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Coupling of Capillary Electrophoresis with Nuclear Magnetic Resonance Spectroscopy for the Analysis of Pharmaceutical and Environmental Relevant Compounds
107 Seiten, Note: 1,0
Chemie - Analytische Chemie
List of Symbols
Motivation and Introduction
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
2.2 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
3.1 Reagents and Chemicals
3.2 Instrumentation and Materials
3.2.1 CE System
3.2.2 Lab-Scale NMR Systems
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
4.1 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
5 Conclusion and Outlook
7.1 Concentration of the Uric Acid and Xanthine Mixtures
7.2 Acquired data for T1 calculations on the 500 MHz NMR system
Statement of Authorship
illustration not visible in this excerpt
Figure 2.1: Chemical structures of the compounds used in this study: a) uric acid, b) 1-methyluric acid, c) 3,7-dimethyl uric acid, d) caffeine, e) xanthine, f) theophylline, and g) theobromine
Figure 2.2: Chemical structures of the fluorinated compounds used in this study: a) TFA, b) PFPA, c) PFOA
Figure 2.3: Schematic diagram of a typical CE set-up
Figure 2.4: Separation mechanism based on interactions of analytes with micelles in MEKC [36]
Figure 2.5: Indirect UV detection in a separation medium [36]
Figure 2.6: Energy level diagram for spin ½ states in a magnetic field
Figure 2.7: A block diagram of the pulsed NMR apparatus [42]
Figure 2.8: CapNMR -FlowProbeTM and the microcoil [45]
Figure 2.9: Left: Picture of a 0.7 T and a 1.8 T Aster Enterprise magnet with size scale. Right: Picture of a superconducting 500 MHz NMR magnet
Figure 2.10: Schematic diagram of a saddle coil (left) and a solenoid coil (right) [57]
Figure 2.11: Picture of the coupled portable NMR and CE system
Figure 3.1: Picture of the portable laser-lathe lithography microcoil NMR system with a 1.8 T Aster Enterprise magnet, a homebuilt probe with a translation stage for its positioning and a Tecmag spectrometer
Figure 3.2: a) Schematic overview of the laser-lathe lithography microcoil manufacturing process [60]. b) Resulting laser-lathe lithography 1 mm OD coil on a capillary (size comparison to a human eye)
Figure 3.3: Schematic pulse sequence acquired with the NTNMR software: pw = pulse width, rd = ring down, ad = acquisition delay, Acq. Time = acquisition time
Figure 3.4: Schematic diagram for the setup of the large-scale coupled CE-NMR system [52]
Figure 3.5: Schematic diagram of the setup for the portable CE-NMR system
Figure 3.6: Schematic figure of the capillary within a capillary configuration
Figure 4.1: Sample Mixture 2 with seven xanthines and uric acids separated with Buffer 1 (10 mM phosphate, pH 4.60) in a 50 µm ID capillary (60 cm long, 3 s injection, 0.5 psi)
Figure 4.2: Sample Mixture 2 with seven xanthines and uric acids separated with Buffer 2 (10 mM borate, pH 8.25) in a 50 µm ID capillary (60 cm long, 3 s injection, 0.5 psi)
Figure 4.3: Sample Mixture 1 with seven xanthines and uric acids separated with Buffer 4 (50 mM borate, 100 mM SDS, pH 9.30) in a 50 µm ID capillary (58.5 cm long, 4 s injection, 0.6 psi)
Figure 4.4: Sample Mixture 1 with seven xanthines and uric acids separated with Buffer 5 (24 mM borate, 46 mM SDS, 10 % methanol, pH 8.20) in a 50 µm ID capillary (58.5 cm long, 4 s injection, 0.6 psi)
Figure 4.5: Sample Mixture 2 with seven xanthines and uric acids separated with Buffer 3 (10 mM phosphate, 10 mM borate, 45 mM SDS, pH 8.25) in a 50 µm ID capillary (58.5 cm long, 4 s injection, 0.6 psi)
Figure 4.6: Sample Mixture 2, 3, and 4 with seven xanthines and uric acids separated with Buffer 4 (50 mM borate, 100 mM SDS, and pH 9.30) in a 50 µm ID capillary (58.5 cm long, 4 s injection, 0.6 psi)
Figure 4.7: Chemical structure and theoretical prediction of the chemical shifts of caffeine
Figure 4.8: 1H NMR spectrum acquired with the flow-through microprobe of 0.25 mM caffeine diluted in D2O (dynamic flow rate 5 µL/min, 600 MHz NMR spectrometer)
Figure 4.9: Chemical structure and theoretical prediction of the chemical shifts of SDS
Figure 4.10: 1H NMR spectrum acquired of the 50 mM borate buffer diluted in D2O with 4 mM and 8 mM SDS concentration. Left: dynamic flow-through microcoil (dynamic flow rate 5 µL/min and 600 MHz NMR spectrometer). Right: static microliter NMR (500 MHz NMR spectrometer).
Figure 4.11: The electropherograms of a 0.218 mM TFA and 0.757 mM acetic acid mixture at 214 nm with 5 mM phthalic acid buffers and different HTAB concentrations: Buffer 1: 0.675 mM, Buffer 2: 0.268 mM, Buffer 3: 0 mM (20 kV, 4 s injection with 0.6 psi).
Figure 4.12: Electropherograms of a 0.598 mM DFA and 0.473 mM TFA mixture at 214 nm with 5 mM phthalic acid buffers and 0.268 mM HTAB using 10 kV, 20 kV, and 30 kV separation voltages (4 s injection with 0.6 psi).
Figure 4.13: Electropherograms of a sample mixture containing 1 drop of TFA diluted in 1 mL separation buffer (5 mM phthalic acid buffer with 0.268 mM HTAB) using 20 kV and a 4 s and 8 s injection with 0.6 psi
Figure 4.14: Electropherograms of a c real(acetic acid) = 1.4 mM, c real(TFA) = 0.5 mM, c real(DFA) = 0.6 mM and c real(TFMHB) = 0.3 mM sample mixture and a separation buffer (5 mM phthalic acid buffer with 0.268 mM HTAB) using 20 kV and a 4 s injection
Figure 4.15: Electropherograms of a 2.85 mM TFA sample using a 20 kV separation voltage for 2 min and stopping the run for different amounts of time, before the sample is pushed out with a 50 mbar pressure (4 s injection with 0.6 psi).
Figure 4.16: Electropherogram of a 0.476 mM TFA sample using an alternating voltage polarity switching (20 kV, 4 s injection with 0.6 psi).
Figure 4.17: Electropherogram of 3.56 M TFA using a simple 5 mM phthalic acid buffer as BGE, a 30 mbar pressure run and detecting the signal at different wavelengths (4 s injection with 0.6 psi).
Figure 4.18: Absorbance spectrum of 3.56 M TFA (red = high absorption, green = low absorption)
Figure 4.19: Reconstruction of the 3.56 M TFA double peak in phthalic acid buffer using different experiments (30 mbar pressure, 4 s injection with 0.6 psi).
Figure 4.20: Last delay time determination of FC-43 (10 runs for each delay time and 32 scans)
Figure 4.21: Comparison of FC-43 19F NMR spectra using a) 1024 and b) 2048 acquisition points
Figure 4.22: Curve fitting according to Equation (8) to calculate T1 of FC-43
Figure 4.23: 19F NMR spectra of 0.88 M TFA (64 scans)
Figure 4.24: Correlation of the accumulated amounts of scans with the average SNR of FC-43
Figure 4.25: FC-43 long-term measurement for the fluorine nLOD under comparable conditions. Data obtained with 16 scans and a 1 s delay time
Figure 4.26: Comparison of portable (left side) and high field (right side) 19F NMR spectra of a) TFA, b) FC-43 and c) PFPA acquired under comparable conditions, see Chapter 3 and 32 scans for the portable system
Figure 4.27: Predictions of the portable 19F NMR spectra of a) FC-43, and b) PFPA based on the large-scale spectra (left side) and analysis of the individual peaks (right side).
Figure 4.28: Electrophoretic current in the CE capillary which induces a second magnetic field and perturbs the B1 field of the RF coil
Figure 4.29: Magnetic susceptibility effects of the microcoil affect the sample in the capillary [68]
Figure 4.30: Injection modes for CE with a sample pre-concentration for electrokinetic injections
Figure 4.31: Electropherogram of 9 mM TFA with negative electrokinetic and pressure injection using a 5 mM phthalic acid pH 5.3 (200 µm ID capillary and 150 cm long)
Figure 4.32: Electropherogram of 1.8 M TFA and 0.6 M PFPA with negative electrokinetic injection (30 kV and 30 s) using a 5 mM phthalic acid pH 5.3 and a) a 30 mbar pressure run and b) a combined voltage-pressure run (200 µm ID capillary and 150 cm long)
Figure 4.33: 19F NMR spectra (32 scans, pulse width = 0.6 µs) of a) 3.5 M TFA, b) 1.15 M PFPA, and c) a PFPA/TFA mixture
Figure 4.34: a) The SNR of 19F NMR spectra (32 scans, pulse width = 0.6 µs) of the PFPA and TFA mixture are plotted versus the monitored time in s and b) three 19F NMR spectra are presented for each spectra type
Figure 4.35: Electropherogram of 6.74 M TFA and 1.15 M PFPA with 5 mM phthalic acid pH 5.3 during a 30 mbar pressure run (200 µm ID capillary, 150 cm long) using individual pressure and electrokinetic injection as shown in Table 4.9
Figure 4.36: 19F NMR spectra (16 scans) of TFA and PFPA acquired during the continuous flow run with a pressure injection (50 mbar for 4 s)
Figure 4.37: 19F NMR spectra (16 scans) of TFA and PFPA acquired during the continuous flow run with a voltage injection (15 kV for 240 s)
Figure 4.38: Relative concentration profiles of a 16.67 mm long plug of sample as a function of time. The diffusion coefficient of water at room temperature was used
Figure 4.39: SNR of 6.74 M TFA are acquired (16 scans, last delay = 1 s, except of first run with 15 s) using static and dynamic acquisition parameters. During dynamic and static runs the whole capillary is flushed with the sample, in contrast to the dynamic plug flow. For the dynamic plug flow, just one measurement was done
Figure 4.40: a) Sum of the spectra shown in Figure 4.41, b) Sum of the spectra shown in Figure 4.41 which are all manually shifted to overlap centered
Figure 4.41: FC-43 19F NMR spectra taken with a 1.8 T Aster Enterprise Ni-Co-V magnet (28 scans, last delay = 0.5 s, 1 h delay time between the 16 individual runs, SNR = 16 to 18)
Figure 4.42: TFA 19F NMR spectra taken with 1.8 T Aster Enterprise Ni-Co-V magnet (32 scans, last delay = 1s) a) Temperature variations observed greater than 5°C in a 10 min interval, b) no temperature shifts
Figure 4.43: Acquired frequency points (in ppm) versus monitored temperature using a SmCo magnet and a FC-43 sample
Figure 4.44: SmCo temperature controlled magnet with FC-43 spectra (16 scans, last delay time = 2 s) through the day a) with frequency drifts and b) with frequency shifted drifts
Figure 4.45: a) Total spectral average of the data points in Figure 4.44 for the temperature shifted and the non temperature shifted data. b) Acquired FC-43 frequency data versus temperature
Figure 4.46: Correlation of the normalized temperature and frequency shift of the FC-43 spectra acquired in Figure 4.44 with the time
Table 3.1: Chemical formula, the chemical name, their purification grade and the company of purchase
Table 3.2: CE buffers used for the xanthine and uric acid separations
Table 4.1: Calibration parameters of the Sample Mixtures 2,3, and 4 (SD = standard deviations)
Table 4.2: Concentration of HTAB and phthalic acid forming the buffer matrix
Table 4.3: Average tmigrate and area out of 3 runs with a 0.218 mM TFA and 0.757 mM acetic acid mixture
Table 4.4: Average tmigrate and area out of 3 runs of TFA and acetic acid at different concentrations
Table 4.5: Main characteristics of the observed UV/VIS peaks in Figure 4.4
Table 4.6: Average SNR of 10 runs (noise range = 700 ppm) measured for two TFA concentrations with the CPMG (4 loops) and a one pulse experiment (acquisition parameters see Chapter 3.2.3)
Table 4.7: Average SNR and peak height of 5 runs with 16 scans (noise range = 600 ppm) measured for FC-43 with 1024 and 2048 data acquisition points (acquisition parameters see Chapter 3.2.3)
Table 4.8: T1 for TFA, FC-43, and PFPA measured with the 500 MHz NMR and FC-43 measured with the portable system using a one pulse experiment with different delay times
Table 4.9: Results of SNR, Sm, and nLOD measurements on the portable NMR system for PFPA, FC-43 and TFA with the 150 MHz high frequency cut-off filter, 32 scans, 1 s last delay time, and 8.2 µs pulse (bold numbers are explained in the text)
Table 4.10: Values of SNR, and nLOD measurements on the portable NMR system for TFA with the 150 MHz high frequency cut-off filter, and 8.2 µs pulse (bold numbers are explained in the text)
Table 4.11: Calculation of the minimum sample amounts for PFPA and TFA which are detectable with the assumptions: SNR = 3 and tacq = 32 s (including 1 s last delay time)
Table 4.12: Time table for the pressure and the electrokinetic injection methods
Table 4.13: Calculated times of the pressure and the electrokinetic injection for the plug being in the microcoil (capillary length to detector 141.5 cm and capillary length to coil 65 cm)
Table 4.14: Calculated widths of the TFA plug out of the peak width in the UV/VIS electropherogram and the time of the acquired scans in the NMR spectra
Coupling capillary electrophoresis (CE) with nuclear magnetic resonance (NMR) provides a remarkable and forward-looking prospect in the modern, analytical chemistry. Thanks to the cooperation of Prof. Dr. Vogt from the University of Hanover in Germany and Dr. Greg Klunder from Lawrence Livermore National Laboratory in USA, the unique opportunity revealed to participate in a research project funded by the U.S. Government to develop the hyphenation of these two analytical techniques. This research was financially supported by the German Academic Exchange Service (DAAD) during the stay abroad.
