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Direct estimation of ethanol as a negative peak

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from alcoholic beverages and fermentation broths

Cite this: DOI: 10.1039/c6ay01075j
by reversed phase-HPLC†
M. Nisha,a Mukund Shankar,b Nagasathiya Krishnan,c Lilly M. Saleena,a Mathur Rajeshb
and M. Vairamani*d

Received 12th April 2016
Accepted 24th April 2016
DOI: 10.1039/c6ay01075j
www.rsc.org/methods
A relatively simple and straightforward technique utilizing reversed phase liquid chromatography with
a PDA-UV detector was used for the detection and quantification of ethanol. Samples of alcoholic
beverages and fermentation broths were injected without any prior chemical pre-treatment or structural
modification. The C18 column was equilibrated with water as a mobile phase having a high UV absorbing
compound, acetone, added in an optimized percentage. Ethanol when injected resulted in a negative
peak within 4 minutes of runtime, and the absolute area/height of the peak was used for quantification.
The method was validated for linearity with the detection ranging from 0.1–100% v/v. Intra-day and
inter-day precision experimental results were within acceptable limits. The accuracy and repeatability of
this method can reduce the dependency of using specific columns and detectors for the analysis of ethanol.

respectively.1 Use of methyl ethyl ketone as an UAC for ethanol

Introduction
Indirect ultraviolet (UV) determination of analytes of biological
samples, solvents and impurities in pharmaceutical drugs
using Reversed Phase (RP)-HPLC is a relatively older tech-
nique.1–4 The technique relies on detecting compounds with low
UV absorbing properties and the detection is facilitated by
adding a minimal quantity of a high UV absorbing compound
(UAC) to the mobile phase which equilibrates itself throughout
the column and sets a high UV absorbing background.4 When
a low UV absorbing sample is injected, a positive or a negative
peak at the retention time (RT) corresponding to the sample
emerges. An UAC peak generally referred to as the system peak
also appears with opposite polarity to that of the sample due to
disturbance in the UAC equilibration on the column.5 The
polarities of the sample peak vary with respect to the system
peak based on its relative charge and relative RT in comparison
to the eluting UAC.6
UACs such as 1-phenthyl-2-picolinium and nicotinamide are
used for the analysis of carboxylic acids and aliphatic alcohols,
a
Department of Bioinformatics, School of Bioengineering, SRM University,
Kattankulathur, Tamil Nadu, India
b
Department of Chemical Engineering, School of Bioengineering, SRM University,
Kattankulathur, Tamil Nadu, India
c
Department of Biotechnology, School of Bioengineering, SRM University,
Kattankulathur, Tamil Nadu, India
d
School of Bioengineering, SRM University, Kattankulathur-603203, Kancheepuram
District, Tamil Nadu, India. E-mail: dean@biotech@ktr.srmuniv.ac.in; Tel: +91–44–
27417812
† Electronic supplementary information (ESI) available: Standard methanol
quantication and analysis. See DOI: 10.1039/c6ay01075j
detection in alcoholic beverages is reported.7 It highlights the
feasibility of using a C18 column in HPLC with methanol : water in
a ratio of 30 : 70 as the mobile phase with methyl ethyl ketone
added in small quantities. The linear range of detection was
limited to a maximum of 40% ethanol. Other polar UACs such as
acetone and methyl isopropyl ketone were also suggested. However
acetone's full potential as an UAC has not been reported yet.
The present work explores the possibility of using acetone as
an UAC in detecting ethanol in two types of samples. The
mobile phase used in the study is water with acetone added in
an optimized percentage. Water in general has a very low UV
cut-off but acetone has a broader UV absorbance range of 190-
330 nm. Ethanol absorbs UV only between 190 and 210 nm.8,9
Negative peaks in a chromatogram are generally observed when
the UV absorbance drops below the calibrated mobile phase
baseline.10 Thus in the present study, UV for detection is
deliberately chosen with a wavelength above the cut-off of
ethanol and in the absorbing range of acetone. When acetone in
water as the mobile phase is equilibrated on the column, a high
UV absorbing background is set. Ethanol absorbs less UV
compared to the high UV absorbing background which will
reverse its peak polarity to negative. No additional UAC or
buffers have been used in ethanol quantication because that
will lead to a possible increase in the number of retained system
peaks and thereby reduce the sensitivity of the detection.11
Experimental
This article does not contain any studies with human partici-
pants or animals performed by any of the authors.

