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Multidimensional and Multimodal Separations by HPTLC in Phytochemistry
Lukasz Ciesla and Monika Waksmundzka-Hajnos
Abstract HPTLC is one of the most widely applied methods in phytochemical analysis. It is due to its numerous advantages, e.g., it is the only chromatographic method offering the option of presenting the results as an image. Other advantages include simplicity, low costs, parallel analysis of samples, high sample capacity, rapidly obtained results, and possibility of multiple detection. HPTLC provides identification as well as quantitative results. It also enables the identification of adulterants. In case of complex samples, the resolving power of traditional one- dimensional chromatography is usually inadequate, hence special modes of devel- opment are required. Multidimensional and multimodal HPTLC techniques include those realized in one direction (UMD, IMD, GMD, BMD, AMD) as well as typical two-dimensional methods realized on mono- or bi-layers. In this manuscript, an overview on variable multidimensional and multimodal methods, applied in the analysis of phytochemical samples, is presented.
The amount of herbal drugs used worldwide has risen dramatically in the recent years. Herbal medicinal products, traditional Chinese medicines (TCM), nutraceu- ticals, and natural health products are only few from among the variety of plant drugs present in the market (Reich and Schibli2008). Botanicals are believed to be very safe and at the same time not very effective drugs, but nothing could be farther from the truth. Many herbal medicines may cause severe side effects when over- dosed or not properly prepared. It is also of great importance to control the amount of active ingredients within the herbal drugs, as their proper amount is responsible for the curing or health-promoting effects (Cies´la and Waksmundzka-Hajnos 2009a). However, the quality control of herbal medicines is completely different
L. Ciesla and M. Waksmundzka-Hajnos (*)
Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Lublin, Lubin, Poland
e-mail: monika.hajnos@am.lublin.pl
MM. Srivastava (ed.),High-Performance Thin-Layer Chromatography (HPTLC), DOI 10.1007/978-3-642-14025-9_5,#Springer-Verlag Berlin Heidelberg 2011
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from the analytical evaluation of synthetic drugs. The entire plant extract should be regarded as an active component; thus, methods established for quality control of single active substance are insufficient in case of highly complex samples such as plant preparations or any other plant-derived products.
Multidimensional chromatography is often a good choice in case of very com- plex samples, offering many advantageous features in the analysis of medicinal plants (Cies´la and Waksmundzka-Hajnos2009b). In multidimensional separation, a sample is first subjected to separation via one method, and then the separated compounds are further resolved by at least one additional independent method (Poole et al.1989). According to Giddings, two conditions should be fulfilled to classify a chromatographic method as a multidimensional one (Giddings 1990).
First of all, the separation mechanisms of the applied steps must be orthogonal, and secondly, the resolution gained in the first dimension may not be lost in any of the subsequent steps. The condition of orthogonal systems means the use of systems characterized by dissimilar retention mechanisms (Daszykowski et al.2008). There are several techniques applied in the identification of such systems, for example, Daszykowski et al. used principal component analysis and hierarchical clustering methods for identification of similar and orthogonal systems for 2D-TLC separa- tions of flavonoids (Daszykowski et al.2008).
