Food Chemistry 217 (2017) 756765

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

A comprehensive approach for milk adulteration detection using

inherent bio-physical properties as Universal Markers: Towards a
miniaturized adulteration detection platform
Suryasnata Tripathy, Aniket Ramesh Ghole, Khandavalli Deep, Siva Rama Krishna Vanjari,
Shiv Govind Singh
Indian Institute of Technology Hyderabad, Telangana 502285, India

a r t i c l e

i n f o

Article history:
Received 28 April 2016
Received in revised form 5 September 2016
Accepted 6 September 2016
Available online 9 September 2016
Chemical compounds studied in this article:
Starch (PubChem ID: 439341)
Boric acid (PubChem ID: 7628)
Ammonium sulphate (PubChem ID:
6097028)
Sodium hydroxide (PubChem ID: 14798)
Sodium carbonate (PubChem ID: 10340)
Sodium bicarbonate (PubChem ID: 516892)
Allantoin (PubChem ID: 204)
Formalin (PubChem ID: 712)
Urea (PubChem ID: 1176)
Semicarbazide (PubChem ID: 5196)
Benzoic acid (PubChem ID: 243)
Cyanuric acid (PubChem ID: 7956)
Melamine (PubChem ID: 7955)

a b s t r a c t
This paper proposes a novel milk quality detection approach based on utilization of inherent biophysical
properties as markers for adulteration. Unlike the traditional adulterant-specific approaches, this
method is generic and universal. It exploits the change in innate milk properties, such as electrical conductivity and pH, upon addition of adulterants as a transduction mechanism for detecting milk adulteration. In this work, adulteration with more than 10 commercially known hazardous adulterants is
detected by monitoring the changes in milk electrical conductivity and pH. The electrical parameters
for pure milk were standardized using AC impedance-spectroscopy with glassy carbon working electrode
and platinum counter/reference electrode at a potential of 0.3 V and in the frequency range of 1 Hz
1 MHz. The experiments were repeated using gold-electrodes fabricated on glass-substrate as a first step
towards developing a miniaturized platform. The concept of a unified-universal-marker for successful
prediction of adulteration is accentuated in this work.
2016 Elsevier Ltd. All rights reserved.

Keywords:
Milk adulteration
Bio-physical properties
pH
Impedance spectroscopy
Unified-universal-marker

1. Introduction
In recent times, availability of quality milk has become a major
problem across the world, specifically in the Indian subcontinent,
owing to unwarranted milk adulteration practices. Challenges
associated with milk adulteration are wide felt due to the lack of
proper device and methodology to tackle the problem at hand.
Corresponding author.
E-mail addresses: suryasnata.tripathy@gmail.com (S. Tripathy), ee13m1022@
iith.ac.in (A.R. Ghole), ee14mtech11017@iith.ac.in (K. Deep), svanjari@iith.ac.in
(S.R.K. Vanjari), sgsingh@iith.ac.in (S.G. Singh).
http://dx.doi.org/10.1016/j.foodchem.2016.09.037
0308-8146/ 2016 Elsevier Ltd. All rights reserved.

Dilution of milk by water to increase the overall volume for commercial purposes is the most common form of milk adulteration
(Afzal, Mahmood, Hussain, & Akhtar, 2011; Mabrook & Petty,
2002, 2003a; Shaikh, Soomro, Sheikh, & Khaskheli, 2013). However
milk diluted with fresh water is not very harmful for the consumer,
apart from the nutritional issues. The problem has become very
severe with the availability of synthetic milk in the market
(Sadat, Mustajab, & Khan, 2006). Commercially available milk is
found to be adulterated with a number of chemicals such as
hydrated lime, sodium hydroxide, sodium carbonate, sodium
bicarbonate, hydrogen peroxide, formalin, sugar, urea, benzoic
and salicylic acids, tertiary nitrogen compounds, borax and boric

