1H Eastern Analytical Symposium

qNMR of Alcoholic Beverages – Detailed Chemical Fingerprint Information for Quality Control and Process Understanding November 13-15, 2017
Crowne Plaza Princeton Conference Center
John C. Edwards, Process NMR Associates, Danbury, CT 06810 USA Princeton, New Jersey

Abstract: 1H qNMR can be utilized to yield quantitative component distributions of various alcoholic beverages that can then be utilized for product consistency, product authenticity, and mandated regulatory labelling. Malt, Dextrin, and Carbohydrate Chemistry: The Figure to the right shows the
The qNMR internal standard approach utilized in our laboratory will be detailed and several examples of QA/QC or manufacturing process understanding will be presented. NMR analysis provides details on ethanol expansion of the anomeric hydrogen region of the NMR spectrum covering a
production in fermentation processes as well as information on methanol and fusel alcohols. Various polyols can also be identified and quantified such as glycerol and 1,3-propandiol. Organic acid distributions are readily range malt contents provided by 23 commercial beers. This area of the
a(1-4) Glucans
quantified providing lactic, acetic, succinic, malic, tartaric and citric concentrations that are of particular interest to sour beer and cider-makers from both a quality and sensory perspective and to other beverage spectrum provides the quantitative fingerprint of the carbohydrate and malt- Miller High Life
manufacturers in the context of troubleshooting off-flavors. NMR also provides information on the yeast and bacterial uptake of amino acids throughout the fermentation process. It also allows for quantitation of sugars, oligosaccharide chemistry of the beer. The 4.9 to 5.5 ppm region of the Longtrail Mostly Cloudy
complex sugars and malt dextrin chemistries throughout the fermentation and ageing processes of various alcoholic beverage types. These chemistry observations are useful in the context of wild fermentations which are spectrum contains signals from a-glucan malto-oligosaccharides and limit North Coast Old Rasputin
becoming quite prevalent and allow the conversion of sugars by different yeast and bacterial mixtures to be followed as well as providing an understanding of the residual carbohydrate chemistry in final products such as dextrin containing a(1-4) and a(1-6) glycosidic linkages. The 4.4 to 4.75 ppm
Newburgh Berliner Weiss
beer, mead, wine and fruit containing beverages. NMR also allows for accurate ethanol analysis of sugar and lipid-containing liquer drinks that can be difficult to analyze by traditional approaches. region contains signals from b-glucan carbohydrate components. In finished
beers the concentration of these components gives an indication of the Newburgh Winter Spruce Porter
Sample Preparation: The only sample preparation step to be taken is a de-gassing procedure which we perform by repeated incomplete conversion of starch to yeast fermentable carbohydrates. Amstel Lite
Organic Acid Analysis – Useful particularly in Beer spoilage and Sour Beer Brewing, Ageing & Blending: Differences in the distribution of starch-derived oligosaccharides reflect the
agitation in a vortex mixer. Coors Banquet
Original beer sample analysis – 175ml of degassed was added to a 5mm NMR tube followed by 100ml of internal standard solution
The figure above is a superposition of 23 beer NMR spectra showing the part of the proton spectrum use of different malting conditions, mashing protocols, yeast strains, and
prepared such that the concentration was 10mg per 100ml. Finally, 375ml of D2O (99.8%D) was added to the sample and the tube where signals from alcohols, acids, and amino acids are observed. The assignments of the various enzymatic processes that effect the degree of starch breakdown. In the Moosehead
capped and agitated for 5 seconds. chemical components are labelled. Different hydrogen (proton) chemistry is observed on the X-axis analysis of mash and wort the ratio of fermentable sugar to non-fermentable Miller Lite
Freeze dried beer samples – 1000ml of degassed beer was placed in a 2 dram vial and freeze dried in a Virtis Benchtop K. The entire showing CH/CH2/CH3 signals that appear as single, double, triple and quadruple peaks depending on sugar can be calculated. The degree of polymerization of malto-
freeze dried sample was then dissolved in 650ml of D2O and 100ml of maleic acid internal standard solution (10mg/100ml) added the molecular structure of the molecule. Note that the NMR spectrum axis shows units of ppm – this Dog Fish Head Noble Rot
and the sample agitated before transfer to a 5mm NMR tube.
oligosaccharides can be determined and the relative distribution of
is not a weight value but rather a normalized frequency scale which allows spectra from different oligosaccharide components can be obtained. The effect of mashing and North Coast Bro Thelonius Abbey Ale
NMR instruments to be directly compared. The data shown in the plot to the right shows the organic boiling conditions can be monitored and the concentration of various malt Founders Blushing Monk
