porous ■ Be sure to connect an in-line filter or a guard column (product no. 18762) to protect the
packing. Large biomolecules that
cannot penetrate the pores of the column (A set of connection tubing is a standard accessory to the guard column)
packing material elute first from the
column. These large biomolecules
are said to be excluded from the
packing; they flow with the mobile
phase in the interparticle space of
Detector:
the packed column. Smaller mole-
cules can partially or completely
■ For UV detectors, use micro flow cells or low dead volume type cells. Low dead volume
enter the packing particles. Because
these smaller molecules have to flow type cells are effective in high-sensitivity analysis. Use of standard cell is also possible
through the interparticle space, as
well as through the pore volume, for 4.6 mm ID columns. However, theoretical plates will be reduced.
they will elute from the column after
the excluded sample components.
TSK-GEL® Columns for SEC SEC is a very simple method for
separating biomolecules, because it Sample:
TSKgel SW-Series is not necessary to change the com-
position of the mobile phase during ■ Sample injection volume should be 1 - 10 µl. sample load should be 100 µg or smaller.
TSKgel PW-Series elution. However, the separation
TSKgel Alpha-Series capacity of this method is limited. For
a baseline separation it is necessary If help is needed, contact our technical support specialists
TSKgel SuperAW-Series that the molecular weights of the bio-
to offer you assistance at +49 (0)711 13257-0.
TSKgel H-Series molecules differ at least 10 to 20 %.
Table 8
SuperSW brochure 28.11.2007 12:38 Uhr Seite 1

High resolution SEC with TSKgel SuperSW

Features & Benefits of TSKgel SuperSW columns

4 µ m particle size featuring superior resolution and highest sensitivity


Lo w non-specific adsorption
High reproducibility due to w ell-defined pore size distribution
> 30.000 theoretical plates/colu m n (4.6 m m ID)
Microbore colu m ns for increased sensitivity and reduced buffer consu m ption

Size exclusion chromatography (SEC) TSKgel S W-type packings feature lo w adsorption


and w ell-defined pore size distribution. It is the
SEC separates m olecules based on their size, or leading SEC colu m n series for HPLC due to its
m ore precisely, their hydrodyna mic volu m e. It is excellent resolution.
usually applied to large m olecules such as proteins
or industrial poly m ers. W hen an aqueous eluent is High resolution SEC
used, SEC is also referred to as gel filtration
chro m atography (G FC). Speed and resolution is an increasing de m and in
liquid chromatography. The need for high sensitivity
SEC is a w ell-kno w n technique for the separation applicable to trace analysis, is increasing as sam ple
and purification of biopoly m ers because of its size or sa m ple concentration beco m e limited. To
effectiveness and non-denaturing m obile phase m eet the needs of high sensitivity and high
conditions. It is popular a m ong bioche mists for resolution protein analysis Tosoh Bioscience
the isolation of proteins, re m oval of aggregates, developed TSKgel SuperS W colu m ns packed with
desalting or characterization of w ater-soluble 4 µ m spherical silica particles. Co m pared to the
poly m ers used in food products, paints, pharm a- w ell established TSKgel S W XL (5 µ m) colu m ns,
ceutical form ulations and the like. W hile soft SuperS W colu m ns sho w higher resolution due to
packing m aterials such as dextran or agarose w ere a 50 percent increase in theoretical plate nu m bers
e m ployed as stationary phases for early G FC, (Table 1).
porous silica particles with high m echanical
Co m parison of TSKgel SuperS W3000 and
strength also have co m e to be e m ployed for SEC Dextran/A garose-type resin
in high perform ance liquid chro m atography (HPLC).
100
TSKgel SuperS W3000
Tosoh Bioscience TSKgel S W and S W XL series are
80
silica SEC phases with pore size distributions suited
to protein separations. A hydrophilic diol-type
60
bonded phase shields the silica surface fro m
Intensity

interacting with protein sa m ples.


40

4 5
Co m petitor product
Particle Colu m n size G uaranteed 20 2 3
1
size theoretical
(µ m) plates
0
0 10 20 30 40 50 60 70
TSKgel 4 4.6 m m ID 30,000 (min)
SuperS W2000 x 30 cm L
Figure 1
TSKgel 4 4.6 m m ID 30,000 Column: A. TSKgel SuperSW3000, 2 mm ID x 30 cm L;
SuperS W3000 x 30 cm L B. Competitor product, 3.2 mm ID x 30 cm L
Sample: 0.2 µL, 1. thyroglobulin, 1.0 mg/mL; 2. γ-globulin, 2.0 mg/mL;
TSKgel 5 7.8 m m ID 20,000 3. ovalbumin, 2.0 mg/mL; 4. ribonuclease A, 3.0 mg/mL;
G2000S W XL x 30 cm L 5. p-aminobenzoic acid, 0.02 mg/mL
Flow rate: A. 65 µL/min N = 30.000; B. 40 µL/min N = 11.000
TSKgel 5 7.8 m m ID 20,000 Temperature: 25°C
G3000S W XL x 30 cm L Detection: UV @ 280 nm

