Hindawi

International Journal of Analytical Chemistry

Volume 2018, Article ID 4605373, 9 pages
https://doi.org/10.1155/2018/4605373

Research Article
Two-Dimensional Thin Layer Chromatography-Bioautography
Designed to Separate and Locate Metabolites with
Antioxidant Activity Contained on Spirulina platensis

Margarita Cid-Hernández,1 Fernando Antonio López Dellamary-Toral,2

Luis Javier González-Ortiz,1 María Judith Sánchez-Peña,1
and Fermín Paul Pacheco-Moisés 1
1
Departamento de Quı́mica, Centro Universitario de Ciencias Exactas e Ingenierı́as, Universidad de Guadalajara,
Blvd. Marcelino Garcı́a Barragán 1421, 44430 Guadalajara, Jalisco, Mexico
2
Departamento de Madera, Celulosa y Papel, Universidad de Guadalajara, km 15.5 de la Carretera Guadalajara-Nogales,
45220 Zapopan, Jalisco, Mexico

Correspondence should be addressed to Fermı́n Paul Pacheco-Moisés; [email protected]

Received 21 February 2018; Revised 30 April 2018; Accepted 20 June 2018; Published 5 July 2018

Academic Editor: Bogusław Buszewski

Copyright © 2018 Margarita Cid-Hernández et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Spirulina platensis contains several biologically active compounds, some of them with antioxidant activity. Nevertheless, not all
of these compounds have been identified to date. As a first step to achieving such identification, a methodology to perform two-
dimensional thin layer chromatography bioautographies on silica gel thin layer chromatography plates was proposed. Starting with
a reference binary system, 5 other binary systems were tested, in which the relative polarity was systematically increased. To further
improve the separation behavior, a phase modifier (NH4 OH) was used. The best separation results were obtained with the isopropyl
alcohol/ethyl acetate/NH4 OH ternary system. This experimental system allowed four well-resolved spots showing antioxidant
activity as well as two additional areas with mixtures containing antioxidant compounds. Although the proposed methodology
was designed with a specific application, it would be predictable that its field of use could be considerably greater, making the
convenient modifications on the solvent polarity and “masking level” produced by the ammonium derivatives.

1. Introduction However, it is clear that such activity has been produced

by relatively complex mixtures that contain an unidentified
Spirulina platensis is a cyanobacterium that has been used number of components, most of which could possess low
in Mexico and other countries since ancient times [1, 2]. or negligible antioxidant activity. Due to the high number
Recently, it has attracted worldwide attention due to its poten- of components potentially present in Spirulina samples, it
tial as a protein source [2, 3] and its therapeutic properties would be predictable that, to take advantage of beneficial
(e.g., immunomodulatory functions) [4]. Spirulina contains properties of Spirulina, the isolation and characterization of
several known antioxidants, e.g., chlorophyll a, carotenoids, its components as much as possible will be necessary.
phycocyanins, glutathione, tocopherol, and others [5–7]. The Thin layer chromatography (TLC) is known as a very
relative amount of these compounds, as well as the possible useful technique, utilized to separate complex mixtures,
presence of some additional ones, mainly depends on the whose behavior depends on the balance of hydrophobic,
environmental conditions during its culture [8–11]. The hydrophilic, and steric interactions, as well as hydrogen
antioxidant activity of Spirulina platensis or its extracts has bonding, occurring between the analytes and the mobile
been reported for in vitro [9–12] and in vivo [13, 14] models. and stationary phases [15]. The most used stationary phase
2 International Journal of Analytical Chemistry

