RESEARCH ARTICLE | DECEMBER 14 2018

Determination of Rhodamine-B and Amaranth in snacks at

primary school Sukolilo district of Surabaya-Indonesia by
thin layer chromatography 
Widya Wahyu Tonica; Maretna Fitri Hardianti; Surya Aji Prasetya; Orchidea Rachmaniah 
AIP Conf. Proc. 2049, 020043 (2018)
https://doi.org/10.1063/1.5082448

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03 October 2024 10:42:21

 Determination of Rhodamine-B and Amaranth in Snacks at
Primary School Sukolilo District of Surabaya-Indonesia by
Thin Layer Chromatography

Widya Wahyu Tonica, Maretna Fitri Hardianti, Surya Aji Prasetya, and Orchidea
Rachmaniaha)

Chemical Engineering Department, Faculty of Industrial Technology, Institut Teknologi Sepuluh Nopember,
Sukolilo, Surabaya, Indonesia, 6011
a)
Corresponding author: orchidea@chem-eng.its.ac.id

Abstract. Rhodamine B and Amaranth, banned artificial food red G\HV ZHUHGHWHUPLQHG IURPWKHSULPDU\VFKRRO¶VIRRG
stall snacks such as sausage, beef burger, and tomato sauce. Both of them are commonly used for coloring textiles, paper,
soap, leather, drugs, etc. Hence, they are toxic and carcinogenic for human. Unfortunately, they can still be found in

03 October 2024 10:42:21

FKLOGUHQ¶VVQDFNVGXHWRW KHH[FLWLQJVWDELOLW\RIFRO
RUDQGORZSULFH
) RUWKDW
UHDVRQ 5KRGDPL QH%DQG$PDUDQWKZHUH
WHVWHGIURPFKL OGUHQ¶VVQDFNVDW6XNROLO R'LVW ULFW
ZLWKKXJHQXPEHURISUL PDU\VFKRROVFDSULYate and state primary
schools. Initially, a survey was conducted; revealing the presence of the food stall at the studied primary schools and
identifying kind of snacks. Only sausage, beef burger, and tomato sauce were sampled because of containing red color
and favorites amongst the snacks. In case of dyes extraction, ethanol (70 v/v) was used as a solvent combining with
vortex and centrifugation method. Moreover, ethanol (EtOH): ammonia (19:1, v/v/v/v); butanol: EtOH: H2O: Ammonia
(50:25:25:10, v/v/v/v); 1,2 propane-diol: Ammonia (4:2, v/v); EtOH: Ammonia (9:2, v/v), and EtOH: Ammonia (8:4,
v/v) were optimized as a mobile phase. Revealing that EtOH: Ammonia (9:2, v/v) is the most appropriate mobile phase
for qualitatively separating of Rhodamine B, Amaranth, Red Allure, Erythrosine, and Ponceau 4R. The result showed that
51% of elementary schools at Sukolilo district, Surabaya allow the food stall presence around the school area, while the
remaining not. None of the samples contain Rhodamine B. While amaranth is found in some of the comparable ketchup
products. Therefore, periodical inspection by an authorized body is needed to keep the producer, i.e. food industry,
strictly fit the regulations. In case of red dyes, Erythrosine is the mostly used in the ketchup, while Ponceau 4R and red
allure are only used in some of the ketchup and corned beef.

Keywords: Red dyes; Rhodamine B; Amaranth; Sausage; TLC.

INTRODUCTION
Preservatives, antioxidants, dyes/colorings, sweetener, and flavorings are the commonly food additives. Food
additive means any substance which is intentionally added to food in small amount but not consumed as a food
itself. The addition intends to conserve food properties, improving food presentation as well as increasing appetite.
Therefore, contaminants and additional compounds for maintaining and improving food nutrients are not included in
the term. In case of food dyes, particularly it solely improve the food performance 1, i.e. visual appeal, consequently
enhancing appetite 2.
Many people find their sense of taste is affected by the color of the food. Moreover, color may also affect both
real and perceived nutritional value such high intensity of yellow color of both fruits and vegetables are accurately
perceiving high content of carotene; influencing flavor identification and product acceptability. Hence, food dye is
important 2. Food manufacturers use food performance, i.e. food colorant, for attracting consumers 3; due to

