MicroFlow NMR™ FAQ Frequently Asked Questions about MicroFlow NMR: 1) Do I need to position my sample management/separation system as close to the NMR magnet as possible?. There are certainly advantages to maintaining a reasonable distance between the magnet/probe and the samp le management system. However, these constraints are significantly relaxed at the capillary scale, primarily due to the excellent fluidic integrity that is maintained using a capillary (75 µm i.d./360 µm o.d.) feed line. A fundamental limit in conventional systems is the amount of peak broadening (sample dilution/mixing) that occurs in the transport tubing. This limitation often requires that the sample management system and magnet/probe be positioned to within 1-2 meters of one another, and even then with measurable degradation in peak integrity. In stark contrast, at the capillary scale, the effect of 5 meters of capillary is virtually unnoticeable in terms of peak broadening. 2) Is it cost-effective to exclusively run deuterated solvents in a capillary NMR flow system? Yes! A 24 hour run of non-deuterated acetonitrile in a conventional scale LC-NMR system (1 mL/min) is approximately $16/day, with 720 mL of total solvent consumed. A 24 hour run of deuterated acetonitrile in a capillary LC-NMR flow system (5 µL/min) is approximately $10/day, with about 3.5 mL of solvent used. Concerning flow injection systems, a conventional-scale 96-well plate will typically require greater than 150 mL of solvent, whereas capillary-scale typically requires approximately an order-of- magnitude less solvent, < 20 mL/plate. Both flow injection estimates include needle and port rinsing. 3) Isn’t the achievable S/N in NMR directly related to sample volume? Isn’t “bigger” better? The S/N in NMR is actually directly related to the quantity of sample (mass, moles, or number of spins) being investigated. In many cases, intelligent sample preparation (for example, preconcentration) can provide a higher concentration of sample for NMR detection using a smaller RF coil that is of greater mass sensitivity, resulting in an effective increase in S/N. In other cases, the simple ability to accomplish useful NMR with a 100- fold reduction in solvent consumption justifies the move to the capillary scale. Fluidic advantages provide significant additional incentive to operate in the capillary regime. 4) Why should I consider small sample volumes? Benefits of small sample analysis include: a) In sample- limited applications (e.g. natural product research, impurity analysis, metabonomics, etc.), where only a small mass is available, analys is at microliter scale provides higher spectral quality and greater S/N than can be obtained by diluting to larger volumes for conventional analysis.
b) Reduction or elimination of the need for solvent suppression via the economic use of deuterated solvents. c) More effective utilization of solvent suppression due to higher signal- to-solvent ratio (relative to conventional probes), resulting in higher spectral purity/quality. d) In protein applications, decreasing the quantity needed from expression systems may significantly reduce both cost and the time required to obtain data. e) In applications where multiple analyses are required, the ability to obtain data from small amounts of material avoids the need for additional sample for NMR, thereby saving both time and money. f) Disposal is more economical and less complicated when using small volumes. 5) Do I need to worry about solvent or sample compatibility? Protasis/MRM probes utilize a completely fused silica transport system, that is, all wetted surfaces are fused silica. While no sample transport medium is 100% compatible with all known chemicals, fused silica provides one of the most robust and workable solutions for most samples and solvents encountered in NMR analysis, including DMSO. 6) Is the flowcell interchangeable by the customer? No, and it is not necessary. Due to the flow-through design of the Protasis/MRM flowcell, clogging or permanent damage will be infrequent, provided that the rules of proper fluidic management are maintained (e.g. in- line filters are employed, solvent miscibility rules are followed when performing solvent change, sample is rinsed from probe prior to probe removal and storage, etc.) In the infrequent case that the flowcell requires replacement, Protasis/MRM offers a return-to- factory service policy. For customers on maintenance agreement, the probe can be diagnosed, repaired, and returned within two weeks (USA; slightly longer international) from the time the probe is received by Protasis/MRM. 7) How long does it take from the time of order to get a Protasis/MRM probe? Typical lead-time from the time of order to the time of delivery is 3 months, although in some cases it can be much quicker. 