Standard Material – Internal Standard: Maleic Acid (99.0%) – Aldrich – Lot#SLBC1970V - 10mg/100ml solution in D2O
acid distribution obtained on 23 commercial beer samples. components quantified at different time points in the process allowing
(99.9%D) Left Hand Milk Stout Nitro
We have utilized the Organic Acid profiles to determine if unexpected lactobacillus activity has optimization of time and energy use. As an example of the variability that is
occurred during the mashing, fermentation or ageing of various beer styles. It can be utilized to Bells 2 Hearted Ale
Experimental: 1H NMR experiments were carried out on a Varian Mercury MVX-300 equipped with a 5mm Varian ATB probe seen in different commercial beers the figure above shows the a(1-4) glucan
operating at a resonance frequency of 299.67 MHz, Experiments were performed with a p/3 pulse with an 8 kHz spectral width obtain organic acid profiles for 1/2/3 year old geuze blending. The glycerol conversion metabolism of component comparison for 23 commercial beers with a breakdown into Shed Mountain Ale
collecting 64k over an 8 second acquisition time and with a 7 second relaxation delay. 64 transients were averaged to produce the wild yeast and bacteria strains can also be readily observed by the observation of 1,3-propandiol after linear and branched dextrins of differing degrees of polymerization (Dp) Troegs Java Head
final spectrum for analysis. In the final data processing the maleic acid resonance at 6.4 ppm was normalized to 10 so that a direct high conversion of malt to alcohol. The detailed chemistry is also of interest in the development of
calculation could be made of all measured components on a mg/L basis. Analysis can be performed effectively on 200-800 MHz
ranging from 3 to 9 monomer units. Non-fermentable sugars such as lactose Keegan Ales Joe Mama Milk
wild fermentation understanding as it provides a detailed chemistry window of the action of various can be identified and quantified.
NMR systems. Analysis takes 15 minutes.
yeast and bacteria strains on standard worts. Founders Rubeus
Calculations: The following volatile components were always obtained on a 175 ml beer sample as the freeze drying process Left Hand Milk Stout Nitro
Alcohol and Diol Analysis: The NMR analysis is routinely utilized to obtain the ethanol content of
would compromise the amount present: ethanol, acetic acid, fusel alcohols (isobutanol, isopentanol (isoamyl alcohol), and 1- Red Hook Seedy Blonde Apple Ale
propanol) ethyl acetate, methanol. The calculation utilized to quantify these components was: degassed beer samples. The analysis can be utilized to quantify ethanol across the entire range
expected values from 0.05 %ABV in non-alcoholic beers up to 50%ABV produced by specialized Broken Bow Broken Heart Stout
Component mg/L = 0.99 * 10 mg * ((Icomp / Ncomp) / 50) * (Mwcomp / 116.1) * (1,000,000 / 175) Eq. 1
brewing yeasts. The response is entirely linear and results correlate closely with other techniques Bacchus - Kilowatt - Oak Aged Sour
Where, wt of maleic acid (MA) = 10mg, Icomp = Integration of component resonance, Ncomp = number of protons integrated,
such as head space GC, HPLC, ASBC distillations, and density meter-based approaches. The figure to
IMA/NMA = 50 (MA integral set as 100), Mwcomp = molecular weight of component molecule, MW of MA = 116.1 amu, the Bacchus - Obolus - Gose
1,000,000/175 factor rectifies the volumetric component of the calculation to allow mg/L to be calculated. 0.99 represents the the right shows a comparison of the NMR calculated ethanol content compared to the bottle or
99% purity of the maleic acid standard. tapdeclared values for the 23 beers presented here. The technique can also quantify methanol, fusel 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000
The following non-volatile components were always obtained on a 1000 ml freeze dried sample: lactic acid, succinic acid, malic
alcohols (1-propanol, isobutanol, isopentanol) as well as diols such as 1,3-propandiol, 2,3-butandiol,
and finally glycerol produced during the fermentation process.
Organic Acid Distribution CONCENTRATION (MG/L)
acid, citrate, various amino acids, glucose, malt and carbohydrates, glycerol. The calculation utilized to quantify these
components was: a(1-4) Linear Dp 3-4 a(1-4) Linear Dp 5-8 a(1-4) Near Branch Dp 6 a(1-4) Near Branch Dp 9
Miller High Life
Component mg/L = 0.99 * 10 mg * ((Icomp / Ncomp) / 50) * (Mwcomp/ 116.1) * (1,000,000 / 1,000) Eq. 2 Amino Acids and Nucleotides: The NMR technique can be used to quantify a large number of amino
acids and nucleotides providing information on yeast accessible nitrogen and information on the Longtrail Mostly Cloudy
Where, wt of MA = 10 mg, Icomp = Integration of component resonance, Ncomp = number of protons integrated, IMA/NMA = 50 (MA
protein content of the beers. A graphic is provided showing the quantification of a large number of North Coast Old Rasputin Amino Acid Distributions
Beer
integral set at 100), Mwcomp = molecular weight of component molecule, MW of MA = 116.1 amu, the 1,000,000/1,000 factor
