7
26
Chemistry Letters Vol.38, No.7 (2009)
High-sensitivity HPLC Quantification of Nonfluorescent but Photolabile Analyte
through Photoreversion in Fluorescence Detector
1
2
3
ꢀ4
Masaki Nishijima, Takehiko Wada, Koushi Nagamori, and Yoshihisa Inoue
Center for Advanced Science and Innovation, Osaka University, 2-1 Yamada-oka, Suita 565-0871
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai 980-8577
1
2
3
Osaka Service Center, JASCO, 4-12 Banzai-cho, Kita-ku, Osaka 530-0028
Department of Applied Chemistry, Osaka University, 2-1 Yamada-oka, Suita 565-0871
4
(Received April 21, 2009; CL-090399; E-mail: inoue@chem.eng.osaka-u.ac.jp)
COOH
COOH
Ultrahigh sensitivity chiral HPLC analysis of nonfluorescent
COOH
h
ν
>320 nm
R2
R1
+
R4
R3
cyclodimers of 2-anthracenecarboxylic acid (AC) was achieved
with much improved accuracy and reproducibility through a
nonconventional on-detector photoreversion/re-excitation/de-
tection mechanism. Excitation of cyclodimers at 254 nm in the
detector cuvette caused photoreversion to AC, in situ excitation
of which led to strong fluorescence at longer ꢀ. The sensitivity
was enhanced by a factor of ca. 2000 compared to usual UV de-
tection.
hν
254 nm
AC
1: R1 = COOH, R2 = H
2*: R1 = H, R2 = COOH
3*: R3 = COOH, R4 = H
4: R3 = H, R4 = COOH
Scheme 1. Photocyclodimerization of 2-anthracenecarboxylic
acid (AC) upon irradiation at >320 nm and photoreversion of
cyclodimers upon irradiation at 254 nm.
4
1
.4 10
.2 10
1
4
4
1
High-performance liquid chromatography (HPLC) is one of
the most prevalent instruments for analyzing a variety of inor-
ganic, organic, and biological species. In particular, chiral HPLC
has become more popular in recent years and a number of chiral
stationary phases are now available for the separation of diverse
6
5
5
5
5
1
10
1 10
8 10
6 10
4 10
8000
2
*
5
4
4
4
4
6000
1
8
6
4
10
10
10
10
4
4
000
000
0
3*
1
2
chiral compounds. Simultaneously, a range of optical detectors,
AC
sensing the change in refractive index, UV–vis absorption, circu-
lar dichroism, and fluorescence, have been developed for quan-
titative analysis with better sensitivity, accuracy, and reproduci-
bility. Among them, fluorescence (FL) detection is practically
the most sensitive and can analyze a trace amount (down to
sub-pg) of sample with high signal-to-noise ratio, as long as
the analyte fluoresces in reasonable quantum efficiency. How-
ever, if the analyte is nonfluorescent or poorly fluorescent, FL
detection becomes useless unless the analyte is labeled with a
fluorescent tag.2
UV2
2
10
FL 0
2 10
AQ
0
UU VV 11
0
20
40
60
80
100
120
140
Retention time/min
Figure 1. Typical chromatograms of a photolyzate, which con-
tains AC, cyclodimers 1–4, and by-product 2-carboxyanthraqui-
none (AQ), recorded by a series of UV1 (254 nm), FL (excitation
at 254 nm, emission at 420 nm), and UV2 (386 nm) detectors
(from bottom to top).
In our study to reveal the detailed mechanism of supramo-
lecular [4 þ 4] photocyclodimerization of 2-anthracenecarbox-
3
ylic acid (AC) with serum albumins, we intended to precisely
determine the relative yield and enantiomeric excess of photo-
cyclodimers at very low conversions. For that purpose, we em-
ployed an FL detector in chiral HPLC analysis to find a remark-
able enhancement of sensitivity, accuracy, and reproducibility,
which is assignable not to the cyclodimer fluorescence but to
the fluorescence of AC generated by photoreversion upon exci-
tation of the cyclodimers in the detector cuvette. This new detec-
tion method, utilizing a series of photochemical and photophys-
ical on-detector events, i.e. the photoreversion of cyclodimers to
AC followed by the re-excitation and fluorescence of produced
AC, provides us with a powerful and practical tool for signifi-
cantly expanding the range of fluorescence detection without
using tedious pretreatment or fluorophore-labeling techniques.
A THF solution of AC (7.5 mM) was irradiated for 12 h
under Ar at >320 nm with a 300-W high-pressure Hg lamp fitted
with a uranium filter to give a photolyzate (Scheme 1), which
was subjected to chiral HPLC analysis on a JASCO LC-2000
Plus equipped with a tandem column of Cosmosil 5C18-AR-II
(4:6 mm ꢁ 150 mm, Nacalai) and Chiralcel OJ-RH (4:6 ꢁ 150
ꢂ
mm, Daicel) at 35 C. The column was eluted with a 64:36
(v/v) mixture of deionized water and acetonitrile containing
ꢃ1
0.1% trifluoroacetic acid at a rate of 0.5 mL min . The effluent
from the column was analyzed by a triple detection system com-
posed of the first UV–vis (UV1; JASCO UV-2075; detection at
254 nm), fluorescence (FL; JASCO FP-2025; excitation at 254
nm and emission at 420 nm), and the second UV–vis (UV2;
JASCO UV-970; detection at 386 nm) detectors. The wave-
lengths were carefully chosen and set at 254 nm for detection
of aromatics by the UV1 detector. The excitation for the FL de-
tector was set at 254 nm to achieve the photoreversion of cyclo-
4
,5
dimers and excitation of regenerated AC and at 420 nm for de-
tection of regenerated AC. The UV2 detector was set at 386 nm
for selective detection of regenerated AC. The photoreversion
occurred not in UV1 but in the FL detector, because of the in-
tense Xe lamp compared to the D lamp used in the UV detector.
2
Copyright ꢀ 2009 The Chemical Society of Japan