Separation and identification of mass-limited chemical samples is the key to understand the complex nature of pharmaceutical and environmental systems. There is a long history of coupling a non-destructive detection, such as NMR spectroscopy to high efficiency microseparation techniques, such as CE, capillary liquid chromatography (cLC) or capillary high performance liquid chromatography (cHPLC). Whereas liquid chromatography (LC)-NMR was considered to be an exotic technique in the late 1970s, today multiple LC-NMR systems are installed world-wide and LC-NMR is an established analytical technique in biomedical, pharmaceutical, and environmental analysis. Despite the fact that high performance liquid chromatography (HPLC), using analytical columns for mg and µg sample amounts, is currently the most widely employed technique in separation science, several very important related techniques, among them the electrodriven separation techniques, such as CE, have to be considered. Nanoliter NMR techniques employ capillaries, consume less solvent, and trace analysis of e.g. natural products where just a small amount of sample is available, will be used in the near future to an increasing extent [1,2].
These hyphenated techniques have revolutionized the ability to separate and identify components in small sample volumes both online and in fractionation experiments [3] and it is an appealing research field with continuously grows in areas of new hardware and methodological improvements. Using this coupled system, complete structural elucidation of complex analyte mixtures separated during an electrophoretic process can be performed using NMR as an on-line detector. However, NMR is inherently less sensitive than other analytical techniques even though the availability of superconducting magnets is increasing, and advances in electronic components as well as Fourier transform (FT) instrumentation are developed constantly. Although sensitivity remains an issue for large-scale, on-line NMR detection, capillary NMR spectroscopy using microcoils has emerged as a major breakthrough for increasing the mass-sensitivity of NMR spectroscopy, since the limit of detection is proportional to the coil diameter. With the advances of sample pre-concentration using CE as an on-line technique, dilute µL samples can be concentrated into nL volume bands. This approach furthermore serves sample handling for the smallest-volume and highest-mass-sensitivity probes [1,4,5].
Since the demand for portable chemical analysis with information-rich spectroscopic methods and structure elucidation is getting more important, a further development is the miniaturization of the magnet enabling the possibility of a truly portable NMR system. Combining microcoil technology with a compact permanent magnet has the added benefit of reducing the cost, maintenance, and space requirements of the NMR system and enabling portability. This portable NMR sensor, coupled to the rapid CE separation system could facilitate high-throughput and on-site identification of nanoliter amounts of solution [1,6,7].
The anthropogenic introduction of chemicals and pharmaceuticals into the environment is considerable. Once present in the environment, they are subjected to transport processes in air, water and soil and they may degrade into toxic transformation products. Especially the input of fluorinated substances is a matter of environmental concern due to their potential to accumulate in some aquatic ecosystems and their environmental persistency. The special properties of the fluorine ion, such as strong electronegativity, small size and the low polarisability of the C–F bond, can have considerable impact on the behavior of a molecule in a biological environment [8-10].
Moreover, fluorinated drugs are getting more and more popular so that among the numerous marketed pharmaceuticals in the world, more than 150 drugs are fluorinated compounds [11]. These fluorine-organic compounds are of great pharmaceutical interest, have inherent biological activity, and the introduction of fluorine into biologically active compounds improves its pharmacological properties. These pharmaceuticals have applications as cytotoxic drugs in cancer research; they are used as antidepressants, or as artificial blood substances. For the analysis of these analyte classes 19F and 1H NMR offer an unsurpassed speciation method for determining compound chemistry or for example post purification contamination of fluorinated substances in pharmaceutical drugs [1,8].
Currently purified peptides undergo a separation technique, followed by fraction collection, NMR, and mass spectrometry (MS) analysis. Relatively large samples are needed for typical laboratory NMR measurements. Normally, relative small amounts of pharmaceuticals or environmental compounds have to be separated and analyzed so that CE and on-line portable NMR offer a low-cost high-throughput detection method that requires only nanoliter quantities for analysis. Time and reagent costs can be dramatically reduced by using small-scale methods for drug screening. In environmental applications, costs can again be reduced by screening samples on-site with portable technologies [1,8,10].
To serve and realize these above stated advantages of a hyphenated CE-NMR system, the aim of this master thesis is the coupling of a CE instrument to both a large-scale NMR system as well as a portable system.
The goal of this experimental research was the hyphenation of CE to micro- and nano-volume NMR and the optimization of CE separation parameters for different classes of substances. Two separate technical approaches were used to investigate different classes of compounds:
1.) CE was coupled to a large lab-scale 600 MHz NMR detector using a capillary NMR microprobe which has a flow-through detection cell with an active volume of approximately 2.5 µL. This NMR probe was tuned for the detection of 1H protons. The selected compounds of interest were a mixture of xanthine (caffeine, theobromine, theophylline, xanthine) and uric acids (uric acid, 3,7-dimethyluric acid, 1-methyluric acid) which are components of various therapeutically classes of drugs. Since in certain cases the presence of small amounts of impurities in these pharmaceutical formulations may also play a significant role in drug toxicity it is important to monitor those substances. For the CE analysis micellar electrokinetic chromatography (MEKC) was used, to separate weakly charged ions more efficiently. Different parameters, e.g. buffer compounds, pH, buffer concentration, surfactant concentration, separation voltage and capillary length, were adjusted to provide an optimized separation method which can be used for the CE-NMR coupling.
2.) In the second approach, CE was coupled to a portable NMR detector, using a small permanent magnet (1.8 T) and a laser-lathe lithography produced microcoil wrapped around a capillary with observable detection volumes in the nL-range. This probe was tuned to 19F fluorine so that trifluoroacetic acid and longer chain perfluorinated acids were studied. This class of compounds is of considerably interest due to its potential to accumulate in some aquatic ecosystems and their persistence, since these substances are widely spread in environmental products. For on-line coupling, different CE and CE-NMR detection parameters were investigated to optimize the whole separation system. These parameters and aspects include buffer systems with different concentrations of the surfactant, coated capillaries, larger internal diameter (ID) capillaries, stop flow and continuous flow NMR detection, different NMR acquisition parameters, system peak evaluations, the sample plug diffusion in the capillary, high sample concentration on the ultraviolet/visible (UV/VIS) detection, and on-line stacking methods.
In this experimental research work two different classes of chemical compounds were analyzed which have garnered interest during the last years, with regard to various aspects of environmental or pharmaceutical concerns.
Caffeine and other related compounds like theobromine or theophylline (see Figure 2.1) are naturally components of the daily foodstuffs (e.g. tea, coffee, soft drinks) and are added during their processing. The actual concentrations of these substances play an important role because they determine the stimulating or sedative effects of food and beverages. This means that the stimulating effects of e.g. tea and coffee are due to its natural xanthine's content. These xanthines have a wide range of therapeutic activities; for example: theophylline is a bronchodilator, respiration stimulant, and smooth muscle relaxant; caffeine is a central nervous system stimulant; and theobromine as well as theophylline are diuretics [12-14].
Furthermore uric acids and xanthines are a family of compounds widely used in pharmaceutical drug preparation, to compensate for the effect of the main compounds of the drug ingredients on either blood pressure or other body functions. So the exact monitoring of these chemicals is important for the quality control of those drugs [15,16].
The metabolism of caffeine is complex, with its metabolic profile varying from species to species. Caffeine undergoes oxidative demethylation and hydroxyllation reactions in human and various other species to yield a number of urinary excretion products. Xanthine and uric acid are among these products and they are therefore present in human plasma and urine, due to their contents in products for human use. Also methyluric acids are metabolites of methylated purines such as caffeine, theobromine, and theophylline. Methyl uric acids have similar properties like uric acid, including low solubility in water solutions, thus, they may play a role in the processes of urinary stone formation [17-19].
This example shows that these substances have a great influence on human health and well being. Their separation and quantification is of great importance in the study of therapeutic and metabolic problems related to their presence. The quality control of diary products and beverages must therefore include the evaluation of the total amount of xanthines and the quantification of each isomer, by rapid and reproducible procedures. This means that accurate, reliable analytical methods have to be developed [12,13,19].
Different methods to measure xanthines and uric acids for example in plasma or food and beverages have been proposed, but just a few of them are able to quantify and separate the analytes simultaneously. Currently available methods are based on reverse phase HPLC and gas chromatography (GC) as stand alone techniques as well as both coupled to MS. However, they all require time-consuming sample preparation, long analysis time, and expensive material [12,19].
Another approach is the use of a 1H NMR method for the identification and determination of caffeine and theophylline in pharmaceutical preparations and human plasma as illustrated by Talebpour et al. [20]. Albert et al. [21] published a CE-NMR method for the analysis of food and pharmaceuticals which allows the analysis of even complex mixtures in a NMR experiment. The substances were separated and analyzed by continuous flow CE-NMR. Up to now CE and MEKC (see Chapter 2.2.1) have been wide spread to determine caffeine and related compounds in food and beverage samples [12,13,22] as well as in pharmaceutical formulations [15,23,24], because a complete separation in a very short time is possible [16,17,22]. Unita et al. [19] recently published a CE-MS method for the analysis of caffeine and its metabolites in urine. This method was able to separate all 12 compounds of interest at the baseline with a concentration of 360 nM for Caffeine in less than 30 min [12,15,19].
This shows that CE is an established method for the analysis of these compounds and with the hyphenation to NMR a new field of analysis is established.
Perfluorinated organic compounds, such as trifluoroacetic acid (TFA) and longer chain perfluorinated carboxylic acids (PFCA) are ubiquitous in the environment at relatively low concentrations. These substances are a matter of great concern, due to their potential to accumulate in aquatic ecosystems, their wide dissemination and their environmental persistence. Major interest in these substances has arisen from the aspect that TFA is the primary and persistent degradation product of the chlorofluorocarbons (CFC) replacement gases. It has been suggested that TFA in the environment results from both, the atmospheric hydroxyl radical-mediated degradation processes of the CFC replacement gases and from the degradation of fluoropolymers in high temperature applications, such as the combustion of polytetrafluoroethylene (PTFE) [25].
TFA has many notable characteristics which make it extremely valuable and essential. On the one hand TFA (see Figure 2.2) is a very strong carboxylic acid, approximately 100.000 times more acid than acetic acid [26], because of its three fluorine atoms, which are the most electronegative atoms. Due to its acidic strength TFA is very useful in organic chemistry, since synthesis and the resulting end products can be manipulated easily. On the other hand TFA is widely used in pharmaceutical applications and pharmaceutical industry for example in peptide or small protein synthesis, and purification processes as ion pairing agent. Since TFA is corrosive and toxic, it can strongly alter biological actions during clinical studies. Thus it is important that any residual TFA is removed from the peptide prior to the final formulation. Therefore, development of reliable and efficient analytical methods for determine fluorinated compounds is extremely essential (see Chapter 2.1.2.1) and it is crucial to monitor for TFA in environmental risk assessments and in products due to the toxicity effects of TFA binding to metabolic proteins in the kidney, liver, and other organs [8,9,25].