Table 1 Specifications of TSKgel SuperSW series compared to TSKgel SWXL


series

TSKgel SuperSW columns 1 www.tskgel.com


SuperSW brochure 28.11.2007 12:38 Uhr Seite 2

TSKgel SuperSW Series

Co m pared to polysaccharide based gel filtration Figure 2 de m onstrates the superior sensitivity
m edia the increase in resolution, sensitivity and reached with TSKgel SuperS W3000 co m pared to
speed is even higher. Figure 1 co m pares the a TSKgel G3000S W XL colu m n of the sa m e length
separation of a protein standard on a dextran/ but larger inner dia m eter. TSKgel SuperS W can
agarose resin to a TSKgel SuperS W3000 colu m n. yield peak heights approxim ately 4 tim es that of
TSKgel S W XL due to do w nsizing in colu m n
Increased detection limits dia m eter and increased theoretical plates. Table 2
sho ws the detection limits for m ajor proteins. The
To further im prove perform ance, TSKgel SuperS W high sensitivity allo ws for analysis of nanogra m
m edia are packed into colu m ns with sm aller inner sa m ple a m ounts.
dia m eter (1, 2, 4.6 m m ID). The sm aller colu m n
dia m eters are one reason for increased peak Separation range of TSKgel SuperSW series
heights. In addition, the high resolution of the
4 µ m TSKgel SuperS W resins and accordingly the TSKgel SuperS W colu m ns are available in tw o
sm aller peak widths further increase peak height, pore sizes, 125 Å (TSKgel SuperS W2000) and
provided the HPLC syste m is optimized with regard 250 Å (TSKgel SuperS W3000) covering different
to dead volu m e. separation ranges. Table 3 sho ws the separation
Co m parison of TSKgel SuperS W3000 and G3000S W XL ranges for polyethylene glycol (PE G), dextran and
for the separation of proteins typical proteins. Figure 3 shows the SEC calibration
curves of TSKgel SuperS W series for standard
A. G3000SWXL B. SuperSW3000
5
proteins.
Rs: 10.7
50 50
4 M olecular w eight separation range
40 40 Rs: 4.0
Rs: 3.5
TSKgel SuperS W2000 TSKgel SuperS W3000
30 30 3
2
Rs: 3. 0
Rs: 8. 4 5
Polyethylene glycol 500 - 15,000 1,000 - 35,000
20 Rs: 3.3 4 20
3 1
Dextran 1,000 - 30,000 2,000 - 70,000
2
10
1 10 Protein 5,000 - 150,000 10,000 - 500,000
0 0
3 5 7 9 11 13 15 3 5 7 9 11 13 15
Minutes Minutes
Table 3 Molecular weight separation range of TSKgel SuperSW series

Figure 2
Column: A.TSKgel G3000SWXL, 7.8 mm ID x 30 cm L; Protein calibration curves for TSKgel SuperS W
B. TSKgel Super SW3000, 4.6 mm ID x 30 cm L 10.000.000
Sample: 5 µL of a mixture of 1. thyroglobulin, 0.5 mg/mL (660,000 Da);
Protein
2. γ-globulin, 1.0 mg/mL; (150,000 Da); 3. ovalbumin, 1.0 mg/mL
(43,000 Da); 4. ribonuclease A, 1.5 mg/mL (12,600 Da); 1.000.000
1
5. p-aminobenzoic acid, 0.01 mg/mL (137 Da) TSKgel SuperS W3000
Elution: 0.1 M Na2SO4 in 0.1 M phosphate buffer with 0.05% NaN3, pH 6.7 2

Flow rate: 1.0 mL/min for G3000SWXL; 0.35 mL/min for SuperSW3000 3
100.000
Temperature: 25°C 4
Detection: UV @ 220 nm
M olecular w eight

10.000 TSKgel SuperS W2000 6


TSKgel SuperS W TSKgel G3000S W XL
Flo w cell Standard cell Micro flo w Standard cell
1.000
(lo w dead cell (lo w dead
volu m e type) volu m e type) 7

Light path 100


length 10 m m 4 mm 10 m m

Thyroglobulin 70 ng 300 ng 200 ng


10
γ-globulin 50 ng 100 ng 100 ng 1.5 2 2.5 3 3.5 4 4.5
Bovine seru m Elution volu m e (ml)
albu min 70 ng 300 ng 200 ng
O valbu min 50 ng 100 ng 100 ng Figure 3
M yoglobin 15 ng 50 ng 30 ng Column: TSKgel SuperSW Series, 4.6 mm ID X 30 cm L
Sample: Standard proteins (5 µL, 0.1g/L each); 1. thyroglobulin 2. γ-globulin
Colu m n: TSKgel SuperS W, 4.6 m m ID x 30 cm L 3. bovine serum albumin 4. ß-lactoglobulin 5. lysozyme
Eluent: 0.2 m ol/L phosphate buffer, pH 6.7 6. cytochrome C 7. glycine tetramer
Detection: U V @ 220 n m Eluent: 0.2 mol/L phosphate buffer (pH 6.7)
Flow rate: 0.35 mL/min
Table 2 Detection limit for proteins (S/N=3) Detection: UV @ 280 nm

TSKgel SuperSW columns 2 www.tskgel.com


SuperSW brochure 28.11.2007 12:38 Uhr Seite 3

TSKgel SuperSW Series


P/N Colu m n size Min. theoretical Asy m m etry factor Flo w rate
plates

18675 4.6 m m ID x 30 cm L 30,000 0.70-1.60 M ax. 350 µL/min


(M ax. 12 M pa)
21485 2.0 m m ID x 30 cm L 25,000 0.70-1.60 M ax. 75 µL/min
(M ax. 12 M pa)
21845 1.0 m m ID x 30 cm L 18,000 0.70-1.60 M ax. 20 µL/min
(M ax.12 M pa)

Table 4: Specifications of TSKgel SuperSW3000 series

Estimation of sensitivity (standard proteins)