is the silica gel [16–18], which contains Si atoms bonded multicomponent spots presence, it is commonly preferred to
to none, one, or two hydroxyl groups [19]. Silanol groups consider it as well resolved, only those spots clearly separated
(Si-OH) present on silica can be reversibly dehydrated, (e.g., more distributed spots through the plate) that present
producing a siloxane group (Si-O-Si) from two Si-OH groups retention factors not very near to zero or the unit [25, 29].
[16]. Therefore, silica surface must simultaneously contain TLC bioautography (TLC bio) is an effective and inex-
both groups, being their relative amount dependent on the pensive technique that combines the chromatographic sepa-
amount of the cationic groups which came from the phase ration with in situ localization of compounds with biological
modifier (e.g., ammonium hydroxide or salts), which are activity [30–32]. The reaction between the 1,1-diphenyl-2-
interacting with the silanol groups, decreasing the retention picryl-hydrazyl (DPPH) radical and an active compound
trend of the stationary phase toward the metabolites that is a method commonly used to determine its antioxidant
are being separated by the TLC technique [20]. Since only activity, which has been used to directly locate those types
the Si-OH groups are considered as strong adsorption sites of compounds on TLC plates [33–37]. The characteristic
[17, 21], the retention behavior can be externally modified reaction of this technique produces a pale yellow on the spots
(e.g., when water molecules are absorbed, preferentially on that contain compounds with antioxidant activity [34, 38].
the Si-OH groups) [22]. Thus, depending on the stationary Zarzycki et al. [33, 39] implemented a one-dimensional
phase characteristics, as well as the mobile phase and sample TLC technique (1D-TLC) to separate the chemical compo-
compositions, the phase modifiers (e.g., ammonium salts nents present in four pharmaceutical formulations of Spir-
or derivatives) may interact with the Si-OH groups and, ulina platensis. The cyanobacterium samples were extracted
therefore, modify the analytes retention behavior, especially with methanol, acetone, or tetrahydrofuran, obtaining the
with polar and basic compounds [18, 23]. best results with methanol [39, 40]. For all extracts, the spots
In certain complex systems (e.g., those containing com- on TLC plates were initially visualized using natural light
ponents covering a wide spectra of polarities), to improve the and, to visualize additional spots, TLC plates were exposed
separation, it could be useful to simultaneously use two differ- to iodine vapors. Unfortunately, iodine vapors can react
ent chromatographic systems (e.g., different pairs of station- with some metabolites, interfering with their antioxidant
ary phase/mobile phase). That is, a normal stationary phase activity, therefore impeding their evaluation with the DPPH
(silica gel) developed with a nonaqueous mobile phase, which technique.
separates preferably nonpolar components and, in parallel, a The objective of this study was to provide a simple and
reverse stationary phase (octadecyl silica) developed with an cheap 2D-TLC biomethod for separation of compounds with
aqueous phase, used to separate preferably polar components antioxidant activity contained in Spirulina platensis. The ease
[24, 25]. However, it is clear that this methodology duplicates of sample preparation, the quickness of this method, and the
the human and material cost of the process, which is an repeatability of the retention factors are the major novelties
important factor to be considered when the objective is to of this work.
obtain a considerable mass of the isolate components (e.g.,
to analyze them). An alternative methodology requires the 2. Experimental
use of a dual phase TLC plate, which have allowed complex
mixtures to be separated, where great separation selectivity 2.1. Materials and Reagents. Methanol (MeOH; Golden Bell,
was reached [24, 25]. Dual phase TLC plates contain a narrow analytical grade), acetone (AcO; Karal, purity: 90%), iso-
zone of SiO2 and a wide zone of octadecyl silica (or vice propyl alcohol (IOH; Fermont, purity: 99.9%), ethyl acetate
versa). Unfortunately, at least in North America, these multi- (EA; Karal, purity: 99.5%), and n-hexane (n-Hx; Fisher
K dual phase TLC plates are no longer available. Therefore, Chemical, purity: 99.9%) were distilled prior to use. Ammo-
an alternative approach to effectively separate the compounds nium hydroxide aqueous solution (Fermont, concentration:
present in complex mixtures is to modify the interactions of 25-30%) and 1,1-diphenyl-2-picrylhydrazyl (DPPH; Sigma-
the stationary phase, by means of the addition of a modifier Aldrich, purity: 97%) were used as received. TLC aluminum
to the mobile phase. Some amines have been used as phase sheets were 20 cm × 20 cm (Merck; 1 mm thick, silica gel
modifiers, since they are able to mask silanol groups, reduce 60 F254), which were heated during 30 min at 100∘ C in an
the silanophilic interactions with analytes, and, therefore, oven (Felisa, Model 292) and maintained on a glass desiccator
increase their retention factors (RF ) [20, 26, 27]. Furthermore, until their use. Spirulina platensis used for the experiments
it has been reported that the chromatographic resolution is was purchased from Natura Vitalis GmbH in the form of
strongly affected by the pKa of the amine used. In fact, the tablets (Original spiruletten-1700 tablets; 400 mg of Spirulina
use of basic systems promotes the separation of closely related platensis/tablet).
compounds with minor structural differences [28].
On the other hand, since component(s) that are prac- 2.2. Extract. 25 g of S. platensis tablets was crushed to fine
tically incompatible(s) with the mobile phase remain very powder using mortar and pestle and transferred into an
close to the bottom of the thin layer chromatograms, the Erlenmeyer flask containing 500 mL of MeOH. This mixture
presence of spots containing two or more compounds at was allowed to macerate for 48 hours under constant stirring.
RF values near to zero could be possible. An equivalent During maceration, the sample was protected from light and
statement can be expressed about components that are very kept under a nitrogen atmosphere. The crude extract was
compatible with the mobile phase, which migrate up to the filtered and concentrated with a rotary evaporator (Buchi R-
top of the chromatogram (RF =1.0). Therefore, to avoid the 3) to reduce the final volume to 125 mL.
International Journal of Analytical Chemistry 3

Table 1: Relative volumetric proportion for the mobile phases used to perform the 1D-TLCs and 2D-TLCs.

