Synthesis of Semi Interpenetrating Network Hydrogel

Beads for the Purification of Anthocyanin From Red

Cabbage

Presented by :
B. Shankaranarayanan
M. S. (By Research)
Research Laboratory, Department of Biotechnology
Sri Venkateswara College of Engineering (Autonomous)

Guided by:
Prof. E. Nakkeeran
Professor & Head
Department of Biotechnology
Sri Venkateswara College of Engineering (Autonomous)
Sriperumbudur Tk – 602 117

1. Addition of Gelatin to a
preheated solution of Sodium
alginate maintained at 800C to
form a homogenous mixture.
Synthesis of semi interpenetrating network
(SIN) hydrogel beads
GRAPHICAL SUMMARY
3. Soaking of the beads in
formaldehyde solution to improve Hydrogel
the physical properties of the
beads Column
PURIFIED
JUICE
2. Drop wise addition of the
homogenous solution to
Calcium chloride solution under
constant mixing at 190rpm to Peristalitic Pump
form the hydrogel beads.
FEED
Purification of Anthocyanin
using SIN hydrogel beads
 ANTHOCYANINS

 Anthocyanins are glycosylated poly-hydroxy and poly-methoxy derivatives of 2-

phenylbenzopyrylium salts that are water soluble in nature.
 They are water soluble phenolic compounds having low molecular weight widely distributed in
fruits and vegetables.
 The water soluble nature facilitates its incorporation into aqueous food systems as food
colourants.
 Anthocyanins are beneficial to health as potent anti-oxidants and are known to produce a
variety of biological functions ranging from induction of insulin production in isolated
pancreatic cells, reduction in inflammatory responses to protection against age related brain
function decline.
 Anthocyanins are present in fruits and vegetables such as strawberries, raspberries,
mangoes, figs, red cabbages, purple sweet potatoes etc.
 Red cabbage is a rich source of phenolic compounds and anthocyanins constitute a major
part.
 Anthocyanins present in the red cabbage (Brassica oleracea) are unique as they exhibit
different colours at different pH.
 The colours vary from red at Acidic range to blue and green at neutral and basic ranges
respectively.
 HYDROGELS

 Hydrogels are three dimensional cross linked polymeric networks that are insoluble but
swell when in contact with water or biological fluids.
 They are capable of retaining large amounts of water and it is the property of the hydrogel
which makes it useful in various industries including pharmaceuticals (Sharma et al.,
2014).
 However, the normal hydrogels are highly sensitive towards external conditions such as
change in temperature, pH etc. (Nesrinne et al., 2013).
 Two or three polymers in the form of hydrogels are fused to form a semi-interpenetrating
network to enhance their mechanical strength.
 In the present work, a semi interpenetrating network hydrogel bead was formed by fusing
sodium alginate with gelatin.
 Gelatin was used as a cross linker because, alginate as such beads lack physical stability
and melt under pressure and on reuse (Rakmai et.al., 2015).
 Also the unique physical properties of gelatin such as its melting point close to
physiological temperature, helps to strengthen the alginate beads and thereby adding
physical stability (Mogharabi et.al., 2012).
AIM
 To identify the important parameters and its effects in the purification of
anthocyanin from red cabbage using semi interpenetrating network
gelatin-alginate hydrogel beads.

OBJECTIVES
To identify the various parameters involved in purification of
anthocyanin from red cabbage using semi interpenetrating network
hydrogel beads.
Optimization of parameters using design of experiments to obtain high
purity anthocyanin.
Process Modelling through Breakthrough Analysis.
 SYNTHESIS OF SEMI INTERPENETRATING
NETWORK HYDROGEL

 Gelatin solution of various concentration ranges was

added drop wise over a pre heated solution of sodium
alginate (at various concentration ranges).
 The resulting solution was added drop wise using a tube
over a solution of calcium chloride solution under
constant mixing at 190rpm.
 The resulting beads were soaked for 24hrs in
formaldehyde solution to improve the cross linking.
 The resulting beads were collected and were loaded
onto the column.

