EXPT 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY: DEVELOPMENT OF METHOD
METHODOLOGY OF THE EXPERIMENT High performance liquid chromatography is suitable for
moderate analysis condition and no need to vaporize the sample like gas chromatography. The instrument are set up in the way of detector distance used in 254nm. It is direction of propagation in consecutive of the wave in the detector. The rate of flow are used as 1.5 mL/min in the column. The mobile phase using polar which is acetonitrile and deionized water with the ratio (50:50). The sample are injected. Later, the arrangement of the mobile phase are altered with the most used in experiment with ratio (70:30). To investigate the component in the mixture using selected high performance liquid chromatography or HPLC specification, each of the components are injected individually in automatic injection. For the gradient elution to do the separation, need to improvise the ratio of the column by conducting the gradient elution method. Figure 1 STANDARD MIXTURE gradient elution (injection 1)
Gradient Program Time (min) %ACN:%Water
0 – 1.8 50:50
1.8 – 3.0 70:30
3.0 – 8.0 85:15
Figure 2 STANDARD MIXTURE gradient elution (injection 2) Gradient Program Time (min) %ACN:%Water
0 – 1.8 50:50
1.8 – 8.0 70:30
Figure 3 STANDARD MIXTURE ISOCRATIC ELUTION 70%: 30% ACN: Water Figure 4 STANDARD ELUTION Individual standard CAFFEINE at 70%: 30% ACN: Water Figure 5 STANDARD ELUTION Individual standard ACETONE at 70%: 30% ACN: Water Figure 6 STANDARD ELUTION Individual standard METHYL BENZOATE at 70% ACN: 30% Water
Figure 7 STANDARD ELUTION Individual standard PHENATOLE at
70% ACN: 30% Water Figure 8 STANDARD ELUTION Individual standard PHENANTHRENE at 70% ACN: 30% Water Figure 9 STANDARD MIXTURE ISOCRATIC ELUTION, 50% ACN: 50% Water Pea Retation time Width k