NMR Sample Preparation

Under any circumstances, it is not allowed to prepare the sample at the NMR room: use your lab, or the special lab room at Wolf building.
In NMR, unlike other types of spectroscopy, the quality of the sample has a profound effect on the quality of the resulting spectrum. So that the sample you prepare gives a spectrum in which useful information is not lost or obscured, you must follow a few simple rules.

Use the Correct Quantity of Material

For 1H spectra of organic compounds (except polymers) the quantity of material required is about 5 to 25mg. It is possible to obtain spectra from smaller quantities, but at very low concentrations, the peaks from common contaminants such as water and grease tend to dominate the spectrum. 13C is six thousand times less sensitive than 1H, and a good rule of thumb is to provide as much material as will give a saturated solution. If about 0.2 to 0.3 millimoles can be dissolved in 0.6ml, the spectrum will take no more than about half an hour to record. If the quantity of material is halved, the data accumulation time will be quadrupled. You should be aware that if you make up a sample at high concentration for 13C, and then record a 1H spectrum from it, the increased solution viscosity may result in a spectrum that has broader lines than you would get from a more dilute solution.

Remove All Solid Particles

Solid particles distort the magnetic field homogeneity because the magnetic susceptibility of a particle is different from that of the solution. A sample containing suspended particles thus has a field homogeneity distortion around every single particle. This causes broad lines and indistinct spectra that cannot be corrected. So that there are no solid particles in your samples, you must filter ALL samples into the nmr tube. You should filter samples through a small plug of glass wool tightly packed into a Pasteur pipette. If the plug is not tight enough, the filtration will be ineffective; if it is too big, some of your sample will remain trapped in it. Do not use cotton wool, since most NMR solvents dissolve material from it, which can easily be seen in 1H spectra. After filtration the sample should be as clear as water though, of course, not necessarily colorless

Make Samples to the Correct Depth

In the magnet, the main field direction is vertical, along the length of the sample. Each end of the sample causes a major distortion of the field homogeneity, which is corrected using the spectrometer's shim controls. A partial correction of the field homogeneity is done for every sample, and takes a few minutes. A complete correction takes many hours using a high quality test sample. Achieving optimal line shapes in the NMR experiment depends critically on the amount of solvent used. In general, what is needed is for a solvent column to extend the rf coils-length both above and below the coil (i.e., total height = 3 times the coil length). Different probes have different coil lengths, so there is no universal guidline to apply. The recommendation is:

                  Bruker 5 mm probes, coil length = 12 mm => 36 mm solvent length => 0.5 mL solvent
                  Varian 5 mm probes, coil length = 16 mm => 48 mm solvent length => 0.65 mL solvent.

Shorter samples are very difficult to shim, and cause considerable delay in recording the spectrum. Samples that are too long are also difficult to shim and are a waste of costly solvent. However, it was found that using less solvent than the above-recommended volumes can be safely done, but only within certain limitation, and with a price to paid of increasing the shimming effort needed to achieve the desired linewidth. Going less than 0.35 mL on Bruker 5 mm probe will almost certainly prevent optimal line shapes from being achieved.
In case of limited amount of an expensive sample, see section 9, When Sample Amount is Limited. You should check your sample length using a ruler. After preparation, you should ensure that the cap is pushed fully onto the tube to minimize solvent loss through evaporation.

Use Deuterated Solvents

Samples must be prepared using at least 5-10% deuterated solvents (solvents that contain deuterium in place of hydrogen). The NMR signal from the deuterium nuclei is called the NMR lock and is used by the spectrometer for stabilization. In case you are not interested in observing protons that exchange with the solvent, use a “100%” deuterated solvent.

Use Clean Tubes and Caps

NMR tubes are available from the stores, and after use they should be cleaned and dried (see Cleaning Procedures for NMR Sample Tubes). There are several vendors that sell similar products, but according to our experience, Wilmad tubes give consistently good results. Tubes must be capped, and caps should be treated the same way as tubes. You must not use NMR tubes with a chipped or broken top because they are dangerous, and very likely to splinter lengthwise.
Wilmad NMR tubes for high-resolution NMR:

           For 250 MHz work:
                        507-PP-7 recommended, $6.60

          For 400/500 MHz work:
                        507-PP-7 routine work, $6.60
                        528-PP-7 recommended, $9.10
                        535-PP-7 best, $16.60