Gelatin + Sodium alginate [Gelatin-Sodium Alginate] [Gelatin-Calcium-Alginate] + Na

Heat (80ᵒC)
EXTRACTION OF ANTHOCYANIN FROM RED
CABBAGE

Red Cabbage Shredded Red

Cabbage Blanching of
shredded Red
Cabbage for 2 h

Chilled at 3oC

Red Cabbage Juice Extraction of juice using 0.1% Apple Vinegar

PURIFICATION OF ANTHOCYANIN USING SIN
HYDROGEL

 A 30 cm long glass tube of 2 cm diameter was used as

the column to carry out the experiments.
 35 g of synthesized hydrogel beads of various
compositions and diameters were taken and loaded into
the column.
 Glass wool was placed at both ends to prevent the
expansion of the hydrogel beads under vacuum.
 Silicone tubing coupled with a peristaltic pump was
connected to both the ends to facilitate the transport of
the red cabbage juice to the column.
 Prior to the experiment, water was passed through the
column for 20 min to evacuate air bubbles and impurities
present on the surface of the hydrogel bead.
 500 ml of red cabbage juice was passed from the bottom
using a peristalitic pump at a desired flow rate.
 Effluent samples were collected at regular intervals from
the top of the column. All the tests were performed at
room temperature
 ESTIMATION OF ANTHOCYANIN
CONCENTRATION
 Anthocyanin concentration was determined using pH
differential method by using the following equation
(Lee et al 2005):
A x MW x DF x L
 Anthocyanin Concentration ( mgΤl) = x 1000

 where,
 Absorbance (A) = (A530 − A700)pH 1.0 − (A530 − A700)pH 4.5 ,
 MW is molecular weight of anthocyanin (449.2 g/mol),
 DF is dilution factor,
 ε is the extinction coefficient (26,900 L/cm mol) and
 L is the path length (1 cm) (Hrazdina et al 1977).
ESTIMATION OF ANTHOCYANIN PURITY
 The purity of anthocyanin was determined using the
following equation (Lee et al 2005):

CV
 Purity=
W

 where, C is the concentration of anthocyanins in the

solution, V is the volume of the sample obtained and
W is the total weight of solution obtained.
 ESTIMATION OF CARBOHYDRATE
ELIMINATION CAPACITY
 The total carbohydrate content was estimated using
phenol sulphuric acid method.
 The carbohydrate elimination capacity of the hydrogel
beads was calculated using the following equation
(Chandrasekhar et al 2012):

C0− Ce
 qe mgΤg = x Vi
W
 where,
 qe is the carbohydrate elimination capacity,
 Co and Ce are the initial and final carbohydrate concentrations,
 Vi is the volume of solution used and
 W is amount of hydrogel beads used to carry out elimination.
 THOMAS MODEL
 The Thomas Model is used to identify the effect of feed
flow rate on carbohydrate elimination capacity of
hydrogel bead column.
 The model is designed based on the following
assumptions:
 Negligible axial and radial dispersion in the packed bed column
 The elimination is described by a pseudo second order reaction
rate
 constant column void fraction
 Constant physical properties of the composition of the solution
passed through
 isothermal and isobaric process conditions
 negligible intra particle diffusion and external resistance during
the mass transfer process
 Thelinearized form of the Thomas model is
expressed as:
C0 Kth qow
 ln −1 = − Kth C0 t
Ct v
 where,
 kTh (ml/(min.mg)) is the Thomas rate constant,
 q0 (mg/g) is the equilibrium carbohydrate uptake per g of
the hydrogel bead,
 C0 (mg/L) is the influent carbohydrate concentration,
 Ct (mg/L) is the effluent concentration at time t,
 w (g) is the mass of hydrogel beads used and
 ν (ml/min) represents the flow rate.
 The values of q0 and kTh are determined using the
intercept and slope of the linear plot between ln[(C0/Ct)-1]
against time (t).
 YOON - NELSON MODEL
 The Yoon Nelson model assumes that rate of decrease in
the probability of elimination of carbohydrates is
proportional to the product of probability carbohydrate
elimination and the probability of carbohydrate
elimination breakthrough on the adsorbent.
 The linearized form of the model is expressed as:
Ct
 ln = KYN t− KYN
C0−Ct
 where, kYN (L/min) is the rate velocity constant,
 t(min) is the time in required for 50% carbohydrate
elimination breakthrough.
 The values of t and kYN are identified from the slope and
intercept of the linear plot of ln [Ct / (C0−Ct)] against
sampling time (t).
 CARBOHYDRATE HOLDING CAPACITY
 The maximum carbohydrate holding capacity for
retention of carbohydrates was calculated using the
area under the curve of the plot between rate of
carbohydrate elimination with respect to time.
 The area under the curve was calculated using the
following equation:
Q ttotal
 qtotal = ‫׬‬ Cad dt
1000 0
 Where, Ce, ttotal and Q are the eliminated
carbohydrate concentration (mg/l), total flow time
through the column and volumetric flow rate
(ml/min), respectively.
 The amount of carbohydrate eliminated was found
using the following equation:
 Ce = Co − Ct

 where, Co and Ct are the concentration of

carbohydrates in the feed and effluent, respectively.
DISSOLUTION AND HYDRATION RATE TESTS

