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Research Article
The New TLC Method for Separation and Determination of
Multicomponent Mixtures of Plant Extracts
Elhbieta Matysik,1Anna Wofniak,2Roman Paduch,3Robert Rejdak,2
Beata Polak,4and Helena Donica5
1Department of Analytical Chemistry, Medical University of Lublin, Chod´
zki 4A, 20-093 Lublin, Poland
2Department of General Ophthalmology, Medical University of Lublin, Chmielna 1, 20-079 Lublin, Poland
3Institute of Microbiology & Biotechnology, Department of Virology & Immunology, Maria Curie Sklodowska University,
Akademicka 19, 20-033 Lublin, Poland
4Department of Physical Chemistry, Medical University of Lublin, Chod´
zki 4A, 20-093 Lublin, Poland
5Department of Biochemical Diagnostics, Medical University of Lublin, Staszica 16, 20-081 Lublin, Poland
Correspondence should be addressed to Beata Polak; beatapolak@umlub.pl
Received November ; Revised January ; Accepted January
Academic Editor: Vito Verardo
Copyright © El˙
zbieta Matysik et al. is 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.
e new mode of two-dimensional gradient thin layer chromatography (MGD-D TLC) has been presented. Short distance
development of sample in the rst dimension leads to formation of the preconcentrated narrow zones. ey are consecutively
separated in the second dimension with the mobile phase gradient in several steps of development until the eluent reaches the
further end of the chromatographic plate. e use of the above-mentioned technique allows isolating and then identifying the
compounds of various polarity from the multicomponent mixture. e practical application of two-dimensional gradient thin layer
chromatography has been performed for isolation of the two plant (Juniperus and ymus)oilscomponentsastheexamplesoftest
mixtures. e experiments have been carried out with the use of silica gel plates as well as a normal phase condition. e results
of solute separation with isocratic one-dimensional thin layer chromatography system have been compared with those of two-
dimensional gradient system. It has been observed that application of the latter mode leads to almost triplicated number of zones
in comparison with the former one. It is purposeful to apply the proposed mode to control the purity of the dominant component
or components of the mixture.
1. Introduction
e separation and determination of compounds in mul-
ticomponent mixtures, that is, plant extracts, are usually
performed with the use of high performance liquid chro-
matography, HPLC (e.g., [, ]), or planar chromatography
(e.g., [–]). e sample complicity oen involves application
of two-dimensional system in both techniques.
e comparison of solute and standard retentions (both
measured as the partition coecient, 𝑘) achieved during
two individual analyses is the principal to the compound
identication in D-HPLC. Similarly, in D-TLC, the spots of
two mixtures (plant extract and standards) are separated with
the use of two chromatographic plates [–]. Subsequently,
thesamplecomponentsareidentiedbycomparisonoftheir
retardation factor (𝑅𝐹)valueswiththoseoftheseparated
standards.
e aforementioned technique has some drawbacks
owing to the dierences in conditions of the separation pro-
cess onto two chromatographic plates, what may cause errors
in identication of the components of the plant extract. It
especially concerns compounds with similar polarity. us,
zones of determined solutes may be contaminated with other
compounds.
It is more rational to separate the plant extract and the
mixture of standards parallel with the use of a single plate
and identical conditions [, ]. e simultaneous separation
of two samples (extract and the mixture of standards) applied
Hindawi Publishing Corporation
Journal of Analytical Methods in Chemistry
Volume 2016, Article ID 1813581, 6 pages
http://dx.doi.org/10.1155/2016/1813581
Journal of Analytical Methods in Chemistry
onto the distinct ends of the chromatographic plate has been
presented in []. Authors have separated both zones in two
directions. e chromatographic plate containing solutes is
developed in the rst direction using a rst mobile phase.
Subsequently aer eluent evaporation from the adsorbent
layer, the chromatogram is developed in the perpendicular
direction from two short sides of the plate (e.g., with the use
of the horizontal chamber). Aer the second development,
the separated extract and separated mixture of standards
form mirror images. Both types of spots (separated sample
compounds and standards) migrate identical distance from
the line dividing the chromatogram onto two parts [].