Longer chain perfluoroacetic acids, including perfluoropentanoic acid (PFPA) and perfluorooctanoic acid (PFOA) (see Figure 2.2), have attracted attention due to their detection in human and animal samples as illustrated by Renner [27]. As early as the 1960s, organic and inorganic fluorine were observed in human blood [28]. However, relatively little is known of their environmental behavior, and the risk to human health posed by exposure to these compounds remains to be elucidated, as addressed by DePierre [25].
Current interest is focused on the longer chain PFCAs since they have been incorporated into many name-brand chemicals (Teflon and Gore-Tex) intended for human use. Further applications are the use as corrosion inhibitors, anti-wetting agents, surfactants, and in fire extinguishers. [8,25,27].
Several analytical techniques have been developed to separate and monitor TFA and longer chain PFCAs including GC, MS, ion chromatography (IC), CE, and 19F NMR. However, the choice of the appropriate analytical technique is predetermined by several factors, such as the concentration of TFA or PFCA in the original matrix and the necessity for derivatization [25].
The fact that TFA and the PFCAs are ionized completely under almost all environmental conditions necessitates their derivatization for GC based analytical techniques. Though their anionic character has made them ideal for electrospray-MS or MS/MS based techniques in the negative-ion mode making it possible to attain a low detection limit [25].
The analysis of fluorinated compounds at low concentrations in environmental matrices requires extensive concentration, sensitive methods and in some cases additional pre-concentration techniques. For the longer chain acids, pre-concentration generally relies on liquid-liquid or solid phase extraction based methods and, for TFA, anion-exchange or evaporation of water. These off-line techniques have the disadvantages that it is relatively time consuming and is not convenient when many analyses have to be carried out simultaneously. Using the CE separation and detection technique it allows on-line sample pre-concentration (see Chapter 2.2), so that no additional methods have to be utilized [25].
The great advantage of using CE, 19F NMR and IC for the direct analysis of TFA and PFCAs in aqueous media is that no derivatization step is required which reduces the time required for a set of analyses and makes the overall process more efficient. 19F NMR has been widely used to investigate the chemistry of longer chain PFPAs in different matrices, such as human blood. More recently, 19F NMR was employed for the determination of longer chain perfluoro acids, where quantization was based on the peak area of the terminal CF3 group [25].
These techniques do, however, have the drawback that they are typically an order or two less sensitive than their GC or MS (MS/MS) counterparts. They therefore always require a substantial pre-concentration step to achieve comparable results for the analysis of typical environmental concentrations [25].
Capillary Electrophoresis (CE) is a powerful analytical technique which is widely used for the separation and analysis of ionic and uncharged substances. Its great advantages are the capability of solving many analytical problems rapidly and economically, with high separation efficiency, while using particularly small sample and buffer volumes of a few nL and µL, respectively. Furthermore, CE is a modern separation method which has the unique feature of pre-concentrating the analyte on-line by different electrostacking methods. Due to the above mentioned benefits CE is an attractive approach for the analysis of pharmaceutical drugs in the clinical and forensic laboratories. However, the major advantage of CE for drug analysis is the power of MEKC which is able to separate uncharged drug mixtures (see Chapter 2.2.1). Thus, CE analysis has met guarded interest as a complimentary tool to HPLC [29-31].
In CE electrically charged substances move in a conductive buffer medium under the influence of an electric field which is applied across the capillary. Due to the voltage, and the chargeable fused silica capillary walls the electrolyte buffer migrates in the direction of the corresponding electrode creating an electroosmotic flow (EOF) in this direction. On the other hand, the sample compounds migrate because of their varying sizes and charges, which lead to a difference in the electrophoretic mobility (µEP) of the individual substance. The effective mobility of each individual compound therefore is the sum of the EOF and the µEP leading to a separation of the analytes [30,31].
A schematic set-up of a capillary electrophoresis system is shown in Figure 2.3. The main components of the system are a sample vial, inlet and outlet buffer reservoirs, a narrow bore capillary, Pt electrodes, a high-voltage power supply, and a detector (typically UV/VIS absorption).
As determined by the acidic properties of the silanol groups due to the pH of the buffer electrolyte, the EOF in the fused silica capillaries is usually directed towards the cathode. This direction of the flow favors a rapid analysis of cationic analytes. However it is counterproductive for the analysis of anionic analytes, such as TFA or difluoroacetic acid (DFA). For the analysis of these anionic substances the direction of the EOF has to be reversed, so that it migrates towards the anode. Reijenga et al. [32] were the first who reported the reversal of the EOF direction in fused silica capillaries after the addition of cationic detergents in low concentrations, such as hexadecyltrimethyl-ammoniumbromide (HTAB). Those long-chain alkyl ammonium salts will dynamically cover the walls of the capillary so that they are not charged [30].
Using CE as a pre-concentration technique sensitivity enhancement can be achieved by either electrokinetic injection or by isotachophoresis (ITP). Applying electrokinetic injection two phenomena are observed. First, the sample components from a relatively broad sample segment can be compressed into very narrow zones before the actual separation begins. Second, the improved sensitivity is based on the ability to extract a significant amount of analyte ions from the bulk of the sample solution. This means that sensitivity enhancements of several hundred times can be achieved for low concentrated samples. Employing ITP two electrolytes are used and the sample is injected at the concentration boundary dividing the two buffer solutions. Once the high voltage is applied, the sample ions will search their mobility position located between the two buffer regions. Due to the formed zone boundaries between the individual compounds, the substances are pre-concentrated in these narrow plugs, leading to high efficient separations [29-31].
To quantify the separation of the different peaks in a CE experiment the resolution (R) is determined. The resolution of two separated species, 1 and 2, is defined in Equation 1, whereas a baseline resolution is achieved when R = 1.5.
As a separation technique CE is limited to the separation of charged substances which exhibit differential electrophoretic mobility. Neutral molecules migrate with the same velocity as the solvent under the influence of EOF. In order to take advantage of the high separation efficiencies obtained by CE for the separation of neutral molecules buffer modifications have to be done. Terabe et al. [33] were the first who described the use of micelles in carrier electrolytes and established the MEKC. This electrophoretic mode can be considered as a separation technique occupying some intermediate position between CE and cLC due to its special separation mechanism. CE is a one-phase process with separation based on differences in µEP. In contrast to CE, in MEKC the analytes are distributed between two phases, an aqueous electrophoretic phase and a micellar phase, which is a pseudo-stationary. These two phases are established by micelle-forming agents, such as sodium dodecylsulfate (SDS), which are added above their critical micellar concentration (CMC) to the carrier background electrolyte (BGE). Above the CMC the surfactants spontaneously form micelles with a diameter of 3 to 6 nm, due to the balance between entropy and enthalpy [30,34]
An MEKC analysis is performed in equipment designed for CE (see Figure 2.3). Figure 2.4 illustrates the separation principle of neutral molecules, employing an anionic surfactant and a fused silica capillary. A high-voltage electric field is applied along the capillary, causing a movement of the aqueous solution towards the cathode by EOF, while the anionic micelles in the BGE are retarded by the electrophoretic migration to the anode. If the EOF is higher than the electrophoretic mobility of the micelles, they will move in the same direction as the bulk of the buffer, but with a slower velocity. Analyte molecules will be distributed between the aqueous and the micellar phase due to their differences in hydrophobicity. Therefore, depending upon their partition coefficients, the sample compounds will migrate with different mobilitis, so that a separation of even uncharged molecules is possible. Very polar molecules, which do not interact with micelles, will migrate first, while highly hydrophobic, non-ionic compounds, which are completely included in the micelles, will elute at the same retention time as the micelles. For all the other analytes, a partition process is vital for the separation and they will migrate with zone velocities between those of the two phases. The migration order is determined by the differential partitioning of the analytes between the micelles and the aqueous phase [14,30,34,35].
With the ability to separate uncharged or weakly charged substances MEKC is an important tool for the analysis of pharmaceuticals, such as caffeine or theobromine, because the partition between the aqueous and micellar phases can increase the selectivity and allows the separation of weakly charged ions and uncharged molecules with very similar mobilitis [29].
Detection in CE seems to impose difficult problems, so that advances in detector technology have played a major role in the success of CE. Up to now a wide variety of detection techniques are available including optical (UV/VIS, thermooptical, fluorescence, and chemiluminescence), radiochemical and electrochemical (potentiometric, conductivity, and amperemetric) devises, MS, and since a few years, nuclear magnetic resonance spectroscopy (see Chapter 2.4) [29].
The above mentioned difficulties arise from the very small sample volumes which do not exceed 100 nL and the extremely short light paths of less than 100 µm in the capillary. However, even though the sample volumes are very small, due to the lack of longitudinal diffusion in CE, the analytes zones arrive at the detector in essentially the same volume as at the beginning of the separation. Moreover, due to electrostacking the initial volume can be a factor of ten to hundred smaller than the original sample volume. The different detection methods benefit from these advantages so that common HPLC detectors can be used for small volumes [29,30].
The simplest and most common detection technique introduced to CE, and for the determination of the studied samples in this research work, is the indirect and direct UV/VIS detection. Applying this on-column detection technique the majority of substances can be detected because they absorb somewhere in the UV region and this technique most often does not require chemical modification of the samples prior to the analysis. The primary disadvantage of absorbance detection for CE is the relatively poor limit of detection (LOD) which is generally in the µmol range. This poor LOD is caused by geometric constrains imposed by the small light path through the capillary and the inherently insensitive nature of absorbance detection [29].
Two modes of detectors can be applied. The fixed-wavelength detector is known for its simplicity and ruggedness, whereas the diode array detector as multi-wavelength detector has the ability to monitor several wavelengths at the same time so that additional spectral information is reported. For a quantification of the substances Lambert-Beer Law is applied. According to this law there is a linear relationship between the concentration of the sample and the absorbance, based on the optical path length [29].
For the detection of the aromatic purines the direct detection mode is used. In this mode the absorption of the analyte is detected within a BGE which does not have a significant absorption at the analyzing wavelength, e.g. borate or phosphate buffer. However, for the determination of the fluorinated substances the indirect UV/VIS detection has to be utilized. Indirect UV/VIS detection makes use of BGE compounds, such as phthalic acid, which exhibit a relatively high level of UV light absorption. As illustrated in Figure 2.5 the absorbance of the BGE is large in the absence of an analyte. If a sample plug passes through the detection element, the originally high level of the absorbance signal decreases due to the dilution of the high UV absorbing compound, and a peak is detectable [29,30].
