Despite breakthroughs in MAS NMR hardware and experimental methodologies sensitivity remains a major challenge for large and complex biological systems. Here, we report 3-4 fold higher sensitivities obtained in heteronuclear-detected experiments, using a novel HCN CPMAS probe, where the sample coil and the electronics operate at cryogenic temperatures, while the sample is maintained at ambient temperatures (BioSolids CryoProbe™). Such intensity enhancements permit recording 2D and 3D experiments for large assemblies that are otherwise time-prohibitive, such as 2D 15 N-15 N proton-driven spin diffusion and 15 N-13 C double cross polarization to natural abundance carbon experiments. The benefits of CPMAS CryoProbe-based experiments are illustrated for assemblies of kinesin Kif5b with microtubules, HIV-1 capsid protein assemblies, and fibrils of human Y145Stop and fungal HET-s prion proteins -demanding systems for conventional MAS solid-state NMR and excellent reference systems in terms of spectral quality. We envision that this probe technology will be beneficial for a wide range of Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Significant, 3-4 fold sensitivity gains are obtained experiments with a novel CPMAS CryoProbe.The benefits of CPMAS CryoProbe are demonstrated in heteronuclear-detected 2D and 3D experiments on five challenging biological assemblies.High-quality 2D NN and NCA/NCO spectra are obtained on U-15 N-CA tubular capsid assembly containing carbons at natural abundance.

The superior mass sensitivity of micro-coil technology in Nuclear Magnetic Resonance (NMR) Spectroscopy provides potential for the analysis of extremely small mass-limited samples such as eggs, cells, and tiny organisms. For optimal performance and efficiency, the size of the micro-coil should be tailored to the size of the mass-limited sample of interest, which can be costly as mass-limited samples come in many shapes and sizes. Therefore, rapid and economic micro-coil production methods are needed. One method with great potential is 5-axis Computer Numerical Control (CNC) micro-milling, commonly used in the jewelry industry. Most CNC milling machines are designed to process larger objects and commonly have a precision >25 µm (making the machining of common spiral micro-coils, for example, impossible). Here, a 5-axis MiRA6 CNC milling machine, specifically designed for the jewelry industry, with a 0.3 µm precision was used to produce working planar micro-coils, microstrips, and novel micro-sensor designs, with some tested on the NMR in less than 24 hours after the start of the design process. Sample wells could be built into the micro-sensor and could be machined at the same time as the sensors themselves, in some cases leaving a sheet of Teflon as thin as 10 µm between the sample and sensor. This provides the freedom to produce a wide array of designs and demonstrates 5-axis CNC micro-milling as a versatile tool for the rapid prototyping of NMR micro-sensors. This approach allowed the experimental optimization of a prototype microstrip for the analysis of two intact adult Daphnia magna organisms. In addition, a 3D volume slotted tube resonator was produced that allowed for 2D 1 H-13 C NMR of D. magna neonates and exhibited 1 H sensitivity (nLOD ꙍ 600 = 1.49 nmol s 1/2 ) close to that of double striplines, which themselves offer the best compromise between concentration and mass sensitivity published to date.

Polyolefins are important and broadly used materials. Their molecular microstructures have direct impact on macroscopic properties and dictate end-use applications. 13 C NMR is a powerful analytical technique used to characterize polyolefin microstructures, such as long-chain branching (LCB), but it suffers from low sensitivity. Although the 13 C sensitivity of polyolefin samples can be increased by about 5.5 times with a cryoprobe, when compared with a conventional broadband observe (BBO) probe, further sensitivity enhancement is in high demand for studying increasingly complex polyolefin microstructures. Toward this goal, distortionless enhancement by polarization transfer (DEPT) and refocused insensitive nuclei enhanced by polarization transfer (RINEPT) are explored. The use of hard, regular, and new short adiabatic 180°1 3 C pulses in DEPT and RINEPT is investigated. It is found that RINEPTs perform better than DEPTs and a sensitivity enhancement of 3.1 can be achieved with RINEPTs. The results of RINEPTs are further analyzed with statistics software JMP and recommendations for optimal usage of RINEPTs are suggested. An example of analyzing saturated chain ends in an ethylene−octene copolymer sample with a hard 180°1 3 C RINEPT pulse is demonstrated. It is shown that the experimental time can be further reduced in half because of faster proton relaxation, where the total experimental time is about 580 times shorter when compared to using a conventional method and a 10 mm BBO probe. A naturally abundant nitrogen-containing polyolefin is analyzed using 1 H− 15 N HMBC and, to our knowledge, is the first 1 H− 15 N HMBC presented in the field of polyolefin characterization. The relative amount of similar nitrogen-containing structures is quantified by two-dimensional integration of 1 H− 15 N HMBC. Two pragmatic technical challenges related to using high-sensitivity NMR cryoprobes are also addressed: (1) A new 1 H decoupling sequence Bi_Waltz_65_256pl is proposed to address decoupling artifacts in 13 C{ 1 H} NMR spectra which contain a strong 13 C signal with a high signal-to-noise ratio (S/N). (2) A simple pulse sequence that affords zero-slope spectral baselines and quantitative results is presented to address acoustic ringing that is often associated with high-sensitivity cryoprobe use.

