Determination of the Solubility of Salicylic acid, Acetylsalicylic acid, and o-Anisic acid in Protonated Chloroform using 1H NMR

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.

Results

Identification of Peaks:
Identification Spectrum - EXP034ASA
Identification Spectrum - EXP034SA
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

Spreadsheet:
EXP034 Spreadsheet
EXP034Continued Spreadsheet

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
Analytical_Scale_2
Analytical_Scale_3
Computer_with_Delta
Pasteur_Pipet
Microsyringe
NMR_external_reference_setup
NMR_external_reference_setup_close_up
Salicylic_Acid
Acetylsalicylic_Acid
o-Anisic_Acid
Acetylsalicylic_acid_after short equilibration
o-Anisic_acid_after short equilibration
Salicylic_acid_in NMR_sample
Salicylic_acid_after equilibrating and removing some supernatant
o-Anisic asic after weekend equilibrationSalicylic acid after weekend equilibration
Acetylsalicylic acid after weekend equilibration

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 34.1: Solubility Measurements of reference 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 34.2: Solubilities of ASA, SA, and o-AA for saturated 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
Table 34.3: Solubilities of ASA, SA, and o-AA for equilibrated solutions

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.

Conclusion
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

1.30.09 CST

Preparation
16.06 - Added 94 uL water to capillary tube using microsyringe
16.50 - Photos of equipment
16.54 - Automatic gradient shim using pre-made deuterated chloroform sample (Z1 = (-1 279), Z2 = (-977), Z3 = (-293), Z4 = (936))

Acetylsalicylic Acid Reference
17.37 - Weighed empty NMR tube (3.088 1 g)
17.39 - Added acetylsalicylic acid and weighed (3.123 5 g)
17.42 - Added protonated chloroform and weighed (4.289 5 g)
17.42 - Sonicated 10 min
17.55 - Reweighed (4.289 4 g)
17.57 - NMR run autogaining (Gain = 7)

Salicylic Acid Reference
17.45 - Weighed empty NMR tube (3.091 3 g)
17.48 - Added salicylic acid and weighed (3.123 8 g)
17.50 - Added protonated chloroform and weighed (4.230 8 g)
17.52 - Added protonated chloroform and weighed (6.201 7 g)
17.53 - Sonicated 10 min (43 degrees Celsius)
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.
18:12 - NMR run autogaining (Gain = 7)

o-Anisic Acid Reference
18.15 - Added water to sonicator
18.25 - Weighed empty NMR tube (3.091 0 g)
18.29 - Added o-anisic acid and weighed (3.178 0 g)
18.32 - Added protonated chloroform and weighed (4.441 3 g)
18.33 - Sonicated 10 min (30 degrees Celsius)
18.43 - Removed from sonicator (40 degrees Celsius)
18.44 - Reweighed (4.441 5 g)
18:46 - NMR run autogaining (Gain = 7)

Saturated Acetylsalicylic Acid
18.37 - Weighed empty vial (13.527 0 g)
18.40 - Added acetylsalicylic acid and weighed (14.126 0 g)
18.50 - Added protonated chloroform and weighed (18.070 3 g)
18.53 - Sonicated 10 min (25 degrees Celsius)
19.03 - Removed from sonicator (31 degrees Celsius)
19.03 - Sat equilibrating for 117 min
21.00 - Transferred supernatant to NMR tube using Pasteur pipet
21.04 - NMR run autogaining (Gain = 7)
21.15 - Equilibrated for about 67 hours

Saturated Salicylic Acid
18.54 - Weighed empty vial (14.451 9 g)
18.56 - Added Salicylic acid and weighed (15.084 9 g)
19.02 - Added protonated chloroform and weighed (18.863 8 g)
19:03 - Sonicated 21 min (31 degrees Celsius)
19.24 - Removed from sonicator (35 degrees Celsius)
19.24 - Sat equilibrating for 66 min
20.30 - Transferred supernatant to NMR tube using Pasteur pipet
20.32 - NMR run autogaining (Gain = 7)
20.35 - Photos
20.40 - Equilibrated for about 67 hours

Saturated o-Anisic Acid
19:03 - Weighed empty vial (13.847 7 g)
19.05 - Added o-Anisic acid and weighed (14.742 3 g)
19.15 - Added protonated chloroform and rusty orange color observed
19.20 - Sample discarded
19:35 - Weighed empty vial (20.019 1 g)
19.38 - Added o-Anisic acid and weighed (20.869 2 g)
19.40 - Added protonated chloroform and weighed (25.798 6 g)
19.42 - Added o-Anisic acid and weighed (26.298 2 g)
19:43 - Sonicated 12 min (33 degrees Celsius)
19.55 - Removed from sonicator (37 degrees Celsius)
20.16 - Added o-Anisic acid and weighed (26.319 2g)
20.18 - Added o-Anisic acid and weighed (26.733 2 g)
20.21 - Sonicated 11 min (32 degrees Celsius)
20.32 - Removed from sonicator (37 degrees Celsius)
20.32 - Sat equilibrating for 17 min
20.49 - Transferred supernatant to NMR tube using Pasteur pipet
20.53 - NMR run autogaining (Gain = 7)
20.58 - Equilibrated for about 67 hours
[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.

02.02.08 CST

Equilibrated NMR Runs
15.45 - Automatic gradient shim using pre-made deuterated chloroform sample (Z1 = (-1 289), Z2 = (-962), Z3 = (-202), Z4 = (691))
15.50 - Photos of Equilibrated Solutions
15:55 - Added 94 uL of water to orginal capillary tube
16:02 - o-Anisic acid NMR run autogaining (Gain = 6)
16:15 - Salicylic acid NMR run autogaining (Gain = 7)
16:24 - Acetylsalicylic acid NMR run autogaining (Gain = 7)


References [please add all these references to SolubilitySum spreadsheet JCB]They should be added now.

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

Solubility of Acetylsalicylic Acid in Chloroform (solubility data viewer)

Solubility of Salicylic Acid in Chloroform (solubility data viewer)

Solubility of o-Anisic Acid in Chloroform (solubility data viewer)

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

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]. 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.
Calculations
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).
Integral ratios were calculated for each of the solute's unsaturated reference spectrum as follows:
Integral Ratio = g solute from unsaturated reference / unsaturated solute peak against water peak
The observed mass of the solute is calculated as follows:
g solute observed = Integral Ratio of unsaturated reference * saturated solute peak against water peak
Weight Percent (wt%) = total mass of solute / total mass of solution * 100%.

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.]


ASA Reference
SA Reference
o-AA Reference
Saturated ASA
Saturated SA
Saturated oAA
Equilibrated ASA
Equilibrated SA
Equilibrated oAA
ASAHba
SAHaa
oAAHca
SASAHba
SSAHaa
SoAAHca
EASAHba
ESAHaa
EoAAHca
ASAHbb
SAHab
oAAHcb
SASAHbb
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
oAAHfb
SASAHfb
SSAHcb
SoAAHfb
EASAHfb
ESAHcb
EoAAHfb
ASAHga
SAHda
oAAHga
SASAHga
SSAHda
SoAAHga
EASAHga
ESAHda
EoAAHga
ASAHgb
SAHdb
oAAHgb
SASAHgb
SSAHdb
SoAAHgb
EASAHgb
ESAHdb
EoAAHgb
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).
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.
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).
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]
[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].
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.


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,
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]