Analytical Chemistry Chemical Cytometry Quantitates Superoxide

FL, USA) with three-dimensional adjustment capabilities. A
cylindrical cathodic/detection reservoir (2 cm diameter ×1 cm
height) contained Pt wires (1 mm in diameter, 99.9% purity),
serving as the counter electrode for amperometric detection and
the cathode for electrophoresis. An Ag/AgCl (3 M NaCl) reference
electrode was placed vertically into the reservoir, whereas the
BDD electrode was inserted upward from the reservoir’s bottom
and sealed with epoxy (the working reservoir volume was ∼3 mL).
The micromanipulator and a laboratory jack (to which the
reservoir was solidly mounted) were attached to a solid breadboard
to prevent movement during alignment. The capillary outlet
was aligned to the detecting electrode using the micromanipulator
with the aid of a surgical microscope (World Precision Instruments).
The capillary outlet was adjusted until it touched the
electrode surface (evident by a slight bend in the capillary
observed by microscopic inspection) and it was then backed off
25-30 µm using the micromanipulator’s z-control. The BDD
electrode was connected to an electrochemical workstation
(CHI660C, CH Instruments, Austin, TX, USA) consisting also of
a platinum wire (1 mm in diameter) as counter electrode and an
Ag/AgCl (3 M NaCl) electrode as reference electrode. The BDD
electrode (3 mm in diameter, 0.1% doped diamond) was purchased
from Windsor Scientific (Slough, Berkshire, U. K.). The analytes
are detected by a boron doped diamond (BDD) electrode which
is positioned close to the capillary outlet. The BDD electrode is
advocated in this work due to its stable low background current
and a wide applied potential window. BDD is also resistant to
fouling due to the hydrogen surface termination and sp 3 carbon
bonding (no extended pi-electron system). 14a,b BDD can be
considered as general-purpose working electrodes with a broad
array of applications for use with HPLC and CE. Pioneering work
in the early 1990s was conducted by Swain and co-workers 14c,d
and the Fujishima group. 14e
The electrophoretic separation was conducted at -10 kV
(reversed polarity) unless otherwise stated. A plastic cap with a
central hole of ∼1 mm was firmly attached to the surface of the
BDD electrode to reduce the active sensing area. The analyte
sample was injected electrokinetically for 5sat-10 kV. Peak
identification was based on the migration time of a single standard
with that of unknown peaks. However, if the resolution between
any peak pair was low, then peak identification was performed
by spiking both solutes individually. The pKa values and aqueous
solubility of the analytes were obtained using the ACD/
Structure Designer software (Advanced Chemistry Development,
Toronto, ON, Canada). The degree of ionization was
estimated as pH ) pKa+ log [A - /AH] for acid and pH )
pKb+log [BH + /B] for base (the Henderson-Hasselbalch
equation).
RESULTS AND DISCUSSION
Performance of the PDDA Coated Capillary. PDDA was
firmly adsorbed on the inner walls of the capillary via ionic
interactions between the negatively charged SiO - of fused silica
and the quaternary ammonium groups of the polymer. Indeed,
immersion of a substrate (glass, quartz, silica wafer, gold, silver,
and even Teflon) into an aqueous 1% solution of this positively
charged polymer results in the strong adsorption of a mono-
6898 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
Figure 1. Electropherograms obtained using a PDDA coating
capillary (50 µm id and 45 cm effective length) for the separation of
20 µM IXS, 25 µM VMA, 25 µM HVA, 25 µM TRP, 100 µM
isoproterenol (ISP), 100 µM normetanephrine (NMN), 100 µM
epinephrine (EP), 50 µM 5-hydroxytryptamine (5-HT), 125 µM 4-hydroxy-3-methoxybenzylamine
(HMBA), and 75 µM tryptamine (TA).
The running buffer consisted of 50 mM H3PO4-Tris, (a) pH 3, (b) pH
4, and (c) pH 5. The separation voltage was applied at -10 kV with
an injection time of 5s at -10 kV. BDD at +1.0 V vs Ag/AgCl, 3 M
NaCl.
layer (1.6 nm) of PDDA on the substrate. 15,16 The adsorption
of a PDDA thin film on a glass substrate was also reported
elsewhere. 17 Notice also that the charge of PDDA is not pH
dependent, as reflected by constant EOF in the range pH 2-8
provided the capillary is coated with high-molecular weight
PDDA. 18 Thus, PDDA with MW of 200 000-350 000 was used in
this study for coating the capillary.
At -10 kV, except for the epinephrine (EP) and normetanephrine
(NMN) pair, all analytes were baseline resolved when 50 mM
H3PO4-Tris pH 3.0 was used as the running buffer (Figure 1,
curve a). On the basis of the calculated pKa values for the analytes
(Table 1), fully deprotonated and highly negatively charged IXS
exhibited high electrophoretic mobility and migrated concomitantly
with EOF as the first peak in the electropherogram. The
carboxylate/carboxyl ratio, estimated as 10 (pH-pK a) ,is∼0.16 and
0.04 for VMA and HVA, respectively. Thus, VMA should
emerge before HVA and slightly ahead of EOF. At pH 3, the
neutral form of TRP should be predominant (neutral TRP/TRP +
) 4.2);therefore, it should migrate very closely to EOF and
trail behind both VMA and HMA. The EP-NMN pair was
slightly split further by conducting the separation at pH 4 with
improved detection sensitivity but the running time was also
slightly longer and VMA emerged very close to IXS (Figure 1,
curve b). The run was lengthier at pH 5, with only 4 discernible
peaks emerging in the electropherogram as HVA comigrated with
VMA and TRP became more neutral at this pH and emerged far
behind the HVA peak. The remaining analytes acquired more
negative charges and interacted strongly with positively charged
PDDA and were not eluted after 1200 s into the experiment. The
detection sensitivity was also greatly compromised at this running
(15) Kotov, N. A.; Harazsti, T.; Turi, L.; Zavala, G.; Geer, R. E.; Dekany, I.;
Fendler, J. H. J. Am. Chem. Soc. 1997, 119, 6821–6832.
(16) Moriguchi, I.; Teraoka, Y.; Kagawa, S.; Fendler, J. H. Chem. Mater. 1999,
1, 1603–1608.
(17) Hrapovic, S.; Liu, Y.; Enright, G.; Bensabaa, F.; Luong, J. H. T. Langmuir
2003, 19, 3958–3965.
(18) Wang, Y.; Dubin, P. L. Anal. Chem. 1999, 71, 3463–3468.