Separation and identification of mass-limited chemical samples is the key to understand the complex nature of pharmaceutical and environmental systems. High efficiency separation techniques, such as capillary electrophoresis (CE), coupled to a non-destructive, information-rich detection, such as nuclear magnetic resonance (NMR) spectroscopy, have revolutionized the ability to separate and identify components in small sample volumes. Using this hyphenated system, structure elucidation of analytes separated during an electrophoretic process can be performed using NMR as an on-line detector. Although sensitivity remains an issue for on-line NMR detection, capillary NMR spectroscopy using microcoils has emerged as a major breakthrough for increasing the mass-sensitivity of NMR spectroscopy, because the limit of detection is proportional to the coil diameter. A further development is the miniaturization of the magnet enabling the possibility of a truly portable NMR system. This portable, low-cost NMR sensor, coupled to the rapid CE separation system could enable high-throughput and on-site identification of nanoliter amounts of solution. Furthermore, CE can provide on-line pre-concentration via electrokinetic injection or other stacking techniques to increase the sample concentration.
In this research, coupling CE to a laboratory-scale NMR and to a portable NMR system are investigated, with the emphasis on the development of the miniaturized system. For the experiments with the large-scale 1H NMR system, a group of selected uric acids and xan- thines are studied since those compounds are representative of various classes of therapeutical drugs. The portable NMR instrument incorporates lithographically patterned microcoils and a small 1.8 T permanent magnet to measure 19F NMR spectra for the analysis of trifluoroacetic acid (TFA) and longer chain perfluorinated carboxylic acids (PFCAs). Our results demonstrate that coupling CE to a portable NMR system is feasible and can provide a low cost method to obtain structural information about the samples of interest. The results confirm that it was possible to acquire congruent sample data using the ultraviolet/visible (UV/VIS) detector of the CE and the 19F NMR detector. To obtain data, CE and NMR conditions were optimized and different modes of data acquisition were investigated.
Table of Contents
1 AIMS OF THE STUDY
2 STATE OF THE ART
2.1 SUBSTANCES FOR THE ANALYSIS
2.1.1 Uric Acids and Xanthine
2.1.1.1 Analytical Determination
2.1.2 Perfluorinated Organic Compounds
2.1.2.1 Analytical Determination
2.2 CAPILLARY ELECTROPHORESIS
2.2.1 Micellar Electrokinetic Chromatography
2.2.2 Detection Methods in CE
2.3 NUCLEAR MAGNETIC RESONANCE
2.3.1 19F NMR
2.3.2 Mass Sensitivity and Limits of Detection
2.3.3 Large-Scale NMR
2.3.4 Microcoil NMR
2.3.5 Portable NMR System
2.4 ON-LINE CE-NMR
3 EXPERIMENTAL
3.1 REAGENTS AND CHEMICALS
3.2 INSTRUMENTATION AND MATERIALS
3.2.1 CE System
3.2.2 Lab-Scale NMR Systems
3.2.3 Portable NMR System
3.3 METHODS FOR CE-NMR AND CE SEPARATIONS
3.3.1 Data Acquisition for CE and Lab-Scale CE-NMR
3.3.2 Data Acquisition for CE and Portable CE-NMR
4 RESULTS AND DISCUSSION
4.1 COUPLING OF CE WITH LAB-SCALE NMR SYSTEM
4.1.1 CE Separation for Xanthine and Uric Acids
4.1.1.1 Buffer Optimization and Substance Identification
4.1.1.2 Calibration of the Xanthines and Uric Acids
4.1.2 NMR Data Acquisition with the Dynamic, Flow-through Microprobe
4.1.3 Coupling of CE to the Flow-through Microprobe
4.1.4 Summary of Chapter 4.1
4.2 COUPLING OF CE WITH PORTABLE NMR SYSTEM
4.2.1 CE Separation of Fluorinated Organic Compounds
4.2.1.1 System Peaks
4.2.1.2 Comparison of Uncoated and Coated Capillaries
4.2.1.3 Continuous Flow Compared with Stop Flow Mode
4.2.1.4 CE Separation and Detection with High Sample Concentrations
4.2.2 Data Acquisition with the Portable CE-NMR System
4.2.2.1 Optimization of the NMR Acquisition Parameters
4.2.2.2 Determination of T1
4.2.2.3 Mass Sensitivity and Limit of Detection Measurements
4.2.2.4 Resolution and Prediction of 19F NMR Data
4.2.2.5 Challenges of Coupling CE to Portable NMR
4.2.2.6 Pre-concentration during the CE Injection
4.2.2.7 Stop Flow Data Acquisition
4.2.2.8 Continuous Flow Data of Pressure and Electrokinetic Injection
4.2.2.9 Temperature Stability
4.2.3 Summary of Chapter 4.2
5 CONCLUSION AND OUTLOOK
Research Objectives and Core Themes
This thesis aims to develop a hyphenated analytical system by coupling capillary electrophoresis (CE) with both large-scale and portable nuclear magnetic resonance (NMR) systems to achieve efficient separation and structural identification of mass-limited samples in pharmaceutical and environmental applications.
