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Tosoh bioscience
ANALYSIS
PROCESS
INSTRUMENTATION
Tosoh
customer magazine
Reversed phase (RP) chromatography is one of the most frequently used
chromatographic modes for analytical separations. It is mostly used for
the analysis of small molecular weight compounds, but there are also
various standard applications for the separation of biomolecules, such
as proteins. Conventional reversed phase HPLC packing materials with
80-140 Å pore sizes are not generally suitable for the analysis of large
intact proteins, as the analytes are not able to access the surface area
within these pores. Silica based TSKgel TMS-250 with 250 Å pores
and polymer based TSKgel Octadecyl-4PW (500 Å) and Phenyl-5PW
(1000 Å) have been widely applied for the reversed phase separation of
intact proteins. Now a wide pore silica based butyl/C4 column, the TSKgel
Protein C4-300, completes the existing range of TSKgel reversed phase
protein columns.
TSKgel Protein C4-300 columns are available in 2 and 4.6 millimeter
inner diameter and 5, 10 and 15 cm column length. The pore size of
300 Å is 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.
Latest surface modification techniques and endcapping of residual silanol
groups reduce undesirable secondary interactions and peak tailing. The
chromatogram below shows the separation of a mixture of standard
proteins on the new C4 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 C4 column compared to 18.6 on
the competitor C4 column. Further, the TSKgel column shows higher
theoretical plates and less peak tailing, especially for BSA (Peak 4), 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 long column and increasing the
flow rate to 3 mL/min (see below). The backpressure remains below 150
bars allowing the use of standard HPLC systems. The long time 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. After 1,000 hours the number of theoretical plates did not change
at all and also retention time of standard proteins only slightly decreased
when compared to the initial separation.
The silica based wide pore TSKgel Protein C4-300 HPLC column is suitable
for highly-efficient, reversed phase separations of proteins such as
separation of recombinant proteins, antibody fragments or PEGylated
proteins.
NEW HPLC COLUMNS FOR PROTEIN ANALYSIS
AT ANALYTICA 2012 TOSOH BIOSCIENCE WILL RELEASE NEW TSKgel HPLC COLUMNS. THEY WILL EXPAND THE RANGE OF REVERSED
PHASE SOLUTIONS FOR PROTEIN ANALYSIS AND ADDRESS THE CURRENT NEEDS FOR INCREASED THROUGHPUT IN BIOANALYSIS. THIS
FOLLOWS ON THE INTRODUCTION OF TSKgel STAT COLUMNS SUITABLE FOR FAST ION EXCHANGE ANALYSIS OF PROTEIN VARIANTS
AND WILL BE CONTINUED SOON WITH THE RELEASE OF NEW SOLUTIONS FOR FAST SIZE EXCLUSION ANALYSIS.
03
WHAT’S NEW
NEW PRODUCTS
Separation of Standard Proteins
Column A: TSKgel Protein C4-300 3 µm (4.6 x 150 mm); B: Competitor C4 3.5 µm
(4.6 x 150 mm); Sample:
[1]
cytochrome c,
[2]
lysozyme,
[3]
BSA,
[4]
alpha-chy-
motrypsinogen A,
[5]
ovalbumin; 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% - 100% B in 45 min;
Flow rate: 1 mL/min; Temp.: 40°C; Detection: UV@210nm
Fast Separation of Standard Proteins
Column A: TSKgel Protein C4-300 3 µm (4.6 x 50 mm); Sample
[1]
cytochrome c,
[2]
lysozyme,
[3]
BSA,
[4]
alpha-chymotrypsinogen A,
[5]
ovalbumin; 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% - 100% B in 5 min; Flow rate: 3 mL/min; Temp.: 40°C; Det.: UV@210nm
20
15
25
30
Time (min)
1
2
A
B
3
4
5
1
3
4
5
2
Time (min)
1
2
3
4
5