Materials
Merck TLC plates (Art. No.: 1.05735.0001, silica gel 60 UV254 pre-coated, 20 × 20 cm, 200 μm layer thickness, plastic-backed, mean particle size: 10–12 μm, particle size distribution: 5–20 μm), Merck HPTLC plates (Art. No.: 1.05548.0001, silica gel 60 UV254 pre-coated, 20 × 20 cm, 200 μm layer thickness, aluminum-backed, mean particle size: 5–6 μm, particle size distribution: 4–8 μm), Merck Lichrospher® HPTLC plates (Art. No.: 1.05586.0001, silica gel 60 UV254 pre-coated, 20 × 20 cm, 100 μm layer thickness, aluminum-backed, mean particle size: 3–5 μm, particle size distribution: 6–8 μm), MN TLC plates (Art. No.:805,023, silica gel 60 UV254 pre-coated, 20 × 20 cm, 200 μm layer thickness, polyester sheet, particle size distribution: 5–17 μm), MN HPTLC plates (Art. No.:818,143, silica gel 60 UV254 pre-coated, 20 × 20 cm, 200 μm layer thickness, aluminum sheet, particle size distribution: 2–10 μm) were gifts from the respective companies. Crude opium powder, morphine hydrochloride, codeine phosphate hydrate, papaverine hydrochloride, noscapine hydrochloride hydrate were gifts from Razi Drug Research Center, Iran University of Medical Sciences, Tehran, Iran. Acetone, toluene, ethanol, and concentrated ammonia solution (32%) were from Merck, Germany.
Solutions
Opium and opium alkaloids reference standards were prepared as previously described (The Japanese pharmacopoeia 2006). Briefly, 0.1 g of powdered opium was added to 5 ml of diluted ethanol (70% v/v) and dissolve by treating with ultrasonication for 10 min. Sufficient diluted ethanol (70% v/v) was added to make 10 ml final volume. The solution was centrifuged (5000 rpm, 5 min) and the supernatant was used for spotting. 25 mg of morphine hydrochloride hydrate, 12 mg of codeine phosphate hydrate, 2 mg of papaverine hydrochloride, and 12 mg of noscapine hydrochloride hydrate were separately dissolve in 25 ml of diluted ethanol (70% v/v), and the resulting solutions were used as the standard solutions 1, 2, 3, and 4 respectively.
Instruments
Flat bottom TLC chamber for development of 20 × 20 cm TLC plates (CAMAG, Switzerland), a dual-wavelength (254/366 nm) UV cabinet (CAMAG, Switzerland), Linomat 5 (CAMAG, Switzerland), a TLC Scanning Densitometer (Shimadzu CS-9000, Japan) and 4 decimal place analytical balance (Sartorius, Entris124-1S, Germany) were used in this study.
Measurements
2 × 2 cm piece of each of 5 plates (Merck TLC, HPTLC, Lichrospher® HPTLC, and MN TLC and HPTLC plates) in quintuplicate plate samples were cut and their silica coating were scraped. The scraped silica samples were used for determination of density, particle size, zeta potential, specific surface, and particle shape.
Mass of each scraped silica sample was measured by weighting with the 4 decimal place analytical balance and the respective density was calculated by division of mass by the volume of silica coating (2 cm × 2 cm × layer thickness in cm). The density for each plate silica sample was repeated in quintuplicate plates and individual measurements were averaged.
Particle size and zeta potential of scraped silica in distilled water were determined by a particle size analyzer (Mastersizer 2000, Malvern Instruments Ltd., Malvern, UK) and a Zetasizer (Nano ZS, Malvern Instruments Ltd., Malvern, UK), respectively. The particle size and zeta potential for each plate silica sample were repeated in quintuplicate plates and individual measurements were averaged.
Specific surfaces of scraped silica by BET method were determined by Nano SORD (Toseye Hesgarsazan Asia Co., Iran). The specific surface for each plate silica sample was repeated in quintuplicate plates and individual measurements were averaged.
Scraped silica samples were photographed by a field-emission scanning electron microscope (FE-SEM) (Hitachi, Model S-4160, Japan) to study the morphology of silica particles.
Initial zones were applied as bands by spraying of 10 μl of standards with a Camag Linomat 5 fitted with a 100-μL syringe and operated with the settings: band length 6 mm, rate of application 4 μl s−1, table speed 10 mm s−1, distance between bands 10 mm, distance from the plate edge 10 mm, distance from the bottom of the plate 1.5 cm.
Plates were developed with a mixture of acetone, toluene, ethanol (99.5% v/v), and ammonia water (28% w/w) (20:20:3:1) as mobile phase to a distance of 10 cm and then air-dried in a flat bottom Camag TLC chamber containing a common filter paper saturation pad in the back wall. The tank was left to equilibrate for 20 min before insertion of the spotted plate with the layer support facing the saturation pad (The Japanese pharmacopeia 2006). The development distance was 10 cm for all TLC and HPTLC plates. Chromatography for each plate was repeated on quintuplicate plates and individual measurements were averaged.
Development time for each plate was measured for quintuplicate plates and individual measurements were averaged.
After development, plates were dried in a stream of cold air for 10 min. Chromatograms were viewed under 254-nm UV light in the Camag UV-viewing cabinet. Chromatograms of the samples were scanned at 254 nm in the single-wavelength, single-beam mode with the TLC Scanning Densitometer with a slit setting of 7 mm length and 1 mm height.
Data for calculation of number of theoretical plates (N; efficiency) and resolution (R) values of seven adjacent largest peaks including migration distance from the origin to the mobile phase front (l), migration distance from the origin to the center of each solute zone (z), and chromatographic zone width in the direction of mobile phase migration (w) were obtained by ruler of PhotoShop CS6 software on each densitogram. N for each spot and R for two adjacent largest peaks were measured in quintuplicate plates and individual measurements were averaged. The equations below were used to calculate N and R, respectively, as previously described (Halkina and Sherma 2006):
$$ \mathit{\mathsf{N}}=\frac{\mathsf{16}\times \mathit{\mathsf{l}}\times \mathit{\mathsf{z}}}{{\mathit{\mathsf{w}}}^{\mathsf{2}}} $$
$$ \mathit{\mathsf{R}}=\frac{{\mathit{\mathsf{z}}}_{\mathsf{1}-}{\mathit{\mathsf{z}}}_{\mathsf{2}}}{\mathsf{0.5}\left({\mathit{\mathsf{w}}}_{\mathsf{1}}+{\mathit{\mathsf{w}}}_{\mathsf{2}}\right)} $$
Subscripts 1 and 2 indicate two adjacent largest peaks.
Statistical analysis
Densities, particle sizes, zeta potentials, specific surfaces, development times, N, and R values of seven adjacent largest peaks of plates were compared by one-way analysis of variance (ANOVA) followed by Scheffe Post-Hoc on SPSS statistical package. P values less than 0.05 were statistically considered significant.