Off-line OPLC

Fig. 10.6. Relalion.ship belwecn the theoretical plate height H) and eluent front velocity (u) asing OPLC with forced flow of the mobile phase / = fully on-line OPLC 2 = on-line sample application, off-line detection J = off-line sample application, on line detection 4 = fully off-line OPLC. Optimum of flow velocity can be reached by using fully off-line OPLC. with a set-up for forced flow of the mobile phase. Reproduced by permission from Ref. [80].

In case of classical, fully off-line OPLC, in the zone under the a front (Fa), the space between the sorbent particles and within the pores is filled partially with air and eluent. This is called the partially wetted zone (Zp ), which sometimes disturbs the separation in this narrow range (26,36). The next zone toward the eluent inlet point is a totally wetted one (Zp ), which is completely filled by the eluent. 

Figure 2 Migration of a front using conventional (TLC) and various off-line OPLC developments. HPTLC silica gel 60 (Merck) is compressed for 10 min with 2.5 MPa prior to development, (a) Whole development eluent, carbon tetrachloride flow rate (OPLC), 0.50 cmVmin temperature, 19.5°C 1, conventional development (normal unsaturated chamber) 2, two-directional linear OPLC 3, circularOPLC 4, one-directional linear OPLC. (b) Initial period eluent, chloroform flow rate (OPLC), 0.325 cm /min 1, conventional development (normal unsaturated chamber) 2, theoretical line of linear OPLC 3, linear OPLC, using rapid eluent admission 4, linear OPLC, using Personal OPLC BS 50 5, proposed place of sample application.

Figure 3 Migration of the solvent fronts and substances during continuous development using OPLC technique (26), Chromatographic conditions CHROMPRES 25 (Labor MIM, Budapest, Hungary) silica gel 60 (Merck) isooctane-THF (100 7.5,v/v) external pressure on the membrane, 2.0 MPa. L, migration distance S, start point I, eluent inlet point 0, eluent outlet point. 1, a front (Fa) 2, front of total wetness (Fm>) 3, P front (Fp) 4, inlet pressure (Pe) 5, curve of eluent volume at outlet (Vf) 6-10, substances separated (6, blue dye, eluting in Fp 7, perylene 8, yellow dye 9, pink dye 10, red dye). Stages of continuous development 1, classical, fully off-line OPLC II, leaving of partially wetted zone III, leaving of secondary fronts IV, equilibration.

It is a well-known fact in classical TLC that the eluent components sorbed strongly by the sorbent sites can cause secondary fronts (Fp, Fy,...) (39) that are independent of F, . This effect can be found during adsorption as well as in reversed-phase development when the eluent consists of solvents of different strength. The effect of this chromatographic solvent demixing is stronger in fully off-line OPLC systems, owing to the total elimination of vapor space, than in chambers with small vapor spaces, e.g., in sandwich chambers. 

The elimination of the vapor phase above the sorbent layer in OPLC may cause disturbances in the retention transfer from TLC to OPLC. (Recall the previous point.) Retention data obtained in fully off-line OPLC can be converted to on-line separation/detection conditions according to the following equation ... 

Reversed-phase ion-pair chromatography can be optimized by fully off-line OPLC (47). Good agreement was found in selectivity between HPLC and OPLC ion-pair systems using the same eluent composition, and this made the modeling of HPLC ion-pair systems by fully off-line OPLC possible (48). 

Selectivity of the mobile phase for coumarins was similar in TLC, off-line OPLC and HPLC... 

In conventional layer chromatography the theoretical plate height (HETP) can be calculated according to Guiochon and Siouffi (49), and it is also applicable to off-line OPLC systems (21). HETP (H) is as follows ... 

The thickness of sorbent layer influences HETP slightly (50). In off-line OPLC, HETP may vary with the linear velocity (50,52), similarly to HPLC. HETP depends on the characteristics of the plate used and decreases in the following order preparative layer, TLC, and HPTLC (26,28). 

The elevation of external pressure decreases the HETP value under off-line OPLC. But it doesn t occur during on-line development (Fig. 7) (52a). The results were similar if 3 pm spherical sorbent layer was used (52b). 

Figure 6 Relationship between theoretical plate height H and eluent front velocity u for different operating modes of OPLC. Operating parameters CHROMPRES 25 external pressure on membrane, 2.8 MPa temperature, 23"C layer, HPTLC silica gel 60 eluent, methylene chloride-ethyl acetate (9 1, v/v) sample, PTH-methi-onine bandwidth deposited and trough width, 0.68 mm migration distance, 175-180 mm for off-line detection and 180 mm for on-line detection. 1, fully on-line OPLC 2, on-line sample application/separation and off-line detection 3, off-line sample application and on-line separation/detection 4, fully off-line OPLC. (Reproduced by permission of Dr. Alfred Huethig Verlag GmbH, from Ref. 42.)...

