EXP034

**Determination of the Solubility of Salicylic acid, Acetylsalicylic acid, and o-Anisic acid in Protonated Chloroform using 1H NMR** media type="custom" key="7609477"media type="custom" key="7609479"media type="custom" key="7609487"media type="custom" key="7609493" **Researcher** David Bulger

**Objective** To determine the solubility of [|salicylic acid], [|acetylsalicylic acid], and [|o-anisic acid] in [|chloroform] using 1H NMR.

**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** Saturated solution of acetylsalicylic acid (0.599 g; 3.32 mmol) and chloroform (3.9443 g; 33.040 mmol) was added to NMR tube labeled saturated ASA. Saturated solution of salicylic acid (0.633 g; 4.58 mmol) and chloroform (3.7789 g; 31.654 mmol) was added to NMR tube labeled saturated SA. Saturated solution of o-anisic acid (1.7847 g; 11.730 mmol) and chloroform (4.9294 g; 41.292 mmol) was added to NMR tube labeled saturated oAA. Temperature was observed to increase from 23 °C to 49 °C during the 10-min sonication when preparing saturated solutions. 1H NMR was performed on the three samples. The samples were equilibrated for 67 hours, and 1H NMR was performed again on the three equilibrated samples.

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

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Identification of Peaks: <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Identification Spectrum - EXP034ASA] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Identification Spectrum - EXP034SA] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Identification Spectrum - EXP034o-AA]

Exported/Converted Spectra: [|ASAreference] [|SAreference] [|o_AAreference] [|ASAsaturated] [|SAsaturated] [|o_AAsaturated] [|ASAequilibrated] [|SAequilibrated] [|o_AAequilibrated] [|Raw JCAMP-DX file for ASAreference] [|Raw JCAMP-DX file for SAreference] [|Raw JCAMP-DX file for o-AAreference] [|Raw JCAMP-DX file for ASAsaturated] [|Raw JCAMP-DX file for SAsaturated] [|Raw JCAMP-DX file for o-AAsaturated] [|Raw JCAMP-DX file for ASAequilibrated] [|Raw JCAMP-DX file for SAequilibrated] [|Raw JCAMP-DX file for o-AAequilibrated]

<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%;">[|EXP034 Spreadsheet] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|EXP034Continued Spreadsheet] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">media type="custom" key="7611309"media type="custom" key="7611285" 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] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Analytical_Scale_2] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Analytical_Scale_3] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Computer_with_Delta] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Pasteur_Pipet] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Microsyringe] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|NMR_external_reference_setup] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|NMR_external_reference_setup_close_up] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Salicylic_Acid] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Acetylsalicylic_Acid] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|o-Anisic_Acid] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Acetylsalicylic_acid_after short equilibration] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|o-Anisic_acid_after short equilibration] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Salicylic_acid_in NMR_sample] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Salicylic_acid_after equilibrating and removing some supernatant] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|o-Anisic asic after weekend equilibration][|Salicylic acid after weekend equilibration] <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Acetylsalicylic acid after weekend equilibration]

Table 34.1: Solubility Measurements of reference solutions
 * Spectrum || Proton || Known Concentration (__M__) || SAMS Solubility (__M__) ||
 * [|ASAreference] || Ha || 0.2410 || 0.2211 ||
 * [|ASAreference] || Hb || 0.2410 || 0.2719 ||
 * [|ASAreference] || He || 0.2410 || 0.2650 ||
 * [|ASAreference] || Hg || 0.2410 || 0.2350 ||
 * [|SAreference] || Ha || 0.1119 || 0.1091 ||
 * [|SAreference] || Hb || 0.1119 || 0.1249 ||
 * [|o_AAreference] || Ha || 0.6177 || 0.4403 ||
 * [|o_AAreference] || Hb || 0.6177 || 0.6593 ||
 * [|o_AAreference] || Hc || 0.6177 || 0.6068 ||
 * [|o_AAreference] || He || 0.6177 || 0.6415 ||
 * [|o_AAreference] || Hg || 0.6177 || 0.6373 ||