Fluorescence detection is the most sensitive detection mode available for CE with LODs in the pmol range. However, this technique can only be applied to a limited number of compounds, which contain fluorophore groups. Since the majority of analytes do not contain any fluorophores, and the derivatizing compounds are encountered with several difficulties only few applications are available with this technique [29,30].
Of all detection devices reported to date, MS clearly has the greatest potential with its advantage of determining the molecular weight and providing structural information. The coupling of CE with MS represents a true two-dimensional separation system and promises to be an extremely powerful detection scheme for micro-scale separations with LODs in the nmol range. Since MS analyzes charged species by their mass-to-charge ratio in the gas phase, these two orthogonal techniques exploit separations based upon ion motion. However, the biggest drawback of this system is the destructive nature of the MS detector. This disadvantage leads to a great interest in the CE-NMR coupling since NMR is able to provide elucidating structural information without destroying the analyte, which is an important aspect when dealing with pharmaceuticals or environmental samples [29,30].
Nuclear Magnetic Resonance (NMR) is an important and powerful spectroscopic technique in the chemist's toolbox to get dynamic and structural information about a substance, encoded in the chemical shifts and coupling constants. Furthermore, NMR is a non-destructive spectroscopy method, which allows the investigation of the electronic environment of individual atoms and the interaction with their surrounding atoms, without changing or consuming the analyte. NMR is different from most other forms of optical spectroscopy, like infrared or visible spectroscopy where the absorption or emission of energy is measured, in that it monitors the response of the nuclei to the frequency as it recovers from a pulse by resonance methods [5,7,25,37-39].
Even though the NMR experiment is intrinsically insensitive in terms of LOD values, which is the greatest disadvantage of this technique, improvements in the signal-to-noise ratio (SNR) are possible. Over the last decades major advances in improving the NMR sensitivity has been in magnet technology, such as, high field, 21.1 T, magnets and cryoprobes to reduce the noise from the NMR coil. Another possibility to improve the rather low sensitivity is by signal acquisition of several spectra in FT experiments and the use of pulses to manipulate the nuclear spins to generate new information by applying hyperpolarisation techniques. An additional approach to overcome this sensitivity drawback, besides the installation of expensive instrumentation or accessories, is the use of highly sensitive microcoils and miniaturization of the probes to reduce the sample coil volume especially when the amount of substance is limited (see Chapter 2.3.4 and 2.3.5) [4,7,25,37-39].
Only those nuclei with an odd number of nucleons (e.g. 1H and 19F) have an intrinsic nuclear magnetic moment and an angular momentum which will interact with an applied magnetic field. This leads, to a nuclear energy level diagram, where the magnetic energy of the nucleus is restricted to certain discrete values according to the rules of quantum mechanics. In non-zero magnetic atoms the nuclei charge is precessing around the nuclear axis which creates a magnetic dipole along this axis. Under a magnetic field (B 0), having a direction that coincides with the axis of the nucleus, 1H and 19F nuclei, having a spin of ½, will form two energy levels, caused by their different spins (m). The energy difference (∆ E) between these two states is very small, of the order of 10-27 to 10-25 J and a slightly higher population of nuclei, typically about 0.001 %, is found in the lower energy state (N β) in accordance to the Boltzmann relation (see Figure 2.6). This very small value in the population difference is the reason for the intrinsic low sensitivity of NMR as a measurement technique, because relatively weak signals are detected, since the NMR signal depends upon the net magnetization (M0) of the sample [4,37-40].
Once those two energy levels have been established by a B 0, it is possible to introduce another magnetic field (B 1) in the form of radio frequency (RF) radiation from a current in a coil wound perpendicular to B0. Under appropriate conditions magnetic nuclei show a characteristic absorption in the magnetic field when the RF electromagnetic (EM) pulse is applied to the sample mixture. So the angel of precession will change and the nuclei are excited to the higher energy level (N α). The absorption of the EM radiation depends on the frequency governed by the characteristics of the sample nuclei. In the fundamental Larmor equation the observation radio frequency (υ) for a particular nucleus can be correlated to the applied B 0 using the magnetogyric ratio (γ). γ is a fundamental nuclear constant, which is characteristic of the particular nucleus and h is the Planck constant.
illustration not visible in this excerpt (2)
Differences in γ lead to different observation frequencies for different nuclei on a particular spectrometer (see Table 2.1). For 1H a υ of 200 MHz is needed at a B0 of 4.7 T to get this system to resonance so that energy is absorbed (see Equation 2). However, a 19F signal at the same field would be observed at 188 MHz, respectively.
Table 2.1: Magnetogyric ratio of the nuclei [37]
If the frequency is in the range of the transition energy (see Equation 3) the absorption is strongest. This absorbed energy is a function of certain nuclei in this molecule and is radiated back out. The energy radiated back out, when the spin relaxes back towards equilibrium, is at a specific resonance frequency which depends on the strength of the magnetic field (see Equation 2 and 3) and other factors. This allows the observation of specific quantum mechanical magnetic properties of an atomic nucleus [37,39].
In order for the spin system to return to thermal equilibrium, after the spin system is disturbed by the RF pulse, there must be an interaction between the spins and the surroundings (lattice) leading to a loss of the excess energy. This rate at which the system returns to equilibrium will depend upon the ability of these interactions to transmit energy out of the system. The resulting peak intensity is proportional to the number of protons involved in this relaxation processes. The spin-lattice relaxation time (T 1) represents the re-establishment of the equilibrium N α and N β state distribution, whereas spin-spin relaxation time (T 2) characterizes the exponential free induction decay (FID) in the receiver coil. Therefore, a NMR spectrum is the plot of the frequencies of the radiation absorption peaks versus the peak intensity. Quantitatively the relative number of different nuclei giving rise to the signal in a spectrum can be determined by integrating the areas under the detected peaks. The peak positions are measured in frequency units from a reference peak and depend on a number of factors, including the intramolecular and intermolecular interaction of the nucleus with other magnetic nuclei in the molecule via bonds or through-space [37,39,40].
Two independent experimental techniques are available for the realization of an NMR experiment. In continuous wave (CW) spectroscopy, the resonance caused by the RF is monitored as either the RF is varied (frequency sweep) or the applied field (field sweep) is changed. In either way, only a single frequency is excited and detected at one moment. Instead of the CW experiment the complete spectrum of frequencies is stimulated by one short duration pulse (typically 1 to 50 µs) of strong RF energy in a FT experiment at a constant field. After the pulse stops, only the B0 influences the magnetization which starts to precess around the axis with the Larmor frequency which is characteristic of the nucleus. Following the perturbation pulse the precessing of the magnetization fades away while the response of the system is measured as a function of time resulting in a FID. The signal is generated subsequently using a mathematically Fourier transformation, converting the time domain into the frequency domain spectrum. The great advantage of using FT experiments is that they allow the fast acquisition of many spectra which are co-added to increase SNR [37,39].
Fluorine containing compounds are widespread in application, e.g. currently used in the development of pharmaceutical drugs, in inorganic chemistry as counter-ligands, and in environmental compounds, as mentioned previously. As a result, many research and industrially relevant compounds can be characterized using 19F NMR since the 19F nucleus, just like the 1H nucleus, has favorable NMR properties. Therefore, 19F NMR spectroscopy is an attractive proposition to determine molecule structural information of fluorine containing compounds. However, since in chemical compounds the element fluorine is not as much widespread as hydrogen, the status of 19F NMR is noticeable lower than 1H NMR [25,41].
The sensitivity of a nucleus to be investigated by NMR depends on the magnitude of its magnetogyric ratio, which determines the energy difference between N α and N β, and its natural abundance. It is fortunate that fluorine has only one naturally occurring isotope, 19F, with an abundance of 100 %. In addition, 19F has a nuclear spin of ½ and its sensitivity is about 0.83 that of 1H. The great advantage of the fluorine NMR in contrast to the proton NMR is the much more extensive range of chemical shifts, which is approximately 100 times greater. For the 1H nuclei the chemical shifts (δ) are within an approximate range of 12 ppm, while 19F has a range of ~1200 ppm (-400 to +800 ppm), thus the smallest structural changes can be accompanied by relatively large frequency shifts. The wide region of chemical shifts for commonly occurring 19F functionalities can be explained with the electronically structure of the element, having low-lying p-orbitals and a large paramagnetic contributions in the shielding constant. The shifts are particularly dependent upon the nature of the other geminal atoms or upon the nature of the atoms attached to the vicinal carbon atoms. Unlike the proton resonance shifts, in which the resonances are predictable, the 19F chemical shifts of fluorocarbons are much more unpredictable, because the influence of the p-electrons is largely depended on the surrounding atoms [25,37,41]. Fluorine can be thought of as a substitute for hydrogen in organic compounds. It is virtually unknown in naturally occurring organic compounds so that the interest in 19F NMR involves synthetic compounds. When using a lab scale 19F NMR system which has the sensitivity and the resolution to show the splitting and the coupling between the F-H atoms the proton-coupled spectra gives a lot of information about the structure of the compound. For the qualitative analysis of a fluorinated compound the combination of a 1H NMR and a 19F NMR spectra is most convincing and more informative than either one is by itself [37].
One of the most important considerations when using an analytical method is the ability to qualitatively identify and quantify the observed sample in the presence of background noise. The sensitivity in NMR is quantified in terms of the SNR and is defined as the ratio of the tallest signal peak height divided by two times of the maximum peak-to-peak amplitude of the root mean squared (RMS) noise (see Equation 4) [5,40].
To improve the SNR for a given experimental setup, FID signals are added together. Since the signals originating from random noise vary in their intensity and phase, true NMR signals are always at the same phase. So the latter are added constructively, while the electronic noise decreases as the square root of the number of FIDs or time (the number of acquisitions is directly proportional to the acquisition time (t acq)). However, this is a very time consuming operation, because a 10 fold improvement in SNR requires a 100 fold increase in experimental time [4,5,40]. SNR is a function of many different variables, including the properties of the NMR detector and aspects related to the nature of the sample nuclei. Instrumental aspects regarding the RF probe design, as well as the length and geometry of the receiver coil (see Chapter 2.3.4). Therefore, a distinction should be made between the total sample volume (V tot) required for an NMR experiment, and the volume of sample observed in the RF coil (V obs).The ratio V obs/ V tot reflects the sample observation efficiency which ideally should be equal 1. However, normally V obs is smaller than V tot because the sample plug is usually bigger than the coil [5,40].
