Assessing the Utility of Fast Fourier Molecular Rotational Resonance Spectrometry (FFMRS) and Triple Quad Mass Spectroscopy (QQQ-MS) in Barrel Aged Spirits Dilution to Avoid Negative Effects of Saponification in Two Distillery Settings

Name *

Robin A Felder

Current Occupation/Title *

Professor of Pathology, Associate Director Laboratory Medicine, The University of Virginia, Department of Pathology.

Chair, Medical Automation.org

Grant Application Title: Assessing the Utility of Fast Fourier Molecular Rotational Resonance Spectrometry (FFMRS) and Triple Quad Mass Spectroscopy (QQQ-MS) in Barrel Aged Spirits Dilution to Avoid Negative Effects of Saponification in Two Distillery Settings

Principal Investigator – Robin A. Felder PhD (Biochemistry)

Master Distiller Monte Piccolo Farm and Distillery

Co-principal investigator – Brooks Pate PhD, Professor of Chemistry (Expert/Inventor of MRRS)

Abstract

Research area: This grant application is related to the outcome and a continuation of the first and second grants awarded by Advisory Committee of Distilling Research.org in 2019 and 2020 to myself to further the advancement of innovation in the craft distilling industry. The need that prompted both the grant submission and its funding was the use of a novel and revolutionary spectrometer to better identify and quantify flavor congeners during the distillation of spirits. Henric Moulin has generously offered to perform the mass spectrometry analysis. Results from these two spectrometers will be compared.

Research question to be tested: This proposal is to determine the effect of different diluting waters (well water,  milliQ deionized 0.22 µm filtered water (250,000 µΩ)) at different dilution rates (time based linear gradient) in pear and apple aged in wood, on saponification and congener profiles.  Congener analysis will be performed by Chirped Pulse Fourier Transform Microwave (CP-FTMW) spectroscopy, also known as Fast Fourier Molecular Rotational Spectroscopy (FMRS).  It is known that FMRS can measure congeners which cannot be measured by Q-Q-Q-MS, which will be performed by Henric Molin.

Rationale for testing the question: We proved in three semesters of use of FMRS that we could not only measure known pear and peach congeners of interest, but also discovered previously unknown congeners significantly affecting peach flavor profiles in Monte Piccolo spirits. Monte piccolo has yet to develop a barreled spirits dilution plan and anticipate that the data from these studies will provide strong scientific evidence for a logical plan for the spectroscopy inspired analysis of the temporal dilution of barrel aged product.

Data/observations expected and their significance to the field of the craft distilling industry: Solid data is anticipated that will guide further studies of spirit dilution rates.  We anticipate that future studies will be funded by commercial sources to more widely disseminate this novel FMRS spectroscopic technique into the craft distiller industry.

 

Background:

It’s been well recognized that adding different kinds of water (e.g. carbonated vs still water) to whiskies prior to drinking can dramatically affect the perception of sweetness, brininess, and grain after taste.  Workshops were held on this topic with tasting opportunities by Academics as far back as 2013 (1).

However, there is growing appreciation to the positive and possibly negative effects of altering not only amount but also the rate at which one adds water to barrel aged whiskey prior to bottling. Nancy Fraley, a well-known blending and distillation consultant for dozens of distilleries was quoted as stating, “After a bourbon has already spent 5, 6, 7, 8 or more years maturing in the barrel, the last thing we want is for all those wonderful flavors and aromas to be destroyed by adding lots of water in a few brief moments.” (2) (3)

When water is added to whisky, brandy and other distilled products rapidly, especially the flavorful esters can convert back to ethanol and acids can convert fatty acids contained in the liquor into soap via hydrolysis (4)(5).  Reduction of the formation of these compounds by gradually diluting the liquor can significantly contribute to the smoothness of a liquor (6).

Determining the molecular structure and concentrations of flavorful free esters and alcohol in liquors can assist in determining if saponification is reducing these beneficial congeners.  Total concentration of soap products, which can reflect light, can be measured using a refractometer but can also be measured by mass spectrometry (7) and flow cytometry.  We anticipate using light scattering to determine the total saponification index of various brandies since these instruments are present in our laboratories at UVA.  Since novel spectroscopy has not been used for these compounds we developed a student exercise in the UVA advanced spectroscopy course (Pate Brooks PhD, Professor of Chemistry) (8-10).  It will be determined which methods are more accurate, facile, and affordable for craft distillers to use in their production process, usually via contract laboratories. However, in the future we anticipate these techniques will be routine in large commercial distilleries.