Table 34.2: Solubilities of ASA, SA, and o-AA for saturated solutions
 * Spectrum || Proton || Solvent || SAMS Solubility (__M__) || External Standard Solubility (__M__) ||
 * [|ASAsaturated] || Ha || Chloroform || 0.3524 || 0.3843 ||
 * [|ASAsaturated] || Hb || Chloroform || 0.3636 || 0.3209 ||
 * [|ASAsaturated] || He || Chloroform || 0.3569 || 0.3235 ||
 * [|ASAsaturated] || Hg || Chloroform || 0.3401 || 0.3468 ||
 * [|SAsaturated] || Ha || Chloroform || 0.2183 || 0.2080 ||
 * [|SAsaturated] || Hb || Chloroform || 0.2301 || 0.1926 ||
 * [|o_AAsaturated] || Ha || Chloroform || 2.0678 || 3.3841 ||
 * [|o_AAsaturated] || Hb || Chloroform || 1.9326 || 2.0039 ||
 * [|o_AAsaturated] || Hc || Chloroform || 1.8826 || 2.1185 ||
 * [|o_AAsaturated] || He || Chloroform || 1.9559 || 2.0966 ||
 * [|o_AAsaturated] || Hg || Chloroform || 1.9205 || 2.0622 ||

Table 34.3: Solubilities of ASA, SA, and o-AA for equilibrated solutions
 * Spectrum || Proton || Solvent || SAMS Solubility (__M__) || External Standard Solubility (__M__) ||
 * [|ASAequilibrated] || Ha || Chloroform || 0.3378 || 0.3595 ||
 * [|ASAequilibrated] || Hb || Chloroform || 0.3636 || 0.3142 ||
 * [|ASAequilibrated] || He || Chloroform || 0.3446 || 0.3050 ||
 * [|ASAequilibrated] || Hg || Chloroform || 0.3547 || 0.3547 ||
 * [|SAequilibrated] || Ha || Chloroform || 0.2147 || 0.2008 ||
 * [|SAequilibrated] || Hb || Chloroform || 0.2052 || 0.1676 ||
 * [|o_AAequilibrated] || Ha || Chloroform || 1.3905 || 2.1766 ||
 * [|o_AAequilibrated] || Hb || Chloroform || 1.6883 || 1.7935 ||
 * [|o_AAequilibrated] || Hc || Chloroform || 1.6395 || 1.8912 ||
 * [|o_AAequilibrated] || He || Chloroform || 1.6821 || 1.8393 ||
 * [|o_AAequilibrated] || Hg || Chloroform || 1.6605 || 1.8229 ||

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**Discussion** The SAMS measurements were compared with the known concentrations of the reference solutions as shown in Table 34.1. The acetylsalicylic acid reference was measured by SAMS with an average percent error of 3.0%. The salicylic acid reference was measured with an average percent error of 4.6%. And the o-anisic acid was measured with an average percent error of 3.7%. The reference solutions acted as the reference external standard solution for the measurements by the External Standard Method.

The solubility of the saturated solutions were measured by both SAMS and External Standard methods as shown in Table 34.2. Acetylsalicylic acid's solubility in chloroform was measured to be 0.35 +/- 0.01 __M__ by SAMS and 0.34 +/- 0.03 __M__ by the External Standard Method. Salicylic acid's solubility was measured at 0.224 +/- 0.008 __M__ by SAMS and 0.20 +/- 0.01 __M__ by the External Standard Method. The solubility of o-anisic acid in chloroform was measured to be 1.67 +/- 0.07 __M__ by SAMS and 2.4 +/- 0.7 __M__ by the External Standard Method.

The solubility of the solutions left to equilibrate were measured by both SAMS and External Standard methods as shown in Table 34.3. The solubility of acetylsalicylic acid was measured at 0.35 +/- 0.01 __M__ by SAMS and 0.33 +/- 0.03 __M__ by the External Standard Method. Salicylic acid's solubility was measured at 0.210 +/- 0.007 __M__ by SAMS and 0.18 +/- 0.02 __M__ by the External Standard Method. The solubility of o-anisic acid was measured at 1.6 +/- 0.1 __M__ by SAMS and 1.9 +/- 0.2 __M__ by the External Standard Method.

<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%;">SAMS seemed to measure solubility with results very similar to the External Standard Method and precision even better than the External Standard Method in this experiment. The solubility of acetylsalicylic acid was measured to be 0.35 +/- 0.1 __M__. The solubility of salicylic acid was measured to be 0.21 +/- 0.01 __M__. And the solubility of o-anisic acid was measured to be 2.4 +/- 0.8 __M__.


