NMR样品管.doc

How to make an NMR sampleSafety warning: Some of the procedures described here are potentially dangerous. If you are unfamiliar with laboratory safety, please ask a qualified chemist for help.Samples for full service should be sufficiently soluble in the solvent requested. If not submitting for full service then please prepare your samples as follows.NMR tubesSamples for NMR analysis should be submitted in 5 mm NMR tubes In cases of low solubility or low sensitivity for 13Carbon, 15Nitrogen, 31Phosophorus and 7Lithium NMR it may be desirable to submit samples in 10 mm NMR tubes for 400 MHz analysis. Samples for (39K) potassium and (183W) tungsten NMR may be submitted in 10 mm tubes for 300 MHz analysis.Select the right type of NMR tube. For 300 MHz NMR tubes of the quality of Wilmad 507-PP-7, Aldrich series 300 or Kontes grade 3 are sufficient. For 400 MHz NMR use Willmad 528-PP-7, Aldrich series 400 or Kontes grade 6. (For 11B NMR use 528-PP-QTZ. For 10 mm tubes use 513-PP.) If the sample dissolves glass (e.g. HF) use a PTFE insert.Please label your tubes clearly with an appropriate concentric label.Examples of incorrect and correct NMR tube labelsSolventWe do not have the equipment for solid state NMR so samples must be submitted in solution.Solvent selection is dictated by a number of factors.1) It is general practice to use deuterated solvents for two reasons. a) The deuterium can be used for field frequency lock, making spectral resolution easier to assure and thereby saving you valuable service time. b) Solvents containing regular hydrogen yield large signals that may swamp those of your sample: the situation is made worse by saturation effects that broaden large signals. We recommend the use of deuterated solvents wherever possible.2) The sample must be sufficiently soluble to yield an NMR spectrum. For 1H and 1H observed NMR, it is recommended to dissolve between 2 and 10 mg in between 0.6 and 1 mL of solvent so that the sample depth is at least 4.5 cm in the tube. For high molecular weight samples, more concentrated solutions are sometimes recommended. However, too concentrated a solution leads to lower resolution due to saturation and/or increased viscosity. For 13C NMR five times the concentration is recommended. (For 10 mm tubes use between 40 and 250 mg of sample in between 2.7 and 4 mL of solvent.) If you do not have enough sample or cannot achieve high enough solubility, it may still be possible to obtain a spectrum although it is recommended that you request 400 MHz service and the spectrum will take longer to acquire. We have observed 1H signals with less than 0.1 mg of sample and 13C signal with less than 1 mg of sample.3) To achieve maximum resolution, choose a solvent with low viscosity. However, achieving higher resolution is more difficult for the operator and likely to increase the service time required. For example, a sample in diethyl ether-d10 will yield a sharper spectrum but may take longer to run than one in DMSO-d6.4) Consider the cost of the solvent. D2O and CDCl3 are the most commonly used solvents in our laboratory because they are the cheapest and yield satisfactory results most of the time.5) If the solvent signals interfere with the sample signals consider a different solvent, changing the temperature or, if the 13C sidebands are overlapping, decoupling the carbon.6) If doing variable temperature studies, take into account the liquid range of the solvent. We do not have the equipment to observe a signal in frozen solvent although depression of the freezing point is possible in narrow tubes. We do have ways of working at above the boiling point.CleanlinessHaving selected the solvent and tube one should ensure that the solvent, sample (if not purposely analyzing a mixture), NMR tube (and cap) and anything else that comes in contact with the sample is clean. For routine work, washing the NMR tube using an NMR tube washer with water and/or acetone then cleaning with cotton wool on a stick followed by thorough drying is sufficient. Do not use a brush or abrasive cleaner in an NMR tube as it will damage the tube and reduce spectral resolution.NMR tube washerRegular cleaning is not always sufficient. If the sample is very dilute, impurities may complicate the spectrum. In such a case, run a blank (spectrum of the pure solvent from the same batch). Sometimes, samples are very sensitive to dirt. For example, certain biological samples require sterile conditions. If the tube is very dirty, normal cleaning may not be sufficient for regular samples. I such cases, NMR tubes (not caps) may be left to soak in aqua regia or sulfurchromic acid (CARE!) for between half an hour and two days then washing several times with distilled water. (Some spectroscopists believe that it is impossible to completely remove the paramagnetic sulfurchromic acid afterwards but I have seen no evidence for this under very demanding conditions provided that the tube is washed at least seven times with distilled water.) The tube should be dried lying on its side (to prevent warping) at up to 150oC for a couple of hours. If the tube is not required immediately then remove it from the oven and return it an hour before use.Sample filtrationIt is important that the solution be free of precipitate, dust, fibers, etc. This can be achieved by filtering the sample. Make up slightly more sample than is required. Pack a small piece of cotton or glass wool tightly into a Pasteur pipette and wash with a small amount of solvent. Filter the solution into the NMR tube using a teat if necessary to force the sample through.Filtering an NMR sampleAir sensitive and high-pressure samplesAn inert atmosphere or vacuum is required if the sample is air sensitive, or a relaxation (T1 or T2) measurement is desired. If NOE is being sought then using an inert atmosphere increases sensitivity. Oxygen in the air is slightly paramagetic so reduces relaxation times and reduces NOE hence the need for removing oxygen in some cases.The simplest way of achieving this is to make up the sample under argon and running the spectrum immediately. It is not recommended that such samples be submitted to our NMR service as we cannot guarantee to run them quickly enough. Special NMR tubes are commercially available for sealing samples under vacuum or an inert atmosphere such as Wilmads 528-JY (513-7JY for 10 mm) tubes. An alternative is to attach a glass tube to the top of a regular NMR tube with a constriction as shown below. Care must be taken to maintain concentricity. The sample may be made up on a vacuum line and the tube then cut with a flame. A partial pressure of inert gas makes cutting easier but with practice it can also be done with a vacuum. To remove all gas, the sample should be frozen pumped and thawed repeatedly. To thaw the sample heat the outer layers to well above the melting point quickly otherwise the tube may break.High-pressure samples up t