Lab 3: Selectivity of UV-visible analysis of anthocyanins from different fruit juices

Introduction

Anthocyanins are natural colorants that responsible for most of the red, purple or blue
colours in fruit, vegetables, legume, flower and other plant (Markakis, 1982). It belongs
to a class of phenolic compounds called flavonoids (Sass-Kiss et al 2005). To quantify and
identify the content of anthocyanin in different fruits, there are several analysis methods
had been introduced. These analysis methods include UVvisible absorption spectroscopy,
high performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear
magnetic resonance spectroscopy (Jungmin et al 2008).

In this study, UV-Visible analysis with different absorbance spectra is using to identify
anthocyanin pigments which undergo reversible structural transformation with change in
pH. In fact, the pigmentation of anthocyanins is pH dependent. Wavelength of light to
measure was chosen critically. Its maximum absorbance wavelength in the visible range
from 400nm 700nm. There are also many health benefits provided by anthocyanins such
as reduction of coronary heart disease, improve visual acuity and antioxidant activities
(Giusti & Wrolstad 2001).

Objectives

To determine the concentration of anthocyanins in dragon fruit and grape juice

To understand spectrophotometric analysis
To study pH differential method

Materials

12x test tubes, red grape juice, dragon fruit juice, mixture of red grape juice and dragon
fruit juice, potassium chloride solution (1M, adjusted to pH 1), sodium acetate buffer (1M,
adjusted to pH 4.5)

1
Methods

1. 2 ml of potassium chloride solution (1M and pH 1) was added to 1 ml of samples in

triplicate:
- Grape juice
- Dragon fruit juice
- Mixture of grape juice and dragon fruit juice
2. The absorbance of 520 nm and 700 nm were measured. It is then zeroed against a
sample blank comprising of 2 ml of sodium acetate buffer (1 M and pH 4.5) with the
same amount of sample.
3. The absorbance measured at 700 nm were subtracted with the absorbance measured at
520 nm. The concentration of anthocyanin using the molar extinction coefficient of
cyaniding-3-glucoside (26,900) were calculated. This allowed us to get the
concentration in terms of cyaniding-3-glucoside equivalent (CGE).
4. The total anthocyanin content of fruit juices were expressed as mg of CGE per litre
juice. The molecular weight of cyanidin-3-glucoside is 484.8 g/mol.

Results

Before data handling:

Group 1 2 3 4 5 6 7 8 9
G 0.117 0.077 0.057 0.086 0.077 0.080 0.079 0.083 0.074
pH1
D 0.193 0.140 0.166 0.163 0.193 0.157 0.166 0.191 0.169
520nm
M 0.142 0.127 0.112 0.118 0.129 0.109 0.134 0.139 0.123
G 0.081 0.021 0.010 0.058 0.007 0.009 0.014 0.008 0.019
pH1
D 0.054 0.019 0.025 0.043 0.014 0.016 0.014 0.020 0.024
700nm
M 0.055 0.018 0.029 0.054 0.008 0.015 0.027 0.011 0.022
G 0.037 0.024 0.010 0.019 0.032 0.030 0.030 0.033 0.028
pH4.5
D 0.284 0.171 0.167 0.158 0.194 0.178 0.189 0.218 0.170
520nm
M 0.113 0.093 0.090 0.113 0.113 0.096 0.109 0.119 0.101
pH4.5 G 0.038 0.005 0.003 0.013 0.004 0.006 0.005 0.004 0.003

2
 700nm D 0.027 0.006 0.004 0.005 0.004 0.008 0.004 0.002 0.003
M 0.034 0.004 0.005 0.006 0.002 0.005 0.012 0.003 0.004
Table 1

After data handling:

Group 1 2 3 4 5 6 7 8 9
G 0.077 0.086 0.077 0.080 0.079 0.083 0.074
pH1
D 0.166 0.163 0.157 0.166 0.169
520nm
M 0.127 0.112 0.118 0.129 0.134 0.139 0.123
G 0.021 0.010 0.007 0.009 0.014 0.008 0.019
pH1
D 0.019 0.025 0.014 0.016 0.014 0.020 0.024
700nm
M 0.018 0.029 0.015 0.027 0.011 0.022
G 0.037 0.024 0.019 0.032 0.030 0.030 0.033 0.028
pH4.5
D 0.171 0.167 0.194 0.178 0.189 0.218 0.170
520nm
M 0.113 0.093 0.090 0.113 0.113 0.096 0.109 0.119 0.101
G 0.005 0.003 0.013 0.004 0.006 0.005 0.004 0.003
pH4.5
D 0.006 0.004 0.005 0.004 0.008 0.004 0.002 0.003
700nm
M 0.004 0.005 0.006 0.002 0.005 0.012 0.003 0.004
Table 2

G=Grape, D=Dragon fruit, M=Mixture

Before data handling

pH1

Juice Mean of data Standard Percentage Relative

Deviation Standard Deviation/ %
(RSD)
Grape juice 0.0747 0.0171 22.89%
Dragon fruit juice 0.1708 0.0182 10.66%

3
Grape + Dragon
0.1258 0.0115 9.14%
fruit juice
Table 3 measured at 520nm

Juice Mean of data Standard Percentage Relative

Deviation Standard Deviation/ %
(RSD)
Grape juice 0.0252 0.0262 103.97%
Dragon fruit juice 0.0254 0.0139 54.72%
Grape + Dragon 0.0265 0.0173 65.28%
fruit juice
Table 4 measured at 700 nm

pH 4.5

Juice Mean of data Standard Percentage Relative

Deviation Standard Deviation/ %
(RSD)
Grape juice 0.0236 0.0089 37.71%
Dragon fruit juice 0.1921 0.0388 20.20%
Grape + Dragon
0.1052 0.0104 9.89%
fruit juice
Table 5 Measured at 520 nm

Juice Mean of data Standard Percentage Relative

Deviation Standard Deviation/ %
(RSD)
Grape juice 0.009 0.0113 125.56%
Dragon fruit juice 0.007 0.0077 110.0%

4
 Grape + Dragon 0.0083 0.01004 120.96%
fruit juice
Table 6 Measured at 700 nm

After Data Handling

pH 1

Juice Mean of data Standard Percentage Relative

Deviation Standard Deviation/ %
(RSD)
Grape juice 0.0711 0.00986 13.86%
Dragon fruit juice 0.1640 0.00455 2.77%
Grape + Dragon
0.1259 0.00924 7.34%
fruit juice
Table 7 measured at 520nm

Juice Mean of data Standard Percentage Relative

Deviation Standard Deviation/ %
(RSD)

Grape juice 0.0125 0.00556 44.48%

Dragon fruit juice 0.0188 0.00449 23.88%
Grape + Dragon
0.0185 0.00789 42.65%
fruit juice

Table 8 measured at 700 nm

pH 4.5

Juice Mean of data Standard Percentage Relative

Deviation Standard Deviation/ %
(RSD)

5
 Grape juice 0.0253 0.00691 27.31%
Dragon fruit juice 0.1838 0.01812 9.86%
Grape + Dragon
0.1052 0.0104 9.89%
fruit juice
Table 9 measured at 520 nm

Juice Mean of data Standard Percentage Relative

Deviation Standard Deviation/ %
(RSD)

Grape juice 0.0054 0.00325 60.19%

Dragon fruit juice 0.0045 0.00185 41.11%
Grape + Dragon
0.0051 0.00304 59.61%
fruit juice
Table 10 measured at 700 nm

Calculation

Before data handling

Grape juice

1. 520nm, pH1
Mean
0.117+0.077+0.057+0.086+0.077+0.080+0.079+0.083+0.074
= 9

= 0.0747

Standard deviation
=
(0.1170.0747)2 +(0.0770.0747)2 +(0.0570.0747)2 +(0.0860.0747)2 +(0.0770.0747)2 +(0.0800.0747)2 +
(0.0790.0747)2 +(0.0830.0747)2 +(0.0740.0747)2
(91)