1st and 2nd ADI Grant Award Progress report: The 1st ADI grant funded the collaboration between Monte Piccolo Distillery and the Department of Chemistry at The University of Virginia (Charlottesville, VA) was focused on testing the ability of a novel spectrometer to measure the molecular content of the heads and hearts of pear eau-de-vie style fresh pear brandy.   Normally, with a pot distillation apparatus, the heads and hearts “cut” is made at sensory discretion of the distiller.  Similarly, with continuous distillation, the parameters that control the determine the distillate quality are set at the distiller’s discretion.  Thus, the distillate can vary greatly based on the olfactory acuity of the distiller, congener content of the fruit due to seasonal variation, fermentation, and the distillation process itself.  Until objective measures of congener concentrations can be made, the industry cannot begin to be able to know which steps in the entire production of spirits (i.e. from raw material to sip) are most critical to control.

Because of the high spectral purity of microwave light sources and high dynamic range, Chirped Pulse Fourier Transform Microwave (CP-FTMW) spectroscopy, also known as Fast Fourier Molecular Rotational Spectroscopy (FMRS), provided us the unparalleled ability to unambiguously determine molecular structure in distillates. Since the rotational transition frequencies of a given species are determined purely by the moments of inertia of the molecule, mass shifts due to isotopic substitution generally cause relatively large shifts in transition frequency with respect to the spectral resolution. The acquisition and assignment of isotopically substituted spectra of a target species allow the determination of accurate experimental molecular structures with precision at the level of a few pico-meters, with application of Kraitchman’s equations or least linear squares fitting.

Work to be Completed:

Methods:

  1. Control/baseline experiments. Obtain 1000ml specimens in triplicate from undiluted twice distilled pear and apple aged 6 years in once used bourbon barrels. Measure congeners by FMRS, and send samples to Enric Molin to perform Q-Q-Q MS. Quantify control/baseline saponification products by right angle light scatter. 2 brandies X triplicate samples = 6 baseline controls = 6 reportable baseline control congener lists in addition to 9 saponification data points. We anticipate these control saponification data points will be negative since water dilution will not be performed. Total specimens will be analyzed in triplicate determined by performing statistical Power Analysis which indicated statistical significance will be achieved.

Total control specimens = 6

  1. Add well water, and milliQ deionized 0.22 µm filtered water (250,000 µΩ), gradually at a rate to bring to 80 proof over a period of 168 hours (14 days) using a calibrated peristaltic pump. Each collection bottle will be equipped with a magnetic stir bar and placed on a magnetic stirrer to provide near instantaneous mixing. I will sample the diluted products (100 mL) at the same time daily and measure saponification by right angle light scatter as well as congeners using FMRS. I will save all samples minus what was used for analysis in dark cool cellar until sensory analysis can be performed at a later date. 2 brandies (apple aged in wood, pear eau-de-vie) X 14 samplings x 2 water sources = 56 saponification data points + 56 congener lists from FMRS analysis.

Total analytical specimens = 112 (of which 56 are saponification and 56 FMRS analysis).

 

Total control (6) + 56 Experimental specimens = 62 which will be analyzed in triplicate and averaged = 186 FMRS and saponification products analyses over a period of one year.

 

Caveats: The quality of the water will have to be highly controlled in order to not influence the outcome.  All samples should be stored in borosilicate analytical grade laboratory glass. We will use the most extensive potable water analysis employed by the state of Virginia ($300 per analysis is budgeted). The results from these experiments will determine if dilution rates will increase saponification and alter congener concentrations. We will use continuous water addition and period sampling with constant stirring so that we are creating a continuous linear dilution gradient to avoid localized instead of generalized events.  However, given the wide variety of waters available in each local across America and the variation in dilution rates employed by American distilleries, we will state unequivocally that each distillery should adopt personalized water choices and dilution plans.  These plans will be able to be developed with insights gained through this proposed research.

Materials, Methods, and timeline:

Month 1 – 3 – Robin Felder PhD (Owner) at Monte Piccolo will collect all specimens on site to assure that there isn’t any interpersonal variation in sampling, quality control, labeling, and tracking (using pre bar coded specimen vials).  Sufficient specimens will be collected to assure our studies are sufficient powered to achieve statistical significance.

Month 4- 6 – Analyze the specimens in triplicate and reported as mean ± standard error, perform data reduction, statistical analysis, and validation.  Flavor analysis will be a topic of another proposal.

Month 6-12 – We will write our scientific articles and submit for consideration for publication in Scientific Spirits.  We will also write, as required, an article for the ADI.

