The Complex Fingerprint Analysis of Alcoholic Beverages

by Quantitative NMR (qNMR)

John Edwards, Ph.D.,

Process NMR Associates, LLC, Poughkeepsie, NY

Presented at Bells Brewery, Comstock MI

October 12, 2018
 On-Line Process NMR

45 Systems Installed 1996-2004

Predominantly in Petroleum
and Petrochemical
 Typical High-End Analysis Performed on Alcoholic Beverages

Good for volatiles Molecules separated by boiling

point and molecular interaction
with coatings present in distillation
column.

Oven used to
temperature ramp
the sample

Liquid Chromatography
Extensive Calibration and
Expensive Standards
Required!!!

Molecules separated by solvent and interaction with column packing material. Different
solvents and columns required to analyze different components – sugars, alcohols, organic
acids often require different tests and instrument set up.
 New Technology - Nuclear Magnetic Resonance (NMR)
Main Chemical Analysis of Molecular Structure Used by Chemists since 1960’s
Technology also Evolved into Magnetic Resonance Imaging during 1970’s
Manufacturers:
NMR Spectrometer Bruker – Germany
JEOL - Japan

No Chemical Standards Required

Molecular Structure Directly Observed
Linear Concentration Response is Guaranteed
No Constant Calibration Necessary !!!

Superconducting Magnet
Superconducting special alloy coil Spectrometer
immersed in Liquid Helium at -269oC Radio-Frequency
and surrounded by a liquid 40-400 MHz
nitrogen dewar at 67K

Human, Yeast, and Bacterial Metabolomics

Cider/Beer/Wine =
Yeast-Bacteria Metabolites + Non-Assimilable Ingredients

Eg. "A primer to nutritional metabolomics by NMR spectroscopy

and chemometrics", F.Savorani,M. A. Rasmussen, M.S.
Mikkelsen, S. Balling-Engelsen, Food Research International,
54(1), 2013, Pages 1131-1145.
Example of Complex Mixture Quantitative Component Analysis – Bruker Winescreener

Information Copied from Bruker Website Materials

Beer
 CH3-CH2-OH
CH3

Splittings = N+1 Rule

Adjacent to 2H = 3 Peaks

CH2
1 2 1

OH

Adjacent to 3H = 4 Peaks

1 3 3 1
Precision of Measurement = 0.5%
 N=3

HO-CH2-CH3

Maleic Acid
Internal
Standard

N=2
 Sigma-Aldrich Organic TraceCERT®
products are characterized by:
•Manufactured according to
ISO/IEC 17025 and ISO Guide 34
•Certified Content by quantitative
NMR (qNMR)

•Superior level of accuracy,

calculated uncertainties and
lot-specific values
•Traceability to NIST
•Comprehensive
documentation delivered
with the product
(certification according to ISO
Guide 31)
 d, 5.32 ppm

Glucose d, 4.1 ppm

d, 5.21 ppm
Sucrose d, 5.4 ppm
Fructose Galacuronic Acid

Lactic Acid
Malic Acid d, 1.41 ppm Acetic Acid Succinic Acid Citric m, 2.94 ppm
m, 2.89 ppm s, 2.08 ppm s, 2.67 ppm

d, 0.88 ppm Ethanol

s, 3.32 ppm 1,3-propandiol

Glycerol Isobutanol Isoamyl alcohol t, 1.17 ppm p 1.76 ppm
Methanol m, 3.61 ppm d, 0.87 ppm
d 1.14 ppm
d, 1.56 ppm
s=singlet
Examples of Major Protons Utilized d=doublet
for 1H Quantitative (qNMR) Analysis t=triplet
p=pentet
of Alcoholic Beverages m=multiplet 2,3-butandiol
Alanine
Quinic Acid
2 x d 1.9 ppm
Calculations:
Standard Solution = 10mg Maleic Acid (MA) per 100ml of D2O Solution
Original Sample: The following volatile components were always obtained on a 175 ml beer sample as the
freeze drying process would compromise the amount present: ethanol, acetic acid, fusel alcohols
(isobutanol, isopentanol (isoamyl alcohol), and 1-propanol) ethyl acetate, methanol. The calculation
utilized to quantify these components was:

Component mg/L = 0.995 * 10 mg * ((Icomp / Ncomp) / 50) * (Mwcomp / 116.1) * (1,000,000 / 175)

Where, wt of maleic acid (MA) = 10mg, Icomp = Integration of component resonance, Ncomp = number of
protons integrated, IMA/NMA = 50 (MA integral set as 100), Mwcomp = molecular weight of component
molecule, MW of MA = 116.1 amu, the 1,000,000/175 factor rectifies the volumetric component of the
calculation to allow mg/L to be calculated. 0.995 represents the 99.5% purity of the maleic acid standard.
Freeze Dried Sample: The following non-volatile components were always obtained on a 1000 ml freeze
dried sample: lactic acid, succinic acid, malic acid, citrate, various amino acids, glucose, malt and
carbohydrates, glycerol. The calculation utilized to quantify these components was:

Component mg/L = 0.995 * 10 mg * ((Icomp / Ncomp) / 50) * (Mwcomp/ 116.1) * (1,000,000 / 1,000)

Where, wt of MA = 10 mg, Icomp = Integration of component resonance, Ncomp = number of protons

integrated, IMA/NMA = 50 (MA 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. 0.995 represents the 99.5% purity of the maleic acid standard.
 Temperature Dependence of the Sour
Mash Technique
Goal:
Does the “magic” temperature of 120°F have a chemical
significance?
Record and quantify sour metabolites & contaminant products
as a function of sour mash temperature
Determine wt% of metabolites using Maleic Acid internal standard,
manual integration, and Mestrelab GSD SMA plug-in
 Lactic Acid
 Acetic Acid (contaminant)
 Succinic Acid
 Ethanol
 γ-Amino Butyric Acid (contaminant)
Absolute Metabolite Proportions
16.00 1.4
Metabolite Weight % vs. Temperature 1.2 Weight % Ethanol

Percent by Mass
1
14.00 0.8
0.6
0.4
12.00 0.2
0
90 110 130
10.00
Temperature (°F)

8.00

6.00

4.00

2.00

0.00
90 95 100 105 110 115 120 125 130 135 140

Lactic Acid Acetic Acid Succinic Acid Poly. (Lactic Acid) Poly. (Acetic Acid) Poly. (Succinic Acid)
1H NMR – Malt/Hot Water Extracts
 Linear Maltodextrins (mg/L)
50000
45000
40000 New England IPA
35000
30000
Differences in Mash Carbohydrate
25000 Chemistry with Turbid Mash
20000
15000
10000
5000
0
Lab Mash #2 3/15 Lab Mash #3 3/15 Standard Mash 3/15 Turbid Mash 3/10 Total Carbs (mg/L)
a(1-4) Linear Dp 3-4 a(1-4) Linear Dp 5-8 160000

a(1-4) Near Branch Dp 6 a(1-4) Near Branch Dp 9 140000

120000
Reducing End Sugar and Glucose Concentration (mg/L) 100000
45000 80000
40000 60000
35000 40000
30000 20000
25000 0
Lab Mash #2 3/15Lab Mash #3 3/15 Standard Mash Turbid Mash 3/10
20000
3/15
15000

