The Complex Fingerprint Analysis of Cider and Other Alcoholic

Beverages by Quantitative NMR (qNMR) Spectroscopy

John Edwards, Ph.D.

Process NMR Associates, LLC, Danbury, CT

Presented at CiderCon 2018, Baltimore MD

February 1, 2018
 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.
 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
 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
 1 N=4
1
Succinic Acid
1

Ethylacetate A B C A N=3
B C N=3
N=2

W
Methanol N=3
W

L-Malic Acid D D N=2

E
E N=1

2 2
Isobutanol 3 4 N=6
2 4 N=2 3 N=1

5
8 N=2 9 N=3 5
Isoamylacetate 6 8 6 7 N=2
5 9 N=6
7 N=1

Citric Acid F F F N=4

Acetic Acid X N=3

Oligomers
Acetaldehyde Oligomers Y Z Y
Z
G N=6
2-3-butandiol H G
G H N=2
H

1-Propanol 11 12 N=2 11 N=2 10

12 N=3
10

9.8 9.6 5.2 5.0 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)
 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)
 Water and Et-OH
1H NMR of Cider Product

Internal
Standard Ethanol
 N=3

HO-CH2-CH3

Maleic Acid
Internal
Standard

N=2
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.
Example of Complex Mixture Quantitative Component Analysis – Bruker Winescreener

Information Copied from Bruker Website Materials

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

54 Apple Juices of Heirloom Variety Apples 47

48
49
Sutton Beauty
Yellow Newtown Pippin
Colamer
47.5
74.2
29.8
9.3
17.0
5.0
0.4
1.1
0.6
102.5
138.8
97.1
112.2
157.0
102.6
47.5
74.2
29.8
159.7
231.1
132.5
10.0
10.4
11.1

Apples Obtained at Cider Days – Franklin MA 51

52
53
Montreal Peach
Goof
Ellisons Orange
72.9
17.7
89.9
10.2
15.8
9.3
0.6
0.6
0.7
144.5
103.7
95.3
155.3
120.1
105.2
72.9
17.7
89.9
228.2
137.8
195.1
9.3
8.8
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
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)
 Nutrient Effect on Cider Chemistry During Fermentation
Grote-009-H_FD
Cider #3 Fructose
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300
Glucose

Sucrose Malic
Acid

Grote-008-H_FD
Cider #2
Parallel Fermentation of Three Ciders from Sample Juice
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300

Grote-007-H_FD
Cider #1 Cider#1 – Nutrient Depleted
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300 Cider #2 – Control
Cider #3 – Nutrient Added

6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 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 0.6
f1 (ppm)
Grote-009-H_FD
Cider #3
YAN Observed in Cider #3
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300
After addition of nutrients
Valine
Alanine Luecine
Glutamic
Isoleucine
Acid

Grote-008-H_FD
Cider #2
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300

Grote-007-H_FD
Cider #1
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300

6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.04.6 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 0.6
f1 (ppm)
Grote-022-H Aromatic Proton Signals from Pure Amino Acids and O-Ferm Nutrient Product
Scott Labs O-Ferm
1H NMR in D2O 100ul MA
JCE-PNA-MercPlus300

HO
13
AminoAcid-L-Tyrosine 12 11
L-Tyrosine in acidified D2O 11,10 8,9
10 9 O
JCE-PNA-MVX300 7
8 5 4 OH
6
3 2

NH2
OH 1
6
O
7
4 9
AminoAcid-L-Tryptophan 9
NH
L-Tryptophan in Acidified D2O 12 2
NH2
JCE-PNA-MVX300 10
5
3 1
15 13 11 14
13 8

15 11
14

12 10
10,11
AminoAcid-L-Phenylalanine 11 8 O
L-Phenylalanine in acidified D2O 6 8,9
9 7 3 OH
JCE-PNA-MVX300 5
4 2 12
NH2
1

NH
10 11
AminoAcid-L-Histidine 9
O
L-Histidine in acidified D2O N 5
JCE-PNA-MVX300 10 8 7 9
3
OH
4 6
2

