Table of Content

Chapter- I Introduction

Chapter- II Materials and Method

 Materials
 Determination of the alcohol content in beverages
 Determination of alcohol content in beverages
 Sample Preparation
 Parametric studies
 Analysis of ethanol containing beverages
 1H NMR Spectroscopy
 Determining alcohol content on distillate by hygrometry

Chapter- III Results and Discussion

 Analysis of ethanol containing beverages

 Bioethanol Estimation
 Experimental variations
 Limitations

Chapter- IV Summary, Conclusion, References

~1~
Abstract

Determination of the ethanol concentration in beverages remains an occa-

sional request to forensic laboratories. While traditionally determined using
headspace gas chromatography (HS-GC), this process involves lengthy run
times and the need for specialized headspace sampling equipment for the GC
system. The work presented here highlights a potential alternative to HS-GC
analysis, using direct analysis in real time mass spectrometry (DART-MS).
By incorporating a simple T-junction to the orifice of the mass spectrometer,
analysis of the headspace of a beverage can be completed, resulting in mea-
surement of the ethanol concentration in a matter of seconds with greater than
99% accuracy. This work presents the development of a method for ethanol
quantitation as well as results from the analysis of both ethanol reference ma-
terial standards and in-house created ethanol-containing solutions to evaluate
the precision and accuracy of the technique. Analysis of ethanolic beverages
via DART-MS is shown to be a rapid and reliable alternative to traditional
analysis by HS-GC.

Keywords: Alcohol, DART-MS, Ethanol, Quantization

~2~
 Chapter- I
Introduction

~3~
Introduction

Determination of the ethanol concentration in beverages remains an occa-

sional request to forensic laboratories. While traditionally determined using
headspace gas chromatography (HS-GC), this process involves lengthy run
times and the need for specialized headspace sampling equipment for the GC
system.

In forensic analyses, the need occasionally arises to determine the concentra-

tion of ethanol in beverages to evaluate, among other things, whether a bever-
age has been tampered with. This analysis is typically completed by
headspace gas chromatography (HS-GC) which can involve lengthy (on the
order of minutes) run times as well as the need for additional equipment to
complete headspace sampling instead of the more common liquid sampling.
This type of analysis is also completed in food safety analyses for investiga-
tive purposes, to determine if adulteration has occurred, or quality control
purposes. Techniques other than HS-GC have been demonstrated in literature
- including Raman spectroscopy , Fourier transform infrared (FTIR) spec-
troscopy, spatially offset Raman spectroscopy, gas chromatography Fourier
transform infrared (GC-IR) [8], and even Schlieren imaging [9].

Direct analysis in real time mass spectrometry (DART-MS) is an ambient

ionization mass spectrometry technique that is witnessing increased use in
forensic analyses. DART-MS is appealing for many applications because it
provides rapid analysis (on the order of seconds) of samples with minimal to
no sample preparation. Previous work has shown the ability of DART-MS to

~4~
analyze compounds such as drugs, explosives, lubricants, paints, and even
woods. Studies have also been completed utilizing DART-MS to analyze
beverages for qualitative purposes to identify compounds of interest such as
caffeine, phthalates, and gamma hydro-xybutyric acid (GHB). While the
compounds of interest analyzed by DART-MS are normally higher in molec-
ular weight than ethanol, recent work has highlighted the ability of this tool
to detect low molecular weight adulterants, such as ammonia, bleach,
methanol, and butanol in beverages. Detection of these compounds, espe-
cially methanol, and others, such as iso-amyl alcohol, are crucial for adulter-
ation detection.

This study builds upon previous work in the qualitative identification of low
molecular weight compounds by DART-MS by extending to quantitative
measurements of ethanol concentration in beverages. Utilizing a simple mod-
ification to the DART-MS interface to collect headspace vapors into the
DART gas stream, a method for determining the ethanol concentration was
created. This method was then used to evaluate whether quantitative informa-
tion could be obtained with samples of known ethanol concentrations. The
ability to re-analyze samples was also evaluated. The method described
herein represents a viable alternative to ethanol quantitation in place of HS-
GC analysis.

Ethanol, commonly called ethyl alcohol, is the principal alcoholic component

in wine produced by fermentation of sugars by yeast. Wine-making is a
rigidly controlled process and the final commercial product is subjected to

~5~
stringent regulations regarding the alcohol content, acidity, sulfur dioxide
quantity, sugar content, quality of the grapes, and amount of preservatives.

Several methods to quantify the alcoholic content in wine to ensure quality of

highest standard are based on chemical (dichromate oxidation), physical (py-
cnometry, densitometry, refractometry, and hydrometry), chromatographic,
and spectrometric analyses. The International Organization of Vine and Wine
(OIV) has set up a compendium of official test methods to analyze wines and
musts that vitivinicultural sectors need to follow to comply with international
standards worldwide. Spectroscopic techniques such as infrared (IR) and
NMR and mass spectrometry offer alternative methods other than traditional
chemical testing and chromatographic methods such as high-performance liq-
uid chromatography (HPLC) and gas chromatography (GC) . Recently, Ra-
man spectroscopy has been explored to quantify ethanol concentration in al-
coholic beverages . To maintain international oenological standards and to
preserve high quality products, methods to determine alcohol content must be
reliable to measure this very important parameter. An additional advantage
would be if the method implemented is officially recognized, but inexpensive
and less prone to human errors.

