Proton NMR Integral Ratio Comparision using 4-Chlorobenzaldehyde in Chloroform-d

Researcher
David Bulger

Objective
To evaluate the integral ratio precision using of proton NMR for solubility determination in non-aqueous solvents using chloroform-d with shimming, 60 sec relaxation time, and 65 536 data points.

Materials and Instrumentation
A JEOL 300-MHz high resolution 5-mm probe NMR was used with no spin and no lock at 22.4 °C. Shimming was performed followed by three NMR runs while the spin was temporarily working at 12-13 Hz with 2.0600 g reagent grade non-deuterated chloroform in an NMR tube. Mass determinations were performed using a 0.1 mg accuracy Sortorius balance. Shimming was performed followed by three NMR runs while the spin was temporarily working at 12-13 Hz with 2.0600 g reagent grade non-deuterated chloroform in an NMR tube. Sonication was performed with a Branson 50/60 Hz Ultrasonic Cleaner.

Experimental
Chloroform (1.9228 g; 1.2992 mL; 16.1 mmol) and 4-chlorobenzaldehyde (14.7 mg; 9.9 μL; 105 μmol) were added in various increments to a 5 mm NMR tube until solute precipitated and mixed by 30 min of sonication. This solution was then analyzed by 1H NMR.

Results
Identification of Peaks on Spectrum 297:
Identification Spectrum

Fixed Spectra:
Spectrum 295
Spectrum 297
Raw JCAMP-DX for Spectrum 295
Raw JCAMP-DX for Spectrum 297

Printed Spectra Integrated with Hd set as norm=1:
Spectrum 295
Spectrum 296
Spectrum 297

Results Spreadsheet:
EXP028 Spreadsheet


Photos:
300_MHz_NMR_1
300_MHz_NMR_2
300_MHz_NMR_3
300_MHz_NMR_4
NMR_Tubes
NMR_Caps
Sample_Preparation
Weighing_Table
Analytical_Scale_1
Analytical_Scale_2
Analytical_Scale_3
Sonicator_Detail
Empty_Sonicator_Back
Empty_Sonicator_Front
Sonicator_with_DI_Water
Computer_with_Delta
Pasteur_Pipet
Chloroform-d
4-Chlorobenzaldehyde
Solid residue

Integration Method
Spectrum
%RSD
intXY.php
Spectrum 295
2.4554
intXY.php
Spectrum 297
2.4997
JEOL
Spectrum 295
1.7189
JEOL
Spectrum 296
0.6756
JEOL
Spectrum 297
1.9631
Table 1: Integral Comparison for 4-Chlorobenzaldehyde

Discussion

While JEOL was able to integrate the spectra, the exported JDX files were also processed in ACD/NMR Processor 12.01 and converted in JSpecView to XY format to be integrated by Dr. Lang's intXY.php. The area beneath the signals from protons Ha, Hb, and Hc of 4-chlorobenzaldehyde were compared using these two methods of integration. Table 1 shows that JEOL performed more consistent integration within JEOL (1.4525% Average RSD) than the intXY.php performed on the converted exported spectra (2.4776% Average RSD). Overall, both methods consistently integrated the spectra of 4-chlorobenzaldehyde.

Since 4-chlorobenzaldehyde was added until precipitation, the solution never seemed to reach saturation even after sonication. The most saturated solution reached was less than 0.08 M. This is even less than the 0.81 M solution formed in EXP017. The Identification Spectrum clearly shows the identity of the compound to be 4-chlorobenzaldehyde. However, the solid resin at the bottom of the solute bottle may indicate some form of polymerization that would leave the majority of the 4-chlorobenzaldehyde that was added insoluble. Maybe a fractionation procedure could be preformed to purify the soluble monomers if this was the case.

Note: When converting spectra using ACD/NMR Processor 12.01, export as JDX with Fixed Length to preserve shifts.

Conclusion
JEOL integrated the original spectra with 1.45 Average %RSD and intXY.php integrated the converted spectra with 2.48 Average %RSD. The insolubility of 4-chlorobenzaldehyde may be attributed to some form of polymerization impurities in the solute.