Figure 7 Effect of external pressure on HETP. (a) Fully off-line OPLC, chromatographic conditions experimental CHROMPRES chamber development distance, 16.6 cm eluent, methylene chloride u. 1,73 cnv min temperature, 26.4 C 1 = CIB A FII 2 = Ariabel red 3 = Butter yellow, (b) Fully on-line OPLC, chromatographic conditions experimental CHROMPRES chamber eluent, methylene chloride u, 48 mm/min temperature, 26.5 C, relative humidity, 68% 1 = PTH-Ala 2 = Butter yellow.

In case of thin column and fully off-line OPLC, the maximum value of spot capacity ( /) is... 

Three factors were studied regarding the resolution using fully off-line OPLC (50). Use of the optimal linear flow velocity in relation to HETP produces the highest resolution. The relationship between resolution and distance of development is approximately linear, and the resolution increases with increasing front distance. The layer thickness shows an optimum value (80-160 pm) in terms of resolution. 

Figure 9 Spot/peak capacity of different OPLC separations (23a). n, spot/peak capacity A/, capacity factor L, distance of development. 1, spot capacity of the sorbent layer (0 < Lf < 180 mm H, 27.5 pm) calculated and measured under fully off-line OPLC conditions 2, peak capacity measured by a combination of on-line and off-line systems 3, peak capacity (L, 180 mm, //, 27.5 pm) calculated and measured under on-line separation-detection OPLC conditions 4, spot capacity of the sorbent layer measured after on-line elution. Sample application used is off-line, and in cases 2, 3, and 4 a prewetting was applied from the outlet direction prior to development. (Reproduced by permission from Ref. 23a.)...

Figure 10 Relationship between Rs (F) and / (average R/value X) for reversed phase chromatography of aldehyde DNPH derivatives 1, off-line OPLC 2, TLC (Reproduced by permission from Ref. 56.)...

In fully off-line OPLC systems, all the principal steps in the chromatographic process, such as sample application, separation, quantitative evaluation and isolation are performed as separate operations. 

In analytical off-line OPLC, several samples can be processed in parallel. The technique offers further advantages, such as that only the spots or bands of analytical interest need to be assessed, quantitative evaluation can be repeated with various detection parameters, and chromatogram spots or bands can be evaluated visually. 

In preparative off-line OPLC, after development the procedures of drying, scraping of the sorbent layer, elution, and crystallization are similar to conventional preparative TLC methods. However, in preparative off-line OPLC, the resolution is considerably increased and thick, fine-particle sorbent layers can also be used. It is possible to isolate only the components of interest from the sorbent layer. 

Combining on- and off-line OPLC increases the efficiency of the OPLC system, providing approximately twice the spot capacity obtained by single systems because the spot and peak capacity are combined (23a). 

D. Elimination of Fronts in Fully Off-Line OPLC and Selection of Eluents... 

Two types of fronts may be formed in fully off-line OPLC the front of total wetness (F ,.) and secondary fronts (Fp, Fy,...). The location of can be modified by the flow rate changing the Ry value of Fnv can be increased or decreased by increasing or decreasing the flow rate (36,37). The total elimination of can be carried out by applying a prerun prior to the separation, in which the components to be analyzed do not migrate and the air is removed from the layer (36). 

Figure 18 Correlations of the retention data between the fully off-line OPLC versus fully on-line OPLC and HPLC. (Reproduced by permission from Ref. 70.)...

Two-dimensional off-line OPLC was used for the perfect separation of five organophosphorus warfare agents in the presence of seventeen pesticides (76) and in case of sixteen closely related coumarins (77). In the latter paper the optimization process of eluent system and its transfer from TLC to 2D-TLC and 2D-OPLC were also be discussed. 

In what follows some experience in comparison of validation characteristics of quantitative TLC and fully off-line OPLC are discussed based on definitions of ICH draft guideline (81). 

The robustness-test of a quantitative off-line OPLC assay-procedure was recently reported (89). The test was performed by fractional factorial design and evaluated by half-normal probability plot. The effects of seven factors were investigated on two levels. The method was found to be robust. 

As with analytical OPLC, off-line and on-line methods can be distinguished in preparative OPLC applications. In the off-line OPLC method, the steps of preparation after development are similar to conventional TLC methods drying, scraping of the sorbent layer, elution, and crystallization. Phorbol diester constituents of croton oil were identified by off-line OPLC separation followed by extraction and chemical ionization mass spectrometry (CI-MS) (90). The on-line method is more effective for preparative applications because time-consuming scraping and elution can be eliminated. 

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