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Franklin Ifeanyichukwu Uba

CHEM 7750 - Separation science


Objective
To develop a new and sensitive CE-ECL
method for the analysis of Enrofloxacin and
Ciprofloxacin in milk samples
CE Principle

http://www.authorstream.com/Presentation/abikesh086-174281-capillary-electrophoresis-education-ppt-powerpoint/
Electro-chemiluminescence
• Excitation results from
electrochemical reaction
• Widely used in clinical analytes
• Ru(bpy)32+; emits photons-620nm
Ru(bpy)32+ + e- Ru(bpy)3+
Ru(bpy)32+ - e- Ru(bpy)33+
Ru(bpy)3+ + Ru(bpy)33+ Ru(bpy)32+* + Ru(bpy)32+
Ru(bpy)32+* Ru(bpy)32+ + hν

Alan lee, et.al. Proc. Of SPIE Vol . 5331, 2004.


Analyte - metabolism

N - dealkylation
N - Alkylation

Enrofloxacin (ENR) Ciprofloxacin (CIP)

• Quinolones – as synthetic antibiotic


• ENR - treatment of respiratory and Gastro-intestinal infection
• CIP – active metabolite
• CIP and ENR amine stimulants
• MRL values for ENR plus CIP in food stuffs (animal origin)
in the range 100–300 μg/kg
ECL Principle
(CIP or ENR) H+
e-
+.
P o r E NR )
(CI
Electrode Ru(bpy)3
2+
.
(CIP or ENR)
e- 3+
Ru(bpy)3
(CIP or ENR)

2+
Ru(bpy) 2+*
Ru(bpy) 3 3

hν λ = 620 nm
Experiment
• Solid phase extraction
• Electro-kinetic injection – HV 10 kV, 20 s, RT
• Uncoated Fused-Silica – 50cm, 50μm, 360μm
• Separation voltage 14 kV
• 15 mM running buffer
• MPI-A CE-ECL detector – 3-electrode system
– Working electrode; Pt wire (300-μm dia.)
– Reference electrode; Ag/AgCl (KCl saturated)
– Counter electrode; Pt wire
– PMT to capture photons (800 V)
Effect of Detection potential
Effect of running buffer pH

a = pH 7
b = pH 7.5
c = pH 8
d = pH 8.5
e = pH 9
Injection time - ECL intensity and Resolution

a = CIP
b = ENR
c = Rs
Linearity

Optimum conditions
Milk Samples

a = Blank Milk
b = Blank milk spiked; 15μg/kg CIP and 15μg/kg ENR;
Conclusions
• ECL detector is sensitive, simple and cheap
detection with a low background noise
• Detection limits of ENR (10 ng/ml) and CIP (15
ng/ml) were achieved
Acknowledgments

• Dr S.D Gilman
• CHEM 7750 Colleagues

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