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Analytical Methods
Chemicals
Acetone and ethanol of HPLC grade and ltered Milli-Q water
for dilutions were used throughout the study.
Instrumentation
HPLC. All the preliminary feasibility and parameter opti-
mizations were performed with an Agilent HPLC 1260
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INFINITY equipped with an Agilent 1260ALS autosampler.
Detection of the samples was carried out through a 13 mL ow
cell with a photodiode array detector (Agilent 1260DADVL).
The detector peak width and the response time were set as >0.1
min (2.0 s response time) (2.5 Hz). A reversed phase Zorbax
Eclipse Plus C18 column with an ID, length and pore size of 4.6
mm, 250 mm and 5 mm respectively was used. All the outputs
were processed through Agilent Chemstation-Open lab so-
ware. Peak areas in arbitrary units and peak heights in milli
absorbance units (mAU) were considered for quantitative
analyses.
Parameters standardized in the Agilent LC system were then
subsequently applied to a Shimadzu UFLC system for all
precision, accuracy, sensitivity, standard calibration and test
sample quantication experiments. The temperature controlled
autosampler (SIL 20AC HT) was maintained at 25
C and
separation was carried out in the same aforementioned C18
column. The data were analysed with Lab Solution soware
version 5.31 (CBM20A). The total runtime was 6 min with peaks
detected with a UV detector (SPD20A).
Gas chromatography-mass spectrometry. Analysis was per-
formed in an Agilent 7890B GC equipped with an autosampler
and a HP-5MS capillary column with an ID, length and lm of
250 mm, 30 m and 0.25 mm respectively. Helium was used as the
carrier gas with 1 mL min1 column ow rate. 1 mL of the
collected sample was injected at a split ratio of 1 : 10 and the
inlet temperature was maintained at 250
C with the septum
purged at 3 mL min1. The column temperature was held at 40


