hilic
61
hilic
Column Operation and Specifications
TSKgel HILIC columns can be operated over a broad range of mobile
phase conditions. Factors to consider when employing these columns
include:
Sample loading capacity is dependent upon the polarity of the mobile
phase. It increases with decreasing mobile phase polarity. For example,
on a TSKgel Amide-80 column the highest loading capacity for mannitol
(200 μg) occurs with a mobile phase of 75:25 acetonitrile/water.
However, <100 μg of mannitol can be loaded in a mobile phase of 65:35
acetonitrile/water. The maximum sample volume for a 4.6 mm ID x 25 cm
L Amide-80 analytical column is 50 μL.
Temperature range: TSKgel Amide-80 columns can be operated over
a temperature range of 10-80°C (10-50°C for Amide-80 3 μm), TSKgel
NH
2
-100 columns in the range of 10-50 °C. In general, retention
times for carbohydrates decrease with increasing temperature,
thereby shortening analysis time. Below certain temperatures some
carbohydrates may elute as split peaks. In this case, column heating or
addition of triethylamine to the mobile phase is required.
Choice of mobile phase: the pH range of TSKgel Amide-80 and NH
2
-100
columns is 2.0-7.5 with a maximum salt concentration of 100 mmol/L.
The columns are stable in 100% organic for normal phase separations;
however, in HILIC mode a combination of aqueous and organic solvents
is necessary in order to create the water-rich surface layer. As the
mobile phase polarity decreases (higher organic content) the sample is
retained longer on the column.
Long Term Stability
The high stability of TSKgel Amide-80 columns is demonstrated in
Figure 2
showing the same analysis after 330, 660 and more than
1000 runs compared to the first injection. Only 5% reduction of column
performance (theoretical plates) is observed after more than 1000
injections.
Figure 3
shows the high stability of TSKgel NH
2
-100 columns.
Compared to the first injection only a slight reduction of retention time
of inositol is observed with the TSKgel NH
2
-100 column after more than
400 hours of flushing with mobile phase.
Column: TSKgel Amide-80 3 µm (2.0 mm ID x 15 cm L)
Eluent : H
2
O/ACN = 15/85; Flow rate: 0.2 mL/min; Inj. volume: 2 µL
Detection : UV @ 254 nm; Temp. : 25 °C; Samples: Uracil (37 mg/L)
figure 2
Durability of TSKgel Amide-80 3 µm
min
Inj. No. 1038
Inj. No. 330
Inj. No. 660
Inj. No. 1
TP = 13,264
(94,5%)
As ‘f = 1.22
TP = 14,024
(99,9%)
As ‘f = 1.16
TP = 14,228
(101,3%)
As ‘f = 1.18
TP = 14,034
As ‘f = 1.14
0
1
2
3
4
5
0
100
200
300
mV
400
500
600
figure 3
Long term stability of TSKgel NH
2
-100 columns
Column:
TSKgel NH
2
-100 3 µm, 4.6 mm ID x 15 cm L
Conventional Amino Column, 4.6 mm ID x 25 cm L;
Eluent: H
2
O/ACN (25/75); Flow rate: 1.0 mL/min; Detect: RI;
Temp.: 40 °C; Injection: 10 µL; Sample: Inositol
TSKgel NH
2
-100 3
m
m
Conventionall Amino Column
