HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC), METHOD DEVELOPMENT

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

MOHAMAD NOR AMIRUL AZHAR BIN KAMIS

STUDENT ID:

2014647344

PARTNERS’ NAMES: 1.

MOHAMAD HAMIZAN BIN MOHD ISA

2.

MOHAMAD SHAFIQ BIN PARMAN

3.

MOHAMAD AZMIZAM BIN MOHAMAD NOOR

DATE OF EXPERIMENT:

6/11/2014

DATE OF SUBMISSION:

8/12/2014

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 analyte that interacts most in the stationary phase will elute later than the analyte 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, methyl benzoate, phenatole and phenanthrene using HPLC by varying the mobile phase composition.

EXPERIMENTAL: a. HPLC was set up with following condition: Detector wavelength: 254nm Mobile phase flow rate: 1.5mL/min Mobile phase: acetonitrile:water b. Effect of mobile phase on HPLC separation: 1. 20μL of standard mixture was injected into the HPLC by using combination of acetonitrile and water (50:50). 2. The ratio of acetonitrile to water was changed for the second injection to 70:30 (ACN:water). 3. The resolution of the two chromatograms was calculated and compared to determine the best composition for that analysis. c. Identification of components in the mixture: 1. Each of the standard compounds (caffeine, methyl benzoate, phenatole and phenantrene) was injected with the optimized HPLC condition to be compared with the retention time of the standard mixture. d. Separation using gradient elution: 1. Gradient elution separation was performed based on the separation by using isocratic elution to improve the efficiency of the column.

RESULT AND DISCUSSION: A. Effects of the variation of composition of mobile phase on resolution (isocratic elution): * Calculation of resolution is based on peak 2 and 3 as references. Composition of mobile phase (ACN:H2O) 50:50 70:30

Retention time of peak 2 and 3 (min) 3.028, 5.170 1.645, 2.172

Base peak width of peak 2 and 3 (min) 0.1581, 0.2412 0.0856, 0.1065

Resolution

22.30 5.49

B. Effects of the gradient elution program: Composition of mobile phase (ACN:H2O) 75:25

Retention time of peak 2 and 3 (min) 1.641, 2.139

Base peak width of peak 2 and 3 (min) 0.0870, 0.1039

Resolution

5.22

C. The retention time of the standard mixture at optimized mobile phase composition: Standard compound Caffeine Methyl benzoate Phenatole phenanthrene

Retention time in standard (min) 0.947 1.649 2.180 4.680

D. Sample calculation: *70:30 (ACN:H2O) – isocratic elution Rs (2,3) = = 5.49 *75:25 (ACN:H2O) – gradient elution Rs (2,3) = = 5.22

Retention time in mixture (70:30)(min) 0.942 1.645 2.172 4.637

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:H 2O) 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 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, secondly is methyl benzoate, phenatole peak and lastly is corresponds to phenanthrene peak.

REFERENCES: 1. Nor’ashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation Methods Laboratory Guide (2nd edition), 3/10/2014. 2. Mardiana Saaid, Gas Chromatography Lecture Notes, 15/10/2014.