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

4 6.887 0.2767

5 26.141 1.3710