The 3rd International Seminar on Chemistry

AIP Conf. Proc. 2049, 020043-1–020043-9; https://doi.org/10.1063/1.5082448
Published by AIP Publishing. 978-0-7354-1775-5/$30.00

020043-1
improvement quality of food perception and appetizing of food 2. Consequently, gaining more sale and profits.
Moreover, the intention of adding food colorant plausibly to: (1) enhance and correction both of natural variations in
food or ingredient colors and color changes/loss during storage, processing, packaging, or distribution; (2)
emphasize associated flavors or preserving unique identifying characteristics, such quickly identifying
pharmaceuticals and dietary supplement; and (2) to protect flavor and vitamins from photo-degradation. However,
natural dyes is still the first choice such carotenoids, chlorophyll, anthocyanin, and turmeric. Unfortunately, due to
natural dyes drawbacks e.g. unstable and altered rapidly during food processing and storage; synthetic dyes become
an alternatives which over much superiority than natural dyes such lower price, high effectiveness, and excellent
stability of color 4.
Period of growth and development of children is occurred during elementary stage of school, ca. 7-12 years,
therefore adequate nutritious food should be consumed with exclusively attention to food safety. At elementary
school, approximately >99% of students consume snacks to fulfil their energy need. Therefore, all school parties, i.e.
school authorities, parents including the government as well as the students itself should have a high awareness
about the quality of snacks in terms of health and safety. Ensuring consume UV¶KHDOWK 
The National Agency of Drug
and Food Control of Republic of Indonesia (Badan Pemeriksaan Obat dan Makanan, BPOM) strictly regulates the
usage of food additives, i.e. food dyes. Regulation of the Minister of Health of Republic Indonesia (PerMenKes) No.
33/2012) regulates eleven synthetic food dyes permitted to add to food e.g. Ponceau 4R Cl No. 16255 (Ponceau 4R),
Erythrosine Cl No. 45430 (Erythrosine), Red Allure Cl No. 16035 (Red Allure), and etc. Unfortunately, still
prohibited dyes such Rhodamine B and Amaranth are founded especially at school snacks which commonly
delivered by food stall. Survey conducted at 4,500 primary schools at 79 districts in 18 provinces in Indonesia
revealed that both 701 and 860 snacks and drinks, respectively, contain Amaranth. Moreover, related survey was
conducted to red colored school snacks at elementary schools in East Jakarta, syrup, bakpao, cotton candy, tomato
sauce, jelly, dodol, sweets, sausage, burger, and kolang-kaling (Arenga pinnata fruit); resulting that 9.09% of snacks
(from 33 samples) contain Rhodamine B 5.
Both Rhodamine B and amaranth are synthetic red dyes, delivering bright reddish violet color, widely used in
many industries such cosmetic, textile, medical as well as paper production. Hence, forbidden use in food

03 October 2024 10:42:21

(PerMenKesNo. 43/2013). Rhodamine B capable of cause irritation to the skin, eyes, gastrointestinal tract as well as
respiratory tract 6. High consumption of Amaranth may lead to hyperactivity and other disturbed behavior especially
in children 7.Consumed food with prohibited synthetic dyes can lead to chromosomes break, high genotoxicity 8,
hypothalamic, pituitary, ovaries, reproductive tract 9, phototoxic, photo allergic reactions 6, breakdown of red blood
cells 1, carcinogenic, teratogenic effects 10. The carcinogenicity, reproductive and developmental toxicity,
neurotoxicity and chronic toxicity towards humans and animals due to consuming chemical carcinogenicity are
experimentally proven 11, though at least two-year time frame of consuming period is needed. Indeed, it is a short
time period.
Moreover, considering of both Fatimah and Dewi 5 results and BPOM surveillance (2008-2010) that 40-44% of
school snack did not meet the requirements; due to the misuse of additives, the presence of microbial contaminants
and/or food additives exceeding the limit. Lack of awareness and responsibilities among food industries, food
distributors, consumers and the regulation bodies such BPOM was indicated in the issues of this food security
(BPOM, 2014).Hence, it is necessary to conduct a survey especially the usage of red dyes on favorite foods or
drinks of the children, i.e. school snacks.
Favorites and red-colored food of elementary student, sausages and burger including their tomato sauce, are
sampled in this current study. A survey was carried out to the food stalls in elementary school at Sukolilo District,
Surabaya, as the first step of study. Overall, they are 20 private elementary schools and 14 state elementary schools;
therefore, in total they are 34 elementary schools were surveyed. This step also indicated elementary schools which
permitted the presence of food stalls in their school area, i.e. in front of the schools fence etc. Moreover, types of
snacks which are sold also indicated at this step. Furthermore, the extraction of Red dyes from the sampled of snacks
was conducted; by mean liquid-solid extraction protocol using ethanol (70%, v/v) as a solvent. Subsequently, a
qualitative analysis of the red dyes was conducted with Thin Layer Chromatography (TLC). In this step, a TLC
mobile phase compositions were varied and tested; finding the best eluent combination to have a well separation of
five different types of the red dyes: Rhodamine-B, Amaranth, Red Allure, Ponceau-4R, and, Erythrosine. The first
two of the red dyes is illegally added to the food while the rest is legally permitted. Lastly, TLC-densitometry
method was used to quantitatively analyze the red dyes in the snacks sample. The results provided by this project
will be valuable for establishing an awareness especially among school authorities, parents, and the government to
food health and security. Moreover, a food health and security socialization considering the project result will be
also conducted in Sukolilo District, Surabaya.