8) What nuclei are currently offered? Current models include: 1) Proton detect (1 H detect with 2 H lock, with optional zgradient), 2) indirect carbon with gradient (ICG, 1 H{13 C} detect with 2 H lock and zgradient, and 3) triple resonance (TXI), 1 H{13 C, 15N} detect with 2 H lock and z- gradient. 9) What sample management options are possible? The options for sample management range from very simple and economical direct injection modules to completely automated liquid handling options and chromatography solutions. The manual injection module provides a cost-effective and highly accurate means of introducing a sample manually to the probe using a syringe and calibrated quantity of push solvent. Many of our natural products customers and academic researchers employ this means of sample introduction. The new- for-2004 Open Access Automation system bundles a CTC (Leap Technologies) liquid handler with Protasis control software to enable well-plate automation at the capillary scale. This system can
be configured for both high throughput and walk- up vial submission from multiple users. Data management is provided with the Protasis Secure Data Server module. Chromatography users can utilize the Waters capillary LC system, with an elution volume (320µm columns) volume- matched to the NMR flowcell in the Protasis/MRM probe. Alternative sample management options are available. Call your sales representative today at 508-481-4163 to determine which configuration is right for your application needs! 10) Will my current liquid handler work with a Protasis/MRM probe? Attainment of high quality NMR performance specifications requires both that the probe be highly sensitive and that the means of introducing the sample to the probe be highly accurate and efficient. Protasis/MRM has worked aggressively to achieve system configurations that provide optimum performance and that can be supported in field operation. The recommended choice in liquid handlers is the CTC (Leap Technologies) liquid handler. This choice is based upon the ability and accuracy of the CTC model to handle capillary-volume samples, and Protasis/MRM has developed an extensive software automation solution to complement this configuration. Protasis/MRM supports the popular Gilson 215 liquid handler as well, but with some compromises in overall system performance relative to the CTC solution. Protasis/MRM staff are available to discuss the differences and performance tradeoffs of these two configurations. 11) Is the Protasis/MRM probe compatible with my Bruker/Varian/JEOL spectrometer? Protasis/MRM probes are compatible with all of the major NMR spectrometer brands. 12) Is the Protasis/MRM probe difficult to shim? No. In fact, ease of use is one of the attributes that our customers vocalize as a strength of this product. Due to the small size of the detection coil and the manner in which magnetic field matching is achieved, you’ll find that the Protasis/MRM probe is generally easier to shim than other conventional NMR probes. 13) What frequencies are offered? Protasis/MRM currently offers 300 – 600 MHz 1 H frequencies, but both 700 MHz and 800 MHz designs are under development with a delivery goal of 1Q05.. 14) Will I need to do anything special to my spectrometer to accommodate a Protasis/MRM probe? The microflow line of probes interface directly to the spectrometer and require nothing beyond what is typical of a new probe. Shim settings, power settings, heater control settings, etc. should be set to the recommended Protasis/MRM values. 15) How do capillary fluidics compare to conventional scale fluidics for NMR flow probes? Capillary-scale fluidics provide a highly accurate and reproducible means of sample introduction to the probe. This contrasts sharply with conventional probes where peak broadening and diffusion can lead to significant sample degradation prior to NMR analysis and where elaborate protocols for sample injection are required to keep mixing at tolerable levels.