10000

5000

0
Lab Mash #2 3/15 Lab Mash #3 3/15 Standard Mash 3/15 Turbid Mash 3/10

a-Glucan Reducing End a-Glucose b-glucose b-glucan Reducing End

 Hop Chemistry

Fast Sampling, Analyses and Chemometrics

for Plant Breeding: Bitter Acids,
Xanthohumoland Terpenes in Lupulin
Glands of Hops(Humulus lupulus),
Killeen, et al,
Phytochem. Anal. 2017, 28, 50–57
Hop Chemistry
Apple Juice Analysis
 Concentration (g/L)
Apple Variety Sucrose Glucose Xylose Fructose Red Sugar Non-Red Sugar Total Sugar Malic Acid
1 Cavelle Blanc 125.9 14.1 0.8 120.2 135.1 125.9 261.0 20.7
2 Fall Pippin 63.5 8.1 0.7 115.0 123.9 63.5 187.4 9.5
3 Dr Mathews 33.6 6.6 0.4 86.2 93.2 33.6 126.8 6.3
4 Chieftan 59.2 7.7 0.6 85.0 93.3 59.2 152.6 8.4
5 Pink Pearl 56.6 7.7 0.6 84.4 92.6 56.6 149.2 14.8
6 Oakin Pin 52.2 17.1 0.4 111.9 129.4 52.2 181.6 25.7
7 King David 48.3 7.7 0.7 100.4 108.8 48.3 157.1 10.8
8 Bennoni 51.7 8.5 1.2 97.7 107.4 51.7 159.2 9.6
9 Green Newtown Pippin 86.6 9.8 0.3 86.7 96.8 86.6 183.4 19.8
10 Ashmaead Kernel 78.8 11.4 0.5 109.3 121.2 78.8 200.0 14.5
11 English Golden Harvey 104.5 11.2 0.4 127.2 138.8 104.5 243.3 22.2
12 Yates 34.9 9.6 0.3 74.2 84.2 34.9 119.0 7.5
13 Spitzberg 61.5 10.5 0.8 123.0 134.3 61.5 195.8 9.5
14 Tompkin's King 59.1 13.3 0.6 137.9 151.7 59.1 210.8 14.9
15 Golden Russet 68.7 17.5 0.7 170.6 188.8 68.7 257.6 13.3
16 Decio 78.5 7.1 0.4 69.9 77.4 78.5 155.9 7.8
17 Edwards VII 49.2 10.1 0.6 137.8 148.5 49.2 197.7 10.5
18 Rhode Island Greening 48.1 9.9 0.3 88.6 98.9 48.1 147.0 9.0
19 Baldwin 105.8 6.1 0.3 81.6 87.9 105.8 193.8 17.6
20 Starr 8.4 6.3 0.4 74.1 80.8 8.4 89.2 9.2
21 Razor Russet 67.3 11.0 0.6 125.7 137.2 67.3 204.5 9.8
22 Gold Rush 62.8 4.9 0.3 67.6 72.8 62.8 135.6 11.0
23 Jewitt Nodhead 45.5 12.1 0.7 131.5 144.4 45.5 190.0 8.9
24 American Beauty Sterling 76.6 8.0 0.5 86.1 94.5 76.6 171.1 13.4
25 Kidds Orange Red 42.5 8.7 0.7 119.2 128.6 42.5 171.1 8.6
26 Opalescent 42.7 9.0 0.7 94.8 104.6 42.7 147.3 8.0
27 Ben Davis 30.4 6.2 0.5 74.9 81.5 30.4 111.9 6.4
28 Chestnut Crab 7.4 7.9 0.6 94.1 102.6 7.4 110.0 9.7
29 Scarlet Crofton 62.9 9.3 0.9 105.9 116.1 62.9 179.0 9.8
30 Quinte 39.3 6.4 0.8 70.9 78.1 39.3 117.4 9.6
31 Barnack 69.8 14.2 0.8 118.2 133.1 69.8 202.9 10.0
32 Sweet Winter Pennock 31.8 7.4 0.3 86.6 94.3 31.8 126.1 6.1
33 Bancroft 56.3 5.3 0.7 116.3 122.4 56.3 178.7 18.7
34 Titus Pippen 33.7 7.2 0.8 101.6 109.6 33.7 143.3 9.5
35 Spy 94.4 14.5 1.0 146.3 161.8 94.4 256.2 14.1
36 Pearl Apple 91.8 8.6 0.7 133.1 142.4 91.8 234.3 20.0
37 Golden Noble 67.1 12.4 1.2 124.6 138.1 67.1 205.3 24.1
38 Devonshire Quarendon 23.0 10.4 0.8 105.8 117.0 23.0 140.0 9.1
39 Virginia Beauty 66.5 17.6 0.8 113.4 131.8 66.5 198.3 13.2
40 Winter Banana 24.1 17.1 0.6 108.7 126.4 24.1 150.5 8.9
41 Marie Joseph 20.0 3.4 0.2 38.5 42.1 20.0 62.1 6.9
42 Mother 70.8 9.2 0.9 119.3 129.3 70.8 200.1 10.8
43 Pitmaston Pineapple 50.6 26.3 1.1 196.2 223.6 50.6 274.2 16.2
44 Roxburg Russet 78.8 16.6 0.7 141.4 158.7 78.8 237.6 14.1
45 Shiawasse Beauty 34.8 5.7 0.6 129.0 135.3 34.8 170.1 13.6
46 Wagener 77.1 9.8 0.5 111.6 121.8 77.1 198.9 13.0
47 Sutton Beauty 47.5 9.3 0.4 102.5 112.2 47.5 159.7 10.0
48 Yellow Newtown Pippin 74.2 17.0 1.1 138.8 157.0 74.2 231.1 10.4
49 Colamer 29.8 5.0 0.6 97.1 102.6 29.8 132.5 11.1
51 Montreal Peach 72.9 10.2 0.6 144.5 155.3 72.9 228.2 9.3
52 Goof 17.7 15.8 0.6 103.7 120.1 17.7 137.8 8.8
53 Ellisons Orange 89.9 9.3 0.7 95.3 105.2 89.9 195.1 10.7
54 Granny Smiths 53.7 9.2 0.1 64.1 73.3 53.7 127.0 18.7
55 Gala 45.7 23.9 0.7 191.2 215.8 45.7 261.5 7.2
56 Fuji 8.3 20.2 0.4 112.2 132.8 8.3 141.1 2.9