 * Log**

<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%;">__Preparation__ <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%;">__Acetylsalicylic Acid Reference__ <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.37 - Weighed empty NMR tube (3.088 1 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.39 - Added acetylsalicylic acid and weighed (3.123 5 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.42 - Added protonated chloroform and weighed (4.289 5 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.42 - Sonicated 10 min <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.55 - Reweighed (4.289 4 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.57 - NMR run autogaining (Gain = 7)

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">__Salicylic Acid Reference__ <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.45 - Weighed empty NMR tube (3.091 3 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.48 - Added salicylic acid and weighed (3.123 8 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.50 - Added protonated chloroform and weighed (4.230 8 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.52 - Added protonated chloroform and weighed (6.201 7 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17.53 - Sonicated 10 min (43 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.03 - Removed from sonicator (49 degrees Celsius)**[Is this heat generated from the sonication directly? JCB]** The heat does appear to be generated from the sonicator directly, since no other heat source was around that I know of. <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18:12 - NMR run autogaining (Gain = 7)

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">__o-Anisic Acid Reference__ <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.15 - Added water to sonicator <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.25 - Weighed empty NMR tube (3.091 0 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.29 - Added o-anisic acid and weighed (3.178 0 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.32 - Added protonated chloroform and weighed (4.441 3 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.33 - Sonicated 10 min (30 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.43 - Removed from sonicator (40 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.44 - Reweighed (4.441 5 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18:46 - NMR run autogaining (Gain = 7)

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">__Saturated Acetylsalicylic Acid__ <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.37 - Weighed empty vial (13.527 0 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.40 - Added acetylsalicylic acid and weighed (14.126 0 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.50 - Added protonated chloroform and weighed (18.070 3 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.53 - Sonicated 10 min (25 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.03 - Removed from sonicator (31 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.03 - Sat equilibrating for 117 min <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">21.00 - Transferred supernatant to NMR tube using Pasteur pipet <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">21.04 - NMR run autogaining (Gain = 7) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">21.15 - Equilibrated for about 67 hours

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">__Saturated Salicylic Acid__ <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.54 - Weighed empty vial (14.451 9 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18.56 - Added Salicylic acid and weighed (15.084 9 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.02 - Added protonated chloroform and weighed (18.863 8 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19:03 - Sonicated 21 min (31 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.24 - Removed from sonicator (35 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.24 - Sat equilibrating for 66 min <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.30 - Transferred supernatant to NMR tube using Pasteur pipet <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.32 - NMR run autogaining (Gain = 7) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.35 - Photos <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.40 - Equilibrated for about 67 hours

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">__Saturated o-Anisic Acid__ <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19:03 - Weighed empty vial (13.847 7 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.05 - Added o-Anisic acid and weighed (14.742 3 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.15 - Added protonated chloroform and rusty orange color observed <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.20 - Sample discarded <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19:35 - Weighed empty vial (20.019 1 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.38 - Added o-Anisic acid and weighed (20.869 2 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.40 - Added protonated chloroform and weighed (25.798 6 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.42 - Added o-Anisic acid and weighed (26.298 2 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19:43 - Sonicated 12 min (33 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19.55 - Removed from sonicator (37 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.16 - Added o-Anisic acid and weighed (26.319 2g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.18 - Added o-Anisic acid and weighed (26.733 2 g) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.21 - Sonicated 11 min (32 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.32 - Removed from sonicator (37 degrees Celsius) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.32 - Sat equilibrating for 17 min <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.49 - Transferred supernatant to NMR tube using Pasteur pipet <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.53 - NMR run autogaining (Gain = 7) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20.58 - Equilibrated for about 67 hours <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**[Make sure to measure the exact volume when transferring to NMR tube - otherwise you can't get molar value JCB]** For this experiment, I would have to measure the exact volume of the reference run solutions. This would mean repeating the reference runs and possibly using a volumetric flask rather than a vial. I plan on measuring the volume in future experiments.

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">02.02.08 CST

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">__Equilibrated NMR Runs__ <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">15.45 - Automatic gradient shim using pre-made deuterated chloroform sample (Z1 = (-1 289), Z2 = (-962), Z3 = (-202), Z4 = (691)) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">15.50 - Photos of Equilibrated Solutions <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">15:55 - Added 94 uL of water to orginal capillary tube <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">16:02 - o-Anisic acid NMR run autogaining (Gain = 6) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">16:15 - Salicylic acid NMR run autogaining (Gain = 7) <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">16:24 - Acetylsalicylic acid NMR run autogaining (Gain = 7)

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**References** **[please add all these references to SolubilitySum spreadsheet JCB]**They should be added now.