16) What is the total volume of sample that I need to inject? The Protasis/MRM family of microflow probes is designed to accommodate specific volumes of samples. At the present time, Protasis/MRM offers both a 5 µL NMR flowcell and a 10 µL NMR flowcell. Generally, these are the target values that should be used for determination of injection volume, i.e. one should aim to fill the NMR flowcell for optimum S/N per unit injected volume. However, this does not preclude the injection of smaller or larger volumes for analysis. 17) What is the total quantity of sample that I need to inject? Sample quantity depends upon the intended application, sample type (e.g. molecular weight), and other sample parameters such as solubility and viscosity. In general, nanomoles and micrograms of small molecule samples will be sufficient to obtain 1D NMR spectra in under one minute. Multidimensional and heteronuclear experiments typically require 50-100 micrograms of similar sample type for target acquisition times of several hours. 18) How do I load my samples into the probe? There are multiple means of sample introduction, and the choice of which means is right for you depends upon your sample, your intended application, and your budget. The simplest and most efficient means of sample introduction is generally direct injection into the probe using a calibrated syringe and push solvent. Protasis/MRM offers a 2-syringe with valve manual injection module that can be positioned at the base of the magnet or secured via Velcro straps to the leg of the magnet for this purpose. Automation and well plate NMR is accommodated using the Protasis/MRM Open Access NMR system. This system provides liquid handling robotics using a CTC (Leap Technologies) liquid handler that interfaces to Protasis fluidic pumps, all running under Protasis automation software. Chromatographic solutions are accommodated using the Waters capillary LC system. Bruker fully supports the combination of the Waters CapLC and Protasis/MRM probe under Hystar automation, whereas the other NMR vendors utilize Waters MassLynx software for chromatographic control. 19) Why does Protasis/MRM use sucrose S/N specifications rather than the more traditional ethylbenzene standard? Concentration sensitivity is defined as the S/N that can be achieved by filling a probe with a sample of known concentration. Mass sensitivity is defined as the S/N that can be achieved with a known mass in the probe. The Protasis/MRM family of microflow probes are designed for enhanced mass sensitivity, and therefore we typically specify the quantity of sample being injected when computing S/N, although in some instances a less accurate assessment of concentration sensitivity can provide meaningful information concerning probe-to-probe reproducibility (e.g. during the quality control phase of manufacturing). Sucrose has been used recently as a specification standard for masssensitive probes, due in part to the ability to rapidly assess the (single) anomeric proton doublet for purposes of both S/N (height) and resolution (degree of splitting of the doublet).
20) Isn’t a flowprobe more susceptible to problems than a standard tube probe? In some ways, flowprobes constitute a simpler mechanical design than tube probes. There is not a need for a mechanical spinning apparatus, and the RF coil can generally be designed to more closely conform to the sample, providing higher fill factor and greater mass sensitivity. Flowprobes do require a well- engineered fluidic system to ensure that the samples reaching the probe are of high quality and without particulates that could potentially block the flowpath. This requires that some fundamental laws of fluidic management are followed. For example, the use of in- line filters is essential. In manual injection applications, it is beneficial for the user to pre- filter solvents and samples that have potential for particulates. In Protasis/MRM automation systems, specific algorithms are incorporated into the sample introduction process to provide periodic filter maintenance to ensure longer filter lifetime and minimize the risk of clogging. Finally, there are rinsing and cleaning procedures recommended by Protasis/MRM that can be easily performed by the user, to ensure continued high integrity of the NMR flowcell. 21) Does the CapNMR probe accommodate stopped- flow or on- flow detection? The Protasis/MRM family of microflow probes accommodate both on- flow and stoppedflow measurements. The automation systems provided by Protasis/MRM provide accurate peak parking in the NMR flowcell for extended NMR acquisition. The feed lines leading into the NMR flowcell are of sufficiently small inner diameter as to effectively limit diffusion to the flowcell itself. This ensures that the sample can remain in the NMR flowcell for days or even weeks without penalty.
22) Can the CapNMR probe accommodate salty samples? The CapNMR accommodates salty samples without significant penalty in S/N. The use of 0.5M salt solution results in a reduction in S/N by less than 10%. 23) Can the CapNMR probe perform direct carbon detection? The standard carbon nuclei microflow probes are specified as indirect detection probes, but many of our users have found that direct carbon detection with these probes is quite useful. Contact us today if you have interest in this aspect of microflow probe capability. 24) Are there plans to add other nuclei to the product offering? Protasis/MRM is always interested to hear feedback from current and potential customers. Contact us today to let us know if you have interest in a specific probe configuration that would help to address your application needs.