S. Tripathy et al. / Food Chemistry 217 (2017) 756765

acids (Handford, Campbell, & Elliott, 2016). The purpose of milk

adulteration with these adulterants has been diverse, such as to
alter the overall composition or acidity, to work as preservatives
in milk, for extension of shelf life, to increase the overall nitrogen
content etc. (Singh & Gandhi, 2015). Table 1 (Supplementary material S1) provides a detailed list of the commonly used adulterants
and their impact on milk and milk properties. Most of these adulterants are hazardous and cause irreversible damage to the organs
(Chen, 2009; Handford et al., 2016; Hau, Kwan, & Li, 2009). The
Indian Council of Medical Research in an earlier report (Toteja,
Dasgupta, Saxena, & Kalra, 1993) had mentioned that detergents
in milk caused food poisoning and gastrointestinal complications
while the other synthetic compounds caused impairments, heart
problems, cancer and sometimes turned out to be fatal. The implications of consuming adulterated milk are far more complicated
and long lasting in case of children. It is imperative to detect the
milk adulteration just prior to consumption. Detection methodologies that are amenable to miniaturization would enable bulk
production of low cost disposable devices for milk adulteration
detection.
The general milk adulteration detection techniques involve
chemical tests that are cumbersome, require laboratory setup
and are not comprehensive. Furthermore, such methods require
an elaborative approach with separate tests to be performed for
identification of individual adulterants. The economic aspects of
such methods can always be debated. The most commonly used
instrument which checks for the level of adulteration in a given
sample of milk is the lactometer. It works on the principle of density variation of the milk samples upon adulteration. However,
alternative methods are being adapted to adulterate milk with a
carefully chosen combination of adulterants without altering the
density of milk, thereby limiting the efficiency of lactometer.
Several sophisticated methods such as Surface Enhanced Raman
Scattering (Kim, Barcelo, Williams, & Li, 2012), Enzyme Linked
Immunosorbent Assays (Hurley, Coleman, Ireland, & Williams,
2004), Near Infrared Spectroscopy (Borin, Ferrao, Mello, Maretto,
& Poppi, 2006), capillary electrophoresis (Muller et al., 2008), Liquid Chromatographymass spectrometry analysis (Czerwenka,
Muller,
&
Lindner,
2010),
High
Performance
Liquid
Chromatography-mass spectroscopy (Abernethy & Higgs, 2013),
Macro-scale Raman Chemical Imaging (Qin, Chao, & Kim, 2013)
and gas chromatography-mass spectroscopy (Dai et al., 2010) are
reported to be used for milk adulteration detection. For detection
of cow milk samples adulterated with milk from other animal species, several immunological techniques and techniques based on
polymerase chain reaction (PCR) have been reported in the literature (Bania, Ugorski, Polanowski, & Adamczyk, 2001; Borkova &
Snaselova, 2005). Adulteration detection methods based on sensitive two-site ELISA (Levieux & Venien, 1994), DNA-based screening
(Klotz & Einspanier, 2001) and capillary electrophoresis (Cartoni,
Coccioli, Jansionowska, & Masco, 1999) have also been reported
previously for similar applications. The possibility of combining
Kjeldahl and classical spectrophotometric methods for desired
screening of milk or whey protein adulteration with urea, melamine and ammonium sulphate has been discussed by Finete,
Gouvea, Marques, and Netto (2013). However such methods
mostly require high end equipments and are also adulterant specific in general. Detection of adulteration for multiple adulterants
with such techniques can be very time consuming and cost
ineffective.
A great deal of research work has also been carried out in the
past targeting electrical conductivity of milk for detection of adulteration. OConner (1979) reported an automated impedance measurement method for bacteriological quality determination of raw
milk. Veiga and Bertemes-Filho (2012) provided a bioelectrical
impedance analysis of bovine milk with a different content of fat.

