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TOSOH BIOSCIENCE
TOSOH BIOSCIENCE
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
Solvent compatability 100% polar 50% polar 100% polar, and nonpolar
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)
* 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.
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
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
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
SEC
TSKgel SW SERIES APPLICATIONS
SEC
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.
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
Fr4
SEC-MALS-UV-RI ANALYSIS OF MAB AGGREGATES 10
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.
mV
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
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)
14 aggregates
100
0.2 mol/L
13
1.0 mm ID X 30 cm L
12
50
Intensity
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)
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SEC
TSKgel SuperSW SYSTEM REQUIREMENTS
SEC
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.
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
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)
SEC
TSKgel SW SERIES ORDERING INFORMATION
SEC
ORDERING INFORMATION
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
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)
SEC
TSKgel PW SERIES
SEC
FIGURE 13
CALIBRATION CURVES
FIGURE 12
Protein calibration curves on TSKgel PWXL columns
107
106 1 2 5 3 4
Molecular weight (Da)
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
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
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
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)
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)
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).
FIGURE 18
SEC
TSKgel PW SERIES ORDERING INFORMATION
SEC
ORDERING INFORMATION
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
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
TABLE 6
ORDERING INFORMATION
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
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
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As SEC is a partition chromatography, sample load on the For TSKgel SW columns mobile phases a buffer concen-
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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
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
2010 TOSOH CELEBRATES ITS 75TH YEAR IN BUSINESS WITH THE OPENING OF FIVE NEW PLANTS, AND CONTINUED RAPID EXPANSION IN CHINA
ANALYSIS
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
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
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
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.
Figure 4
Ordering information
Part-No Description Matrix Housing Dimensions
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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
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.
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)
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
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
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
THF
Detergents
up to 0.1% SDS
up to 1% Tween, Triton
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
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
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
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.
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
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
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
TSK-GEL® PWXL-CP
Size Exclusion Chromatography Columns for Cationic Column Recovery
Polymer Analysis G3000 PWXL-CP 100,2%
Particle size 7 µm 10 µm 13 µm
Separation range 200 - 50 000 400 - 500 000 1 000 - 10 000 000
(Da), (PEO, PEG)
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
FIGURE 2
Ordering information
TSKgel PWXL-CP
F08L05A
Column Flyer
NEW universal steric exclusion columns
PW, PWXL
*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
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 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
Company Brochure
Title
Department
Column Selection Guide
on CD-ROM NEW! Address / Building
TSKgel SuperAW
City / Post Code / Country
Separation Report
Tel.
Fax
F06L02A
SW cover.qxd 05.10.2007 17:21 Uhr Seite 1
TOSOH BIOSCIENCE
Separations Business Unit
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
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
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
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
0
Microbore TSKgel SuperSW columns 0 5 10 15 20
Tim e (min)
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
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
Hardware requirements
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
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
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
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
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
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
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%
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.
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
4
HIGHLIGHTS 4
Column TSKgel SuperSW mAb HR TSKgel SuperSW mAb HTP TSKgel UltraSW Aggregate
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
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
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
F13L04A