EXP031

**Solubility of Benzoic Acid in Chloroform using 1H NMR** media type="custom" key="7607757"media type="custom" key="7607761" **Researcher** David Bulger

**Objective** To confirm the solubility of benzoic acid in chloroform using 1H NMR.

**Method**

NMR experiments were conducted on a [|JEOL_300MHz_NMR] equipped with a [|5_mm NMR_tube] capped with a [|single seal cap]. All experiments were carried out at approximately 293.2 K using auto-gain with spin off. The proton spectra were acquired using a 45-degree pulse with a relaxation delay of 5 seconds, 1 scan with a resolution of 77.97 mHz. Shimming of spectra used JEOL automatic gradient shimming with deuterated chloroform spinning at 15 Hz. Mass determinations were performed using a 0.1 mg accuracy [|Sortorius balance]. A saturated solution of benzoic acid ([|ChemSpider]) in protonated chloroform ([|ChemSpider]) was made and the supernatant was transferred to 3 NMR tubes using [|Pasteur_Pipet] followed by addition of capillary tubes filled with 94 uL water using a [|Microsyringe] ([|NMR_external_reference_setup]). Reference NMR run performed first with deuterochloroform, then with protonated chloroform.

**Results**

Identification of Peaks on Solubility_35: [|Identification Spectrum]

Spectra: [|Solubility_15] [|Solubility_34] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_35] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Raw JCAMP-DX file for Solubility 15] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Raw JCAMP-DX file for Solubility 34] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Raw JCAMP-DX file for Solubility 35]

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Spreadsheet: <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|EXP031 Spreadsheet] media type="custom" key="7608299" Photos of Equipment: [|300_MHz_NMR_1] [|300_MHz_NMR_2] [|300_MHz_NMR_3] [|300_MHz_NMR_4] [|NMR_Tubes] [|NMR_Caps] [|Weighing_Table] [|Analytical_Scale_1] [|Computer_with_Delta] [|Pasteur_Pipet] [|Microsyringe] [|NMR_external_reference_setup] [|NMR_external_reference_setup_close_up] [|Benzoic_Acid] [|Benzoic_Acid_top]

Table 31.1: Solubility Results for Benzoic Acid in Chloroform
 * Spectrum || Proton || SAMS Solubility (__M__) || External Standard Solubility (__M__) ||
 * __<span style="color: #800080; font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_15] __ || Ha || 1.8889 || 1.8893 ||
 * __<span style="color: #800080; font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_15] __ || Hb || 1.5685 || 1.5682 ||
 * __<span style="color: #800080; font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_15] __ || Hc || 1.4970 || 1.4968 ||
 * __<span style="color: #800080; font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_15] __ || Hd || 1.4926 || 1.4928 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_34] || Ha || 1.9043 || 1.9045 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_34] || Hb || 1.5130 || 1.5135 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_34] || Hc || 1.5033 || 1.5029 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_34] || Hd || 1.4747 || 1.4750 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_35] || Ha || 1.7483 || 1.7479 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_35] || Hb || 1.5440 || 1.5441 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_35] || Hc || 1.4649 || 1.4647 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility_35] || Hd || 1.5183 || 1.5181 ||

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**Discussion** <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The solubility of benzoic acid in chloroform was measured by NMR with three replicates using the SAMS method with and without an external standard (Table 31.1). Both methods were shown to give almost identical results: 1.59 +/- 0.16 __M__ for SAMS and 1.59 +/- 0.16 __M__ for External Standard.

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**Conclusion** <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The solubility of benzoic acid in chloroform was measured to be 1.59 +/- 0.16 __M__ by both SAMS and External Standard methods.

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**Log**

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">1.23.09 CST

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">11.30 - Reference sample preparations <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">11.40 - Weighed benzoic acid (0.116 2 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">11.45 - Weighed deuterated chloroform (1.306 6 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">11.50 - Mixed by hand <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">11.55 - Capillary tube filled with water using microsyringe <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">11.57 - NMR run (Solubility_12) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Note: This reference was not used because it used deuterated chloroform.

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.26 - Filled 3 capillary tubes with water <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.31 - Labeled 3 NMR tubes <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.47 - Weighed vial (48.046 6 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.49 - Added benzoic acid and weighed (49.149 7 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.52 - Added protonated chloroform and weighed (52.141 0 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.55 - Added benzoic acid and weighed (53.313 7 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.04 - Added chloroform and weighed (57.445 8 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.08 - Added benzoic acid and weighed (58.263 6 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.10 - Added chloroform and weighed (61.104 4 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.09 - Shimmed (Z1 = (-1 279), Z2 = (-981), Z3 = (-159), Z4 = (790)) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.11 - Transferred supernatant to NMR tubes and capillary tubes added <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.23 - NMR run [|Solubility 15] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.30 - NMR run [|Solubility 34] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.42 - NMR run [|Solubility_35] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.30 - Photos taken of equipment and reagents

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">1.30.09 CST

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">16.06 - Added 94 uL water to capillary tube using microsyringe <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">16.50 - Photos of equipment <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">16.54 - Automatic gradient shim using pre-made deuterated chloroform sample (Z1 = (-1 279), Z2 = (-977), Z3 = (-293), Z4 = (936)) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">16.58 - Weighed empty NMR tube (3.086 8 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.01 - Added benzoic acid and weighed (3.099 7 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.02 - Added benzoic acid and weighed (3.114 3 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.04 - Added protonated chloroform and weighed (4.627 6 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.12 - Sonicated for 10 min <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.24 - Added capillary tube and NMR run (Gain = 6) with zero_region ([|Benzoic Acid Reference])

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**References**

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|1) 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]

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**[Please also add measurements like these directly in to the** [|**SolubilitySum spreadsheet**] **as well. That way you can refer to all references for a solubility with a link like this:** [|**http://oru.edu/cccda/sl/solubility/ugidata.php?solute=salicylic%20acid&solvent=ethanol**] **JCB]**They should be added now.