- Hyphenation of CE with micro- and nano-volume NMR spectroscopy.
- Optimization of CE separation parameters for xanthines, uric acids, and fluorinated compounds.
- Development of portable NMR systems using lithographically patterned microcoils.
- Investigation of on-line sample pre-concentration techniques for CE-NMR.
- Evaluation of data acquisition modes including stop-flow and continuous-flow analysis.
Excerpt from the Book
2.4 On-Line CE-NMR
NMR spectra exhibit intrinsically high information content in the acquired spectra of unknown sample mixtures which leads to challenging structure elucidation, because NMR signals might overlap. For that reason the experimental protocol typically requires a sample separation prior to the NMR measurements. If the mixture can be separated and analyzed by a two dimensional, on-line coupled chromatographic or electrophoretic techniques the resulting NMR spectra get less complex, structure elucidation gets easier, fewer transfer steps are necessary and no time-consuming pre-separation techniques have to be used. The small detection volumes associated with nano-volume microcoil NMR make it a feasible alternative for coupling NMR with a nano-volume separation technique like CE. Although the CE-NMR coupled technique using microcoil technology has an improved mass-sensitivity when compared to laboratory-scale systems, the concentration-sensitivity is poor. To maximize this, ITP is the most promising system of microcoil CE-NMR which can be used since it is an on-line pre-concentration and separation method (see Chapter 2.2). Furthermore, ITP provides a precise technique to position the sample plug in the active volume of the coil so that even smaller coils, in the nL-range, can be used to gain a higher mass-sensitivity. Due to these developments, pharmaceutical industries have already been stimulated and use this technique to solve problems in biochemistry and drug metabolism [1,3,4,25,52,61,62].
Summary of Chapters
AIMS OF THE STUDY: Defines the research goal of hyphenating CE to micro/nano NMR and optimizing parameters for different substance classes.
STATE OF THE ART: Reviews existing literature on xanthines, perfluorinated organic compounds, CE, NMR, and the development of on-line CE-NMR hyphenation.
EXPERIMENTAL: Details the reagents, instrumentation (including specific CE and NMR setups), and methodologies used for data acquisition.
RESULTS AND DISCUSSION: Presents experimental findings on coupling CE with lab-scale and portable NMR, focusing on separation optimization, data acquisition modes, and temperature stability.
CONCLUSION AND OUTLOOK: Summarizes the feasibility of the hyphenated system and suggests future improvements regarding sensitivity, resolution, and integration.
Keywords
Capillary Electrophoresis, Microcoil 1H NMR, Portable 19F NMR, On-Line CE-NMR, Perfluorinated Carboxylic Acids, Trifluoroacetic Acid, Xanthine, Uric Acids, Mass Sensitivity, Structural Elucidation, Sample Pre-concentration, NMR Spectroscopy, Analytical Chemistry, Hyphenated Techniques
Frequently Asked Questions
What is the primary objective of this research?
The research focuses on the hyphenation of capillary electrophoresis (CE) with both large-scale and portable nuclear magnetic resonance (NMR) spectroscopy to enable efficient separation and structural identification of mass-limited chemical samples.
What are the core thematic fields covered in this study?
The study spans analytical chemistry, pharmaceutical drug analysis, and environmental monitoring, specifically examining the coupling of micro-separation techniques with microcoil-based NMR detection.
What is the primary Forschungsfrage (research question)?
The work investigates the feasibility of coupling CE to different NMR systems and how to optimize separation parameters and acquisition modes to overcome sensitivity and resolution limitations for structural elucidation.
Which scientific methods are primarily utilized?
The study utilizes capillary electrophoresis (CE) as the separation method and nuclear magnetic resonance (NMR) spectroscopy (using both 1H and 19F nuclei) as the detection method, supported by mathematical models for sensitivity and diffusion calculations.
What is the main focus of the experimental section?
The experimental section covers the preparation of buffer systems for xanthine/uric acid and fluorinated compound separation, the setup of lab-scale and portable NMR instruments, and the investigation of dynamic/static data acquisition parameters.
Which keywords best characterize this work?
Key terms include CE-NMR hyphenation, microcoil NMR, mass sensitivity, perfluorinated compounds, on-line pre-concentration, and portable analytical instrumentation.
How does the portable NMR system overcome its size limitations?
The portable system uses compact permanent magnets (1.8 T) combined with lithographically manufactured microcoils wrapped around the capillary, which allows for small detection volumes and reduced cost/maintenance compared to superconducting magnets.
How is the temperature stability of the portable NMR addressed?
Temperature fluctuations in the portable magnets are managed by using a temperature controller, and software-based frequency shifting is employed to correct for temperature-induced drifts in the NMR spectra.
- Quote paper
- Joana Diekmann (Author), 2009, Coupling of Capillary Electrophoresis with Nuclear Magnetic Resonance Spectroscopy for the Analysis of Pharmaceutical and Environmental Relevant Compounds, Munich, GRIN Verlag, https://www.hausarbeiten.de/document/169088
