Automatic Titration Journaling

[This will turn into a more comprehensive guide to titration.]

I recently acquired a high end automatic titrator from Hanna Instruments. The goal is to perform a wide variety of acid titrations related to distilled spirits production. Esters are an extension of these acid determinations. I had previously been exploring old fashioned manual titration with a tall buret but was not mastering it quickly. Manual titration is tricky and requires both skill and pretty much a knowledge of the end result before you even start so it can be completed in a reasonable amount of time. Automatic titration removes the skill component, but you still need a good idea of what your results are going to be so you can develop a suitable method.

What I’ve found is that I need a lot of methods. I am investigating:

various substrates, molasses, fruit juices (molasses would be by grams)
mashes, especially “thick mashes” treated with lime (25.0 ml sample size)
slaked lime – calcium hydroxide (titrated with HCl) (0.050 g sample size)
fermentations (25.0 ml sample size)
dunder (25.0 ml sample size)
vinegar (2.0 ml sample size)
original spirits total TA, both new make and aged (where they accumulate fixed acidity from the barrel)
original spirits fixed acidity (25 ml sample)
orginal spirits volatile acidity (arrived at by subtraction)
birectifier fractions TA (I don’t think these need to be known individually, but rather organoleptic experience tied to total volatile acidity)
ester determinations, both of original spirits and birectifier fractions

Titratable acidity (TA) is far more easy to interpret than pH data for many tasks. Although they do go hand in hand, one can not substitute for the other. Wineries regularly measure TA as well as perform other titrations such as free sulfur or yeast available nitrogen. Wineries typically do not use chromatography because the data is harder to interpret and expensive to generate. Fine wine was born in the lab and what many of the early Napa pioneers had in common was that they were all lab guys and knew titration inside and out. New American distilleries are in a similar position where lab skills will enhance quality and would benefit from returning to titration. Automatic titrators are quite expensive so we need to explain the value proposition and reduce all the learning curves. What we may end up doing is laying a foundation so budget conscious distillers can start manually and eventually upgrade.

One advantage that titration holds for the distillery (paired with the birectifier) over chromatography, besides being cheaper with less learning curve, is that the methods can be used across various distillery processes. One type of chromatogarphy may be required for a distillate while another for a ferment, multiplying the expense. Some forms of spectroscopy even rely on models built using titration. The complexity can spiral out of control fast. Much historic data was also performed by titration so there is opportunity to help contextualize the spirits we are producing today.

For the distillery, pretty much everything can be calculated as acetic acid, but measures will need different scaling such as g/L or mg/L and mg/100ml absolute alcohol. Spread sheets can make quick work of this and automatic titrator methods can also do a lot of the math for you.

In the olden days, titrations were conducted to a fixed endpoint using an indicator that changed color at pH 8.2. A number higher than pH 7.0 is chosen because the salts formed at the equivalence point retain a slight charge (strong base/weak acid). Automatic titration holds the promise of using an algorithm to calculate the real equivalence point which is close to 8.2 but differs slightly. In theory, this would improve accuracy. In practice, I’ve had problems with this concept and the titrator has gotten confused and destroyed my sample (however, you can look through the report log and salvage an answer). I can easily fall back on a fixed 8.2 end point while I slowly learn more about the algorithm (all the wine protocols I see for automatic titration use a fixed 8.2 end point). For ester determinations, where you neutralize the sample before saponifying, an 8.2 end point needs to be chosen. There are more idiosyncrasies to learn than I thought, but they are all doable.

To get the best of the technology and maximize accuracy, every task needs a method that uses the ideal sample size and corresponding titrant concentration. You also need to learn about maintaining your pH probe at a higher level of accuracy than most distillers are used to, but those skills will serve anyone well and Hanna has wonderful articulate guides that demystify probes and best practices.

Ester determinations are an extension of acid titration. To perform them, a sample has its free acids first neutralized then the sample is saponified which entails adding a measured excess of sodium hydroxide (NaOH) which will split the acids from the alcohols in the esters. The acids get bound as salts. To measure these acids bound as salts, there are two options:

Go forwards with NaOH → then go somewhat backwards with acid to the equivalence point ← (8.2) and calculate the difference.

