Jun 22 Nov 24 May 04 Nov 13 May 10 Feb 19 Jan 15 Oct 25 Aug 24 Chromatography is the scientific technique of separating a substance into its various components and compounds for individual identification. Planar chromatography is one branch of the discipline, defined by having the stationary phase of the process take place on a plane. This is in contrast to column chromatography, whereby the stationary phase takes place inside a tube. Column chromatography can take the form of packed column, where the entire volume of the tube is filled with the entire substance, or open tubular column, where an open, empty space is left for the mobile phase to travel.
There are two main branches of planar chromatography: paper chromatography and thin-layer chromatography TLC. As can be seen from planar chromatograms presented in Fig. This compound is slightly eluted from the start line R F ranging from 0. This observation is in agreement with our basic retention concept strong interaction of low retarded cyclodextrin molecules with analyte , which is occurring in mobile phase.
Similar phenomenon was not observed under HPLC conditions: no massive decrease of the peaks area for separated 1-acenaphthenol enantiomers at subambient temperatures was observed. It should be noted that contrary to column chromatography, a non-forced flow planar chromatography, due to the presence of gas phase, is strongly affected by several additional parameters.
To eliminate potential instrumental problems, which may cause the phenomenon observed, planar chromatographic experiment was repeated using different reversed stationary phase HPTLC RP W; wettable with water octadecylsilane and equipment conditions providing high stability of given temperature and horizontal positioning of the plate during chromatographic run microchamber unit working within insulated, temperature-controlled metal oven [ 50 ].
Accordingly to the plates Certificates of Analysis, provided by Merck to each plates batch, the retention of testing marker cholesterol is different. Similarly, for plain binary mobile phase studied in our experiment, retention of acenaphthenol is stronger on TLC plates Fig.
As can be seen from the spots pattern presented in Fig. Accordingly to data presented in Fig. Therefore, this system provides the principal requirement of low retention of macrocyclic additive acting as complexation agent.
Using such experimental setup, the R F values exceeding 0. Under temperatures investigated, the run time involving 50 mm microplates 45 mm developing distance varied between 7 to 25 min, despite mobile phase composition with or without cyclodextrin additive; Fig.
Moreover, using different stationary phases C and C , we checked if there is the peaks area decreasing phenomenon at subambient temperatures, indicating potential strong adsorption of the 1-acenaphthenol fraction on the column front no elution from the column that is equivalent of strong retention on TLC plate.
Similar linear behavior of 1-acenaphthenol was observed using planar chromatography systems as described above. This phenomenon was observed before and documented experimentally for various low-molecular mass chiral and achiral molecules including polycyclic aromatic hydrocarbons, steroids, and bisphenols chromatographed under similar conditions [ 9 , 41 — 43 ].
As can be seen from plots a2 and b2 presented within Fig. Observed variations of the peak areas can be associated with UV-Vis spectra changes of 1-acenaphthenol-CD complex at different temperatures due to the fact that UV detector cell of this HPLC system was not temperature controlled. In such case, temperature of mobile phase inside detector cell can be affected by temperature of mobile phase pumped from HPLC column no heat stabilizer was placed between column thermostatic jacket and detector flow cell.
Effect of temperature on UV-Vis spectra was observed before for several molecules including dyes, alcohols, anti-inflammatory drugs, and polycyclic aromatic hydrocarbons, where both hypsochromic and bathochromic shifts as well as hyperchromic and hypochromic effects were reported [ 40 , 43 , 66 , 67 ]. These phenomena are commonly used for calculation of binding constants of supramolecular complexes [ 3 ]. From practical point of view, any loss in the peak area may affect the accuracy and robustness of quantification protocol for components of interest, which are analyzed with help of cyclodextrin-modified liquid chromatographic system.
Therefore, we strictly inspected all previously generated data, in term of this problem. There is also a difference in acenaphthenol quantity injected into both systems.
Particularly, 0. It should be noted that in both cases, investigated chromatographic systems were not overloaded spot or peaks tailing were not observed but initial local concentration of 1-acenaphthenol can be significantly lower on column than on plate. UV-Vis spectrophotometric data presented in Fig. This was detected by increase of baseline level within whole UV-Vis spectrum range Fig. This effect is time-dependent as demonstrated on graph A in Fig.