          For 800 MHz work:
                       535-PP-7 recommended, $16.60
                       541-PP-7 better, $27.50
                       542-PP-7 best, $37.50

Label Your Samples

This is best done with a permanent marker directly on the top of the tube, or on the cap. If you use a sticker or a piece of tape, your label must stick smoothly on the tube. Do not leave a flap. Remember that the tube has to spin at 20Hz (1200rpm) while it is in the magnet

Use an internal reference

Usually, a small amount of reference is added to the solvent by the supplier. However, the amount of TMS or any other reference material that is required for a 1H spectrum is far less than can be added after the sample has been prepared. One drop of TMS in a sample causes serious problems due to distorted baseline and exceeded dynamic range. Even the standard amount of TMS added to a bottle of CDCl3 is too much. One should typically add about 2-3 mLs of CDCl3 containing TMS to a bottle that does not contain TMS and then use that bottle for sample preparation. This provides a small TMS signal; you never want your reference signal to be taller than your solvent signal. Alternatively, the residual protons in the deuterated solvent may be used as a secondary reference. For samples in D2 O, DSS or TSP is used as an internal reference. Remember that the chemical shift of water is highly temperature (and pH) dependent.

Degassing Samples

Some samples need to be degassed or have oxygen removed. The only effective way of doing this is by using the Freeze-Pump-Thaw technique, at least three cycles. It is sometimes sufficient to flush the space above the sample surface with nitrogen. This should be done with great care to avoid blowing the solution out of the tube. Do not bubble nitrogen through the solution in an NMR tube. This wastes costly solvent through evaporation, and is not an effective method of removing oxygen.

When Sample Amount is Limited

In cases you have a limited amount of your sample, you can reduce the above-mentioned recommended solvent volume by a factor of 3 by using special NMR tubes. Susceptibility inserts tubes (e.g. Shigemi ) allow the solvent volume to be reduced by removing the susceptibility gradients normally occurring at the solvent-air interface. When using a Shigemi tube, the solvent volume should equal the length of the rf coil. When using a Shigemi tube, the expected factor of 32 (=9) improvement in experimental time for identical S/N is achieved (an overnight experiment in a normal tube gives the same S/N as a 1.5 hr experiment with a Shigemi tube). Shigemi tubes are generally regarded as optimum for precision/best quality work (e.g. when needing water suppression). The disadvantages are the slightly higher cost (glass is more chemically durable, however), and susceptibility matching of the solvent, as shown below.
Susceptibility Inserts ( Shigemi ):

             D2O BMS-005V $75.00 (per set)
             DMSO DMS-005V $85.00 (per set)
             CDOD MMS-005V $75.00 (per set)
             CDCl3 CMS-005V $75.00 (per set)

modified from Alan S.F. Boyd's document dated 4 October, 1995 (NMR Services at Heriot- Watt University Chemistry Dept., Edinburgh, Scotland). And Charlie Fry, U Wisc-Madison

NMR Tube Specifications and Quality (NMR-001: taken from Wilmad)

Introduction

The purpose of an NMR tube is to confine a liquid sample in a perfectly cylindrical volume in a magnetic field while being spun at 25Hz. Since this description is based on mechanics, it is not surprising that NMR tubes have been described by structural parameters for many years. The degree to which an NMR sample tube approaches the ideal mechanical structure defines the quality of the NMR tube. But since words like 'Camber' and 'Concentricity' aren't associated by spectroscopists with familiar NMR performance criteria like spectrometer 'Sensitivity' or 'Resolution,' it is helpful for those who use NMR tubes to understand how the structural specifications published by Wilmad relate to NMR instrument performance. If your prestige as a researcher depends on the excellence of your NMR results, understanding the connection between these two terminologies will prove a valuable asset to your research efforts.
What follows is an explanation about each structural parameter used to describe Wilmad NMR tubes, why each is important, and how each relates to NMR instrument performance. The explanation will be limited to empirical terms. Wilmad tubes are grouped into structural Quality Classes and each is given a name, like Royal Imperial. The minimum tolerances which Wilmad NMR tubes in each Quality Class must meet are defined in our printed catalogs.

