Regarding the separating behavior of the starting system (a system of 2D-TLC using the same
mobile phase in both developments), as it could be expected [42, 43], these paration quality was improved by the application of the second development.
The program of the
mobile phase was set up as follows: the concentration of [(N[H.sub.4]).sub.2]C[O.sub.3] was 5 mM for 2 min followed by a linear increase to 50 mM in 10 min.
From different compositions the
Mobile phase selected was Potassium hydroxide buffer: Acetonitrile = (60: 40).
Theoretical description of the effects of the
mobile phase on the retention, selectivity, and resolution of sample components enables prediction and optimization of gradient operation conditions and significantly reduces the number of experiments and time necessary for high-performance liquid chromatography (HPLC) separation method development.
*
Mobile phase A: CX-1 pH-gradient buffer A (pH 5.6) diluted 10x in deionized water
The
mobile phase under gradient mode was a mixture of acetonitrile--0.2% phosphoric acid (pH was adjusted to 3.0 with triethylamine).
Mobile phase components were degassed using Super Sonic X-3 Sonicator before use.
Accurately weigh 25mg + 2mg of d-ephedrine in a 100 ml volumetric flask, dissolve and dilute to volume with
mobile phase.
Into a series of 10 mL measuring flasks, accurately measured aliquots of standard NC solution in the concentration range of (0.005-40 [micro]g/mL) and completed to volume with the
mobile phase. All samples were filtered through 0.45-um sample filters (RC 25, Sartorius AG, Germany) prior to injection into HPLC system.
Accurately weighed amount of metformin HCl std about 25 mg was dissolved in
mobile phase as a diluent and make up to50 ml and 5 ml of its volume to 100 ml with diluent the concentration prepared approximately 25 ppm.
Mobile phase A consisted of 1 L of water mixed with 0.25 mL of TFA and 10 mL of propionic acid;
mobile phase B was as described above.
The use of high separation temperatures lowers the viscosity of the
mobile phase, to drive the chromatographic separations at high volume and speed.