rectifies the volumetric component of the calculation to allow mg/L to be calculated. 0.99 represents the 99% purity of the amino acids in the 23 commercial beers presented here. Newburgh Berliner Weiss Miller High Life
maleic acid standard.
Newburgh Winter Spruce Porter Longtrail Mostly Cloudy
Nutritional Information: As the detailed chemical fingerprint has been produced it Amstel Lite North Coast Old Rasputin
Ethanol Content (v/v) by NMR Compared to Label Calories per 12 Oz Beer - Component Breakdown
is a straightforward calculation to provide calorie values for the beers that are
Coors Banquet Newburgh Berliner Weiss
analyzed. The contribution of the individual beer components Miller High Life
(Ethanol/Carbs/Amino Acids/Organic Acids) to the total calorie count can also be Moosehead Miller High Life Newburgh Winter Spruce Porter
Longtrail Mostly Cloudy
provided. Another aspect of NMR analysis that we are currently investigating is Miller Lite Longtrail Mostly Cloudy Amstel Lite
North Coast Old Rasputin
the simultaneous observation of sodium in beer samples to obtain a quantitative North Coast Old Rasputin Coors Banquet
Dog Fish Head Noble Rot Newburgh Berliner Weiss
value for sodium for labelling. (23Na is a highly NMR active nucleus that can be Newburgh Berliner Weiss Moosehead
North Coast Bro Thelonius Abbey Ale Newburgh Winter Spruce Porter
quantified down to ppm levels in a relatively short time with minimal sample Newburgh Winter Spruce Porter Amstel Lite Miller Lite
preparation). Founders Blushing Monk Amstel Lite Coors Banquet Dog Fish Head Noble Rot
Left Hand Milk Stout Nitro Coors Banquet Moosehead
Adjunct Chemistry: The 1H NMR analysis can also be utilized to observe the North Coast Bro Thelonius Abbey Ale
Bells 2 Hearted Ale Moosehead Miller Lite
quantity of adjunct chemistry present in a beer sample. Beer that contains wine Founders Blushing Monk
must will show observable quantities of malic acid and tartaric acid. Apple ales Shed Mountain Ale Miller Lite Dog Fish Head Noble Rot
Dog Fish Head Noble Rot North Coast Bro Thelonius Abbey Ale Left Hand Milk Stout Nitro
will also show quantifiable malic acid content. Cocoa nibs provides a source of Troegs Java Head
North Coast Bro Thelonius Abbey Ale Founders Blushing Monk Bells 2 Hearted Ale
Theobromine that can be analyzed by the NMR, and coffee addition provides
Keegan Ales Joe Mama Milk Founders Blushing Monk Left Hand Milk Stout Nitro Shed Mountain Ale
caffeine that can be observed and quantified. Addition of rice and other sources
of carbohydrate can be observed in changes of dextrin character. The rich detail of Founders Rubeus Left Hand Milk Stout Nitro Bells 2 Hearted Ale Troegs Java Head
this region allows differences/similarities in original and residual dextrin character Left Hand Milk Stout Nitro Bells 2 Hearted Ale Shed Mountain Ale Keegan Ales Joe Mama Milk
to be studied eg. Beer vs Sake Red Hook Seedy Blonde Apple Ale Shed Mountain Ale Troegs Java Head Founders Rubeus
Troegs Java Head Keegan Ales Joe Mama Milk
Hops and Lipids: 1H NMR approaches have been developed to investigate the hop Broken Bow Broken Heart Stout Left Hand Milk Stout Nitro
Keegan Ales Joe Mama Milk Founders Rubeus
and lipid component chemistries. However these approaches are complicated by Bacchus - Kilowatt - Oak Aged Sour Left Hand Milk Stout Nitro Red Hook Seedy Blonde Apple Ale
Founders Rubeus
the very low concentrations and variable solubilities of these two components. Bacchus - Obolus - Gose Red Hook Seedy Blonde Apple Ale Broken Bow Broken Heart Stout
These approaches are the subject of ongoing research. Left Hand Milk Stout Nitro
Broken Bow Broken Heart Stout Bacchus - Kilowatt - Oak Aged Sour
0 2000 4000 6000 8000 10000 12000 Red Hook Seedy Blonde Apple Ale
Bacchus - Kilowatt - Oak Aged Sour Bacchus - Obolus - Gose
Nutritional Values: The detailed chemical breakdown obtained by this NMR CONCENTRATION (MG/L) Bacchus - Kilowatt - Oak Aged Sour
1H NMR spectra of a number individual molecular components found in fermented beverages. Molecular assignments of the various NMR peaks that represent Bacchus - Obolus - Gose
analysis allows the calorific values, total weight of carbohydrates and amino acids Bacchus - Obolus - Gose 0 1000 2000 3000 4000 5000
proton chemistries present in each molecule are show along with the molar number of protons that each peak represents. These pure component spectra of
and actual ethanol %ABV to be calculated. Lactic Acid Succinic Acid Acetic Acid Malic Acid 0 50 100 150 200 250 300 CONCENTRATION (MG/L)
individual molecules found in beer are superimposed on each other in the final NMR spectrum. However, a knowledge of these component spectra allows the 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
identification of unique NMR signals for most molecular components allowing the identification and quantification of the components present in the complex Pyruvic Acid Pyruvic Hydrate Citric Acid Formic Acid Ethanol Cal/12 Oz Carbs Cal/12 Oz Amino Acids Cal/12 Oz Organic Acids Cal/12 Oz Histidine Uridine Tryptophan Phenylalanine Tyrosine Gallic Acid GABA Proline Alanine Valine
mixture that is the beer. Ethanol v/v Label Ethanol v/v NMR