020043-2
 MATERIALS AND METHODS
Standard compounds of Rhodamine B, Amaranth, and Red Allure are commercially purchased from Sigma-
Aldrich (India) in pro analyst grade whereas both Erythrosine and Ponceau 4R in pharmaceutical grade from Morton
Chemical (Netherlands). Silica gel 60 F 254 20×20 cm in size (Merck, Darmstadt, Germany) are used for qualitative
analysis of dyes. In case of solvents such ethanol (EtOH), ammonia (25%, v/v), and butanol (BuOH) are purchased
from Merck (Darmstadt, Germany) in pro analyst grade. A spectrophotometer U99LV*( 1 ( 6<6Π7KHUPR-
Scientific, USA) were used to quantify the red dyes.

Method of Survey
Sukolilo District consists of eight of sub-district, i.e. Klampis Ngasem, Keputih, Medokan Semampir, Menur Pumpungan,
Nginden Jangkungan, Semolowaru, Semampir, and Medokan. Totally, they are 19 private elementary schools and 14 state
elementary schools; therefore, in total they are 33 elementary schools were surveyed.
Initially, a survey was conducted at mid-April 2018; revealing the presence of the food stalls in the elementary school at
Sukolilo District, Surabaya (Table 1). This step also indicated elementary schools which permitted the presence of
food stalls in their school area, i.e. in front of the schools fence etc. as well as types of snacks which were sold.

Preparation of Stock Solution Standard of Red Dyes

Stock solutions of dyes (1 mg/mL) were prepared by serial dilution. Each of the dye, i.e. Rhodamine B,
Amaranth, Red Allure, Erythrosine, and Ponceau 4R, was accurately weighted 5 mg and diluted up to 100 mL of
EtOH 70 % (v/v). Subsequently, a mixture of standard dyes solution was prepared by pipetting 1 mL of each
standard dye solution and diluting up to 10 mL with EtOH 70 % (v/v); hence 1 mg/mL concentration of each dye is
obtained. The prepared standards solution is stored in dark for further use.

Extraction of Red Dyes

03 October 2024 10:42:21

Analysis of synthetic red dyes was performed from three products, i.e. sausage meat, beef burger, and tomato, all
of them are no brand and sold by food stall. All of snacks are sampled from food stalls in elementary school at
Sukolilo District, Surabaya (May 2018).
Three grams (3 g) of each sausage, beef burger or tomato are subsequently chopped, homogenized with 5 mL of
EtOH 70 % (v/v), vortexed within 10 min, and centrifuged (3000 rpm, 10 min). The obtained clear solution was
further separated by means a filtration. The filtrate was further concentrated by evaporating the solvent before using
Thin Layer Chromatography (TLC).
Confirming the legalized red dyes, various legalized brand of sausage meat, beef burger, and tomato, which was
identified either by halal logo of Indonesian Ulema Council (Majelis Ulama Indonesia, MUI), PIRT-code (Izin
Pangan Industri Rumah Tangga, PIRT), or BPOM registered number, were bought (June 2018) from traditional
market at Keputih, Sukolilo. We also bought no brand products with good-looking packaging, checking whatever
they contained Rhodamine B and Amaranth or not. Moreover, the contained red dyes were extracted follow the
previous described method and qualitatively identified with thin layer chromatography (TLC).

