Micro-thin-layer chromatography in two-dimensional (2D-mTLC) mode in normal- (NP) and reversed-phase (RP) systems by use of cyanopropyl-bonded stationary phases was applied to make fingerprints of 11 species of Mentha genus and two finished pharmaceutical products. Non-aqueous eluents were used in the NP systems. Mixtures of acetonitrile with water and methanol with water were used in the RP chromatographic systems. Optimization of one-dimensional systems was performed by determining RM vs. composition of mobile phase dependencies for standards occurring in various Mentha sp. On the basis of these dependencies, the most selective chromatographic systems for each run were chosen. Then most selective eluents were applied to optimize two-dimensional systems by creating RF in NP systems vs. RF in RP systems correlations. The best two-dimensional systems were chosen on the basis of R2 values for RF vs. RF correlations (the lowest values of R2 coefficients). The 2D-mTLC optimized systems were applied to separate phenolic compounds and make fingerprints of the examined plant materials.

Introduction

The use of mint species in traditional and conventional medicine is mostly due to the presence of two classes of secondary metabolites: monoterpenoids in essential oils and different structural types of phenolic compounds. Essential oils are known to act as antimicrobial, antispasmodic, carminative and antiviral agents. In addition, essential oils of several mint species have been recently qualified as natural antioxidants. However, since oil composition is highly variable, the pharmacological activity strongly depends on certain variety. In contrast, composition of phenolic constituents is relatively stable within species. The most important phenolic compounds in Mentha species are flavonoids. Mints are characterized by the presence of specific lipophilic aglycones. Phenolic compounds of mints are found to exhibit a wide range of pharmacological activities: antioxidant, antiulcer, cytoprotective, hepatoprotective, cholagogue, chemopreventive, anti-inflammatory, antidiabetic, etc. However, besides healing properties some mint species can exhibit an adverse effect on human health (13).

The genus Mentha includes 25–30 species that grow in the temperate regions of Eurasia, Australia and South Africa (4). The mint species have a great importance, both medicinal and commercial. Indeed, leaves, flowers and stems of Mentha spp. are frequently used in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavour. In addition, Mentha spp. has been used as a folk remedy for treatment of nausea, bronchitis, flatulence, anorexia, ulcerative colitis and liver complaints due to its antinflammatory, carminative, antiemetic, diaphoretic, antispasmodic, analgesic, stimulant, emmenagogue and anticatarrhal activities (13).

The micro-thin-layer chromatography (TLC) technique can be applied to generate fingerprints of complex mixtures, which are present in biological or environmental samples. In addition, miniaturized planar chromatography allows fast sample separation and low mobile phase consumption. This method can be considered as an environmentally friendly and green chemistry focused analytical tool, supplementary to analytical protocols involving column chromatography.

Modern high-performance planar chromatography including micro-TLC is a very useful technique for separation of biological samples, generally due to the high-sensitivity analysis, short analysis time, low consumption of eluent and a small amount of the sample (58).

The TLC technique has been used previously for the determination of qualitative and quantitative composition of Mentha sp. TLC is often applied in the analysis to determine antioxidant activity and to generate fingerprints of various Mentha sp. (5, 912).

Polar-bonded stationary phases (cyanopropyl, aminopropyl and diol) are the special types of stationary phases which can be used in both normal-phase (NP-TLC) and reversed-phase (RP-TLC) systems. In this case, two-dimensional thin-layer chromatography can be performed without technical problems of connection of various types of stationary phases (e.g., silica–RP phases). It makes possible the separation of multicomponent natural mixtures on one plate by use of non-aqueous and perpendicularly aqueous eluents (various properties and selectivities) (1319).

The main aim of this work is the examination of the possibility of use of this method as the instrument for comparison of the composition of Mentha sp. extracts (fingerprints).

Experimental

Plant material was collected from the following eleven species of Lamiaceae family: Mentha piperita var. Zgadka, Mentha piperita var. Cernolistnaja (Pharmacognosy Garden of Medical University of Lublin), Mentha suaveolens var. Variegata, Mentha arvensis L., Mentha piperita L., Mentha spicata var. Crispa, Mentha aquatica L., Mentha piperita var. Perpeta, Mentha spicata L., Mentha longifolia L. (Botanical Garden of Maria Curie-Sklodowska University in Lublin) and Mentha longifolia (collected in Ostrowsko, southern Poland, during summer 2012). The study also included two finished pharmaceutical products: mix of Mentha species fix (Manufacturer 1, Poland and Manufacturer 2, Poland).

