Micropreparative chromatography

The optimization of preparative and even micropreparative chromatography depends on the choice of an appropriate chromatographic system (adsorbent and eluent), sample application and development mode to ensure high purity, and yield of desirable compounds isolated from the layer. For the so-called difficult separations, it is necessary to perform rechromatography by using a system with a different selectivity. But it should be taken into account that achievement of satisfactory results frequently depends on a compromise between yield and the purity of the mixture component that is being isolated. 

PLC is used for separations of 2 to 5 mg of sample on thin-layer chromatography (TLC) plates (0.25-nun layer thickness) or high-performance TLC (HPTLC) plates (0.1-mm thickness). In these instances, the method is termed micropreparative TLC. The isolation of one to five compounds in amounts ranging from 5 to 1000 mg is carried out on thicker layers. PLC is performed for isolation of compounds to be used in other tasks, i.e., further identification by various analytical methods, such as ultraviolet (UV) solution spectrometry [1] or gas chromatography/mass spectrometry (GC/MS) [2], obtaining analytical standards, or investigations of chemical or biological properties [3]. 

Males et al. [103] used aqueous mobile phase with formic acid for the separation of flavonoids and phenolic acids in the extract of Sambuci flos. In a cited paper, authors listed ten mobile phases with addition of acids used by other investigators for chromatography of polyphenolic material. For micropreparative separation and isolation of antraquinone derivatives (aloine and aloeemodine) from the hardened sap of aloe (Liliaceae family), Wawrzynowicz et al. used 0.5-mm silica precoated plates and isopropanol-methanol-acetic acid as the mobile phase [104]. The addition of small amounts of acid to the mobile phase suppressed the dissociation of acidic groups (phenolic, carboxylic) and thus prevented band diffusions. 

Rose, D.J., Opiteck, G.J. (1994). Two-dimensional gel electrophoresis/liquid chromatography for the micropreparative isolation of proteins. Anal. Chem. 66(15), 2529-2536. 

Using amylose tris-3,5-dimethylphenylcarbamate as the chiral selector in enantioselective high-performance liquid chromatography, micropreparative resolution of the DHA racemate was achieved and the chromatographic behaviour in enantio-GC could be defined by coinjecting these references of definite chirality (Fig. 17.4) [13]. 

In our laboratory, an on-flow LC-NMR-MS screening (Figure 5.1.1) was applied to both saponin fractions which were not separated into pure compounds by classical column chromatography and further to total asterosaponin fractions obtained by the micropreparative technique, matrix solid-phase dispersion (MSPD) extraction [45] (see Figure 5.1.2). The LC-NMR-MS hyphenation is set up in the widely used parallel configuration of NMR and mass spectrometer (Figure 5.1.3). Typically, absolute amounts of asterosaponin mixtures of about 500 xg - 1 mg are injected onto the column. 

As an instrumental approach to conventional electrophoresis, capillary electrophoresis offers the capability of on-line detection, micropreparative operation and automation (6,8,45-47). In addition, the in tandem connection of capillary electrophoresis to other spectroscopy techniques, such as mass spectrometry, provides high information content on many components of the simple or complex peptide under study. For example, it has been possible to separate and characterize various dynorphins by capillary electrophoresis-mass spectrometry (33). Therefore, the combination of CE-mass spectrometry (CE-MS) provides a valuable analytical tool useful for the fast identification and structural characterization of peptides. Recently, it has been demonstrated that the use of atmospheric pressure ionization using Ion Spray Liquid Chromatography/ Mass Spectrometry is well suited for CE/MS (48). This approach to CE/MS provides a very effective and straightforward method which allow the feasibility of obtaining CE/MS data for peptides from actual biological extracts, i.e., analysis of neuropeptides from equine cerebral spinal fluid (33). 

The elution method involves scraping off the separated zones of samples and standards and elution of the substances from the layer material with a strong, volatile solvent. The eluates are concentrated and analyzed by use of a sensitive spectrometric method, gas or liquid column chromatography, or electroanalysis. Scraping and elution must be performed manually because the only commercial automatic micropreparative elution instrument has been discontinued by its manufacturer. The elution method is tedious and time-consuming and prone to errors caused by the incorrect choice of the sizes of the areas to scrape, incomplete collection of sorbent, and incomplete or inconsistent elution recovery of the analyte from the sorbent. However, the elution method is being rather widely used (e.g., some assay methods for pharmaceuticals and drugs in the USP Pharmacopoeia). 