Thin Layer Chromatography (TLC) Analysis

Red dyes identification was conducted by chromatographic method qualitatively meaning Thin Layer
Chromatography (TLC). TLC plates (10 cm ×10 cm), Silica gel 60 F 254, was prepared, marked 1.5 cm and 0.5 cm,
respectively, from the base of plate and top of the plate. While, 1.5 cm each from the right and left side of plate. Ten
microliter (10µL) extract of each sample solution (previously described) was applied to the TLC plate in duplicates
as well as the standards solution. In case of mobile phase of TLC, various composition of mobile phase was
observed, finding the optimum separation of five different kinds of red dyes, i.e. Rhodamine B, Amaranth, Red
Allure, Erythrosine, and Ponceau 4R. They were EtOH: Ammonia (19:1, v/v/v/v); BuOH: EtOH: H2O: Ammonia
(50:25:25:10, v/v/v/v); 1,2 propane-diol: Ammonia (4:2, v/v); EtOH: Ammonia (9:2, v/v), and EtOH: Ammonia
(8:4, v/v). Subsequently, a retardation factor (Rf) value of each spot was calculated right after the elution was
finished.

஽௜௦௧௔௡௖௘௧௥௔௩௘௟௟௘ௗ௕௬௖௢௠ ௣௢௡௘௡௧
Rf = (1)
஽௜௦௧௔௡௖௘௧௥௔௩௘௟௟௘ௗ௕௬௧௛௘௦௢௟௩௘௡௧

020043-3
Quantification of the Red Dyes by Spectrophotometry UV-Vis
The identified spot of the red dyes subsequently quantified with UV-Vis spectrophotometry. Therefore, a
calibration curve of each of red dyes need to be done previously; meaning for Rhodamine B, Rhodamine B,
Amaranth, Red Allure, Erythrosine, and Ponceau 4R. Initially, sample solution of the red dyes was scanning using
*( 1 ( 6<6Œ  899LVSpectrophotometer (Thermo-Scientific, USA) at the range of wavelength 400–700 nm (0.5
nm interval), finding the maximum wavelength of the dye (λmax).
Calibration Curve was prepared by serial dilution of a standard stock solutions of red dye. Each of standard stock
solution was made by accurately weighted 3 g and diluted up to 5 mL of EtOH 70 % (v/v), hence 0.6 g/mL was
obtained. The calibration was conducted using 11 concentrations (7.0, 6.0, 5.0, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, and
0.50 ppm) which is made in triplicate.

RESULT AND DISCUSSION

Results of the Survey

Identification of prohibited dyes (Rhodamine B and Amaranth) was performed on children's snacks at 33
elementary schools in Sukolilo district, East Surabaya (Table 1). Most of the elementary school are located at sub-
district Keputih (24%), followed with Menur Pumpungan (21%), Klampis Ngasem, Nginden, and Semolowaru with
3%; while the rest are Medokan Semampir, Semampir and Medokan with 3% of each.
A survey was previously conducted at mid of April 2018, identifying type of snacks sold around both of public
and private elementary schools in Sukolilo district, East Surabaya. Not all the elementary schools has the food stall
either in front of or near the school area. The survey results shows that 48% of schools (16 from total 33 elementary
schools at Sukolilo District) allow the food stall sell snacks around the school area, while the remaining (52%) do
not. Mostly, ca. 64%, food stalls are allowed in the area of the state elementary school (while the rest of them are not
(Fig. 1). Type of snacks which are sold were also identified; they are: meatball (1); small meatball (2); ice tea (3);

03 October 2024 10:42:21

sausage (4); pempek (savory fishcake made from fish and tapioca) (5); papeda (egg with flavoring) (6); waffle (7);
milk (8); toys (9); sempol (mashed chicken, flour, and flavoring) (10); crispy tofu (11); hot dog (12); egg rolls (13),
and pisang molen (fried banana wrapped in stripe of wheat flour dough) (14). However not all these snacks are
sampled only those which may contain red dyes. Seventeen of samples were sampled and identified further for the
contained red dyes (Table 2).

