How to cite:
Yusuf Irfan, Andini Putri Riandani, Anita Suri, Mutiah Aulia Amali (2024) Determination of Beta-
carrot Levels of Campolay Fruit (Pouteria campechiana) by method HPLC, (06) 08,
E-ISSN:
2684-883X
DETERMINATION OF BETA-CARROT LEVELS OF CAMPOLAY FRUIT
(POUTERIA CAMPECHIANA) BY METHOD HPLC
Yusuf Irfan, Andini Putri Riandani, Anita Suri, Mutiah Aulia Amali
Universitas Pelita Bangsa, Indonesia
Email: [email protected], [email protected],
Abstract
Besides its sensory characteristics, campolay fruit has the potential to be functional food. The
yellow color of campolay fruit indicates the presence of carotenoids. One highly beneficial
group of carotenoids is beta-carotene, which is abundantly available in agricultural products
and makes this compound one of the most advantageous components in the prevention and
treatment of various types of eye diseases. HPLC, which stands for High-Performance Liquid
Chromatography, is a chemical analysis technique used to separate, identify, and measure the
components in a solution mixture. The beta-carotene testing process in campolay fruit is
conducted using High-Performance Liquid Chromatography (HPLC) equipment with a visible
light detector at a wavelength of 446 nm. From the HPLC analysis, the regression equation
obtained is y = 366032x - 6388. From the standard beta-carotene solution, a linear
relationship between absorbance and concentration was obtained with a correlation coefficient
(r) of 0.9996. The peak area of the campolay fruit extract was obtained. The sample
concentration is calculated by comparing the peak area of the sample with the peak area of the
standard beta-carotene, resulting in the beta-carotene concentration of the campolay fruit
extract using the high-performance liquid chromatography method. Based on the beta-
carotene concentration of the campolay fruit obtained from this study, it can be concluded that
the beta-carotene concentration of the campolay fruit is 0.15 mg/L.
Keywords: Beta-carotene, campolay, HPLC, tropical fruit
INTRODUCTION
Campolay fruit is classified as a rare fruit that has not been widely used and cultivated
by people in Indonesia (Do et al., 2023). This causes not many people to consume this fruit
due to limited information about its characteristics. The flesh of a ripe campolay fruit will be
yellow to orange, the flesh is soft, has a strong aroma, and tastes sweet (Aminullah, Purba,
Rohmayanti, & Pertiwi, 2020) (Mangunsong, Assiddiqy, Sari, Marpaung, & Sari, 2019).
JOURNAL SYNTAX IDEA
p–ISSN: 2723-4339 e-ISSN: 2548-1398
Vol. 6, No. 08, Agustus 2024
Determination of Beta-carrot Levels of Campolay Fruit (Pouteria campechiana) by Method
HPLC
Syntax Idea, Vol. 6, No. 08, Agustus 2024 3451
Figure 1. Campolay Fruits and Pulp
(Personal Documentation, 2024)
In addition to its sensory characteristics, according to several research results, this fruit
can have the potential to be a functional food. The yellow color of the campolay fruit is an
indicator of the presence of carotenoid content in the fruit. Carotenoid compounds are
substances that function as precursors for the formation of vitamin A (Dzulhijjah, Sarli, &
Shabayek, 2022) (Juniarti & Hasnelly, 2016). Of the many groups of carotenoid compounds,
only three types of carotenoids correlate with the formation of vitamin A (retinol) in the
human body. The three types are α-carotene, β-carotene and β-cryptoxanthin (Pertiwi,
Rohmayanti, Aminullah, Apriani, & Silpia, 2022). Of the three types of carotenoid
compounds, β-carotene is the compound that has the best function as a precursor to vitamin A
or provitamin A. β-carotene is also a carotenoid component that is available in large quantities
in agricultural products which makes it one of the most beneficial components in the
prevention and treatment of several types of eye diseases (Syukri, 2021) (Adyas & Dzulhijjah,
2022).
HPLC stands for High-performance liquid chromatography or High Performance
Liquid Chromatography (KCKT) is a chemical analysis technique used to separate, identify,
and measure the content of components in a mixture of solutions. In HPLC, the mixture of
solutions to be analyzed is injected into a column containing a stationary phase, which can be
resin or small particles. The solution is then passed through a column with the help of a phase
of motion or solvent (mobile phase)(Adiyaman [2016] Identification and quantification of
poly phenolic compounds in underutilized fruits (Star fruit and egg fruit) using HPLC.pdf,
n.d.) (Mudrikah, Hidayah, Amelia, & Helsen, 2024).