The TSKgel SuperS W series has the sa m e pore 140
sizes as the co n ventional TSKgel S W XL series with 5

equivalent grade. Therefore it has similar calibration 120 4


curves and separation ranges as w ell. Thus m ethod
100
transfer fro m conventional SEC to high resolution 2 3

SEC is very straight forw ard. In general, TSKgel 80 1


Intensity

SuperS W 2000 is suited for the separation of


60
proteins with m olecular w eights of 150,000 Da or
sm aller. TSKgel SuperS W3000 can be used for the 40
1.0 m m ID

separation of proteins with m olecular w eights up to 2.0 m m ID


500,000 Da. 20
4.6 m m ID

0
Microbore TSKgel SuperSW columns 0 5 10 15 20
Tim e (min)

If sa m ple a m ount is limited a reduction of colu m n Figure 4


inner dia m eter can enhance sensitivity. To m eet Column: TSKgel SuperSW3000, 1.0, 2.0, 4.6 mm ID x 30 cm L
Sample: 1. tyroglobulin (1.0 g/L), 2. γ-globulin (2.0 g/L),
these requests TSKgel SuperS W3000 colu m ns are 3. ovalbumin (2.0 g/L), 4. ribonuclease A (3.0 g/L),
available with 2 and 1 m m ID. Table 4 sho ws the 5. p-aminobenzoic acid (0.02 g/L)
specifications of the TSKgel SuperS W3000 colu m ns. Eluent: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4 + 0.05% NaN3
Flow rate: 16 µL/min (1 mm ID), 65 µL/min (2 mm ID), 350 µL/min (4.6 mm ID)
Applications Inj.volume: 0.2 µL
Temp.: 25 °C
Detection.: UV @ 280 nm, cell vol. 2 µL (4.6 mm ID), 35 nL (1.0, 2.0 mm ID)
The sm all particle size and w ell-defined pore sizes
of T SKg el S u p erS W colu m ns pro vid e fast Figure 4 sho ws the levels of sensitivity w hich can
separations with guaranteed efficiencies of 30,000 be reached with se mi-micro or micro colu m ns. In
theoretical plates per 30 cm colu m n (4.6 m m ID). the emerging research fields of proteo mics, limited
This is the reason w hy TSKgel SuperS W colu m ns sa m ple a m ount is an issue for m ost of the
are widely used for peptide and protein analysis in separations. In such cases enhancing detection
research and develop m ent. W henever li mited limits by using a micro colu m n can increase the
sa m ple a m ount is an issue, TSKgel SuperS W nu m ber of hits.
colu m ns are the first choice for gel filtration HPLC
analysis. In addition to the increased sensitivity, the SEC analysis of antibodies
narro w bore colu m n design involves a re m arkable
reduction in solvent consu m ption. Therm ally induced denaturation or aggregation of
therapeutic antibodies can be a significant problem
Selection of column dimension d urin g differe nt sta g es of its pro d uctio n a n d
form ulation, since aggregates affect the efficiency
As a result of sm aller particle size and accordingly of th e for m ulatio n. T h us th e q u a ntificatio n of
higher nu m ber of theoretical plates, sensitivity is aggregates is an important parameter in the quality
increased w hen using TSKgel SuperS W colu m ns control analysis of biophar m aceuticals. Using
co m pared to TSKgel S W or S W XL colu m ns. TSKgel SuperS W3000 colu m ns the a m ounts of
Sensitivity can be further enhanced by reducing tri-, di- a n d m o n o m ers of Ig G m o n oclo n al
inner dia m eter of SuperS W colu m ns. antibodies can be m onitored.

TSKgel SuperSW columns 3 www.tskgel.com


SuperSW brochure 28.11.2007 12:38 Uhr Seite 4

Applications
Separation of proteins with am m oniu m formate
Q uantification is facilitated by using sm aller inner eluent on TSKgel SuperS W3000
dia m eter colu m ns since peak height is significantly 350

increased (Figure 5).


0.1 m ol/L
300
Phosphate buffer
Estimation of sensitivity (Ig G)
250
A m m oniu m form ate
70

60

Intensity (m V)
200 0.4 m ol/L
50
Intensity

40
1.0 m m ID X 30 cm L
150
30 0.3 m ol/L
2.0 m m ID X 30 cm L
20
4.6 m m ID X 30 cm L 100
10
0.2 m ol/L
0
0 5 10 15
50
min

0.1 m ol/L
14 0
aggregates 0 5 10 15
Tim e (min)
13
1.0 m m ID X 30 cm L Figure 7 Same conditions and sample as in figure 4.
12
Intensity

2.0 m m ID X 30 cm L
11 SEC-ESI-MS analysis of proteins
4.6 m m ID X 30 cm L
10

Hyphenated separation techniques like HPLC-M S


0
0 5 10 15 or HPLC-ELSD allo w sensitive analysis of sa m ples
min
with very lo w analyte concentrations. M oreover
Figure 5 M S/M S detection is a po w erful tool to provide
Column: TSKgel SuperSW3000, 1.0 mm ID x 30 cm L further structural information about the compounds.
Sample: IgG (mouse, mAb, 1.0 g/L)
Eluent: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4 + 0.05% NaN3
These detection methods require the use of volatile
Flow rate: 16 µL/min buffer syste ms because the solvent m ust be
Inj.volume: 0.2 µL evaporated before the sam ple m olecules enter the
Temp.: 25 °C detection syste m. For LC/M S analysis TSKgel
Detection.: UV @ 280 nm, cell vol. 2 µL (4.6 mm ID), 35 nL(1.0, 2.0 mm ID) SuperS W colu m ns can be run with formate buffers
as mobile phase, instead of the com mon phosphate
Separation of Ig G and albu min on buffers. Figure 7 demonstrates that at least 300 m M
TSKgel SuperS W3000 a m m o niu m for m ate is n ecessary to reach
separation efficiencies co m parable to 100 m M
120
1 phosphate buffer.
100
Protein analysis under denaturing conditions
80
2 S o m e S EC se p aratio ns re q uire d e n aturin g
60 co n ditio ns like so diu m d o d ecylsulfate (S D S)
(m V)

