On-line NMR detection

Another CE separation method that has been adapted to on-line NMR detection for trace level separations is capillary isotachophoresis [22]. In this case, after the separation, the analyte bands are slowly moved through the capillary until they lie directly within the coil. Precise positioning of the analyte bands in the NMR detection coil can be difficult. A recent enhancement is the use of several NMR detection coils on a single separation capillary [23], In this way, the first coil acts as a scout coil and is optimized for sensitivity (not necessarily linewidth) to locate the analyte band as it moves through the coil. After an analyte band is detected, the flow is stopped after the appropriate time-interval so that the analyte bands are now located in the second coil, which is used to acquire high-resolution NMR spectra. 

DL Olson, TL Peck, AG Webb and JV Sweedler. On-line NMR detection for capillary electrophoresis applied to peptide analysis. Peptides Chem Struct Biol PTP Pravin, RS Hedges, (eds.). Mayflower Scientific, Ltd. 730-731, 1996. 

The major advantage of on-line NMR detection as opposed to off-line detection for liquid chromatography (LC) are improved chromatographic resolution, consistent response, and rapid data acquisition. The disadvantages include poorer sensitivity due to limited measurement time and a flow-rate dependence of the NMR linewidth [Webl]. Following the first stopped flow design in 1978 [Wat 1 ], an on-line system had been developed within a year [Bayl], and the techniques have been improved continuously since then [Webl]. 

The direct coupling of HPLC with NMR spectroscopy has waited for a number of technological developments to make it a feasible routine technique. Since then, major advances have made the routine use of on-line NMR detection of HPLC fractions a useful adjunct to the armoury of analytical methods. " Experiments can be carried out in one of three modes, direct on-line NMR detection of the HPLC eluent (on-flow), a stopped-flow approach or finally the eluted fractions can be stored in capillary tubes for later recall for detailed NMR spectroscopic studies. No compromise needs to be made in the chromatographic conditions, and programmed gradient elution profiles can be accommodated. The extension of the method to nanolitre scale separations has also been discussed. ... 

The direct coupling of HPLC with NMR spectroscopy has required a number of technological developments to make it a feasible routine technique and now on-line NMR detection of HPLC fractions is a useful adjunct to the armoury of analytical methods. 

In this chapter, spectroscopy is an umbrella term for a variety of complementary methods such as tJV/VIS, IR, luminescence, and NMR, with the object of examining mainly polymer additives in solution after extraction but usually before a chromatographic separation. On-line spectroscopic detection hyphenated to chromatography is dealt with in Chapter 7. 

The first practical example of an on-line SFC-1 NMR separation was recorded by Dorn and co-workers [16] (Figure 7.2.14). Since up to 90% of a fuel is aliphatic, SFC-NMR on-line analysis is the matter of choice for separation and identification. Figure 7.2.14 shows a fuel mixture of isooctane, n-hexane, -nonane, dodecane, and n-hexadecane, separated by SFC and detected by on-line NMR spectroscopy. The SFC separation was accomplished with a flow rate of 2.0ml/min, a C18 250 x 4.6 mm column, operated at an isobaric pressure of 100 bar and a temperature of 323 K, using CO2 with 1% (w/w) CD3CN as solvent. Each NMR spectrum consists of 20 co-added transients at an acquisition time of 1 s per transient. The total separation occurred within 5 min. The first eluting isooctane can be easily identified by the methylene-to-... 

To date, two arrangements of NMR coils, the solenoidal radio frequency (RF) coil (Figure 2A) and the saddle-type (Helmholtz) RF coil (Figure 2B) have been employed as on-line NMR detectors with CE and CEC. Theoretical studies have shown that reduction of the diameter of the RF coils increases the coil sensitivity [32], The miniaturized versions of saddle types are commonly used in commercial probes. As a major development, a saddle coil which houses 1.7-mm-diameter sample tubes has been introduced by Varian. Another significant contribution is the designing of an inverse coil to accommodate 3-mm-diameter sample tubes with a detection volume of 60 pL and a total volume of 140 pL [33], Fabrication procedures hinder further reduction of diameter of saddle-type coils that are optimized for sample volumes smaller than —1 pL. 

Online detection using 4H nuclear magnetic resonance (NMR) is a detection mode that has become increasingly practical. In a recent application, cell culture supernatant was monitored on-line with 1-dimensional NMR for trehalose, P-D-pyranose, P-D-furanose, succinate, acetate and uridine.33 In stopped-flow mode, column fractions can also be analyzed by 2-D NMR. Reaction products of the preparation of the neuromuscular blocking compound atracurium besylate were separated on chiral HPLC and detected by 4H NMR.34 Ten isomeric peaks were separated on a cellulose-based phase and identified by online NMR in stopped-flow mode. 

FTIR in multiply hyphenated systems may be either off-line (with on-line collection of peaks) [666,667] or directly on-line [668,669]. Off-line techniques may be essential for minor components in a mixture, where long analysis times are required for FT-based techniques (NMR, IR), or where careful optimisation of the response is needed. In an early study a prototype configuration comprised SEC, a triple quadrupole mass spectrometer, off-line evaporative FTIR with splitting after UV detection see Scheme 7.12c [667]. Off-line IR spectroscopy (LC Transform ) provides good-quality spectra with no interferences from the mobile phase and the potential for very high sensitivity. Advanced approaches consist of an HPLC system incorporating a UV diode array, FTIR (using an ATR flow-cell to obtain on-flow IR spectra), NMR and ToF-MS. 

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. 

Al MAS NMR has been demonstrated to be an invaluable tool for the zeoHte sdentist It provides a simple and direct way to quantify the proportions of A1 in four [Al(4)j, five [Al(5)j and six [Al(6)j coordinations. Quantitative determination of these species is an important issue in catalysis, and major effort is devoted on this topic. As mentioned already, for A1 only the central transition (-i-half to —half selective exdtation ) is detected. The central transition is unaffected by first order quadmpolar interaction, but the presence of second order effects causes broadening and complicates the quantitation of the A1 species. Usually hydrated samples and short radiofrequency pulses are employed for quantitative determination of framework and extra framework aluminum species. It is uncertain whether hydration changes the coordination of A1 species. Certain extra framework A1 can have very large quadmpolar interactions resulting in very broad lines ( NMR invisible ) [155, 202]. Unlike Si NMR, Al has a short relaxation time due to its quadmpolar nature, and the Al NMR spectrum with good signal to noise can be obtained in a relatively short time. 

---