ION EXCHANGE COLUMNS - page 19

15 Tosoh bioscience ANALYSIS
IEC
IEC
TSKgel NPR SERIES
TSKgel DEAE-NPR, DNA-NPR and SP-NPR are packed with
2.5 μm particles. High column efficiency coupled with low
sample capacity restricts the application of these columns
to fast analysis and micro-scale preparative isolation.
The DNA-NPR column is a longer version of the DEAE-NPR
column that allows improved resolution of oligonucle-
otides, including those amplified by PCR. Small guard col-
umns are available to protect the DNA-NPR and DEAE-NPR
columns.
Applications of TSKgel NPR ion Exchange
Columns
TSKgel DEAE-NPR and DNA-NPR Anion Exchangers
Because of their small particle size, non-porous resin (NPR)
columns excel in rapid separations of large biomolecules
such as DNA digests. A chromatogram of a standard Hae III
digest of pBR322 DNA on TSKgel DEAE-NPR, protected by
a guard column, is shown in Figure 18.
To achieve better resolution for PCR fragment analysis we
recommend the use of TSKgel DNA-NPR columns, which are
7.5 cm long and 4.6 mm wide, providing higher efficiency
in a longer column.
TSKgel SP-NPR Cation Exchanger
TSKgel SP-NPR columns provide fast separations due to
their small spherical particles. A purity check of adeno-as-
sociated virus, commonly used in gene therapy research,
on a TSKgel SP-NPR column is shown in Figure 19. This 10
minute HPLC method replaces an existing assay that took
two days.
Higher resolution and faster analysis on TSKgel DEAE-NPR
51 57 64 80 89
104 123 124
184 213234
192
267
434
540504
458
587
2 5 10 13
Minutes
Column:
Sample:
Buffer A:
Buffer B:
Elution:
Flow Rate:
Pressure:
Temperature:
Detection:
TSKgel DEAE-NPR, 4.6mm ID x 3.5cm, with guard
column, 4.6mm ID x 0.5cm
Hae III digest of pBR322 DNA,
(base pair number for each peak is indicated)
0.02mol/L Tris-HCl, pH 9.0
Buffer A plus 1.0mol/L NaCl
15min linear gradient from 48% to 65% buffer B
1.5mL/min
2000psi
40º C
UV @ 260nm
higher resolution and faster analysis on
TSKgel deae-npr
Column: TSKgel DEAE-NPR, 4.6 mm ID x 3.5 cm L, with guard column,
4.6 mm ID x 0.5 cm L; Sample: Hae III digest of pBR322 DNA, (base pair
number for each peak is indicated); Buffer A: 0.02 mol/L Tris-HCl, pH 9.0;
Buffer B: Buffer A plus 1.0 mol/L NaCl; Elution: 15 min linear gradient from
48 % to 65 % buffer B; Flow rate:1.5 mL/min; Pressure: 14 Mpa; Temp.: 40 C;
Detection: UV @ 260 nm
figure 18
Analysis of purified AAV with TSKgel SP-NPR
-5
0
5
10
15
20
2.5 5 7.5 10 0
Minutes
mAU
Column:
Sample:
Elution:
Flow Rate:
Detection:
TSKgel SP-NPR, 4.6mm ID x 3.5cm
purified adeno-associated virus
A. 50mmol/L HEPES, 1mmol/L EDTA, 5mmol/L MgCl, pH 7.5;
B. 50mmol/L HEPES, 1mmol/L EDTA, 5mmol/L MgCl, pH 7.5 with
0.5mol/L NaCl; linear gradient from 20% to 100% B in
10 column volumes
1mL/min
UV @ 280nm
analysis of purified AAV with TSKgel SP-NPR
Column: TSKgel SP-NPR, 4.6 mm ID x 3.5 cm L; Sample: purified adeno-
associated virus; Elution: A. 50 mmol/L HEPES, 1 mmol/L EDTA, 5 mmol/L
MgCl, pH 7.5; B. 50 mmol/L HEPES, 1 mmol/L EDTA, 5 mmol/L MgCl, pH
7.5 with 0.5 mol/L NaCl; linear gradient from 20 % to 100 % B in 10 column
volumes; Flow rate: 1 mL/min; Detection: UV @ 280 nm
figure 19