When we first started mixing nicotine we based our calculations on pure math.  This seems theoretically logical in assumption if all methodology is accurate in implementation.  So the idea was to prove or disprove just this.  In essence we set out to self regulate our methods and procedures to ensure that we were producing an accurate concentration of nicotine in every product.  To do so we purchased a nicotine concentration test kit on the internet which we found on ECF.  It seemed simple enough, but how did we know if this test was giving accurate results?  So first we started out to "test the test".

Testing the Test

Detail:

            The test consists of measuring nicotine concentration using a simple reagent called Bromothymol Blue to titrate the nicotine solution to a pH level of 6.  What does this mean?  Bromothymol Blue has a pH sensitivity of 6 to 7.6.  At 7.6 or above the solution is blue.  As it hits 7.6 and decreases in pH the color changes to a blue green and then to a yellow when it hits its bottom limit of sensitivity of 6.0.  Nicotine by nature has a pH of around 10.2.  So when diluted with vegetable glycerin or propylene glycol the pH of the solution is still above 7.6.  So a solution of .12N Sulfuric Acid (H₂SO₄) can be used to titrate the sample to a pH of 6.  The amount of Sulfuric Acid used can then be calculated, when multiplied with a constant, to mathematically measure the mg concentration of the nicotine.  The constant (19.47) we used was from the test kit.

Defining the Test:

            Start with a 1 mL sample of VG or PG nicotine in a graduated cylinder.  Then add 2 drops of Bromothymol Blue, and then topped off with distilled water to make a 3 mL solution.  (If testing a nicotine sample over 30 mg use 4 drops of Bromothymol Blue.  If testing a nicotine sample over 100 mg use 8 drops of Bromothymol Blue.)  Then add small amounts of .12N Sulfuric Acid with stirring until the color of the solution starts to change.  Continue until the solution is a golden yellow.  As the solution nears the end of the test and gets closer to the desired color change decrease the amount of Sulfuric Acid to just a drop at a time to ensure you don't move past the end titration point.  Measure the final volume of the solution and subtract out the original 3 mL to give you the resulting volume of Sulfuric Acid used.

            So our first step was to break down the test to see if any one variable would change the outcome of the test.  This was broken down into the following areas.

1. Amount of distilled water used.
2. Volume of sample used.
3. Number of drops of Bromothymol Blue used.
4. Type of Bromothymol Blue used.
5. Testing with a pH meter.

Amount of Distilled Water Used

Hypothesis – The amount of distilled water used with the sample will not affect the results of the test because distilled water has a neutral pH.

Methodology – Run a test on the same bottle of nicotine with different amounts of distilled water to see if there is a change in the results of the test.

            We started by making a 50 mL bottle of 24 mg VG Nicotine.  We then ran the test with three 1 mL samples adding 4 drops of Bromothymol Blue and topping to 3 mL of total solution with distilled water which equated to about 2 mL of distilled water.  We ran the same test 3 times on the same 24 mg VG bottle but this time adding closer to 6 mL of distilled water which gave a volume of 7 mL.  We then titrated the samples with .12N Sulfuric Acid.  The results are below:

2 mL Distilled Water	Sample 1	Sample 2	Sample 3
Beginning Volume (mL)	3	3	3
Ending Volume (mL)	4.3	4.4	4.3
H2SO4 Used (mL)	1.3	1.4	1.3
Mg (x 19.47)	25.31	27.25	25.31

6 mL Distilled Water	Sample 1	Sample 2	Sample 3
Beginning Volume (mL)	7	7	7
Ending Volume (mL)	8.2	8.3	8.3
H2SO4 Used (mL)	1.2	1.3	1.3
Mg (x 19.47)	23.36	25.31	25.31

Conclusion – There is no significant change with differing volumes of distilled water used in the test.  However having a little more distilled water does make the solution thinner making it easier and quicker to mix the solution since vegetable glycerin is a relatively viscous solution by itself.

Volume of the Sample Used

Hypothesis – By increasing the amount of the sample used to test, the accuracy would increase.

Methodology – From a 50 mL bottle of 24 mg VG, we tested 1, 2, and 3 mL samples.  Use corresponding multiplicative factors corresponding to the nicotine sample size.

1 mL sample                19.47

2 mL sample                9.735

3 mL sample                6.49

Note that the numbers for the 2 and 3 mL samples are derived by dividing the constant for the 1 mL sample of 19.47 by 2 and 3 respectively.  With a larger sample you will have more nicotine in the sample thus increasing the amount of sulfuric acid needed in the titration by 2 and 3 times.  Hence the constant used as a multiplicator is decreased to ½ and 1/3 of the original number.

