Coupling of Capillary Electrophoresis with Nuclear Magnetic Resonance Spectroscopy for the Analysis of Pharmaceutical and Environmental Relevant Compounds E-Book

Joana Diekmann

• Herausgeber: GRIN Verlag
• Sprache: Englisch

Veröffentlichungsjahr: 2011

Beschreibung

Master's Thesis from the year 2009 in the subject Chemistry - Analytical Chemistry, grade: 1,0, University of Hannover (Anorganische Chemie), language: English, abstract: 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.

Leseprobe

Table of Content

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

2.3.2 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 T 1

4.2.2.3 Mass Sensitivity and Limit of Detection Measurements

4.2.2.4 Resolution and Prediction of 19 F 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

6 REFERENCES

7 APPENDIX

7.1 CONCENTRATION OF THE URIC ACID AND XANTHINE MIXTURES

7.2 ACQUIRED DATA FOR T 1 CALCULATIONS ON THE 500 MHZ NMR SYSTEM

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This work was carried out between 30. March and 29. September 2009 at the following two

institutions:

Lawrence Livermore National Laboratory

Physical and Life Sciences Directorate

11. May 2009 to 7. August 2009

Chemical Sciences Division

7000 East Avenue

Livermore, CA 94550

USA

www.llnl.gov

Leibniz University Hannover

Faculty of Natural Sciences

30. March 2009 to 8. May 2009 and 11. August 2009 to 29. September 2009

Institute of Inorganic Chemistry

Department of Analytical Chemistry

Callinstraße 1

30167 Hannover

Germany

www.analytik.uni-hannover.de

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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, informationrich 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 preconcentration 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-scale1H NMR system, a group of selected uric acids and xanthines 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 measure19F NMR spectra for the analysis of trifluoroacetic acid (TFA) and longer chain perfluorinated carboxylic acids (PFCAs). These fluorinated com-pounds are of environmental concern, due to their potential to accumulate in some aquatic ecosystems and their environmental persistence. Since it has been shown that TFA potentially has toxic effects, it is important to monitor the environment and the products intended for human use, such as rain proofed Gore-Tex garments, for perfluorinated compounds. 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 the19F NMR detector. To obtain data, CE and NMR conditions were optimized and different modes of data acquisition were investigated.

Keywords:Capillary Electrophoresis, Microcoil1H NMR, Portable19F NMR, On-Line CE- NMR, Perfluorinated Carboxylic Acids, Trifluoroacetic Acid, Xanthine, Uric Acids

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Die Trennung und Identifizierung von sehr kleinen Mengen chemischer Proben ist notwendig um komplexe pharmazeutische und ökologische Systeme zu verstehen. Hoch effiziente Trennverfahren, wie z.B. Kapillarelektrophorese (CE), gekoppelt mit einer nichtdestruktiven, informationsreichen Detektion, wie Kernspinresonanz (NMR) Spektroskopie, haben die Fähigkeit, kleine Probenvolumina zu trennen und zu identifizieren. Mit diesem gekoppelten System ist die Strukturaufklärung von Analyten, die in einem elektrophoretischen Lauf getrennt werden, mittels NMR als on-line Detektor möglich. Obwohl die Empfindlichkeit für die on-line NMR-Detektion ein Problem bleibt, stellt die Kapillar-NMR-Spektroskopie mit Mikrospulen einen wichtigen Durchbruch für die Erhöhung der Massenempfindlichkeit in der NMR-Spektroskopie dar, da die Nachweisgrenze proportional zum Spulendurchmesser ist. Eine weitere Entwicklung ist die Miniaturisierung des Magneten, um ein portables NMR-System zu realisieren. Dieser tragbare, kostengünstige NMR-Sensor, der an das CE Trennsystem gekoppelt ist, könnte ein Hochdurchsatz-Verfahren und eine lokal flexible Bestimmung von Proben im Nanoliter-Maßstab ermöglichen. Darüber hinaus bietet CE eine on-line Aufkonzentration mit Hilfe von elektrokinetischer Injektion oder anderen Stapeltechniken, um die Probenkonzentration zu erhöhen. In diesem Forschungsbericht werden beide Ansätze, die Kopplung von CE mit einem NMR-Gerät im Labormaßstab und mit einem tragbaren NMR-System untersucht, wobei der Schwerpunkt der Arbeit auf dem miniaturisierten System liegt. Für die Versuche mit dem großen1H-NMR-System, wird eine Gruppe von ausgewählten Harnsäuren und Xanthinen untersucht, da diese Verbindungen Vertreter von verschiedenen Klassen therapeutischer Arzneistoffe sind. Das tragbare NMR-Gerät enthält lithographisch angefertigte Mikrospulen und einem kleinen 1,8 T Permanentmagneten, um19F-NMR-Spektren für die Analyse von Trifluoressigsäure (TFA) und längeren perfluorierten Carbonsäuren (PFCAs) zu messen. Diese fluorierten Verbindungen sind ökologisch bedenklich, da sie sich in aquatischen Ökosystemen anreichern können und in der Umwelt nicht abbaubar sind. Aufgrund der toxischen Wirkung von TFA, ist es wichtig die Umwelt und Produkte, die für den menschlichen Gebrauch bestimmt sind, wie z.B. wasserfeste Gore-Tex Bekleidung, auf perfluorierte Verbindungen zu überwachen.