C for 5 min and ramped further to 100
C at 10
C min1. The
mass spectrometer (Agilent 5977A MSD) was scanned between
40 and 100 amu at a rate of 1.562 u s1 [N ¼ 2] with a solvent
delay of 0.5 min. The total runtime was 5 min.
Method optimization
The PDA detector was scanned across wavelengths ranging from
190 to 400 nm with 235 nm used for all concerned experiments.
Water was used as a mobile phase with acetone added in an
optimized percentage. Acetone percentages were varied from 1
to 10% v/v from which 4% (v/v) acetone in water was used in all
the experiments. The standard ow rate was set at 1 mL min1.
Standard ethanol solutions were prepared in water (0.1–100% v/v)
for all analyses and validation experiments. Injection volumes
were varied from 1 to 20 mL and optimized for different ethanol
concentration ranges based on the sensitivity.
Method 1. An injection volume of 10 mL for 0.1–5% v/v
ethanol concentrations was considered.
Method 2. An injection volume of 1 mL for >5% v/v ethanol
concentrations was considered.
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Peak conrmation
Since the study involved only three chemical entities, in every
run, the peak patterns detected will represent acetone, water or
ethanol singly or in combination. The method is acceptable if
there are no interfering peaks in the retention time of the
analyte peak. Pure water, ethanol and acetone samples were
injected individually into the HPLC system in separate runs
(mobile phase: 4% acetone in water) and their retention times
were conrmed. For further validation of acetone and ethanol,
the peaks emerging at the RT 3.14  0.13 min and 3.49  0.17
min were collected and the samples were subjected to GC-MS.
Water was not conrmed because the mass spectrometer was
scanned only from 40 to 100 amu.
Method validation
Data analysis. All statistical analyses were independently
performed for both the methods (methods 1 and 2) using
Microso Excel 2010 and Minitab Release-14.
Linearity. Linearity between the concentration and its cor-
responding response (peak area) values arising from triplicate
injections for each ethanol concentration was analysed through
the least-square method. The analysis of variance (ANOVA) test
was performed to establish the lack of t and statistical
signicance of the regression equation.12–14
Precision, accuracy and recovery. The closeness between the
estimated value and its corresponding actual observed mean
values was calculated. Precision with repeatability (5 consecu-
tive hours) and interday variations (5 consecutive days) was
analysed by calculating the relative standard deviations (%
RSDs). Accuracy and precision were checked in intermittent
concentrations in the linear range (three injections per
concentration) for the instrument. The recovery percentage for
the mean observed values to the known standards was subse-
quently calculated.13,15,16
Limits of detection and quantication. The minimum
detectable range was calculated to be 3.3v/a and the minimum
quantiable limit was calculated to be 10v/a where ‘v’ is the
standard deviation and ‘a’ is the slope of the calibration plot as
per ICH guidelines.13,15,17,18
Test sample analysis
Alcoholic beverages. The need for sample preparation may
purely depend on the nature of the analyte.
For commercial alcoholic beverages such as whiskey and
vodka no sample preparation is suggested. In method 1, 1 mL of
clear (vodka) and coloured (whiskey) alcoholic beverages were
directly injected, quantied and compared with the claimed
values as per the label contents.
Bioethanol from fermentative broth. Clostridium thermo-
cellum DSM 1313 was cultivated in standard DSM media with
1% cellulose and anaerobically fermented for 120 hours at 58
 1
C.19 The nal hour broth samples were analysed for bio-
ethanol. Fermentative broths are known to contain UV
absorbing compounds such as proteins. To avoid any such
interference, 50 mL of centrifuged samples were distilled
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Technical Note
until a constant weight of 10 g was collected. 10 mL of the
distillate was immediately subjected to analysis in method 2.
Standards were also prepared as per the above procedure. The
bioethanol from the fermented broth sample was also ana-
lysed with a GC-FID (Chemito GC 7610 system, 10% SE30
column)20 to compare the values produced from the current
method.
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Results and discussion
Peak conrmation
Under the optimized method conditions, a negative peak cor-
responding to ethanol was observed at a retention time (RT) of
3.49  0.17 min as conrmed by GC-MS. A co-eluting peak
always preceded this negative analyte peak. One of the co-
eluting peaks corresponded to a negative peak of water at 3.13 
0.02 min. This was ascertained by injecting pure water into the
optimized aqueous mobile phase containing 4% acetone
(Fig. 1). The other co-eluting peak corresponded to a positive
peak of acetone at a RT of 3.14  0.13 min as conrmed by GC-
MS and is the system peak. However, it is noteworthy that all the
three peaks never emerged together for any of the ethanol
concentrations. Two kinds of peak pairs were always noted
based on the ethanol concentration (Fig. 2 and 3).
(i) A negative peak pair (3.13  0.02 min for water, 3.49 
0.17 min for ethanol) was observed for <3.0% (v/v) ethanol
concentrations.
(ii) A pair of positive and negative peaks (3.14  0.13 min for
acetone, 3.49  0.17 min for ethanol respectively) was observed
for >3.5% (v/v) ethanol.
The ethanol peak was observed for all concentrations (0.1–
100% v/v) which inferred the non-dependence of ethanol on the
system peak.
Method optimization
The developed method is an attempt to utilize a polar solvent
to elute a polar sample in a reversed phase column. Based on
Fig. 1 Relative absorbance of water is low compared to that of the 4% acetone mobile phase. A single negative peak detected when pure water
was injected.
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the peak occurrence, peak separation, and steadiness of the
baseline, 235 nm was selected from the UV scan range. The
selected UV wavelength lies within the UV absorbing range of
acetone and above the cut-off range of ethanol.8,9 Fig. 4 shows
the formation of a negative peak for the analyte (ethanol)
during the detection. The quenching effect is due to drop in
the mAU value in comparison to the higher UV absorbing
mobile phase (4% v/v acetone). An ideal chemical component
such as water having low UV absorbing properties, high
polarity and miscibility with solvents such as acetone and
ethanol is a suitable candidate for a mobile phase.21 Its inter-
action with the hydrophobic C18 column is at the minimum
and provides a background with least noise. It also allows
lower polar compounds (acetone and ethanol) to partially
interact with the column components and allows separation
and detection.22 The optimization of acetone as an added UAC
was performed for better negative peak emergence, peak
separation with constant RT and a steady baseline. Lower
acetone concentrations (1–3%) gave poorly resolved broadened
peaks. Higher acetone concentrations (5–10%) depicted
a trend of decreased sample RTs within 2 min of runtime. No
sample peaks were detected for acetone concentrations >10%.
The mobile phase was thus optimized at 4% (v/v) acetone in
water. Reports suggest that the sensitivity of indirect photo-
metric estimation depends entirely on the added UAC, which
in turn relies on charge, ion pairing mechanisms, pH, pro-
tolysis and other buffering agents in the mobile phase.2,6,11
However, the present work essentially detects the negative
peak of ethanol directly thus enabling linearity from 0.1 to
100% (v/v). During optimization, injection volumes (>5 mL) for
ethanol concentrations above 25% resulted in either
a deformed system peak or merged system and sample peaks.
Whereas, at lower injection volumes (<5 mL) samples with
ethanol concentrations below 0.5% remained undetected. To
maintain the linearity two injection volumes were thus
considered 10 mL for 0.1–5% (method 1) and 1 mL for
concentrations above 5% (v/v) (method 2).
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Fig. 2 Chromatograms of standard ethanol (0.1–5% v/v). The injection volume is 10 mL.

