Mixture LC-NMR

In cases where 2D NMR experiments are insufficient for a complete analysis of anionic surfactant mixtures, LC-NMR may provide better information. Characterisation of fatty alcohol ethoxylate (FAE) based oligomeric surfactants by on-line 2D (GCOSY, TOCSY and Homo 2DJ) stopped-flow HPLC- H NMR has been described [655,656]. The analysis of a typical mixture comprising three components (PEG and PEOs with different end-groups) is shown in Figure 7.34. In this representation, the 111 NMR frequency domain is in the... 

Simultaneous separation and identification of very complex mixtures LC-NMR... 

A different issue is one that is quite common in the Pharmaceutical industry. A relatively frequent situation that arises is the need to identify a 0.1% impurity from a reaction mixture or metabolism sample. These samples are often quite convoluted in terms of the amount of compounds present as well as the general complexity of the separation, akin to a natural products extract, as can be seen in Fig. 19.19. However, to simplify this scenario to just a two-component mixture is appropriate for this section. Under common LC-NMR systems, it is typically required to have at least 50 pg of material for a complete structure elucidation (to enable the collection of long-range heteronu-clear correlation data, HMBC). Therefore, one must be able to load 50 mg of the mixture on the column. Keep in mind, that if a ID 1H spectrum is all that is needed (in the case of a regiochemical issue in an aromatic system) this task becomes more amenable. The point trying to be made is that LC-NMR is a fantastic technique, but it must be used in... 

While the early days of LC-NMR and LC-NMR-MS were plagued by the poor sensitivity of the NMR spectrometer, the recent probe design advances have provided a means to potentially overcome this hurdle. As reported in the literature, it is possible to get both ID and 2D homo-nuclear and heteronuclear correlation data on sub micrograms of materials in quite complex mixtures utilizing cryogenic flow-probes in tandem with SPE peak trappings [98]. While these technologies are still in their infancy, they have the potential to revolutionize LC-NMR as a structure elucidation technique. 

N. Blechta et al. [63] used LC-NMR experiments with H- Si indirect detection to analyze mixtures of siloxan polymers. Other studies take advantage of the unique ability of NMR to study dynamic processes like isomerization, for example, the interconversion of rotational isomers or enol-keto tautomers [64,65]. 

The coupling of chromatographic techniques such as HPLC with NMR, LC-NMR can, in principle, provide the molecular structures of compounds in mixtures (extracts) in just one online experiment. The use of LC-NMR in the flavonoid field has been reviewed by... 

In LC-NMR this problem does not arise in the same way as it does in standard sample changer automation systems, where a change between such different solvents as water and chloroform is possible. In LC-NMR, the samples are eluted from a column in a mixture of solvents, in which only the relative composition changes ... 

The situation in LC-NMR is different. A solvent mixture is used and in most cases two or more solvents are suppressed. The solvents which are delivered from the LC pump are not completely isolated from the humidity of the ambient air. Additional water is brought into the system via the acetonitrile, the sample itself and any additives such as ammonium acetate which are used in the undeuterated form. Therefore, the HDO signal is relatively strong and has to be suppressed as well. 

Figure 2.6 NMR spectra obtained under LC-NMR conditions (a) an unsuppressed spectrum showing acetonitrile ( 2.0ppm), methanol-CIT ( 3.3 ppm) and -OH signals ( 4.2 ppm) (b) the corresponding ID proton spectrum acquired with (WET) solvent suppression (on the three solvent lines a digital solvent filtering procedure was also applied). The sample used was 140 xg of cortisone in a acetonitrile/methanol/D20 (5 5 1) solvent mixture...

In LC-NMR/MS analysis, the original sample is a mixture, and the actually measured peak represents only a fraction of this. The sample amount in this fraction is unknown, and only a rough estimation based on the response of the UV detector is possible. 

The extension of an LC-NMR system to include mass spectrometry has been in application for several years [35,36]. By directly coupling a mass spectrometer to an LC-NMR system it is possible to obtain valuable mass spectral data. Configuring the system to have the sample reach the mass spectrometer before it reaches the NMR flow-cell enables the mass spectrometer to be employed as an experimental control device for analysing complex mixtures. Mass spectrometry is an ideal detector, provided that the molecules of interest are ionizable. It provides data rapidly and can thus yield valuable information on parent or daughter ion masses prior to initiating time-consuming NMR experiments. This synergy is not possible when the instruments are not directly coupled. 

Moreover, such conventional off-line approaches often result in the reisolation ( replication ) of already known compounds. Here, LC-NMR offers an unique insight into the composition of mixtures at an early stage of the analytical process for identifying ( dereplicating ) unwanted or already known compounds and thus guide the targeted isolation of potentially new substances. 

While important early publications demonstrated the suitability of LC-NMR to examine mixtures of isomerisation products of natural product standards that were exposed to light [4] or heat [5], the application of LC-NMR to natural products extracts was first presented with the characterisation of sesquiterpene lactones from the Mexican plant Zaluzania gray ana [6], In this chemotaxonomic investigation, a new lactone was already identified in onflow mode. 

In our laboratory, an on-flow LC-NMR-MS screening (Figure 5.1.1) was applied to both saponin fractions which were not separated into pure compounds by classical column chromatography and further to total asterosaponin fractions obtained by the micropreparative technique, matrix solid-phase dispersion (MSPD) extraction [45] (see Figure 5.1.2). The LC-NMR-MS hyphenation is set up in the widely used parallel configuration of NMR and mass spectrometer (Figure 5.1.3). Typically, absolute amounts of asterosaponin mixtures of about 500 xg - 1 mg are injected onto the column. 

Figure 5.2.3 depicts the HPLC chromatogram of a tomato peel extract monitored by UV absorbance at 469 nm. The separation was performed on a 150 x 4.6 mm C30 column (ProntoSil, 3 xm, 200 A, Bischoff, Germany) at room temperature and a flow rate of 1 ml/min with a binary mixture of acetone/ water, developed for LC-NMR experiments. The 50-min gradient elution was performed in four steps, i.e. (1) an initial 3 min with 75/25 (v/v) acetone/water, (2) a 24-min gradient to 100% acetone, (3) an isocratic step from 27 45 min with 100% acetone, and (4) a 2-min gradient back to the initial conditions. 

It is interesting that, in analytical chemistry, besides the efforts to increase the sample throughput and to decrease the detection limits, another trend can be observed which is directed to the analysis of more and more complex mixtures without laborious sample preparation and separation steps. This development was triggered by the requirements of bio- and environmental analysis and is closely connected to the development of multidimensional analytical methods, as well as hyphenated techniques which provide much more selectivity than one-dimensional analytical methods. Among the range of hyphenated techniques, those which combine a high separation efficiency with a maximum of structural information are of particular importance. These are hyphenated techniques such as GC-MS, LC-MS, LC-NMR and LC-NMR-MS. 

Effluents, released from textile companies, may contain dyes and auxiliaries used in the textile industry. The dyes themselves often form complex mixtures that contain considerable quantities of manufacturing precursors and by-products. However, for non-target analysis not only the large variety of compounds but also the large differences in the volatility, solubility and polarity of individual components pose problems. Most of the dyes are nonvolatile or thermally unstable. Thus, in recent years predominantly LC-MS techniques have been used for the analysis of dyes [10]. However, the combined use of LC-NMR and LC-MS offers extended possibilities which are illustrated by the analysis of an untreated waste water sample from a textile company [11],... 

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