Such technique permits identifying the compounds of both
samples by direct comparison.
e method described above sometimes fails to achieve
complete separation of spots, especially those containing
dominant and minor components. Moreover, there is poor
separation of solutes of similar retention. is fact makes the
qualitative and quantitative analyses of the mixture impossi-
ble.
Authors propose the modication of the above-men-
tionedmethod.enewtechniqueleadstothepreconcen-
tration of the solute zones during two-dimensional plate
developments and they are consecutively separated with the
useofthemobilephasegradientsandtheprogressivechange
of the development distance.
New method has been successfully veried by the sep-
aration of two plant extracts (Juniperi Oleum and ymi
Oleum).
2. Experimental
Chromatography was performed on cm × cm glass
HPTLC plates coated with silica gel Si F254 (Merck, Darm-
stadt, Germany). Essential oils (Juniperi Oleum and ymi
Oleum) were obtained from Profarm Sp. z O.O. (Lębork,
Poland) and dissolved in toluene to furnish .% v/v solu-
tions.
e organic solvents (toluene, ethyl acetate, and meth-
anol) and sulphuric acid were purchased from Avantor
Performance Materials (Poland, Gliwice), while the vanillin
was obtained from Merck (Darmstadt, Germany), whereas
anise aldehyde was received from Sigma-Aldrich (St. Louis,
MO, USA).
𝜇L of the toluene solutions of essential oils (Juniperi
Oleum and ymi Oleum)wasappliedascmzoneonthe
10×20 cm chromatographic plates by means of the Hamilton
HPLC syringe.
2.1. Chromatographic Development. Chromatographic plates
aer spot applications were developed in a horizontal Teon
DS L or DS-II-10×20 chamber (Chromdes, Lublin, Poland) in
the rst direction in traditional way with mixture of toluene
andethylacetateastheeluent.edevelopmentdistances
were varied from to cm. en, the mobile phase has been
evaporated. Subsequently, the plate was turned by ∘and
the separated zones were preconcentrated by development
with a strong volatile solvent on the distance from . to
cm from the starting point. e chemical properties of
investigated solutes inuenced the type of eluent applied.
us, the monoterpenes were developed with toluene or ethyl
acetate, whereas more polar compounds, that is, polyphenols,
required stronger eluent, that is, methanol or mixture of
water and methanol. e preconcentration procedure was
repeated until the zones were narrow and compact; aer
each repetition, the plate was dried. en, the plate was
developedinfulldistanceintheperpendiculardirection
[]. All experiments were conducted at room temperature
(∘C).
2.2. Detection of Compounds. e spots of separated com-
pounds were detected with anise aldehyde in sulphuric acid
(. mL of anise aldehyde, mL glacial acetic acid, mL
methanol, and mL % sulphuric acid) or with vanillin
ethanol solution ( g of vanillin, mL % ethanol, and
mL % sulphuric (VI) acid).
e plate was heated to ∘Cuntilthecolourspots
became visible.
e spots were detected with Desaga (Heidelberg, Ger-
many) CD densitometer (slit size . mm ×. mm; 𝜆=
560nm).
3. Results and Discussion
e new method, presented in experimental part proce-
dure, has been applied to separate compounds of two
essential oils, that is, Juniperus and ymi. According to
the literature, both investigated mixtures contain solutes
of various polarities (e.g., monoterpenes, sesquiterpenes,
and monoterpene alcohols) [–]. Various chromatographic
techniques, that is, GC [] or TLC with special detection
mode (BioArena [], MS [], and bioautography []), have
been employed for determination of Juniperus and ymi
oil compounds. However, till today, there is no information
on applying the multiple gradient development for this
purpose.