Nuclear magnetic resonance (NMR) spectroscopy, a nondestructive noninva-

sive technique that provides a direct relationship between the measured spec-
tra and chemical structure, is the primary standard and routine method for
molecular structure characterization. The capability of NMR for quantitation
(qNMR) to simultaneously detect a large number of compounds in a complex
mixture without separation proves to be advantageous as a method to use for

~6~
analysis of natural products including food, fruit juices, or alcoholic bever-
ages . In particular, 1 H NMR methodologies have been applied to determine
chemical constituents (phenolics, sugars, and organic acids) and to measure
the amount of alcohol for authenticating the quality of wine. NMR analysis
has been applied even in full intact wine bottles to preserve the integrity of
this valuable commodity . The objective of this study was to investigate the
use of a low-field 45 MHz NMR spectrometer to determine the alcohol con-
tent of several alcoholic products. Benchtop NMR spectrometer has been
used in teaching organic chemistry laboratory techniques and is gaining pop-
ularity due to ease and cost-effectiveness in its utility including in vivo imag-
ing applications compared to high-field NMR instrumentation

~7~
 Chapter- II
Materials and Method

~8~
Materials and Method

Materials

Commercially available alcoholic beverages were obtained from local com-

mercial vendors and analyzed for alcohol content. Fourteen different alco-
holic samples used were Banana liqueur (KY, USA), Black Magic rum (KY,
USA), Fleischmann’s vodka (LA, CA, USA), Smirnoff vodka (Norwalk, CT,
USA), Stolichnaya vodka (Latvia), Three Olives vodka (Rochester, NY), Dr.
McGillicuddy’s root beer liquor (WI, USA), Merlot red wine (Modesto, CA),
Cruzan Key Lime rum (IL, USA), Pinot Noir (Modesto, CA, USA), Canadian
ice wine (Niagara on the Lake, ON, Canada), Tour de Soleil Rosé (Côtes de
Gascogne, France), Beringer White Zinfandel (Napa, CA, USA), and
Moscato white wine (Modesto, CA, USA). Absolute ethanol (200 proof) used
as calibration standard was obtained from Mallinckrodt Chemicals (St. Louis,
MO, USA). Acetic acid and acetonitrile were obtained from Sigma Aldrich
(St. Louis, MO, USA). Deuterated water (D2O, 99.9% D) was purchased
from Cambridge Isotope Laboratories (Tewksbury, MA, USA).

Distilled beverages

Distilled beverages (also called liquors or spirit drinks) are alcoholic drinks
produced by distilling (i.e., concentrating by distillation) ethanol produced by
means of fermenting grain, fruit, or vegetables. Unsweetened, distilled, alco-
holic drinks that have an alcohol content of at least 20% ABV are called spir-
its.[16] For the most common distilled drinks, such as whiskey and vodka,
the alcohol content is around 40%. The term hard liquor is used in North
~9~
America to distinguish distilled drinks from undistilled ones (implicitly
weaker). Vodka, gin, baijiu, shōchū, soju, tequila, whiskey, brandy and rum
are examples of distilled drinks. Distilling concentrates the alcohol and elimi-
nates some of the congeners. Freeze distillation concentrates ethanol along
with methanol and fusel alcohols (fermentation by-products partially re-
moved by distillation) in applejack.

Fortified wine is wine, such as port or sherry, to which a distilled beverage

(usually brandy) has been added. Fortified wine is distinguished from spirits
made from wine in that spirits are produced by means of distillation, while
fortified wine is wine that has had a spirit added to it. Many different styles
of fortified wine have been developed, including port, sherry, madeira,
marsala, commandaria, and the aromatized wine vermouth.

~ 10 ~
Determination of the alcohol content in beverages

Distillation method Accurate alcoholic beverages analysis ensures excellent

beverages quality. Therefore this analysis is an essential part of the daily
work in the labs in the breweries, vineyards and distilleries. Knowledge of
the alcohol content is necessary to ensure that the beverage conforms to the
label declaration of alcohol content and to establish the basis for the payment
of tax. Distillation is the most important method of separation and purifica-
tion of liquids. In the simplest case of distillation liquid is heated to boiling
and produced vapor is condensed in a condenser and collected as a distillate.
Distillation when only one phase is in the motion – vapor – is called simple
distillation. Distillation when portion of condensed vapor still flow down into
the distillation flask is called rectification. Rectification is performed using
fractionating column.

Many substances form azeotrope mixtures, which at definite composition

show maximal or minimal boiling temperature. Azeotrope mixture cannot be
~ 11 ~
separated by distillation because vapor and liquid phase have the same com-
position. The example of azeotrope mixture is exactly water-ethanol mixture.
The mixture containing 95 % by mass of ethanol boils at the lower tempera-
ture i.e. 78,15 °C, than pure water (100 °C) or pure ethanol (78,3 °C). The
following diagram shows the phase equilibrium between liquid and vapor in
the function of concentration for water-ethanol mixtures.

Suppose, we are going to distill a mixture of water and ethanol with composi-
tion C1. It will boil at a temperature given by a liquid curve and produce a
vapor with composition C2. When a vapor condenses it will still have the
composition C2. If we reboil this new mixture it will produce a new vapor
with composition C3. If we carried on with this boiling-condensing-reboiling
procedure we end up with a vapor with a composition of 95 % by mass of
ethanol. If we condense this vapor we gat a liquid with this same composi-
tion. Because of liquid and vapor curves meet at this point, each time when
we boil the liquid with the composition of 95 % by mass of ethanol, the con-
densed vapor still has the same composition of 95 % by mass of ethanol. It
means that it is impossible to get pure ethanol (100 %) by distilling any mix-
ture of water-ethanol containing less than 95 % by mass of ethanol. This par-
ticular mixture of ethanol and water boils as if it were a pure liquid. It has a
constant boiling point and the vapor composition is exactly the same as the
liquid. This liquid is known as an azeotrope. In the consequences of this for
fractional distillation of dilute aqueous solutions of ethanol we cannot get
pure ethanol, but only a product with a maximum concentration of 95 % of

~ 12 ~
ethanol. What we can get it is a pure water, of course if we earlier ethanol
rich vapor given off from the distillation flask.

Pure ethanol can be produce only by the fractional distillation of ethanol-wa-

ter mixtures containing more than 95 % by mass of ethanol. In this case we
get a distillate containing 95 % of ethanol in the collecting flask and pure
ethanol in the boiling flask.

Shot sizes

Shot sizes vary significantly from country to country. In the United Kingdom,
serving size in licensed premises is regulated under the Weights and Mea-
sures Act (1985). A single serving size of spirits (gin, whisky, rum, and
vodka) are sold in 25 ml or 35 ml quantities or multiples thereof.[35] Beer is
typically served in pints (568 ml), but is also served in half-pints or third-
pints. In Israel, a single serving size of spirits is about twice as much, 50 or
60 mL.

