IEC
50
TOSOH BIOSCIENCE
TSKgel SuperQ-5PW and DEAE-5PW
Figure 4
shows the analysis of a 16-mer morpholine oligonucleotide
on TSKgel SuperQ-5PW column using a NaCl gradient in a 10 mmol/L
sodium hydroxide mobile phase.
Figure 5
shows the fractionation of high molecular weight E. coli RNA
on TSKgel DEAE-5PW, effectively utilizing the large 100 nm pores of this
base resin.
TSKgel DEAE-NPR and DNA-NPR
Because of their small (2.5 µm) 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 6
.
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.
Column: TSKgel SuperQ-5PW, 7.5 mm ID x 7.5 cm L;
Sample: 16-mer morpholine oligonucleotide, AAG AAG AAG AGG GGA G;
Sample load: 0.5 O.D. (optical density); Mobile phase: A: 10 mmol/L NaOH; B:
10 mmol/L NaOH with 1 mol/L NaCl; Gradient: Initial: 0 % B, 40min: 50 % B, 41
min: 100 % B, 46min: 100% B; Flow rate: 1 mL/min; Detection: UV @ 254 nm
Column: TSKgel DEAE-5PW, 6mm ID x 15cm; Sample: total E. coli RNA
Elution: 300 min linear gradient from 0.3 mol/L to 1.0 mol/L NaCl in 0.1 mol/L
Tris-HCl, pH 7.6; Flow rate:1.0mL/min; Detection: UV @ 260 nm
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: 2000 psi; Temp.: 40 °C;
Detection: UV @ 260 nm
figure 4
Analysis of synthetic oligonucleotide on TSKgel SuperQ-5PW
figure 5
Large pore TSKgel DEAE-5PW resolves high MW RNA
figure 6
Higher resolution and faster analysis on TSkgel DEAE-NPR
Column:
TSKgel SuperQ-5PW, 7.5mm ID x 7.5cm
Sample:
16-mer morpholine oligonucleotide,
AAG AAG AAG AGG GGA G
Sample load:
0.5 O.D. (optical density)
Mobile phase:
A: 10mmol/L NaOH
B: 0 mol/L NaOH with 1mol/L NaCl
Gradient:
Initial: 0% B
40min: 50% B
41min: 100% B
46min: 100% B
Flow Rate:
1 mL/min
Analysis of synthetic oligonucleotide on TSKgel SuperQ-5PW
Minutes
40
30
20
10
0
Large pore TSKgel DEAE-5PW resolves high MW RNA
Minutes
0
40
80
4S tRNA
5S rRNA
16S rRNA
23S rRNA
High MW
Impurities
Column:
Sample:
Elution:
Flow Rate:
Detection:
TSKgel DEAE-5PW, 6mm ID x 15cm
total E. coli RNA
300min linear gradient from 0.3mol/L to 1.0mol/L NaCl
in 0.1mol/L Tris-HCl, pH 7.6
1.0mL/min
UV @ 260nm
Higher resolution and faster analysis on TSKgel DEAE-NPR
51
57
64
80
89
104
123
124
184
213
234
192
267
434
540
504
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
Applications of TSKgel ANION exchange columns