Outside Diameter (OD) and Inside Diameter (ID) A measure of the distance across the center of the tube from the outer surface (for OD) and inner surface (for ID), diameters are measured over a large number of points along the length and around the circumference of the tube. Best described as a range into which all measurements fall, Wilmad gauges these dimensions fluimetrically. Meeting stringent standards for OD and ID assures the sample tube defines a precisely reproducible cylindrical volume for the sample within the Rf coil. Diameters vary for Wilmad NMR tubes over a range that is usually <0.001" (0.026mm). Failure to adhere to strict diameter tolerances can produce a diversity of undesirable performance characteristics in NMR spectroscopy. If the ID is too large, vortex plugs and coaxial inserts can move during spectral accumulation. If the ID is too small, finned vortex plugs might actually burst the tube, making sample loss, possibly inside the probe, a catastrophic possibility. When the OD is too small, the tube might slip in the spinner turbine. This can cause spinning wobble, a major source of modulation sidebands. Or the sample might shift in the probe or fall completely through the probe when pneumatically lowered into the magnet. In combination with other imperfections in an NMR tube, a tube with an OD that is too large could make contact with the probe insert, damaging the probe, a costly repair. When the failure to meet OD or ID tolerances is localized in a small area of the tube, portions of the sample might fall outside the 'perfect' cylindrical volume and be exposed to a portion of the magnetic field which may not be homogeneous with the remaining portions of the sample within the 'perfect' cylinder. You'll know this is the case if you must perform extensive shimming from one sample to another. In some cases, you may not be able to achieve satisfactory resolution with further adjustments in magnetic field homogeneity.

Concentricity A measure of the lack of wall uniformity, concentricity can be thought of as the degree to which the cylinders defined by the inner and outer surfaces of the tube are parallel and overlap.
Determined using an Indicating Gauge, this tolerance is reported as a deviation (±) or Total Indicator Reading (TIR or the absolute variation in wall thickness) as measured at the ends of a tube during rotation. Concentricity is zero for a 'perfect' tube. But tubes which conform to TIR tolerances between 0.006" (0.15mm) and 0.0005" (0.013mm) will spin reliably and provide increasingly improved spectral resolution. Failure to conform to concentricity tolerances causes portions of the sample to fall outside the 'perfect' cylindrical volume, exposing that portion to a magnetic field which may not be homogeneous with portions of the sample within the 'perfect' cylinder. This leads to modulation sidebands, which can quickly become significant.

Camber A measure of the lack of straightness of a tube, camber is determined with indicating gauges by measuring the deflection at the middle of the tube held at the ends and rotated. Recorded as a deviation (±) or Total Indicator Reading (TIR or the absolute value of the deflection range), tubes with deviations <0.0021" (0.053mm) in Camber can be expected to spin reliably, barring deviations in other specifications. Higher frequency NMR spectrometers have placed greater importance on the Camber of tubes in recent years and stricter tolerances have been required to meet the needs of spectroscopists fortunate enough to be using the most sophisticated NMR spectrometers. Modulation sidebands are prominently seen when tubes with poor Camber are used. When camber becomes marked, contact of the spinning tube with the probe insert is likely. Instances of probe damage have been noted when 'cheap' NMR tubes were used. Since only 300µ separates the tube and insert in the most commonly used probes, meeting strict Camber tolerances is the most important criterion in maintaining the integrity of the probes of an NMR Spectrometer and avoiding costly and time-consuming probe repairs.

Proper Cleaning Pr ocedures for NMR Sample Tubes (NMR-010: from Wilmad)

Wilmad NMR tubes are not 'analytically clean' when delivered to you. So if your NMR samples require scrupulously clean glass, follow the procedures below for Difficult Cleaning Problems to assure your sample purity is never jeopardized. Since NMR tubes are formed over a metal mandrel and certain organic lubricants are used, these cleaning steps will assure that any trace organic or inorganic residues from these procedures is removed.
When you invest in high quality precision NMR Sample Tubes, you expect high resolution and
sensitivity. Proper cleaning procedures can help you preserve the quality of your investment. Since the purpose of an NMR Sample Tube is to confine a liquid sample in a perfectly cylindrical volume within the spectrometer probe, the degree to which the tube accomplishes this determines the quality of the sample tube. Improper cleaning can damage NMR tubes and reduce your apparent spectrometer performance.
You should never use a brush or other abrasive materials to clean NMR tubes. Scratches on the inside surface of the tube allow a portion of the sample to extend beyond the perfect cylinder defined by the NMR tube. Because the portion of your sample which fills a scratch on the inner surface of a tube experiences a different magnetic field than the rest of the sample, lines will broaden and resolution will deteriorate when you use scratched tubes. And you'll see a reduction in apparent spectrometer performance, unless you reshim your spectrometer for each sample. That's a tedious procedure your investment in high quality tubes was designed to eliminate to begin with.
Proper cleaning of NMR tubes can be easy or difficult, depending on your sample. We'll start with simple cleaning situations and move to the harder cleaning problems. Because even difficult cleaning procedures end with a proper rinsing, explained under Simples Cleaning of NMR Tubes, you should be familiar with both cleaning procedures.