Fig. 3 Chromatograms of standard ethanol (10–100% v/v).The injection volume is 1 mL.

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column, which may migrate through the column pores as an

isolated pocket resulting in an early emergence.23 Since the
standard ethanol samples were prepared in an aqueous solution,
the presence of this negative peak at 3.13  0.02 min is very
evident. The height and area of the water peak are inversely
related to the increasing concentrations of ethanol. However, this
tendency is observed only in <3.0% (v/v) ethanol.
Acetone peak. The injection of pure acetone into the aqueous
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mobile phase containing 4% acetone resulted in a single posi-

tive peak at 3.14  0.13 min (Fig. 5). The positive peak indicates
a higher absorbance of absolute acetone compared to that of 4%
acetone as the mobile phase (Fig. 1). The area of the system

Table 1 Data analysis of method 1a

Method 1
Fig. 4 The UV absorbance (A235) value of 4% acetone in water solution
was considered as a reference and values of absolute acetone, ethanol Injection volume 10 mL
and water were read. Absolute acetone had the highest absorbance Linear range (% v/v) 0.1–5
whereas ethanol and water gave negative absorbance relative to the Replicates 3
4% acetone solution. It infers that any ethanol solution will have lower Regression equationb Y ¼ 29 843X  177
absorbance relative to the 4% acetone and vice versa for any acetone Correlation coefficient R2 0.996
solution above 4%. This justifies the negative peaks for ethanol and
water and positive peaks for acetone. Source of variation

Residual
Regression error Lack of t Pure error
Peak occurrences
Degrees of 1 19 5 14
Water peak. It is noteworthy that a negative peak at 3.13  freedom
0.02 min appears when pure water was injected into an optimized Sum of squares 2.01  1010 7.76  107 2.77  107 4.98  107
aqueous mobile phase (Fig. 1). A similar observation was re- Mean sum of 2.01  1010 4.08  106 5.55  106 3.56  106
ported by Jan Sbrek et al. (2005), where they injected pure water squares
F value 4917.55 1.56
into a RP-HPLC system containing an aqueous mobile phase with
acetate buffer, copper acetate and alkyl sulfonates.6 The negative p 0.235
polarity of the water peak is due to the lower UV absorbance of F critical 2.31 (a ¼ 0.1)
water than that of the 4% acetone mobile phase (Fig. 4). There is No evidence of lack of t
no corresponding system peak for water since the aqueous a
ANOVA results for linearity assessment with weighted regressions. b Y:
solvent is not adsorbed onto the column. The high polarity of negative peak area, X: ethanol concentration (% v/v).
water resists itself from interacting with the hydrophobic

Fig. 5 Relative absorbance of absolute acetone is high compared to that of the 4% acetone mobile phase. A single positive peak detected when
absolute acetone was injected.