containing eluents. In other cases the form ulations


40
of biopharm aceuticals contain so m e detergents
20 (e.g. Tween 20 or Triton). TSKgel SuperS W columns
can be operated under these conditions although
0 certain a m ounts of the detergent will stick to the
colu m n, effecting colu m n lifetim e and the future
-20
use of the colu m n. If analysis under denaturing
5 10 15 20 25
conditions w as perform ed once, the affected
Tim e (min) colu m n should be used with detergent containing
Figure 6 eluents only. Regular m aintenance of the colu m n,
Column: TSKgel SuperSW3000, 4.6 mm ID x 30 cm L
Sample: 5 µL mouse ascites: 1. IgG; 2. albumin
the use of guard colu m ns and m onitoring of the
Eluent: 50 mM phosphate buffer + 100 mM Na2SO4, pH 6.7 colu m n status by analysing control sa m ples are
Flow rate: 0.2 µL/min reco m m ended as w ell.
Temp.: 25 °C
Detection.: UV @ 280 nm

TSKgel SuperSW columns 4 www.tskgel.com


SuperSW brochure 28.11.2007 12:38 Uhr Seite 5

Hardware requirements

Optimization of HPLC equipment Colu m n efficiency starts deteriorating re m arkably


w hen the volu m e of the tubing exceeds 10 µl (e.g.
O ptimizing the HPLC syste m to minimize extra 0.1 m m ID x 150 cm L). Shortening of tubings of
colu m n peak broadening is strongly reco m m ended 0.1 or 0.125 m m inner dia m eter to the minim u m is
to reach the highest separation po w er with a TSKgel often better than using long tubings with sm aller
SuperS W SEC colu m n. This m eans minimization of inner dia m eters. The backpressure increases with
dead volume and adjustment of sample concentration sm aller inner dia m eters and the syste m beco m es
and injection volu m e. Key co m ponents of the HPLC m ore susceptible to w ards clogging.
syste m with regard to dead volu m e reduction are
the void volu m e of tubings, the cell volu m e of the Cell volume of the detector
detector cell and the void volu m e of the injection
unit. HPLC syste ms designed for use with m odern The detector cell volu m e also contributes to the
sub 2 µ m HPLC colu m ns exhibit extre m ely sm all dead volu m e of the syste m and might im pair peak
dead volu m es. Currently these syste ms are not resolution. Co m pared to a semi-micro detector cell
evaluated for hig h resolution SEC use. Using with 2 to 3 µl cell volume, the standard cells of most
co m m on SEC buffers with such a syste m might high end HPLC instru m ent’s U V or PD A detectors,
result in a high syste m backpressure or increased having cell v olu m es of 10-12 µl and sm all inner
risk of clogging. In w orst case the pressure could diameter inlet capillaries do not have a big influence
exceed the pressure limits of TSKgel SuperS W on the nu m ber of theoretical plates. The increase
colu m ns. We reco m m end to carefully evaluate the in efficiency by using a sm aller cell is belo w 5 %.
system’s dead volu me and the system’s backpressure O n the other hand the path length of se mi-micro
at the flow rates used for SEC analysis. or micro cells is often shorter than for standard
cells. Consequently so m e 40 to 60% loss of
Void volume of the tubing sensitivity might be the price for higher resolution
(Table 2). For m ost separations with 4.6 m m ID
The volu m e of tubing betw een injector and colu m n, TSKgel SuperS W columns a 10 µl standard detector
and colu m n and detector influences the diffusion cell is a good choice.
within the tubing and the colu m n efficiency.

Recommended flow cells for common HPLC systems for UV /PDA detection
Detector m odel/Colu m n ID 4.6 m m ID 4.6 m m ID (m ax. 1 m m ID
(m ax. sensitivity) resolution) & 2 m m ID

A gilent Technologies Standard cell, 14 µl Se mi-micro cell, 5 µl


1200 V W D SL G1314C #018 G1314C #016

A gilent Technologies Standard cell, 13 µl Se mi-micro cell, 5 µl Micro cell, 2 µl


1200 D A D SL G1315C #018 G1315C #016 G1315C #010

Dionex UltiM ate Standard cell, 11µl Micro cell, 1.4 µl U-Z-Vie w Micro 180 nl
V W D-3100/-3400 6074.0250 6074.0260 6074.0290

Dionex UltiM ate Standard cell, 13 µl Se mi-micro cell, 3.1 µl


PD A-3000 6080.0210 6080.0230

Waters 2489 U V/VIS Standard cell, 10 µl Microbore cell 2.6 µl


WA S081140 WAT081159

Shim adzu Pro minence Standard cell, 12 µl Se mi-micro cell, 2.5 µl Dionex U-Z Vie w Micro,
U V/U V-VIS SPD-20A/-20AV Incl. 228-45605-91 140 nl; 160239