Figure 1. Percentage of private and state elementary schools at Sukolilo District, East Surabaya (insert pie diagram: shown
percentage of state elementary schools which allowing food stalls in their school area).

Finding the Best Mobile Phase for Qualitative Identification of Red Dyes with Thin Layer Chromatography
(TLC)
Considering qualitative identification of red dyes with thin layer chromatography (TLC), retardation factor (R f)
value is important. Therefore, we tried several combination of mobile phase to have the best separation of the
studied red dyes: Rhodamine B, Amaranth, Red Allure, Erythrosine, and Ponceau 4R.
Fatimah and Dewi 5 used different combination of mobile phase for separating Rhodamine B, Karmoisin,
Erythrosine, and Ponceau 4R with paper chromatography method, Whatman paper (No.1). They were 2% NaCl
solution in 50% ethanol (EtOH), methyl ethyl ketone: acetone: H2O (7:3:3, v/v/v), and butanol (BuOH): acetic acid:
H2O (40:20:24, v/v/v). The results shown that the last combination was separated four different dyes well. However,
2% NaCl solution in 50% ethanol, Karmoisin and Erythrosine shown closed position of R f and methyl ethyl:
acetone: H2O (7:3:3, v/v/v), did not elute Ponceau 4R at all.

020043-4
 Table 1. Elementary Schools at Sukolilo District, East Surabaya
Type of
No Elementary School Name Address Sub-district
School
1 SDN Klampis Ngasem I/246 JL.Arif Rahman Hakim No.99-C Klampis Ngasem State
2 SDN Klampis Ngasem II/511 JL.Klampis Anom XI No.1 Blok G Klampis Ngasem State
Perum Wisma Mukti
3 SDN Klampis Ngasem III/512 JL. Manyar Kertoarjo III No. 107 Klampis Ngasem State
4 SDN Klampis Ngasem V/230 JL. Manyar Tirtoyoso Selatan I/1 Klampis Ngasem State
5 SD Raden Paku Jl. Klampis Ngasem 88 A Klampis Ngasem Private
6 SDN Keputih 245 JL. Arif Rahman Hakim No.1 Keputih State
7 SD Bunga Bangsa Jl. Marina Emas Barat I/69 Keputih Private
8 SD Kristen PETRA 5 Jl. Galaxy Klampis Utara No. 1-3 Keputih Private
9 SD Mabadiul Ulum Jl. Rodah Sekolah No. 1 Keputih Private
10 SD VITA Jl. Arif Rahman Hakim 189-191 Keputih Private
11 SD YAPITA Jl. Arif Rahman Hakim 19 Keputih Private
12 SD Islam Terpadu Al Uswah Jl. Kejawan Gebang No. 6 Keputih Private
13 SDS Muhammadiyah 26 Jl.KH. Ahmad Dahlan No. 2 Keputih Private
14 SDN Medokan Semampir I Medokan Semampir Gg Kelurahan Medokan Semampir State
No 124
15 SDN Menur Pumpungan Menur Pumpungan 28 Menur Pumpungan State
16 SDN Menur Pumpungan IV/236 Nginden Semolo No. 23 Menur Pumpungan State
17 SDN Menur Pumpungan V/510 Jl. Manyar Rejo I / 37 Menur Pumpungan State
18 SD Al Amin Pumpungan Masjid 15 Menur Pumpungan Private
19 SD Surabaya Montenssori School Jl. Manyar Kartika Timur D Menur Pumpungan Private
20 SD Wachid Hasjim 2 Jl. Menur II / 68 Menur Pumpungan Private
21 SDK Santa Melania Jl. Pumpungan III 11 A Surabaya Menur Pumpungan Private
22 SDN Nginden Jangkungan I/247 Jl. Nginden Baru VI No. 30 Nginden State

