HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): METHOD DEVELOPMENT PDF

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Summary

Experiment 2-chm510-ANALYTICAL SEPARATION METHOD
OBJECTIVE
To optimize a separation of a mixture of five compounds using HPLC by verifying the
mobile phase composition....

Description

CHM510 (ANALYTICAL SEPARATION METHOD) (EXPERIMENT 2) HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): METHOD DEVELOPMENT GROUP: AS2222A1 (GROUP 4) GROUP MEMBERS:

NUR FADHLI BIN ABDULLAH

2020980931

NURUL AFIQAH BINTI MOHD HARIS

2020974799

FATIN SYAHIRA BINTI AHMAD

2020980415

NOR AMIERA SYAFIAH BINTI SHAMSUL KAMAL

2020970661

LECTURER’S NAME

:

DR. NUR NADIA BINTI DZULKIFLI

DATE OF EXPERIMENT :

19/11/2020

DATE OF SUBMISSION

26/11/2020

:

OBJECTIVE To optimize a separation of a mixture of five compounds using HPLC by verifying the mobile phase composition. INTRODUCTION High Performance Liquid Chromatography (HPLC) is basically a highly improved form of column liquid chromatography. A liquid sample is injected into a stream of solvent (mobile phase) flowing through a column packed with a separation medium (stationary phase). The different component in the mixture passes through the column at different rates due to differences in their portioning behaviour between stationary phase and mobile phase. Compounds are separated by injecting a plug of sample onto the column. The injector allows a convenient and controlled introduction of sample directly onto the column, while the detector indicates when a particular analyte (also called a band) elutes (or leaves the column). A detector and a computer are connected to the HPLC instrument in order to signal when the eluents are coming off the column and fractions should be collected. In this experiment, reversed phase chromatography is used which the stationary phase is non-polar and the mobile phase is relatively polar. The stationary phase is non-polar hydrophobic packing with octyl or octa decyl functional group bonded to the silica gel and the mobile phase is polar solvent. The polar compound will eluted first in this mode and the non-polar will retained for longer time. Changes in the mobile phase composition can be done by isocratic elution whereas the mobile phase is constant or by gradient elution which shows the changes in the mobile phase during separation either continuously or in steps in order to separate wide range of polarity. Isocratic elution is a single composition of solvents is used for the duration of the separation. Later eluting peaks are broader than earlier eluting peaks because of dispersion. Steps must be taken to periodically flush the column at higher solvent strength to clean it of intractable materials that build up from sample injections. Gradient Elution is the composition of solvents that is either continuously or stepwise modified. In fact, the peaks are sharper throughout the chromatography compared to isocratic

elution. There may be some separations that cannot be accomplished by using isocratic elution. Chromatogram run times can be shorter compared to isocratic elution. METHODOLOGY a) Instrument set-up (may vary depending on instrument):

Detector wavelength

: 254 nm

Flow rate

: 1 mL min -1

Mobile phase

: acetonitrile: water

b) Effect of mobile phase on HPLC separation:

1. The instruments to use a mobile phase ratio of acetonitrile: water (50:50 v: v) was set up and the sample was injected. 2. The mobile phase then was changed to 70:30. Then, the best composition of mobile phase is chosen.

c) Identification of components in the mixture:

1. Each component was injected individual to identify the component mixture using the selected HPLC conditions.

d) Identification of components in methyl esters mixture:

1. Based on the separation, a gradient elution separation was performed to improve the efficiency of the column.

RESULTS AND CALCULATION

I.

Effect of the mobile phase on HPLC separation (isocratic elution) Composition of mobile phase (ACN: H O) 50:50 2

Peak

1 and 2 2 and 3 3 and 4 4 and 5

Retention time (min) Base peak width (min) Resolution, Rs

1.135 and 1.356 1.356 and 3.964

0.1674 and 0.1319 0.1319 and 0.2201

3.964 and 6.887

0.2201 and 0.2767

6.887 and 26.141

0.2767 and 1.3710

1.4768 14.8182 11.7673 23.3708

Average Resolution

1.4768 14.8182 11.7673 23.3708

Composition of mobile phase (ACN: H O) 70:30 2

Peak

Retention time (min) Base peak width (min) Resolution, Rs

1 and 2 1.169 and 1.234 2 and 3 3 and 4 4 and 5

0.1435 and 1.1069

1.234 and 2.089

1.1069 and 0.1247

2.089 and 2.800

0.1247 and 0.1303

2.800 and 6.377

0.1303 and 0.5229

0.5192 7.3834 5.5765 10.4713

Average Resolution

0.5192 7.3834 5.5765 10.4713

II.