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peak (acetone) was also proportional to the increasing ethanol
concentrations and was detected only at >3.5% v/v concentra-
tions of ethanol. Since ethanol and acetone are polar solvents,
the incoming ethanol molecules may replace the bound acetone
molecules in the column. This may be due to various contrib-
uting factors such as individual hydrophobicity and dipole–
dipole interactions among them.24 As mentioned by Arvidsson
et al.11 any disturbance in the equilibrium will cause a change in
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propose that ethanol does an equimolar replacement of acetone
from the column. This justies a proportional change in the
system peak intensity and also its prior emergence to the
negative ethanol peak. The variation observed in the system
peak intensity was due to the concentration of the desorbed
UAC at the ow cell.
Ethanol peak. The negative peak at 3.49  0.17 min that
appeared for 100% ethanol injection was conrmed to be due to

the concentration of the retained mobile phase components ethanol by GC-MS. The late emergence of the negative ethanol
(acetone) resulting in their migration as separate zones peak can be attributed to a new equilibrium state which is
throughout the column. Since acetone and ethanol both have achieved soon aer disturbance of the previous equilibrium by
polar and non-polar ends with similar polarity indices, we desorption of the mobile phase component (acetone) by
ethanol. The process of achieving this state may occur in several
different ways,6 out of which the process of solvation and
Table 2 Data analysis of method 2a desorption of the analyte molecule by the incoming mobile
phase component is relevant here. The as-obtained peak
Method 2 polarities are in accordance with the published report where the
Injection volume 1 mL sample peak is negative when it emerges aer the system peak
Linear range (% v/v) 10–100 and when the sample is uncharged or has the same charge as
Replicates 3 the UAC. However, the ion pairing mechanism as mentioned in
Regression equationb Y ¼ 32 516X + 19 629 the article11 may not be relevant to our study, since the analyte
Correlation coefficient R2 1
and the UAC both essentially are polar solvents.
Source of variation
Method validation
Residual
Regression error Lack of t Pure error Both the UV response (negative mAU) and the peak area were
proportional to the increasing ethanol concentrations (Fig. 2
Degrees of 1 13 3 10 and 3). The regression coefficients of the linear equation were
freedom
determined and are mentioned in Tables 1 and 2. Based on the
Sum of squares 1.69  1013 7.32  109 1.65  109 5.67  108
Mean sum of 1.69  1013 5.63  108 5.51  108 5.67  108 residual plot of method 1 weighted regression was considered.
squares The residual plots for method 2 showed a homoscedastic spread
F value 30 006 0.97 and no weighted analysis was performed. A linear relationship
between the analyte concentration and its corresponding
p 0.444
response for both the methods was established with no
F critical 2.73 (a ¼ 0.1)
No evidence of lack of t evidence of lack of t with p $ 0.1 from the analysis of variance
a b
(Tables 1 and 2).
ANOVA results for linearity assessment. Y: negative peak area, X:
The limit of detection was calculated to be 0.062% and the
ethanol concentration (% v/v).
limit of quantication was 0.19%. However, 0.1% (v/v) was
observed to lie within the linear quantiable range with no

Table 3 Validation results for methods 1 and 2. Calibration curve for method 1 with weighted linear regression

Inter-day variationsa Intra-day variationsb

Ethanol
Standard concentration Mean Standard
Mean (area a.u) deviation % RSD % accuracy % recovery (% v/v) (area a.u) deviation % RSD % accuracy % recovery

Method 1
2981.00 140.68 4.72 96.30 103.84 0.10 2964.80 184.06 6.21 95.28 104.96
15 133.40 190.85 1.26 97.46 102.61 0.50 15 083.52 103.68 0.69 97.76 102.29
30 495.00 331.31 1.09 96.19 103.96 1.00 30 255.40 692.21 2.29 96.09 104.06
152 239.40 1462.39 0.96 97.90 102.14 5.00 154 254.60 2008.61 1.30 96.51 103.61

Method 2
345 668.42 3559.12 1.03 99.88 99.88 10.00 344 385.22 2137.40 0.62 99.73 100.27
823 402.60 3277.72 0.40 98.84 98.85 25.00 823 199.72 5757.19 0.70 98.86 98.88
1 669 659.60 10 621.72 0.64 99.09 100.92 50.00 1 660 322.40 11 271.35 0.68 98.53 100.46
3 236 016.80 22 298.87 0.69 99.29 99.30 100.00 3 248 955.20 26 180.64 0.81 99.25 99.25
a
Mean of 5 intermittent injections (each triplicate) on a daily basis. b Mean of 5 intermittent injections (each triplicate) on an hourly basis.