Simple Cleaning of NMR Tubes

When cleaning your NMR tubes is as simple as rinsing the tube with water or an organic solvent, you can rinse them one at a time. Your main concerns, then, are what to do with the rinsate. And, if you're using Acetone, also preventing dermatitis that results when oils are removed from your skin by this potent solvent.
If you rinse a lot of tubes, there are apparatuses available that will make your job much simpler. Tube washers, listed in the Wilmad NMR Catalog as Solvent Jet Cleaners, provide an easy way to clean either one or five tubes at a time. Using a vacuum flask and aspirator, solvent recovery is simple. And your hands won't be so easily dried out by solvents, either. A final rinse with Acetone is frequently used to remove the last organic contents from the tube. When your sample is to be dissolved in water or D2O, a final rinse with distilled water is usually adequate. You may want to take steps to remove traces of water from the surface of the tube. Follow the procedures for deuterium exchange, below.

Difficult Cleaning Problems

Tubes left with samples in them for a period of time frequently present a more challenging cleaning problem. Sample degradation or precipitation can cause material to adhere to the inner walls of the tube. Rinsing the tube doesn't always remove this adhered material. So Wilmad recommends using strong mineral acids such a concentrated or, in severe cases, fuming Nitric Acid soaks of 1-3 days, as needed. Nitric Acid can oxidize many organic chemicals and dissolves most inorganic materials, as well. Wilmad doesn't recommend using Chromic Acid, since residual Chromium can often adversely affect NMR experiments. Chromic Acid, while a stronger oxidizer, can leave paramagnetic Chromium VI behind, which can be removed only with repeated soaks with Nitric Acid. Copious rinsing of NMR tubes washed in acids is required to assure removal of residual acids. A final rinse with distilled water or Acetone is also appropriate.
Tubes which contained polymeric samples can be even more difficult to clean. When the polymers are natural products, like proteins and polysaccharides, strong acid soaks will usually be sufficient. However, when dealing with synthetic polymers, the challenge is more severe, since many polymers are inert to acids or insoluble in organic solvents by design.
Although polymers may not readily dissolve in solvents, it may be possible to soften them by soaking the tubes in a solvent that swells the polymer. Then a pipe cleaner might be sufficient to remove the softened material. It may take some experimentation to find the solvent combination that works best with your polymer system.
Agitation in an Ultrasonic bath with an appropriate solvent can also help dislodge stubborn sample residues. However, you should take precautions to assure that NMR tubes don't touch, since contact and vibrations can fracture delicate thin wall tubes. Wilmad offers a special tube rack for use in its Ultrasonic bath that prevents such destructive contact between tubes.

Removing Water from NMR Tubes

Drying tubes at elevated temperatures can reshape and ruin precision NMR tubes. If you dry tubes in an oven, Wilmad recommends placing tubes on a perfectly flat tray at 125° C for only 30-45 minutes. Better is the use of a vacuum oven that will remove water at lower temperatures. In a flat position, tubes that do reshape could be out-of-round and may not fit the spinner turbine as well. But they'll not affect the spectrometer probe adversely. Tubes placed in an oven in a beaker, flask, or tube rack can bend, increasing Camber (lack of straightness)1. Bent tubes may still fit the spinner turbine, but can damage or break the NMR probe insert, a costly repair with many probes.
But even drying at high temperatures doesn't remove water chemisorbed to the surface of the tube. Thus, the preferred method of water removal is chemical, not physical, treatment. In most cases, it is the protic content of water that must be avoided. So Wilmad recommends exchanging the protons of chemisorbed water with a deuterated solvent such as D2O prior to a short drying period in the oven. A bottle of D2O that isn't being used any longer is perfect for this purpose.
When water chemically degrades your samples, then removal of water is essential. Here, reaction of the water with a hydride solution can be used, with caution. After rinsing the hydride solution, a final rinse with very dry Acetone can be used to remove rinse solvent prior to oven drying. Cap tubes promptly to avoid absorption of moisture when removing dry tubes from the oven.