 The stability of the hydrogel beads was tested using

dissolution and hydration rate tests.
 In the dissolution test, 10g of beads were placed in
10ml of HCl and NaOH solutions prepared at various
pH ranges.
 The weight of the beads were determined at regular
intervals.
 Therate of dissolution of the beads was identified
using the following equation:
Wf
 Dissolution % = x 100
Wi
 where, Wi and Wf denote the initial and final weights
of the hydrogel beads, respectively.
 To determine the hydration rate of the beads, 10 g of
hydrogel beads were taken and subjected to drying
at room temperature at 121°C.
 The rate of hydration of the beads was calculated
using the following equation:
Wh − Wd
 Hydration Rate % = x 100
Wh
 where, Wh is the weight of the beads before drying
and Wd is the weight of dehydrated beads.
 Hydration rate indicates the mass and water
contents in the hydrogel beads.
 Smaller the hydration rate, greater the mechanical
strength of the hydrogel beads.
Results and Discussion
 Effect of Hydrogel Composition and
Feed Flow Rate on Anthocyanin Purity
 Sum of Contribution
Source DF Co-Efficient P- Value
Squares (%) ANALYSIS OF
Constant - 1.1271 - - 0.000 VARIANCE
Main Effects  Two-way interactions between
A 1 -0.1210 0.46883 26 0.022 gelatin and calcium chloride and
B 1 -0.0645 0.13331 7 0.195 three-way interactions between
C 1 -0.0076 0.00183 0 0.876
gelatin, sodium alginate and
E 1 0.0353 0.03995 2 0.470
2-Way Interactions flow rate were significant in

AB 1 0.1061 0.36024 20 0.041 affecting anthocyanin purity.

AE 1 0.1680 0.01146 1 0.003
BC 1 -0.0189 0.10603 6 0.697  The main effect of formaldehyde
BE 1 0.0576 0.17149 9 0.697 and its interaction effects with
3-Way Interactions other parameters showed high
ABC 1 -0.0732 0.01528 1 0.654
degree of insignificance.
ACE 1 -0.0219 0.14649 8 0.011
BCE 1 0.1368 0.18964 10 0.175
 Hence, eliminated from the
4-Way Interactions
ANOVA table. The result
ABCE 1 -0.0770 0.18964 10 0.126
confirmed the inert nature of
Residual
17 - 0.18964 - - formaldehyde
Error

Total 29 - 1.83419 - -
72
R-Sq - - - -
%
 Considering the co-efficient of significant parameters,
purity was modeled using the following equation:
 Purity Fold = 1.1271 - 0.1210 A + 0.1061 AB + 0. 1680
AE - 0.0219 ACE
 NORMAL
PROBABILITY
PLOT
 The type of effect induced by the
significant parameters on purity
of anthocyanins was found using

the normal probability plot.

 Two way interactions between

gelatin and calcium chloride and

three way interactions between
concentration of gelatin and

sodium alginate and flow rate

showed positive effect of above
70% in affecting the purity of

anthocyanins

 Concentration of gelatin showed

negative effect on the purity

OPTIMIZATION USING
DESIRABILITY
FUNCTION
 Two-fold increase in purity of
anthocyanin was obtainable
with a desirability value of 1
when

 Red cabbage was passed in

column with a flow rate of 5
ml/min through hydrogel
column prepared with 1%
gelatin combined with 5%
sodium alginate and 1%
calcium chloride.
CARBOHYDRATE
ELIMINATION
CAPACITY
 The elimination capacity correlates
with the properties of the hydrogel
beads towards retaining

carbohydrates. Physical features

of the hydrogel also played an
important role in process of

elimination of carbohydrates

 Maximum carbohydrate elimination

capacity of 35 mg/g was obtained

when feed flow rate was
maintained at 5 ml/min and the

hydrogel bead was prepared using

1% gelatin, 5% sodium alginate
and 1% calcium chloride.
 EFFECT OF BEAD
Experimental levels of Factors used to SIZE AND REFLUX
Estimate Effect of Bead Size and Reflux Rate RATE ON PURITY
FOLD
Range and level
Independent Variables when hydrogel beads were
-1 +1 synthesized with bead size
Bead Size 400 µm 3 mm of 400 µm and when no
refluxing was carried out, a
Reflux Rate 1 5
two fold increase in purity
was seen with a maximum
Effect of Bead Size and Reflux Rate on Purity carbohydrate elimination
and Carbohydrate Elimination Capacity capacity of 15.3 mg/g.