= 0.0171

6
 %RSD
0.0171
= 0.0747 x 100

= 22.89%

2. 700nm, pH1
Mean

0.081+0.021+0.010+0.058+0.007+0.009+0.014+0.008+0.019
= 9

= 0.0252

Standard deviation
=
(0.0810.0252)2 +(0.0210.0252)2 +(0.0100.0252)2 +(0.0580.0252)2 +(0.0070.0252)2 +(0.0090.0252)2 +
(0.0140.0252)2 +(0.0080.0252)2 +(0.0190.0252)2
(91)

= 0.0262

%RSD
0.0262
= 0.0252 x 100

= 103.97%

3. 520nm, pH4.5
Mean

0.037+0.024+0.010+0.019+0.032+0.030+0.030+0.033+0.028
= 9

= 0.0236

Standard deviation

7
 =
(0.0370.0236)2 +(0.0240.0236)2 +(0.0100.0236)2 +(0.0190.0236)2 +(0.0320.0236)2 +(0.0300.0236)2 +
(0.0300.0236)2 +(0.0330.0236)2 +(0.0280.0236)2
(91)

= 0.0089

%RSD

0.0089
= 0.0236 x 100

= 37.71%

4. 700nm, pH4.5
Mean

0.038+0.005+0.003+0.013+0.004+0.006+0.005+0.004+0.003
= 9

= 0.009

Standard deviation

=
(0.0380.009)2 +(0.0050.009)2 +(0.0030.009)2 +(0.0130.009)2 +(0.0040.009)2 +(0.0060.009)2 +
(0.0050.009)2 +(0.0040.009)2 +(0.0030.009)2
(91)

= 0.0113

%RSD

0.0113
= x 100 = 125.56%
0.009

Dragon Fruit Juice

1. 520nm, pH 1
Mean

8
 0.193+0,140+0.166+0.163+0.193+0,157+0.166+0.191+0.169
= 9

= 0.1708

Standard deviation

=
(0.1930.1708)2 +(0.1400.1708)2 +(0.1660.1708)2 +(0.1630.1708)2 +(0.1930.1708)2 +(0.1570.1708)2 +
(0.1660.1708)2 +(0.1910.1708)2 +(0.1690.1708)2
(91)

= 0.0182

%RSD

0.0182
= 0.1708 x 100

= 10.66%

2. 700nm, pH 1
Mean

0.054+0.019+0.025+0.043+0.014+0.016+0.014+0.020+0.024
= 9

= 0.0254
Standard deviation
=
(0.0540.0254)2 +(0.0190.0254)2 +(0.0250.0254)2 +(0.0430.0254)2 +(0.0140.0254)2 +(0.0160.0254)2 +
(0.0140.0254)2 +(0.0200.0254)2 +(0.0240.0254)2
(91)

= 0.0139

%RSD

0.0139
= 0.0254 x 100

9
 = 54.72%

3. 520nm, pH 4.5
Mean

0.284+0.171+0.167+0.158+0.194+0.178+0.189+0.218+0.170
= 9

= 0.1921

Standard deviation

=
(0.2840.1921)2 +(0.1710.1921)2 +(0.1670.1921)2 +(0.1580.1921)2 +(0.1940.1921)2 +(0.1780.1921)2 +
(0.1890.1921)2 +(0.2180.1921)2 +(0.1700.1921)2
(91)

= 0.0388

%RSD
0.0388
= 0.1921 x 100

= 20.20%

4. 700nm, pH 4.5
Mean

0.027+0.006+0.004+0.005+0.004+0.008+0.004+0.002+0.003
= 9

= 0.007
Standard deviation

=
(0.0270.007)2 +(0.0060.007)2 +(0.0040.007)2 +(0.0050.007)2 +(0.0040.007)2 +(0.0080.007)2 +
(0.0040.007)2 +(0.0020.007)2 +(0.0030.007)2
(91)

= 0.0077

10
 %RSD

0.0077
= x 100
0.007

= 110.0%

Mixture fruit juice

1. 520nm, pH 1
Mean

0.142+0.127+0.112+0.118+0.129+0.109+0.134+0.139+0.123
= 9

= 0.1258

Standard deviation

=
(0.1420.1258)2 +(0.1270.1258)2 +(0.1120.1258)2 +(0.1180.1258)2 +(0.1290.1258)2 +(0.1090.1258)2 +
(0.1340.1258)2 +(0.1390.1258)2 +(0.1230.1258)2
(91)