Multidimensionality can be realized in gas chromatography; however, in case of liquid chromatography, it is not a simple task. Planar chromatography gives a possibility of performing multidimensional separations with the use of several techniques. 2D-TLC has several unique features and thus is quite often a choice in the analysis of complex natural mixtures (Cies´la and Waksmundzka-Hajnos 2009b). First of all, multidimensional TLC is the only real multidimensional method in which, after the first separation in the first direction, all compounds can be passed to a second direction (Nyiredy2001). Sophisticated equipment is not required, as in case of LCLC separations, what is more plate is used once only, there is no need to worry about the adsorbed constituents that may cause column contamination. There is no need to perform complicated clean-up procedures, and multiple detection can be used to analyze the wide spectrum of compounds, which is impossible to realize in the sequential mode of HPLC. The sample preparation step does not have to be modified even if one wants to focus on different substance classes present in the extract. Proper chromatographic systems have to be chosen in order to focus on the desired constituent group (Cies´la and Waksmundzka-Hajnos 2009a). Multidimensional planar chromatography has also several noticeable dis- advantages. The amount of the analyzed samples is considerably reduced in MD- PC (multidimensional planar chromatography), when compared to one-dimen- sional technique, as only one sample per plate can be analyzed. Multidimensional techniques are usually more time-consuming than one-dimensional methods, and if there is no significant improvement, when compared to one-dimensional mode, they simply do not pay off. Sometimes, change in chromatographic conditions may bring satisfying results without the need to apply multidimensional separation, as in the case of fatty oils’ resolution, according to the European Pharmacopoeia
(Reich and Schibli2008). In case of multidimensional methods, there is always a possibility of artifact formation, due to chemisorption or decomposition during chromatography. There are also several limitations as far as the solvents used are considered. Very polar and nonvolatile solvents should be avoided as they are difficult to be removed from the adsorbent, e.g., dimethyl sulfoxide, acetic acid, trimethylamine, as well as ion-pair reagents and nonvolatile buffer components (Poole and Poole1995). In case of quantitative analysis, HPLC still remains a better alternative to TLC. Special modes of development can be classified as following
a) Repeated multiple development techniques in one direction
l Unidimensional Multiple Development (UMD)
l Incremental Multiple Development (IMD)
l Gradient Multiple Development (GMD)
l Bivariant Multiple Development (BMD) and its automated version – Auto- mated Multiple Development (AMD)
b) Multidimensional techniques
l Comprehensive 2D planar chromatography realized on one adsorbent or on bilayer plates
l Targeted (selective) 2D planar chromatography – only chosen spots, sepa- rated after the first development, are subjected to further analysis
l Modulated 2D planar chromatography – first eluents of decreasing strength are applied in the perpendicular direction and the sample is developed several times with solvent mixture of different selectivity at constant eluent strength
l Graft TLC – the analyzed compounds are transferred from the first adsorbent to another and redeveloped in orthogonal system
l Combination of MD-PC methods
l Coupling of two chromatographic techniques realized in on- or off-line modes, e.g., HPLC–TLC, TLC–GC, TLC–MS, etc.
Techniques commonly applied in the analysis of phytochemical samples are discussed in the subsequent sections.
Multiple Development Techniques
In the chromatography of complex mixtures, the main problem is to improve resolution (Waksmundzka-Hajnos and Cies´la 2009). This might be achieved by increasing separation efficiency. Besides, for the analysis of complex mixtures, it is advantageous to obtain (if possible) a large spot capacity. The aforementioned goals can be achieved with the application of multiple development techniques in one direction. The observed improvement in separation for multiple development tech- niques, when compared to traditional approach, is due to the spot reconcentration mechanism. Each time the solvent front traverses the sample, it compresses the zone in the direction of development (Fig. 5.1) (Poole and Poole 1995). Due to the
fact that lower edge of the spot is overtaken earlier than the upper one, by the mobile phase front, spot becomes narrower. The constructional constrains causes slit-scanning densitometers are difficult to be used to scan the plate after two- dimensional separation. The application of multiple development techniques in one direction relieves the problems associated with detection (Poole et al.1989).
There are several factors that can be varied and adjusted in every single develop- ment to obtain desired separation, e.g., plate length, time of development, composi- tion of the mobile phase, the number of developments, etc. What is more, detection can be repeated after every single step, using different detection conditions and detection modes. Constituents with comparable polarity are difficult to separate with the use of multiple development techniques, as they tend to migrate together from the origin and need long development distance for satisfying resolution.
Multiple chromatographic developments in one direction should rather be reserved for those samples where only modest increase in resolution is needed (Poole and Poole1995).