Or you can go forwards with NaOH → then go backwards with acid the same distance ←. All acids are now free again and you can proceed → as in a normal acid titration (your results however will need to be calculated with the molecular weight of ethyl acetate).

The first method is called back titrating and was probably the most common. Sometimes this was performed alongside a blank. The blank was important in case your acid concentration was not precisely standardized (not we can buy standardized solutions affordably and there are new ideas in standardizing). The blank may also point to odd reactions between other compounds you did not anticipate, but this was more important before we had so much basic science to draw from (Some wines protocols for volatile acidity add a small amount of hydrogen peroxide and I still need to figure out why).

To undertake all this work, we have just a few limitations to consider. We can probably only work with two NaOH concentrations, 0.1N and 0.05N. Kervegant seems to imply that everyone worked with 0.1N NaOH and sometimes it was written in different ways such as 1/10N or called deci-normal. Arroyo may actually have worked with something less concentrated for birectifier fractions such as 0.01N. The Hanna titrator has a 25 ml buret while traditional manual burets are often 50 or 100 ml. The Hanna buret can both refill itself and apply extra small doses, but at the minimum, a best practice is to use a sample size large enough to use at least 10% of the buret. This rule of thumb helps frame the math. Either increase your sample size or decrease the concentration of your NaOH.

Some limitations pop up. I thought I could measure the TA of the birectifier stillage with the same concentration NaOH as birectifier fractions 6,7,8, but I cannot because fixed acidity of aged samples can be very significant. Fixed acidity (which applies to aged spirits) likely needs its own method and should be calculated from original spirit and not from birectifier stillage.

Right now, I’m leaning on dropping measurement of birectifier stillage and instead switching to more measures from an original spirit such as total acids, fixed acids, and volatile acids. This may be a better point of measure because typically there is a larger sample size available. I’ve developed a method to measure the TA of birectifier fractions, 6,7,8, but it may just be more useful to know the volatile acidity of the original spirit.

The Hanna titrator can use multiple pumps, but I only own one because an additional unit costs $800. This limits the method for ester determinations. Back titrating becomes a non option because it would require a pump dedicated to acid. In theory this is no problem, but I’m still working on becoming confident in my results. I think 15 ml is the best excess of 0.1N NaOH for a 50 ml original spirit sample, but I need to do a little more leg work from the historic data. Liquid handling decisions also need to be made. Should I use a volumetric transfer pipet or a 0.001 gram scale and assume the density of both 0.1N NaOH and 0.1N HCl is 1.000? To use the scale, I would use my adjustable 5.0 ml pipet (5000μl) and use its screw mechanism to go drop by drop until I hit the mark. Practice will reveal the best method.

For protocols to draw from, there is Kervegant, the A.O.A.C. as well as some great literature from India. For the birectifier fractions, we can draw from Arroyo’s own data tables. We can take his results and calculate backwards what concentrations of NaOH he must have used from a likely sample size. What I’m starting to realize is that he may have done his birectifier distillations in triplicate to accumulate enough sample size and that his work is not so much to be duplicated but drawn from. His foundation shows that we don’t need to first neutralize birectifier fractions 1,2,3 for an ester determination because the free acids content should be near the margin of error. It also shows that we may not derive value from the TA of fractions 6,7,8, instead we should calculate the total acidity, free acidity, and volatile acidity of the original spirit sample. He also shows us a ratio of low value (ethyl acetate) to high value (long chain) esters that may be very useful when comparing the Jamaican vinegar process and the Arroyo process. Are Jamaican total ester numbers useful if they are mainly junk esters?

For something like vinegar that is quite acidic, we can take a starting sample like 10.0 ml (I settled on 2.0 ml), see how much of the buret we use and grow or shrink the sample size until we use a reasonable portion of our buret to be confident in the accuracy.

To jump from purchased 0.1N NaOH to either 0.05N or 0.01N, we can simply use class A volumetric flasks and distilled water. It is easy to make quick work of diluting standard solutions. For extra accuracy, Hanna provides protocols to standardize your NaOH. If it is really 0.101N and not 0.100N, you can specify the correct number for your calculation.