03 October 2024 10:42:21

Jangkungan
23 SDN Nginden Jangkungan II/611 Jl. Nginden Baru 6/30 Nginden State
Jangkungan
24 SD Islam Baitul Mukmin Jangkungan 1 No. 20 A Nginden Private
Jangkungan
25 Nginden
SD Kristen Anak Bangsa Jl. Manyar Kartika Timur No.6 Private
Jangkungan
26 SDS I Darussalam Jl. Nginden II/18A Nginden Private
Jangkungan
27 SDN Semolowaru I/261 Semolowaru 179 Semolowaru State
28 SDN Semolowaru II/262 Semolowaru 179 Semolowaru State
29 SDN Semolowaru IV/614 Jl. Semolowaru Timur II No. 1 Semolowaru State
30 SD Islam Raden Patah Semolowaru Utara II No.16-20 Semolowaru Private
31 SD Kreatif An Nur Jl. Semolowaru 96-98 Semolowaru Private
32 SD Al Falah Jl. Medokan Semampir Masjid No. 42 Semampir Private
33 SDS Sekolah Alam Insan Mulia Jl. Medokan Semampir Indah No. 99- Medokan Private
101

In our study, five different combination of mobile phase were studied; they were: EtOH: Ammonia (19:1, v/v)
(E1); BuOH: EtOH: H2O: Ammonia (50:25:25:10, v/v/v/v) (E2); 1,2 propanediol: Ammonia (4:2, v/v) (E3); EtOH:
Ammonia (9:2, v/v) (E4), and EtOH: Ammonia (8:4, v/v) (E5). The retardation factor (R f) values of five different
red dyes is shown at Table 3 while it spot visualization is shown at Fig. 2; seems that both mobile phase of E4 and
E5 are well separated all the red dyes (Table 3). However, when it was eluted at E5 of mobile phase Ponceau 4R
and amaranth have coincide spot visualizations, hence they Rf value are comparable (Fig. 2). Either increasing or
decreasing ethanol or ammonia volume ratio will not enhance the separation, therefore EtOH: Ammonia (9:2, v/v) is
the most appropriate mobile phase for separating all the red dyes (Fig. 3).

020043-5
 Table 2. Name of Snacks were Sold at The Surveyed Elementary Schools at Sukolilo District, East Surabaya
Sample Code
SDN SDN SDN
SDN SDN Menur SDN Menur SDN
Name of Snacks Klampis Klampis Klampis SD
Semolowaru Pumpungan Pumpungan Keputih
Ngasem Ngasem Ngasem YAPITA
IV/614 V/510 IV/236 245
I/246 III/512 II/511
Sempol ketchup A
Meatball ketchup B D a7 a9
Grilled meatball
C
ketchup
Ketchup E
Papeda F
Roasted sausage G
Fried meatball
a1
ketchup
Fried intestine
a2
seasoning
Fried sausage a3
Corned beef a4
otak-otak ketchup a5
Cireng ketchup a6
egg rolls ketchup a8
Sausage ketchup a10

Table 3. Retardation factor (Rf) value of studied red dyes eluted with different mobile phases
Retardation factor (Rf) value

03 October 2024 10:42:21

Mobile phase, code Rhodamine Red Ponceau
Erythrosine Amaranth
B Allure 4R
EtOH: Ammonia = 19:1 (v/v), E1 0.56 0.63 N.D N.D 0.38
BuOH: EtOH: H2O: Ammonia = 50:25:25:10 N.D 1.15 N.D 0.92 1.53
(v/v/v/v), E2
1,2 propanediol: Ammonia = 4:2 (v/v), E3 N.D N.D N.D N.D 0.80
EtOH: Ammonia = 9:2 (v/v), E4 0.89 0.83 0.73 0.79 0.76
EtOH: Ammonia = 8:4 (v/v), E5 0.92 0.88 0.80 0.83 0.80

N.D = not detected

E E5

Figure 2. TLC plate visualization of standard of Red dyes using EtOH: Ammonia (9:2, v/v) (E4) and EtOH: Ammonia (8:4, v/v)
(E5) as mobile phase. Rhodamine B (line 1); Red allure (line 2); Ponceau 4R (line 3); Erythrosine (line 4), Amaranth (line 5), and standard
mixture of five different of red dyes (line 6).

020043-6
 Figure 3. TLC plate visualization of standard of Red dyes with variance composition of EtOH: Ammonia: 9:2 v/v (E4), 9:3 v/v
(E41), and 9:4 v/v (E42). Rhodamine B (line 1); Red allure (line 2); Ponceau 4R (line 3); Erythrosine (line 4), Amaranth (line 5), and
standard mixture of five different of red dyes (line 6).