Each component in the solution mixture interacts with the stationary phase and the
motion phase in different ways, resulting in the separation of the components. Eventually,
these components can be detected and their concentrations measured using sensitive detectors,
such as ultraviolet detectors or mass detectors.
The high-performance liquid chromatography technique is a liquid-liquid
chromatography method that can be used for both imaging analysis and quantitative analysis.
Quantitative analysis with high-performance liquid chromatography techniques is based on
standard area measurements. With the knowledge of beta carotene levels with analysis using
HPLC instruments that have accurate results, it is hoped that it can be a reference for the
utilization and development of campolay fruit processing technology
RESEARCH METHOD
1. Tool
Yusuf Irfan, Andini Putri Riandani, Anita Suri, Mutiah Aulia Amali
3452 Syntax Idea, Vol. 6, No. 08, Agustus 2024
The tools used include a 0.45 micron filter, glassware, filter paper, micropipettes, and
analytical scales. The analysis instruments consisted of HPLC Shimadzu LC20AT,
LiChrospher®100RP-18 column endcapped (5 μm) 25cm long, inner diameter 4mm
(E.Merck), KLT silica gel plate 60 F254, Chamber, Microscale (Sartorius Dragon),
Blender (Philips), Rotavapor (Buchi V-800), Separator funnel, Glass tools, Whatman filter
paper No. 40.
2. Material
The chemicals used are standard beta carotene (quality p.a. from Merck), acetonitrile
(HPLC grade, Merck), isopropanol (HPLC-grade, Merck), nitrogen gas (technical),
methanol (p.a, Merck), petroleum ether (p.a, Merck), distilled water, potassium hydroxide
(p.a, Merck), sodium sulfate anhydrous (p.a, Merck), chloroform p.a, ethyl acetate p.a,
methanol p.a, antimony trichloride.
3. Prepare sample
A quantity of the pulp of the campolay is mashed in a blender, then weighed in as
much as 50 grams, put into a sealed Erlenmeyer covered with aluminum foil on the outside
and protected from light. Ethyl acetate solvent was added, beaten for 30 minutes with a
magnetic stirr, and filtered with a Buchner funnel. The non-polar part is taken and
subsequently used for quantitative testing.
4. Method of Determining Optimum Conditions HPLC/KCKT
Determination of maximum absorption wavelength β-carotene: A total of 20 mg of
β-carotene isolate is put into a 50-mL flask, dissolve and dilute with chloroform until mark.
Then pipetted a total of 2.5 mL inserted into the flask 10-mL diluted with chloroform until
the mark. Next, a spectrum was created using a UV-Vis spectrophotometer at a wavelength
of 420-500 nm.
5. Identification of β-Carotene in Campolay
Extract/Isolate Manufacturing
Fresh campolay fruits that have been cut into pieces and pureed, weighed in the
amount of 100 g (campolay fruit), 50 g blended using mineral water solvent, then
filtered. The residue is rinsed with Filtrate obtained given calcium salt, centrifuged at
3000 rpm for 15 minutes. The residue as pellets is beta carotene, concentrated using
rotavapor at 400 C. Stored in cold temperatures.
a) Solution manufacturing
Preparation of raw solution: A quantity of 10 mg of β-carotene is put into a 50-mL
flask, dissolved and diluted with chloroform to a mark. Then pipetted a 2.5 mL amount
is inserted into a 10-mL flask diluted with chloroform up to the mark line (Standard beta
carotene).
b) How to identify
A number of test solutions were injected as much as 20 μl into the KCKT device,
then the retention time was compared with the standard retention time of β-carotene.
6. Quantitative analysis by high-performance liquid chromatography
a) System conformity test The system conformity test is carried out to determine whether
tools, methods and conditions form a single analysis system. A total of 20 μl of β-
carotene raw solution was injected 5 times into the KCKT device, then the peak area
Determination of Beta-carrot Levels of Campolay Fruit (Pouteria campechiana) by Method
HPLC
Syntax Idea, Vol. 6, No. 08, Agustus 2024 3453
was measured with optimal KCKT conditions, then the relative standard deviation value
was calculated.
b) Determination of β-carotene levels in campolay fruit extract by KCKT. The test solution
is sonicated for 10 minutes. Each of them was injected as much as 20 μl into the KCKT device
and the peak area was measured with the optimal KCKT condition.
