[ Pobierz całość w formacie PDF ]
.The result is a set ofNMR spectra for certain selected peaks of the chromatogram.The samples remain static in the flow cell and the conditions will remainstable during the whole NMR experiment.The parameters can be preciselyadapted for the measurement of each individual sample.These include, inparticular, the homogenization of the magnetic field and the adjustment ofthe solvent suppression parameters.While the NMR experiment for a certain peak is being performed, furtherpeaks remain in the chromatographic system.Diffusion may occur and willbroaden these peaks and therefore decrease the concentration or even destroythe separation of two closely eluting peaks.This effect is dramatically reduced ifsolvent gradients are used.During the measurement of early peaks, the wholesystem is filled with a solvent mixture where later (analyte) peaks are not dissolved but adhere to the stationary phase of the column.Diffusion isminimized so that gradient separations can be often extended to run times ofseveral hours.This is illustrated in Figure 2.2.In trace (b), a test chromatogramis shown.In the second separation (a) a higher sample amount was injected and(a)(b)0 5 10 15 20 25 30 35 40Time (min)Figure 2.2 Effect of the stop-flow procedure on a chromatographic separation: (a) chroma-togram with 14 stops for NMR measurements of 0.5 1 h each, with a total run time of 9.5 h;(a) uninterrupted chromatogram24.30(HOLD)18.90(HOLD)18.35(HOLD)30.70(HOLD)23.71(HOLD)32.12(HOLD)33.45(HOLD)22.71(HOLD)33.00(HOLD)14.96(HOLD)18.06(HOLD)20.85(HOLD)11.66(HOLD)15.92(HOLD) LC NMR: Automation 27the chromatographic experiment was stopped and continued for a total of 14times.Through the duration of the NMR experiments of 0.5 1 h each, the totalrun time of the chromatogram was extended to ca.9.5 h.Observe that theseparations for the peaks at 33.0 and 33.48 min, which had the longest residualtimes on the column, are comparable to those of the uninterrupted chromato-gram.The volume of the NMR detection cell is relatively large (30 240 "l) whencompared with the peak volumes and other void volumes in the chromato-graphic system.This leads to a considerable broadening of the peaks when theypass the NMR detection cell.It takes a long time until a peak is completelywashed out of the flow cell, i.e.a tailing is observed.This is especially criticalwhen traces of a high-concentration first peak interferes with the spectrum of aminor compound.2.2.3 LOOP STORAGE/LOOP TRANSFERIn this case, the eluent is directly flowing from the chromatographic system intoa storage device.As only selected peaks are measured in the NMR system theseparation is monitored, in parallel, with an LC detector (typically a UVdetector).A peak is selected from the chromatogram recorded by the LCdetector, and the storage loop is isolated after a certain time delay which isnecessary to allow the peak to move from the detector into the loop.Withoutinterrupting the separation, further peaks can be trapped in the subsequentstorage loops.At a later stage, after the separation is finished, the loop contents aretransferred in arbitrary order into the NMR spectrometer.Now, all kinds of1D and 2D NMR measurements can be carried out.The result is a set of NMRspectra for certain selected peaks of the chromatogram.As the peaks are collected in the sample loops, the separation is not influenced by the overallprocess, no start/stop disturbances, nor diffusion due to waiting times can occur.The transfer process is completely independent from the NMR measurements.This means that the samples can be prepared for the NMR measurements whilethe NMR spectrometer is used for other purposes.It is even possible to performthe chromatographic experiments in a separate laboratory.Once the peaks are stored and isolated in the loops, the measurementtimes for the individual NMR experiments are not limited by diffusion effects.The separation conditions are typically tested and planned with aqueoussystems, and a relatively low on column loading.For the best quality spectra,the NMR stage requires the use of D2O, instead of H2O, which will lead to adifferent pH value.This different solvent system, and the fact that a differentchromatographic system is used for the LC NMR experiments and the devel-opment of the separation, will lead to differences in the observed chromato-grams [ Pobierz całość w formacie PDF ]
