HPTLC is a straightforward analytical technique that offers numerous advantages. While the technique follows the same concept of separating mixture components between two phases (mobile phase and stationary phase), it differs from other liquid chromatographic techniques in the fact that a gas phase is present during and, indeed, influences the development process [1]. This property has always posed a challenging issue for controlling the outcome of the separation. Moreover, the fact that a broad spectrum of solvents can be used means gas phase control holds great promise for resolving complex matrix separations.
In order to investigate this, the Module DEVELOPMENT (a component of the CAMAG® HPTLC PRO System [2]) was employed in this study. The Module not only allows the generation and introduction of a gas phase of varying composition into the development chamber but also provides control over the timing and power settings of the pump used to build up the gas phase. The Module is equipped with three separate solvent bottles that enable the generation of gas phase from either the same solvents used for plate development or from different solvents. Additionally, the Module can be configured to introduce the generated gas phase at two distinct stages, prior to the start of the development (referred to as pre-conditioning) and/or during the development process (referred to as conditioning). These features provide useful tools to control the gas phase throughout the development process.
This study aims to investigate whether it is possible to manipulate the gas phase to attain a desired chromatographic separation. To achieve this objective, we sought to control the gas phase in a way that we can obtain RF values based on the Universal HPTLC mix (UHM), a mixture of chemicals for system suitability testing, that are comparable to (ΔRF ≤ 0.05) those previously measured using the ADC 2 [3].
Standard solutions
The ready to use solution of UHM was prepared in house according to [4] and applied on track 8 (middle track) of each plate.
Chromatogram layer
HPTLC plates silica gel 60 F254 (Supelco), 20 × 10 cm are used.
Sample application
2.0 µL of UHM solution are applied as bands with the HPTLC PRO Module APPLICATION, 15 tracks, band length 8.0 mm, distance from the left edge 20.0 mm, track distance 11.4 mm, distance from the lower edge 8.0 mm. The default settings of methanol as sample solvent are used. The first rinsing step (bottle 1 solvent) is done with methanol – acetonitrile – iso-propanol – water – formic acid 250:250:250:250:1 (V/V) and the second rinsing step (bottle 2 solvent) is done with methanol – water 7:3 (V/V).
Chromatography
The plates are prepared in the HPTLC PRO Module DEVELOPMENT, using an activation step at 33 % relative humidity for 10 min with a saturated MgCl2 salt solution. Throughout all experiments, the developing solvent used is ethyl acetate – toluene 1:9 (V/V) with a migration distance of 70 mm. The solvents used for generating a gas phase, the power pump values and durations of the gas phase for both pre-conditioning and conditioning are differently optimized according to each approach.
Documentation
Images of the plates are captured with the TLC Visualizer in UV 254 nm.
Results and discussion
It is known that the gas phase surrounding the HPTLC plate during the development process can significantly influence the chromatographic separation. The HPTLC PRO Module DEVELOPMENT has a unique chamber design compared to the chambers used in the ADC 2 or for manual development. These differences can lead to changes in the rate of evaporation and the concentration of the developing solvent, which may result in differences in the pattern of separations. Therefore, it may be expected that the RF values measured using the HPTLC PRO Module DEVELOPMENT will exhibit some deviations from those obtained through ADC 2 or manual development.
To explore the effects of the gas phase on compound separations, our goal in this study was to achieve RF values similar to those obtained using the ADC 2 method.
Key aspects of the study involve:
- Examining the impact of gas phase composition, while keeping the developing solvent and activation constant at 33 % rH.
- Setting a limit of ΔRF at ± 0.05, meaning that the absolute difference between RF values in the ADC 2 and the HPTLC PRO Module DEVELOPMENT should not exceed 0.05.
An initial experiment (HPTLC PRO M1) was conducted without using pre-conditioning or conditioning. In comparison to the ADC 2 results, the overall RF values were different. However, compounds [e], [f], and [h] exhibited average RF values within the specified control limits. Notably, ΔRF was higher for compound [g] (~ 0.06).
These findings suggest that the migration pattern for all compounds does not behave uniformly. The development without the use of the gas phase leads to increased RF values for compounds in the lower half of the plate, and to decreased RF values for compounds in the upper half of the plate.