Shim adzu Pro minence Standard cell, 10 µl Se mi-micro cell, 2.5 µl Dionex U-Z Vie w Micro,
PD A SPD-M20A Incl. 228-45605-92 140 nl; 160239

V WR LaChro m Elite U V/U V-VIS Standard cell, 13 µl Se mi-micro cell, 3.2 µl Micro cell, 0.9 µl
L-2400/2420 890-0500 890-0504 890-0506

V WR LaChro m Elite D A D Standard cell, 13 µl Se mi-micro cell, 3.2 µl Micro cell, 0.9 µl
L-2450 890-0550 890-0554 890-0556

Table 5

TSKgel SuperSW columns 5 www.tskgel.com


SuperSW brochure 28.11.2007 12:38 Uhr Seite 6

Hardware requirements
Effect of sam ple load
In case that semi-micro (2 m m ID) or micro colu m ns
(1 m m ID) are used, w e strongly reco m m end
adjusting the cell volu m e accordingly. Table 5 160.0

sho ws the reco m m ended flo w cells for the m ost 140.0 TSKgel SuperS W3000
frequently used HPLC syste ms. 120.0 TSKgel G3000S W XL

100.0

HETP (µm)
Injector 80.0

60.0
The m axim u m nu m ber of theoretical plates in
40.0
isocratic HPLC separations is alw ays reached using
a lo w diffusion ty pe m anual injector like the 20.0

Rheodyne 8125. A general-purpose injector like the 0.0


0.1 1 10 100 1.000 10.000
Rheodyne 7125 will lead to the loss of 10% in Sa m ple loading (µg)
efficiency. All kinds of auto m ated HPLC injectors
will deteriorate colu m n efficiency as w ell. Due to Figure 9
practical reasons, auto-sa m plers are no w adays Column: TSKgel SuperSW series, 4.6 mm ID x 30 cm L,
standard in HPLC syste ms. All the m ore it is TSKgel SWXL series, 7.8 mm ID x 30 cm L
im portant to select an auto-sa m pler capable of Sample: Bovine serum albumin
Eluent: 0.2 mol/L phosphate buffer, pH 6.7
trace injection m ode. Flow rate: 0.35 mL/min (TSK-GEL SuperSW series)
1.00 ml/min (TSK-GEL SWXL series)
Dead volu m e of the outlet capillary should be Temp.: 25 °C
mini mized to the utm ost (as short as possible, Detection.: UV @ 280 nm, micro flow cell
ID < 0.1 m m). Figure 8 sho ws the effect of injector
tubing on colu m n efficiency for a 1 m m ID colu m n. Sample load and injection volume

Although the height equivalent to a theoretical


Influence of tubing (injector to colu m n) plate (HETP) is sm all in TSKgel SuperS W series it
is obvious that it increases at high sa m ple loads.
34.000
Figure 9 shows that sam ple load should not exceed
32.000
100 µg for a TSKgel SuperS W3000 colu m n of 4.6
m m ID x 30 cm L. O n the other hand the injection
Theoretical plates

30.000
0.050 m m ID
28.000 volu m e itself is a critical para m eter.
x
0.075 m m ID
26.000
x 0.130 m m ID
24.000 Effect of injection volu me on a 2 m m ID
TSKgel SuperS W3000 colu m n
x

22.000
x
20.000
6000
0 100 200 300 400 500 600 700
Tubing length (m m)
5000
Figure 8
Theoretical plates

Column: TSKgel SuperSW3000 1.0 mm ID x 30 cm L


Eluent: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4 + 0.05 % NaN3 4000
Flow rate: 16 µL/min
Inj.volume: 0.2 µL 3000
Temp.: 25 °C 1 µl
Detection.: UV @ 280 nm 2000 5 µl
Sample: p-Aminobenzoic acid (20mg/L) 10 µl
Tubing: ID (mm) L (mm) Vol. (nL) 1000
0.050 200 393
400 785 0
600 1178 0 1 2 3 4 5
0.075 200 883 Conc. of Ig G (g/l)
400 1766
600 2469 Figure 10
0.130 200 2653 Column: TSKgel SuperSW3000 2.0 mm ID x 30 cm L
400 5307 Sample: IgG (mouse, mAb) 1, 5, 10 µl
600 7960 Eluent: 0.1 M phosphate buffer + 0.1 M Na2SO4 + 0.05 % NaN3 (pH 6.7)
Flow rate: 0.065 mL/min
Temp.: 25 °C
Detection.: UV @ 280 nm

TSKgel SuperSW columns 6 www.tskgel.com


SuperSW brochure 28.11.2007 12:38 Uhr Seite 7

Hardware requirements
Van Deemter curve
As for all HPLC applications injection volu me should
25
be as small as possible. If injection volu me exceeds
20 µl o n a 4.6 m m ID colu m n, a considerable 2.0 m m ID x 30 cm L
20
deterioration of colu m n efficiency is observed for 1.0 m m ID x 30 cm L