= 0.0115

%RSD

0.0115
= 0.1258 x 100

= 9.14%

2. 700nm, pH 1
Mean
0.055+0.018+0.029+0.054+0.008+0.015+0.027+0.011+0.022
= 9

= 0.0265

11
 Standard deviation

=
(0.0550.0265)2 +(0.0180.0265)2 +(0.0290.0265)2 +(0.0540.0265)2 +(0.0080.0265)2 +(0.0150.0265)2 +
(0.0270.0265)2 +(0.0110.0265)2 +(0.0220.0265)2
(91)

= 0.0173

%RSD
0.0173
= x 100
0.0265

= 65.28%

3. 520nm, pH 4.5
Mean

0.113+0.093+0.090+0.113+0.113+0.096+0.109+0.119+0.101
= 9

= 0.1052

Standard deviation

=
(0.1130.1052)2 +(0.0930.1052)2 +(0.0900.1052)2 +(0.1130.1052)2 +(0.1130.1052)2 +(0.0960.1052)2 +
(0.1090.1052)2 +(0.1190.1052)2 +(0.1010.1052)2
(91)

= 0.0104

%RSD
0.0104
= 0.1052 x 100

= 9.89%

12
 4. 700nm, pH 4.5
Mean

0.034+0.004+0.005+0.006+0.002+0.005+0.012+0.003+0.004
= 9

= 0.0083

Standard deviation

=
(0.0340.0083)2 +(0.0040.0083)2 +(0.0050.0083)2 +(0.0060.0083)2 +(0.0020.0083)2 +(0.0050.0083)2 +
(0.0120.0083)2 +(0.0030.0083)2 +(0.0040.0083)2
(91)

= 0.01004

%RSD

0.01004
= x 100
0.0083

= 120.96%

After data handling

Grape Juice

1. 520nm, pH1
Mean

0.077+0.086+0.077+0.080+0.079+0.083+0.074
= 7

= 0.0711

Standard deviation

(0.0770.0711)2 +(0.0860.0711)2 +(0.0770.0711)2 +(0.0800.0711)2 +

(0.0790.0711)2 +(0.0830.0711)2 +(0.0740.0711)2
= (71)

13
 = 0.00986

%RSD
0.00986
= x 100
0.0711

= 13.86%

2. 700nm, pH 1
Mean

0.021+0.010+0.007+0.009+0.014+0.008+0.019
= 7

= 0.0125

Standard deviation

(0.0210.0125)2 +(0.0100.0125)2 +(0.0070.0125)2 +(0.0090.0125)2 +

(0.0140.0125)2 +(0.0080.0125)2 +(0.0190.0125)2
= (71)

= 0.00556

%RSD
0.00556
= x 100
0.0125

= 44.48%

3. 520nm, pH4.5
Mean

0.037+0.024+0.019+0.032+0.030+0.030+0.033+0.028
= 8

= 0.0253

14
 Standard deviation

(0.0370.0253)2 +(0.0240.0253)2 +(0.0190.0253)2 +(0.0320.0253)2 +(0.0300.0253)2 +

(0.0300.0253)2 +(0.0330.0253)2 +(0.0280.0253)2
= (81)

= 0.00691

%RSD

0.00691
= x 100
0.0253

= 27.31%

4. 700nm, pH 4.5
Mean

0.005+0.003+0.013+0.004+0.006+0.005+0.004+0.003
= 8

= 0.0054

Standard deviation

(0.0050.0054)2 +(0.0030.0054)2 +(0.0130.0054)2 +(0.0040.0054)2 +(0.0060.0054)2 +

(0.0050.0054)2 +(0.0040.0054)2 +(0.0030.0054)2
= (81)

= 0.00325

%RSD

0.00325
= x 100
0.0054

= 60.19%

Dragon Fruit Juice

15
1. 520nm, pH1
Mean

0.166+0.163+0,157+0.166+0.169
= 5

= 0.1640

Standard deviation

(0.1660.164)2 +(0.1630.164)2 +(0.1570.164)2 +

(0.1660.164)2 +(0.1690.164)2
= (51)