High-performance thin-layer chromatography (HPTLC) CN F254s 10 cm × 10 cm plates (Merck, Darmstadt, Germany), cut to the 5 cm × 5 cm squares, were used in all tests. All solvents (propan-2-ol, ethyl acetate, acetonitrile (ACN), n-heptane and methanol) were of analytical grade and purchased from Polish Reagents (POCh, Gliwice, Poland). To receive non-aqueous phases, propan-2-ol was mixed with n-heptane (5), and distilled water was mixed with ACN or methanol to obtain aqueous solvent for 2D-HPTLC. ACN and distilled water (concentrations: 20, 30, 40, 50, 60 and 80% (v/v)) were prepared to optimize the separation of test substances in aqueous systems using CN-bonded chromatographic plates as the stationary phase.

All test substances (rutin, diosmin, rosmarinic acid, apigenin, eriocitrin, luteolin, luteolin 7-glugoside, isorhoifolin, narirutin, naringenin, caffeic acid) were acquired from various manufacturers (Sigma-Aldrich, Fluka). Naturstoff reagent (2-(diphenylboryoxy)-ethylamine and PEG4000) was produced by Merck and was composed of a 5% methanolic solution of polyethylene glycol (PEG) and 1% methanolic solution of 2-(diphenylboryoxy)-ethylamine.

Twenty grams of each Mentha sp. L. were weighed, finely divided and closed in a paper case. Each material was extracted in the Soxhlet apparatus on a water bath during 8 h using chloroform to isolate chlorophylls. After that, dried plant materials (in the paper case) were again extracted for 8 h by a portion of 200 mL of methanol. After extraction methanol was evaporated on a water bath under reduced pressure (0.9 atm). The dry residue was dissolved in methanol and filled with the same solvent in a 25-mL flask. The prepared extracts were stored in a refrigerator and then examined in all experiments.

The 0.1% (v/v) solution of the mixture of standards (5 µL) and 5 µL methanolic solutions of extracts were spotted manually by glass capillaries 0.5 cm from each edge of the chromatographic plate and developed in two directions by the use of DS-II horizontal developing chambers (Chromdes, Lublin, Poland). In the first direction the plate was developed by the use of non-aqueous solvent at a distance of 4.5 cm after conditioning in eluent vapours for 20–30 min to avoid the demixing effect. After development, by drying in air the same plate was developed in the perpendicular direction to the first one by use of aqueous eluent. In RP systems plates were developed without conditioning. After drying, the plates were sprayed with Naturstoff reagent (Merck, Darmstadt, Germany) using the Merck TLC sprayer and photographed under a Camag Cabinet UV lamp at 365 nm by the use of a Kodak 14 mpx camera. Examined standards are listed in Table I.

Table I.

The RF Values of Standards on CN-silica Plates Using Suitable Mobile Phases

Lp Substance Manufacturer RF
iPrOH–n-Hp
(4 : 6 v/v) 
RF
ACN–H2O
(3 : 7 v/v) 
RF
EtOAc–n-Hp
(8 : 2 v/v) 
RF
MeOH–H2O
(5 : 5 v/v) 
Rutin Sigma-Aldrich 0.18 0.32 0.17 0.5 
Diosmin Sigma-Aldrich 0.09 0.18 0.12 0.4 
Rosmarinic acid Fluka 0.27 0.49 0.56 0.89 
Apigenin Sigma-Aldrich 0.40 0.02 0.6 0.13 
Eriocitrin Sigma-Aldrich 0.11 0.39 0.27 0.57 
Luteolin Sigma-Aldrich 0.40 0.06 0.58 0.18 
Luteolin 7-glucoside Sigma-Aldrich 0.19 0.18 0.27 0.33 
Isorhoifolin Fluka 0.14 0.20 0.12 0.37 
Narirutin Sigma-Aldrich 0.11 0,29 0.18 0.47 
10 Naringenin Sigma-Aldrich 0.33 0.06 0.64 0.16 
11 Caffeic acid Fluka 0.44 0.43 048 0.69 
Lp Substance Manufacturer RF
iPrOH–n-Hp
(4 : 6 v/v) 
RF
ACN–H2O
(3 : 7 v/v) 
RF
EtOAc–n-Hp
(8 : 2 v/v) 
RF
MeOH–H2O
(5 : 5 v/v) 
Rutin Sigma-Aldrich 0.18 0.32 0.17 0.5 
Diosmin Sigma-Aldrich 0.09 0.18 0.12 0.4 
Rosmarinic acid Fluka 0.27 0.49 0.56 0.89 
Apigenin Sigma-Aldrich 0.40 0.02 0.6 0.13 
Eriocitrin Sigma-Aldrich 0.11 0.39 0.27 0.57 
Luteolin Sigma-Aldrich 0.40 0.06 0.58 0.18 
Luteolin 7-glucoside Sigma-Aldrich 0.19 0.18 0.27 0.33 
Isorhoifolin Fluka 0.14 0.20 0.12 0.37 
Narirutin Sigma-Aldrich 0.11 0,29 0.18 0.47 
10 Naringenin Sigma-Aldrich 0.33 0.06 0.64 0.16 
11 Caffeic acid Fluka 0.44 0.43 048 0.69 