[|Solubility of benzoic acid in chloroform](solubility data viewer)

**Acknowledgements:**

The auther wishes to thank Dr. Robert Stewart for his advice throughout the course of the experiment including reference #3 and for the use of the NMR and other equipment, Dr. Jean-Claude Bradley for his help with the interactive NMR spectra, Dr. Reed for the use of the department camera, and Dr. Antony Williams for reference #1.
 * Past Material **

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**Calculations**

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The solute and the chloroform peaks' integrations were analyzed against the external water reference integration per hydrogen as follows: (area / # H's) / (water area / 2 H's).

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Weight Percent (wt%) of entire solution = total mass of solid added / total mass of solution * 100%.

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Integral ratio was calculated for the unsaturated reference spectrum as follows:

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Integral Ratio = wt% of unsaturated reference / (unsaturated solute peak against water peak / unsaturated chloroform peak against water peak).

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The solubility is calculated in wt% as follows:

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">wt% observed = Integral Ratio of unsaturated reference * (saturated solute peak against water peak / saturated chloroform peak against water peak).

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Interactive Spectra (these are very large files (800K) and may require [|increasing JAVA memory] and closing all browser windows before attempting to view each spectrum) and Interactive Spectra Screenshots of Integrations (Benzoic Acid Reference: Minimum Y = 0, Integral Factor = 150, Integral Offset = 0; Solubility 15, 34, 35: Minimum Y = 0.1, Integral Factor = 50, Integral Offset = 0):


 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Benzoic Acid Reference] || [|Solubility 15] || [|Solubility 34] || [|Solubility_35] ||
 * || [|015Ha] || [|034Ha] || [|035Ha] ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|BARHb_a] || [|015Hb_a] || [|034Hb_a] || [|035Hb] ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|BARHb_b] || [|015Hb_b] || [|034Hb_b] ||  ||
 * || [|015Hc_a] || __ 034Hc_a __ || [|035Hc_a] ||
 * || [|015Hc_b] || [|034Hc_b] || [|035Hc_b] ||
 * || [|015Hd_a] || [|034Hd_a] || [|035Hd_a] ||
 * || [|015Hd_b] || [|034Hd_b] || [|035Hd_b] ||
 * [|BARHe_a] || [|015He_a] || [|034He_a] || [|035He_a] ||
 * [|BARHe_b] || [|015He_b] || [|034He_b] || [|035He_b] ||
 * [|BARHf_a] || [|015Hf_a] || [|034Hf_a] || [|035Hf_a] ||
 * [|BARHf_b] || [|015Hf_b] || [|034Hf_b] || [|035Hf_b] ||

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**[Well done on finding a reference #2 for the solubility of benzoic acid in chloroform - how does your measurement compare? JCB]** <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The weight percent was found to be [|16.5 wt%] at 22 degrees Celsius. This is greater than the 12.5 wt% from the [|"Solubilities of Inorganic and Organic Compounds" (Reference 3)] at 25 degrees Celsius. [|Reference 2] showed the solubility at 20 degrees Celsius to be 12.5 g/ 100 mL. The Merck Index showed the solubility to be 22 g/ 100 mL at room temperature.

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**[Could you mention exactly how (and how long) you mixed the solutions? We have found that mixing must be very thorough to get reproducible values. JCB]** The solution was mixed by hand for approximately 3 min after each of the chloroform additions.**[That is not enough mixing - in the future you should vortex (or sonicate) for at least 10 mins after the last addition of solute.JCB]**

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**[The log doesn't say how much water was added - are you assuming that the water and the entirety of your sample are being integrated?]** The capillary tube was filled to about half an inch from the top of the capillary tube. This prevented the water from changing the solubility while providing an external reference to compare the sample peaks with. Both the water and the sample were integrated manually. The microsyringe measured the amount of water inserted into the capillary tube to be 94 uL.

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The solubility of benzoic acid in chloroform was determined to be approximately 16.5 g benzoic acid/ 100 g solution of protonated chloroform and benzoic acid. **[Please explain to me what you mean by wt%, how/whether this is different from g solute/100g solvent and why you are reporting 16.5 wt% three paragraphs above and 12.5g/100g here? Finally, is there a reason not to convert g/100g to molarity? -BH]** Thank you for pointing that out. I copied the wrong number from the discussion and stated the solvent rather than the solution. What I mean by weight percent is really just g solute/ g solution * 100%. 16.5 g benzoic acid/ 100 g solution with protonated chloroform is the same as 16.5 wt%. I find weight percent a little more concise than g solute/ 100 g solution. The solubility was not converted to molarity because I did not measure the volume of the solution and do not know the density of the solution. <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**[Note that the primary format of reporting our solubilities is molar so you should design your experiment to report it that way. You can do that if you transfer over a measured volume of saturated solution to the NMR tube and add an internal standard. Since you didn't do that you'll have to convert over from mass solute/mass solution to molar using [|some approximations].** <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**The [|SolubilitySum spreadsheet] already has columns ready to process that conversion when necessary - but if you are designing the experiment you shouldn't have to JCB]**