NH2
1

9.2 9.1 9.0 8.9 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7
f1 (ppm)
Aliphatic Peak Region for Amino Acids Compared to Cider #3 and Nutrient
Ala IsoLeu, Leu
Val, IsiLeu

Grote-022-H Glu Thr

Scott Labs O-Ferm
Glu Leu

O CH3
1 8

AminoAcid-L-Valine 2 6 7 8
6 HO 3 CH3
4 7

H2 N
5 OH
3 1
2
AminoAcid-L-Threonine H3 C
1
4 NH2
5

O 6
O 7
1
OH
AminoAcid-L-Leucine
8 9
2 6
CH3 7,6 8
HO 4 7 9
3
O H3 C
CH3 1 8
H2 N
5 8
2 5 CH3
3 7
9 9 8
AminoAcid-L-IsoLeucine HO
4

5 7 7 NH2
6
O
7
4 OH
6
AminoAcid-L-Glutamic Acid H2 N 2

5 3 1
3 5

8 O
9 O
HO 1
10
AminoAcid-L-Alanine 6 2 CH3
4 6
HO
3

NH2
5

Grote-009-H_FD Val, IsoLeu

Glu Ala Thr
Cider #3 Glu Leu
Leu

2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7
f1 (ppm)
 Gly
Mal Succinate

Fru
Suc Glu

Cider #1
NMR Data over Fermentation
 Gly
Mal Succinate

Fru
Suc Glu

Cider #2
NMR Data over Fermentation
 Gly Mal Succinate

Fru

Suc Glu

Cider #3
NMR Data over Fermentation
Comparison of Sugar Data for 3 Ciders over Course of Fermentation
Spanish and Wild Fermentations
 Ac Lac

NY Farmhouse Barrel Aged Succinate

Methanol

Fusel

Breton Cidre

Mal

NY Dry Cider Gly

Back Sweetened

Examples: ciders with different chemical characteristics – sugar concentration + malolactic

 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)
 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
Cyzer
Residual Sugars

Honey
Sugar Distribution
Cyzer and Mead - High Concentrations of Unused Nutrient Amino Acids
 Mystery Solved?
Batch 2 – supposedly the same cider.
Lactic
Contains twice as much sorbate.
Batch 2 – “good” Was Batch 1 under-additized causing continued
Fermentation and explosions?

New Mystery – why has malolactic fermentation

taken off – juice handling
More Sorbate
Acetic

Malolactic
Batch 1 – “good”

Mystery: Obvious reduction of sugars and

Batch 1 – Exploding Can production of more ethanol observed and
compared to “good” can – didn’t explain exploding can

Production and Quality Issues

Production and Quality Issues
Examples of Sugar Analysis of Commercial Ciders – Part of Dryness Scale?
Organic Acids Distribution
Beer
Residual Malt Chemistry – Non-Assimilable Complex Carbohydrates
Branching Chemistry Obtained – Correlated to Beer Style
Kombucha
Is Kombucha typically alcoholic – Yes.
Difficult to keep live SCOBY fermentation below 0.5% ABV
Official Brewery Operation is Suggested
Can be as alcoholic as beer …. and is unregulated
Bottle Bombs and Pregnant Women
Liquor, Liquers, Wine, Sake, Mead
Sugar Chemistry of Popular Liquors
 Vodka or highly
distilled spirits
show no fusels

Fusels

Fats from cream component

Analysis of ANY Alcoholic Beverage
Vinegars
Acetic Acid
Sugar Acetylations

Residual
Ethanol
 qNMR Developments
• Bruker have developed the NMR WineScreener – which can provide fingerprint
chemistry analysis, grape variety, vintage and geographical origin.
• NMR also used to confirm origin and authenticity of Honey – Honeyscreener
• 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

* 5th qNMR Summit – Tokyo, January 29-30, 2018

Japanese, European and US Pharmacopoeial Authorities working to establish quantitative NMR as ISO method

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

Questions ?
Acknowledgements:

William Grote and his collaborators in the Nutrition study - Tom Bell, Holly Schmidt & Doug
Schmidt (see Nutrients session 3:30 on Friday)

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