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evidence of lack of t. The usage of lower measurement viz. For precision analysis the use of a non-thermostated auto-
0.1% may purely depend on an agreement between the labo- sampler introduced errors in the analysis (data not shown).
ratory, end user's data and the intended purpose.13 Since the study involved volatile solvents such as acetone and
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Fig. 6 Chromatograms of test samples (A) whiskey, (B) vodka, and (C) bioethanol fermentation broth.

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Analytical Methods
ethanol the use of a temperature controlled autosampler is
crucial. Repeatability was thus achieved with a temperature
controlled autosampler; with the % RSDs of intra-day and inter-
day precision experiments falling within the acceptable limits.
The experiment also gives information on the errors which
seem to decrease proportionately with increasing ethanol
concentrations. % accuracy and % recovery mentioned in Table
3 also take into account the systematic errors.
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Test sample analysis
Injection of commercial alcoholic beverages, both clear and
coloured, gave clean chromatograms which are benecial for
easy detection and quantication. The percentage of ethanol
content detected in the two commercial alcoholic beverages
whiskey and vodka was 43.03  0.25% (v/v) and 43.01  0.10%
(v/v) respectively. The labelled values for both the samples are
42.8% (v/v) of ethanol. Samples derived from the anaerobic
fermentation broth of Clostridium thermocellum were checked
for the presence of bioethanol at the end of a 120 hour batch
and was found to be 0.114  0.01% (v/v). The result was
comparable to the mean value as obtained with the GC-FID
(0.117% v/v). The results are also in agreement with the pub-
lished data.19 When the fermentative broth was centrifuged,
ltered and injected directly, the presence of certain early
eluting positive peaks was observed but did not interfere with
the concerned peaks. However, this will purely depend on the
nature of the fermentative medium composition. It is suggested
to perform a simple distillation as mentioned in the “Experi-
mental” section to avoid the interference of all the non-essential
positive peaks (Fig. 6).
Feasibility of methanol quantication and analysis
The developed method also could be extended to the analysis of
methanol in ethanol. The presence of methanol in spurious
non-commercial liquors is a global concern causing serious
health issues in consumers.25 Trials for the detection of meth-
anol separately and in combination with ethanol have been
attempted. Standard methanol in water (0.1–100% v/v) was
injected under the same conditions followed for the ethanol
quantication. A positive peak at 2.77  0.01 min varied linearly
for all methanol concentrations singly or in combination with
ethanol. Marginal shis in the RTs were observed even for the
characteristic peaks of ethanol (ESI†). Further investigations
into peak trends and their behaviours and method validation
will be done in future.
Conclusion
Direct identication of a negative peak linear to ethanol
concentration is studied and conrmed in the present work.
Even though linearity in the ethanol quantication was
observed from 0.1–100% (v/v), the use of two injection volumes
for two sets of concentration ranges (0.1–5% and >5–100%) is
suggested to avoid peak splitting and peak merging at higher
ethanol concentrations. This method proves to be an alternative
to other ethanol detection techniques in chromatography. This
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Technical Note
approach is simple, user-friendly and does not require any
chemical modication or the use of any secondary UACs or
costly reagents. A HPLC instrument with a thermostated auto-
sampler is suggested for accurate and precise results. The
method is applicable for the detection of ethanol in fermenta-
tive samples and alcoholic beverages and its avenue can be
widened by analysing its feasibility in medical and pharma-
ceutical samples. The feasibility of methanol quantication was
also studied with one of the representative peaks linear across
all methanol concentrations. The scope of this study can be
extended to higher alcohols as well.
Conflict of interest
The authors declare that they have no conict of interest.
Acknowledgements
Support by the Department of Science and Technology (DST)
under Women Scientist Scheme A (SR/WOS-A/LS-196/2011(G)),
New Delhi for author N. M. is gratefully acknowledged. We also
extend our acknowledgement to SRM University for providing
instruments and facilities.
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