<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%;">[|Solubility of Acetylsalicylic Acid in Chloroform] (solubility data viewer)

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility of Salicylic Acid in Chloroform] (solubility data viewer)

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Solubility of o-Anisic Acid in Chloroform] (solubility data viewer)

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

**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 his help with the interactive NMR spectra, Dr. Hal Reed for the use of the department camera, and Dr. Antony Williams for reference #1.


 * Past Discussion **


 * Method**

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">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 22 degrees C using auto-gain with spin off. The proton spectra were acquired using a 45-degree pulse with a relaxation delay of 5 seconds [**I assume you are not using any steady state scans? ChemSpiderman**], JEOL automatic zero filling [**zero filling to what level? 2X? 4X?**], and 1 scan with a resolution of 77.97 mHz (about 64 000 x_points) [**Is it appropriate to quote to this accuracy? What is thedigital resolution overall?**]. Shimming of spectra used JEOL automatic gradient shimming with deuterated solvent spinning at 15 Hz **[Based on this spectrum I think that autoshim is failing or autophase is off...this one looks like Z2 is too high: http://onschallenge.wikispaces.com/file/view/ASAHfb.png].** <span style="color: black; font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Mass determinations were performed using a 0.1 mg accuracy [|Sortorius balance]. A saturated solution was made **[this is crucial, so you need to be more specific about how this was done, and in particular how you know it to be saturated -BH]** by adding solute to approximately 6 mL chloroform (note: there may be very small amounts of ethanol and water in the chloroform) until a layer at least 1 cm thick was observed in the vial after at least ten minutes of sonication and the supernatant was transferred to the NMR tubes using [|Pasteur_Pipet] followed by addition of capillary tubes filled with 94 uL water using a [|Microsyringe] ([|NMR_external_reference_setup]). Samples were sonicated with a thermometer. Reference NMR runs performed with protonated chloroform. <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%;">Two solute peaks were chosen based on how clear (how distinct and separate from other peaks - complete lorenzian tails) the peak **[Please clarify what you mean by "Clear" - ChemSpiderman]** is and what the peak corresponds to. The solute peaks' integrations as found using the JSpecViewer as shown under the interactive spectra section with screenshots of the manually obtained start and end points of the integral with the individual calculations in the __ spreadsheet __ [**How do you obtain the integral values - do you measure them yourselves with ruler and pen or use computer extracted integral values. How do you set the start and end point of the integral? Automatically or manual?** **ChemSpiderman**] 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%;">Integral ratios were calculated for each of the solute's unsaturated reference spectrum as follows: <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Integral Ratio = g solute from unsaturated reference / unsaturated solute peak against water peak <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The observed mass of the solute is calculated as follows: <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">g solute observed = Integral Ratio of unsaturated reference * saturated solute peak against water peak <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Weight Percent (wt%) = total mass of solute / total mass of solution * 100%.

<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) with Screenshots of Integrations (Integration Parameters: Minimum Y = 0, Integral Factor = 150, Integral Offset = 0):**[This was done just to salvage your results the first time you put up 800K files - going forward go back to creating 200K files JCB]** For the equilibrated spectra, I decided to be consistent and use the high resolution (around 64 000 x_points) **[By high resolution do you mean highly digitized? Resolution in NMR generally refers to linewidth. Do you apply any line broadening to these spectra? I believe that you have Truncation issues in the spectra because of the wiggles in this portion of the spectrum : http://onschallenge.wikispaces.com/file/view/ASAHba.png However, I'd need to see the whole spectrum. I see similar issues here: http://onschallenge.wikispaces.com/file/view/SAHbb.png. ChemSpiderman]**. In future experiments, I will decrease the resolution to what was used in EXP017. [**I encourage you to post the actual spectra on ChemSpider and I will provide you with the appropriate Javascript to embed the spectrum in this page. The technology is already built... ChemSpiderman**] The whole spectra can be found in JSpecViewer below in the top row of the table. Here are the two spectra you referred to above: [|ASA Reference] and [|SA Reference]. Are you able to access these spectra? If not, I can also post the JCamp files. **[I need the JCAMP spectra please, or even the original JEOL files that I can process. Please feel free to send to my email account. AJW.]**