The shape of a glass can have a significant effect on how much one pours. A
Cornell University study of students and bartenders' pouring showed both
groups pour more into short, wide glasses than into tall, slender glasses.[36]
Aiming to pour one shot of alcohol (1.5 ounces or 44.3 ml), students on aver-
age poured 45.5 ml & 59.6 ml (30% more) respectively into the tall and short
glasses. The bartenders scored similarly, on average pouring 20.5% more into
the short glasses. More experienced bartenders were more accurate, pouring
10.3% less alcohol than less experienced bartenders. Practice reduced the ten-
dency of both groups to over pour for tall, slender glasses but not for short,

~ 13 ~
wide glasses. These misperceptions are attributed to two perceptual biases:
(1) Estimating that tall, slender glasses have more volume than shorter, wider
glasses; and (2) Over focusing on the height of the liquid and disregarding the
width.

Determination of alcohol content in beverages

Distillation method. The base of the method of the determination of alcohol

content in beverages is a precise measurement of volume (or weight) of a de-
gassed and filtered sample of beverage. The sample is quantitatively trans-
ferred to the distillation apparatus, then the distillation is performed and the
obtained alcohol fraction is filled up to the original volume (or weight) with
distilled water. In this way a liquid is obtained which has the same alcohol
concentration as the original sample but no longer has extract. The alcohol
concentration in % by vol. or in % by mass is determined highly accurately
via density or refractive index measurements and an appropriate alcohol con-
centration tables. The alcoholic strength by volume is the number of liters of
ethanol contained in 100 liters of beverage, both volumes being measured at a
temperature of 20 °C. It is expressed by the symbol '% by vol'. Homologues
of ethanol, together with the ethanol and ethanol homologues in ethyl esters
are included in the alcoholic strength since they occur in the distillate.

Methods of measurements of alcohol content in distillate. The alcohol content

in a distillate can be determined using [8, 9, 12]:

• Pycnometer – determination of density or specific gravity of distillate –

method basing on a precise measurement of a mass of liquid,

~ 14 ~
• Areometer (hydrometer), alcoholometer – determination of density,
specific gravity or alcohol concentration of distillate – method basing
on buoyancy,
• Hydrostatic weighing – determination of density of distillate – method
basing on buoyancy,
• Digital density meter – determination of density of distillate – measure-
ments of the frequency of oscillation of the u-shaped tube filled with a
sample,
• Refractometer – measurement of the refractive index of a distillate,
spectrometer – measurement of the alcohol-specific range of the near
infrared (nir) spectrum

~ 15 ~
Sample Preparation

NMR samples for internal standard method were prepared by measuring 900 
μL of alcoholic samples and 100 μL of glacial acetic acid into a small capped
3 mL vial for a total volume of 1 mL. Five different mixtures were prepared.
The solutions were mixed well and immediately used for analysis in a 45 
MHz NMR spectrometer. For comparison, NMR samples were prepared for
400 MHz NMR spectrometer by placing 100 μL of the acetic acid-alcohol
sample mixture into a 5 mm NMR tube combined with 600 μL of D2O. Addi-
tionally, acetonitrile was also used as an internal standard instead of acetic
acid. NMR samples for the calibration curve were prepared by mixing 10–
90% of absolute ethanol and a fixed quantity of acetic acid (10%) as internal
standard for a total volume of 1 mL in milliQ water. Samples for standard ad-
dition method were prepared with absolute ethanol concentration ranging
from 0 to 50% volume/volume being added into the mixture. Preparation of
six samples for the standard addition method is shown in Table 1.

~ 16 ~
Table 1
Preparation of samples for standard addition method. Internal standards:
acetic acid or acetonitrile.

% EtOH Sample Internal standard H2O Total volume

EtOH (mL) (mL) (mL) (mL) (mL)

0 0 0.5 0.1 1.4 2.0

10 0.2 0.5 0.1 1.2 2.0
20 0.4 0.5 0.1 1.0 2.0
40 0.8 0.5 0.1 0.6 2.0
50 1 0.5 0.1 0.4 2.0

Instrumentation

~ 17 ~
Samples were analyzed using a DART-MS with an in-house built headspace
configuration, a schematic of which is shown in Fig. 1. The mass spectrome-
ter, a JEOL JMS-T100LP (JEOL USA, Peabody, MA, USA), was coupled
with a Vapur® interface (IonSense, Saugus, MA, USA). To direct the pull of
headspace of the sample into the Vapur interface, the ceramic tube of the Va-
pur interface was shortened to 30 mm and a Swagelok (Solon, OH, USA) T-
junction was fitted to the end of the ceramic tube, which allowed uninter-
rupted flow of the DART gas while also allowing for the placement of the 2.0
mL glass vial directly underneath. Placement of the vial directly under the
junction allowed for an approximately 2 mm air gap between the vial and
junction. This configuration promoted the entrainment of the headspace from
the vial into the DART gas stream. Analysis was completed by placing un-
capped vials under the T-junction and allowing the headspace to be sampled
for 8 s to 10 s. Helium (99.999% purity) was used as the DART source gas
for all analyses. Unless otherwise stated, relevant instrument parameters in-
cluded a DART exit grid voltage of 150 V, a 350 °C DART gas temperature,
and a Vapur flow rate of 3.5 L min−1. Mass spectrometer settings included:
operation in positive ionization mode, a 110 °C Orifice 1 temperature, 100 V
ion guide voltage, 25 V orifice 1 voltage, 20 V ring lens voltage, 5 V orifice 2
voltage, 2300 V detector voltage, and a mass scan range of m/z 20 to m/z 150
at0.25 s scan−1. Polyethylene glycol 600 (PEG) was used for mass calibra-
tion.

~ 18 ~
Ethanol quantitation was accomplished by integrating the peak areas of ex-
tracted ion chronographs of m/z 47 ([Ethanol + H]+) and m/z 61 ([IPA + H]
+) using MassCenterMain software. Integration was completed using the in-
tegrate function of the software. The ratio of the peak areas (m/z 47: m/z 61)
was then compared to a 9-point calibration curve to obtain the concentration
of ethanol in the solution. The ethanol dimer peak of m/z 93 ([2 Ethanol + H]
+) and the ethanol-IPA complex peak of m/z 107 ([Ethanol + IPA + H]+)
were also monitored but were ultimately not used for quantitation.