Mobile phase Solvent 1(𝛿1 ∗): Solvent 2 (𝛿2 ∗): modifier Relative volumetric proportion
MP-1 AcO (20.3): n-Hx (14.9): non-used 3:7:0
MP-2 AcO (20.3): n-Hx (14.9): non-used 1:1:0
MP-3 AcO (20.3): EA (18.6): non-used 1:1:0
MP-4a IOH (23.5): EA (18.6): non-used 0.16:1:0
MP-4b IOH (23.5): EA (18.6): non-used 1:1:0
MP-4c IOH (23.5): EA (18.6): non-used 1:0.16:0
MP-5a IOH (23.5): EA (18.6): NH4 OH∗∗ 0.16:1:0.25
MP-5b IOH (23.5): EA (18.6): NH4 OH∗∗ 1:1:0.25
MP-5c IOH (23.5): EA (18.6): NH4 OH∗∗ 1:0.16:0.25
∗
Solubility parameter (𝛿i ; expressed in (J/cm3 )1/2 ) [38]. ∗∗pKb = 4.75.

2.3. TLC Chromatography. TLC strips (2 cm × 10 cm) and seconds. Then, the first photographic record was taken (t0 );
TLC plates (20 cm × 20 cm) were used for 1D- and 2D- subsequently, more photographs were taken each hour, for 12
TLC, respectively. Nine different solvent systems (Table 1) hours. Additional pictures were taken every 12 hours up to 48
were tested as mobile phases (MPs); the MP-1 was reported hours and, for the MP-5c/MP-4a system (1st development/2nd
previously by Zarzycki et al., which was considered as the development), some extra photographs were collected at
starting mobile phase [39, 40]. longer times. Nevertheless, only the images taken at t0 and
For 1D-TLC, a cylindrical glass chamber (10 cm x 11 cm; when the spots showed maximum intensity (tF ; for the MP-
D x H) was used; its temperature was kept at 30± 1∘ C. Ten 1/MP-1 system, tF =6 h and, for the MP-5c/MP-4a one, tF =7
𝜇L of the methanolic extract was spotted near the bottom days) are shown.
of the TLC strip. Then, the solvent of the applied extract
was evaporated completely at room conditions. 15 min before
the TLC strip was developed, ten mL of the correspon- 3. Results and Discussion
dent MP was poured inside the chamber. Immediately, the
3.1. 1D-TLCs. In order to separate the compounds of the
developed strips were dried at room temperature and were
methanolic extract of Spirulina platensis, a “family” of 1D-
photographed under visible light (VL) or ultraviolet light
TLCs was prepared. For this purpose, just as starting point,
at 366 nm (UVL366 ). A Chromato-Vue CC-20 ultraviolet
the mobile phase used by Zarzycki et al. [39] was used,
chromatography viewer, equipped with a UV filter from
performing a systematic modification of such mobile phase
Ultra-Violet Products Inc., was used to allow the direct
to increase its relative polarity. The relative polarity of mobile
observation of the irradiated strips.
phases was qualitatively estimated considering the solubility
For 2D-TLCs, 100 𝜇L of the methanolic extract was
parameters of the correspondent pure solvents (𝛿); such
spotted near the bottom of the TLC plate; the solvent
criteria are commonly used to design binary solvents with
of the applied extract was evaporated completely at room
a gradual decreasing of its solvation capacity [41]. This
temperature. A standard TLC glass chamber (rectangular
methodology considers that when the relative amount of
TLC developing tank complete from Aldrich; 27 cm x 26.5
the solvents is kept constant, using a solvent with a higher
cm x 7.0 cm; L x H x W) was placed inside a recirculating
solubility parameter instead of another with a lower 𝛿 value
water bath and kept at 30 ± 1∘ C; the temperature inside the
produces a binary solvent with a higher polarity. Similarly, the
TLC chamber was monitored continuously to avoid thermal
polarity of the binary system increases as the relative content
variations. 100 mL of the corresponding mobile phase was
of the solvent with higher 𝛿 value increases [41].
added to this chamber. At the end of the first development,
Representative photographs of the TLC strips developed
the plate was dried at room temperature and again placed in
with the indicated mobile phases are shown in Figure 1.
the chamber in a perpendicular direction from the original,
Under visible light, eleven spots were obtained when the
to be developed in a second dimension. To finish the process,
MP-1 was used; this result was equivalent to the reported
the plate was dried at room temperature and the plates were
previously [39], demonstrating that such experiment was
photographed under VL and UVL366 as above.
successfully reproduced. Besides, six additional spots were
To corroborate the repeatability of the chromatographic
observed under UVL366 . In contrast, only five additional
procedure, the 2D-TLCs were performed by quintuplicate
spots were reported by Zarzycki when observed after iodine
obtaining repetitive results, evaluated by their respective RF
vapor exposure [39]. Nevertheless, since the well-resolved
values.
spots are especially important to this work, to establish the
number of this type of spots is relevant. Thus, when analyzing
2.4. 2D-TLC Bioautographic Assay. In order to locate spots the chromatograms obtained with the MP-1 (Figure 1) and
with probable antioxidant activity, the 2D-TLC plates were their respective retention factors (Table 2), it was observed
carefully dipped for 3-5 seconds in a methanolic solution that only three well-resolved spots were obtained (since for
of DPPH (0.25 mM) and dried at room temperature for 30 spot # 1, RF = 0.97, there is uncertainty about whether that spot
4 International Journal of Analytical Chemistry