757

Impedance spectroscopy and dc conductivity measurement of

whole milk, low fat milk and fat free bovine milk were carried
out using an FPGA based impedance measuring system comprising
of a digital oscilloscope and a current source. Mabrook and Petty
(2003a) reported a novel technique for detection of added water
to full fat milk using single frequency admittance measurements.
Liang, Zhang, and Qin (2009) reported a molecularly imprinted
polymer based potentiometric sensor for determination of melamine in milk. The total analysis period for the reported sensor
was reported to be less than 15 min including the sample pretreatment time. A water compatible molecularly-imprintedpolymer using cyromazine as a mimic template was reported by
He et al. (2009) for solid phase extraction of melamine from feed
and milk samples. Liu et al. (2011) reported a poly (paraaminobenzoic) acid film modified glassy carbon electrode based
electrochemical system for determination of melamine in milk.
Given a plethora of milk adulterants, adulterant specific
approach is not practically viable. An alternate approach for detection of milk adulteration, which is not adulterant-specific rather is
comprehensive and generic in nature, is imperative for development of a single device/sensor platform to detect all the known
adulterants in milk. Such a miniaturized platform would avoid
manipulation and tampering, as milk samples can be easily examined at several points in the supply chain for detection of adulteration without the need of any complex laboratory setup. In that
regard, we propose to use certain unique bio-physical properties
of milk as parameters or markers for adulteration detection. The
markers are expected to get affected or altered in presence of adulterants in a manner liable to be detected by an appropriate sensing
mechanism. The basic difference in the proposed method and the
existing adulterant specific approaches is the fundamental principle of looking at milk properties rather than targeting the individual adulterants separately. A brief description of the proposed lab
on chip platform using bio-physical properties as markers for adulteration detection can be found in the Supplementary material S2.
In this paper, the efficacy of the electrical conductivity and the
pH of milk as potential markers are demonstrated. The presence of
various ions in milk accounts for its electrical conductivity. The
conductivity can be easily monitored by applying a voltage across
two electrodes dipped in a milk sample and measuring the current,
or vice-versa. Addition of ionic adulterants is expected to alter the
milk conductivity by providing additional charge carriers to the
colloidal entity. Increase in milk acidity by adulteration would also
alter the conductivity by altering the amount of dissolved ionic
content. Similarly, adulteration detection by monitoring the
changes in pH is also quite feasible as addition of basic or acidic
chemicals is likely to alter the overall acidity of milk. As reported
in the literature, pH of fresh cow milk ranges from 6.6 to 6.8
(Atasever, Erdem, & Altop, 2010; Corredig & Dalgleish, 1996; Lu
et al., 2013). Adulteration detection by correlating the changes in
the selected markers to the presence of several commercially
known adulterants (individually as well as for a combination of
more than one) is reported in this work. Though adulteration of
milk without altering these markers individually is possible by a
suitable combination of adulterants, it is difficult to completely
undo the changes in both the markers simultaneously. Hence, we
propose to use both these markers together as a unified marker
for adulteration detection.
2. Materials and methods
2.1. Materials
All the chemicals (urea, boric acid, benzoic acid, sodium hydroxide, sodium carbonate, sodium bicarbonate, ammonium sulphate,
salicylic acid, semicarbazide, formalin, cyanuric acid, allantoin,

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S. Tripathy et al. / Food Chemistry 217 (2017) 756765

starch and melamine) used in this study were purchased from

Sigma Aldrich, USA and were used as purchased without any extra
purification. Deionized water used in this study was obtained from
a Millipore water purifier system. The glassware used for sample
preparation and storage were obtained from Hychem Laboratories
and were cleaned using standard protocols.
2.2. Sample preparation
Fresh cow milk samples from several local sources were collected for establishing the threshold values for pure milk impedance and pH. Once such limiting values were established, each
collected sample was tested first before being accepted for further
study and was discarded if there was any unacceptable degree of
departure from the pre-decided thresholds. To prepare the test
samples, defined addition of adulterants to 10 ml of fresh milk
was performed and the mixture was stirred gently to allow proper
dissolution of the added chemicals and to avoid clot formation.
When not in use, the fresh and the adulterated samples were covered and kept under refrigeration at 48 C to avoid any damage to
the sample properties due to external ambient. Prior to any measurements, the samples were brought to the room temperature
(25 C). The samples were left at the normal room temperature
ambient for several minutes to allow the temperature and other
components to stabilize. The sample volume was kept constant
at 10 ml throughout all the experiments in order to maintain a
standard testing procedure. All the experiments were carried out
at room temperature.
2.3. Electrode fabrication
The gold electrodes for impedance spectroscopy were fabricated on glass slides using standard microfabrication steps. The
glass slides were cleaned using Piranha Cleaning followed by
cleaning with acetone, isopropyl alcohol and de-ionized water.
The cleaned slides were blown-dry using N2 and then heated at
220 C for 15 min for removing the moisture-content. Subsequently, the substrates were subjected to optical-lithography for
developing the desired electrode pattern using a positivephotoresist and a photomask. Metal deposition on the patterned
substrates was carried out using a multisource DC sputtering system (AJA International, USA). The metal deposited substrates were
subsequently subjected to a lift-off process for obtaining the
desired electrode pattern. The process flow for electrode fabrication and the fabricated electrode dimensions are shown in Supplementary material S3. The thickness of the metal layer was 200 nm
(Titanium 20 nm + Gold 180 nm) which ensures a minimal drop of
voltage between the pads and the tip of the electrodes. For each
measurement, an identical portion of the electrodes was dipped
in the sample under test.
2.4. Impedance spectroscopy
Impedance spectroscopy is a widely used and an effective technique for characterizing biological samples based on their electrical response over a particular frequency range. In general, an
alternating voltage is applied to the desired sample and both the
in-phase and the out-of-phase currents, related to the resistive
and capacitive behavior of the sample respectively, are monitored
over a specified frequency range using a two or three electrode system. Application of dc voltage for such measurements results in
electrolysis and polarization of the electrodes, reducing the current
through the external circuit to zero over time (Mabrook & Petty,
2003b). The electrical impedance between two electrodes
immersed in an electrolyte (such as milk, in this study), can be
modeled using a series and parallel combination of different

