4. Acquisition

Time requirement: 1.5 h

Sample: Host: β-cyclodextrin hydrate (CD), Guest: 4-t-butylpyridine (TBP).

Solvent stock solution

Prepare 20 ml of a 50 mM phosphate buffered D₂O solvent stock by dissolving 142 mg (1 mmol) Na₂HPO₄. Add 1–3 drops of concentrated H₃PO₄ to adjust the pH to 7.0 using a digital pH meter. The use of a buffer is absolutely necessary in order not to be tricked by the pH-dependence of the chemical shifts.

10 mM CD stock solution

113.5 mg (0.1 mmol) CD is weighted using an analytical balance and dissolved in 10 ml of the above described buffered D₂O solution using an analytical volume flask.

10 mM TBP stock solution

7.39 μl TBP (6.76 mg, 0.05 mmol) were syringed into an analytical volume flask containing 5 ml of the above described buffered D₂O solution. The CD and TBP stock solutions should have equal concentration as closely as possible. This can be checked by NMR, taking equal amounts and comparing the integrals.

Reference System

Since the different guest and host concentrations may influence the reference signal, prepare a sealed melting-point capillary with acetone and insert this into the sample tubes. On modern digital instruments you may instead use the spectrometer reference value by assuming that the lock signal is not shifted in the different samples.

Sample preparation

Prepare 9 NMR tubes according to the following table. The principle of continuous variation (Job's method [2]) is used so that in all samples the condition (5) holds:

[H] + [G] = constant(5)

Sample	V_CD [μl]	V_TBP [μl]	x_CD
1	0	600	0
2	75	525	0.125
3	150	450	0.25
4	225	375	0.375
5	300	300	0.5
6	375	225	0.675