Analysis of specimens: The FMRS specimens will be analyzed at BrightSpec LLC, which has two FMRSinstruments performing contract analysis.  These research full spectrum spectrometers are designed to identify any and all relatively small compounds in volatile liquids (such as the effluent from a still).  For example, fruit esters in brandies are generally under 11 carbons and thus are much easier and faster to quantify (real time) than compounds in pharmaceuticals. Q-Q-Q-MS (Mass spectroscopy) is the current gold standard analytical method, which we anticipate will be superseded by FMRS in some industries due to its relative rapid analysis and simplicity of direct vapor headspace sampling without the need of gas or liquid chromatography or any other specimen pre-analytical cleanup.

Budget justification (Budget at the end of this proposal):

Supplies:

  1. Bar coded specimen vials will be provided by the funds from this grant.
  2. Specimen analytical fees will be paid to Bright Spec who will not charge overhead. Saponification analysis by alternative methods will be done on refractometers and a flow cytometer since Monte Piccolo has its own laboratory and these instruments are not expensive to operate due to lack of need for dedicated columns and buffers.

Interpretation of results: We will continue as per our current protocol and use standard purified materials (e.g. acetaldehyde, methanol, butanol, propanol, 2,4-ethyl decadieneoate, etc.) obtained commercially from Sigma Aldrich and Perfumers Supply House. We will use our previously demonstrated capabilities to focus on measuring saponification profiles of both distiller’s products after dilution in a linear continuous gradient.    We anticipate measuring the most abundant and flavor influencing saponification products per specimen yielding thousands of data points that will need some extensive computer time for application of Kraitchman’s equations and least linear squares fitting for accurate determination of molecular structures with precision at the level of a few picometers.

Specimens will be made available to professional tasters as those relationships are established through offering co-authorships on published papers.

If successful with the findings how should research process further or what will this contribute to the distilling field?

We have demonstrated in the 2016 – 2018 academic years that FMRS can be instrumental in improving the quality control of craft level distilled spirits.  Based on this preliminary data and resulting publication (10) (follow on papers in preparation), we will be applying for grants from the National Science Foundation.  Dr. Felder will ultimately retire full time into his craft distillery and use his scientific knowledge to further art and science of craft distilling thanks to the generosity of the ADI and its nonprofit granting program. With introductions from the ADI Dr. Felder would like to introduce the FMRS technology to several influential industrial scale distilleries (e.g. Makers Mark and Gallow) in order to accelerate wide spread adoption.

Budget:

Specimen collection disposables: gloves (prevent human contamination), screw cap tubes, pipettes, marking pens, sequentially labelled bar codes, specimen racks (for repository use for reanalysis if necessary) =                                                                                                     $600

Congener analyses  (BrightSpec, LLC specimen rate, no indirect costs included). ~$22/specimen x 186 specimens  =                                                                                                $4,100

Water analysis –  2 specimens at $300 each =                                                                    $600

Mailing of specimens –we will bundle the specimens for bulk shipment, 4 shipments @ $25/shipment =                                                                                                                       $100

Labor– no charge                                                                                                       Total  $5,404

References:

  1. https://www.alcademics.com/2013/07/a-whisky-and-water-tasting.html
  2. Emen J., Distiller Blog February 6, 2020. https://blog.distiller.com/proofing-whiskey/#:~:text=When%20a%20spirit’s%20alcohol%2Dby,it%20besides%20starting%20from%20scratch.
  3. Bell D. “Design your whiskey to be great, don’t just hope for greatness.” Excerpted from his second book, Fire Water. From Artisan Spirit Magazine. Summer 2015 https://issuu.com/artisanspiritmag/docs/artisanspirit_issue011_web/41
  4. https://en.wikipedia.org/wiki/Saponification#:~:text=Saponification%20is%20a%20process%20that,least%2010)%20e.g.%20sodium%20palmitate
  5. Charalambous, G., phenolic, Sulfur, and Nitrogen Compounds in Food Flavors. SSN:

1947-5918, Date: 06/1976.

  1. Kew, W., Goodall, I., Clarke, D. & Uhrín, D. Chemical diversity and complexity of scotch whisky as revealed by high-resolution mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 200–213 (2017).
  2. https://www.theverge.com/2017/8/17/16088926/whiskey-alcohol-dilution-water-drinking-chemistry
  1. West, Channing, Miller, Thomas, Khan-Rafii, Noah, Plunkett, Emily, Chmielinski, Alexander, Hurst, John, Delaney, Thomas, Preston, Charles, Pate, Brooks, Neill, Justin L., Felder, Robin A.

Rotational Spectroscopy of Flavor Compounds in Peach Brandy for Process Monitoring in Craft Distilleries. 74th International Symposium on Molecular Spectroscopy DOI: 10.15278/isms. 2019.TL07

  1. http://faculty.virginia.edu/bpate-lab/cpftmw.html
  2. Felder RA, Distiller Magazine Winter, Vol 15, Issue 3, 2020, pp 86-88