A few odd tasks stand out. Powdered lime needs to be titrated with an acid which for practical purposes should be performed manually in a tall buret. Slaked lime is limestone baked to drive off CO2, but gradually the CO2 is reabsorbed. Lime will need to be tested to understand to what degree it is still active. Understanding the equivalence of your lime may reduce a lot of headaches when trying to apply it to a large scale process. This can be used in conjunction with an acid profile of a substrate so it is known what amount of TA corresponds to a increment of pH. We ferment via pH and pretty much adjust via TA. Their correlation is not exactly the easiest thing to intuit. Arroyo style ferments need to be continuously adjusted to maximize enzyme activity. Titration will help this be performed quickly, accurately, and efficiently.

These are some preliminary thoughts and eventually this will turn into a comprehensive guide.


An appropriate sample for slaked lime is 0.050 grams to 0.250 grams and titrated with 0.1N HCl. Slaked lime is calcium hydroxide which has a molecular weight of 74.093 g/mole. The formula is Ca(OH)2 which means there are two hydroxide ions and that the equivalent weight is half the molecular weight. If 1 Molar calcium hydroxide is 74.093 g/L, 1 Normal would be 37.047.

A 0.250 gram sample would theoretically require 67.48 ml of our buret with 0.1N HCL. A 0.050 sample would require 13.496 ml.

In practice, I used even more titrant, but I don’t really understand why. Purity could change the result, but I would think skew it in the other direction. I added 50 ml of distilled water to my powder then two drops of 8.2 indicator. In the last third of the titration, the color would change to clear, but then become pink again after many minutes of stirring. This could be the point where CO2 is being slowly driven off the last amount of calcium hydroxide. It took me roughly a half hour to complete the titration. For batician preparation, it may be necessary to account for the entire potential equivalence, while for adjusting fermentation pH, it may be okay to account for the free calcium hydroxide and assume any calcium carbonate is bonus time release that will benefit the active ferment (that’s a theory).

As far as my own calculation notes so far, birectifier fractions need to be calculated as mg/mL for their sample size (typically 15). This result gets multiplied by 25 (mL of a birectifier fraction) and this represents the amount from a 100 mL absolute alcohol sample.

For original spirit samples, you start with a sample such as 25, 50, or 100 ml but when the method asks for the analyte size size, you provide the absolute alcohol scaled from the sample so that the result will be in terms of absolute alcohol. For example, inputting a 100 mL sample of 40% ABV spirits, you will tell the titrator you are analyzing 40 mL. These results will be calculated in the traditional mg/100 mL absolute alcohol.

You name your data in GLP data before you start your titration. There appears to be no way to go back and modify this after the fact without exporting the data to your PC. If the text ends with a numerical digit, it will be advanced by one for every titration. (Example, “fraction 1, fraction 2, fraction 3”)

What I’d like to see is a titration emulator on the PC to help you with setup and give more background on the options. This would be like CAD/CAM software that machinists use for simulations, but be vastly simpler. I’m simply trying to learn this and gain confidence, but using up samples that took hours to generate. I also have historic data I want to run through calculators I wish already existed and I want to build intuition for methods.


Some calculation advice you see in the educational literature for manual titration is to know the grams / mole of the common acids, the moles per equivalent, and then finally the grams of acid per particular molarity/normality of your NaOH concentration.

We are primarily dealing with acetic acid which is 60.05196 g/mole. It is 1 mole per equivalent.

Tartaric acid differs, and though it has a molecular weight of 150 g/mole, it is 1 mole per 2 equivalents. This means the gram per equivalent is only 75.

Ethyl acetate for ester determinations is 88.11 g/mole and its mole per equivalent is 1.

Manual titration operators know their gram results in terms of the NaOH they are using. For every mL of titrant there is a corresponding amount of acid:

1mL of 1.0N NaOH = 0.06 grams acetic acid.
1mL of 0.1N NaOH = 0.006 grams acetic acid.
1mL of 0.05N NaOH = 0.003 grams acetic acid.
1mL of 0.01N NaOH = 0.0006 grams acetic acid.

1mL of 1.0N NaOH = 0.088 grams ethyl acetate.
1mL of 0.1N NaOH = 0.0088 grams ethyl acetate.
1mL of 0.05N NaOH = 0.0044 grams ethyl acetate.
1mL of 0.01N NaOH = 0.00088 grams ethyl acetate.