Thin-layer chromatography (TLC) is mainly applied in micropreparative taxoids separation [2-4]. Silica gel 6OF254 preparative plates are usually applied for this purpose. The problem of taxoids separation involves not only their similar chemical structure (e.g., paclitaxel versus cephalomannine) but also, due to different coextracted compounds usually encountered in crude yew extracts (polar compounds such as phenolics and nonpolar ones such as chlorophylls and biflavones), the separation is very difficult. The common band of paclitaxel and cephalomannine was satisfactorily resolved from an extraneous fraction in isocratic elution with ethyl acetate as a polar modifier [4] and n-heptane-dichloromethane as the solvent mixture and it was of suitable purity for high-performance liquid chromatography (HPLC) quantitative determination. 

In a series of papers by Vigh and co-workers [143-145], it has been shown that displacement chromatography may allow micropreparative (1 mg) separations of enantiomers to be performed with somewhat better chemical and enantiomeric recoveries than in the overloaded elution mode. However, this technique does not offer principal solutions to the above-mentioned inherent problems of the elution batch mode, and is not discussed in detail here. 

In order to determine the sequence of amino acids in a peptide, initially it was subjected to partial hydrolysis. Thereby fragments were formed, smaller peptides the sequence of which could be more easily established. After micropreparative separation, e.g. by paper chromatography, the fragments were hydrolyzed and analyzed for amino acid composition both before and after treatment with nitrous acid. The amino add missing in the treated, desaminated, sample, was its N-terminal residue. The sequence of the whole peptide was then reconstructed through the appropriate combination of the structurally identified peptide fragments. A typical example, the eluddation of the structure of gramiddin S, is described on p. 205. 

Rotation planar chromatography (RPC), as with OPLC, is another thin-layer technique with forced eluent flow, employing a centrifugal force of a revolving rotor to move the mobile phase and separate chemical compounds. The RPC equipment can vary in chamber size, operative mode (analytical or preparative), separation type (circular, anticircular, or linear), and detection mode (off-line or online). The described technique was applied in analytical and micropreparative separation of coumarin compounds from plant extracts. 

Fig. 5 (A) Densitogram obtained from micropreparative zonal chromatography of a mixture of quaternary alkaloids on silica plate with toluene/EtOAc/MeOH (83 15 2) as mobile phase. Detection by UV at A = 254 nm. (B) Densitogram obtained from micropreparative zonal chromatography of a quaternary alkaloid mixture on silica plate. Gradient elution with T/EtOAc/MeOH, ra=l 75 25 5, n = 2 70 20 10, n — 3 70 15 15, n = 4 EtOH/CHCla/AcOH (67 30 3) as mobile phases. Detection by UV at A = 254 nm.

Tuzimski, T. and Soczewinski, E. 2004. Use of database of plots of pesticide retention (RF) against mobile-phase compositions for fractionation of a mixture of pesticides by micropreparative thin-layer chromatography, Ghromatographia, 59 121-128. 

RPC involves the use of centrifugal force to accelerate the flow of solvent from the feed-point at the center to the periphery of a rotating plate. Up to 72 samples can be separated and quantified in situ by analytical RPC. One sample is applied as a circle for micropreparative and preparative RPC, for which separations can be carried out off-line or on-line with elution from the layer and recovery in a fraction collector (48). A variety of N- and S-type chambers with the prefix designations N (normal), M (micro), U (ultramicro), and C (column) are used for RPC, differing mostly in the size of the vapor space (153). Nyiredy (45) has described commercial instruments (Chromatotron and Rotachrom) and the various modes of RPC and covers preparative layer chromatography, including RPC, in Chapter 11 of this Handbook. 

Ivory s group published very interesting examples of micropreparative enantioseparations based on the FCCE principle very similar to that described in [15]. This group also developed an electrophoretic separation system shown in Fig. 6 [91, 92, 94] as an analogue of the simulated moving bed (SMB) system well known in chromatography [95]. 

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