second-step
before the injected sample needed to sure,ensure there are no air bubbles inside the system, by opening the purge valve anti-clockwise 1.5 round which means the column will become offline, then select purge off inside the computer to start to pump mobile phases inside the system for 30-second - 5 ml per 1 min. After that close purge valve. Then select start pump, the mobile phase to the column.

Third-step- adjust the parameters
A-used C- 18 reverse column hydrophobic, length of column 150 mm.
B-the mobile phase was composed of 50% water and acid and 50% acetonitrile.
E-The temperature of the mobile phase and sample are room temperature.
C-change, the flow rate of the mobile phase to 1.2 ml/min
D-select wavelength to 220 because it is lambda max of Flucloxacillin
g- The volume of the sample was 20 μL because of Dionex limited volume.


Four-stepFourth-step
preparation sample to injection to HPLC.
A- clean syringe by using a solvent of the sample, which is water.
After that, use some amount of sample to the clean the syringe to make sure there are no participateparticles or contamination in the syringe which may affect to a UV detector.
Five-stepFifth-step
use Chromeleon software to run sample inside HPLC.
steps to use Chromeleon software:
1-Opens1-Open the programme
B-select date then chick addsclick add. changeChange, the flow rate of the mobile phase to 1.2 ml/min after that save thanthen chick compromisation
D-s wavelength to 220 because it is lambda max of Flucloxacillin
C- select 6 min for one round to run the sample inside HPLC.
E-injected sample manually moves knob to make a sure injection is done quickly to avoid back pressure.
The volume of the sample was 20 μL.
SixSixth step - RESULT
foundfind the result of the sample by selecting an instrument to see diagram