The LOD is defined as the smallest measure that can be detected with reasonable certainty and is several orders of magnitude poorer if compared with other analytical techniques [4]. LOD and the mass sensitivity (S m), calculated to determine the probe sensitivity if used as a detector on a mass limited sample, have to be defined in terms of sample concentration (c) which resides in the V obs of the coil. Additionally this parameter has to be time-normalized, because a number of acquisitions must be accumulated to give a meaningful result giving the normalized LOD (nLOD) (see Equation 5 and 6) [4,5,40].
The nLOD for each analyte is derived from a combination of the measured sensitivity of the available NMR spectrometer coupled with t acq for each analysis and will be variable dependent upon the nature of the fluorine reporter group. Furthermore the nLOD also allows the user to compute the approximate mass of sample needed to acquire a desired SNR for a specific spectral peak using a particular probe and t acq [5,25,40].
The most important parts of the NMR spectrometer are the magnet, the RF transmitter with frequency-field stability and field homogeneity, and the computer interface. In large-scale NMR investigations the sample, most often a solution in a deuterated solvent, is contained in a glass sample tube (5 mm outer diameter (OD), 17 cm long) which is placed in the probe. The sample is spun around its vertical axis in order to improve the field homogeneity. Furthermore, this probe contains the RF coil, which generates a linear field RF oscillation and which detects the stimulated signal. This coil is situated perpendicular to the axis of the spinning sample tube. After amplification and transmission of the signal to a plotter, the spectrum can be recorded and the resonance frequencies measured. In this arrangement, see Figure 2.7, the tube is spinning at a right angle to the z axis which is horizontal [37-39].
Today, high frequency (200 to 600 MHz) NMR instruments are used, which are operated in the pulsed FT mode with higher resolution and sensitivity. Since the energy gap between the two energetic levels (N α and N β) is getting bigger with a stronger magnetic field, superconducting electromagnets are used. Here the magnetic field is generated by a superconducting coil which is held at the temperature of liquid helium. With this technique magnetic fields of up to 17.5 T can be achieved so that the sensitivity of the NMR experiment can be significantly improved [38,39].
Gottfried Wilhelm Leibniz Universität Hannover (Anorganische Chemie)
Joana Diekmann (Autor)
This Master Thesis was awarded with the "E.ON Future Award 2010" and it was carried out at the Leibniz University Hannover, Faculty of Natural Sciences, Institute for Inorganic Chemistry, Department of Analytical Chemistry, in cooperation with the Lawrence Livermore National Laboratory, Physical and Life Sciences Directorate, Livermore, CA, USA.
Capillary Electrophoresis Microcoil 1H NMR Portable 19F NMR On-Line CE- NMR Perfluorinated Carboxylic Acids Trifluoroacetic Acid Xanthine Uric Acids
Joana Diekmann (Autor), 2009, Coupling of Capillary Electrophoresis with Nuclear Magnetic Resonance Spectroscopy for the Analysis of Pharmaceutical and Environmental Relevant Compounds, München, GRIN Verlag, https://www.hausarbeiten.de/document/169088
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text:
Charleston Physicians
Charleston SC Doctors, Physicians & Hospitals | Charleston Physicians.com
2023 Best in Health Voting
Loren B. Frankel MD, Palmetto Women's Health
She is the mother of three young children and the wife of a brain surgeon. How does she find time to attend to the patients at her own obstetrics and gynecology practice?
"I manipulate things to get the most," explains Loren Frankel, M.D. of Palmetto Women's Health in Mount Pleasant, South Carolina. "Because I own the practice, I work the schedule so that I'm home most weekends and on Fridays. My daughters are excited because I'm even able to go on field trips with them sometimes."
Finding the balance between work and family can be a challenge for Dr. Frankel because her patients often need her services at unpredictable times.
"Trying to protect myself from the unplanned is hard in my line of work," Frankel admits, explaining that her patients' needs sometimes conflict with family plans.
To "help someone at a special moment in their lives. It's wonderful to provide the actual listening that some patients need."
"Our children are very resilient. They understand that we have important jobs that aren't the typical nine-to-five workplace."
Because Loren Frankel's parents take an active part in the children's lives and are available as back-up when needed, the family is able to handle the challenges of a household where both parents are practicing physicians.
"Our children are willing to make sacrifices and we try to make up for it when we can," Frankel explains.
Women In Health: Dr. Jeris Cox, Adore Fertility Jeris Cox, M.D., of Adore Fertility, ended up in South Carolina by chance, but the choice was all hers to…
Dr. Frankel has been in private practice since 1998, ultimately opening her own practice in Mount Pleasant in 2005. She has admitting privileges with Roper St. Francis Healthcare and East Cooper Medical Center.
Although well-established and successful, Frankel is aware of the difficulties that face her practice and the medical field as a whole in the future.
"There are many changes that will need to be made to ensure that doctors continue to practice. With malpractice rates as high as they are, it costs so much money to run a business, and insurance companies don't want to pay, patients don't want to pay and it adds a lot of extra stress on the day to day practice."
Like many women who choose to become physicians and have families, Dr. Frankel has endured criticism for her choices.
"It has come from different people including some PTA moms but I just have to shake it off. I've worked for men who didn't get that I had a family to go home to but then again, during my residency, one male doctor was my champion. He delivered my first child."
Despite these issues, Frankel still regards the best moments in her career as being able to "help someone at a special moment in their lives. It's wonderful to provide the actual listening that some patients need."
She has the best of both worlds and encourages other young women to understand that it is possible to have a successful career and a family.
"Go into medicine with reasonable expectations. Even in the most difficult fields, you can always find a balance and make it work."
For more information or to set up an appointment contact Loren at Palmetto Women's Health, 1300 Hospital Drive, Suite 380, Mt. Pleasant, SC 29464 or call (843) 884-2206 or visit www.pwhealthmp.com.
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Welcome to Locked in the NOC (Network Operations Center) list, part 3. If you were to build a managed services provider (MSP) business, these are the folks you'd want to kidnap and LOC in your NOC -- to gather their technical, business, sales, marketing and community insights. Forever.
The Locked in the NOC list is a spin-out of our MSPmentor 250 list. What's the difference?
Welcome to Locked in the NOC (Network Operations Center) list, part 3. If you were to build a managed services provider (MSP) business, these are the folks you'd want to kidnap and LOC in your NOC — to gather their technical, business, sales, marketing and community insights. Forever.
The Locked in the NOC list is a spin-out of our MSPmentor 250 list. What's the difference?
Now, let's get to the honorees.
Sabhlok oversees two of the world's top companies in the managed and cloud services markets. ManageEngine is one of the leading providers of IT management and automation software for MSPs. Similarly, Zoho is one of the top providers of SaaS and cloud applications.
Sandiford has continued to evolve Level Platforms' Managed Workplace platform to help the company's global partner base; those partners, in turn, are growing their businesses. With Managed Workplace 2012, Sandiford and his team introduced new Mobile Device Management capabilities that allow MSPs to monitor and secure smartphones and tablets, as well as fully integrated Network Operations Center and Help Desk Services to allow MSPs to deliver 24x7x365 remediation and support offerings. But the bigger story here involves Sandiford successfully positioning Level Platforms to build relationships with Intel, Symantec and other leading IT vendors.
Schafran has over 25 years of experience both running and consulting with IT services firms. He positioned Cogent Growth Partners as a buy-side M&A firm focused on IT services. His expertise covers all aspects of IT services, including strategy, operations, marketing, technology, HR Management and business governance.
Scott has pioneered numerous business models in the MSP market for more than a decade. He currently leads TUC brands — which is acquiring and integrating MSPs to build one of North America's leading IT service providers. Scott's current focus includes strategy; the company's technology roadmap; along with mergers and acquisitions. A career entrepreneur, Scott has launched three companies over the past 18 years focused on business-to-business services, including two market-defining firms in the managed IT services industry — including N-able Technologies. He also serves on the MSPAlliance board of directors.
Shapero, a Kaseya veteran, has helped hundreds of MSPs to build and shape their businesses. Most recently, he launched ClikCloud — which offers digital marketing services to MSPs. Check the speaking circuit and you'll often see Shapero on the agenda for CompTIA-oriented eduational sessions.
MJ Shoer, President, Jenaly Technology Group, Inc.
Shoer successfully guided Jenaly Technology Group to it's 15th anniversary in 2012. The company has seen continual growth in the business year over year, most notably in net earnings over time. He is deeply active with CompTIA, ASCII Group, Robin Robins and ConnectWise. He also volunteers his time locally, chairing a local Crime Stoppers Board that serves 12 local communities. Under Shoer's leadership, Jenaly has also become one of the founding partners of TOGL, an OS33 Master MSP in the cloud services space.
Jeff Shreeve, CEO, StorageCraft Technology Corp.
Shreeve has quietly led StorageCraft to one of the biggest software success stories in the MSP market. StorageCraft has doubled its revenues (at a minimum) in each of the past three years (2008-2011), building OEM relationships with many of the industry's top BDR solutions providers. Shreeve also positioned StorageCraft to work incredibly closely with VMware long before most MSP-centric software companies were in the virtualization market.
Sorensen is best-known for building a successful managed services business while also growing HTG Peer Groups. More recently Sorensen has been driving life-work balance awareness with an emphasis on making sure entrepreneurs don't lose site of the family lives and spousal relationships. Currently, following a successful pilot effort, Sorensen is preparing the 2013 launch of the Heartland Leadership Group –which drives solution provider success through executive coaching.
With over 20 years of software product development experience, Sutherland guides Kaseya's product development and architecture team. Roughly two years ago he played a key role in Kaseya's push beyond on-premises software to cloud-based IT automation software. He also positioned Kaseya to aggressively push into the mobile device management (MDM) market in 2011, giving MSPs the ability to maintain Apple iOS and Google Android devices.
Under Van Etten's watch, Azaleos was named the 28th fastest growing technology company in North America in 2011, and 10th fastest in its primary industry (software), by Deloitte. He grew revenues by more than 100 percent in 2010 and 2011. At last count, Azaleos had more than one million seats under management for its managed UC services across Fortune 500, federal government, and medium business customers.
mindSHIFT, a Best Buy company, acquired White Glove Technologies in mid-2012, at which time Wald exited the company to enjoy some time off. Wald grew White Glove's revenues 67% in 2011 vs. 2010. Wald has delivered similar results previous years. He is a member of Ingram Micro's Services Advisory Council and weighs in on discussions regarding future cloud services and platforms.
Wensley drives the continuing growth and expansion of Level Platforms community of solutions providers and MSPs. As of August 2012, Level Platforms had partners in over 30 countries adopting Managed Workplace as the platform of choice for managed services. Wensley in 2012 organized a roadshow that attracted 1400 attendees in North America, the UK and Ireland. He was nominated to CompTIA's Board of Directors in 2011.
Wong has spent 22 years designing and developing systems and software products for the computer security and software industries. As co-founder and CTO, his current Kaseya responsibilities include all aspects of the architecture of the Web-based remote PC management system, and the software design and implementation of the client/server components of the system.


