That is, a normal stationary phase (silica gel) developed with a nonaqueous mobile phase
, which separates preferably nonpolar components and, in parallel, a reverse stationary phase (octadecyl silica) developed with an aqueous phase, used to separate preferably polar components [24, 25].
However, most of the mobile phases
including phosphate buffer for anion-exchange chromatography or carbon rich mobile phase
for reversed phase chromatography were used.
In 1000 ml volumetric flask 600 ml of Potassium Hydroxide buffer was transferred and then 400 ml Acetonitrile to prepare a mobile phase
The aim of this part of the study was to obtain experimental data on different C18 columns, and to find the stationary phase and mobile phase
effects on protein sorption.
* Mobile phase
B: CX-1 pH-gradient buffer B (pH 10.2) diluted 10x in deionized water
The powder was dissolved and made up to volume with mobile phase
. 5 mL solution was withdrawn and transferred into another 25 mL volumetric flask.
The separation was achieved with an analytical column ChromolithAE Performance RP-18e (100 4.6mm), by Merck KGaA (Darmstadt, Germany)) with an isocratic mobile phase
of acetonitrile: mixed phosphate buffer (pH 2.5, 10mM) (pH adjusted with phosphoric acid); (30:70, v/v) at a flow rate of 2mL/min.
Accurately weigh about 66mg + 5mg of l- ephedrine sulfate standard in a 20ml volumetric flask, add 2ml of 1N Sodium hydroxide and dissolve, pipette 10ml of the mobile phase
and 1ml of the Stock impurity solution and shake well.
Into a series of 10 mL measuring flasks, accurately measured aliquots of extracted NC solution in the concentration range of (5-40 [micro]g/mL) and completed to volume with the mobile phase
. All samples were filtered through 0.45-[micro]m sample filters (RC 25, Sartorius AG, Germany) prior to injection into HPLC system under the above chromatographic conditions.
The pH 3.0 of the buffer and C18 bonded stationary phase has played key role in achieving the peak shape of metformin HCl and degradation product using a mobile phase
containing a 0.025 M dibasic potassium phosphate and 0.005 M hexane sulphonic acid sodium salt with ratio of acetonitrile 93:7 as a mobile phase
stressed samples of bulk were injected onto the above chromatographic system.
The only sample preparation step was the addition of mobile phase
B to the samples.
Waters has recently introduced an automated HPLC system with the Empower and DryLab softwares which provide not only a web-based platform for Millenium data acquisition software, but also a development tool to optimize the temperature, pH, as well as the mobile phase
composition and gradient.