Table 2: Retention factor (RF ) values of spots observed in the 1D-TLC plates, developed using the indicated mobile phases.

MP-1 MP-2 MP-3

VL UVL366 VL UVL366 VL UVL366
1 0.97 12 0.76 1 0.98 7 0.97 1 0.84 8 0.84
2 0.93∗ 13 0.66 2 0.93 8 0.79∗ 2 0.82 9 0.71∗
3 0.90∗ 14 0.47 3 0.87∗ 9 0.70∗ 3 0.78 10 0.62∗
4 0.84∗ 15 0.41 4 0.76∗ 10 0.26∗ 4 0.68∗ 11 0.36∗
5 0.79 16 0.37 5 0.07 11 0.08 5 0.10∗ 12 0.13∗
6 0.69 17 0.07 6 0.00 12 0.03 6 0.04 13 0.04
7 0.58 13 0 7 0.00 14 0
8 0.50
9 0.11
10 0.04
11 0.00
MP-4a MP-4b MP-4c
VL UVL366 VL UVL366 VL UVL366
1 0.92 7 0.92 1 0.83 8 0.83 1 0.89 8 0.81∗
2 0.84 8 0.79∗ 2 0.77∗ 9 0.71∗ 2 0.87 9 0.59∗
3 0.51∗ 9 0.23∗ 3 0.69∗ 10 0.53 3 0.77∗ 10 0.39∗
4 0.43∗ 10 0.19∗ 4 0.32 11 0.33 4 0.70∗ 11 0.18∗
5 0.07 11 0.04 5 0.11∗ 12 0.22∗ 5 0.49∗ 12 0.00
6 0.00 12 0.00 6 0.06 13 0.07 6 0.04
7 0.00 14 0 7 0
MP-5a MP-5b MP-5c
VL UVL366 VL UVL366 VL UVL366
1 0.94 16 0.92 1 0.96 16 0.93 1 0.96 16 0.93
2 0.92 17 0.44∗ 2 0.93 17 0.70∗ 2 0.93 17 0.70∗
3 0.88 18 0.37∗ 3 0.91 18 0.46 3 0.91 18 0.59∗
4 0.80∗ 19 0.29∗ 4 0.58∗ 19 0.38∗ 4 0.77∗ 19 0.49∗
5 0.41∗ 20 0.26∗ 5 0.54∗ 20 0.28 5 0.74∗ 20 0.43∗
6 0.36∗ 21 0.16∗ 6 0.50∗ 21 0.16∗ 6 0.68∗ 21 0.39∗
7 0.32∗ 7 0.42 22 0.09∗ 7 0.52∗ 22 0.32∗
8 0.24∗ 8 0.32∗ 23 0.00 8 0.50∗ 23 0.27
9 0.20∗ 9 0.26 9 0.48∗ 24 0.17∗
10 0.13 10 0.21∗ 10 0.42∗ 25 0.04∗
11 0.11∗ 11 0.18∗ 11 0.38∗
12 0.10∗ 12 0.14∗ 12 0.30∗
13 0.09∗ 13 0.07 13 0.28
14 0.04 14 0.03 14 0.11∗
15 0.00 15 0.00 15 0.00
∗
Spots considered as well resolved.