TradeGecko receives an overwhelming response from investors and media at the inaugural JFDI Demo Day. Check out our pitch video. It will also give you a better understanding of why we are building TradeGecko and where we are going with it.


text:
Q: If $\frac ab= \frac bc= \frac cd$ then $(a^2+b^2)(c^2+d^2)=(ab+cd)^2$ [sic?] This is a basic algebra question. I found in class 9 math book and it is little tricky for me.

If $$\dfrac ab= \dfrac bc= \dfrac cd$$
  prove that  $$(a^2+b^2)(c^2+d^2)=(ab+cd)^2$$
Note (by @Blue). As observed in comments, the problem is incorrect as stated. It becomes valid if $(ab+cd)^2$ is replaced by $(ac+bd)^2$, but it is not clear if this is the source's intention.

A: Using complex numbers and their absolute value one can transform
$$
(a^2+b^2)(c^2+d^2)=|a-ib|^2|c+ id|^2=|(ac+bd)+i(ad-bc)|^2,$$
so with $ad=bc$ the right side is pretty fixed.

A: As noted in comments, the formula is wrong (or was, until I just corrected it).
Let $\alpha$ be the common ratio, so that $a=\alpha^3d,\ b=\alpha^2d,\ c=\alpha d$.  Then
$$(a^2+b^2)(c^2+d^2)=((\alpha^3d)^2+(\alpha^2d)^2)((\alpha d)^2+d^2)\\=(\alpha^6d^2+d^2)(\alpha^4d^2+d^2)=\alpha^4(\alpha^2d^2+d^2)(\alpha^2d^2+d^2)\\
=[\alpha^2(\alpha^2d^2+d^2)]^2=[\alpha^4d^2+\alpha^2d^2]^2\\
=[(\alpha^3d)(\alpha d)+(\alpha^2d)d]^2=(ac+bd)^2$$

