EXPERIMENT 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): METHOD DEVELOPMENT

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CHM510
ANALYTICAL SEPARATION METHOD



EXPERIMENT 2:
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): METHOD DEVELOPMENT



OBJECTIVE
To optimize a separation of a mixture of 5 compounds using HPLC by verifying the mobile phase composition.



INTRODUCTION
High Performance Liquid Chromatography is a type of separation analysis that uses liquid mobile phase and liquid stationary phase. The separation occurs based on the differences in the polarity of the analytes. The analytes that interacts most in the stationary phase will elute later than the analytes that interact least in the stationary phase. Reversed phase chromatography is used in which the stationary phase is non-polar while the mobile phase is the polar mixture. The changes in the mobile phase polarity will affect the interaction between the analyte with the stationary phase and also affect the efficiency of the separation. Changes in the mobile phase composition can be done either by isocratic elution whereas the composition of the mobile phase is constant throughout analysis or by gradient elution in which the composition of the mobile phase is change during separation either continuously or in step in order to separate wide range of polarity of the analytes. The objective of this experiment is to optimize a separation of caffeine, acetone, methyl benzoate, phenatole and phenanthrene using HPLC by varying the mobile phase composition.




PROCEDURE

The instrument wavelength is set up to 254 nm. The flow rate is set 1.5 mL min-1. Acetonitrile and water are used as the mobile phase. The instrument is set to use a mobile phase ratio: water (50: 50 v: v) and the sample is injected. Then, the mobile phase composition is changed to 70:30. Then, the best composition of mobile phase is chosen. Each component is injected individual to identify the component mixture using the selected HPLC conditions. Based on the separation, a gradient elution separation is performed to improve the efficiency of the column.


1.      Effect of the variation of composition of mobile phase on resolution (isocratic elution);
Composition of Mobile Phase (ACN : H2O)
Injection
Retention Time of Peak 1 and Peak 2 (min)
Base Peak Width of Peak 1 and Peak 2 (min)
Resolution
Average Resolution
50:50
1
0.968, 1.103
0.0682, 0.0549
2.19
2.19
70:30
1
0.951, 1.042
0.0524, 0.0486
1.80
1.80
2
0.951, 1.043
0.0521, 0.0504
1.80



2.      Effect of the gradient elution program (ACN : H2O);
Composition of Mobile Phase (ACN : H2O)
Retention Time of Peak 1 and Peak 2 (min)
Base Peak Width of Peak 1 and Peak 2 (min)
Resolution
75:25
0.948, 1.099
0.0598, 0.0460
2.85



3.      The retention time of compound in standard mixture at optimized HPLC condition (70:30) of (ACN : H2O);

Standard Compound
Retention Time in standard (min)
Retention time in mixture (min) for 1st Injection
Retention time in mixture (min) for 2nd Injection
Caffeine
0.953
0.951
0.952
Methyl Benzoate
1.659
1.653
1.656
Phenatole
2.195
2.181
2.186
Phenanthrene
4.761
4.677
4.680
Acetone
1.045
1.042
1.043






DISCUSSION
Composition of the mobile phase is strongly affected the retention time of the analytes to be eluted throughout the column. Since it is a reversed phase partition liquid chromatography, the stationary phase is a polar while the mobile phase liquid is non-polar. Strong eluent strength tends to decrease the retention time of the analytes. Strong eluent strength is where the composition of the organic solvent used in the ratio is higher than the water. In comparison between 50: 50 (ACN: H2O) and 70: 30 (ACN: H2O), 70: 30 ratio has higher eluent strength so that the analytes come out earlier than the analytes that used mobile phase composition of 50:50. The separation quality is depending on the resolution between peaks that ideally 1.5. Resolution that is higher than 1.5 will produce good separation between peaks but needs longer retention time for the last analyte to be eluted. Low resolution than the ideal value of 1.5 produces inadequate separation between peaks or in other words, the peaks is overlap between them. There is different is happening when the gradient elution mode is used. Gradient elution mode is used when there are wide polarities of compounds to be separated. Gradient elution changes the polarity of mobile phase composition throughout analysis either continuously or in step during the separation. Gradient elution mode will decrease the retention time for the analytes to be eluted throughout the column. The gradient elution will give better separation, high efficiency and good resolution in separating the mixture with wide polarities. The qualitative analysis was done to identify the components in the mixture by comparing the peaks in the mixture with the peaks of the standard compound. There is caffeine indicated for the first peak followed by acetone, methyl benzoate, phenatole and phenanthrene




CONCLUSION
The optimized mobile phase composition for the separation of the mixture is 70:30 (ACN: H2O). Higher composition of organic solvent will increase the solvent strength that will shorten the analysis time. The first peak is corresponds to caffeine and then are followed by acetone, methyl benzoate, phenatole and phenanthrene peak.




REFERENCE
  1.   https://chem.libretexts.org/LibreTexts/University_of_California_Davis/UCD_Chem_115_Lab_Manual/Lab_2%3A_High_Performance_Liquid_Chromatography
  2.         Nor’ashikin S., Ruziyati T., Mardiana S.(2012), Analytical Separation Methods Laboratory Guide (2nd edition).
  3.            http://hiq.linde gas.com/en/analytical_methods/liquid_chromatography/high_performance_liquid_chromatography.html

Faizzarul Mohd Fadzli

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