= 0.00455

%RSD
0.00455
= x 100
0.1640

= 2.77%

2. 700nm, pH 1
Mean

0.019+0.025+0.014+0.016+0.014+0.020+0.024
= 7

= 0.0188

Standard deviation

(0.0190.0188)2 +(0.0250.0188)2 +(0.0140.0188)2 +(0.0160.0188)2 +

(0.0140.0188)2 +(0.0200.0188)2 +(0.0240.0188)2
= (71)

= 0.00449

16
 %RSD
0.00449
= x 100
0.0188

= 23.88%

3. 520nm, pH4.5
Mean

0.171+0.167+0.194+0.178+0.189+0.218+0.170
= 7

= 0.1838

Standard deviation

(0.1710.1838)2 +(0.1670.1838)2 +(0.1940.1838)2 +(0.1780.1838)2 +

(0.1890.1838)2 +(0.2180.1838)2 +(0.1700.1838)2
= (71)

= 0.01812

%RSD
0.01812
= x 100
0.1838

= 9.86%

4. 700nm, pH 4.5
Mean

0.006+0.004+0.005+0.004+0.008+0.004+0.002+0.003
=
8

= 0.0045

Standard deviation

(0.0060.0045)2 +(0.0040.0045)2 +(0.0050.0045)2 +(0.0040.0045)2 +(0.0080.0045)2 +

(0.0040.0045)2 +(0.0020.0045)2 +(0.0030.0045)2
= (81)

= 0.00185

%RSD

17
 0.00185
= x 100
0.0045

= 41.11%

Mixture

1. 520nm, pH1
Mean

0.127+0.112+0.118+0.129+0.134+0.139+0.123
= 7

= 0.1259

Standard deviation
(0.1270.1259)2 +(0.1120.1259)2 +(0.1180.1259)2 +(0.1290.1259)2 +
(0.1340.1259)2 +(0.1390.1259)2 +(0.1230.1259)2
= (71)

= 0.00924

%RSD

0.00924
= x 100
0.1259

= 7.34%

2. 700nm, pH 1
Mean

0.018+0.029+0.008+0.015+0.027+0.011+0.022
= 7

= 0.0185

Standard deviation

(0.0180.0185)2 +(0.0290.0185)2 +(0.0080.0185)2 +(0.0150.0185)2 +

(0.0270.0185)2 +(0.0110.0185)2 +(0.0220.0185)2
= (71)

= 0.00789

18
 %RSD

0.00789
= x 100
0.0185

= 42.65%

3. 520nm, pH4.5
Mean

0.113+0.093+0.090+0.113+0.113+0.096+0.109+0.119+0.101
= 9

= 0.1052

Standard deviation
=
(0.1130.1052)2 +(0.0930.1052)2 +(0.0900.1052)2 +(0.1130.1052)2 +(0.1130.1052)2 +(0.0960.1052)2 +
(0.1090.1052)2 +(0.1190.1052)2 +(0.1010.1052)2
(91)

= 0.0104

%RSD

0.0104
= 0.1052 x 100

= 9.89%

4. 700nm, pH 4.5
Mean

0.004+0.005+0.006+0.002+0.005+0.012+0.003+0.004
= 8

= 0.0051

Standard deviation

(0.0040.0051)2 +(0.0050.0051)2 +(0.0060.0051)2 +(0.0020.0051)2 +(0.0050.0051)2 +

(0.0120.0051)2 +(0.0030.0051)2 +(0.0040.0051)2
= (81)

19
 = 0.00304

%RSD

0.00304
= x 100
0.0051

= 59.61%

Total anthocyanins content

Grape Juice
[(0.07110.00986)(0.02530.00691)](484.8)(5)(1 103 )
= (26900)(1)

(0.061240.01839)(484.8 103 )(5)

= 26900

= 3.8613 CGE/litre

Dragon Fruit Juice

[(0.16400.00455)(0.18380.01812)] (484.8 )(5)(1 103 )
= 26900
(0.159450.016568)(484.8 103 )(5)
= 26900

