Application Note 10: Column
Chromatography
Column chromatography is a universally used technique in chemistry laboratories inwhich compounds are purified from mixtures on the basis of some physicochemical
property. Thin-layer chromatography (TLC) is the traditional method of determining thecorrect solvent system in which to perform column chromatography, and analyzing theelution profile once the procedure has started. Small samples (~ 1 μL) of each fraction
are sequentially spotted onto a plate of the same stationary phase medium as used in thecolumn.
We demonstrate an alternative procedure to TLC, employing direct analysis of the eluentsusing 1H NMR spectroscopy.

A mixture of benzyl alcohol, benzaldehyde, methyl benzoate and ibuprofen (Figure 1) was purified on silica
gel using dichloromethane as the mobile phase. Test tube fractions were collected at 3 minute intervals,
from which 0.5 mL aliquots were analysed directly using the Spinsolve (Figure 2).

benzyl alcohol

ibuprofen

benzaldehyde

methyl benzoate

mixture12.0 11.5 11.0 10.5 10.0 9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0 5.5
f1 (ppm)

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

Figure 1: 1D proton NMR spectra of the mixture to be purified and the consituent compounds in dichloromethane,obtained using the Spinsolve.

Collect column fractions in test tubes

Transfer contents from the test tube to theNMR tube

Press Start

Insert sample in to the Spinsolve

Figure 2: Process for acquiring spectra of chromatography fractions.

31

26

21

16

116

1

12.0

11.5

11.0

10.5

10.0

9.5

9.0

8.5

8.0

7.5
7.0
f1 (ppm)

4.0

3.53.0

2.5

2.0

1.5

1.0

0.50.0Figure 3: Elution profile of silica gel flash column. Samples were taken directly from test tubes (obtained using theSpinsolve in dichloromethane).

From Figure 3 it can be seen that benzaldehyde and methyl
benzoate co-elute (fractions 9-11), followed by ibuprofen (fractions26-28) and benzyl alcohol (fractions 29-35). Using NMR to monitor
the chromatography has several advantages that traditional TLC
doesn’t offer. Firstly, the TLC plate does not allow the determinationthat benzaldehyde and methyl benzoate co-eluted (Figure 4), unlesseach of the pure compounds were analysed separately which is
not possible in all situations, for example if both compounds werethe product of a reaction. Secondly, proton NMR can not only be
Figure 4: Silica TLC plate of theused to determine when compounds are eluting, as TLC does, but
mixture run with dichloromethane
it also gives structural information about what is eluting. In this set
and visualized with UV light.
of compounds, each has a characteristic functional group that is notpresent on any of the other molecules, thus the appearance of thisfunctionality in the 1D proton NMR spectrum allows the compound to be identified easily. Thesecompounds, however, are structurally similar and can therefore not be distinguished on a TLC plate byacid charring or UV development.

Monitoring column chromatography in this way would not be achievable on a high field instrument for anumber of reasons. One is that a high field instrument requires a deuterated solvent to lock to the sample
which introduces significant cost. It also takes significantly longer to run each sample (it takes time toshim and lock on every sample) such that one column would take a day to collect the 1D proton data for.
The proton NMR spectra in Figure 3 took 10 seconds to acquire each spectrum, and with minimal sample
preparation between spectra, all 35 samples took less than 15 minutes to acquire. It was estimated thatthe time to prepare NMR samples from the fractions was comparable to preparing but not running a TLC
plate.

Related Applications
The Spinsolve benchtop NMR system solves many of the issues associated with hyphenated NMR
techniques, such as LC-NMR (the most common). Currently, LC-NMR systems use a deuterated solvent
for the chromatography so that the sample can be analysed by a high field NMR spectrometer. This
introduces a significant cost, not only the expense of running a high field instrument but also the cost of
deuterated solvents required. The Spinsolve does not require deuterated solvents and is a fraction of thecost of a high field instrument to run. Combining the Spinsolve with an LC set-up would provide a more
automated system than that described in this note.
Conclusion
We have demonstrated here how the Spinsolve can be used to monitor flash column chromatography.
Using NMR spectroscopy to analyse the elution profile provides advantages over the traditional TLC
method as you gain information about when compounds are eluting and something about its structure.
In this experiment, each compound was able to be identified by a characteristic peak in the 1D proton
NMR despite being structurally related. Chromatography monitoring by NMR is possible using a Spinsolve
system as it does not require deuterated solvent to lock the sample and the spectra are acquired
significantly quicker than on a high field instrument.

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