TSKgel SuperS W 2000 (80 µl for TSKgel


15

HETP (µm)
SuperS W3000).
10
The influence of injection volu m e is even higher
when using microbore TSKgel SuperS W colu m ns.
5
Figure 10 de m onstrates that a certain increase in
sa m ple concentration does not harm the efficiency
0
of a microbore colu m n if the injection volu m e is 0 50 100 150 200 250 300
small. O n the other hand an increase of the injection Linear velocity (cm/h)
Figure 11
volu m e itself has a re m arkable effect. Column: TSKgel SuperSW3000, 1.0 mm ID x 30 cm L
TSKgel SuperSW3000, 2.0 mm ID x 30 cm L
In general the sample load should be less than 100 µg Sample: p-Aminobenzoic acid (20 mg/L)
as total a m ount and less than 10 µl as injection Eluent: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4 + 0.05% NaN3
volu m e for a 4.6 m m ID TSKgel SuperS W colu m n. Detection.: UV @ 280 nm
Temp.: 25 °C
Inj.volume: 0.2 µL (1.0 mm ID), 1.0 µL (2.0 mm ID)
Mobile phase

The eluent plays an i m portant role in SEC The appropriate flo w rate for TSKgel SuperS W
separations. W hen denaturing eluents are used, colu m ns is up to 0.4 ml/min for a 4.6 m m ID
the exclusion limit for proteins beco m e sm aller column, up to 75 µl/min for a 2 m m ID column, and
since they lose their co m pact globular structure. up to 20 µl/min for a 1 m m ID colu m n respectively.
Proper selection of eluting conditions is necessary If higher resolution is required the flo w rate can
to m aximize m olecular sieving m echanisms and be lo w ered.
to minimize secondary effects, such as ionic and
hydrophobic interactions betw een the sa m ple and Recovery of protein
the colu m n packing m aterial. U nder conditions of
high ionic strength (> 1.0 M), h y drophobic TSKgel SuperS W series is capable of obtaining high
interactions m ay occur. U nder lo w ionic strength protein recovery even in trace analysis with sam ple
(<0.1 M), ionic interactions are m ore likely to occur. load of 1 µg or lo w er. Table 6 sho ws the recovery
In general, the use of relatively high ionic strength of proteins at sa m ple concentrations of 20 µg/mL
buffers is reco m m ended for m ost applications. A (sa m ple load 100 ng). M ost proteins are recovered
neutral salt, such as sodiu m sulfate, is often added quantitatively with TSKgel SuperS W series, but it
to increase ionic strength. is im portant to make sure that samples in small
concentrations are not adsorbed to the HPLC system
If hydrophobic interaction occurs betw een the (injector, tubing etc.) itself. Similar sam ples should
sa m ple and the m atrix, up to 100% w ater soluble be injected several times before measurement so
organic, such as acetonitrile, acetone, m ethanol or that the adsorption point within the syste m is in-
ethanol, can be added to the m obile phase. If m ass activated in advance when trace analysis is performed.
spectro m etric detection is applied it is necessary to
change to a volatile buffer syste m. TSKgel SuperS W2000 TSKgel SuperS W3000
Thyroglobulin 86% 97%
Flow rate dependence γ-globulin 90% 90%
BS A 99% 86%
The effect of flo w rate on HETP depends on particle O valbu min 97% 98%
size of packing m aterials, sa m ple m olecular size,
Ribonuclease A 86% 87%
eluent viscosity, etc. Since the particle size of
M yoglobin 93% 96%
TSKgel SuperS W is sm all, it has sm all HETP
Cytochro m e C 85% 90%
throughout a broad range of flo w rates (Figure 11).
Lysozy m e 93% 89%

Table 6 Recovery of proteins

TSKgel SuperSW columns 7 www.tskgel.com


SuperSW brochure 28.11.2007 12:38 Uhr Seite 8

Ordering Information

Conclusion

TSKgel SuperS W series is a group of colu m ns in In order to exert the better perform ance of TSKgel
w hich particle size and colu m n size of the S u p erS W series, th e use of e q uip m e nt w ith
con ventional TSKgel S W XL series have been minimized dead volu m e is reco m m ended. Table 7
reduced and at the same time to im prove resolution su m marizes the cautions in using TSKgel SuperS W
and sensitivity. As additional benefit of the narro w series colu m ns. U nder ideal conditions with a
colu m n dia m eters buffer co nsu m ption is proper sam ple preparation and the use and regular
considerably reduced. TSKgel SuperS W series is exchange of guard colu m ns a long colu m n lifetim e
ideal for sa m ple-limited applications because it ca n b e achie v e d. F or m icro a n d se m i m icro
m aintains high recovery even for sa m ple injection colu m ns a line filter instead of a guard colu m n is
at a lo w concentration. It is therefore suited to trace reco m m ended to keep dead volu m e lo w.
analysis of biopoly m ers by SEC.
Based on their high efficiency TSKgel SuperS W2000
As a result of the high m anufacturing quality of and TSKgel SuperS W3000 colu m ns are ideally
TSKgel SuperS W resins these colu m ns sho w an suited for all highly sensitive gel filtration analysis
extre m ely lo w a m ount of colu m n bleeding. Hence in the fields of biotechnology, proteo mics and in
they can be used for SEC separation follo w ed by quality control of lo w dose biopharm aceuticals.
m ass spectro m etric detection as w ell.