Selected Beer References: 1) “Quality control of beer using high-resolution nuclear magnetic resonance spectroscopy and multivariate analysis”, D W Lachenmeier, et al., Eur Food Res Technol, 220, 215–221 (2005), 2) “Quantification of organic and amino acids in beer by 1H NMR spectroscopy”, L I Nord, et al., Anal Chem, 76 (16), 4790–4798 (2004), 3) “Application of quantitative nuclear magnetic resonance spectroscopy to biological acidification of barley mashes”, A Dicaprio, J C Edwards, J Inst Brewing, 120(3), 207-211 (2014),
4) 1H NMR spectroscopy for profiling complex carbohydrate mixtures in non-fractionated beer”, B O Petersen et al., Food Chem, 150, 65–72 (2014) 5) “Development of brewing science in (and since) the late 19th century: molecular profiles of 110–130 year old beers”, A Walther et al., Food Chem, 183, 227–234 (2015), 6) "Beer metabolomics: molecular details of the brewing process and the differential effects of late and dry hopping on yeast purine metabolism", C W Bamforth et al., J. Inst. Brew., 122, 21–28 (2016)

Small Organic Acid Analysis of Commecial Ciders

Cider Making Process
Racking Filtration 10000

MaloLactic Centrifugation
8000
Fermentation

6000
Extended Time Back
Esp. High Acidity
Sweetening
Sorting Racking
4000
Initial
Sweating
Fermentation
Washing Pasteurization 2000
saccharomyces
And
Addition of SO2 Packaging 0
Reduces wild fermentations
Grind
To Blending
Pomace

Filtration
Pressing and
Cooling Magners Cider – assignments of the components quantified in the 1H qNMR analysis – spectrum was Stella Artois Cidre – assignments of the components quantified in the 1H qNMR analysis – spectrum was
obtained with water suppression on a 175ml aliquot of freshly degassed cider Sucrose, glucose and obtained with water suppression on a 175ml aliquot of freshly degassed cider. Spectrum shows low lactic
fructose are observed in the sample and relatively high quinic acid indicating considerable tannin content and only fructose and glucose are observed. Tannin content id observed to be relatively low.
content. Lactic Acid mg/L Succinic Acid mg/L Acetic Acid mg/L Malic Acid mg/L
The chemistry is remarkably similar regardless of the beverage being analyzed.
Sugar Analysis of Commercial Ciders
90000

80000

70000

60000

50000

40000

30000

20000

10000

0
Live culture in sweetened fermented teas often leads to high alcohol content in carelessly stored
products leading to bottle explosions and inadvertent consumption of alcohol by the public
(pregnant women, recovering alcoholics beware).

1H NMR spectrum of a dry cider with relatively high tannin content showing no residual sugar 1H qNMR spectrum of cider allowed to ferment to ultimate completion yielding the highest
present in the cider. High conversion of malic acid to lactic acid is seen with high lactic possible ethanol content. The malolactic fermentation continued and produced lactic acid and
content and a low concentration of malic acid. This indicates that malolactic fermentation left no residual malic acid. The yeast then continued to ferment and turned the glycerol of the
occurred during the manufacturing process of this cider. The residual signal in the 3.5-3.8 cider into 1,3-propandiol.
ppm region is due almost completely to glycerol (see Figure 5 below – peaks in the 3.5-3.6
ppm region can be utilized to quantify glycerol if identified in the spectrum). Sucrose mg/L Glucose mg/L Fructose mg/L