resistive and reactive elements representing the faradaic and the

mass-transfer processes associated with the system (Franks,
Schenker, Schmutz, & Hierlemann, 2005).
The initial studies for impedance analysis of the pure and the
adulterated samples were performed using an Electrochemical
Workstation (Model-660E, CH Instruments, USA) with a 3 mm
Glassy carbon working electrode, a platinum wire counter/reference electrode and the milk sample as electrolyte, in a frequency
range of 1 Hz1 MHz. A schematic representation of the experimental setup has been provided in the Supplementary material
S4. For every analysis, the glassy carbon working electrode was
carefully polished using alumina slurry, pre and post measurement. The cleaning of electrodes was strictly monitored to avoid
any unintended interference in the impedance measurements.
Subsequently, two-electrode ac impedance spectroscopy at a constant frequency of 100 kHz was carried out using gold electrodes
fabricated on glass substrates such that a lab-on-chip platform
can be developed in future. In all these cases, the impedance spectroscopy was carried out with a supply voltage of amplitude
300 mV and an ac perturbation of amplitude 10 mV. Each measurement was repeated several times for verification of accuracy of the
results.
2.5. Measurement of pH
Milk pH measurement for detection of adulteration was carried
out using a handheld pH meter (Waterproof pHTestr-30, Eutech
Instruments, Singapore). The pH meter was calibrated in regular
intervals using three standard pH solutions of pH 4, 7 and 10
respectively. The pH values of pure and adulterated samples were
recorded by dipping the pH sensitive electrode in to 10 ml of sample volume.
3. Results and discussion
3.1. Milk electrical conductivity as a marker for adulteration detection
The effectiveness of electrical conductivity of milk as a potential
marker for detection of adulteration was investigated in this study.
The efficiency of any marker can be estimated based on its sensitivity with respect to the adulterants (i.e. the number of adulterants
that can be successfully detected in milk by monitoring any particular marker) and the limit of detection with respect to sensing (i.e.
the minimum amount of adulterant that should be added in to
milk in order to induce an appreciable order of change in any marker which can be accurately sensed). For validation of efficiency of
electrical conductivity of milk as a potential marker, the limit of
detection (LOD) for each adulterant under study was determined.
The concept of LOD has been used differently in this work in addition to the conventional meaning of the word. As the pure milk
samples under study have a range of impedance values, it is difficult to determine a fixed impedance value as the reference point
against which the LOD for adulterated samples can be determined.
Thus, for determining the LOD in case of impedance measurements, we have imposed the criterion that for any adulterated
sample, a minimum of 25 X deviation from the pure milk impedance would be considered as a detectable change. Addition of
any amount of adulterant resulting in an impedance change of less
than 25 X has been considered as undetectable adulteration in this
study.
The threshold for differentiating pure milk from the adulterated
sample was established by measuring the impedance of 15 milk
samples collected from different local sources. It is well known
from literature that the impedance of cow milk varies across breed
and also in accordance with seasonal changes, composition,

S. Tripathy et al. / Food Chemistry 217 (2017) 756765

storage condition, lactation period, health condition and nutrition

(Fernandez-Martin & Sanz, 1985; Sheldrake, Hoare, & Mcgregor,
1983; Yarabbi, Mortazavi, Mehraban, & Sepehri, 2014). Hence,
impedance of the locally available fresh milk (used in this study)
may differ from the data previously reported in literature
(Bertemes-Filho, Valicheski, Pereira, & Paterno, 2010; Das,
Goswami, & Biswas, 2014). However as the sole purpose of the
study was to investigate the effectiveness of milk impedance as a
marker for adulteration detection, the locally available pure milk
was considered as the standard reference sample. The standardization of milk impedance is presented in Fig. 1. As shown in Fig. 1(a),
the impedance of milk samples is very high in the low frequency
range because of the reactances associated with it. In the higher
frequency range, the reactances have negligible effect on milk
impedance and hence a constant resistive behavior is observed,
as shown in Fig. 1(b). Most of the locally available samples were
observed to have an impedance value between 620 and 650 X, in
the high frequency range. This impedance value was selected to
be the reference or threshold for differentiating pure and adulterated sample. Any fresh sample with impedance values outside the
selected range was considered to be already tampered with and
was not selected for further investigation. As shown in Fig. 1(c),
the impedance of cow milk collected on five different days from
the same source also showed some variation. Hence selection of
standard sample for the proposed study was done carefully after
thorough experimentation.
Fig. 2 shows the variation in milk impedance for adulterants
such as tap water, sodium hydroxide, sodium carbonate, formalin,
allantoin, ammonium sulphate, benzoic acid and cyanuric acid. In
case of tap water, the impedance of adulterated sample was found
to be higher than that of the pure sample because the inherent

