On-line HPLC-NMR

In on-line HPLC-NMR coupling, the commonly recorded nuclei are and 19F, because their natural abundances are 99.9 and 100%, respectively. Thus, a direct monitoring of chromatographic separations is possible, as outlined earlier in Chapter 1. Indirect access to the information content of 13C NMR spectra is obtained in the stop-flow mode, where inverse detected 1H, 13C correlation spectra can be recorded. The acquisition of these type of 2D-spectra relies on the fact that a direct proton carbon connectivity via scalar coupling is present. Quartenary carbons without any directly attached protons are not detected. Thus, it is of major interest to record 13C NMR spectra which reveal all possible information within a coupled LC experiment. 

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. 

For structural identification of the fractions, the XH-NMR spectrometer was directly coupled via capillary tubing to the chromatograph. The injection of the sample into the HPLC system was automatically initiated by the NMR console via a trigger pulse when starting the acquisition of NMR data. Using an appropriate pulse sequence, both solvent resonances (ACN at 2.4 ppm and water at 4.4 ppm) could be suppressed simultaneously. As a result of the on-line HPLC-NMR experiment, a contour plot XH chemical shift vs. retention time was generated (see Fig. 39). Due to the efficient solvent suppression, the obtainable structural information relates to the entire chemical shift region. From the contour... 

Fig. 39. Contour plot of chemical shift vs. retention time and chemigram of the on-line HPLC-NMR analysis of a technical poly(ethylene oxide) (from [210] with permission)...

Our on-line GPC/NMR system consists of a JASCO TRI ROTAR-V chromatograph and a JEOL JNM-GX500 spectrometer (a 16-bit A/D converter was installed). A 2- or 3-mm (i.d.) glass tubing with tapered structure at both ends is employed as an NMR observation flow cell (Fig. 30) the detection volumes are 0.060 and 0.140 ml, respectively. A similar type of flow cell was used in the on-line HPLC/NMR experiments.340,341 The tapered structures at both ends are needed to prevent turbulent flow in the inlet path of the cell, which would cause broadening of the elution band. When a cell without tapering was used, some broadening of the band was observed.334... 

Segmuller, B.E., Armstrong, B.L., Dunphy, R., and Oyler, A.R., 2000, Identification of autoxidation and photodegradation products of ethynylestradiol by on-line HPLC-NMR and HPLC-MS, J. Pharm. Biomed. Anal. 23, 927-937. 

On-line HPLC-NMR separations can be conducted either in the continuous-flow or stopped-flow mode. In the continuous-flow mode, the chromatographic effluent resides for a distinct time within the flow-cell volume defined by the r.f coil. This residence time r is defined by the ratio between flow-cell volume and flow rate. It should be in the range of 3 to 5 s [3]. Otherwise severe distortions of the NMR signal line widths could occur. In the stopped-flow mode the peak maximum is transferred to the NMR flow cell and 2D NMR spectra can be recorded without any time restrictions. 

Figure 7-2 Experimental arrangement for on-line HPLC-NMR coupling.

Provided the available amount of substances is in the upper pg range, on-line HPLC-NMR separation is a feasible method for structure identification. Figure 7-3 shows the H NMR spectrum of a mixture of five isomers of vitamin A acetate. 

Without any knowledge of chemical shifts and coupling constants for these isomers, full assignment of the H NMR spectrum would be impossible. Employing a cyanopropyl-modified silica gel, an on-line HPLC-NMR separation was performed with two 250 x 4.6 mm HPLC columns using hexane as eluent at a flow rate of 0.3 ml/min [6], Eight transients were coadded, defining a time resolution of 8 s. 

On-line HPLC-NMR offers unique opportunities for analyzing complex polymer systems. Using a selective chromatographic technique, the sample can be separated according to the chemical structure. The structural analysis of the polymer can then be conducted by on-line H-NMR. Because of sensitivity enhancements and inq)roved solvent suppression techniques of the NMR, it is possible to work under experimental conditions which are common in liquid chromatography of polymers, i.e. sufficiently high flow rate, moderate sample concentration, protonated solvents, and on-flow detection. 

In reeent years, tire use of elevated temperatures has been reeognised as a potential variable in method development. Witlr inereased temperature, aqueous-organie mobile phases separations ean improve, viseosity deereases and diffusion inereases so baek pressures are redueed. At higher temperatures (usually with superheated water > 100 °C under modest pressures) water alone ean be used as the mobile phase and eair provide unique separation opportunities. The absenee of an organie solvent enables the use in HPLC of alternative deteetors sueh as FID or on-line LC-NMR using deuterium oxide as the eluent. 

