Direct stop-flow sample

Both the capillary LC and NMR are controlled by the interface software, which enables the operator to use the UV-detector output for peak selection. Only peaks of interest can be subjected to NMR analysis, while minor or unimportant compounds can be directed to waste. NMR acquisition can take place in either on-flow or stop-flow mode. The combination of capillary LC and NMR is suitable for sample-limited applications (e.g., proteomics) and allows for low nanograms detection. 

The stop-flow technique is the simplest and least expensive method of injection. The flow of the mobile phase is first stopped, either by turning an on—off valve in the line before the column (with constant-pressure systems), or by stopping the pump (with constant-flow systems). The column then returns rapidly to atmospheric pressure, and the sample can be injected directly on to the column with a normal low-pressure syringe. 

The mass spectrometry portion of the analysis was carried out by coupling a Bruker Esquire ion-trap mass spectrometer to the LC-NMR system with a 20 1 splitter. The major portion of the flow was directed to the NMR system while the minor fraction went to the mass spectrometer. The system was plumbed such that the sample reached the mass spectrometer and the UV detector at the same time. In this configuration, it is possible to use the mass spectrometer as an intelligent detector, thus allowing stop-flow experiments to be initiated on the basis of observed molecular ions or daughter ion fragments. Data were acquired with electro-spray ionization (ESI) in the positive-ion mode. 

Frequently industrial hygiene analyses require the identification of unknown sample components. One of the most widely employed methods for this purpose is coupled gas chromatography/ mass spectrometry (GC/MS). With respect to interface with mass spectrometry, HPLC presently suffers a disadvantage in comparison to GC because instrumentation for routine application of HPLC/MS techniques is not available in many analytical chemistry laboratories (3). It is, however, anticipated that HPLC/MS systems will be more readily available in the future ( 5, 6, 1, 8). HPLC will then become an even more powerful analytical tool for use in occupational health chemistry. It is also important to note that conventional HPLC is presently adaptable to effective compound identification procedures other than direct mass spectrometry interface. These include relatively simple procedures for the recovery of sample components from column eluate as well as stop-flow techniques. Following recovery, a separated sample component may be subjected to, for example, direct probe mass spectrometry infra-red (IR), ultraviolet (UV), and visible spectrophotometry and fluorescence spectroscopy. The stopped flow technique may be used to obtain a fluorescence or a UV absorbance spectrum of a particular component as it elutes from the column. Such spectra can frequently be used to determine specific properties of the component for assistance in compound identification (9). 

Figure 10 (A) H NMR spectrum of the trace impurity sample (200 pM atenolol and 200 mM sucrose in 50% TE/D20) from 5-mm probe. The expanded and vertically increased area is shown. Microcoil H NMR spectra shown in (B)-(D) recorded and processed with identical parameters. (B) Static NMR spectrum obtained with direct injection of 25 mM atenolol to the NMR microcoil. S/N of atenolol methyl peak is 21. (C) On-flow cITP-NMR spectrum of atenolol sample band at peak maximum during analysis of the trace impurity sample (200 pM atenolol and 200 mM sucrose in 50% TE/D20). No sucrose peaks can be observed. S/N atenolol methyl peak is 34. (D) Stopped-flow cITP-NMR spectrum of sucrose at peak maximum from the same experiment as in (C). (Adopted with the permission from Ref. 41. Copyright 1998 American Chemical Society.)...

The direct coupling of liquid chromatography with proton NMR has been attempted numerous times. Early experiments of coupled HPLC- H-NMR were conducted in a stop-flow mode or with very low flow rates [193-195]. This was necessary to accumulate a sufficient number of spectra per sample volume in order to improve the signal-to-noise ratio. Other problems associated with the implementation of on-line HPLC-NMR have included the need for deuterated solvents. However, with the exception of deuterium oxide, the use of deuterated eluents is too expensive for routine analysis. Therefore, proton-containing solvents, such as acetonitrile or methanol, must be used. To get rid of the solvent signals in the spectra, the proton NMR signals of the solvents have to be suppressed. 

Recently, NMR spectrometers directly coupled with LC systems have become commercially available. Spectra can be acquired in either of two modes, continuous or stopped flow. In continuous flow mode the spectrum is acquired as the analyte flows through the cell. This method suffers from low sensitivity since the analyte may be present in the cell for only a brief period of time, but it has the advantage of continuous monitoring of the LC peaks without interruption. Fig. 12A shows a contour plot of the continuous flow NMR analysis of a mixture of vitamin A acetate isomers.Fig. 12B shows the spectra taken from slices through the contour plot. These plots highlight the olefinic region of the spectra which provided ample information for the identification of each of the isomers. With very limited sample quantities, the more common method of LC-NMR analysis is stopped flow. Here the analyte peak is parked in the flow cell so any of the standard NMR experiments can be run. 

On-flow HPLC-NMR analysis can also be performed when sufficient material is available. It involves collecting the NMR data continuously as the sample passes through the probe. This is the most efficient method for stmcture evaluation by HPLC-NMR. The NMR data are represented in a 2-D plot where the x direction contains chemical shift information and they direction is representative of the LC retention time. The individual spectra can be extracted from the ID slices along the x axis if so desired. The resolutions in the individual spectra are of somewhat lower quality than in the stop-flow method however, the introduction of the second dimension allows for easy stmcture assignment even for overlapping peaks in the LC separation. As seen in Fig. 19, the on-flow HPLC-NMR characterization shows four distinct sets of resonances. 

The master gas chromatograph is a Varian 2760 instrument with thermal conductivity and flame ionization detection. A second Varian 2760 gas chromatograph (GC-2) serves for analysis of samples from two sources, pyrolysis products from the CDS 820 and from the structural determination function of the CDS 1200. The latter instrument (Chemical Data System) is a functional group and elemental analyzer which generates a vapor-phase thermolytic dissociation pattern for functional group analysis and also performs elemental analysis. The effluent from the master GC is split so that 10% of it is directed to the detector and 90% of it to the CDS 1200. A stop-flow valve admits one... 

There are a few ways of linking the techniques of LC-MS and LC-NMR. The most common method is in a parallel mode by splitting the flow, e.g. 50 1, so as to direct the majority of it to the NMR due to its relative insensitivity. This means that the analytes are detected simultaneously by both detectors and possibly also by UV, which may actually be used as the trigger to begin detection by the NMR and MS modules. Alternatively, the rapidly acquired MS data can be used to direct the NMR experiments or vice versa. A second method of interfacing the two techniques is to use the serial mode or stopped flow mode, which enables more sensitive NMR experiments to be carried out. A recent development in stopped flow NMR is the inclusion of in-line solid phase extraction (SPE) after the LC. The SPE acts as a fraction collector for individual compounds. This trapping/ washing step can improve sensitivity several fold. A third method is fraction collection, where samples from the LC are collected in a loop for analysis later, perhaps after certain data have been reviewed. 

The advantages of directly interfacing HPLC separation with NMR analyses have so far been limited by the time and amount of material required to obtain various NMR spectra. However, two different types of HPLC interface have been developed for LC-NMR analysis. In stopped-flow techniques, the material of interest is directed into an NMR sample cell within the magnetic field after HPLC separation has occurred, and the pumps are then stopped to allow the necessary NMR experiments to be performed. The second type of... 

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