759

ionic concentration was reduced upon dilution. The limit of detection for addition of water was found to be 5% by volume. In case of
NaOH, the limit of detection was as low as 0.3 mg/ml. Addition of
NaOH below this threshold did not result in any significant
changes in the electrical impedance of milk. The concentration of
NaOH in the adulterated milk samples was varied up to 1.5 mg/
ml and with increase in the concentration beyond the reported
LOD there was a decrease in the overall impedance of the adulterated sample, as shown in Fig. 2(b). For low concentrations of the
adulterant in milk (up to 0.7 mg/ml), rapid change in the overall
impedance of the adulterated sample was not observed. However,
for higher concentrations (beyond 1 mg/ml), there was significant
deviation in the electrical impedance of the adulterated sample
from that of the pure milk. For sodium carbonate and formalin,
the limit of detection was found to be 0.05 mg/ml and 10% (volume/volume) respectively. Addition of both sodium carbonate
and formalin enhanced the conductivity of milk. Below the specified LOD, addition of formalin did not alter the electrical behavior
of milk significantly. In case of allantoin, benzoic acid and cyanuric
aid, with increase in the concentration of the adulterants, reduction in the conductance of milk was observed. Essentially, in the
colloidal entity, electrical conduction is influenced by mass transfer or diffusion along with the dissolved ionic content. Addition
of non-ionic adulterants may inhibit efficient mass transfer in the
bulk sample thereby influencing the conductive property. In case
of acidic or basic adulterants, the dissolved ionic content in milk
changes as the overall acidity of milk affects the protein coagulation thereby accepting/releasing mineral salts from/to the colloidal
phase. An increase or decrease in dissolved ionic content in the
serum depending upon formation or dissolution of protein particles directly affects the electrical behavior of milk. Addition of

Fig. 1. Standardization of pure milk impedance: (a) Variation of milk impedance across 15 samples in the frequency range of 1 Hz1 MHz (b) Variation of milk impedance
across 15 samples in the frequency range of 100 kHz1 MHz. (c) Variation of milk impedance for 5 samples, collected from a single source, in the frequency range of 100 kHz
1 MHz.

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S. Tripathy et al. / Food Chemistry 217 (2017) 756765

Fig. 2. Impedance spectroscopy results in the frequency range of 100 kHz1 MHz for milk sample adulterated with (a) Tap water, (b) Sodium Hydroxide, (c) Sodium
carbonate, (d) Formalin, (e) allantoin, (f) ammonium sulphate, (g) benzoic acid, and (h) cyanuric acid. [In case of Formalin (d), the legend enlists the amount of adulterant
added in 10 ml of pure milk.]

ammonium sulphate enhances milk conductivity as shown in Fig. 2

(f). The limit of detection in case of ammonium sulphate was found
to be 1 mg/ml.
Similar experiments were performed for other adulterants such
as urea, starch, boric acid, semicarbazide and sodium bicarbonate

and the results for the impedance spectroscopic studies are shown
in Supplementary material S5. The impedance values for adulterated milk samples containing urea up to 0.2 mg/ml did not provide
any appreciable deviation from that of pure milk sample. So the
proposed impedance spectroscopy based adulteration detection

S. Tripathy et al. / Food Chemistry 217 (2017) 756765

method could not distinguish between pure milk and adulterated

milk sample with urea content up to 0.2 mg/ml. However for
higher urea content, the impedance of adulterated milk sample
was found to be significantly different from that of pure milk. In
case of starch, the limit of detection was found to be 0.12 mg/ml.
Addition of starch in fresh milk increased the overall impedance
and at higher frequencies, a change in resistance in excess of
200 X (as compared to that of pure sample) was observed for
starch concentration 0.15 mg/ml. In case of semicarbazide and
boric acid, the lowest detection limit was found to be 5 mg/ml
and 0.1 mg/ml respectively. Addition of small amount of semicarbazide did not induce any significant changes in the fresh milk
impedance. Hence it is not possible to detect milk adulteration
with small quantities of semicarbazide using the proposed impedance spectroscopy based technique. In case of boric acid, the
impedance values for the adulterated sample do not follow a particular pattern with increase in the amount of adulterant. Up to an
adulterant concentration of 0.5 mg/ml, the high frequency impedance of the adulterated sample increases with increase in amount
of adulterant added as compared to that of pure sample. However,
with increase in adulterant concentration beyond 1 mg/ml, the
high frequency impedance of the adulterated sample decreased
as compared to that of the pure sample. It is a difficult task to comprehend the explicit chemistry behind such a behavior. Our