Multiple development techniques are used not only to enhance the chro- matographic result but also to focus the application zone into the shape of a thin band to improve the resolution of the separation. These techniques may also serve as methods to separate the analyte from the matrix, and a very large sample can be concentrated into a thin starting zone. For removing the matrix, two modes can be applied – more polar analytes remain at the start, while nonpolar excipients migrate with the mobile phase (as in the case of the analysis of furanocumarin fraction from different fruits of theApiaceaefamily (Waksmundzka-Hajnos and Wawrzynowicz Fig. 5.1 The illustration of spot reconcentration mechanism. With permission from Poole et al.
(1989)
1992)), or the very polar matrix (e.g., carbohydrates) remains at the start, while the sample components are separated.
Unidimensional Multiple Development
UMD is a technique that consists of the repeated development of the same plate, with a mobile phase of constant composition, for the same distance (Fig. 5.2) (Nyiredy 2001). Immediately after the mobile phase has reached the specified developing distance, and before the next development, the plate must be removed from the chamber and quickly dried. The most common means of drying is cold air from a hair dryer, especially in case of rather volatile mobile phases. In case of more polar mobile phases, warm air may be used. However, in case of very volatile or labile constituents (e.g., essential oils), the warm air should not be used as it may lead to compound losses. The drying step is also critical and thus should always be validated, in case of quantitative analysis.
This mode of multiple developments is recommended in case of constituents having lowRFvalues in the investigated chromatographic system. It is due to the fact that differences betweenRFvalues are increased for compounds in the lowerRF
range and reduced for those in the upper range (Poole et al.1993). For obtaining good results, when UMD is to be applied, mobile phase of low eluent strength should be used. The retention factor of a solute afternidentical development steps (RFn), during UMD, can be calculated from the equation:
RFnẳ1ð1RFịn
whereRFis the retardation factor of the solute after a single development.
UMD has been successfully applied for the analysis of quaternary alkaloid fraction fromChelidonium majus(Waksmundzka-Hajnos et al.2002) or the alka- loid fraction fromFumaria officinalis(Jo´z´wiak et al.2000). In both cases, the band
Fig. 5.2 Schematic representation of UMD technique. Symbols:ddistance,mphmobile phase.
Adopted from Nyiredy (2001)
resolution markedly increased. The application of UMD in case of F. officinalis resulted in the complete resolution of the main alkaloids present in the extract (Fig.5.3). UMD has also been used hand in hand with other techniques for the resolution of complex mixtures.
Although the application of the UMD technique may bring considerable improvement in the analysis of natural mixtures, there are several cases when it may lead to erroneous results. First of all, the use of multiple development, on silica, should be avoided in case of very labile constituents, which may be oxidized on the surface of polar adsorbents, e.g., carotenoids (Poole and Poole1995). The European Pharmacopoeia method for the identification ofAngelica sinensisand its common adulterationLevisticum officinalerequires double development (Reich and Schibli 2008). Additional spots appearing after the second development are due to the
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Fig. 5.3 Fumaria officinalisisoquinoline alkaloid separation obtained after one-, two-, and three- fold development. With permission from Jo´z´wiak et al. (2000)
formation of artifacts. Although the second development seems to bring significant improvement, it really causes the method to be inadequate for fingerprint develop- ment. Changing chromatographic conditions also may be more beneficial than applying multiple development techniques. For example, fingerprint construction of lavender oil on TLC plates requires double development, while on HPTLC plates, only one development is needed and the separated zones are sharper when compared to those on TLC plate (Reich and Schibli2008). Changing the application mode also was proven to produce better results when compared to multiple developments in case of fatty oils analysis. In this case, UMD was really needed to focus the spots on bands (Reich and Schibli2008).
Incremental Multiple Development
In this method, the development distance is increased during each step, with the use of the mobile phase of constant composition (Fig.5.4) (Nyiredy2001). When the increment is constant,RFvalue of a single compound can be calculated using the equation:
RlinFnẳ1 ð1RFị1 ð1RFịn nRF
whereRFis the retardation factor of the solute after a single development.