RESULTS AND DISCUSSION
Measurement of absorbance of beta carotene raw solution with a concentration of 0.08
ppm; 0.1 ppm; 0.15 ppm; 0.2 ppm; 0.25 ppm; 0.5 ppm; 0.75 ppm; 1 ppm; 2.5 ppm; and 5 ppm
at a maximum wavelength of 446 nm was injected as much as 20 μl into the KCKT device
using the chloroform:methanol (95:5) motion phase and a flow rate of 1.0 ml/min with a
visible light detector at a wavelength of 446 nm. So that the area and retention time are
obtained.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Yusuf Irfan, Andini Putri Riandani, Anita Suri, Mutiah Aulia Amali
3454 Syntax Idea, Vol. 6, No. 08, Agustus 2024
(i)
(j)
Figure 1. Raw Beta Carotene Chromatograph using HPLC (a) Concentration 0.08 ppm;
(b) Concentration 0.1 ppm; (c) Concentration 0.15 ppm (d) Concentration 0.2 ppm (e)
Concentration 0.25 ppm; (f) Concentration 0.5 ppm; (g) Concentration 0.75 ppm; (h)
Concentration of 1 ppm; (i) Concentration 2.5 ppm; (j) Concentration 5 ppm
Tabel 1. Hasil Kurva Kalibrasi Standar
C.
Standar
Antara
(mg/L)
V.
Standar
(mL)
V.
Akhir
(mL)
C. Deret
Standar
(mg/L)
Retention
Time
(menit)
Area
C.
Injeksi
(mg/L)
Residual
(%)
10,88
0,080
10,00
0,087
8,13
30232,94
0,10
14,91
10,88
0,100
10,00
0,109
8,14
37563,87
0,12
10,33
10,88
0,150
10,00
0,163
8,13
53761,29
0,16
0,66
10,88
0,200
10,00
0,218
8,10
72245,16
0,21
-1,31
10,88
0,250
10,00
0,272
8,08
91557,78
0,27
-1,65
108,84
0,050
10,00
0,544
8,05
209945,67
0,59
8,61
108,84
0,075
10,00
0,816
8,02
262957,53
0,74
-9,85
108,84
0,100
10,00
1,088
8,02
402430,76
1,12
2,62
108,84
0,250
10,00
2,721
8,02
978957,50
2,69
-1,06
108,84
0,500
10,00
5,442
8,04
1991336,37
5,46
0,29
Slope
366031,98
Intercept
-6387,99
0,9996
R
0,9998
Figure 2. Standard Solution Calibration Curve Beta Carotene
Determination of Beta-carrot Levels of Campolay Fruit (Pouteria campechiana) by Method
HPLC
Syntax Idea, Vol. 6, No. 08, Agustus 2024 3455
The measurement of beta carotene levels of campolay fruit samples was carried out 2
times at a maximum wavelength of 446 nm injected as much as 20 μl into the KCKT device
using the motion phase of chloroform:methanol (95:5) and a flow rate of 1.0 ml/min with a
visible light detector at a wavelength of 446 nm so that the area was obtained.
(a)
(b)
Figure 3. Beta Carotene Chromatography of Campolay Fruit Samples Using HPLC
Chromatograph Connected 1; (b) Chromatograph Connected 2
Based on the measurement of beta carotene levels of campolay fruit samples carried out
at a maximum wavelength of 446 nm, chromatographs were obtained as shown in Figure 2
and the results are presented in the following table.
Table 2. Results of Measurement of Beta Carotene Area Area in Fruit Campolay
No
Sampel
Bobot (g);
Volume
(mL)
FP
V.
Akhir
(mL)
RT
(menit)
Area
C. Injeksi
(mg/L)
Kadar
(mg/Kg
: mg/L)
1
Ulangan 1
2,0848
1
10
8,18
5219,19
0,03
0,15
2
Ulangan 2
2,0192
1
10
8,18
5220,68
0,03
0,15
Beta carotene is a red-orange pigment that is very abundant in plants and fruits. Beta
carotene is one of the antioxidants that can prevent diseases, especially degenerative diseases
(Anjo et al., 2021). In this study, the flesh of campolay fruit (Pouteria campechiana) which is
extracted with ethyl acetate solvent. Naturally, beta carotene is abundant in orange, red to
dark green fruits and vegetables. The physical color indicator is found in campolay fruit.