761

presumption is that up to a particular limit, addition of boric acid

does not alter milk acidity significantly and only affects the bulk
diffusion. After a particular concentration, addition of boric acid
may alter the overall ionic content of milk serum by affecting the
overall milk pH thereby disturbing the stability of protein complexes. For adulteration with sodium bicarbonate, the limit of
detection was found to be 0.03 mg/ml with the overall conductivity increasing with increase in the adulterant concentration. However, in case of melamine, no significant change in impedance was
observed for lower concentrations. For higher concentrations, the
experimental results obtained do not seem to follow a particular
pattern and the impedance values corresponding to a particular
adulterant concentration was variable for different observations.
An in-depth understanding of behavior of melamine in milk is
required to account for the above mentioned behavior of a milkmelamine system.
The experiments were also performed using gold electrodes
fabricated on a glass substrate using standard micro fabrication
technique. The sample volume and sample preparation process
were identical to the previously described procedure which was
followed for impedance spectroscopy with glassy carbon and platinum electrodes. The experimental findings are presented in Fig. 3.
The reason behind performing the experiments at a constant high
frequency value was to realize a lab-on-chip impedance

Fig. 3. Variation of Normalized change in impedance with concentration of adulterants in milk samples adulterated with (a) Salicylic acid, (b) Sodium hydroxide, (c) Benzoic
acid, (d) Starch, (e) Urea, (f) Ammonium Sulphate, (g) Cyanuric acid, and (h) Allantoin.

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S. Tripathy et al. / Food Chemistry 217 (2017) 756765

measurement setup for adulteration detection in future using milk

impedance as a marker. As shown in the figure, at higher frequencies, measurement of milk impedance gives a clear indication of
quality of the sample under test. Normalization of impedance has
been done to highlight the deviation of impedance of the adulterated sample from that of fresh milk as it is essential to have a measurable/detectable deviation from the threshold value in order to
develop a lab-on-chip read-out circuit with absolute precision.
The normalized change in impedance can be defined as:

Normalized change in Impedance

jZjs  jZj0
jZj0

where jZjs and jZj0 represent the magnitude of the impedance for
adulterated and pure milk samples respectively.
With the fabricated gold electrode, no significant deviation was
observed in relation to the limit of detection for different adulterants as compared to the results obtained using a glassy carbon
working electrode. The limit of detection for different adulterants
is tabulated in Table 1.
3.2. Milk pH as a marker for adulteration detection
The effectiveness of milk pH as a potential marker for adulteration detection was also investigated in this work. Detection of
adulteration using pH has not been a very popular approach among
researchers as it is quite easy to nullify the effects of addition of
adulterants on milk acidity. However, it is still very convenient
to estimate the purity of a milk sample from its pH when the adulterants are basic or acidic in nature. Chemicals that do not affect
the pH of milk cannot be detected by monitoring this marker.
We therefore intend to use milk pH as a potential marker in conjunction with electrical conductivity of milk. The idea being in case
of adulterants that do not affect the pH of milk, there will be some
alteration in the electrical properties or vice versa.
For standardization of pH range for fresh milk, samples were
collected from 15 healthy cows belonging to different local sources
and their pH was measured repeatedly over a period of several
days. Most of the samples were found slightly acidic with a pH
range of 6.66.9. To study the variation of pH for a single sample,
milk samples were collected from a single cow for a period of
8 days and the corresponding pH values were monitored. Such a
study was performed in order that a proper threshold could be
established. As shown in Fig. 4(a), there was not any significant
variation in the pH value across different samples. After conducting
standardization experiments for a period of two weeks, the threshold range for pH value of fresh milk was fixed between 6.6 and 6.9.
Post standardization of pH, adulterated samples were prepared by
Table 1
Limit of Detection (LOD) for different adulterants using milk electrical conductivity as
the marker for adulteration detection.
Name of the Adulterant

LOD (with Glassy carbon

and Platinum Electrode)

LOD (with Gold

electrode)

Tap water
Urea
Sodium hydroxide
Sodium carbonate
Ammonium sulphate
Allantoin
Sodium bicarbonate
Starch
Benzoic Acid
Boric Acid
Salicylic Acid
Cyanuric Acid
Semicarbazide
Formalin

5% (v/v)
0.5 mg/ml
0.3 mg/ml
0.05 mg/ml
1 mg/ml
0.05 mg/ml
0.03 mg/ml
0.12 mg/ml
0.06 mg/ml
0.1 mg/ml
0.1 mg/ml
0.1 mg/ml
5 mg/ml
0.1 ml/ml (v/v)