~ 19 ~
Parametric studies

For the optimization of DART gas stream temperature, DART exit grid volt-
age, and Vapur flow rate a 300 V ion guide voltage and 20 V orifice 1 volt-
age was used. A 20 V orifice 1 voltage was used for the optimization of the
ion guide voltage. A 100 V ion guide voltage was used for the optimization
of the Orifice 1 voltage.

Analysis of ethanol containing beverages

For the analysis of ethanol containing beverages, identical settings as above

were used except for a 100 V ion guide voltage, and a 30 V orifice 1 voltage.

~ 20 ~
1H NMR Spectroscopy

About 200–300 μL of the alcohol-acetic acid mixture was injected into the in-
let port of the 45 MHz NMR instrument. Sample loop holds about 20 μL of
liquid. A sample of approximately ten times the sample loop volume is in-
jected to ensure that sample to be analyzed is inside the loop, and droplets of
liquid drained out of the outlet port. 1H NMR spectra were obtained on a pi-
coSpin 45 MHz NMR spectrometer equipped with a fixed magnet. Shimming
on water was utilized prior to spectral acquisition. One-dimensional one-
pulse 1H NMR spectra generated as exponentially decaying sine wave or free
induction decay (FID) were acquired using the one-pulse sequence available
in the picoSpin pulse script library. FIDs were collected at 298 K by employ-
ing a relaxation delay of 8 s, a pulse width of 90° (43 μs), and 16 scans. 1H
NMR spectra were also obtained on a 400 MHz Bruker Avance NMR spec-
trometer equipped with an autosampler. One-dimensional one-pulse 1H NMR
spectra were acquired using the one-pulse sequence in the Bruker pulse se-
quence library. FIDs were collected at 298 K by employing a relaxation delay
of 1 s, a pulse width of 90° (14.9 μs), and 16 scans. Both FIDs were apodized
with an exponential multiplication prior to Fourier transformation. Chemical
shifts (δ) are given in ppm relative to acetic acid (or acetonitrile) set at 2.04 
ppm. Processing was performed using ACD/Labs NMR Processor Version
12.01 and TopSpin 2.1.6 processing data software for the 45 and 400 MHz
NMR instruments, respectively.

~ 21 ~
Determining alcohol content on distillate by hydrometry:

i. Rinse a hydrometer cylinder or suitable measuring cylinder with a

small portion of the distillate. Discard the rinse liquid.
ii. Transfer the remaining distillate to the cylinder.
iii. Rinse the alcohol hydrometer with a small portion of the distillate.
iv. Gently lower the hydrometer into the distillate, while gently ‘spinning’
the hydrometer stem. Ensure the hydrometer is floating freely in the
distillate.
v. Record the density of the wine (or the alcohol content if hydrometer
gives direct alcohol reading) at the point corresponding to the base of
the meniscus. 6. Record the temperature of the distillate – ideally hy-
drometer readings should be taken at 20°C, however a temperature ad-
justment calculation can be made if necessary.

~ 22 ~
 Chapter- III
Results and Discussion

~ 23 ~
Results and Discussion

An NMR spectrum is a plot of the radio frequency applied against absorption,

and an NMR signal is referred to as a resonance expressed as chemical shift
in ppm, which is independent of the spectrometer frequency [34]. Important
features of an NMR spectrum that provide information pertaining to the
structure of the molecule include chemical shift, multiplicity, peak integra-
tion, and coupling constant. Chemical shift depends on the environment of
the nucleus (such as hydrogen or H) being observed. Hydrogens in the vicin-
ity of electronegative atoms (O, N, and halogens) experiencing reduced elec-
tron density will be deshielded and will resonate downfield or at higher ppm.
In addition, protons in aromatic compounds (benzene) and alkenes (double
bonds) are downfield shifted as a consequence of induced magnetic field due
to circulating electrons or anisotropic effects. Multiplicity or spin-spin split-
ting refers to the interaction of the nucleus with adjacent nuclei, or a hydro-
gen with neighboring hydrogens, and can be evaluated empirically by the ()
rule in which the resonance peak is split into () components, where refers to
the number of equivalent hydrogens () on the adjacent atom(s). The observed
multiplicity which is dependent on the number of adjacent hydrogens will
give rise to splitting patterns designated as singlet (no adjacent hydrogen),
doublet (1 adjacent H), triplet (2 adjacent H’s), quartet (3 adjacent H’s), and
so forth. Integration refers to the area under the peak(s) or multiplets which is
a measure of the number of hydrogens in that particular resonance or chemi-
cal shift. The coupling constant (or value) is a measure of the interaction or
coupling of adjacent nuclei so that hydrogens separated by three bonds will

~ 24 ~
be coupled to each other and will have the same coupling constant or value.
For ethanol (CH3CH2OH), there are three unique hydrogens which will ap-
pear as three separate NMR signals: CH3 (methyl) at 1.18 ppm, CH2 (methy-
lene) at 3.64 ppm, and the OH (hydroxyl) at 4.80, which overlaps with water
peak. Splitting pattern for ethanol will be triplet for CH3, quartet for CH2,
and singlet for OH, with peak integration ratio of 3 : 2 : 1 corresponding to
CH3, CH2, and OH, respectively. Hydroxyl proton will appear as a broad
singlet in both 45 and 400 MHz spectrometers due to presence of water and
residual hydrogens in deuterated solvents. For absolute or ultra-pure anhy-
drous ethanol, splitting of the OH peak is observed in the 45 MHz NMR
spectrometer used for this study [35] since the liquid sample is run neat and
not dissolved in deuterated solvent which can cause broadening of the hy-
droxyl group in the sample due to proton exchange. value for the coupling of
CH3 and CH2 is ~6 Hz.

