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Chemistry Letters Vol.32, No.1 (2003)
Ru(bpy)33þ Electrochemiluminescence Detection of Aliphatic and Aromatic Amines with Diketene
Kazuo Uchikura
College of Pharmacy, Nihon University,
7-7-1, Narashinodai, Funabashi, Chiba 274-8555
(Received August 28, 2002; CL-020732)
A new derivatization method for the detection of aliphatic
and aromatic amines, based on electro-chemiluminescence
(ECL) with Ru(bpy)3 is proposed. Sample amines were
derivatized to acetoacetylamide-type derivatives using diketene.
to stand at room temperature for 5 min for aliphatic amines and
90 min for aromatic amines. Twenty microlitres of the reaction
mixture was injected onto the HPLC apparatus. The ECL
intensity was measured using a Ru(bpy)32þ ECL detectionHPLC
system4 consisting of an intelligent micropump (Model-301,
FROM, Tokyo, Japan), a Rheodyne sample-injection valve
(Cotati, C.A., U.S.A.), anECL detector (COMET-3000KANA-
GAWA, Kawasaki, Japan) and a recorder (Chromatopac CR-5A,
Shimadzu, Kyoto, Japan). During current controlled (galvano-
static) electrolysis, the current was maintained at 80 mA. These
components were connected with stainless steel tubing (0.3 mm
i.d.). The mobile phase was a mixture of 0.1 M acetic acid and
acetonitrile (7 : 3, v/v), and the flow rate was 0.5 ml/min. The
reagent solution was 0.1 M sulfuric acid containing 0.3 mM
Ru(bpy)3Cl2, and the flow rate was 0.3 ml/min. The column was a
Spherisorb ODS 5 (150 mm  0:4 mm).
2þ
The derivatives were detected by reversed-phase HPLC with ECL
2þ
detectionusing Ru(bpy)
.
3
Amines are probably the most heavily studied group of
reagents for chemical derivatization because of their biological,
industrial, and environmental importance.1 The development of
new method for their quantitation is therefore necessary.
Chemiluminescence is an attractive detection technique for
HPLC due to its very low detection limits and wide linear working
range, whilst using relatively simple instrumentation. It has been
found to be an important new detection system for
analytical applications. The tris(2,20-bipyridine)ruthenium(II)
(Ru(bpy)32þ) ECL system is useful as a selective and sensitive
The derivatizationreactionfor the primary amien with
diketene is shown in Scheme 1.
2
detectionmethod for oxalate, indoles,3 diketones,4;5 amino
acids6;7 and trialkylamines.8 We previously reported onthe ECL
R-NH2
+
(CH2CO)2
R-NHCOCH2COCH3
9
detectionof alicyclic tertiary amines
at the pmol level using
Scheme 1.
Ru(bpy)32þ, although the ECL intensity of aliphatic and aromatic
primary amines could only be detected at a level approximately
1000-times lower thanthat of tertiary amines. We have reported
that ꢀ-diketone4 was detectable with a high degree of sensitivity
and the detection of carbonyl compounds5 with methylmalonic
acid has been developed. Recentry, a labeling reagent for
carboxylic acid and amines, having tertiary amine moiety, was
developed.10 As the background noise increased with increasing
pH, it is desirable for it to be derivatized to emit ECL at a low pH
range.
The conditions of the derivatization reaction, such as the pH,
diketene concentration, reaction temperature and reaction time
were optimized in terms of the ECL intensity of samples of
cyclohexylamine and 4-chloroaniline. The effect of pH on the
derivatization reaction was investigated over a range of pH values
from 6.0–13. In the pH range 9.0–13, diketene was degraded to an
ECL active substance that interfered with the measurements. The
amount of degradation decreased with a decrease in the pH, and
accordingly the pH 6.0–8.5 was perferred. The derivatization
reaction of cyclohexylamine with diketene apparently occurred
eveninthe pH range from 6.0 to 8.5 at room temperature. The
reaction time and temperature was investigated (Figure 1). A
higher temperature allowed the ECL intensity to develop slightly
more rapidly. The ECL intensities reached a plateau at 5 min for
cyclohexylamine and at 90 min for 4-chloroaniline. The effect of
the concentration of the diketene was also investigated. The
amount of derivativeproduced increasedwithincreasing diketene
concentration till it was over a three–fold molar excess with the
amine, when it reached a plateau. The pH of the ECL reaction had
an effect on the ECL intensity in the Ru(bpy)32þ ECL system. The
effects of pH on the ECL intensity and noise were investigated for
a pH range from 1–2.5, which was controlled by the addition of
sulfuric acid (Figure 2). The ECL intensity increased with
decreasing pH, but the noise was found to be a minimum at pH
1.5. The inclusion of an organic modifier also affected the ECL
intensity of all the amines investigated. It increased with
increasing concentration of acetonitrile. The ECL intensity of
amines was measured by FIA on an HPLC minus the column. The
ECL intensity of amines is shown in Table 1. The intensity of n-
Inthis paper, we report a new method for the detectionof
amines after derivatization. Sample amines were modified to
acetoacetylamide-type derivatives using diketene. The deriva-
tives were detected by reversed-phase HPLC with ECL using
2þ
Ru(bpy)3
.
Chemicals and standard solutions used were as follows.
Ru(bpy)3Cl2 was obtained from Sigma (St. Louis, M.O., U.S.A.)
and usedwithout furtherpurification. Diketene was obtained from
Wako Pure Chemical Industries Ltd. (Osaka, Japan). Sample
amines were obtained from Tokyo Chemical Industry Co., Ltd.
(Tokyo, Japan). All of the other chemicals and solvents were of
guaranteed grade. Water was de-ionized and distilled using a
hard-glass vessel. A stock standard solution (100 mM) of the
amines was prepared by dissolving them in methanol. A working
solutionwas prepared by appropriate dilutionof the stock solution
before use with a mixture of water and methanol (1 : 1, v/v).
The derivatizationprocedure was as described below. Five
ml of a mixture of 0.1 M borate buffer (pH = 7.0) and acetonitrile
(1 : 1, v/v), 10 microliters of the sample amines and 10 microliters
of diketene were added in a tube and mixed. The mixture was left
Copyright ꢀ 2003 The Chemical Society of Japan