Log

1.16.09 CST

15.40 - Photos taken of equipment and reagents.
16.15 - Weighed tube.
16.20 - Added chloroform-d and weighed.
16.30 - Shimmed sample.
17.00 - NMR Run
17.45 - Weighed sample.
17.49 - Added 4-chlorobenzaldehyde and weighed.
17.50 - Sonicated for 30 min (sample felt warm afterwards with condensation inside of tube).
18.21 - NMR Run 295
18.56 - Weighed sample.
18.57 - Added 4-chlorobenzaldehyde and weighed.
19.00 - Sonicated for 30 min.
19.10 - Shimmed sample.
19.30 - NMR Run 296
19.57 - Weighed sample.
20.00 - Added 4-chlorobenzaldehyde and weighed.
20.03 - Sonicated for 30 min.
20.33 - Shimmed sample.
20.43 - NMR Run 297

1.19.09

16.00 - Solid residue observed in NMR tube.

1.23.09

20.30 - Photo of solid residue taken (Solid residue).

References

Lin, M., Tesconi, M., Tischler, M., Use of H NMR to Facilitate Solubility Measurement for Drug Discovery Compounds, International Journal of Pharmaceutics (2008), doi:10.1016/j.ijpharm.2008.10.038

Acknowledgements:

The author wishes to thank Dr. Robert Stewart for his advice throughout the course of the experiment and for the use of the NMR and other equipment, Dr. Jean-Claude Bradley for setting up the server for the interactive NMR spectra, Dr. Antony Williams for his advice on improving the H NMR peaks and providing the reference listed, Dr. Hal Reed for the use of the camera, and Dr. Andrew Lang for recommending the posting of pictures of the experiment.

Previous Questions:


It was expected that the integral ratios of the last two samples would be close to equal. With only two samples, it was found that there is a 95% chance of future samples to have intregral ratios of (2.36 ± 0.39) * 10^(-3). Solid residue noticed after 68 hours (Solid residue).

The 95% chance of future samples to have intregral ratios of (2.36 ± 0.39)_*_10^(-3) was much better than the (1.04 ± 0.27) * 10^(-2) in EXP017. [Can you explain this a little more explicitly? Are you saying that one of the aromatic H's is integrating 2.36:1 to the aldehyde? If so which peaks specifically? JCB] The ratio was calculated as follows: (grams solute/(integral of Ha/integrate of Hd(1))). Ha and Hd refer to the corresponding peaks in the Identification Spectrum. The number of samples needs to be increased to confirm the integral ratios. Increasing the number of scans may further increase the precision. Sonication might have warmed the sample after 30 min. A thermometer needs to be placed in the sonicator to determine how much the temperature increased. Condensation was observed in the tube and the sample lost approximately_0.4_mg after sonication. As the boiling point of chloroform-d is 334 K, the sonication most likely did not heat the sample to boiling. As the solubility would change, any increase in heat during sonication needs to be avoided. Adding cool water to the warm water in the sonicator at regular intervals may counter the heating. Vortexing in a separate container rather than sonicating could be another way to avoid the heating. However, unless a syringe is used, it is likely that some of the volatile solvent will evaporate during the transfer to the NMR tube.[You can use a Pasteur pipette to transfer chloroform but you must be quick. JCB] Good point.

The 4-chlorobenzaldehyde coming out of solution does not seem to be attributed to not enough time sonicating, since it had been sonicated for a total of 90 minutes. At the low concentration used, it was expected to dissolve quickly. It might have dissolved and oxidized forming the residue. [If it had oxidized how would that change the NMR? JCB] In EXP017, the 4-chlorobenzaldehyde came out of solution less than an hour after sonication. The oxidation product would most likely be 4-chlorobenzoic acid. The problem is that the predicted spectra for 4-chlorobenzoic acid is pretty much the same as 4-chlorobenzaldehyde without the aldehyde peak at ~10 ppm. Both show a doublet of doublets between 7.5 and 8.0 ppm. Also, the chloroform peak's downfield lorenzian tail is in that region. Since there is an aldehyde peak at ~10 ppm, some 4-chlorobenzaldehyde was present. However, the 4-chlorobenzoic acid could change the solubility.


The deuterated chloroform, shimming, and increased relaxation time and number of data points increased the precision, as the relative standard deviation fell from 37.1% in EXP017 to 11.9%. However, until solubility determinations are performed, accuracy cannot be acounted for.