is a pure component or a mixture [28, 29]; therefore, this spot Besides, to make a pseudo-reverse phase that allows com-
was not considered well resolved). When the behavior shown pounds with a high polarity to be resolved more adequately, a
by the other binary mobile phases is considered (Figure 1 small amount of phase modifier (NH4 OH) was added to the
and Table 2), it can be observed that a considerably higher last three systems (MP-4a, b, and c), which promoted a con-
number of well-resolved spots was obtained, the best results siderable improvement in the chromatographic separation
being with the MP-4c, where 7 well-resolved spots can be behavior (Figure 1). Thus, the number of total spots (T), as
observed. Thus, although the highest number of total spots well as the number of well-resolved spots (W) obtained with
was obtained with the MP-1, the highest number of well- phases MP-5a (T=21 and W=13), MP-5b (T=23 and W=11),
resolved spots (the goal of this work) was obtained with the and MP-5c (T=25 and W=18), was noticeably higher than
MP-4c, followed by the mobile phases MP-4a and MP-4b. the ones obtained with the equivalent phases without phase
International Journal of Analytical Chemistry 5

MP-1 MP-2 MP-3 MP-4a MP-4b MP-4c MP-5a MP-5b MP-5c

VL ５６， 366 VL ５６， 366 VL ５６， 366 VL ５６， 366 VL ５６， 366 VL ５６， 366 VL ５６， 366 VL ５６， 366 VL ５６， 366

1
2 7
8

3
4

9
10

5 11
6 12

Figure 1: Representative photographs of 1D thin layer chromatograms for a methanolic extract of Spirulina platensis, developed with the
indicated mobile phases and visualized under visible light (VL) or under ultraviolet light (UVL366 ).

modifier (MP4a: T= 12 and W=5; MP-4b: T=14 and W=5; exhibits 28 spots, with fifteen of them being considered well-
MP-4c: T=12 and W=7), which demonstrate the utility of the resolved spots, it can be affirmed that its separation behavior
NH4 OH presence in the system (Table 2). A global analysis represents a considerable improvement on the chromato-
of the 1D-TLC results shows that, with the MP-5c, a suitable graphic resolution, when it is compared to the starting system
balance of the interactions among the solvent system, the (MP-1/MP-1; 20 total spots and 10 well-resolved ones).
stationary phase (partially masked by the NH4 OH), and the
different metabolites was obtained, allowing for their gradual 3.3. 2D-TLC Bioautographic Assays. Since the main goal of
separation. this work is to identify well-resolved spots with antioxidant
activity, in a preliminary step, the yellowish characteristic
3.2. 2D-TLCs. To further improve the resolution of the produced by the DPPH technique was looked for only on the
components contained in the methanolic extract of Spirulina well-resolved spots (Table 3). Thus, in Figure 2 it is observed
platensis, a wide experimental set of 2D-TLCs was performed, that only spots # 6 and # 8 are useful for providing material
obtaining the best results with the system that used in the first susceptible to being used in a subsequent identification
development (1st ) the mobile phase named MP-5c and, in the procedure (e.g., well-resolved and containing compounds
second one (2nd ), the MP-4a. Thus, in Figure 2 representative with antioxidant activity). In an equivalent analysis, but with
photographs of chromatographic plates developed by 2D- the MP-5c/MP-4a system, a higher number of useful spots
TLC are presented, which used the following solvent systems: were identified, specifically, spots #12, #14, #16, and #29. It
(a) 1st : MP-1 and 2nd : MP-1 (herein referred to as the starting is important to mention that spot #29 could be observed
system) and (b) 1st : MP-5c and 2nd : MP-4a (herein referred neither with visible light nor with UV light, but it could be
to as the “best tested system”). Further, in Table 3 the observed when its component(s) reacted as a consequence
corresponding RF values for such experimental systems are of the DPPH addition. Taking into account only the above-
presented. mentioned information, the improvement reached with the
Regarding the separating behavior of the starting system proposed 2D-TLC bioautographic assays is evident.
(a system of 2D-TLC using the same mobile phase in both A more in-depth analysis of the MP-5c/MP-4a system
developments), as it could be expected [42, 43], the separation showed in Figure 2 that although spots # 4 (green), #6 (more
quality was improved by the application of the second devel- intense orange), and #9 (less intense orange) are qualitatively
opment. Thus, in the 1D-TLC that used the MP-1, seventeen distinguishable by their respective colors, they exhibit a
spots were obtained, but only three well-resolved spots (Fig- considerable overlapped area (spot #4 with #6 and #6 with
ure 1), whereas in the 2D-TLC, 20 total spots could be assessed #9); therefore, they were considered not well-resolved spots.
and ten of them were considered well resolved (Figure 2). Nevertheless, after DPPH application, spot #6 appeared very
In addition, a comparative analysis of the separating quickly (in the photo taken starting the process (t0 ) a weak
behavior of the last mentioned systems (Figure 2 and Table 3) yellowish color could be assessed), hinting toward a strong
shows that since the “best tested system” (MP-5c/MP-4a) antioxidant activity occurring in such spot. In the case of spot
6 International Journal of Analytical Chemistry

