79
rpc
RPC
HIGHLIGHTS
Ideal for the separation of proteins
Endcapping ensures low peak tailing
Small particle size for high theoretical plate numbers
Short column for fast separations available
TSKgel Protein C
4
-300 columns are designed for the optimal recovery
and resolution of proteins such as recombinant proteins, antibody
fragments or PEGylated proteins. The 30 nm (300 A) pore size of the
TSKgel Protein C
4
-300 columns are ideal for the separation of proteins.
A particle size of 3 μm and optimized ligand density and alkyl length
result in better protein and peptide resolution compared to other
leading RP-C4 HPLC phases.
The C4 short alkyl chain ligand and its controlled bonding density
provide moderate hydrophobicity to the stationary phase, which results
in protein separations with high recovery and less peak tailing.
Applications
Figure 1
shows the separation of a mixture of standard proteins on
the TSKgel Protein C
4
-300 column compared to a competitor column
with 3.5 μm particle size. The resolution between cytochrome c and
lysozymes reaches 24.8 on the TSKgel Protein C
4
-300 column compared
to 18.6 on the competitor C4 column. Furthermore, the TSKgel column
shows higher theoretical plates and less peak tailing, especially for
BSA (Peak 3), and also a better resolution of minor peaks.
For high speed separations, the analysis time can be reduced by more
than eighty percent when using the short 5 cm TSKgel Protein C
4
-300
column and increasing the flow rate to 3 mL/min (
Figure 2
). The
backpressure remains below 15 MPa, allowing the use of standard
HPLC systems. The long term stability of the new C4 phase in acidic
solution was tested by flushing the column with 30% acetonitrile, 0.2%
TFA (4 times the standard TFA concentration) at 40 °C. There was no
change in theoretical plates even after 1,000 hours of run time under
this chromatographic condition. Also retention times of standard
proteins didn’t have significant loss when compared to the initial values.
RP Columns for Biomolecules TSKgel Protein C
4
-300
figure 1
Comparison of standard protein separation
10
15
20
25
30
Retention time (minutes)
A
1
2
3
4
5
B
Detector response (AU)
figure 2
High speed separation of proteins
Columns:
A. TSKgel Protein C
4
-300, 3 µm, 4.6 mm ID × 15 cm,
B. Competitor A, 3.5 µm, 4.6 mm ID × 15 cm
Mobile phase: A: H
2
O/CH
3
CN/TFA = 90/10/0.05 (v/v/v),
B: H
2
O/CH
3
CN/TFA = 20/80/0.05 (v/v/v)
Gradient: 0 min (0%B) 45 min (100%B), Flow rate: 1.0 mL/min;
Detection: UV @ 210 nm, Temperature: 40 °C; Injection vol.: 10 µL
Samples: 1. cytochrome C, 2. lysozyme, 3. BSA, 4.
α
-chymotrypsinogen A,
5. ovalbumin (each 2 µg/10 µL)
Column:
TSKgel Protein C
4
-300, 3 µm, 4.6 mm ID × 5 cm
Mobile phase A:
H
2
O/CH
3
CN/TFA = 90/10/0.05 (v/v/v)
Mobile phase B:
H
2
O/CH
3
CN/TFA = 20/80/0.05 (v/v/v)
Gradient: 0 min (0%B) 5 min (100%B)
Flow rate: 3.0 mL/min, Detection: UV @ 210 nm
Temperature: 40 °C, Injection vol.: 10 µL
Samples: 1. phenylalanine, 2. cytochrome C, 3. lysozyme, 4. BSA,
5.
α
-chymotrypsinogen A, 6. ovalbumin (each 0.2 g/µL)
0
1
2
3
4
5
2
3
4
5
6
1
Retention time (minutes)
Detector response (AU)
