HPTLC developing solvents

Practical aspects of TLC method development comprise (i) searching for a suitable developing solvent (ii) optimising the visualisation and evaluation process and (iii) method validation. Table 4.34 lists the main features of HPTLC. 

Valproic acid was determined in plasma by treatment with 2,4-dibromoacetophe-none or 2-bromoacetonapththalone and with dicyclohexano-18-crown-6 and heated at 70 °C for 40 min. The solution was subjected to TLC on C8F octyl plates or to high performance TLC on RP 8 254 S or Kieselgel 60 F254 plates with developing solvents of aq. 63% ethanol, aq. 73% ethanol or CHCl3-cyclohexane (2 1), respectively, with detection at 280 or 254 nm for the naphthoylmethyl or phenacyl derivative, respectively. The limits of detection were 9.7 and 4.9 pg/mL of valproic acid for the TLC RP 8 and HPTLC RP 8 plates, respectively. Recovery was 84-92.74% [28],... 

Once the samples on the HPTLC plate have dried, the plate is inserted into a twin trough chamber such as the one shown in Fig. 13.4 for development. Approximately 10 ml of developing solvent is placed on both sides of the tank, and a piece of blotter paper is placed on the trough opposing the HPTLC plate. The blotter paper ensures a saturated environment for the plate to develop. 

Detection in HPTLC, unlike that in HPLC, is a static process, being completely separated from chromatographic development. Consequently, the selection of the mobile phase does not limit the choice of the detector. For example, UV absorbing solvents cannot be used with UV detectors in HPLC. In HPTLC the solvent is completely evaporated between development and measurement so that it does not influence the detection process. 

It is important in minimising longitudinal diffusion, the second term to ensure that the tank is saturated with eluant vapour and that the developed plate is removed before the eluant reaches the top of the plate. Failure to follow these procedures will lead to spot broadening due to diffusion of the analyte concentration zone. These effects are proportional to the time spent in the mobile phase and hence with HPTLC, where solvent flow is low due to small particle size, shorter run times are employed, less spot broadening occurs and hence resolution is higher. 

Cellulose hydrolysis of 6 and 7. A solution of 6 and 7 (2 mg) in 2 ml 0.1 M HOAc-NaOAc buffer (pH 4.5) was incubated at 40 C with cellulase (1 mg) for 0.5 h. Partially hydrolysed products were identified by comparison with authentic samples on silica gel HPTLC with Et0H-Et0Ac-H20 (20.10 0.1) as developing solvent. 4 h later all glycosides were hydrolyzed and only glucose and aglycone were detected. 

Other special devices, which are of historical interest, include a special chamber for short bed continuous development. In this technique, the plate extends though a slit out of the actual chamber allowing the developing solvent to evaporate. Separation of very similar compounds can thus be achieved at low R values. The U-chamber according to Kaiser was a special device for circular and anticircular HPTLC. 

High-performance silica is used in high-performance thin-layer chromatography (HPTLC). HPTLC differs from normal TLC in that the size of the absorbent (usually silica) is only 5 pm, with a narrow distribution. This enables HPTLC to give better separations compared with TLC, which uses a standard silica, and, moreover, HPTLC requires a smaller sample size and has a lower detection limit compared with conventional TLC. HPTLC plates of varying sizes are commercially available and of late have found considerable applications in the field of lipids. Weins and Hauck (1995), in their survey of TLC, conclude that the use of HPTLC plates increased by 30% over the period 1993-95. An excellent application of HPTLC is illustrated in Fig. 1.1 for the separation of neutral and complex lipids. Yao and Rastetter (1985) have achieved separation of more than 20 lipid classes of tissue lipids on HPTLC plates using four developing solvents. 

Fig. 10 Horizontal developing chamber (Camag). 1 HPTLC plate (layer facing down), 2—glass plate, 3—reservoir for developing solvent, 4— glass strip, 5—cover plate, 6—conditioning tray.

The lipids were separated on a high-performance thin-layer chromatography (HPTLC) plate. The developing solvent was a mixture of chloroform, methanol, and 0.2% aqueous CaCl2. Orcinol reagent and Dittmer s reagent were used to detect glycolipids and phospholipids. 

TLC coupled to ESI was used for the analysis of monomeric flavanols and proan-thocyanidins from standard stock solutions and from extracts of natural samples. Specifically, standard solutions of (-)-epicatechin, (+)-catechin, procyanidin B2, (-)-epigallocatechin, and extracts of pomegranate peel and juniper seeds were developed on HPTLC cellulose plates and HPTLC silica gel 60 plates. Effects of eluent flow, sorbent material, and developing solvent on TLC-ESI mass spectra were studied. A CAMAG TLC-MS interface was used for the elution of compounds from HPTLC plates into the ESI source. For silica plates, it was necessary to use an HILIC guard column mounted between the CAMAG TLC-MS interface and ion source. This need was caused by the presence of stationary phase impurities in the... 