P/N Colu m n Colu m n size Min. theoretical plates

18674 TSKgel SuperS W2000 4.6 m m ID x 30 cm L 30,000


21845 TSKgel SuperS W3000 1.0 m m ID x 30 cm L 18,000
21485 TSKgel SuperS W3000 2.0 m m ID x 30 cm L 25,000
18675 TSKgel SuperS W3000 4.6 m m ID x 30 cm L 30,000
18762 Super S W G uardcolu m n, 4 µ m, for P/N 18674 and 18675

TSKgel SuperSW columns 8 www.tskgel.com


SW cover.qxd 05.10.2007 17:21 Uhr Seite 2

Why we are different Notes to be made in using TSKgel SuperSW series

Tosoh Bioscience is a global leader in the field of bioseparations


Tosoh Bioscience provides comprehensive technical and regulatory support
■ Reduce peak broadening in tubing, detector, etc.
Tosoh Bioscience offers the broadest range of columns for aqueous and organic SEC
■ Take care of sample overloading
Tosoh Bioscience is the leading manufacturer of silica based SEC columns
■ Take care of flow rate of pumping system since the required flow rate is low

Size Exclusion Chromatography


Tubing:
Sample typical ■ Use 0.1 mm ID tubing. It is recommended that the total tubing length is 100 cm or shorter
application chromatogram
■ Connection pipe set type L (product no. 018186; 0.1 mm ID x 40 cm L, 2 pieces) available,
buffer/low salt
concentration connection surface (both ends) with fine-cut finishing

■ Sections requiring 0.1 mm ID tubing

a) Between injection valve/column inlet, or auto-sampler/column inlet

b) Between column outlet/detector inlet (tubing on inlet side of the detector)

Pumping system:
■ Pumping system should be applicable to semi-micro HPLC
Elution
Size Exclusion Chromatography
■ Flow rate should be 0.01 - 0.35 ml/min
buffer/low salt (SEC)
concentration
is the general name for the chromato-
graphic mode also referred to as gel Injector:
permeation chromatography (GPC)
for non-aqueous elution systems or ■ Low diffusion type injector (Reodyne 8125) is recommended
gel filtration chromatography (GFC)
for aqueous systems. SEC is a
method in which components of a
mixture are separated according to Guard column:
their molecular size, based on the
flow of the sample through a porous ■ Be sure to connect an in-line filter or a guard column (product no. 18762) to protect the
packing. Large biomolecules that
cannot penetrate the pores of the column (A set of connection tubing is a standard accessory to the guard column)
packing material elute first from the
column. These large biomolecules
are said to be excluded from the
packing; they flow with the mobile
phase in the interparticle space of
Detector:
the packed column. Smaller mole-
cules can partially or completely
■ For UV detectors, use micro flow cells or low dead volume type cells. Low dead volume
enter the packing particles. Because
these smaller molecules have to flow type cells are effective in high-sensitivity analysis. Use of standard cell is also possible
through the interparticle space, as
well as through the pore volume, for 4.6 mm ID columns. However, theoretical plates will be reduced.
they will elute from the column after
the excluded sample components.
TSK-GEL® Columns for SEC SEC is a very simple method for
separating biomolecules, because it Sample:
TSKgel SW-Series is not necessary to change the com-
position of the mobile phase during ■ Sample injection volume should be 1 - 10 µl. sample load should be 100 µg or smaller.
TSKgel PW-Series elution. However, the separation
TSKgel Alpha-Series capacity of this method is limited. For
a baseline separation it is necessary If help is needed, contact our technical support specialists
TSKgel SuperAW-Series that the molecular weights of the bio-
to offer you assistance at +49 (0)711 13257-0.
TSKgel H-Series molecules differ at least 10 to 20 %.
Table 8
ANALYSIS

TSKgel® SuperSW mAb HTP/HR


TSKgel® UltraSW AGGREGATE
INTRODUCTION The calibration curve of TSKgel SuperSW mAb HR
is most similar to the one of TSKgel G3000SWXL, the
Aqueous size exclusion chromatography (SEC) is the current industrial standard for antibody analysis. The
method of choice for the analysis of protein fragments, new columns are highly reproducible. The high Batch-
monomers, and aggregates under non-denaturing condi- to-Batch stability is proved in Figure 2 for TSKgel Super
tions. Based on the flow of the sample through a porous SW mAb HR. The lifetime of the columns can be further
stationary phase SEC separates molecules according to improved when using the corresponding guard columns.
their size, or more precisely, their hydrodynamic volume.
In aqueous elution systems SEC is also referred to as
CALIBRATION CURVE
gel filtration chromatography (GFC). TSKgel G3000SWXL
columns have been the industry’s standard for quality 7
control of monoclonals by SEC for decades. Based on the SuperSW mAb HTP
1 SuperSW mAb HR
proven proprietary surface technology of the renowned
6 UltraSW Aggregate
TSKgel SW series, a new series of silica-based SEC G3000SWXL

columns was engineered to provide shorter analysis time 2


5
or higher resolution for antibody analysis. 3
Log MW

4
HIGHLIGHTS 4

Optimized for antibody analysis 3


Small particle size for UHPLC use
Highest resolution with SuperSW mAb HR 5
2
Fast separation with SuperSW mAb HTP
Higher molecular weight range with
UltraSW Aggregate 1
0 2 4 6 8 10 12 min
Figure 1
FEATURES
Columns: TSKgel SuperSW mAb HTP 4.6 mm ID × 15 cm, TSKgel Super-
The new series of dedicated SEC columns for mAb ana- SW mAb HR, TSKgel UltraSW Aggregate, TSKgel G3000SWXL (all 7.8 mm
ID × 30 cm)
lysis delivers significant advancements in resolution. It Mobile phase: 0.2 mol/L phosphate buffer, pH 6.7 0.05% NaN3;
comprises of three different columns and their guards. Flow rate: 1.0 mL/min, 0.35 mL/min (SuperSW mAb HTP); Temperature: 25°C;
Table 1 summarizes the characteristics of the mAb SEC Detection: UV 280 nm; Inj. vol.: 10 µL, 5 µL (SuperSW mAb HTP )
1. Thyroglobulin (MW 640,000), 2. γ-Globulin (MW 155,000),
columns and Figure 1 shows the calibration curves 3. Ovalbumin (MW 47,000), 4. Ribonuclease A (MW 13,700)
and the molecular weight range of the three columns. 5. p-Aminobenzoic acid (MW 137)