Figure 1 shows the NMR spectra of alcoholic samples acquired with 45 MHz
benchtop and 400 MHz NMR spectrometers. The triplet assigned to the
methyl (CH3) group at 1.18 ppm was the peak used for quantification from
the formula shown in (1), wherein the amount of ethanol, which is the main
component, is calculated directly from the NMR spectrum. This is based on
standard analytical procedures for determining analyte concentrations [13–
33, 36]. The singlet for the methyl peak at 2.04 ppm corresponding to acetic
acid or acetonitrile was used for calibration and as reference set to 3 for peak
integration to determine the alcohol content of the samples. The methylene
(CH2) peak tends to have an overlapping peak with the alcoholic product

~ 25 ~
probably due to sugar molecules and other dissolved solutes and hence is not
utilized for analysis. Determination of alcohol content in alcoholic beverages
is shown as follows

Figure 1

1H NMR spectra of alcoholic products acquired with a 45 (a–d) and 400 (e–
h) MHz NMR spectrometer. (a, e) Black Magic rum; (b, f) Smirnoff vodka;
(c, g) Dr. McGillicuddy’s root beer liqueur; (d, h) Tour de Soleil Rosé.
Triplet at 1.16 and quartet at 3.56 ppm correspond to hydrogens in methyl
(CH3) and methylene (CH2) groups, respectively, in ethanol, while the sin-
glet at 2.04 ppm corresponds to the methyl peak of acetic acid. Acetic acid
CH3 peak was used for peak integration.

~ 26 ~
Determination of the percent alcohol in the sample was a straightforward cal-
culation based on the average of 5 different sample preparations from the
same alcoholic product. Acetic acid is utilized as the internal standard for
quantification by peak integration [16, 28] since it is readily available, cheap,
stable, nonvolatile, and soluble in aqueous ethanolic solutions. The reference
signal for the methyl group at 2.04 ppm is a singlet and widely separated
from the ethanol peaks. Acetonitrile was also used as an internal standard uti-
lizing the methyl peak also at 2.04 ppm. In addition, one sample for each al-
coholic product was run in a 400 MHz NMR spectrometer (see Figure 1) and
the results obtained for the benchtop NMR are comparable.

Obviously, higher resolution is observed for 400 MHz than 45 MHz as shown

in Figure 1. Sample preparations for low- and high-field strength instrumen-
tations are different. Less sample preparation is involved for the benchtop in-
strument, while for the high-field NMR instrument the alcohol-acetic acid
mixture is dissolved in deuterated water (D2O) for locking purposes. Alco-
holic products that are thick and milky such as Tequila Rose and Chiata Rum
cannot be determined for the 45 benchtop NMR due to difficulty in injecting
the samples.

Table 2 summarizes the results obtained for the determination of ethanol con-
tent of several commercially available alcoholic beverages using both 45 and
400 MHz instruments. The methodologies used to determine the alcohol con-
tent involve the use of an internal standard and standard addition method. The
use of an internal standard in which acetic acid or acetonitrile was added di-
rectly to the sample increases reliability and accuracy of the calculations for

~ 27 ~
the determinations used for both instruments. Standard addition method as
shown in Figure 2 includes adding known quantities of ethanol to correct for
matrix effects. Both methods (internal standard or standard addition method)
require the use of an internal standard to calibrate for peak integration. The
internal standard addition method involves less sample preparation and would
be the preferred method of choice if substantial amounts of samples were to
be determined. Linearity curve for standard concentrations ranging from 10
to 90% ethanol in water with acetic acid as reference is shown in Figure 3 for
the 45 MHz 1H NMR spectrometer. External standard method using a cali-
bration curve such as shown in Figure 3 with the use of acetic acid for peak
integration produced erroneous calculations of the alcohol content and was
not utilized for this study. Final determination of the alcohol content was pri-
marily based on the peak integration of methyl (CH3) peaks referenced to
acetic acid or acetonitrile using (1) since this NMR spectral region was ob-
served to be significantly less affected by overlap caused by other compo-
nents in the sample compared to the methylene (CH2) region, where other
peaks were observed (see Figure 1). A reference or standard added internally
will be needed always to quantify the amount of alcohol. Acetic acid and ace-
tonitrile were chosen as the internal standards due to cost, molecular structure
similarity to ethanol, and ease of preparation since these are liquid standards
compared to 3-(trimethyl-silyl)-1-propane sulfonic acid sodium salt (DSS) or
(CH3)3Si (CD2)2CO2Na, sodium 3-trimethylsilyl-propionate-2,2,3,3-d4
(TMSP), both of which are solids. No significant amount of acetic acid was
detected in the alcoholic beverages that were used in this study as shown in
Figures 4(a) and 4(c) in which alcoholic beverages were run as neat samples

~ 28 ~
in the 45 MHz spectrometer and confirmed when the same samples (dis-
solved in D2O) were run in the 400 MHz instrument (see Figures 4(b) and
4(d)). If acetic acid were present in significant amount in the alcoholic bever-
age, then the amount of acetic acid originally present would be calculated by
running two samples: one with acetic acid standard of known amount and the
other without acetic acid standard. The peak area for the sample with added
acetic acid standard would be greater than without it. Consequently, the
amount of ethanol present can be corrected accordingly.

~ 29 ~
Table 2

Percent alcohol content of alcoholic beverages using 45 and 400 MHz NMR

spectrometers. Standard errors were calculated for 5 determinations per-
formed for the 45 MHz instrument. HOAc = glacial acetic acid; ACN = ace-
tonitrile; IS = internal standard; SA = standard addition method.