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.The result is a set ofNMR spectra for certain selected peaks of the chromatogram.The samples remain static in the flow cell and the conditions will remainstable during the whole NMR experiment.The parameters can be preciselyadapted for the measurement of each individual sample.These include, inparticular, the homogenization of the magnetic field and the adjustment ofthe solvent suppression parameters.While the NMR experiment for a certain peak is being performed, furtherpeaks remain in the chromatographic system.Diffusion may occur and willbroaden these peaks and therefore decrease the concentration or even destroythe separation of two closely eluting peaks.This effect is dramatically reduced ifsolvent gradients are used.During the measurement of early peaks, the wholesystem is filled with a solvent mixture where later (analyte) peaks are not dissolved but adhere to the stationary phase of the column.Diffusion isminimized so that gradient separations can be often extended to run times ofseveral hours.This is illustrated in Figure 2.2.In trace (b), a test chromatogramis shown.In the second separation (a) a higher sample amount was injected and(a)(b)0 5 10 15 20 25 30 35 40Time (min)Figure 2.2 Effect of the stop-flow procedure on a chromatographic separation: (a) chroma-togram with 14 stops for NMR measurements of 0.5 1 h each, with a total run time of 9.5 h;(a) uninterrupted chromatogram24.30(HOLD)18.90(HOLD)18.35(HOLD)30.70(HOLD)23.71(HOLD)32.12(HOLD)33.45(HOLD)22.71(HOLD)33.00(HOLD)14.96(HOLD)18.06(HOLD)20.85(HOLD)11.66(HOLD)15.92(HOLD) LC NMR: Automation 27the chromatographic experiment was stopped and continued for a total of 14times.Through the duration of the NMR experiments of 0.5 1 h each, the totalrun time of the chromatogram was extended to ca.9.5 h.Observe that theseparations for the peaks at 33.0 and 33.48 min, which had the longest residualtimes on the column, are comparable to those of the uninterrupted chromato-gram.The volume of the NMR detection cell is relatively large (30 240 "l) whencompared with the peak volumes and other void volumes in the chromato-graphic system.This leads to a considerable broadening of the peaks when theypass the NMR detection cell.It takes a long time until a peak is completelywashed out of the flow cell, i.e.a tailing is observed.This is especially criticalwhen traces of a high-concentration first peak interferes with the spectrum of aminor compound.2.2.3 LOOP STORAGE/LOOP TRANSFERIn this case, the eluent is directly flowing from the chromatographic system intoa storage device.As only selected peaks are measured in the NMR system theseparation is monitored, in parallel, with an LC detector (typically a UVdetector).A peak is selected from the chromatogram recorded by the LCdetector, and the storage loop is isolated after a certain time delay which isnecessary to allow the peak to move from the detector into the loop.Withoutinterrupting the separation, further peaks can be trapped in the subsequentstorage loops.At a later stage, after the separation is finished, the loop contents aretransferred in arbitrary order into the NMR spectrometer.Now, all kinds of1D and 2D NMR measurements can be carried out.The result is a set of NMRspectra for certain selected peaks of the chromatogram.As the peaks are collected in the sample loops, the separation is not influenced by the overallprocess, no start/stop disturbances, nor diffusion due to waiting times can occur.The transfer process is completely independent from the NMR measurements.This means that the samples can be prepared for the NMR measurements whilethe NMR spectrometer is used for other purposes.It is even possible to performthe chromatographic experiments in a separate laboratory.Once the peaks are stored and isolated in the loops, the measurementtimes for the individual NMR experiments are not limited by diffusion effects.The separation conditions are typically tested and planned with aqueoussystems, and a relatively low on column loading.For the best quality spectra,the NMR stage requires the use of D2O, instead of H2O, which will lead to adifferent pH value.This different solvent system, and the fact that a differentchromatographic system is used for the LC NMR experiments and the devel-opment of the separation, will lead to differences in the observed chromato-grams [ Pobierz całość w formacie PDF ]