My understanding is that 1.0N is never used so much as purchased standardized to dilute yourself with class A volumetric flasks.

One thing this means is that when you are working, the most important measure is your end volume of NaOH and its concentration. If your automatic titrator had the incorrect formula, you can also go back and fix the math yourself.

By the above math, 15 ml of 0.1N NaOH can salt 0.132 grams of ethyl acetate per sample. So how large should the sample be? And to what degree does the NaOH benefit the reaction from being in excess? [A.O.A.C gives the advice: “Reject determinations in which excess 0.1N NaOH is <2ml, or > 10ml.”] This may have to be considered for very high ester rums.

Resources (1). Resources (2).


As we accumulate data, we have some ideas to consider. According to the Technology of Wine Making (4th edition), new wine should have 0.04 g/100 ml of volatile acidity and a sound aged wine less then 0.07. The California legal maximum for volatile acidity are 0.110 g/100 ml. How will heavy rum ferments compare?

Volatile acidity in wines is measured after distillation with a cash still (I do not own one yet). 25 ml is used for a wine and 300 ml for a distillate. Normality of the NaOH is recommended to be between 0.01N and 0.05N

It is a valid technique to measure either fixed acidity or volatile acidity by subtracting one or the other from the total titratable acidity, but they must be in the same units (i.e. as acetic).

A method for fixed acids is evaporation of the same, followed by addition of distilled water, followed by another round of evaporation.

Technology of Wine Making (4th edition) says a 25 ml brandy sample can be titrated with 0.1N NaOH. It also says fixed acids can be determined in 25 or 50 ml of brandy by evaporating to dryness with a steam bath (to prevent decomposition) followed by oven drying at 100°C (I’ll use my food dehydrator, but you can also put a sous-vide stile PID on a toaster oven). The dried sample is dissolved in neutral spirit of the same ABV and volume as the original sample followed by more distilled water.

A.O.A.C. 10th edition (1965)

Total acids— 25 ml sample / 0.1N NaOH
Fixed acids— 25-50 ml sample / 0.1N NaOH one evaporation followed by 100°C oven (same as Technology of Winemaking)
Volatile acids = Total acids – Fixed acids.

Esters— 100 ml sample. / 0.1N NaOH. “Reject determinations in which excess 0.1N NaOH is <2ml, or > 10ml.”


Vinegar turns out to be quite acidic and to use only one buret with 0.1N NaOH, I’m only using a 2.0 ml sample size. A 6% vinegar can use as much as 21.00 ml of the buret.

For thick mashes of roughly 55 brix, I’m using 25.0 ml sample size and using roughly 4.0 ml 0.1N titrant. This protocol only titrates to pH 6.2 and spits out a g/L number of slaked lime that can be added to adjust the thick mash to that point. I had a lot of trouble with this and kept having to adjust the molecular weight to get closer. All the theoretical numbers I had did not work. I eventually manually got the thick mash to 6.2pH keeping track of the lime with my analytic balance because the numbers are quite small then I kept changing the molecular weight of the method until it conformed to my number. I still have to test it across multiple molasses types. For our thick mash, we adjust up to pH 6.2, heat to 80°, centrifuge, then adjust it back down typically with sulfuric acid.


For washes of roughly pH 4.3, I’m using 25.0 ml sample size and using roughly 19.3 ml 0.1N titrant (4.6478 g/L as acetic).


For fixed acidity, I dehydrated 50 ml of original spirit (measured via volumetric flask with rinsing) to dryness in a canning jar with an excalibur food dehydrator. When it was dry, I added distilled water and dehydrated it again to blow off any residual volatile acids. Keep in mind, a safe way to work with heating devices and not forget them is to use a count down timer on the outlet. I reconstituted the volume to roughly 100 ml to cover the pH probe and stirred until the pH reading dropped to its lowest point. It can start as high as 7.0+ and drop to the 4.0’s as the acids dissolve. I regretted titration with 0.1N NaOH and wish I used 0.05N or 0.01N. If the sample volume is no big deal, it may even be helpful to increase the size to 100 ml and rapidly dry with a double boiler. The dehydrator is only nice because its easy to load with samples and walk away from, however it is best run overnight. Even though the pH can start in the 4.0’s because distillates have very little buffering, it can climb rapidly with an incredibly small amount of titrant. The last sample I ran, only used 0.6 ml of 0.1N titrant. We want the results in mg/100ml of absolute alcohol so even though you use 50 ml of original spirit the calculation will be scaled by the ABV. In my automatic method, I have it set up for a manual volume extra. 50 ml is multiplied by the ABV and that number is entered, thus the final calculation is perfectly scaled.