e plate containing the zones is developed in the rst
direction with isocratic elution (mixture of toluene and ethyl
acetate) at the rst stage of experiments. Juniperi oil contains
essential amount of monoterpene alcohols [] which requires
more polar mobile phase (toluene and ethyl acetate; : v/v)
in comparison to the ymi oil (toluene and ethyl acetate;
. : .% v/v). e results of separation of Juniperi Oleum
and ymi Oleum are presented as photos in Figures and
, respectively. However, the purity of separated zones is
unknown. erefore, the development of the chromatogram
in the perpendicular direction with gradient of the mobile
phase is undertaken. e development programs for eluents
realized with MGD-D TLC have been determined exper-
imentally and are presented in Tables and for Juniperi
Oleum and for ymi Oleum, correspondingly. Decreasing of
the separation distances and simultaneously increasing of the
mobile phase polarity during the multiple plate developments
enhance the separation of the polar compound zones on one
hand and keep nonpolar compound zones separated, on the
other hand.
Journal of Analytical Methods in Chemistry
T : e program used for four-step gradient elution for the
development in second dimension for Juniperi Oleum.
Mobile phase
Step
number
Step
number
Step
number
Step
number
Tolu e n e . . . .
Ethyl acetate . . . .
Development distance (cm) . . . .
e composition is given in %v/v.
T : e program used for ve-step gradient elution for the
development in second dimension for ymi Oleum.
Mobile phase
Step
number
Step
number
Step
number
Step
number
Step
number
Toluene . . . . .
Ethyl acetate . . . . .
Development
distance (cm) . . . . .
e composition is given in %v/v.
e separation of Juniperi Oleum with MGD-D-TLC
technique is presented in Figure (a). e application of
new mode of development leads to achieving ve addi-
tional zones of compound number from isocratic system
(Figure ) denoted as a, b, c, d, e, and f (densi-
togram, Figure (b)). Also, compound number from one-
dimensional development (Figure ) turns to be the mixture
since ve additional spots are detected (see densitogram in
Figure (c)).
Correspondingly, ymi Oleum is analysed with the
newtechnique.eenhancementofthezonenumbersis
presented in Figure (a). Some of the identied solutes from
one-dimensional development (Figure ) such as compounds
number (thymol, 𝑅𝐹= 0.61), number (,-cyneole,
𝑅𝐹= 0.55), number (linalool, 𝑅𝐹= 0.37), and number
(borneol,𝑅𝐹= 0.24) are separated into additional zones
(number into (a) and (b); numbers and into extra
zones; number into extra zones). e only pure zone
turns to be carvacrol (compound number ). Overlaid of
ymi Oleum separation densitograms obtained with MGD-
D TLC technique is presented in Figure (b).
e essential dierences between single, isocratic devel-
opment and MGD-D-TLC of investigated oils are sum-
marizedinTablesand.eapplicationofMGD-D
TLC to Juniperi Oleum separation brings about the enhance-
ment of number of separated spots from (from D,
isocratic development) into . What is more, the same eect
was observed for the second separated oil. Only zones
were obtained by simple isocratic TLC separation of ymi
Oleum whileinthenewtechniquethenumberincreasedto
.
e detailed description of the novel MGD-D TLC
techniquehasbeenpublishedinPolishPatent[].
F : D separation of Juniperi Oleum compounds. e mobile
phase: toluene and ethyl acetate ( : v/v). e zones were der iva-
tized with the anise aldehyde-sulphuric (VI) acid reagent.
T : e comparison of eciency of JuniperiOleum separation
by means of two methods: isocratic elution and MGD-D TLC.
Compounds applied in order of decreasing 𝑅𝐹value and increasing
polarity.
