Chemistry Reference
In-Depth Information
group, and interact with ionized groups of the analytes. Because of the electrostatic interaction between the
analytes and the polymer modified stationary phase, the retentions are bigger at low temperature. By raising
the column temperature, the cation-exchange group may have been immersed in the hydrophobic polymer
chain, and the reduced electrostatic interaction may have caused decreased retention times.
Another separation method using a cationic monomer,
N,N
-dimethylaminopropylacrylamide (DMAPAAm),
instead of AAc, was also developed. A novel method for the qualitative analysis of adenosine nucleotides
(AMP, ADP and ATP) and synthetic oligonucleotides was proposed, utilizing the pH- and a temperature-
responsive polymer [42], P(NIPAAm-
co
-BMA-
co
-DMAPAAm) modified HPLC stationary phase (Figure
20.9b). On this column at pH 3.0, which is lower than the p
K
a
values of the adenosine nucleotides [AMP
(3.80), ADP (3.9), ATP (4.1)], the retention times at low temperature were small, and decreased with an
increase in the temperature. The hydrophobic interaction should contribute to the retention under the specific
condition. The electrostatic interaction may not have predominated, because the adenosine nucleotides were
undissociated. On the other hand, at pH 4.5, which is higher than p
K
a
values, the retention times increased
below the LCST, while they decreased above the LCST. Because the adenosine phosphate groups are
deprotonated in pH 4.5 condition, the electrostatic interaction between the adenosine nucleotides and the
polymer stationary phase should cause increased retention below the LCST. Because the polymer chain is in
an extended at a temperature lower than that of LCST, the DMAPAAm moiety should act as an anion-
exchange group and interact with the ionized group of the adenosine nucleotides. By raising the temperature,
the anion-exchange group may be immersed in the hydrophobic polymer chain, and the reduced electrostatic
1
4
2
3
CO
2
H
10°C
NH
2
H
L-Tryptophan (1)
3
4
2
1
HO
NH
2
Serotonin (4)
N
H
30°C
3
HO
NHCOMe
2
4
1
N
H
N
-Acetylserotonin (2)
MeO
NHCOMe
50°C
H
Melatonin (3)
0
10
20
30
40
Retention time (min)
Figure 20.10
Chemical structures and chromatograms of a mixture of tryptophan (1),
N
-acetylserotonin (2),
melatonin (3) and serotonin (4). HPLC condition: column, P(NIPAAm-
-AAc) modified column (4.6 mm
i.d. × 150 mm); mobile phase, 10 mM CH
3
CO
2
NH
4
(pH 6.0); flow rate, 1.0 ml min
−1
; detection at UV 275 nm.
co
-tBAAm-
co