5% (v/v)
0.5 mg/ml
0.5 mg/ml
0.05 mg/ml
1 mg/ml
0.1 mg/ml
0.03 mg/ml
0.3 mg/ml
0.1 mg/ml
0.1 mg/ml
0.1 mg/ml
0.1 mg/ml
5 mg/ml
0.15 ml/ml (v/v)

adding known amount of adulterants to 10 ml of fresh (raw) milk

and subsequently their pH values were measured.
As shown in Fig. 4(bf), for addition of basic and acidic adulterants the pH of milk changed drastically. Determination of LOD was
performed by monitoring the concentration of adulterants present
in the adulterated sample which alters the overall pH beyond the
specified range for unadulterated milk. Addition of any amount
of adulterant which did not result in a change in milk pH beyond
the specified safe range was categorized as undetected adulteration. In case of sodium hydroxide, a detection limit of 0.5 mg/ml
was obtained whereas the LOD for sodium bicarbonate was found
to be 0.7 mg/ml. Addition of these adulterants below the above
mentioned limiting values did not alter the milk pH sufficiently
enough for prediction of tampering. In case of acidic adulterants
such as boric acid and salicylic acid, the limits of detections were
0.3 mg/ml and 0.5 mg/ml respectively. Similar experiments were
performed for other adulterants such as tap water, urea, starch
and sodium carbonate. Pure water has a neutral pH and hence is
not expected to alter the pH of milk. Interestingly, tap water is
not absolutely pure and behaves acidic or basic depending on composition. Upon addition of tap water, we have found significant
variation in milk pH with a limit of detection of 5 percent by volume. However, such a result is not constant and repeatable as
the acidity of tap water is not constant and it varies from source
to source. In case of adulteration with urea and starch, as shown
in Fig. 4(f) and (g) respectively, for adulterant concentrations ranging from 0.1 mg/ml to 5 mg/ml, no significant change in milk pH
was observed. In case of urea, for most of the adulterated samples,
the pH values were found to be within the range of pH values specified for pure milk. Even for adulterant concentration of 5 mg/ml,
the pH value of the adulterated sample was close to 6.5. Likewise,
addition of starch did not result in any drastic deviation of the pH
of the adulterated sample from that of the pure milk. For most of
the adulterated samples used in this study, the pH values were
close to 6.5. Similar results were obtained in case of semicarbazide
where addition of the adulterant does not induce any significant
alteration of milk pH. It was quite palpable that certain adulterants
do not affect the acidity of milk and hence their presence in milk
cannot be detected using pH as a marker. For sodium carbonate,
as shown in Fig. 4(h), the limit of detection was found to be
0.3 mg/ml.

3.3. A unified universal marker

Accurate detection of milk adulteration using any of the proposed markers is not feasible for reasons described before. In order
to strengthen the proposed idea of using multiple markers as a unified universal marker, we have monitored the effect of multiple
adulterants on the selected bio physical properties. As shown in
Fig. 5(a), addition of 1.2 mg/ml of NaOH alters the impedance of
milk significantly which can be easily identified by appropriate
measuring techniques. However, addition of 1 mg/ml benzoic acid
to the same sample almost nullifies the change in milk impedance.
Alternatively, if 1 mg/ml ammonium sulphate is added to the sample containing 1.2 mg/ml NaOH, the impedance value is reduced
further causing no harm to the detection process. But in addition
to ammonium sulphate, if starch is added to the sample, the milk
impedance increases again and approaches the threshold limit
selected for fresh samples. Increase in starch concentration to
5 mg/ml reverses the polarity of change in milk impedance for
the same sample and increases it significantly as compared to that
of pure sample. Similarly, as shown in Fig. 5(b), by the addition of
water in to a milk sample adulterated with 0.1 ml/ml formalin
(10%, volume/volume) the changes in conductance of milk can be
compensated. Such experimental results clearly indicate the

S. Tripathy et al. / Food Chemistry 217 (2017) 756765

763

Fig. 4. (a) Standardization of milk pH; Variation of Milk pH with addition of (b) Sodium hydroxide, (c) Sodium bicarbonate, (d) Boric Acid, (e) Salicylic acid, (f) Urea, (g) Starch
and (h) Sodium carbonate.