This technique has been successfully applied, for example, for the fractionation of alkaloids in extract ofF. officinalis(Waksmundzka-Hajnos and Jo´z´wiak2008), as well as separation of furanocoumarins fromHeracleum sphondyliumfruit extract (Wawrzynowicz et al.1998). In the first case, the extract was developed on silica plate with the use of eluent: PrOH/CH3OH/CH2Cl2(4:1:5), 20 mm increment was applied. Seven constituents were separated with the use of the aforementioned conditions (Fig.5.5). The use of IMD enabled the resolution of three structurally similar furanocuomarins: xanthotoxin, bergapten, and phellopterin.
Fig. 5.4 Schematic representation of IMD technique. Symbols:ddistance,mphmobile phase.
Adopted from Nyiredy (2001)
Gradient Multiple Development
The definition of GMD is as following: it is a method in which each stage of rechromatography is performed with a mobile phase of increasing strength, while the development distance remains constant (Fig.5.6) (Nyiredy2001). This method is especially beneficial in case of samples containing substances spanning a wide polarity range. The difficulty in the separation of samples containing components of widely different polarities is difficult because of the “general elution problem”
(Matysik and Soczewin´ski1988a). In TLC separation of plant extracts, gradient elution markedly improves the separation of spots owing to stronger displacement effects under condition of numerous adsorptions – desorption processes. According to the above definition, in GMD technique, first the apolar constituents are chro- matographed over the entire plate with the use of the weakest mobile phase. This step is followed by detection, and after that, the plate is developed for the second time, for complete resolution of more polar compounds. In order to do that, mobile
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Fig. 5.5 Fumaria officinalis isoquinoline alkaloid fraction developed with the use of IMD technique with an increment of 20 mm. With permission from Waksmundzka-Hajnos and Jo´z´wiak (2008)
phase characterized by the higher eluent strength is used. After the densitometrical evaluation, the plate is developed with eluent of the highest eluent strength. Despite the ability to resolve compounds differing in polarity, this method is quite lengthy as the plate is developed several times over the entire length, and detection is repeated as many times as the number of developments, which can also be consid- ered as an advantage. The method has been successfully applied for the complete resolution of natural complex mixture containing furanocoumarins (apolar com- pounds), flavonoid aglycones (medium polar), and flavonoid glycosides (polar compounds) (Nyiredy2001).
Although in its classical version, the definition of GMD includes only methods in which the analyzed sample is developed on the entire plate length, a method proposed by Matysik and Soczewin´ski can also be described as a multiple gradient method (Matysik and Soczewin´ski 1988b). In this technique, chromatogram is developed stepwise with the use of eluent of increasing polarity, on such distances that the constituents resolved in earlier steps do not migrate with the solvent front during the consecutive step and remain resolved until the last development. The number of gradient steps depends on the sample character as well as the polarity of its constituents and eluent polarity. The actual position of each band, after each development, should be checked under UV or densitometrically.
The increase of eluent strength of the mobile phase passing through the partly separated starting spot causes that consecutive sample components reach the optimal range in order to increaseRFvalues. Because the lower edge of the spot is overtaken earlier than the upper edge, by the front of the mobile phase, the spot becomes narrower than the starting band.
Gradient developments can be realized in two modes: stepwise and semicontin- uous (Matysik and Soczewin´ski1988c). In the first case, mobile phase fractions, of increasing strength, are poured into eluent reservoir in horizontal DS chamber, after the previous eluent has been completely exhausted. More sophisticated equipment is needed for the realization of semicontinuous gradient. Such equipment was introduced by Soczewin´ski and Matysik: portions of increasing eluent strength are placed in a pipe – they can be divided from each other by air bulbs and then slowly pressed to the eluent distributor (Matysik and Soczewin´ski1988c).
