JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

2-02
Development of Zwitterionic Monolithic Column for
Hydrophilic Interaction Liquid Chromatography and
its Application to the Separation of Catecholamines
and Related Compounds
Y. Kamiya a) , S. Shin a) , A. Sabarudin a) , T. Umemura a) , Y. Ueki b) and M. Tamada b)
a) EcoTopia Science Institute, Nagoya University,
b) Environment and Industrial Materials Research Division, QuBS, JAEA
1. Introduction
Monolithic columns have attracted increasing attention
and interest in the last decade, due to the low flow-resistance
and excellent mass transfer. The authors have so far been
researching monolithic columns, and have succeeded in
developing organic polymer-based monolithic columns for
reversed phase liquid chromatography and ion
chromatography. For the purpose of further expansion of
column choice, in the present study, the development of a
zwitterionic monolithic column for hydrophilic interaction
liquid chromatography (HILIC) was attempted by -ray
induced polymerization, and its separation performance was
investigated.
2. Experimental
0.7 g of [2-(Methacryloyloxy) ethyl]dimethyl-(3-sulfopropyl)ammonium
hydroxide (SPE) and 0.3 g of ethylene
dimethacrylate (EDMA) were dissolved uniformly into 2.0 g
of methanol. After degassing with nitrogen for 5 min, the
monomer solution was immediately filled into a silicosteel
tubing (0.5 mm i.d. × 100 mm long), whose inner surface
was pretreated with 3-methacryloxypropyltrimeth-oxysilane
to provide anchoring sites for the polymer. The tube was
sealed with septa at both ends, and then irradiated at 10 kGy
(dose rate: 5 kGy/h) using -rays from a 60 Co source at 0 °C.
After polymerization, the resulting monolithic column was
washed with ethanol and with water, and provided as the
zwitterionic monolithic column for HILIC.
3. Results and Discussion
The zwitterionic monolithic column possessing adequate
separation efficiency and high permeability was prepared by
-ray induced polymerization of SPE and EDMA in the
silicosteel tubing. Observation by scanning electron
microscopy revealed that the radiationally-produced
zwitterionic monolithic column had a more uniform 3D
framework structure than the thermally-produced
zwitterionic monolithic column. The radiationally-
produced zwitterionic monolith was composed of
interconnected homogeneous globules of ~2 m in diameter
and the through-pores of ~2 – 10 m. Figure 1 shows the
separations of common anions (IO 3 - , NO2 - , NO3 - ) on the
radiationally- and thermally-produced zwitterionic
monolithic column. As can be seen in Fig. 1, the column
efficiency of the radiationally-produced zwitterionic
JAEA-Review 2010-065
- 42 -
monolithic column was five times higher than that of
thermally-produced zwitterionic monolithic column. This
result could be ascribed to the improvement of specific
surface area that contributed to separation of sample and the
suppression of sample diffusion within the column by the
use of more uniform monolith. As a practical application,
the separation of urinary catecholamines and related
compounds (creatinine; Cre, homovanilic acid; HVA,
vanillylmandelic acid; VMA) was attempted by using the
zwitterionic monolithic column, and the results are shown in
Fig. 2. When laboratory reagents were used as analyses, a
series of analyses were baseline separated. Furthermore,
three compounds in a human urine sample could be
separated and its chromatogram provided good analytical
signal and enough sensitivity for peak identification.
(A)
(B)
(A)
(B)
0.05 Abs.
0.05 Abs.
0 1 2 3 4
Retention time / min
Fig. 1 Separations of three anions on (A) radiationally-
and (B) thermally-produced zwitterionic monolithic
columns.
0.1 Abs.
0.002 Abs.
(1)
(1)
(2)
(3)
(1)
(2)
(1)
(2)
(2) (3)
(3)
(3)
(1) IO 3 -
(2) NO 2 -
(3) NO 3 -
(1) Cre
(2) HVA
(3) VMA
0 2 4 6 8
Retention time / min
Column size: 0.5 mm i.d. ×
100 mm long, mobile phase:
water, flow rate: 12 L/min
(corresponding to a linear
velocity of 1 mm/s),
pressure drop: (A) 0.5 MPa
and (B) 0.3 MPa, sample:
10 mM each of IO3 - , NO2 -
and NO3 - , sample volume:
0.2 L, column temperature:
20 °C, UV detection at
210 nm.
Column size: 0.5 mm i.d. ×
100 mm long, mobile phase:
acetonitrile–5 mM
ammonium acetate (80:20,
v/v), flow rate: 12 L/min
(corresponding to a linear
velocity of 1 mm/s),
pressure drop: 0.3 MPa,
sample: (A) 10 mM each of
laboratory reagents and (B)
human urine sample (5 times
dilution), sample volume:
0.2 L, column temperature:
20 °C, UV detection at
214 nm.
Fig. 2 Separations of catecholamines and related
compounds in a human urine.