The challenge now is to control the retention of each of the four compounds individually on the plate solely based on gas phase control.
HPTLC chromatogram of UHM after development. 1: Experiment ADCRF 2, 2: Experiment HPTLC PRO M1
Based on this initial information, three methods were developed to evaluate the effect on the ΔRF.
Methods used to study active gas phase control
The method HPTLC PRO M2 focused on using the same solvent for both, the developing solvent and for gas phase generation. Initially, conditioning with the developing solvent was employed. However, the experiment revealed that initiating conditioning at various migration distances (while keeping the pump power constant) significantly affected the RF values of individual substances.
For example, beginning conditioning at either 0 or 30 mm resulted in a substantial reduction of the RF value for zones located in the upper part of the plate, while starting conditioning at 50 mm exhibited less impact on these zones. Consequently, we decided to initiate conditioning after 50 mm, leading to an improvement of the RF values for most zones, except for compound [f], which required the use of a pre-conditioning step. Previous studies have shown that conditioning in normal phase HPTLC usually increases the RF values and pre-conditioning lowers them.
Ultimately, increasing the pre-conditioning duration from 10 to 30 s corrected the RF value for compound [f], but this came at the expense of reduced RF values for compounds [g] and [h].
Those data highlight the various parameters that can be used to regulate the gas phase. It also reveals that substances respond differently to each given experimental condition, indicating that the chemical properties of the compounds play a role in regulating the gas phase.
HPTLC chromatograms show results obtained at different conditions with method HPTLC PRO M2
Similar optimization processes were employed in the other two approaches (optimization data not shown). However, in these two approaches we demonstrated how to control the gas phase with solvents that are different from the developing solvent. One approach involved entirely different solvents, adopted from [3] (referred to as HPTLC PRO M3), while the other maintained the same composition but different solvent proportions (referred to as HPTLC PRO M4).
Notably, the fourth approach (HPTLC PRO M4), which uses ethyl acetate – toluene 3:7 (V/V) for pre-conditioning, yielded the most favorable outcome. In this approach, no conditioning is required and in contrary to the common tendency for pre-conditioning to decrease RF values (due to the known building of virtual fronts), our study revealed an anomalous outcome where RF values for compounds other than [g] experienced an increase in RF value. By exploring these alternative solvent combinations, we can expand our understanding about the effect of the gas phase composition and its subsequent impact on chromatographic performance.
HPTLC chromatograms of the UHM after development with different conditions (A): track 1: ADC 2 (standard conditions), track 2: HPTLC PRO M1, track 3: HPTLC PRO M2, track 4: HPTLC PRO M3, track 5: HPTLC PRO M4; Control chart for ΔRF (B)
RF values obtained from methods conducted in this study
Conclusion
This study emphasizes the essential role of the gas phase in regulating the development process and extends its significance beyond the establishment of standardized chromatographic procedures for HPTLC analysis.
Furthermore, this study shows, that it is possible to control the gas phase. By optimizing the composition of the gas phase, the pump power used to build up the gas phase, and the duration of the gas phase using the HPTLC PRO Module DEVELOPMENT, we demonstrated how the control of the gas phase allows the customization of the retention of each of the target compounds in specific regions of the chromatogram. This results in the achievement of the desired separation pattern through three distinct approaches.
This groundbreaking work highlights the critical role of the gas phase in controlling the development process, introducing new possibilities for strengthening and enhancing the selectivity of the gas phase on the development. These concepts, previously not fully explored, represent a significant step towards a deeper understanding of the complexities involved in pre-conditioning and conditioning processes within Thin-Layer Chromatography systems.
Literature
[1] E. Reich et al., High-Performance Thin-Layer Chromatography for the Analysis of Medicinal Plants (2007).
[2] CAMAG CBS 123. Introducing CAMAG HPTLC PRO.
[3] T. K. T. Do et al. J. Planar Chromatogr. - Mod. TLC (2022) 299
[4] T. K. T. Do et al., J Chromatogr A (2021) 1638
Further information is available on request from the authors.
Contact
Dr. Ehab Mahran, CAMAG, Sonnenmattstrasse 11, 4132 Muttenz, Switzerland, ehab.mahran@camag.com