Light scattering from the solid supramolecular complex crystals can be also visualized using laser beam as can be seen from the upper picture in Fig. Close inspection of centrifuged solid particles by optical microscopy bottom picture in Fig. We demonstrated that solubility changes of studied supramolecular complex and kinetics of solid complex precipitation as well as differences in total analysis time between TLC and HPLC separation may trigger strong retention of 1-acenaphthenol in planar chromatographic systems.
Such disadvantage can be eliminated using, e. Moreover, supramolecular complex precipitation combined with plates or bars solid phase extraction can be applied as very selective method for low-molecular mass components fractionation or separation involving micro-chromatography or microfluidic devices. It has been demonstrated that contrary to column chromatography, where analytes must be eluted from the column to reach the detection place, classical non-forced flow planar chromatographic system can be very useful tools for examining the behavior of analytes that are strongly adsorbed by the stationary phase retarded, chemisorbed, or precipitated within or close to the start line.
National Center for Biotechnology Information , U. Analytical and Bioanalytical Chemistry. Anal Bioanal Chem. Published online Mar Zarzycki 2. Author information Article notes Copyright and License information Disclaimer. Zarzycki, Email: lp. Corresponding author. This article has been cited by other articles in PMC. Electronic supplementary material The online version of this article doi Introduction Cyclic oligosaccharides form a wide group of low-molecular mass donut-like-shaped compounds.
Experimental Reagents and materials 1-Acenaphthenol racemic mixture was obtained from Aldrich-Chemie Steinheim, Germany. Column chromatographic separations were carried out using two HPLC systems consisting of: i.
Open in a separate window. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.
References 1. Villiers A. Fermentation of starch by the butyric ferment. Compt Rend. Example of Planar chromatography : Thin Layer chromatography, paper chromatography. Different types of Filter Paper are used as per the requirement in Paper chromatography. Column Chromatography:. In column chromatography the stationary phase is packed or coated into the column, Hence it is known as the column chromatography.
The column of the chromatography may be the packed column or the capillary column depends on the requirement. Normally in gas chromatography, capillary columns are used. Example of column chromatography : Gas chromatography, High-performance Liquid chromatography, Liquid chromatography. As the figure shows, the TLC separation at room- and low-temperature regions is baseline.
It is noteworthy that the planar technique provides chromatograms with good peak distribution regardless of temperature. This is a consequence of both Van't Hoff plot trajectories and the low retention of the components of interest. The calculated minimum and maximum values of capacity factors for estrogens are 0. Hence, the phenomena of nonlinearity between the raw retention factor and retardation factor values described by equation 1 and visualized in Figure 1 can affect significantly the final result of chromatogram symmetry of estrogens separated by the planar method.
Figure 4: Influence of temperature on the a selectivity, b separation, and c peak distribution of the HPLC circles and TLC black dots systems investigated. The high capability of planar chromatographic systems to separate complex mixtures composed of relatively weakly retarded solutes is mainly driven by the nonlinear relationship between. We demonstrated that using the same type of stationary and mobile phase composition, this problem can be fixed easily if the column technique is replaced by a simple nonforced isocratic planar method.
This behavior can be explained easily, taking into account the effect of nonlinear independence between the raw retention factor and retardation factor values described by equation 1. Surprisingly, under similar reversed-phase experimental conditions, the separation of estrogens by the column technique was incomplete. Our observation might be valuable for scientists seeking an inexpensive, simple, and robust separation technique, which is appropriate for prepurification and quantification of a battery of low-retarded compounds in complex mixtures.
Particularly, this methodological approach can be applied for the metabolomics technology platforms that involve LC techniques for separation, detection, and quantification of steroids in biosamples. They also would like to thank the Gladys M. Brawn Memorial Gift Committee for financial support. Crecelius, M. Clench, and D. Brzezinka and N. Bertram, J. Marengo, E. Robotti, P. Righetti, and F. Antonucci, J.
Drouen, H. Billiet, P. Schoenmakers, and L. Haddad, A. Billiet, and L. Zarzycki and H. Lamparczyk, Chromatographia 48 , — Kuronen , P. Volin, and T. Laitalainen, J. Zarzycki, K.
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