Results

On the basis of experiments conducted previously in the Department of Inorganic Chemistry in Lublin by our research team, the choice of optimal eluent systems for the separation of phenolic fractions was made (5). Our study included two species of mint with two finished pharmaceutical products. Previous results were so satisfying that the same systems have been used for the analysis of more mint species. The best results were obtained for systems with 40% propan-2-ol (iPrOH) in n-heptane (n-Hp) for the non-aqueous mobile phase in NP-TLC systems. We used also our previous results with 50% methanol (MeOH) in water for aqueous mobile phase in RP-TLC systems (5).

Figure 1a shows RM vs. log c dependencies for investigated test compounds for ACN in water used as aqueous mobile phase in RP-TLC systems. This dependence makes it possible to choose the best system for analysis. The system in which RM values for investigated compounds are the most different for the given concentration of the mobile phase is the most selective and it is most suitable for the separation of these compounds. The optimal RP aqueous eluent was 30% ACN in water. Figure 1b shows RM vs. log c dependencies for investigated standards for ethyl acetate (EtOAc) in n-heptane (n-Hp) used for the non-aqueous mobile phase in NP-TLC systems in system II. It was concluded that optimal NP non-aqueous eluent was 80% ethyl acetate in n-heptane. The RF values of standards in the above eluent systems are presented in Table I.

Figure 1.

Rm vs. c% relationship for tested compounds in the system: (a) cyano-bonded stationary phase—ACN + water as mobile phase, and (b) cyano-bonded stationary phase—ethyl acetate + n-heptane as mobile phase. Numbers in the legend as in Table I.

Figure 1.

Rm vs. c% relationship for tested compounds in the system: (a) cyano-bonded stationary phase—ACN + water as mobile phase, and (b) cyano-bonded stationary phase—ethyl acetate + n-heptane as mobile phase. Numbers in the legend as in Table I.

RF vs. RF dependencies were plotted to optimize two-dimensional thin-layer chromatographic systems. The orthogonal systems with the lowest correlation coefficients (R2 values) are the best for two-dimensional separations, because points corresponding to test substances are placed on the whole chromatographic plate. However, there is not just one factor which decides about the separation effect. Figure 2a shows the plots of RF vs. RF for the system I: the first direction of development—40% iPrOH + n-heptane and the second direction of development—30% ACN + water. Figure 2b shows the plots of RF vs. RF for the system II: first direction of development—80% EtOAc + n-heptane and the second direction of development 50% MeOH + water.

Figure 2.

RF vs. RF relationship for the 2D-TLC system with cyano-bonded stationary phase and (a) 40% iPrOH + n-heptane in the first direction of development, 30% ACN + water in the second direction of development. (b) 80% EtOAc + n-heptane in the first direction of development, 50% MeOH + water in the second direction of development. Numbers as in Table I. This figure is available in black and white in print and in color at JCS online.

Figure 2.

RF vs. RF relationship for the 2D-TLC system with cyano-bonded stationary phase and (a) 40% iPrOH + n-heptane in the first direction of development, 30% ACN + water in the second direction of development. (b) 80% EtOAc + n-heptane in the first direction of development, 50% MeOH + water in the second direction of development. Numbers as in Table I. This figure is available in black and white in print and in color at JCS online.

Simulated separations achieved by the use of two systems (I: 40% iPrOH + n-heptane/30% ACN + water and II: 80% EtOAc + n-heptane/50% MeOH + water) were satisfactory and these eluents were applied for the separation of Mentha extracts. On the basis of these experiments, the two-dimensional separations of standards as well as of extracts of mint species herbs were performed.