% al- % alco- % alco- % alco- % alco-

%
cohol hol hol hol hol
alco-
Alcoholic 400  45  45  45  45 
hol
beverages MHz MHz MHz MHz MHz
on
HOA HOAc ACN HOAc ACN
label
c IS IS IS SA SA

Banana 49.03   53.69   54.56   56.46  

49.5 46.80
liqueur 0.24 0.19 0.46 0.58
Black
48.81   49.71   41.40   47.41  
Magic 47 46.07
0.19 0.34 0.48 0.35
rum
Fleis-
39.15   40.04   44.56   44.62  
chmann’s 40 37.07
1.13 2.07 0.28 1.42
vodka
Smirnoff 43.39   42.21   42.05   44.50  
40 37.83
vodka 0.15 0.25 0.78 0.49
Stolich-
37.84   38.99   44.81   44.61  
naya 37.5 37.33
0.90 0.04 0.86 0.51
vodka
Three 33.54   32.39   30.04   34.05  
30 28.05
Olives 0.26 0.62 0.59 0.42

~ 30 ~
vodka
Dr.
McGillicu
20.51   21.47   22.26   22.25  
ddy’s root 21 22.15
1.10 0.60 0.35 0.56
beer
liqueur
Cruzan
26.33   20.34   22.94   23.96  
Key Lime 21 20.50
0.21 0.45 0.25 0.31
rum
Merlot red 13.36   13.41   15.89   15.32  
13 11.30
wine 0.09 0.15 0.32 0.31
11.47   11.25   10.22   11.58  
Pinot Noir 11.5 10.13
0.07 0.11 0.37 0.39
Canadian 11.87   11.38   9.10  0. 12.46  
11 10.23
ice wine 0.10 0.04 53 0.30
Tour de
9.94  0. 11.51   13.51   14.49  
Soleil 11 11.50
28 0.43 0.20 0.76
Rosé
Beringer
9.26  0. 13.10   10.92   13.13  
White 10 10.56
32 0.24 0.21 0.22
Zinfandel
Moscato
9.09  0. 9.69  0. 10.62   10.40  
white 9 7.69
28 06 1.50 0.90
wine

~ 31 ~
To obtain accurate peak integration for quantitative NMR experiment, a re-
laxation delay of approximately value is typically utilized. refers to a time
constant or relaxation time for the nucleus to fully relax between pulses and
return to equilibrium. Each nucleus in a molecule will have different values
due to different magnetic environments.

A relaxation delay of 8 seconds was used for the 45 MHz spectrometer. How-
ever, a 1-second relaxation delay was used to reduce run time for 400 MHz
FT instrument since an insignificant difference of about 0.1–0.3% was ob-
served when the value was changed from 1-second to 8–10-second delay.

Based on the results of this study, internal standard method performed in the
low-field 45 MHz benchtop NMR spectrometer produced percent alcohol lev-
els that are more comparable to the label than the standard addition method
and therefore was the method utilized to compare with the high-field 400 
MHz NMR spectrometer. Results obtained for 45 MHz and 400 MHz spec-
trometer differ slightly, most likely due to variation in the sample prepara-
tion. Samples for the 400 MHz were 100 μL aliquots derived from the sam-
ples prepared for the 45 MHz and the aliquot diluted with deuterated water.
Discrepancy in exact volume measurements could have introduced this dif-
ference.

The most important advantage for using the 45 MHz benchtop NMR spec-
trometer over the high-field instrument is the cost. Low-field fixed magnet
NMR compared to superconducting high-field spectrometer requires less
maintenance because cryogens are not needed avoiding additional task for

~ 32 ~
personnel to manage the instrument. NMR tubes and deuterated solvents re-
quired for field/frequency stabilization for high-field instrumentation are not
necessary reducing additional expenses incurred to run the samples.

Quantitation & limits of detection

Once an optimal method was established, efforts were made to identify the
limit of detection (LOD) of ethanol in water mixtures. Calculation of LOD
was completed using the ASTM E2677 method [23], which provides a 90%
confidence for the LOD. To accomplish this, ten replicates of samples at 0%
v/v, 0.1% v/v, 1.0% v/v, and 10.0% v/v were analyzed. Using the ASTM
E2677 LOD calculator [24], an LOD90 of 0.15% v/v ethanol in water was
calculated.

~ 33 ~
Additional studies were completed to evaluate the ability to quantitate
ethanol concentrations. Using a series of ethanol solutions from 0% v/v to
100% v/v, a linear response with a R2 value of greater than0.997 (Fig. 4) was
obtained. A y-intercept very close to zero was also observed, indicating the
absence of any significant background at m/z 47 for ethanol. Deviation from
the regression line was more evident in the mid-range ethanol concentrations
(10% v/v to 50% v/v). This slight deviation is likely caused by the volume
compression of ethanol–water mixtures [21,22] which would cause a greater
number of ethanol molecules to be transferred in the 1 mL aliquot than if the
pure liquid (water or ethanol) were transferred. The volume compression ef-
fect of the ethanol-water mixture was not accounted for in these curves. Ad-
ditional aliquots from the same calibration curve were re-analyzed one week
and up to 2 weeks after preparation to see if the calibration curve could be
reused. It should be noted, however, that the response was different for the
different analysis days. This may be a function of laboratory environmental
conditions (i.e., temperature or humidity) and suggests the need for the cali-
bration curve (or an abbreviation thereof) to be sampled during the same time
that the unknowns are analyzed.

Analysis of ethanol containing beverages

To evaluate the applicability of this method to quantitate unknowns, three

ERMs, beverages with analytically certified ethanol concentrations, were
tested. These ERMs were chosen to span the range of ethanol concentrations
and included lager (reported concentration of 5% v/v), wine (15% v/v), and
brandy (40% v/v). Samples were spiked with isopropanol as the internal stan-

~ 34 ~
dard and at least ten replicates were analyzed. The ratio of ethanol to iso-
propanol was then fitted to up to three different calibration curve windows
(0% to 10% v/v, 0% to 50% v/v, and 0% to 100% v/v), when applicable, to
determine if there was any effect from the range of the curve that was used
due to the volume compression of the mid-range concentrations. Table 1
shows the average measured ethanol concentration of the ERMs using each
of the three calibration curves. For the lowest curve, only the lager was quan-
titated as it was the only standard within the 0% v/v to 10% v/v range. In all
instances, the calculated concentrations fell within 1% v/v of the documented
concentrations, and the relative standard deviation of replicate measurements
was below 15%, with higher relative variability observed for the low ethanol
concentration lager. Additionally, there did not appear to be a dependence on
the calibration curve range with respect to accuracy of the measurement, indi-
cating that any calibration curve would be suitable for the measurement pro-
vided the unknown concentration fell within that range and that the volume
compression could be considered negligible for this type of analysis. Devia-
tions from the reported concentration may be due to variations in the calibra-
tion curve or due to differences caused by the process used to certify the
ethanol concentration (distillation followed by measurement of the apparent
density in air by pycnometry at 20 °C and compared to the “Laboratory Alco-
hol Table’ Reference RDC80/267/04). This study also highlighted that the
presence of complex matrices did not affect the ability to quantitate the solu-
tion even though aqueous calibrants were used. A representative mass spec-
trum of the wine ERM is presented in Fig. 5A.