redpajama_set_name: RedPajamaStackExchange


text:
Q: Менять родительскую переменную в дочернем компоненте В родительском компоненте есть переменная которая отвечает за состояние checkbox и нужно менять ее в дочернем компоненте при нажатии на checkbox, подскажите что я делаю неправильно и как можно это исправить?
РОДИТЕЛЬСКИЙ ЭЛЕМЕНТ

<template>

    <form @submit.prevent="sendMail">

        <button type="submit">Send</button>
        <CheckBoxInput
          model="mail.newsletterSub"
          id="contact-terms"
          :label="checkboxOptions.label"
          :dark="checkboxOptions.dark"
        />

    </form>

</template>

ДОЧЕРНИЙ CHECKBOX

<template>
  <div :class="[`checkbox-input`, `${dark ? 'checkbox-input_dark' : ''}`]">
    <input :id="id.replace(' ', '')" type="checkbox" v-model="model" />

      {{ label }}

</template>

A:

const CheckBox = Vue.component("CheckBox", {
  template: `

   <input type="checkbox" v-model="value"></input>Checkbox

`,
  props: {
    checkBox: {
      type: Boolean
    },
  },
  model: {
    event: "change",
  },
  data() {
    return {
      value: this.checkBox,
    };
  },
  watch: {
    value: function(e) {
      this.$emit("change", e);
    },
  },
});

const app = new Vue({
  el: "#app",
  components: {
    CheckBox
  },
  template: `<CheckBox v-model='value' :checkBox="value"></CheckBox>`,
  data() {
    return {
      value: true,
    };
  },
  // для визуализации изменений в родительском компоненте
  watch: {
    value: function(e) {
      console.log(e);
    },
  },
});

A: Для реализации такой логики вам необходимо обратиться к разделу документации о работе с emit для настройки v-model. Ниже пример использования данного подхода (может возникнуть вопрос о разнице между эвентами change и input, тут данный хороший ответ):

const CheckBox = Vue.component("CheckBox", {
  template: `

        <input type="checkbox" v-model="localValue"/>
        Checkbox

  `,
  model: {
    event: 'change',
    prop: 'value',
  },
  props: {
    value: {
      type: Boolean,
      required: true,
    },
  },
  computed: {
    // Объявляем вычисляемое свойство с геттером и сеттером
    // чтобы не делать промежуточную локальную переменную и вотчер
    localValue: {
      get() {
        return this.value;
      },
      set(newValue) {
        this.$emit('change', newValue);
      },
    },
  },
});

const app = new Vue({
  el: "#app",
  components: {
    CheckBox,
  },
  template: `

      <CheckBox v-model='value'></CheckBox>
      Result: {{ value }}

  `,
  data() {
    return {
      value: true,
    };
  },
});

Ответ основан на ответе участника Дмытрык, данный выше и содержит некоторые правки для оптимизации кода и ознакомительную информацию.