Isocratic elution MGD-D TLC
Number of band 𝑅𝐹
. Band number 1→1a
. Band number 2→2a,2b,2c,2d,2e
. Band number 3→3a,3b,3c,3d
. Band number
. Band number
. Band number 6→6a,6b,6c,6d,6e,6f
. Band number
. Band number 8→8a
. Band number 9→9a,9b,9c,9d,9e
. Band number
. Band number
. Band number
4. Conclusions
e successful application of the above-mentioned tech-
nique for separation of Juniperi Oleum and ymi Oleum
as model complex mixtures makes it promising to further
investigations. Continuation of this study will consist in
spectrophotometric estimation of purity of separated zones.
e presented results indicate that a determination of the
purity of separated zones in planar chromatography cannot
be based on a simple isocratic technique. Application of
two-dimensional multiple gradient development leads to the
isolation of minor compound in the presence of dominant
one. Additionally, changes of the development distances
makethistechniquepromisingforseparationofpolarcom-
pounds. New method enables also more reliable estimation
of the pharmacological properties of the components (e.g.,
lipophilicity). What is more, compact, concentrated zones of
Journal of Analytical Methods in Chemistry
(a) (b)
F : e D-TLC separation of ymi Oleum compounds. e mobile phase: toluene and ethyl acetate (. : . v/v). e two modes of
compounds derivatization: (a) anise aldehyde-sulphuric (VI) acid reagent and (b) vanillin in ethanol reagent.
13a
13
1211
10
9e
9d
9c
9b
9a
98a
8
7
6f
6e
6d
6c
6b
6a 6
5
4c
4b
4a
4
3d
3c
3b
3a
3
2e
2d
2c
2b
2a
2
1a
1
(a)
6e 6f
6d
6c
6b
6a
−50.0
0.0
50.0
100.0
150.0
AU
20.0 30.0 40.0 50.0 60.0 70.0 80.010.0
(mm)
(b)
9e
9d
9c
9b
9a
9
20.0 30.0 40.0 50.0 60.0 70.0 80.010.0
(mm)
−25.0
0.0
25.0
50.0
75.0
100.0
125.0
AU
9e
9
9d
9d
9c
9
9
9b
9
9a
9a
9
9
(c)
F : (a) MGD-D TLC separation of Juniperi Oleum compounds. e program of eluent for second dimension development is presented
in Table . e spots were derivatized with the anise aldehyde-sulphuric (VI) acid reagent. (b) Densitogram of the separation of compound
(𝑅𝐹= 0.43 from isocratic elution) into additional bands by means of MGD-D-TLC. Peaks with e and f were not visible with the applied
wavelength ( nm). (c) Densitogram presenting the separation of compound number (𝑅𝐹= 0.23, from isocratic elution) into additional
bands by means of MGD-D TLC. Peaks with low 𝑅𝐹values, not marked on the densitogram, were not visible with the naked eye. eir
detection was performed by densitometry at the wavelength of nm (compare Figures and ).
Journal of Analytical Methods in Chemistry
(a)
9-11a
8-8a, b, c, d, e
7
5, 6-6a, b, c
4-4a, b, c, d
2, 3-3a, b
1-1a, b, c
0.0
250.0
500.0
750.0
1000.0
1250.0
AU
20.0 30.0 40.0 50.0 60.0 70.0 80.010.0
(mm)
(b)
F : (a) MGD-D TLC chromatogram presenting the separation of ymi Oleum compounds. e eluent gradient program for second
dimension development is presented in Table . Solutes were derivatized with the anise aldehyde-sulphuric (VI) acid reagent. (b) Overlaid
densitograms of the ymi Oleum separations into additional bands by means of MGD-D TLC. e eluent gradient program for second
dimension development from Table .
T : e comparison of eciency of ymiOleum separation
by means of two methods: isocratic elution and MGD-D TLC. e
identication of particular compounds was performed according to
𝑅𝐹values given in the literature.
Isocratic elution MGD-D TLC
Number of
band 𝑅𝐹Compound
. Unknown Band number 1→1a,1b,1c
. Carvacrol Band number
. ymol Band number 3→3a,3b
. ,-Cyneole Band number 4→4a,4b,4c,4d
. 𝛼-Terpineo l Band number
. Linalool Band number
6→6a,6b,6c,6d,6e,6f
. p-Cymene Band number
. Borneol Band number 8→8a,8b,8c,8d
. Unknown Band number
. Unknown Band number
. Unknown Band number 11 → 11a
solutes may be further investigated with the use of various
detectors, for example, mass spectrometry or diode array
spectrophotometry.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
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