~ 35 ~
Table 1

Measured ethanol concentrations for the three ERMs examined. The con-
centrations using different calibration curves are presented. Docu-
mented ethanol concentrations were 5% v/v, 15% v/v, and 40% v/v for
the lager, wine and brandy respectively. The right three columns show
the measured ethanol concentrations 6 months after making the first set
of measurements. Uncertainties represent the standard deviation of ten
replicate measurements.

~ 36 ~
Cal Curve
Measured Ethanol Concen- Repeat Measurement After 6
Range (%
tration (% v/v) Months (% v/v)
v/v)
Lager Wine Brandy Lager Wine Brandy
5.31 ( ± 5.30 ( ±
0% to 10% N/A N/A N/A N/A
0.67) 0.04)
5.24 ( ± 14.89 ( ± 40.13 ( ± 5.25 ( ± 15.32 ( ± 41.69 ( ±
0% to 50%
0.68) 1.92) 1.55) 0.04) 0.05) 0.07)
5.05 ( ± 14.46 ( ± 40.29 ( ± 5.51 ( ± 15.11 ( ± 40.28 ( ±
0% to 100%
0.78) 0.80) 3.80) 0.30) 0.03) 0.68)

Cases in a forensic laboratory may sit for some time before analysis, there-
fore repeat measurements of the same ERMs were made six months after the
initial set of measurements to establish whether the results could be repro-
duced. For these samples a new calibration curve was created, and a new 1
mL aliquot was removed from the original ERM solutions. As shown in Ta-
ble 1, results can be replicated even after a six-month window. Deviation
from the reported ERM value was slightly higher for the 6-month-old sam-
ples than the freshly analyzed samples and may be attributable to the six-
month storage period in the refrigerator.

To expand the investigation into matrix effects beyond the three ERMs,
ethanol was spiked in four common mixing beverages (cola, pineapple juice,
orange juice, and fruit punch) at four different concentrations (5% v/v, 10%

~ 37 ~
v/v, 25% v/v, and 50% v/v). Ethanol was added volumetrically and 1 mL
aliquots of the mixed solutions were placed in 2.0 mL amber vials and spiked
with isopropanol internal standard. Ten replicates of each sample were ana-
lyzed, and the ethanol concentration calculated using a 0% v/v to 50% v/v
calibration curve. The measured concentrations can be found in Table 2.
Much like the ERMs, the measured ethanol concentration fell largely within
± 1% v/v of the prepared concentration. The standard deviation of the repli-
cate measurements increased with ethanol concentration, though the relative
standard deviation remained relatively constant, around 10%.
Ability to re-sample
The final component of the study investigated whether re-sampling of vials
was possible to see if changes in the headspace of the sample occurred with
repeat sampling. To investigate this, aqueous solutions containing 5% v/v and
50% v/v ethanol by volume were created volumetrically and the solutions
were then split into 18 individual vials and spiked with isopropanol as an in-
ternal standard. Five of the vials were uncapped, sampled, and capped at each
timepoint over the course of the 24-hour study while the remaining vials were
sampled at only one of the 12 timepoints. The timepoints for resampling
were: 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, and 24 h.
The ethanol concentration of the single sample vial and the average of the re-
sampled vials were then calculated.

The results of re-sampling study, shown in Fig. 6, show starkly different

trends for the 5% v/v solution and the 50% v/v solution though both show
changes in the headspace do occur with repeat analysis. For the 5% v/v solu-

~ 38 ~
tion, where ethanol was at a lower concentration than the isopropanol internal
standard, the re-sampled vials trended lower than the actual initial concentra-
tion. This is likely due to an increasing fraction of isopropanol in the
headspace of the vial which pushes the calculated ethanol concentration
lower. When the ethanol concentration exceeds the isopropanol concentra-
tion, as was the case for the 50% v/v ethanol solution, the calculated concen-
tration trends significantly higher than the initial concentration, likely due to
the decreasing fraction of isopropanol in the headspace. In both instances, the
number of data points outside the 2σ range (red dotted lines) was higher for
the re-sampled vials than the single sample vial, indicating that repeat analy-
sis of the same vial should be avoided in order to obtain the most accurate
ethanol concentration.

~ 39 ~
Bioethanol Estimation
Bioethanol concentration was estimated either by solvent extraction and
dichromate oxidation method or by Gas Chromatography (GC) method dis-
cuss below. The dichromate reagent was prepared by dissolving fresh 10 gm
potassium dichromate in 100 ml of 5 M sulfuric acid solution [5] . Tri-n-butyl
phosphate [TBP, density = 0.975 to 0.976, solubility in water = 0.028% (w/
v), Sigma] was used to extract ethanol in an aqueous solution. In detail, 1 ml
of TBP and 1 ml of standard solutions; 0% to 5% (v/v) or samples were
mixed in a 2 ml microtube and then vortex vigorously until the reaction mix-
ture was separated into two phases (upper phase-very clear, transparent and
lower phase-turbid). From the upper phase, the 500 μl solution was trans-
ferred to a new 2 ml microtube then 500 μl dichromate reagent was added in
a tube and vortex vigorously again for 10 minutes. The reaction mixture is
separated into two phases (lower phase-blue green color). Dichromate
reagent-containing lower phase (100 μl) was transferred in to the test tube
and diluted up to 10-times with both standard and experimental samples. Op-
tical density (OD) for standard ethanol solution and samples were measured
at 595 nm in a UV spectrophotometer (Thermofisher). Bioethanol content of
unknown sample was estimated from the ethanol standard curve as shown be-
low.