Documentation of chromatographic experiments includes images of plates photographed in under a CAMAG Cabinet UV lamp after derivatization with the Naturstoff reagent at 365 nm by using a Kodak 14 mpx camera.

Discussion

Table II shows a comparison of the presence of substances in various Mentha extracts detected by UV 245, UV 365, Naturstoff reagent on cyano-bonded plates for system I (40% iPrOH + n-heptane and 30% ACN + water). All tested compounds are present in Mentha piperita var. Cernolistnaja and Mentha spicata var. crispa. In extracts of Mentha aquatica L. and Mentha longifolia L. from the natural habitat in Ostrowsko (southern Poland) rosmarinic acid is absent. Four species of mint, such as Mentha arvensis L., Mentha piperita L., Mentha piperita var. Perpeta and Mentha spicata L. contain no rutin and rosmarinic acid. Two finished pharmaceutical products were also tested. The herb of mint produced by Manufacturer 2 contained all selected standards except rosmarinic and caffeic acids. Herb of mint produced by Manufacturer 2 has also no luteolin 7-glucoside and isorhoifolin. Diosmin, apigenin, eriocitrin, luteolin, narirutin and naringenin were identified in all tested extracts, while rutin was present only in three.

Table II.

The Comparison of Presence of Substances in Various Mentha Extracts Detected by UV 245 nm, UV 366 nm, Naturstoff Reagent on Cyano-bonded Plates for System 40% iPrOH + n-heptane and 30% ACN + water

Number of substance 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 
Mentha piperita var. Zgadka   
Mentha piperita var. Cernolistnaja 
Mentha suaveolens var. Variegata  
Mentha arvensis L.   
Mentha piperita L.   
Mentha spicata var. Crispa 
Mentha aquatica L.  
Mentha piperita var. Perpeta   
Mentha spicata L.   
Mentha longifolia L. (UMCS)     
Mentha longifolia (Ostrowsko)  
Extract of Mentha fix (Manufacturer 1)     
Extract of Mentha fix (Manufacturer 2)   
Number of substance 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 
Mentha piperita var. Zgadka   
Mentha piperita var. Cernolistnaja 
Mentha suaveolens var. Variegata  
Mentha arvensis L.   
Mentha piperita L.   
Mentha spicata var. Crispa 
Mentha aquatica L.  
Mentha piperita var. Perpeta   
Mentha spicata L.   
Mentha longifolia L. (UMCS)     
Mentha longifolia (Ostrowsko)  
Extract of Mentha fix (Manufacturer 1)     
Extract of Mentha fix (Manufacturer 2)   

Numbers as in Table I.

In turn Table III relates to system II (80% EtOAc + n-heptane and 50% MeOH + water). Also in these systems, the results of micro-2D-TLC separation can be useful in quality control of herbal material or herbal drugs containing mint. Rutin, diosmin, rosmarinic acid, isorhoifolin, narirutin and naringenin were present in all examined extracts. Only the herb of mint produced by Manufacturer 1 contained caffeic acid. Table III also shows that eriocitrin was found barely in two extracts, whereas luteolin 7-glucoside in four extracts. To illustrate our study the best result in the form of photographs was chosen (Figure 3 for system I and Figure 4 for system II).

Table III.

The Comparison of Presence of Substances in Various Mentha Extracts Detected by UV 245 nm, UV 366 nm, Naturstoff Reagent on Cyano-bonded Plates for System 80% EtOAc + n-heptane and 50% MeOH + water

Number of substance 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 
Mentha piperita var. Zgadka   
Mentha piperita var. Cernolistnaja     
Mentha suaveolens var. Variegata     
Mentha arvensis L.  
Mentha piperita L.    
Mentha spicata var. Crispa    
Mentha aquatica L.    
Mentha piperita var. Perpeta   
Mentha spicata L.    
Mentha longifolia L. (UMCS)   
Mentha longifolia (Ostrowsko)     
Extract of Mentha fix (Manufacturer 1)   
Extract of Mentha fix (Manufacturer 2)    
Number of substance 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 
Mentha piperita var. Zgadka   
Mentha piperita var. Cernolistnaja     
Mentha suaveolens var. Variegata     
Mentha arvensis L.  
Mentha piperita L.    
Mentha spicata var. Crispa    
Mentha aquatica L.    
Mentha piperita var. Perpeta   
Mentha spicata L.    
Mentha longifolia L. (UMCS)   
Mentha longifolia (Ostrowsko)     
Extract of Mentha fix (Manufacturer 1)   
Extract of Mentha fix (Manufacturer 2)    

Numbers as in Table I.