Identification of Red Dyes by Thin Layer Chromatography (TLC)

Red dyes from 17 sampled of snacks were extracted and identified as well as the comparable legalized products,
bought from local traditional market; they were chicken Sausage CHA (H), beef sausage KMB (I), corned beef no
brand (J), chicken sausage KMB (K), chicken sausage no brand (L), ketchup NKS (M), ketchup JPL (N), ketchup
SLW (O), ketchup 3B (P), ketchup SS (Q), and ketchup BGN (R).

Table 4. Identified red dyes both at sampled snacks (A to G and a1 to a10) and comparable legalized products (H to R). Please
refer to Table 2 for code of samples.
Identified Red Dyes
Code of

03 October 2024 10:42:21

Rhodamine Red Ponceau
sample Erythrosine Amaranth
B Allure 4R
A (-) (-) (-) (+) (-)
B (-) (-) (-) (-) (-)
C (-) (+) (-) (+) (-)
D (-) (-) (-) (-) (-)
E (-) (-) (-) (-) (-)
F (-) (-) (-) (-) (-)
G (-) (-) (-) (-) (-)
H (-) (-) (-) (-) (-)
I (-) (-) (-) (-) (-)
J (-) (-) (+) (-) (-)
K (-) (-) (-) (-) (-)
L (-) (-) (-) (-) (-)
M (-) (-) (-) (-) (+)
N (-) (-) (-) (-) (+)
O (-) (-) (-) (-) (+)
P (-) (-) (-) (-) (-)
Q (-) (-) (-) (-) (-)
R (-) (-) (-) (-) (-)
a1 (-) (-) (-) (-) (-)
a2 (-) (-) (-) (-) (-)
a3 (-) (-) (-) (-) (-)
a4 (-) (-) (-) (-) (-)
a5 (-) (-) (-) (-) (-)
a6 (-) (-) (-) (-) (-)
a7 (-) (-) (-) (-) (-)
a8 (-) (-) (-) (-) (-)
a9 (-) (-) (-) (-) (-)
a10 (-) (-) (-) (-) (-)

020043-7
 The spot visualization of red dyes was done by using EtOH: Ammonia (9:2, v/v) (results not shown). The
identified red dyes of all samples including the comparable legalized products are tabled at Table 4. Qualitatively,
only 6 of 28 samples containing red dyes (including the comparable legalized products): A, C, J, M, N, and O ;
while for B, D, E, F, G, H, I, K, L, P, Q, R, a1, a2, a3, a4, a5, a6, a7, a8, a9, and a10 no red dyes were identified
(Table 4). None of the samples contains Rhodamine B. However three of the samples, i.e. M (NKS ketchup), N
(JPL ketchup), and O (SLW ketchup), contains amaranth an illegalized red dyes. They are a ketchup which were
bought at traditional market at Keputih, Sukolilo. Both of ketchup N and O have PIRT and BPOM registered
number but neither for ketchup M. Hence, periodical inspection by an authorized body is needed such BPOM to
keep the products strictly fit the regulations. In case of sample A contains erythrosine, sample C contains both red
allure and erythrosine, while sample J contains Ponceau 4R; all of them are legalized red dyes regarding to
PerMenKes RI Number 722/Menkes/Per/IX/88. The quantitative analysis of the red dyes was further conducted with
spectrophotometry.

Quantitative Quantification of Red Dyes by UV-Vis Spectrophotometry

UV-Vis Spectrophotometry was used to quantify the identified red dyes. Previously, a scanning method of UV-
Vis spectrophotometry was conducted at 400-700 nm of wavelength; obtaining the maximum wavelength ( max) of
each red dyes. They were 542 nm; 525.5 nm; 511.5 nm; 518 nm; and 512.5 nm respectively for Rhodamine B,
Amaranth, Red Allure, Erythrosine, and Ponceau 4R. The Calibration Curve of all the red dyes were shown at Table
5. Applying the obtained calibration curves, the identified red dyes at the 6 samples were quantified. Sempol ketchup
SDN Klampis Ngasem I/246 (A) contains 531.993±4.96 ppm of erythrosine; Grilled meatball ketchup SDN Klampis
Ngasem III/512 (C) contains 498.771±2.67 ppm of red allure and 667.045±6.73 of erythrosine; and corned beef no
brand (J) contains 176.497±0.57 ppm of ponceau 4R. However, all those values were beyond the allowable limit of
the dyes in ketchup according to Regulation of BPOM No. 37/2013, maximum 300 mg/kg for erythrosine, red
allure, and ponceau 4R.
In case of the comparable legalized ketchup, ketchup NKS (M), ketchup JPL (N), ketchup SLW (O), contains
138.846±0.24 ppm, 256.106±4.43 ppm and 233.991±0.87 ppm of amaranth, respectively. Hence, amaranth is not

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permitted as the food additives, i.e. colorant.