Carbohydrate Thus, it was concluded that

Bead Size Reflux rate Purity Fold Elimination Capacity bead size and reflux rate
should be maintained at
(mg/g)
minimal levels to achieve
3 mm 5 1 3.3
greater purity and
0.4 µm 5 2 11.5
0.4 µm 1 2 15.3
carbohydrate elimination.
3 mm 1 1 11
PERFORMANCE MODELING
 THOMAS MODEL
 The column data was fitted to the
Thomas model to determine the
Thomas rate constant and the
maximum solid phase concentration
(qe).

 It was identified that as the flow rate

was increased from 1 to 5 ml/min, the
values of qe and kTh increased.
Maximum solid phase concentration
of 7.19 mg/g of carbohydrates was
obtained when flow rate was
maintained at 5 ml/min.

 This inferred that the driving force for

retention of carbohydrates is the time
of contact between the feed and
hydrogel bead.
Thomas rate constant (x Equilibrium
Flow Rate 10-3) Level of  Further, from the R2 values, it was
Carbohydrate Uptake
(ml/min) Significance identified that stability of the model
(ml/(min.mg)) (mg/g)
increased with increase in flow rate.

 Thus, it was concluded that the

hydrogel bead in a packed bed
1 1.09 0.56 0.91 column should be operated at higher
flow rates to improve the column
performance.
5 1.4 7.19 0.99
 YOON NELSON MODEL

 The effect of interaction between

carbohydrates and the hydrogel
beads was predicted using the
values of Yoon Nelson rate
constant (kyn) and time required
for 50% breakthrough () .

 It was identified that Yoon Nelson

rate constant increased with
increase in flow rate. This
showed the poor carbohydrate
elimination efficiency of the
beads at low flow rate.
Velocity Rate Constant (x
10-3) Level of  Thus, it was concluded from the
Flow Rate (ml/min) Half Life (min)
Significance
(ml/(min.mg)) model that flow rate showed
direct proportionality with
carbohydrate elimination rate.

1 0.17 1805 0.91

5 1.24 175 0.99

MAXIMUM HOLDING
CAPACITY OF PACKED
BED COLUMN
 The maximum carbohydrate
holding capacity of the hydrogel
beads in a packed bed column for
feed flow rate of 5 ml/min was
calculated using the area under
the curve of the plot between rate
of carbohydrate eliminated with
respect to time.

 From the plot, a maximum holding

capacity of 762 mg/l of
carbohydrates was obtained.

 The R2 value of 0.99 showed the

significance of the experiment.
 DISSOLUTION AND HYDRATION RATE TESTS

 The rate of dissolution of hydrogel beads with respect to

change in pH was identified.
 It was found that a change in pH effected dissolution of the
beads and 100% dissolution of the beads was observed at
extreme pH conditions of 1 and 12, respectively.
 In contrast, it was identified that at neutral pH, the beads
remained highly stable indicating the suitability of the beads to
separate impurities from neutral foods.
 Considering the hydration rate, a linear relationship was
observed between drying temperature and hydration rate.
 After a period of 60 min, a maximum of 94% hydration rate
was observed at 121°C. However, at room temperature, no
hydration was observed.
 Hence, it was concluded that the synthesized beads showed
high degree of stability at room temperature.
 CONCLUSIONS
 A two-fold increase in anthocyanin purity could be obtained withcarbohydrate
elimination capacity of 35 mg/g using the developed method.
 This showed the efficiency of the developed method for obtaining pure
anthocyanin extracts from red cabbage juice.
 Further, it was identified that the hydrogel bead size, concentration of the
dopant (gelatin) and the refluxing were inversely proportional to the
response factors.
 The reusability studies on the hydrogel bead packedbed column showed that
it could be used only once without any washing step.
 Breakthrough analysis using kinetic models such as Thomas model and
Yoon Nelson model revealed that flow rate was directly proportional to the
carbohydrate retention.
 Further, the time for 50% breakthrough reduced by 40% when the flow rate
was increased from 1 to 5 ml/min.
 Overall, the hydrogel bead in a packed bed column showed a maximum
carbohydrate holding capacity of 762 mg/l.
 Dissolution and hydration studies revealed the stability of beads under
neutral pH and room temperature.
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 LIST OF PUBLICATIONS

 International Journal
 Balaji Shankaranarayanan & Ekambaram Nakkeeran, 2019,
‘Purification of anthocyanins from red cabbage using semi
interpenetrating network hydrogel beads in a packed bed column’,
Separation Science and Technology, 54(5), 675-682 (Annexure I).
Impact factor (2018) – 1.200.

 Conference Proceedings
 Shankaranarayanan, B & Nakkeeran, E., 2018, ‘Proceedings of
International Conference on Frontiers in Engineering, Applied
Sciences and Technology’, PP. 25-28. ISBN: 978-81-908388-6-3.