54 Apple Juices of Heirloom Variety Apples

Apples Obtained at Cider Days – Franklin MA
Analysis of Other Fruit Juices
 Some Major Components of Cider
D-Fructose
How to ID and Quantify

D-Glucose

Sucrose

Glycerol

Ethanol

5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8
f1 (ppm)
 N=0.53 N=2.02 Total Labile Proton Integral = 7

D-Fructose Single doublet or series of peaks utilized to quantify fructose

D-Glucose

anomeric protons
utilized to quantify
glucose in cider
a b

Sucrose Which fructose peaks are utilized to calculate

the concentration depends on the presence or
Anomeric proton non-presence of sucrose as it has a signal that
utilized to quantify overlaps with this region.
sucrose

5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0
f1 (ppm)
 Red - Fructose
Blue - Glucose
Green - Sucrose

Sucrose

Glucose Fructose

5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1
f1 (ppm)
 Propionic Acid
Spanish Sidra 2

Glycerol
Spanish Sidra 1 Absent
Hydroxypropionic Acid

“American Sidra”

Ethyl lactate
Ethyl acetate 1,3-propandiol 1-propanol

Unique Character of Sidra and Other Wild Fermented Cider – metabolism of glycerol utilization
 Anaerobic Fermentation of Glycerol
Seems to occur in all Sidra and in many wild fermentations
Ethanol Fermentation Process that ferment sugars “to dryness”.
 Chemistry observed in
Honey and Maple Syrup

Honey = Terroir Fingerprint in Residual Sugar Distribution

Non-Fermentable Sugars
Kombucha
Liquor, Liquers, Wine, Sake, Mead
Vinegars
 qNMR Developments
• Bruker had developed the NMR WineScreener – which can provide fingerprint chemistry
analysis, grape variety, vintage and geographical origin.

• Various Summits are being held to encourage the use of qNMR in quality control, purity and
authentication analysis and standard test method development
*1st qNMR Summit - US Pharmacopoeial Convention – Rockville MD October 2016
* 2nd qNMR Summit – BAM, Berlin, Germany, March 15-17, 2017

One Reference of Many:

Quantitative 1H NMR Analysis of Egg Yolk, Alcohol, and Total Sugar Content in Egg Liqueurs
• Monika Hohmann, Verena Koospal, Claudia Bauer-Christoph, Norbert Christoph, Helmut Wachter,
Bernd Diehl, and Ulrike Holzgrabe
• J. Agric. Food Chem., 2015, 63 (16), pp 4112–4119
• DOI: 10.1021/acs.jafc.5b00940

A comprehensive list of NMR articles on alcoholic beverage analysis can be provided upon request
 Thank You for Your Attention

Questions ?

References and Presentations can be found on PNA Website http://www.process-nmr.com

Contact John Edwards - email: john@process-nmr.com Tel: (203) 744-5905