HPTLC quantitation

In addition to the use of disposable pipets for manual apphcation, some workers apply samples for HPTLC quantitative determinations by hand in 50-200 nl volumes, using either an adjustable microhter syringe (>50 p.1) or fixed-volume platinum-iridium capillary (200 nl) fused into a glass tube. Because these devices are reused, they must be ftiUy rinsed between solutions to eliminate any possible memory effect from previous solutions. 

Commercially available pre-coated plates with a variety of adsorbents are generally very good for quantitative work because they are of a standard quality. Plates of a standardised silica gel 60 (as medium porosity silica gel with a mean porosity of 6mm) released by Merck have a specific surface of 500 m /g and a specific pore volume of 0.75 mL/g. They are so efficient that they have been called high performance thin layer chromatography (HPTLC) plates (Ropphahn and Halpap J Chromatogr 112 81 1975). In another variant of thin layer chromatography the... 

In situ quantitation Fluorimetric analysis was made with long-wavelength UV light (2exc = 365 nm, X(, > 430 nm). The detection limit on HPTLC plates that were analyzed in a moist state was 25 ng cholesterol per chromatogram zone (Fig. 1). 

The introduction of the sample into the adsorbent layer is a critical process in HPTLC. For most quantitative work a platinum-iridium capillary of fixed volume (100 or 200 nL), sealed into a glass support capillary of larger bore, provides a convenient spotting device. The capillary tip is polished to provide a smooth, planar surface of small area (ca 0.05 mm2), which when used with a mechanical applicator minimises damage to the surface of the plate spotting by manual procedures invariably damages the surface. 

The aim of microchemical investigations on TLC/HPTLC plates is to provide information concerning identity and/or purity. Quantitation is not usually carried out but is possible at any time. The simple absence of a reaction is frequently taken as evidence of the absence of a particular substance. 

Ritter, W. Post-Chromatographic Derivatizations in Quantitative (HP)TLC , Proc. 2nd Int Synq>. lustrum. High ifomu Thm-Lay Chmmalogr. (HPTLC) p. 100-113, Interlaken, 1982. 

Note Under the conditions employed emetine and cephaeline were not well separated but there was good resolution of the subsidiary alkaloids of the ipecacuanha tincture (Fig. 1). The separation and quantitative determination of the main alkaloids (Fig. 2) can be carried out under the following conditions Ascending, one-dimensional development in a trough chamber with chamber saturation layer HPTLC plates Silica gel 60 (Merck) mobile phase dichloromethane — methanol — ammonia solution (25%) (34+6+1) migration distance 6 cm running time 13 min h/ f cephaeline 65-70 emetine 75-80. 

Ripphahn, J. Advances in Quantitative Analysis by HPTLC, Danube Symp. Chromatogr., 1st, Szeged (Hungary) 1976. 

Anderton, S.M., Incarvito, C.D., and Sherma, J., Determination of natural and synthetic colors in alcoholic and non-alcohohc beverages by quantitative HPTLC, J. Liq. ChromatogK, 20, 101, 1997. 

Patzsch, K., Netz, S., and Funk, W. (1988). Quantitative HPTLC of sugars—Part 2 Determination in different matrices. /. Planar Chromatogr. 1,177-179. 

Modern TLC is thus an effective tool for separation of complex solutions into their components. Analysis can be qualitative or quantitative. When the composition or the analysed solution is known approximately, identification of the substance contained in a particular spot by its positional coordinates is frequently adequate. HPTLC provides the means not only for flexible screening procedures but also for demanding quantitative determinations. HPTLC features highly sensitive scanning densitometry and video technology for rapid chromatogram evaluation and documentation. 

In situ recording and quantitation (automated scanning densitometers) (HPTLC)... 

The main limitation of TLC is its restricted separation efficiency. The separating efficiency (in terms of plates per metre) decreases rapidly over long development distances. That is, highest efficiencies are only achievable within a development distance of approximately 4-7 cm. Therefore, the total number of theoretical plates achievable on an HPTLC plate is limited (about 5000) and inferior to long LC or GC columns. Consequently, complex separations of many compounds are usually not achievable by means of HPTLC. This method is most useful for quantitating only a few components in simple or complex sample matrices. The efficiencies can also be reduced if the plate is overloaded, in an attempt to detect very trace components in a sample. 

The main characteristic features of HPTLC (use of fine particle layers for fast separations, sorbents with a wide range of sorption properties, high degree of automation for sample application, development and detection) are the exact opposite of conventional TLC. Expectations in terms of performance, ease of use and quantitative information from the two approaches to TLC are truly opposite [419], Modern TLC faces an uncertain future while conventional TLC is likely to survive as a general laboratory tool. 

Somsen et al. [796] have reported the use of SERR spectroscopy for the in situ selective determination and semi-quantitative analysis of structurally similar dyes separated by TLC. The limits of identification of the TLC-SERRS method (ca. 5ng applied) were sufficient for acquisition of spectra of impurities present in the certified dye standards. SERRS may also be used for in situ identification of highly fluorescent molecules on HPTLC plates. 

Krzek et al. [35] reported the qualitative identification and quantitative analysis of the mixtures of OTC, tiamulin, lincomycin, and spectinomycin in the veterinary preparations by using TLC/densitometry. As stationary phase, they used precoated TLC aluminum sheets, and the mobile phases were mixtures of 10% citric acid solution, hexane, ethanol (80 1 1, v/v), and n-butanol, ethanol, chloroform, 25% ammonia (4 5 2 5, v/v). The other application of TLC or HPTLC for analyzing OTC in the various samples is summarized in Table 2 [36]. 

TLC and HPTLC can be used for both qualitative and quantitative studies. Five examples where TLC can be utilized qualitatively are discussed in this chapter and include reaction completion, identity testing, Rf determination, overlapping experiments, and TLC/HPLC correlation experimentation. 

Sample application is a decisive step in TLC measurements especially in quantitative analyses. The preparative or analytical character of the separation and the volume and physicochemical properties of the sample solution influence equally the mode of sample application. The concentration of the analyte(s) of interest in the sample frequently determines the volume to be applied on the TLC plate a relatively low concentration of analyses requires a high sample volume. Samples containing analyses liable to oxidation have to be applied in a nitrogen atmosphere. Samples can be applied onto the plates either in spots or in bands. It has been proven that the application of narrow bands results in the best separation. The small spot diameter also improves the performance of TLC analysis. The spot diameter has to be lower than 3 mm and 1 mm for classical TLC and HPTLC, respectively. It has been further established that the distance between the spot of the analyte and the entry of the mobile phase also exerts a marked impact on the efficiency of the separation process, the optimal distance being 10 and 6 mm for TLC and HPTLC plates, respectively. 

A new HP-TLC method has been applied for the quantitative analysis of flavonoids in Passiflora coerulea L. The objective of the experiments was the separation and identification of the compound(s) responsible for the anxiolytic effect of the plant. Samples were extracted with 60 per cent ethanol or refluxed three times with aqueous methanol, and the supernatants were employed for HPTLC analysis. Separation was performed on a silica layer prewashed with methanol and pretreated with 0.1 M K2HP04, the optimal mobile phase composition being ethyl acetate-formic acid-water (9 1 l,v/v). It was established that the best extraction efficacy can be achieved with 60 - 80 per cent aqueous methanol. The HPTLC technique separates 10 different flavonoids, which can be used for the authenticity test of this medicinal plant [121],... 

E. Pastene, M. Montes and M. Vega, New HPTLC method for the quantitative analysis of flavonoids in Passiflora coerulea L. J. Plan. Chromatogr.—Mod. TLC. 10 (1997) 362-367. 

---