Figure 3.

Photographs of chromatographic cyanopropyl plates developed by 2D-TLC mode (I—40% iPrOH–n-Hp; II—30% ACN–water) after derivatization with Naturstoff reagent at λ = 365 nm with (a) separated extract of Mentha suaveolens var. Variegata, and (b) with separated extract of Mentha spicata var. Crispa. Numbers as in Table I. This figure is available in black and white in print and in color at JCS online.

Figure 3.

Photographs of chromatographic cyanopropyl plates developed by 2D-TLC mode (I—40% iPrOH–n-Hp; II—30% ACN–water) after derivatization with Naturstoff reagent at λ = 365 nm with (a) separated extract of Mentha suaveolens var. Variegata, and (b) with separated extract of Mentha spicata var. Crispa. Numbers as in Table I. This figure is available in black and white in print and in color at JCS online.

Figure 4.

Photographs of chromatographic cyanopropyl plates developed by the 2D-TLC mode (I—80% EtOAc–n-Hp; II—50% MeOH–water) after derivatization with Naturstoff reagent at λ = 365 nm with (a) separated extract of Mentha arvensis and (b) separated extract of Mentha piperita var. Perpeta. Numbers as in Table I. This figure is available in black and white in print and in color at JCS online.

Figure 4.

Photographs of chromatographic cyanopropyl plates developed by the 2D-TLC mode (I—80% EtOAc–n-Hp; II—50% MeOH–water) after derivatization with Naturstoff reagent at λ = 365 nm with (a) separated extract of Mentha arvensis and (b) separated extract of Mentha piperita var. Perpeta. Numbers as in Table I. This figure is available in black and white in print and in color at JCS online.

The presence of phenolic compounds of investigated Mentha sp. can be illustrated by use of Naturstoff reagent (2-(diphenylboryoxy)-ethylamine and PEG4000), which was produced by Merck and was composed of a 5% methanolic solution of PEG and 1% methanolic solution of 2-(diphenylboryoxy)-ethylamine. The effect of derivatization (yellow luminescence) in examination of extracts is shown in Figures 3 and 4.

Conclusion

A micro-2D-TLC technique by the use of CN silica plates enables separation of phenolic fractions from Mentha sp. extracts. The advantages of this method are high sensitivity of analyses, short analysis time, low consumption of eluent and a small amount of the sample.

In separation of phenolic fractions of herb material in the two-dimensional mode, the system used consisted of cyanopropyl layers developed with a non-aqueous eluent composed of propan-2-ol or ethyl acetate and n-heptane in the first direction and an aqueous eluent composed of ACN or methanol and water in the second direction.

Research has shown that plants of Mentha sp. contain high amounts of pharmacologically active phenolic compounds. Other Mentha sp. also contain a lot of phenolics and can be useful for preparing herbal medicines or diet supplements. Micro-2D-TLC experiments give us the information about the composition of plant extracts and are helpful in construction of fingerprints of the examined herbs.