            We made a 24 mg 50 mL bottle of VG nicotine and mixed the solution for 10 minutes to make sure the nicotine and VG were fully incorporated.  We then tested each sample size 3 times.

Observation – I had purchased online 2 kits for performing the nicotine test, both from the same source.  Before proceeding I tested the volume of each graduated cylinder and found that they were .1 mL different in volume at the 1 mL mark.  While this seems like a small amount, it would show up as a 10% discrepancy from the start.  I tested the volume of both with a known source and discarded the errant graduated cylinder.  Be sure if you are testing that your equipment is accurate.

1 mL Sample	Sample 1	Sample 2	Sample 3
Beginning Volume (mL)	4.0	4.0	4.1
Ending Volume (mL)	5.2	5.15	5.3
H2SO4 Used (mL)	1.2	1.15	1.2
Mg (x 19.47)	23.36	22.39	23.36

2 mL Sample	Sample 1	Sample 2	Sample 3
Beginning Volume (mL)	5.0	5.1	5.0
Ending Volume (mL)	7.5	7.6	7.7
H2SO4 Used (mL)	2.5	2.5	2.7
Mg (x 9.735)	24.34	24.34	26.28

3 mL Sample	Sample 1	Sample 2	Sample 3
Beginning Volume (mL)	6.0	6.0	6.0
Ending Volume (mL)	9.7	9.9	9.8
H2SO4 Used (mL)	3.7	3.9	3.8
Mg (x 6.49)	24.01	25.31	24.66

Conclusion - It was our conclusion that all of the samples were relatively close in nicotine concentration.  Certainly there was a degree of variability that could be attributed to human error.  For instance, we found that measuring a 1 mL sample of VG nicotine was harder than a 3 mL sample as the meniscus was easier to see and was better formed with the larger volume (at least in the graduated cylinder we were using).  The larger sample size was also more accurate because the amount of Sulfuric Acid used increased and we used a smaller multiplier meaning there was less room for human error because a couple of drops over or under the end titration point was not going to alter the results as much.  For example, .1 mL of sulfuric acid used to titrate with a 1 mL sample changes the mg concentration by 1.947 mg, but on a 3 mL sample .1 mL only changes the concentration by .649 mg.  The amount of sulfuric acid used was quite substantial with a 3 mL sample and have opted for a happy medium using 2 mL samples. 

Number of Drops of Bromothymol Blue Used

Hypothesis – The number of drops of Bromothymol Blue used will not have an effect on the results of the test.

Methodology – Mix a 50 mL bottle of 24 mg VG Nicotine for testing.  Draw a 2 mL sample of VG Nicotine, add 2 drops of Bromothymol Blue and top with about 4 mL of distilled water to a total volume of 6 mL.  Perform the test with 4 drops and 8 drops of Bromothymol Blue.  Repeat each test 2 times.

2 Drops of Bromothymol Blue	Sample 1	Sample 2
Beginning Volume (mL)	6	6
Ending Volume (mL)	8.5	8.5
H2SO4 Used (mL)	2.5	2.5
Mg (x 9.735)	24.33	24.33

4 Drops of Bromothymol Blue	Sample 1	Sample 2
Beginning Volume (mL)	6.1	6
Ending Volume (mL)	8.6	8.4
H2SO4 Used (mL)	2.5	2.4
Mg (x 9.735)	24.33	23.36

8 Drops of Bromothymol Blue	Sample 1	Sample 2
Beginning Volume (mL)	6	6
Ending Volume (mL)	8.5	8.5
H2SO4 Used (mL)	2.5	2.5
Mg (x 9.735)	24.33	24.33

Conclusion – The amount of Bromothymol Blue did not have an effect on the test results.  However, when using 4 and 8 drops it was significantly easier to see the color change of the solution while titrating.  A 4 drop sample is sufficient but I will use 8 drops when testing higher nicotine concentrations.

Type of Bromothymol Blue Used

We were running low on Bromothymol Blue so we went to a pet store and got an aquarium test kit which had a pH indicator for testing for a pH of 6 to 7.6.  We called the manufacturer of the test kit and confirmed that it was indeed Bromothymol Blue.  But this Bromothymol Blue was amber in color.  So we ran some tests to see how it would compare to the Blue colored Bromothymol Blue.

