ANALYSIS
TSKgel® SP-STAT and CM-STAT Columns
INTRODUCTION
Reaction Monitoring
A sample of β-lactoglobulin (5 mg/mL) was reacted with
polyethylene glycol (5 kDa) in a pH 6.5 phosphate buffer.
The formation of PEGylated protein reaction products was
monitored in 5 minute intervals on a 3.5 cm TSKgel SP-STAT
column. As demonstrated in Figure 3, peak areas of mono-,
di-, and tri-PEGylated β-lactoglobulin increased with reaction time, while the area of unreacted β-lactoglobulin
declined.
TSKgel SP-STAT and TSKgel CM-STAT cation exchange
columns allow fast equilibration and analysis, as well as
isolation, of complex biomolecules. Both TSKgel columns
are packed with 7 or 10 µm mono-disperse, non-porous
resin particles of which the surface consists of an open
access network of multi-layered cation exchange groups
(see Figure 1). The innovative bonding chemistry, combined
with a relatively large particle size, result in a respectable
loading capacity and a low operating pressure, attributes
not found in traditional mono-disperse, non-porous resins.
Antibody Analysis
The analysis profiles for five antibodies separated on a
TSKgel CM-STAT column were compared with the profiles
obtained on a competitive WCX column (Figure 4). Similar
or higher resolution profiles were obtained on TSKgel
CM-STAT in approximately half the time.
PRODUCT HIGHLIGHTS
Very efficient chromatography for high as well as low
MW solutes made possible by novel bonding chemistry
and the absence of micro-pores
High speed and high resolution analysis of biomolecules
Higher adsorption capacities and lower pressures
compared to competitive non-porous columns
7 or 10 µm particles for SP and CM chemistries
1
2
APPLICATIONS
3
Fast Separations
(B)
mV
The fast separation of protein standards was investigated
using short cation exchange columns (see Figure 2). A
TSKgel SP-STAT column shows superior resolution, better
peak shape, and a shorter analysis time (< 60 seconds)
compared to a competitive monolithic SP-type column.
Protein
Ionic group
Hydrophilic
chain
(A)
0
0.2
0.4
0.6
0.8
1
1.2
Retention time (min)
Non porous material
Figure 1
Protein
Ionic group
Hydrophilic
Figure 2 A: TSKgel Q-STAT, 10µm, 3.0mm ID x 3.5cm
Columns:
B: ProSwift SCX-1S Monolith, 4.6mm ID x 5cm
Column: A: TSKgel SP-STAT, 10 µm, 3.0 mm ID x 3.5 cm L;
Eluent: A: 20mmol/L
sodium
acetate
(pH5.0)
B: Competitor column
4.6 mm ID
x 5.0 cm
L
Eluent: A: 20 mmol/l
sodium NaCl
acetate
5.0);AB:(pH5.0)
1.0 mol/l
in buffer
B: 1.0mol/L
in (pH
buffer
forNaCl
column
A
A (pH 5.0) for column
A; 1.5 mol/l
in buffer
A (pHfor
5.0)
for column
1.5mol/L
NaClNaCl
in buffer
A (5.0)
column
B B;
Gradient: 0% B (0 min), 100% B (1 min);
Gradient:
B (0min),
BDetection:
(1min) UV @ 280 nm;