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The solubility of acetylsalicylic acid using the Hb peak of the Saturated spectrum was observed to be [|1.43 wt% (0.12 M)], using the Hb peak of the Equilibrated spectrum was observed to be [|1.32 wt% (0.11 M)], using the Hg peak of the Saturated spectrum was observed to be [|1.36 wt% (0.11 M)], and using the Hg peak of the Equilibrated spectrum was observed to be [|1.34 wt% (0.11 M)]. <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The solubility of salicylic acid using the Ha peak of the Saturated spectrum was observed to be [|1.62 wt% (0.18 M)], using the Ha peak of the Equilibrated spectrum was observed to be [|1.51 wt% (0.17 M)], using the Hb peak of the Saturated spectrum was observed to be [|1.92 wt% (0.21 M)], and using the Hb peak of the Equilibrated spectrum was observed to be [|2.10 wt% (0.23 M)]. This compares to the 1.64 wt% in [|Reference 4].**[No that is not the wt% in the ref - that is g/100ml of solution JCB]** Thank you. <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The solubility of o-anisic acid using the Hc peak of the Saturated spectrum was observed to be [|7.07 wt% (0.52 M)], using the Hc peak of the Equilibrated spectrum was observed to be [|5.96wt% (0.44 M)], using the Hg peak of the Saturated spectrum was observed to be [|6.51 wt% (0.48 M)], and using the Hg peak of the Equilibrated spectrum was observed to be [|5.61 wt% (0.42 M)]. <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Note: I am unsure of what References 2, 3, and 5 mean by their ratios. For example, does a solubility of 1:17 mean 1 g solute/ 17 g solution or 1 g solute/ 17 g solvent? **[The ratios would be weight solute/weight solvent. It is helpful to compare all these measurements with the same units. Add these references in molar to the [|SolubilitySum] spreadsheet. To convert g/100g solvent you'll have to use the columns on the right and look up properties on ChemSpider. Let me know if you need help with that. JCB]** <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">**[What information does the reference provide if you are dividing both solute and solvent against it - what does that give you that dividing the solute by solvent integration directly not give you? JCB]** The chloroform signals do not seem trustworthy as other solute signals tend to be around the same ppm. For example, [|ASA's_Hc_signal] contributes to the chloroform peak.**[No I don't see an overlap - but even if there were the solute's integration is insignificant compared to the CHCl3. You actually can't use the water any more reliably than the chloroform since they are roughly the same integration. Try using an internal standard which integrates in the same range as your solute. You won't be able to use water because it is not miscible with chloroform - I suggest acetonitrile. JCB]**. <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">Also, if a deuterated solvent is used, the reference provides the known concentration to compare with. **[What known concentration are you referring to? JCB]** The concentration of the reference run.
 * [|ASA Reference] || [|SA Reference] || [|o-AA Reference] || [|Saturated ASA] || [|Saturated SA] || <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|Saturated oAA] || [|Equilibrated ASA] || [|Equilibrated SA] || [|Equilibrated oAA] ||
 * [|ASAHba] || [|SAHaa] || <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|oAAHca] || [|SASAHba] || <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|SSAHaa] || <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|SoAAHca] || [|EASAHba] || [|ESAHaa] || [|EoAAHca] ||
 * [|ASAHbb] || [|SAHab] || [|oAAHcb] || [|SASAHbb] || <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|SSAHab] || [|SoAAHcb] || [|EASAHbb] || [|ESAHab] || [|EoAAHcb] ||
 * [|ASAHda] || [|SAHba] || [|oAAHda] || [|SASAHda] || [|SSAHba] || [|SoAAHda] || [|EASAHda] || [|ESAHba] || [|EoAAHda] ||
 * [|ASAHdb] || [|SAHbb] || [|oAAHdb] || [|SASAHdb] || [|SSAHbb] || [|SoAAHdb] || [|EASAHdb] || [|ESAHbb] || [|EoAAHdb] ||
 * [|ASAHfa] || [|SAHca] || [|oAAHfa] || [|SASAHfa] || [|SSAHca] || [|SoAAHfa] || [|EASAHfa] || [|ESAHca] || [|EoAAHfa] ||
 * [|ASAHfb] || [|SAHcb] || <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|oAAHfb] || [|SASAHfb] || [|SSAHcb] || [|SoAAHfb] || [|EASAHfb] || [|ESAHcb] || [|EoAAHfb] ||
 * [|ASAHga] || [|SAHda] || <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|oAAHga] || [|SASAHga] || [|SSAHda] || [|SoAAHga] || [|EASAHga] || [|ESAHda] || [|EoAAHga] ||
 * [|ASAHgb] || [|SAHdb] || <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">[|oAAHgb] || [|SASAHgb] || [|SSAHdb] || [|SoAAHgb] || [|EASAHgb] || [|ESAHdb] || [|EoAAHgb] ||