Volatile acidity for a wash would be measured with a cash still, described above, while volatile acidity for a distillate would be measured by subtracting fixed acidity from total acidity. Volatile acidity for a new make is the same as total acidity.

When you measure the pH or TA for a sample of original spirit high in alcohol, you may need to significantly dilute the spirit with distilled water beyond 100 ml. This will protect the electrolyte in the pH probe from becoming dehydrated. UC Davis seems to be liberal with the distilled water and would dilute many of their samples to as much as 200 ml.


We are finally at esters! I had some minor hiccups while practicing this and then the equipment was sometimes tied up by other tasks, but I just got a determination that I feel is accurate. This rum, original spirit, weighed in at 251.54 mg/100 ml absolute alcohol. This was calculated from a 50 ml sample where the sample volume was inputted to the titrator as only the absolute alcohol, so the sample was inputted at 18.5 ml (50 * 0.37 [Australian 37% ABV]).

Remember, our best protocol advice was: Esters— 100 ml sample. / 0.1N NaOH. “Reject determinations in which excess 0.1N NaOH is <2ml, or > 10ml.”

I did not have 100 ml to sample, but will aspire to that when possible. This determination used 5.282 ml of the 25.0 ml buret (0.1N NaOH), which is higher than the 10% rule of thumb to be confident in the results. If this was 0.01N NaOH, we would have used 52.82 ml!

To start the determination, I used a 25 ml volumetric flask which I rinsed with distilled water. This was already a minor mistake and I should have either used two of these flask or a 50 ml version. On the second use, it was not fully dry and tainted the volume to a slight degree. This 50 ml volume was titrated for total acidity to an 8.2 end point. This now neutral sample was transferred to the 500 ml boiling flask with a rinse. 15.0 ml of 0.1N NaOH was measured roughly with a 5.0 ml automatic pipet (which is really pretty good), but fine tuned with the analytic balance to 15.000 grams. This assumes a density of 1.000, but it seems to work better than other liquid handling methods. The 15.0 ml NaOH was transferred to the boiling flask with a rinse of distilled water.

The excess NaOH saponifies the esters with the help of heat. This volume is heated for roughly three hours. The first hour is the heat up time while the second two are the recommendation for saponification. My apparatus is 500 ml double kneck boiling flask (second neck holds a thermometer) and a 250 mm (roughly 24″) air condensor. This is nothing more than a tall glass tube with 24/40 tapered fittings on both sides. They are slightly obscure and I bought two because they are cheap. The mantle has a simple heating controller. For safety, I add a count down timer on the outlet I set to three hours. Protocols I’ve seen mention that you can run this and shut it off and it is perfectly fine to let it sit overnight. To develop an initial heating routine, I like to use a kill-a-watt meter set on watts to determine how much energy is entering the heating mantle. This helps if the dial is analog and hard to read or if the controller is shared with another process. The idea is that only enough energy should be added to keep the liquid just below its boiling point.

Before you transfer the saponified volume to a flask for titration, measure out 15.0 ml of 0.1N HCl into the flask using the same method as the 0.1N NaOH. First, notice we are using Hydrochloric acid instead of Sulfuric which used to be more traditional. Hanna sold standardized HCl so it made sense to use it instead. Second, we are not back titrating, but using the alternative we go forward to saponify, backwards in the same direction, then forwards again a normal acid titration but calculating result, not as acetic acid, but instead as ethyl acetate (swapping the molecular weights). The advantage here is we keep using the automatic titrator with only one pump.

Before the titration starts, the sample must be stirred until the pH drops to its lowest point where the HCl reacts with all the NaOH leaving only the free acids that were formally esters. In the last one I performed, the pH dropped as low as 3.7, but because this is fairly unbuffered, it took only a very small volume of titrant to bring that back up.

Boom esters!

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