= 12.8753 CGE/litre

Grape + Dragon Fruit Juice

[(0.12590.00924)(0.10520.0104)](484.8 )(5)(1 103 )
= 26900
(0.116660.0948)(484.8 103 )(5)
= 26900

= 1.9698 CGE/litre

Discussions
The total anthocyanins content found in grape juice, dragon fruit juice and the
mixture obtained throughout this experiment is shown in Table 2.0 above which is the data
after data handling. The primary pigments of dragon fruit is betalains. After the dragon
fruit being harvested, the chlorophyll will gradually decomposes, other pigments will be

20
synthesized that are responsible for the characteristic red color of the mature dragon fruit,
at this time small amounts of carotenoids appear, reaching a peak in the yellow-pink zones
and it will start to decrease when the fruit reaches a more deeper color, this color contains
a higher concentrations of the betalains and anthocyanins, while the anthocyanins reaches
higher concentrations than the carotenoids and betalains (Vargas et al. 2013). While for
the grapes. The primary pigment found is anthocyanins. The pigments may appear to be
red, purple or blue depending on the pH medium. Anthocyanins will be affected by the
change of pH while betalain (dragon fruit juice) is a stable compound that retains its red-
purple color even pH medium changed (Burin et al. 2010).

The pH differential method has been used by food technologists and horticulturists to
assess the quality of fresh and processed fruits and vegetables. This method can be used
for the determination of total monomeric anthocyanin content, based on the structural
change of the anthocyanin chromophore between pH 1.0 and 4.5. Monomeric
anthocyanins undergo a reversible structural transformation as a function of pH which pH
1.0 are colored oxonium form and pH 4.5 are colorless hemiketal form as shown in figure
1.0. The maximum absorption wavelength of anthocyanins in the visible light range
(400nm-700nm) is 520nm which is proportional to the concentration of pigment. Degraded
anthocyanins in the polymeric form are resistant to color change with change in pH.
Therefore, polymerized anthocyanin pigments are not measured by this method because
they absorb both at pH 4.5 and 1.0 (Lee 2005). From the above result, it is clearly shown
that Grape Juice = 3.8613 CGE/liter, Dragon Fruit Juice = 12.8753 CGE/liter, Grape +
Dragon Fruit Juice = 1.9698 CGE/liter. Dragon fruit juice has the highest content of
anthocyanins while the mixture of both has the lowest content of anthocyanins.

21
Figure 1.0 Predominant anthocyanin structural forms present at different pH levels.

Anthocyanins are highly hygroscopic (high tendency to absorb moisture from the
environment), therefore it is very difficult to obtain pure crystalline form of anthocyanins.
The optimum wavelength to be measured in this experiment is 520nm. However, there is
a reference wavelength used in this experiment, which is 700nm. The reference wavelength
is important when cloudy solutions is tested in this experiment. The reference wavelength
is used in order to improve measurement precision. Based on the displacement effect
between solvent and solute molecules in a solution, the signal at the reference wavelength
is used as an internal reference to correct the spectrum of the sample under test (Chen et al.
2009).

Conclusion

As a conclusion, the anthocyanin content present in different fruit juices can be

measured using spectrophotometry by setting the maximum wavelength of 520nm and
reference wavelength of 700nm. The anthocyanin content can be calculated using the

22
absorbance reading from the result of this experiment by using the formulae below.
10000
Anthocyanin pigment = .

References

Burin, V., Falco, L., Gonzaga, L., Fett, R., Rosier, J. and Bordignon-Luiz, M., 2010.
Colour, phenolic content and antioxidant activity of grape juice. Cincia e Tecnologia
de Alimentos, 30(4), 1027-1032.

Chen, Y., Chen, W., Shi, Z., Yang, Y. and Xu, K., 2009. A Reference-Wavelength-Based
Method for Improved Analysis of Near-Infrared Spectroscopy. Applied Spectroscopy,
63(5), 544-548.

Giusti, M.M. and Wrolstad, R.E., 2001. Characterization and Measurement of

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