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">The solubilities of salicylic acid, acetylsalicylic acid, and o-anisic acid are shown below.


 * **Sample** || **Solute in chloroform** || **Spectrum** || **Peak** || **wt%** || **~ __M__** ||
 * 1b || Acetylsalicylic acid || Saturated || Hb || 1.43 || 0.12 ||
 * 2b || Acetylsalicylic acid || Equilibrated || Hb || 1.32 || 0.11 ||
 * 1g || Acetylsalicylic acid || Saturated || Hg || 1.36 || 0.11 ||
 * 2g || Acetylsalicylic acid || Equilibrated || Hg || 1.34 || 0.11 ||
 * 3a || Salicylic acid || Saturated || Ha || 1.62 || 0.18 ||
 * 4a || Salicylic acid || Equilibrated || Ha || 1.51 || 0.17 ||
 * 3b || Salicylic acid || Saturated || Hb || 1.92 || 0.21 ||
 * 4b || Salicylic acid || Equilibrated || Hb || 2.10 || 0.23 ||
 * 5c || o-Anisic acid || Saturated || Hc || 7.07 || 0.52 ||
 * 6c || o-Anisic acid || Equilibrated || Hc || 5.96 || 0.44 ||
 * 5g || o-Anisic acid || Saturated || Hg || 6.51 || 0.48 ||
 * 6g || o-Anisic acid || Equilibrated || Hg || 5.61 || 0.42 ||


 * [Please convert to molar as described [|here] and add your references to the [|SolubilitySum] spreadsheet so we can easily compare using the [|dropdown menus]. I just added a reference there from the 1919 Seidell book for salicylic acid. JCB]**


 * [I used your integrations for Ha and Hc for saturated SA and got 0.95% - check these over (green section in the spreadsheet) JCB]** I see what you mean by not needing a reference in light of the calculation method you used. However, using that method I got 0.45 wt% rather than the actual 1.04 wt% of the reference SA solution (red section in the spreadsheet). **[That suggests that you might be saturating the chloroform peak and not getting reliable integrations because there is just too much of it. (You also have a huge excess of water with the same problem). I would recommend using an internal standard next time that is closer in amount to your solute so that the peaks can be of comparable intensity. With an internal standard you don't need to rely on the solvent peak at all. 5 microliters of acetonitrile is 0.1 millimole. JCB]** How would the possibility of the acetonitrile internal standard changing the solubility of the solution followed by precipitation of the solute be accounted for? Also, D2O could be added to the external water standard to decrease the peak height if needed.**[You're also diluting the saturated solution as you're adding a very small amount of acetonitrile (5-10 microliters). We've diluted in CDCl3 but you could also try diluting in CHCl3 and see if you peak quality is similar. (see EXP030). I just have concerns about the use of an external standard - how can you be sure that the entirety of the external standard AND your solution are completely within the volume detected by the NMR? If you have some documentation about that it would be helpful. JCB]** In order to make sure the sample and external standard fills the range the NMR detects, a standard gauge is used. So far, there have been no problems with not having enough solution in the NMR tube. As long as the solute concentration and water concentration is uniform throughout the sample, I do not see why the entirety of the sample needs to be detected rather than a fraction.**[But since the water is not actually in the CDCl3 how can you talk about "concentration"? Even if the external reference was completely uniform, you would still have to add an exact amount of solution exactly up to the mark of the water. But the tube does not appear uniform - it looks like it tapers towards the bottom and there is clearly no water in the tube at the very bottom. JCB]** Even if the tube is not uniform and 100% of the water is not is the receptor coil, the water peak should still be consistent. All the water peak is used for is a consistent peak to reference. Just to test the consistency, I might perform several NMR runs with differing known concentrations of a mixture of solutes and check the consistency of the external standard. Also, the internal coax has arrived and might be used to improve the precision of the external standard even more. However, the capillary tube is expected to be consistent enough for this experiment. **[Using an external reference in this way will require you to add exactly the same volume of solution every time and you will have to do a calibration since you don't know exactly how much of the water is exposed to the field. This seems like an awful lot of work when an internal standard gives you the answer directly in one run without needing a calibration JCB]** The same capillary tube was used for each of the NMR runs. The capillary tube was removed and cleaned with chloroform and a KemWipe after each run. I agree that it would be faster with the internal reference, especially since a calibration run is not required.