An efficient NMR approach is described for determining the chemical structures of the monosaccharide glucose and four disaccharides, namely, nigerose, gentiobiose, leucrose and isomaltulose. This approach uses the 1 H resonances of the −OH groups, which are observable in the NMR spectrum of a supercooled aqueous solution, as the starting point for further analysis. The 2D-NMR technique, HSQC-TOCSY, is then applied to fully define the covalent structure (i.e., the topological relationship between C–C, C–H, and O–H bonds) that must be established for a novel carbohydrate before proceeding to further conformational studies. This process also leads to complete assignment of all 1 H and 13 C resonances. The approach is exemplified by analyzing the monosaccharide glucose, which is treated as if it were an “unknown”, and also by fully assigning all the NMR resonances for the four disaccharides that contain glucose. It is proposed that this technique should be equally applicable to the determination of chemical structures for larger carbohydrates of unknown composition, including those that are only available in limited quantities from biological studies. The advantages of commencing the structure elucidation of a carbohydrate at the −OH groups are discussed with reference to the now well-established 2D-/3D-NMR strategy for investigation of peptides/proteins, which employs the −NH resonances as the starting point.

Ethylene–hexene linear low-density polyethylene (LLDPE) is widely used in the packaging industry due to its outstanding mechanical properties. Some LLDPEs contain long-chain branching (LCB), which improves viscoelastic properties. Therefore, it is important to detect and quantify LCB content to enable production of improved LLDPEs for targeted applications. 13C NMR is an intrinsically quantitative method and can measure LCB directly. However, 13C NMR LCB measurement in ethylene–hexene LLDPE has challenges, such as the overlap of the ethylene–hexene–ethylene branch (EHE Br) peak with the LCB methine peak in conventional solvents used for dissolving ethylene–hexene LLDPE. Recently a 1-choloronaphthelane/para-dichlorobenzene-d 4 (PDCB-d 4) (9:1, w/w) solvent was developed to separate the LCB methine peak from the ethylene–octene–ethylene branch (EOE Br) peak in an ethylene–octene copolymer. Here, we explore using this solvent for 13C NMR LCB measurement in ethylene–hexene LLDPE. Based on 13C NMR simulation, a high level of LCB in low hexene content LLDPE might be detected and measured (with some errors due to incomplete separation from the EHE Br peak) with 400 and 500 MHz 10 mm NMR cryoprobes. However, low sensitivity and LCB peak overlap with the EHE Br peak pose even more difficulties for detecting the low level LCB in high hexene content LLDPE. Although 600 and 700 MHz 10 mm NMR cryoprobes provide much better sensitivity and excellent separation of the LCB methine peak from the EHE Br peak, the LCB methine peak overlaps with the EHE Br peak’s downfield 13C satellite. Therefore, a new anti-incredible natural-abundance double-quantum transfer experiment (anti-INADEQUATE) inverse-gated NMR pulse sequence, ainadigsp1d.2, was developed to remove 13C satellite peaks and quantify LCB content in ethylene–hexene LLDPE. The LCB results obtained with this new NMR pulse sequence are in very good agreement with expected values.