Flow rate: A: 2.0 0%
ml/min;
B: 4.73 100%
ml/min;
FlowSamples:
rate: 1. α-chymotrypsinogen
A: 2.0mL/min A; 2. cytochrome C; 3. lysozyme
B: 4.73mL/min
Detection:
UV@280nm
Samples:
1. alpha-chymotrypsinogen A
2. cytochrome C
3. lysozyme
ANALYSIS
50
40
A
45
mV
UV @ 280 nm (1 Abs/1000 mV)
30
Native
40
35
E
20
D
30
C
10
B
Mono- PEG
25
A
0
Di- PEG
20
0
10
20
Retention time (min)
Tri -PEG
15
40
B
15
10
30
5
0
0.5
1
1.5
2
2.5
10
E
mV
Retention time (min)
100
D
5
90
C
Native
B
80
A
0
70
0
20
Peak %
60
40
Retention time (min)
60
80
Columns:
50
A: TSKgel CM-STAT, 7µm, 4.6mm ID x 10cm
B: ProPac WCX-10, 10µm, 4mm ID x 25cm
Eluent:
A: 20mmol/L
(pH6.0)
Column: A: TSKgel CM-STAT,
7 µm,MES
4.6 mm
ID x 10 cm L; B: Competitor
20mmol/L
WCX, 10 µm, 4.0 mm B:
ID x
25 cm L; MES + 0.5mol/L NaCl (pH6.0)
Eluent: A: 20 mmol/L MES
(pH 6.0)
; B: 20 mmol/L
+ 0.5 mol/L
NaCl
(pH 6.0)
Gradient:
A: 10%
B (0min),
30% BMES
(15min),
100%
B (15min),
Gradient: A: 10% B (0 min), 30% B (15 min), 100% B (15 min), 100%
100%
B (17min),
10%
(17min),
10%30%
B (21min)
B (17 min), 10% B (17 min),
10%
B (21 min);
B: B
10%
B (0 min),
B (30
B: 10%
30%
B B(30min),
100%
min), 100% B (30 min),
100%BB (0min),
(32 min),
10%
(32 min),
10% B
B (30min),
(36 min)
Flow rate: A: 1.0 mL/min100%
B: 2.0 B
mL/min;
Temp.:
Detection:
@
(32min),
10%Ambient;
B (32min),
10% B UV
(36min)
280 nm; Inj. Vol.: 20 µL; Sample: monoclonal antibodies (mAb A through E)
Flowrate:
A: 1.0mL/min
B: 2.0mL/min
Temp.:
Ambient
Detection:
UV@280nm
Inj. Vol.:
20µL
Sample:
monoclonal antibodies (mAb A through E)
Figure 4
40
Mono-PEG
30
20
Di -PEG
10
Tri- PEG
0
0
Figure 3
10
20
30
40
50
60
70
Reaction time (min)
Column:
A: TSKgel SP-STAT, 10µm, 3mm ID x 3.5cm
Eluent:
A: 20mmol/L sodium acetate buffer (pH5.0)
Column: TSKgel SP-STAT, 10 µm, 3.0 mm ID x 3.5 cm L; Eluent: A: 20 mmol/l
B: 1.0mol/L
NaCl
ininbuffer
sodium acetate (pH 5.0);
B: 1.0 mol/l
NaCl
bufferAA(pH5.0)
(pH 5.0);
Gradient:
B (2 min);
Flow
rate: 2.0 ml/min; Detection:
Gradient:0% B (0 min),
0%100%
B (0min),
100%
B (2min)
UV
@ 280 nm; Samples:
PEGylated β-lactoglobulin
Flowrate:
2.0mL/min
Detection:
UV@280nm
Sample:
pegylated β-lactoglobulin
Ordering information
TSKgel STAT COLUMNS
Part-NoDescription
Matrix
Housing
Dimensions
0021963
TSKgel SP-STAT, 10 µm
Polymer
Stainless steel
3.0 mm ID x 3.5 cm L
0021964
TSKgel SP-STAT, 7 µm
Polymer
Stainless steel
4.6 mm ID x 10 cm L
0021965
TSKgel CM-STAT, 10 µm
Polymer
Stainless steel
3.0 mm ID x 3.5 cm L
0021966
TSKgel CM-STAT, 7 µm
Polymer
Stainless steel
4.6 mm ID x 10 cm L
Tosoh Bioscience GmbH Im Leuschnerpark 4 64347 Griesheim, Germany
Tel: +49 6155-7043700
Fax: +49 6155-8357900 [email protected]
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