text:
Two die, 3,000 homeless in S.Africa shack fire
Shack fires are a perennial threat to the impoverished residents of sprawling informal settlements such as Blikkiesdorp, pictured here on April 20, 2010 about 25km East of Cape Town (AFP Photo/Rodger Bosch)
Cape Town (AFP) - Two people died and about 3,000 were left homeless when a fire destroyed 800 shacks at the Masiphumelele informal settlement in Cape Town Sunday, officials said.
"Disaster risk management and the South African Red Cross Service are on scene to co-ordinate relief efforts including providing food, baby packs, mattresses and blankets," mayor Patricia de Lille said in a statement.
"At this stage the cause of the fire is unknown and the incident was handed over to the South African Police Service for investigation."
The settlement, near Fish Hoek on the Southern Peninsula, has recently been rocked by vigilante killings carried out by residents who complain of a lack of policing in the area.
Shack fires are a perennial threat to the impoverished residents of sprawling informal settlements near major cities throughout South Africa.
'More aggressive' on China and an eye on domestic terrorism: Biden's intelligence pick testifies to Senate panel
On the eve of Joe Biden's inauguration, members of the Senate Intelligence Committee questioned his pick as director of national intelligence, Avril Haines, on national security issues ranging from China to domestic extremism.
MyPillow CEO Mike Lindell has received a letter from Dominion Voting Systems, warning him that litigation is "imminent" due to his false claims that the company's machines were rigged to change the outcome of the election.Lindell, an enthusiastic supporter of President Trump, has been spreading baseless claims of widespread voter fraud for months. In the letter, Dominion's lawyers told Lindell, "You have positioned yourself as a prominent leader of the ongoing misinformation campaign. Litigation regarding these issues is imminent."Lindell told The New York Times he would "welcome" Dominion to "sue me because I have all the evidence against them. They sent this letter a couple of weeks ago. They're lying, they're nervous because I have all the evidence on them." Lindell did not say why, if he has such evidence, he has kept it to himself this entire time, holding onto it as judge after judge rejected lawsuits filed in an attempt to overturn the election in Trump's favor.More stories from theweek.com Chief Justice John Roberts reportedly wants no part of Trump's impeachment trial Trump's White House staff and alumni are reportedly using the same excuse to skip his big sendoff 5 more scathing cartoons about Trump's 2nd impeachment
The president, who is skipping the inauguration, also urged America to "pray" for Mr Biden's success
The Dutch government will announce further measures to fight the coronavirus this week amid fears that the "British variant" and other new mutations could soon start spreading, the health minister said on Tuesday. The country's Outbreak Management Team had advised the government on Sunday that infections remain worrying and "new variants of the virus are going to contribute to that in the coming weeks", Hugo de Jonge said in a letter to parliament. The government has been considering a night curfew on top of its current lockdown, which includes a ban on public gatherings and the closure of schools, restaurants and non-essential shops.
Twelve U.S. Army National Guard members have been removed from the presidential inauguration security mission after they were found to have ties with right-wing militia groups or posted extremist views. (Jan. 19)
Viktor Orban accused of putting son through Sandhurst at Hungarian taxpayer's expense
Opposition leaders have accused Hungarian prime minister Viktor Orban of putting his son through training at the Royal Military Academy Sandhurst at the expense of the taxpayer. Gaspar Orban, the son of Hungary's authoritarian leader, graduated in December after attending the nine-month course, which local media said cost about £93,000. Viktor Orban has faced frequent accusations of cronyism and of using the levers of state power to benefit and reward those closest to him since becoming prime minister for a second time in 2010. The opposition Democratic Coalition demanded that Viktor and Gaspar Orban, along with Tibor Benko, the Hungarian defence minister, stand before the parliament's defence committee to answer questions over the affair. The party wanted to know why Gaspar could attend Sandhurst at the expense of the "Hungarian taxpayer" and a price that was "unaffordable to Hungarians", the Hungary Today website reported. The Hungarian government told the Daily Telegraph that Gaspar Orban had received no preferential treatment. "The Ministry of Defence rejects the accusations of Left-wing MPs, and wishes to stress that naturally they and their children have the same opportunity as anyone else to apply for membership of the Hungarian Defence Forces, within which they would also be able to train abroad if they meet the relevant requirements," the government said. "Everyone in the Hungarian Defence Forces has the opportunity to apply for various courses and training programmes, both at home and abroad," the government told The Telegraph. "As part of this, in recent years 163 Hungarian military personnel have participated in various training courses in many countries around the world." Hungarian news website Telex published a photo of the younger Mr Orban in a group photograph of Sandhurst's foreign students, and found that his name had appeared on a list of graduates published on January 1. Only three Hungarian students, including Gaspar Orban, benefitted from the training in the elite academy. "The Royal Military Academy Sandhurst offers a number of courses to international students. Last year, three candidates funded by the Hungarian Ministry of Defence graduated from the Academy, alongside individuals from 43 other countries," An Army spokesperson said. A defence cooperation agreement with Hungary includes courses at Sandhurst. There were three self-funded places on commissioning courses in the last 18 months, which were nominated by the Hungarian Ministry of Defence. Gasper Orban, 28, had previously shown little sign that he was interested in a military career before joining the army last year. When he was younger Orban dabbled at becoming a professional football player, allegedly to help please his soccer-loving father. Having failed to make the cut in the professional game, he later founded a Christian community in Hungary having, allegedly, found Christ while teaching football to children in Uganda. Andras Fekete-Gyor, a politician from the liberal opposition party Momentum, said on Facebook that the prime minister should have paid for his son to attend Sandhurst "out of his own" pocket but could not resist the state footing the bill. Members of the ruling Fidesz party have robustly defended Mr Orban. Szilárd Németh, the deputy defence minister, said Mr Fekete-Gyor should join the army. "We will do our best to ensure that Fekete-Gyor becomes a self-sacrificing, absolutely duty-conscious, straightforward, and gallant officer of the Hungarian Homeland, a man who is accustomed to a modest lifestyle and passionate about his future vocation," he said. Fidesz's parliamentary group leader Máté Kocsis said that the prime minister's family had been attacked "for the umpteenth time". Sandhurst counts Prince William and Prince Harry, as well as Henri, Grand Duke of Luxembourg among its former cadets.


text:
Fungi in the intestinal mucosa induce immune signals ( IL-17 cytokines) that impact the nervous system and animal behavior. (Art by Katarina Liberatore)
Gut Brain Connection, Gut Health, Immunity
Gut fungi work in harmony with the brain, immune system to mediate immunity and behavior
by Jocelyn Solis-Moreira
Gut fungi interact with immune cells to protect against intestinal injury and infection, according to a recent study. Additionally, specific gut fungi helps to promote social behavior, expanding on the role of a "gut-immunity-brain axis."
"We have made a direct link between a major immune pathway induced by fungi in the lining of the intestine and signals in the nervous system that impact animal behavior," explains senior author Dr. Iliyan Iliev, associate professor of immunology in medicine in the Division of Gastroenterology and Hepatology at Weill Cornell Medicine, in a statement. Iliev is also a member of the Jill Roberts Institute for Research in Inflammatory Bowel Disease.
The lining of the intestine is constantly juggling the need to absorb water and nutrients from food while also acting as a barrier to stop gut microbes from entering the bloodstream. In a mouse model, researchers identified the location of different fungi species living in the intestine and found certain fungi hung around specific areas near the gut lining. The findings suggest these specific fungi species closely interact with nearby epithelial cells.
Evidence of a fungi-epithelial cell relationship was found when researchers observed mice carrying these fungi species were less likely to have intestinal injuries or have a bacterial infection. "There is this harmony—a kind of communication between or across different types of organisms," says Dr. Iliev.
A separate behavioral experiment in mice also found the presence of specific fungi species was more sociable compared to animals without gut fungi. These effects came from fungi's activation of T cells which secreted immune signaling proteins known as cytokines IL-22 and IL-17.
IL-22 works to improve the intestinal barrier while IL-17 enters the bloodstream and interacts with receptors connected to the brain. Genetically modified mice that did not have the IL-17 receptor in their neurons did not show any increased social behavior in the presence of gut fungi.
The study is available to read in the journal Cell.
Jocelyn Solis-Moreira
Jocelyn is a New York-based science journalist whose work has appeared in Discover Magazine, Health, and Live Science, among other publications. She holds a Master's of Science in Psychology with a concentration in behavioral neuroscience and a Bachelor's of Science in integrative neuroscience from Binghamton University. Jocelyn has reported on several medical and science topics ranging from coronavirus news to the latest findings in women's health.
View Jocelyn's article archive
Your brain's surprising ally when it comes to fighting infection: Your gutJanuary 9, 2023
Strong evidence of connection between gut microbiome and developing Alzheimer'sJanuary 6, 2023
Your body's 'thermostat' relies on gut bacteria12 mins ago
Your body's 'thermostat' relies on gut bacteria


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In an attempt to stop his internet stardom from fading away, a young man agrees to be on a web show giving a retrospective of his work. However, when his old friend joins as an unexpected guest, they take the opportunity to catch up and hash out their left over discrepancies.
- Bonus Short: "THE RACHAEL SHOW"
- The Screenplays for "FALLING OUT" and "THE RACHAEL SHOW"
What's happening on stage while Dane and Will are talking things out? Find out on this bonus sketch where Rachael interviews her first guest: Chef Chris.
David J. Cantu & Tyler W. Moore sit down to talk about their approach to making the film.


text:
+ special guest Slothrust
7:30pm Doors //
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In 2015, Highly Suspect gained national recognition after the release of their debut album Mister Asylum on 300 Entertainment and their singles "Lydia" and "Bloodfeather" topped the rock radio charts and earned them two Grammy nominations for Best Rock Album and Best Rock Song. And now the band is ready to release album three, a manifesto addressing lead singer Johnny Steven's complicated past and attempt to change his future. Titled MCID, the album is not just the third full length project from three guys who approach the rock genre with a hip hop ethos, but it's a family meeting and an apology from singer Johnny Stevens whose lyrics demand accountability from himself and urge him to be more honest and to conquer the demons that made him who he is.
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