This technique was applied for the resolution of a variety of samples, e.g., constituents present in the extract of Radix rhei(Fig.5.7) and Cortex frangulae Fig. 5.6 Schematic representation of IMD technique. Symbols:ddistance,mphmobile phase.
Adopted from Nyiredy (2001)
(Matysik and Soczewin´ski 1996), Herba euphrasiae (Matysik and Toczołowski 1997), Herba convallariae majalis (Matysik et al. 1996), and others. Gradient elution was proven to cause considerable improvement in separation in comparison to isocratic elution. In case of this technique, elution can be performed with the use of gradients, which cannot be applied in case of an automated technique – AMD.
Fig. 5.7 Multiple gradient development ofRadix Rheiextract. The extract was developed in the following mode: (a) 10% AcOEt (ethyl acetate)þCHCl3, 9 cm, (b) 50% AcOEtþCHCl3, 9 cm, (c) 100% AcOEt, 8 cm, (d) 15% MeOH (methanol)þAcOEt, 5 cm. With permission from Matysik and Soczewin´ski (1996)
Fig. 5.8 Schematic representation of IMD technique. Symbols:ddistance,mphmobile phase.
Adopted from Nyiredy (2001)
Bivariant Multiple Development and its Automated Version:
Automated Multiple Development
BMD involves the step-by-step changing of both the development distance and mobile phase composition. The development distance is increased and the eluent strength of the mobile phase reduced for consecutive steps (Fig. 5.8) (Nyiredy 2001). It is a much simpler technique, when compared to classical GMD, as the separation is detected as a single chromatogram. However, it is not suitable for the analysis of very complex samples, due to its limited spot capacity, which is the result of very short developing distance. This technique was proven to be suitable, for example, for resolution of furanocoumarins present in H. sphondylium fruit extract (Wawrzynowicz et al.1998). Mixtures of ethyl acetate withn-heptane, of decreasing eluent strength, were used for the resolution of structural analogs present in the extract.
AMD can be considered as an improved, automated version of BMD. AMD is a technique that uses repeated development of HPTLC plates with decreasing solvent strength on the increasing distance. After each development, the plate is carefully dried by vacuum. The development starts with the most polar solvent (for the shortest development distance) and concludes with the least polar solvent (for the longest migration distance) (Poole et al.1989). Gradient development with linear eluotropic profile leads to a band reconcentration improving the separation. A successful separation depends mainly on the choice of the solvent components, optimization of the shape of the gradient, the stepwise movement of the elution front, and the repeated developments (Pothier and Galand2005). AMD is highly recommended in case of samples containing substances of wide polarity or those being structural analogs. For the best resolution of constituents spanning wide polarity range, steep gradient is especially beneficial, while shallow gradient with small increases of developing distance provides good results in case of the analogs (Reich and Schibli2008). AMD provides a more certain approach to optimizing a gradient separation when compared to other nonautomated TLC gradient methods (Poole et al. 1989). In case of nonautomated gradient elution, the formation of multiple zones of different solvent strength in the direction of chromatography can be observed as a result of solvent demixing (Poole et al.1989). When compared to manual methods, AMD provides a high degree of gradient reproducibility. One of the disadvantages of AMD is the possibility of losing volatile as well as less volatile constituents present in the analyzed samples, during repetitive drying under vacuum (Reich and Schibli 2008). As far as the phytochemical analysis is concerned, AMD has been applied, for example, for separation of phenolic com- pounds in a solvent extract from an acidified aqueous suspension of the herb chamomile (Menziani et al. 1990), opiate alkaloids (Pothier and Galand 2005), and calystegines (Scholl et al.2001). Pothier and Galand give several examples of plant secondary metabolites that have been analyzed with the use of AMD (Pothier and Galand 2005). Gocan et al. describe a simultaneous AMD separation and a comparison with isocratic chromatography for ten plant extracts that contain