To estimate the content of dihydro-2,5-dihydroxy-6-methyl-4//-pyran-4-one (DDMP) saponin and saponin B in selected pea cultivars, an HPTLC method was developed [35]. Silica gel 60 F254 plates were prewashed with methanol and activated at 100°C prior to development in automated multiple development chamber AMD2 using chloroform-methanol-water (55 37 8, v/v). Densitometry, after postchromato-graphic derivatization with anisaldehyde-sulfuric acid, revealed the prevalence of saponin B over DDMP saponin in all pea cultivars. The identities of the two com-ponnds were determined by coupling HPTLC directly to ESI-MS and additionally by offline MALDI-TOF/TOF-MS by application of a purified extract/matrix mixture onto a standard steel MALDI plate. HPTLC plates intended for MS analysis were developed with a modified developing solvent chloroform-methanol-water (6.5 3.5 0.9, v/v). The analytes were eluted from the plates with 0.1% formic acid-acetonitrile (40 60, v/v) by means of TLC-MS interface (CAMAG) and transferred into the ESI-MS system, which operated in positive-ion mode. 

To remove extraneous materials that might be present due to manufacture, shipping, or storage conditions, it is advisable to clean plates by predevelopment with methylene chloride-methanol (1 1) or the developing solvent to be used. This is especially important for trace analysis by quantitative HPTLC. Activation of adsorbents at 70-80°C for 30 minutes can be carried out, although this is not usually necessary for commercial plates unless they have been exposed to high humidity. Reversed... 

The Vario-KS Chamber and HPTLC-Vario-Chamber (Camag) are horizontal chambers that have a wide variety of operational modes and applications. The plate is placed layer down over a tray with various compartments, which can hold different solvents, humidity-control liquids, and volatile acids and bases whose vapors will impregnate and condition or preload the layer. Developing solvent is in a separate tray and is transferred to the layer by a wick. The Vario chamber can be used to test six mobile phases side-by-side on one plate for solvent optimization, to determine if layer preequilibration (preloading) is advantageous, to ascertain if S- or N-chamber configuration is best, and to test different humidity conditions. 

From the diagram in Fig. 1 it can be seen that in HPTLC layers capillary flow chromatography takes place in the fast flow range of the respective developing solvent. From this is derived practically all the advantages of HPTLC over conventional TLC (10) ... 

HPTLC separation material is available in the form of precoated layers supported by glass, plastic sheets or aluminum foil. Although precoated layers of aluminum oxide, cellulose, polyamide, ion exchange materials, reversed phase silica (alkyl bonded) have been commercially available for quite some time, the vast majority of TLC separations is carried out on normal phase silica gel, particularly in quantitative TLC analysis. Recently introduced plates modified with amino, cyano and diol functional groups bonded to the silica can be used as multimodal media. Depending on the developing solvent used, they extend normal phase or reversed phase chromatographic properties. These layers may affect the predominant role of normal phase silica gel in TLC to a certain extent. They are described in Chapter 4 of this book. Also chiral layers for the separation of optical isomers are available and cause increased interest (11). 

Figure 7 Horizontal Developing Chamber schematically (1) HPTLC plate with layer facing downward, (2) counterplate (inserted for development in sandwich configuration, removed for development with group B and C solvents and for humidity control), (3) troughs for developing solvent, (4) glass strips for solvent transfer by capillary action, (S) cover glass plate. The tray beneath counterplate (2) contains any preconditioning liquid that is needed.

CAMAG HPTLC VARIO System HPTLC development with six different solvents tested side by side used to efficiently find optimal developing conditions for chromatography on HPTLC layers. 

Eleven water-soluble dyes were separated by HPTLC on Si 60 gel with methylketone-methanol-aq. 28% NH3 (8 4.1) as the developing solvent. Dyes that were insoluble in aqueous concentrated NH3 were separated on layers of cellulose with butanol-ethylmethyl ketone-aq. 1% NH3-H2O (4 2 1 1) as developing solvent (142). The advantages of the method are shorter analysis time, more data measurement per plate, high resolution, and an improved signal-to-noise ratio compared with those of TLC plates. The theory of this technique is well discussed by Jupille et al. (143) along with some data on the separation of dyes. 

In one paper, RP-18 HPTLC plates were used to separate a-tocopherol from other antioxidants in food samples (64). Developing solvents were methanol-cyclohexane, methanol-water or methanol-water-acetic acid. Eleven antioxidants were resolved in one run, ascorbic acid having a value close to 1 and a-tocopherol being retained at the origin. 

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