Table 5. Calibration Curve of Red dyes (Rhodamine B, Amaranth, Red Allure, Erythrosine, and Ponceau 4R).
Red dyes Calibration Curve* R2 max (nm)
Rhodamine B y = 0.2053x + 0.0087 0.997 542
Amaranth y = 0.0309x + 0.0193 0.999 525.5
Red Allure y = 0.0615x – 0.0122 0.999 511.5
Erythrosine y = 0.0097x – 0.0013 0.997 518
Ponceau 4R y = 0.0276x – 0.0182 0.999 512.5
*y = absorbances; x = dye concentration (ppm)

CONCLUSIONS
There were 33 elementary schools in Sukolilo sub-district and only 48% of them are allowing the food stalls in their
area. Fourteen types of snacks were identified and only those which may contain red dyes were sampled. Hence, 17
samples were collected and identified. Sausage CHA (H), beef sausage KMB (I), corned beef no brand (J), chicken
sausage KMB (K), chicken sausage no brand (L), ketchup NKS (M), ketchup JPL (N), ketchup SLW (O), ketchup
3B (P), ketchup SS (Q), and ketchup BGN (R) were also identified as comparable legalized products. None of the
samples contain Rhodamine B. While amaranth is found in some of the comparable ketchup products (M, N, and O).
Therefore, periodical inspection by an authorized body is needed to keep the producer, i.e. food industry, strictly fit
the regulations. In case of red dyes, erythrosine is the mostly used as a ketchup colorant, while ponceau 4R and red
allure are less. However, all these red dyes are beyond the maximum allowable limit of colorant in ketchup, 300
mg/kg.

ACKNOWLEDGMENTS
The authors are grateful to Ministry of Research, Technology, and Higher Education of the Republic of Indonesia
for their financial support via Student Creativity Program (Program Kreativitas Mahasiswa, PKM) grant 2018.

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 REFERENCES
1. Z. Naseem, K. Imran, K. S. Muhammad, M. Zilwa, A. Naima, Alim-un-Nisa., H. Sajila, M. Shahid, A. Ijaz and
A. Muhammad, International Research Journal of Biological Sciences 6, 23-29 (2017).
2. S. S. A. Soltan and M. M. E. M. Shehata, Food and Nutrition Sciences 03 (07), 897-904 (2012).
3. R. P. Ilham, Asterina and Laila Isrona, Jurnal Kesehatan Andalas 3 (3), 297-303 (2013).
4. A. Ozaki., M. Kitano., N. Itoh., K. Kuroda., N. Furusawa., T. Masuda. and H. Yamaguchi., Food and Chemical
Toxicology 36, 811-817 (1998).
5. N. Fatimah and I. S. Dewi, Farmasi Sains 2, 143-149 (2014).
6. S. Merouani, O. Hamdaoui, F. Saoudi and M. Chiha, Chemical Engineering Journal 158 (3), 550-557 (2010).
7. P. Mpountoukas, A. Pantazaki, E. Kostareli, P. Christodoulou, D. Kareli, S. Poliliou, C. Mourelatos, V.
Lambropoulou and T. Lialiaris, Food Chem Toxicol 48 (10), 2934-2944 (2010).
8. K. Rovina, S. Siddiquee and S. M. Shaarani, Trends in Food Science & Technology 65, 68-79 (2017).
9. D. R. Sulistina, R. Ratnawati and I. W. A. Wiyasa, Asian Pacific Journal of Reproduction 3 (3), 180-183
(2014).
10. E. Baldev, D. MubarakAli, A. Ilavarasi, D. Pandiaraj, K. A. Ishack and N. Thajuddin, Colloids Surf B
Biointerfaces 105, 207-214 (2013).
11. R. Jain, M. Mathur, S. Sikarwar and A. Mittal, J Environ Manage 85 (4), 956-964 (2007).

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