To my knowledge, the NMR does not read the sample above the gauge line. Both the water in the capillary tube and the saturated solution were above the gauge line. Reference 6 states on pages 195-196, <span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">“//Choosing a reference.// Chemical shifts are normally reported relative to a standard reference, e.g. tetramethylsilane (TMS) for 1H and 13C, with positive values at higher frequencies than the reference peak. When reporting results, it is important to specify the reference for each spectrum. However, because of physical or chemical incompatibilities, it is not always possible to include the primary reference in the sample, and a compound with a known chemical shift relative to the primary reference must be used. Solvent resonances are frequently used as secondary reference peaks, and the protio impurities in deuterated solvents are particularly convenient for 1H-NMR spectra (note that these peaks may be multiplets due to coupling to deuterium), e.g. CHCl3 (singlet) in CDCl3 reference compound in a sealed, concentric capillary inside the NMR tube. For nuclei other than 1H or 13C, the 'sample replacement' method is often used. In this case the spectrometer is calibrated with a solution of the standard reference compound immediately before or after measurements on the sample."**[That just means that you can use an external reference to locate the position of a reference peak (which you have to fix going forward before saving your spectra - chloroform is at 7.27ppm) - it does not say that you can reliably integrate that peak. JCB]** You are right, it does not state anything about the quantitative use of the external standard. One planned future experiment is using both an external and internal reference in the same sample to compare the two methods.


 * [The "NMR active volume" is the fraction of the overall sample volume that is contained within the irradiating coil to provide NMR responses. David, please forward a copy of reference 1 to me if you have it. Are they using internal or external references for their work? JCB may also be interested. ChemSPiderman. ]** Reference 1 can be accessed through the link under references (__ Reference 1) __, but I will also e-mail you a copy of it. They are using internal references.


 * (Thanks for forwarding the spectra to me. I have some basic observations. I believe your solubility determinations will be off, maybe a factor of two for the acetylsalicylic acid. Look at the spectrum here: http://www.chemspider.com/Chemical-Structure.2157.html. YOu might need to do Right Click> View> Reverse Plot to get 0ppm to the right. Notice there are no ethyl signals. I believe you have a close to 50/50 ratio of acetylsalicylic acid with some impurity. See the image here: [|ethylsignals.png]. Also, I think you have spectrometer issues...you are picking up noise bursts. Check your filters. Just an observation. See this image: [|noisebursts.png] ChemSpiderman)** I see what you are observing. The vials came straight from the storeroom and could have had some impurities in them. For example, when o-Anisic acid was first added, a rusty color was observed that was not present in the second vial. I plan on repeating the experiment and cleaning the vials again before adding the sample. I will inquire about the noise bursts and filters. The NMR was just checked on in December when the valve problem was fixed. **[Out of interest, do you dry the NMR solvents at all? If not, what is the contribution to the water peak from internal solvent relative to the insert? Can I post the reference H1 NMR spectra to ChemSpider for the NMR Game?]** The NMR solvents used are from the stock. The stock solution usually does have some very small amounts of ethanol and water in it. Good point, I could run the NMR of the solvent to check for water. Yes, you can post the spectra on the NMR Game. That way if a lot of people miss the spectra on the Game, there would be even more reason to check for impurities. In reference to the noise bursts, I was only scanning the sample once in this experiment. If the sample was scanned more times with a higher relaxation time, the noise is expected to decrease.**[The fact that the chloroform has ethanol is not a problem but you need to make a note of it. Probably most of the chloroform measurement do have a small amount of ethanol in them. The advantage of using NMR is that we can be sure that it is there or not. JCB]**