Microcoil NMR probes are well-established for analysis of trace (mass-limited) samples. This “microplug” loading method improves sensitivity by confining analyte to the NMR observed volume. Conventional Flow Injection Methods of Loading Microcoil Flow Cells FIA (miscible carrier) SFA (immiscible carrier) Zero-Dispersion SFA Microcoil NMR flow cell (10 uL) 1 uL dye, flow injection Microcoil NMR flow cell (10 uL) 1 uL dye, Zero-Dispersion SFA Sample Wets Capillary Wall Carrier Wets Capillary Wall Parabolic Flow MRM Probe (air filled) 200µm tubing 5 µL solvent “leader” 2µL sample Chase solvent 20% sample efficiency 10µL syringe, Syringe Pump Dispersion of 2 µL Sample in Flow Injection Dispersion of 2 uL sample injected into MRM ICG Probe Injection Volume (uL) NMR Peak Intensity lead_integral lead_height sample_integral sample_height chase_integral chase_height filter Preparing Trace Samples in 1-2 µL Volumes Dried with keeperDried with no keeper Recovery from FAS-coated glass vial insert. Note DMSO contact angle even as drawn down. 50 ng of anandamide was dried into a vial, resuspended in 2.2 µL DMSO, injected into an MRM ICG probe (1 µL V obs ) and data acquired overnight (16 hr). Peaks labelled “D” are also seen in control acquisition of clean DMSO-d6. Reference spectrum is 4 scans of 10 mM taxol in 5 mm tube. gHSQC of 20 µg Taxol, gHMBC of 50 ug 50 µg of taxol in 2.2 µL DMSO, was injected into an MRM ICG probe gHSQC data above were acquired in 4 hr; gHMBC in 16 hr. Similar gHSQC data acquired in 16 hr from 20 µg. 500 MHz, microcoil probe (MRM ICG), 29 µg/3 µL 900 MHz, 5 mm cryo (CPCTI), 29 ug/600 µL noise S/N=61 S/N=52 Identical aliquots of 29 ug taxol, dried in vial, prepared in indicated volume. 1-scan, 90-degree pulse, 1 Hz lb HOD DMSO ? Comparison of 500 MHz Microcoil with 900 MHz Cryoprobe Identical aliquots of 29 µg taxol were prepared, reconstituted in the indicated volume of DMSO-d6, and single-scan spectra acquired at (A) the 500 MHz microcoil at the Barnett Institute, and (B) the 900 MHz cryoprobe system at UIC (courtesy of Alec Krunic). Note: 1) 2-fold better mass sensitivity of the 900 could be obtained in a 3 mm Shigemi tube. 2) A 600 MHz CPTXI probe gave comparable S/N for this sample as the 900 MHz. 3) Micro-cryo probes (e.g. 1 mm at National Magnet Lab) offer an additional 2-fold. References High Throughput Microcoil NMR of Compound Libraries Using Zero Dispersion Segmented Flow Analysis. Kautz et al. J. Combinatorial Chem. 7: (2005) Samples may be small when FC 43-bracketed: Behnia & Webb, Anal. Chem. 70:5326 (1999) Analysis of 500 ng from single combi-chem bead: Lacey et al., J. Mag. Reson 153:215 (2001) An SFA-PCR method showing low carryover: Curcio & Roeraade, Anal. Chem 75:1 (2003) Zero-Dispersion SFA review: Patton & Wade in “Analytical Instr. Handbook” 1997 Microplug NMR Increasing microcoil NMR sensitivity using segmented flow sample loading, or “Shigemi tubes for microcoil probes” Yiqing Lin and Roger Kautz* The Barnett Institute of Chemical and Biological Analysis Boston, Massachusetts 1 Dept. Pharmacognosy, U. IL Chicago Microplug Sample Loading NMR Observed Volume The direct injection method is to fill the entire flow system, including the dead volume, with sample. The NMR observed volume can be filled with high concentration sample, but the overall sample efficiency is low (observed volume / total volume). In flow injection, the system is filled with solvent. A smaller volume of sample is injected, followed by more solvent to push the sample into the observed volume. In ideal parabolic flow, attempting to push a volume element 10 cm down the capillary results in a 20 cm smear: the fluid touching the wall is stationary, the fluid in the center moves further than 10 cm 1. At right, an “NMR chromatogram” plots the relative concentrations of a 2 µL DMSO sample plug, the DMSO in front of it, and the DMSO behind it, as the sample is injected through the 1 µL NMR observed volume. The sample has been injected directly into the inlet of a microcoil probe, to minimize dead volume (8 µL to fill V obs ). Alternatively, dispersion can be avoided if samples are sandwiched between plugs of an immiscible fluid. Care must be taken to avoid “painting the wall” of the tubing with the sample plug, which consumes about a microliter of sample per meter of 100 µm tubing. This microdrop NMR approach uses zero-dispersion conditions for segmented flow. The tubing and carrier fluid are chosen so that the carrier fluid aggressively wets the tubing wall, relative to the sample. At right, several DMSO plugs separated by an immiscible perfluorocarbon liquid are being pushed through the same 1 µL NMR observed volume as the figure above. These 1 and 3 µL plugs have traversed a 100 µL dead volume. A perfluorocarbon carrier fluid in Teflon tubing achieves the zero-dispersion conditions, and is immiscible with common NMR solvents. 1 NB: for long plugs, this dispersion only affects the ends of the sample volume, so samples can generally be injected through a dead volume equal to the sample volume and maintain their initial concentration at the center of the sample zone. Also, flow injection can take advantage of non-parabolic flow: when fully optimized, the Protasis/MRM automated microcoil loading system can inject 5 uL samples through a 20 µL dead volume and provide 90% of initial sample concentration in the 2 µL microcoil observed volume. Start flow, (Well 74 in probe) Wash Plug Detected Flow Stopped, Well 75 centered in probe Segmented Flow Loading of 1 µL Sample TMSP peak, 2 sec intervals Wash plug Sample plug Previous Sample Wash plug (1 µL) Sample plug (3 µL) Immiscible fluid 1 µL NMR Observed Volume 1 µg/µL beta-methyl-glucoside in D 2 O Microdrop Loading Provides Equivalent Spectra From Smaller Sample Volumes; Proportionally Higher Sensitivity for Trace Samples The spectra above were generated by injecting indicated volumes of a standard solution (all are the same concentration). They show how equivalent spectra can be obtained with 1/5 the sample mass in a 1 µL plug, as by filling the flow cell with a 5 µL plug. For a 1 µg sample dissolved and injected in the indicated volumes, a 1 µL plug would give 5 times the S/N. Note that the Flurocarbon fluid is susceptibility matched to D2O, permitting interpretable spectra (2 Hz linewidth) of a sample volume less than the NMR probe observed volume. (Above spectra obtained with MRM H1 probe.) 1D NMR of 50 ng Using Microdrop NMR