Figure 7.22 shows the H NMR chromatogram (contour plot) of the separation of a 10% phthalate mixture in CH2CI2. The spectrum is almost free from interferences the NMR resolution is excellent, and it is possible to identify all plasticisers even at concentrations as low as 2%, which corresponds to 60 xg per component. In contrast, in on-line HPLC- H NMR separation the regions between 3.9-3.3 and 1.9-1.7 ppm are completely obscured by solvent signals. 

Thermal degradation of Irganox 1076 in air was studied by means of HPLC-UV/VIS and by preparative HPLC-NMR. At 180 °C cinnamate and dimeric oxidation products are formed, and at 250 °C de-alkylation products are observed [660], On-line LC-NMR hardly covers a real need in polymer/additive analysis, as the off-line option is mostly perfectly adequate for that purpose. 

Multiple hyphenation ( hypemation ) provides comprehensive spectroscopic information from a single separation. The first doubly hyphenated HPLC-NMR-MS appeared in 1995 [661], and its value is now accepted meanwhile fully integrated on-line LC-NMR-MS and MSn systems (QMS, QTTMS) are commercially available. On-line LC-NMR-MS coupling is by no means trivial. For example, the sensitivity of NMR is limited, while MS is incompatible with non-volatile buffers. The... 

In summary, NMR spectroscopy is an extremely versatile tool useful that enables researchers to understand the structure of natural products such as carotenoids. For a full structural assignment, the compound of interest has to be separated from coeluents. Thus, it is a prerequisite to employ tailored stationary phases with high shape selectivity for the separation in the closed-loop on-line LC-NMR system. For the NMR detection, microcoils prove to be advantageous for small quantities of sample. Overall, the closed-loop system of HPLC and NMR detection is very advantageous for the structural elucidation of air- and UV-sensitive carotenoids. 

The main prerequisite for on-line LC-NMR, besides the NMR and HPLC instrumentation, are the continuous-flow probe and a valve installed before the probe for the registration of either continuous-flow or stopped-flow NMR spectra. 

For the on-line SFE-NMR experiments, the set-up shown in Figure 7.2.17 can be used. A main pump serves an HP supercritical fluid chromatograph (G1205A), with analytical HPLC columns being used as extraction cells. The continuous-flow NMR cell is connected between the column outlet and the back-pressure regulator. 

Overall, both types of experiment clearly show that the application of polarization transfer experiments has great potential for recording continuous-flow 13C NMR spectra. The further refining and fine-tuning of these types of experiments could lead to practical related applications of on-line HPLC-13C NMR experiments. 

Godejohann, M., Preiss, A., Miigge, C. and Wii-nsch, G, Application of on-line HPLC- H NMR to environmental samples analysis of ground-water near former ammunition plants, Anal. Chem., 69, 3832-3837 (1997). 

The analysis of fatty alcohol ethoxylate based surfactants by on-line HPLC- H-NMR has been described by Schlotterbeck et al. [211]. Using a reversed stationary phase and ACN/deuterium oxide as the eluent, surfactant mixtures were... 

Figure 1. NMR on-flow run of oligostyrene in 100% protonated acetonitrile (with no deuterium lock). Retention time vs. chemical shift. The separation into different chain lengths is apparent. (Reprinted from Polymer, Vol.39, H.Pasch, W.Hiller, R.Haner Investigation of the tacticity of oligostyrenes by on-line hplc/ H nmr , 1515-1523, 1998, with permission from Elsevier Science)...

Figure 3. HPLC-chromatogram of styrene-ethyl acrylate copolymers at different compositions and high conversion. Also shown is the styrene content obtained by HPLC calibration -) and on-line HPLC- H NMR experiments (o). (Reprinted from MacromolChem.Phys, Vol.201, LKramer, W.HiUer, H. On-line coupling of gradient-HPLC and NMR for the analysis of random poly[(styrene)-co-(ethyl acrylate)]s , 1662-1666, 2001, with permission from Wiley-VCH)...

Fig. 27. RPLC chromatograms of poly(S-co-EA)s (SEA) of different composition with high conversion. Styrene contents obtained by HPLC calibration (lines) and on-line HPLC/ H NMR (circles) experiments match each other very well. Column Nucleosil CIS 100 A 250 X4.6 mm. Eluent THF/CH3CN, gradient elution from 10/90 to 100/0 (v/v) in 25 min. Reproduced from [152] with permission...

In many cases functionality does not affect the hydrodynamic volume of the whole polymer chain significantly and SEC can separate them in terms of molecular weight. In order to separate the polymers in terms of functionality, however, IC has to be used. Pasch and Hiller separated a technical oligo(ethylene oxide) by isocratic RPLC with respect to degree of polymerization and functional end groups, and analyzed the chemical structure by on-line H-NMR detection [ 170]. The experiments was conducted under conditions that are common for HPLC... 

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