Based on various sources and research results, beta carotene can prevent cancer and reduce
the risk of lung cancer because it is the main compound that attacks cancer (Awang-Kanak &
Abu Bakar, 2018)
Yusuf Irfan, Andini Putri Riandani, Anita Suri, Mutiah Aulia Amali
3456 Syntax Idea, Vol. 6, No. 08, Agustus 2024
The quantitative analysis process begins with maceration extraction. Extraction is
carried out by maceration because the sample to be studied is the pulp. The flesh of the fruit
was chosen because the beta-carotene to be studied is found in the pulp. In addition, this
extraction method was chosen because it is very simple, and can be used to extract simplicia
with content that does not withstand heating, such as beta carotene. In this process, ethyl
acetate is added to attract the carotenoid compounds. Ethyl acetate is a semi-polar solvent so
that it can attract both polar and nonpolar compounds, has low toxicity, and is easily volatile
so that it can be used for the extraction of carotenoids including beta carotene.
The testing process was carried out with a High Performance Liquid Chromatography
(KCKT) instrument using a mixed chloroform: methanol (95:5) motion phase, flow rate of 1.0
ml/min with a visible light detector at a wavelength of 446 nm. Each sample and as a
benchmark for synthetic beta-carotene made in the form of a solution with a concentration of
0.08 ppm; 0.1 ppm; 0.15 ppm; 0.2 ppm; 0.25 ppm; 0.5 ppm; 0.75 ppm; 1 ppm; 2.5 ppm; and 5
ppm is injected as much as 20 μl into the KCKT device. From the analysis with KCKT, the
regression equation y = 366032x - 6388 was obtained. From the standard solution of beta
carotene, a linear relationship between absorbance and concentration in absorbance
measurement with a correlation coefficient (r) of 0.9996 was obtained. An r value close to 1
indicates that the regression equation is linear (Mangunsong et al., 2019). From the campolay
fruit extract, the area of the peak area is obtained. The sample content was calculated by
comparing the area of the peak area of the sample with the area of the peak area at the
standard beta carotene, so that the beta carotene content was obtained from the pulp extract of
campolay by high-performance liquid chromatography method.
Based on the results of the beta carotene content of campolay fruit obtained from this
study, it can be concluded that the beta carotene content of campolay fruit is 0.15 mg/L. The
carotenoid content in the flesh of campolay fruit is 278.24 μg/g (Puspita, Kurniawan, &
Aiboi, 2019). Puspita, Kurniawan, Aiboi, et al., (2019), menyebutkan jika di dalam sawo
mentega terdapat beragam fraksi-faksi karotenoid seperti; β-karoten, β-kriptosantin,
violasantin, neosantin, ζ-karotenoid. Senyawa-senyawa biokatif tersebut berpotensial sebagai
sumber pro vitamina A (Anjo et al., 2021).
The difference in yield can be caused by various factors, namely differences in soil
conditions, temperature, weather, air humidity from the area of origin of the fruit used. Then
another factor is the difference in the solvent used and the tool to analyze (Mangunsong,
Puspita, Simamora, & Taswin, 2023).
CONCLUSION
Based on the results of the study, it was concluded that the synthetic beta-carotene
comparator raw solution was made in the form of a solution with a concentration of 0.08 ppm;
0.1 ppm; 0.15 ppm; 0.2 ppm; 0.25 ppm; 0.5 ppm; 0.75 ppm; 1 ppm; 2.5 ppm; and 5 ppm is
injected as much as 20 μl into the KCKT device. From the analysis with KCKT, the
regression equation y = 366032x - 6388 was obtained. From the standard solution of beta
carotene, a linear relationship between absorbance and concentration in absorbance
measurement with a correlation coefficient (r) of 0.9996 was obtained. Measurement of beta
carotene levels of campolay fruit samples was performed at a maximum wavelength of 446
nm. Campolay fruit beta carotene level of 0.15 mg/L.
Determination of Beta-carrot Levels of Campolay Fruit (Pouteria campechiana) by Method
HPLC
Syntax Idea, Vol. 6, No. 08, Agustus 2024 3457
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