Mobile Before DPPH

phase addition After DPPH addition

VL t

1D:MP-1
2D:MP-1

UVL  tF

1D:MP-1
2D:MP-1

VL t

1D:MP-5c
2D:MP-4a

UVL  tF

1D:MP-5c
2D:MP-4a

Figure 2: Representative photographs of 2D thin layer chromatograms for a methanolic extract of Spirulina platensis, developed with the
indicated mobile phases and visualized under visible light (VL) and under ultraviolet light (UVL366 ) (central column) and the correspondent
2D thin layer biochromatograms, obtained after DPPH treatment at t0 and tF (right column).

#9, its yellowish color could be assessed only after several containing components with a noticeable global antioxidant
hours, which can be interpreted as an antioxidant activity activity. In addition, due to spot #28 remaining without dis-
weaker than the one shown by component(s) present in spot placement after both chromatographic developments, it was
#6. Finally, spot #4 never showed antioxidant activity. With considered a non-well-resolved spot that possibly contained
such evidence, it could be useful to isolate the global area more than one component. Nevertheless, after the application
visualized in yellow in plate with DPPH to obtain a mixture of the DPPH, a very defined pale yellow spot appeared, which
International Journal of Analytical Chemistry 7

Table 3: Retention factor (RF ) values observed in 2D-TLC plates, developed with the indicated mobile phases.

VL UVL366
Spot MP-1 MP-1 Spot MP-1 MP-1
1 0.74 0.76∗ 12 0.59 0.59∗
2 0.66 0.66∗ 13 0.56 0.56∗
3 0.62 0.62∗ 14 0.52 0.49
4 0.58 0.53∗ 15 0.41 0.38
5 0.55 0.49 16 0.30 0.26
6+ 0.47 0.44∗ 17 0.15 0.15∗
7 0.38 0.37 18 0.06 0.12∗
8+ 0.34 0.30∗ 19 0.04 0.02
9 0.31 0.26 20 0.00 0.00
10 0.03 0.02
11 0.00 0.00
MP-5c MP-4a MP-5c Mp-4a
1 0.79 0.79 19 0.46 0.59
2 0.74 0.86 20 0.46 0.09∗
3 0.68 0.59∗ 21 0.38 0.06∗
4 0.64 0.69 22 0.34 0.35
5 0.64 0.54∗ 23 0.26 0.35
6 0.60 0.71 24 0.26 0.03∗
7 0.60 0.53∗ 25 0.16 0.02
8 0.56 0.88∗ 26 0.11 0.00∗
9 0.56 0.68 27 0.05 0.00∗
10 0.50 0.43∗ 28 0.00 0.00
11 0.45 0.48 29+ 0.20 0.26
12+ 0.45 0.29∗
13 0.38 0.43∗
14+ 0.38 0.18∗
15 0.30 0.09∗
16+ 0.22 0.00∗
17 0.15 0.00
18 0.04 0.00
∗
Spots considered as well resolved.
+
Spots considered as well-resolved and containing compounds with antioxidant activity.

demonstrates that, regardless of the number of components, Data Availability

it is a mixture (or a pure component) potentially useful thanks
to its antioxidant activity. The data used to support the findings of this study are
available from the corresponding author upon request.
4. Conclusion Conflicts of Interest
As a first step in the identification of the antioxidant
The authors declare that they have no conflicts of interest.
compounds contained in methanolic extracts of Spirulina
platensis, a simple and fast 2D-TLC biosystem was developed.
The proposed experimental system allowed a suitable sepa- References
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