Lab 8. Procedure.

You will collect data with a lab partner (who should be noted in your write-up). However, all the data analysis and the write-up should be done without collaboration.

The purpose of this lab is to have hands-on experience with NMR spectroscopy. Three projects will be carried out:

1. Identification of compounds: Using ¹H NMR, spectra of a few molecules should be taken and corresponding compounds identified
2. Slow kinetics: Determination of a proton exchange rate for thiamine hydrochloride using NMR intesity changes with time.
3. Fast kinetics: Study of rotational rates using NMR spectral analysis.

Before the experiment

• Depending on availability we will be using either Unity 400, Gemini-200 or Anasazi FT-NMR. Maunals are a available on the local NMR page maintained by K.Burke.
• This year (2005) it will be Varian Unity 400 spectrometer.
• For preparation to use this spectrometer read the manual . If you plan to use 200 MHz Gemini spectrometer, I recommend you to read through the Beginner's Procedure (our lab adapted copy is here) and a bit lengthier Instructions. There is always the original Operator Manual next to the spectrometer.

The experiments
Section A. Identification of compounds.

• You will have eight out of twelve NMR tubes labeled A, B, C, D, E, F, G, H, I, J, K, and L, which you need to identify using ¹H NMR. They have as a solvent either CDCl₃ or D₂O. The following compounds are among the eight:
  1. methanol (CDCl₃)
  2. ethanol (CDCl₃)
  3. ethylbenzene (CDCl₃)
  4. benzhydrol , (C₆H₅)₂CHOH, (CDCl₃)
  5. acetone (CDCl₃)
  6. anthracene (CDCl₃)
  7. phenanthrene (CDCl₃)
  8. diethylamine (CDCl₃)
  9. 2-propanol (CDCl₃)
  10. 1,4-dimethylbenzene (CDCl₃)
  11. 1,2-dimethylbenzene (CDCl₃)

Section B. Slow kinetics.

• Record room temperature
• Prepare 2 mL of acetate/acetic acid buffer solution in D₂O at pH ~ 4.5 and ionic strength of 0.05M (adjust using NaCl); If you wish, you may do it by the appropriate mixing of NaAc and acetic acid (d₄) in D₂O.
• Dissolve in your buffer approximately 20 mg of thiamine hydrochloride
• Take ¹H NMR in the range 10 ppm to 8 ppm every 3 min (keep track of the time); you may ask the computer to do that time sequensing by typing nt=1 and pad=0,180,180,180,180,180,180,180,180,180,180 It will acquire 11 successive spectra 3 min apart
• Plot all spectra and the integrated intensities of the C-(2)-H (I₂) and C-(6')-H (I_6') hydrogens; later, during analysis, you will plot intensity I₂ normalized on I_6' (I₂/I_6') as a function of time
• Measure pH of your solution using a pH meter
• If time allows, repeat evrything with 0.2 M and 1.0 M of the buffer

Depending on whether we use 400 MHz, 200 MHz or 60 MHz spectrometer, the third part will be one of the following:

Section C (60 MHz). 2-D NMR.

• You will run COSY experiments on a few molecules chosen by your TA (ethanol, 1-propanol...) and explain the observed correlation between the peaks in 2D

Section C (200 MHz). Fast kinetics.

• You should have prepared a sample of deuterated cyclohexane in methylene chloride. Since the cyclohexane is has 0.4% H "impurities", 0.4 x 12 = 4.8% of cyclohexane molecules are actually C₆D₁₁H
• Record the room temperature proton NMR spectrum and identify the proton signal of C₆D₁₁H
• Setup the temperature controller for - 40° C, wait until the temperature stabilizes (about 10-15 min) and record the spectrum again. Here are instructions on how to use the temperature controller, courtesy of K. Burke.
• Repeat the same measurements at approximately every additional 5° lowering the temperature, i.e. at - 45°, C- 50° C, etc. down to -85° C.
• Watch the changes in your spectra. Upon temperature decrease, you should observe that a single starts broadening and eventually splits into two lines, which will individually narrow with further temperature decrease. Note the temperature of coalescence.

Section C (400 MHz). Fast kinetics.

• You should have prepared a sample of 3-dimethyl-aminoacrolein either in CDCl₃, deuterated DMSO or deuterated toluene. Samples in in CDCl₃ and toluene should be in sealed tubes.
• Start with the sample in CDCl₃. Setup the temperature controller for 25° C, wait until the temperature stabilizes (about 10 min) and record the spectrum.
• Repeat the same measurements after raising the temperature by approximately every additional 5-10° , i.e. at 30° C, 40° C, etc. up to 95° C. Don't exceed this temperature unless you use nitrogen gas to heat your sample.
• Watch the changes in your spectra, especially in the range of methyl protons. Upon temperature increase, you should observe for the latter that the two lines start broadening and eventually coalesce into a single line that narrows with further temperature increase. Note the temperature of coalescence. Copy each spectrum in a digital form to a disk.
• If time allows, repeat the measurements in DMSO and toluene.

After you finish

• copy your data on disk
• logout from the spectrometer in accordance with the procedure described in the above mentioned manuals
• clean up after yourself
• show your notebook and spectra to the TA or the instructor before you leave
• transfer you data onto pc using Vhelper ; you may find it very useful to process you data and prepare for your report uisng ACD NMR SpecManager software which we have licensed from the ACD Labs.
• you may also find it helpful to the analysis of spectra from Section C while in class, so that TA can help you eliminate possible problems with fitting

Plan to run sections A, B and C on first, second and third days, respectively.

Last updated 03/18/08.