64
hilic
Applications of TSKgel nh
2
-100 columns
Separation of water soluble vitamins
Figure 7
shows the separation of a standard solution of water soluble
vitamins on a TSKgel NH
2
-100 column compared to a TSKgel Amide-80
column. Dimension (4.6 mm ID x 15 cm L), particle size (3 μm), flow rate
and mobile phase were identical for both columns. The elution order
of the compounds changes when applying the same mobile phase to
both columns: The TSKgel NH
2
-100 column shows stronger retention for
nicotinic acid, vitamin C, and vitamin B12, while retention of vitamin B1,
B2, and pyridoxine is reduced.
Separation of methotrexate and derivatives
Figure 8
compares the separation of methotrexate and its derivatives
(MTXPG2~7) on TSKgel NH
2
-100, 3μm HILIC and TSKgel ODS-100V, 3μm
reversed phase narrow bore columns. Methotrexate, abbreviated MTX
and formerly known as amethopterin is an inhibitor of the folic acid
metabolism. It is used in cancer chemotherapy and as a treatment of
autoimmune diseases. The MTX and polyglutamate derivatives were
eluted in the order of the number of glutamate groups in their mole-
cules on the TSKgel NH
2
-100 HILIC column, but eluted in reverse order
on the TSKgel ODS-100V column. Despite the early elution of MTX and
MTXPG2 on the TSKgel NH
2
-100 HILIC column, the overall separation is
better than what can be accomplished on the C18 column.
figure 7
Separation of water soluble vitamins
Columns:
TSKgel Amide-80 3 µm, 4.6 mm ID x 15 cm L
;
TSKgel NH
2
-100 3 µm, 4.6 mm ID x 15 cm L;
Eluent: 25 mM phosphate buffer (pH 2.5)/ACN=30/70
Flow: 1 mL/min; Temp.: 40°C; Detection: UV @ 254 nm
Sample: Vitamin standard mixture: 1 = Nicotinamide, 2 = Vitamin B2, 3 =
Pyridoxine, 4 = Nicotinic acid, 5 = Vitamin C, 6 = Vitamin B1, 7 = Vitamin B12
Injection: 5 µL
1 4
5
2 3
7
6
1
2
3 4
5
6
7
figure 8
Separation of MTX and derivatives
Column:
A) TSKgel ODS-100V, 3 µm, 2.0 mm ID x 15 cm L;
Mobile phase: a) H
2
O/ACN (90/10) + 0.1% TFA, b) ACN + 0.1% TFA;
B) TSKgel NH
2
-100, 3 µm, 2.0 mm ID x 15 cm L;
Mobile phase: a) H
2
O/ACN (10/90) + 0.1% TFA, b) H
2
O + 0.1% TFA;
Gradient: 0 % B (0 min), 40 % B (15 min), 0 % B (17 min);
Flow rate: 0.20 mL/min; Detection: UV @ 313 nm; Temperature: 40°C;
Injection vol.: 10 µL; Sample: 1. MTX (MTXPG) 2. MTXPG2 3. MTXPG3
4. MTXPG4 5. MTXPG5 6. MTXPG6 7. MTXPG7
A.
M
B.
M
G
Fl
D
T
In
S
0
200
400
600
800
1000
0
2
4
6
8
10
12
Retention time (minutes)
Intensity (mV)
1
A
B
2
3 4
5
6
7
1
2
3
4
5
6
A)
B)