Standard ethanol solutions with water were prepared from 0.0% - 1.0% (v/v)
and 1 - 2 μl was injected into the injection port of the GC and then subjected
to quantitative analysis of ethanol on a GC apparatus (SRI Model 8610A GC
5890 GC, Shimadzu, Japan). Packed column head pressure: 30 psi, column:

~ 40 ~
carbo wax 20 M, 1/8” OD, length = 6 feet, oven temperature: 115˚C, carrier
gas flow rate: 30 - 40 ml/min. Retention time, peak area and height of the in-
jected standard ethanol solution was measured and recorded. That was used
to determine the concentration of unknown experimental samples. In order to
determine the reproducibility for injection method, standard solutions were
injected at least three times. After the standards were completed, we replicate
injections of our unknown samples same as standard solutions. The peak ar-
eas were measured and retention times were adjusted for the ethanol peaks in
each chromatogram. A standard ethanol curve was prepared by plotting the %
of standard ethanol solution on the X-axis and the peak areas on to the Y-
axis. Ethanol concentrations from unknown samples were estimated from the
ethanol standard curve.

~ 41 ~
Experimental variations

The same measurement procedure can be used to determine ethanol concen-

trations in other samples such as various beverages, food and biological sam-
ples including serum, plasma and urine. For strongly colored samples the ab-
sorbance at 340 nm must be taken into account and corrected for. Non-liquid
and turbid samples need ethanol extraction and/or filtration before the assay
can be performed.

Limitations

ADH will also react with certain higher aliphatic alcohols. The highest activ-
ity is found for ethanol, n-Butanol has about 40% and isopropanol about 8%
of activity compared to ethanol (100% activity). The activity for secondary
and branched chain alcohols is negligible.

~ 42 ~
Points to Consider:

 This method is still the internationally accepted standard for alcohol de-
termination. Sugar, which interferes in so many other methods for de-
termining alcoholic strength, is not distillable and has no effect on the
composition of the distillate.
 There are a few minor interferences however, high levels of either
volatile acidity or sulphur dioxide may volatise and potentially affect
the density of the distillate.
 Levels of VA >1.0g/L as acetic acid, or SO2 levels >200mg/L can
cause significant interference.
 Generally the effect of high VA or SO2 is to increase the density of the
distillate, therefore producing lower apparent alcohols.
 Neutralisation of the wine prior to distillation (use NaOH solution) will
negate the interfering effects of volatile acidity and sulphur dioxide.
 Alcohol hydrometers in the range suitable for wine distillate testing
have a very large bulb.
 This is to allow the density scale corresponding to alcohol content to be
sensitive enough for an accurate result with sufficient significant fig-
ures to be obtained (generally 0.1% vol/vol graduations).
 Alcohol hydrometers are very fragile and must be handled with care.
 The cylinder used in the density measurement must be wide enough for
the hydrometer to float freely without touching the sides.

~ 43 ~
 For lower alcohol wines it may be required to distil 500mL and use a
larger cylinder, in order to obtain the depth of solution required for the
hydrometer to float.
 The use of smaller SG hydrometers and attempting to convert to % al-
cohol by calculation is not recommended as the S.G scale is not fine
enough to convert to an accurate, precise result. (The entire range of
wine alcohols may be covered in an S.G. range of only 0.005, for ex-
ample)
 As with all methods involving densitometry, temperature is critical.
Only a very small range of density values correspond to the range of al-
cohol content in wine, therefore in this case it is critical to work at the
correct temperatures to avoid volumetric error. When measuring the
initial volume of the wine to be distilled, and when diluting the distil-
late to volume prior to measuring density, it is imperative that the
working temperature is 20ºC, as this is the temperature that the volu-
metric glassware is calibrated at.
 Even slight variations in temperature can alter the volume enough to
cause a significant density measurement error. Hydrometers may vary
as to which temperature they are calibrated at. In every case, if measur-
ing density at a temperature that differs from that at which the hydrom-
eter was calibrated will require a temperature correction calculation.


~ 44 ~
 Chapter – I V
Summary, Conclusion, Reference

~ 45 ~
Summary
This application note is a simple guide to determine ethanol concentrations
of unknown samples by measuring the . The enzymaticabsorbance at 340
nm with Photopette based assay takes less than 30 min to perform. The
method can be easily adapted by breweries, analytical laboratories or for
school experiments.

Alcohol is a depressant, which in low doses causes euphoria, reduces anxiety,

and increases sociability. In higher doses, it causes drunkenness, stupor, un-
consciousness, or death. Long-term use can lead to an alcohol use disorder,
an increased risk of developing several types of cancer, cardiovascular dis-
ease, and physical dependence. As reported by WHO, alcohol is the highest
risk-group carcinogen, and no quantity of its consumption can be considered
safe; the temperance movement advocates against the consumption of alco-
holic beverages and those who do not use alcoholic drinks are known as tee-
totalers.

~ 46 ~
Conclusion

Benchtop NMR technology provides a cost-effective and low maintenance

equipment compared to the high-field NMR instrumentation and can be a
promising technique in the future for the wine industry to analyze the alco-
holic content to authenticate quality of wine or other alcoholic beverages.
Compared to other techniques whereby correction methods need to be ap-
plied during the analysis due to presence of sugars and other dissolved com-
ponents in the alcoholic products that may lead to unacceptable errors, NMR
technique is a straightforward method for quantification purposes.

DART-MS represents a promising new technique for the quantitation of

ethanol in beverages. Rapid determination of ethanol concentration in bever-
ages can be achieved in a matter of seconds. Quantitation of ERMs with
known ethanol concentrations provided measurements within ± 1% v/v, and
accurate measurements were also shown in several other beverage matrices at
varying concentrations. Some care, however, needs to be taken with this type
of analysis as laboratory conditions may affect the response, leading for the
need of the calibration curve, or a subset thereof, to ideally be run at the same
time as the unknown samples. Repeat analysis of the same sample over time
is also not recommended as the regeneration of the headspace will lead to dif-
ferences in ethanol concentration - skewing higher at larger ethanol concen-
trations and lower at smaller ethanol concentrations. This technique provides
a unique compliment or replacement to current tools such as HS-GC. Future
work is focused on expanding the detection and quantitation capabilities of

~ 47 ~
this technique to include other compounds that may be present in adulterated
beverages, such as methanol, iso-amyl alcohol, and ethyl acetate butanol.

~ 48 ~
References

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 wine, beer and spirits using a flow-through infrared sensor,” Chemistry
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~ 50 ~