difficulties in detecting milk adulteration for multiple adulterants

by solely considering the electrical conductivity as a marker.
In case of adulteration with multiple adulterants, the variation
of milk pH was also investigated for establishing the efficiency of
the proposed marker. As shown in Fig. 5(c), the change in milk
pH on addition of 0.7 mg/ml of boric acid can be nullified by adding
adequate amount of NaOH. Several such combinations of adulterants can be identified with careful experimentation where it is possible to tamper the quality of milk without altering the pH. In such
cases, the measurement of pH is not the ideal option to indicate the
presence of foreign entities in milk.
Alternatively we have also investigated the efficiency of using
both the selected markers as a unified marker for adulteration
detection. As shown in Fig. 5(d), adjustment of pH of adulterated
samples with careful selection of adulterants does not necessarily

roll back the changes in milk impedance. Even for the pH adjusted
sample, the impedance is much higher compared to the threshold
value attributed to fresh milk. Thus by considering milk impedance
as a marker, presence of multiple adulterants in fresh milk can be
detected though the resulting changes in milk acidity is negligible.
Similarly, as shown in Fig. 5(e), we tried to see the effect on milk
pH with a combination of adulterants that cancels the changes in
electrical conductivity of milk. Interestingly, it is still possible to
detect adulteration with formalin and starch by monitoring milk
pH though the same combination cannot be detected by considering electrical conductivity as the marker. Fig. 5(f) shows the scattered plot of Impedance vs pH for both adulterated and pure
samples. It shows a distinct, non-overlapping clustering for
pure milk samples. Any unsupervised clustering algorithms such
as K-means or supervised clustering algorithms such as SVM can

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S. Tripathy et al. / Food Chemistry 217 (2017) 756765

Fig. 5. (a) Variation in milk impedance in presence of multiple adulterants, (b) Effect of simultaneous addition of formalin and water on milk impedance, (c) Bar plot showing
the effect of simultaneous addition of multiple adulterants on Milk pH (d) Bar plot showing adulteration detection by measuring impedance for pH balanced samples; (e) Bar
plot showing adulteration detection by measuring pH for impedance balanced samples; (f) Scatter plot showing variation of impedance with pH for pure and adulterated milk
samples.

be utilized to categorize test samples as adulterated milk or pure

milk. These experimental results strongly support the need to
use multiple markers together as a unified universal marker for
milk quality profiling.
4. Conclusion
In this work, the efficiencies of electrical conductivity and pH of
milk have been investigated for detection of adulteration and milk
quality profiling. We have successfully demonstrated the possibility of detecting the presence of foreign entities in milk by monitoring both of the inherent bio-physical properties. Limit of detection
with respect to a large number of adulterants has been reported in
relation to both the selected markers. The impedance spectroscopic studies were performed using fabricated gold electrodes
as the first step towards realizing a lab-on-chip platform for milk
adulteration detection. The concept of using multiple markers as
a unified universal marker has been laid out and proven with
experimental findings. We have demonstrated a combination of
two markers (Impedance and pH) as a unified mechanism to detect
adulteration of milk immaterial of the adulterants added. The
results suggest that the performance of the combination is far bet-

ter than using the markers individually. Milk samples carefully

adulterated to conceal the changes in any of the two markers have
been identified by monitoring the changes in the other. Milk samples adulterated with Salicylic acid (4 mg/ml) and pH balanced
with subsequent addition of Sodium Hydroxide were identified
as adulterated by monitoring changes in electrical impedance.
Similarly, milk adulteration with Formalin was successfully identified by tracking the changes in pH after the impedance variations
were masked by the addition of starch. Furthermore, for a large
number of milk samples, the impedance values were plotted
against milk pH and the resulting cluster provided a clear distinction between pure and adulterated sample. Hence, combination of
adulterants that does not neutralize the changes in both these
markers simultaneously can be effectively determined using the
proposed approach. However, monitoring a set of biophysical
properties simultaneously would potentially cover the entire gambit of adulterants and thus this combinatory approach makes the
adulteration detection mechanism comprehensive in nature. In
order to create a tamper proof system, the effect of adulterants
on multiple biophysical properties such as turbidity and absorption needs to be analyzed. Despite that, there is still a strong need
to define a safe range corresponding to each selected marker, as a

S. Tripathy et al. / Food Chemistry 217 (2017) 756765

mere deviation from respective thresholds related to fresh milk

does not necessarily guarantee a harmful level of adulteration.
Determination of such safe range for different markers would
essentially enable us to identify the degree of adulteration of any
milk sample, which can be used to successfully profile the quality
of the milk sample. The research aims at simultaneous monitoring
of multiple biophysical properties through a multiplexed lab-onchip platform for milk adulteration detection across the globe, irrespective of the geographical, biological and seasonal variations;
and the present work is a proof of concept in that direction.

Conflict of interest
We hereby declare that there is absolutely no conflict of interest
among the authors regarding the content of this work. We would
also like to ensure that there is no conflict among the authors
regarding the submission of this article to the Journal of Food
Chemistry.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
09.037.
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