Unsere Resultate zeigen, dass die Kopplung von CE mit einem tragbaren NMR System eine kostengünstige Methode bietet, um strukturelle Informationen über die interessierenden Proben zu erhalten. Die Ergebnisse bestätigen dass es möglich war vergleichbare Probendaten mit dem ultraviolett/sichtbaren (UV/VIS) Detektor der CE und dem19F-NMR-Detektor zu bekommen. Für diese Messungen wurden die CE und NMR Parameter optimiert und verschiedene Verfahren zur Messung der Daten angewandt.

Stichwörter:Kapillarelektrophorese, Mikrospulen1H-NMR, Portable19F-NMR, On-Line CE-NMR, Perfluorierte Carbonsäuren, Trifluoressigsäure, Xanthine, Harnsäure

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I would like to thank all the persons who provided support and encouragement throughout the pursuit of this project. In particular:

I would like to express my sincere gratitude to my home supervisor Prof. Dr. Carla Vogt for the continuous support of my study and research. My sincere thanks also go to my LLNL local supervisor Dr. Gregory L. Klunder for offering me the summer internship and the opportunities to work in his group, for his patience, motivation, enthusiasm and immense knowledge.

My special thanks are dedicated to Dr. Julie Herberg and Dr. Kristl Adams for their technical advice with the experimental work, for their encouragement, insightful comments, and excellent support in every sense. Moreover I would like to thank both of them for reading my thesis and providing inspiration. Thank you!!!

I would like to thank Prof. Dr. Helmut Duddeck for being my second supervisor and introducing me into the NMR.

Furthermore I would like to thank Mrs. Marry C. Jessup for arranging my stay at the Lawrence Livermore National Laboratory (LLNL) and the German Academic Exchange Service (DAAD) for the financial support during my researches at the LLNL.

Special thanks to my friends and colleagues of the “AK Analytik” for the joyful time that we have spent together and their encouragement and help in a lot of different ways.

Last but not least I would like to thank my family and friends for the encouragement during my Master study and supporting me spiritually throughout my life.

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BGE background electrolyte CE capillary electrophoresis CFC chlorofluorocarbons cHPLC capillary high performance liquid chromatography cLC capillary liquid chromatography CMC critical micelle concentration CPMG Carr-Purcell-Meiboom-Gill CW continuous wave DFA difluoroacetic acid EM electromagnetic EOF electroosmotic flow FID free induction decay FT Fourier transform GC gas chromatography HPLC high performance liquid chromatography HTAB hexadecyltrimethyl-ammoniumbromide IC ion chromatography ID internal diameter ITP isotachophoresis LC liquid chromatography LOD limits of detections MEKC micellar electrokinetic chromatography MS mass spectroscopy nLOD normalized limits of detections NMR nuclear magnetic resonance OD outer diameter PFCA perfluorinated carboxylic acid PFOA perfluorooctanoic acid PFPA perfluoropentanoic acid PTFE polytetrafluoroethylene RF radio frequency RMS root mean square SD standard deviation SDS sodium dodecylsulfate SNR signal-to-noise ratio TFA trifluoroacetic acid TFMHB 3-trifluoromethyl-3-hydroxybutyric acid UV/VIS ultraviolet/visible

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B0magnetic fieldB1magnetic field of the coilB2second local magnetic fieldcconcentrationcrealactual concentrationdcoilcoil diameterδchemical shift in ppmΔEenergy differenceγmagnetogyric ratiohPlanck constanticurrentmspinsMmagnetizationM0net magnetizationNαhigher Boltzmann energy stateNβlower Boltzmann energy stateυradio frequency µEPelectrophoretic mobilityRresolution R2correlation coefficientSmmass sensitivityT1spin-lattice relaxation timeT2spin-spin relaxation timetacqacquisition timetmigratemigration timeVtottotal sample volumeVobsobserved sample volume in RF coil