Query – Does the amber and blue Bromothymol Blue test the same and what is the difference if any?

Methodology – Starting with a 50 mL bottle of 24 mg VG  nicotine, test using each bottle of Bromothymol Blue.  We used 2 mL samples of VG nicotine 4 drops of Bromothymol Blue and topped up to 5 mL with distilled water.  The test was run twice using each kind of Bromothymol Blue.  The results are below.

4 Drops of Amber Bromothymol Blue	Sample 1	Sample 2
Beginning Volume (mL)	5	5
Ending Volume (mL)	7.5	7.5
H2SO4 Used (mL)	2.5	2.5
Mg (x 9.735)	24.33	24.33

4 Drops of Blue Bromothymol Blue	Sample 1	Sample 2
Beginning Volume (mL)	5	5
Ending Volume (mL)	7.5	7.5
H2SO4 Used (mL)	2.5	2.5
Mg (x 9.735)	24.33	24.33

Conclusion – The results were identical.  So there was no difference in how they perform when used in the test.  The difference between them is that one is protonated and the other is deprotonated.

Testing with a pH Meter

Detail – Our next thought on accuracy was whether testing with a pH meter would yield the same results as testing visually looking for the color change in the Bromothymol Blue.  Since the titration when completed should be a pH of 6, we decided to test by measuring pH and titrating until we hit a pH of 6.  We had purchased an Orion 3 Star portable pH meter with a triode with automatic temperature compensation built in.  The pH meter has a resolution to 3 decimal places (7.000). We calibrated the meter with 4, 7, and 10 pH buffers and then checked the calibration by measuring the 7 pH buffer again which gave a reading of 7.000.  I periodically checked to make sure the calibration was still accurate twice while making this next test each time with the reading on the 7 pH buffer remaining at 7.000.

Hypothesis – The test results for the mg concentration of the sample would be the same as when tested visually.

Methodology – Start with a 24 mg VG nicotine solution.  Test the concentration with a 2 mL sample of VG nicotine and test visually as we have been doing.  Then measure the pH of the solution to see if we were accurate in our visual interpretation of the end point of the titration.  I did this 4 times.  Here are the results of those 4 tests.

Visual Test	Sample 1	Sample 2	Sample 3	Sample 4
Beginning Volume (mL)	4	4	4	4
Ending Volume (mL)	6.5	6.5	6.5	6.5
H2SO4 Used (mL)	2.5	2.5	2.5	2.5
Mg (x 9.735)	24.34	24.34	24.34	24.34
pH of Final Volume	5.585	5.903	4.995	5.325

At this point we were a bit confused by the pH readings until we tested the pH during a titration and watched it change dramatically as the test reached the end point of the titration.  A single drop changed the pH dramatically.  We also realized our dropper bottle was producing way too large of a drop to be accurate.  So we changed our thinking a bit and went to test using the pH meter.  This time we used 3 mL samples for accuracy and we also grabbed a syringe with a 20 gauge luer lock needle tip on it.  This produced a much smaller drop.  Here are the results of the test.

pH Meter Test	Sample 1	Sample 2
Beginning Volume (mL)	10	10
Ending Volume (mL)	13.7	13.6
H2SO4 Used (mL)	3.7	3.6
Mg (x 6.49)	24.01	23.36
pH of Final Volume	6.013	5.923

            Our results in mg content were not drastically different; however we are much more confident that we did not titrate past the end point.  It was much easier to read the pH meter than it was to guess on a subtle change in color from a very light green-blue to all yellow. 

After This Series of Tests our Initial Conclusions are –

• Make sure your measuring equipment is accurate. 
• The amount of distilled water is not a factor but can make mixing the solution easier if you use 3 to 4 mL with a 1 ml sample of VG nicotine and more so if you are using 2 or 3 mL. 
• No matter how much distilled water you use, top to an even increment on the graduated cylinder preferably on an even number.
• The larger the sample of nicotine tested, the more accurate the test.  It just reduces the human error in reaching the end of the titration but uses 2 or 3 times the Sulfuric Acid over a 1 mL sample size.
• The number of drops of Bromothymol Blue does not change the results of the test but 4 drops is easier to see than 2.  If you are testing higher concentrations over 100 mg, use 8 drops.
• Both amber and blue Bromothymol Blue can be used for the test.
• A pH meter will increase accuracy of the test because it removes having to visually evaluate when you have reached the end of the titration.