References

1
Olennikov
D.N.
,
Tankhaeva
L.M.
;
Quantitative determination of phenolic compounds in Mentha piperita leaves
;
Chemistry of Natural Compounds
 , (
2010
);
46
(n
o. 1
):
23
27
.
2
Shan
B.
,
Cai
Y.Z.
,
Sun
M.
,
Corhe
H.
;
Antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents
;
Journal of Agricultural and Food Chemistry
 , (
2005
);
53
:
7749
7759
.
3
Gulluce
M.
,
Sahin
F.
,
Sokmen
M.
,
Ozer
H.
,
Daferera
D.
,
Sokmen
A.
et al
.;
Antimicrobial and antioxidant properties of the essential oils and methanol extract from Mentha longifolia L. ssp. Longifolia
;
Food Chemistry
 , (
2007
);
103
:
1449
1456
.
4
Shaiq Ali
M.
,
Saleem
M.
,
Ahmad
W.
,
Parvez
M.
,
Yamdagni
R.
;
A chlorinated monoterpene ketone, acylated β-sitosterol glycosides and a flavanone glycoside from Mentha longifolia (Lamiaceae)
;
Phytochemistry
 , (
2002
);
59
:
889
895
.
5
Hawrył
M.A.
,
Niemiec
M.A.
,
Waksmundzka-Hajnos
M.
;
Micro-two-dimensional TLC in search of selected Mentha sp. extracts for their composition and antioxidative activity
;
Journal of Planar Chromatography
 , (
2013
);
26
(2)
:
141
146
.
6
Zarzycki
P.K.
;
Simple horizontal chamber for thermostated micro-thin-layer chromatography
;
Journal of Chromatography A
 , (
2008
);
1187
:
250
259
.
7
Zarzycki
P.K.
,
Ślączka
M.M.
,
Włodarczyk
E.
,
Baran
M.J.
;
Micro-TLC approach for fast screening of environmental samples derived from surface and sewage waters
;
Chromatographia
 , (
2013
);
76
:
1249
1259
.
8
Zarzycka
M.B.
;
Wykorzystanie danych retencyjnych uzyskanych przy pomocy techniki mikro-TLC w doborze warunków procesu ekstrakcji do fazy stałej (SPE) prowadzonej z użyciem faz odwróconych (Application of retention data derived from micro-TLC plates for optimization of a reversed phase solid-phase extraction protocol)
;
Camera Separatoria
 , (
2011
);
3
(n
o 1
):
129
145
.
9
Pelter
L.S.W.
,
Amico
A.
,
Gordon
N.
,
Martin
C.
,
Sandifer
D.
,
Pelter
M.W.
;
Analysis of peppermint leaf and spearmint leaf extracts by thin-layer chromatography
;
Journal of Chemical Education
 , (
2008
);
85
(no. 1)
:
133
134
.
10
Al-Bayati
A.F.
;
Isolation and identification of antimicrobial compound from Mentha longifolia L. leaves grown wild in Iraq
;
Annals of Clinical Microbiology and Antimicrobials
 , (
2009
);
8
:
20
26
.
11
El-Desoky
S.K.
,
El-Ansari
M.A.
,
El-Negoumy
S.I.
;
Flavonol glycosides from Mentha lavandulacea
;
Fitoterapia
 , (
2001
);
72
:
532
537
.
12
Zaidi
F.
,
Voirin
B.
,
Jay
M.
,
Viricel
M.R.
;
Free flavonoid aglycones from leaves Mentha pulegium and Mentha (Labiatae)
;
Phytochemistry
 , (
1998
);
48
:
991
994
.
13
Hawrył
M.A.
,
Soczewiński
E.
;
Separation of some flavonoids using RP–HPLC–NP–TLC off-line coupled system
;
Chromatographia
 , (
2000
);
52
:
175
178
.
14
Soczewiński
E.
,
Hawrył
M.A.
,
Hawrył
A.
;
Retention behavior of some flavonoids in 2D-TLC systems on cyano bonded polar phases
;
Chromatographia
 , (
2001
);
54
:
789
794
.
15
Nyiredy
Sz.
,
Szabady
B.
;
The versatility of multiple development in planar chromatography
. In
Kaiser
R.E.
,
Günther
W.
,
Gunz
H.
,
Wulff
G.
(eds).
Dünnschicht-Chromatographie (in memoriam Prof. Dr. Hellmut Jork)
 .
InCom Sonderband
,
Düsseldorf
, (
1996
), pp.
212
224
.
16
Nyiredy
Sz
. (ed.).;
Multidimensional planar chromatography
. In
Planar chromatography, a retrospective view for
 
the third millennium
 .
Springer
,
Budapest
, (
2001
); p.
103
.
17
Hawrył
M.A.
,
Soczewiński
E.
,
Dzido
T.H.
;
Analytical chemistry (Warsaw)
 , (
1999
);
44
:
15
21
.
18
Cieśla
Ł.
,
Bogucka-Kocka
A.
,
Hajnos
M.
,
Petruczynik
A.
,
Waksmundzka-Hajnos
M.
;
Two-dimensional thin-layer chromatography with adsorbent gradient as a method of chromatographic fingerprinting of furanocoumarins for distinguishing selected varieties and forms of Heracleum spp.
;
Journal of Chromatography A
 , (
2008
);
1207
:
160
168
.
19
Cieśla
Ł.
,
Waksmundzka-Hajnos
M.
;
Two-dimensional thin-layer chromatography in the analysis of secondary plant metabolites
;
Journal of Chromatography A
 , (
2009
);
1216
:
1035
1052
.