CHARACTERISTICS OF TSKgel mAb COLUMNS

Column TSKgel SuperSW mAb HR TSKgel SuperSW mAb HTP TSKgel UltraSW Aggregate

Dimension 7.8 mm ID x 30 cm 4.6 mm ID x 15 cm 7.8 mm ID x 30 cm

Theoretical plates ≥ 30.000 ≥ 15.000 ≥ 35.000

Base material Silica gel Silica gel

Particle size 4 µm 3 µm

Separation range
10,000 - 500,000 Da 10,000 - 2,000,000 Da
(globular proteins)

Table 1
ANALYSIS

Each of the new SEC columns is tailored to a specific separa- FAST ANALYSIS OF mAb AGGREGATION
tion problem. TSKgel SuperSW mAb HTP - “HTP” standing
for high throughput - was developed to enable an easy
transfer of HPLC methods based on TSKgel SWXL to fast monomer

UHPLC analysis. Small particle size silica beads are packed


in UHPLC column hardware with 4.6 mm inner diameter.
monomer Flow rate: 0.50 mL/min
This enables to double the throughput without compro- dimer
Pressure: 5.0 MPa
trimer
mising resolution (Figure 3). aggregates
Rs(dimer/monomer)=1.91

TSKgel SuperSW mAb HR - “HR” indicating high resolu- dimer


Flow rate: 0.35 mL/min
Pressure: 3.6 MPa
tion - delivers superior resolution over the whole range trimer
aggregates Rs(dimer/monomer)=2.13
from fragments to aggregates when analysing monoclonals
(Figure 4). It has the same column dimensions - 7.8 mm by 30
0 1 2 3 4 5 6 7 min
cm length - as the established TSKgel G3000SWXL. TSKgel
Ultra SW Aggregate (7.8 mm ID x 30 cm) features a smaller FIGURE 3
particle size and a higher exclusion limit. It offers a wider
Column: TSKgel SuperSW mAb HTP (4.6 mm ID x 15 cm)
separation window in the aggregate region (Figure 4C). Elution: 0.2 mol/L phosphate buffer (pH 6.7) + 0.05% NaN3
Flow rate: 0.50 mL/min, 0.35 mL/min; Detection: UV @ 280 nm
Temp.: 25°C; Sample: monoclonal antibody (mouse-human chimeric
IgG, Erbitux), 5 µL

BATCH TO BATCH REPRODUCIBILITY COMPARISON OF AGGREGATE ANALYSIS

A monomer

5
4 dimer

trimer
1 2 3 aggregates
UV absorbance @ 280 nm (mAU)

Lot.A 5 6 7 8 9 10 11 12 min

B monomer IgG
fragment
dimer

trimer
aggregates

Lot.B

5 6 7 8 9 10 11 12 min

monomer
C dimer

trimer
Lot.C aggregates

4 6 8 10 12 14 16 min 5 6 7 8 9 10 11 12 min

FIGURE 2 FIGURE 4
Column: TSKgel SuperSW mAb HR (7.8 mm ID x 30 cm) Columns: A. TSKgel G3000SWXL, B. TSKgel SuperSW mAb HR,
Eluent: 0.2 mol/L phosphate buffer (pH 6.7)+ 0.05% NaN3 C. TSKgel UltraSW Aggregate; Dimension: 7.8 mm ID × 30 cm;
Flow rate: 0.8 mL/min; Detection: UV @ 280 nm; Inj. volume: 10 µL; Eluent: 0.2 mol/L phosphate buffer (pH 6.7) + 0.05% NaN3
Sample: 1. Thyrobulobulin, 2. γ-Globulin, 3. Ovalbumin, 4. Ribonuclease A, Flow rate: 0.8 mL/min; Detection: UV @ 280 nm; Temp.: 25°C;
5. p-Aminobenzoic acid Sample: monoclonal antibody, (mouse-human chimeric IgG, Erbitux),10 µL

Ordering information
Part-No Description Matrix Housing Dimensions

22854 TSKgel SuperSW mAb HR, 4 µm, 250 Å Silica Stainless steel 7.8 mm ID x 30.0 cm L

22855 TSKgel SuperSW mAb HTP, 4 µm, 250 Å Silica Stainless steel 4.6 mm ID x 15.0 cm L

22856 TSKgel UltraSW Aggregate, 3 µm, 300 Å Silica Stainless steel 7.8 mm ID x 30.0 cm L

22857 TSKgel Guardcolumn SuperSW mAb, 4 µm Silica Stainless steel 6.0 mm ID x 4.0 cm L

22858 TSKgel Guardcolumn SuperSW mAb, 4 µm Silica Stainless steel 3.0 mm ID x 2.0 cm L

22859 TSKgel Guardcolumn UltraSW, 3 µm Silica Stainless steel 6.0 mm ID x 4.0 cm L

TOSOH BIOSCIENCE ZETTACHRING 6 70567 STUTTGART GERMANY T: +49 (0)711 13257-0 F: +49 (0)711 13257-89 SALES-MARKETING.TBG@TOSOH.COM WWW.TSKGEL.COM
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