Capillary electrophoresis for the analyses of amines, aristolochic acids and DNA
Date Issued
2006
Date
2006
Author(s)
Huang, Ming-Feng
DOI
en-US
Abstract
Abstract
This thesis focuses on developing efficient and sensitive capillary electrophoresis-laser induced fluorescence (CE-LIF) techniques for amines, aristolochic acid, and DNA. We developed a novel method for the analysis of amines under acidic conditions by CE in conjunction with indirect laser-induced fluorescence (CE-ILIF). The analysis of six amines by CE-ILIF using a solution, pH 3.5, containing 5.0% methanol, 0.1 mM sulfuric acid, 0.1 mM cresyl violet, and 0.3 mM lithium was complete in 5 min, with the limits of detection (LOD) on the level of µM.To further improve the sensitivity, on-line concentration based on pH junction has
been demonstrated. When injecting the sample prepared in a solution of 0.2 mM sulfuric acid, pH 3.3, at 15 kV for 60 s to the above-mentioned solution, the LODs for the amines down to sub µM and the sensitivity improvements up to 19-fold when compared to that injecting at 15 kV for 5 s.
Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Two predominant forms of AA are 8-methoxy-6-nitrophenanthro-(3,4-d)-1,3-dioxolo-5-
carboxylic acid (AA-I) and 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AA-II). Owing to lack of intrinsic fluorescence characteristics of oxidized AAs
(OAAs), the RAAs reduced from OAAs in 10.0 mM HCl containing iron powder is required prior to CE analysis. The RAAs exhibit fluorescence at 477 nm when excited
at 390 nm. By using 50.0 mM sodium tetraborate (pH 9.0) containing 10.0 mM SDS,the determination of aristolochic acid I and aristolochic acid II by CE-LIF has been
achieved within 12 min. The CE-LIF provides the LODs of 8.2 and 5.4 nM for AA-I and AA-II, respectively. The successful analysis of 61 medicinal samples and dietary supplements shows that the present CE-LIF is practical for the determination of AA-I and AA-II in real samples.
Reproducible, rapid, and high-resolution DNA separations have been achieved using low-viscosity PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO(8 MDa) containing 56-nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp
using 0.05% PEO(2 MDa) containing 13-nm GNPs or 0.05% PEO(4 MDa) containing 32-nm GNPs.
To separate long double strands of DNA by CE, with high efficiency, high speed, simplicity, and reproducibility, we describe a CE technique, which we call nanoparticle-filled CE (NFCE), using polymer-modified gold nanoparticles (GNPPs)
in this topic. The gold nanoparticles were modified with poly(ethylene oxide) via noncovalent bonding to form GNPPs. The separations of high molecular weight (HMW) DNA with sizes ranging from 8.27 to 48.5 kbp and λ DNA (0.12–23.1 kbp) were accomplished within 6 and 5 min, respectively. The separation speed and resolution are greater than those by pulsed CE and slab gel electrophoresis. This is the first example for separating such high DNA molecules by CE-LIF using GNPPs.
The results present in this thesis clearly demonstrate that CE-LIF based techniques are practical for analysis of amines, aristolochic acid, and DNA, with the advantages of rapidity, sensitivity, and reproducibility. The examples of separating DNA using PEO containing GNPs and GNPPs open the avenue of using nanoparticles for improved resolution and reproducibility for analysis of DNA. It is our strongly
belief that the technique should be further applied to analysis of small solutes such as amines and acids and macromolecules like proteins.
Subjects
毛細管電泳
胺
馬兜鈴酸
DNA
Capillary
elecrophoresis
amines
aristolochic acids
SDGs
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-95-D89223027-1.pdf
Size
23.31 KB
Format
Adobe PDF
Checksum
(MD5):217da3bcf3f854a3d46bcd87a1a0cab4