Effect of the mobile phase on HPLC separation (gradient elution)

a) Program 1 Composition of mobile phase (ACN:H2O)

Retention time (min)

Base peak width (min)

Resolution, Rs

Average resolution, Rs

1.136

0.1598

1.278 1.278

0.1013 0.1013

1.0915

1.0915

2.582

0.2222

8.0618

8.0618

2.582 3.489 3.489 5.403

0.2222 0.1507 0.1507 0.2770

4.8646

4.8646

8.9502

8.9502

Peak

Retention time (min)

Base peak width (min)

Resolution, Rs

Average resolution, Rs

1 and 2

1.138 1.278

0.1540 0.0991

1.1063

1.1063

2 and 3

1.278

0.0991

2.582

0.1934

8.9162

8.9162

3 and 4

2.582 3.487 3.487 5.403

0.1934 0.1521 0.1521 0.2758

5.2388

5.2388

8.9554

8.9554

Peak 1 and 2

0 – 1.8 min 50:50

2 and 3

1.8 – 3.0 min 70:30

3 and 4 4 and 5

b) Program 2 Composition of mobile phase (ACN:H2O)

0 – 1.8 min 50:50 1.8 – 8.0 min 70:30 3.0 – 8.0 min 85:15

III.

4 and 5

Modified HPLC mode 70:30 (ACN: H2O), identification of the compounds in the mixture

Standard compound

Retention time in individual standard (min)

Retention time in standard mixture (70:30 ACN:H O) (min)

Acetone

1.234

1.332

Caffeine Methyl benzoate

1.169 2.089

1.121 2.095

Phenanthrene Phenatole

6.377 2.800

6.353 2.812

2

CALCULATION: Calculate resolution, Rs using formula: () ( )

I.

Effect of the mobile phase on HPLC separation (isocratic elution) (ACN: H O) 50:50 2

Peak 1 & 2

(ACN: H O) 70:30 2

Peak 1 & 2

1.4768 Peak 2 & 3 Peak 2 & 3

7.3834 Peak 3 & 4 Peak 3 & 4 11.7673 5.5765 Peak 4 & 5 Peak 4 & 5 23.3708 10.4713

II.

Effect of the mobile phase on HPLC separation (gradient elution) Program 1

Peak 1 & 2

1.0915

Program 2

Peak 1 & 2

1.1063

Peak 2 & 3

Peak 2 & 3

8.0618

8.9162

Peak 3 & 4

Peak 3 & 4

4.8646 Peak 4 & 5

5.2388 Peak 4 & 5

8.9554

DISCUSSION In this experiment, the column used was C18 and the composition of mobile phase relatively polar solvent such as water and acetonitrile. The C18 column is nonpolar column or known as hydrophobic column. From the information, the modes of the separation used was reversed phase. In reversed phase, when the solvent strength was increase, it will reduce the polarity of the compound. So, less polar compound will elute faster compared to polar compound. In order to optimize the separation of mixture of 5 compounds which are caffeine, acetone, methyl benzoate, phenatole and phenantrene using HPLC method development by varying the mobile phase composition. For the mobile phase, a mixture of acetonitrile: water is used. Both isocratic elution and gradient elution are used in this experiment. Isocratic elution was first done for mobile phase composition to identify the components in the mixture. Then, gradient elution was done to improve the efficiency of the column so that the later eluting compounds will elute faster. The composition of mobile phase was set up ACN: H2O 50:50 at the initial process of the separation. Then, the ratio was changed to ACN: H2O 70:30 to observe the separation of the compounds. If the composition of mobile phase was suitable for the compound separation, it can be used in order to get the better separation. The standard mixture was injected into the column and the retention time was analyze to determine the compound by comparing the retention time of standard mixture and the individual standard.

Figure 1 Composition of mobile phase (ACN: H2O) 50:50 At composition of mobile phase (ACN: H2O) 50:50, the resolution for peak 1 and peak 2 is 1.4768. The resolution is almost near 1.5 baseline resolution so the separation between peak 1 and peak 2 is well separated. Meanwhile for peak 2 and 3, peak 3 and 4, peak 4 and 5 resolution are 14.8182, 11.7673 and 23.371, respectively. These resolutions exceed 5.0, so the separation is complete but produce broad peaks. The elution time for the mobile phase composition of (ACN: H2O) 50:50 has a longer elution time for the last or late eluting compound, at the 25th minute.

Figure 2 Composition of mobile phase (ACN: H2O) 70:30

At composition of mobile phase (ACN: H2O) 70:30, the resolution for peak 1 and peak 2 is 0.5192 which is less than 1.0, the separation is not well separated and produce broad peak. However, the resolution for peak 2 and 3, peak 3 and 4, peak 4 and 5 resolutions are 7.3834, 5.5765, and 10.4713, respectively. All three-resolution value for both conditions exceed 5.0 means that the separation between the two peaks is complete but produce broad peaks but the elution time of last eluting compound for the mobile phase composition of ACN: H2O 70:30 is at the 6th minute, so this mobile phase ratio is chosen since it is more suitable. In other words, the mobile phase composition of ACN: H2O 70:30 has shorter analysis time.

Based on the results obtained, ACN: H2O 70:30 gives better separation compared to mobile phase ratio of (ACN: H2O) 50:50 which is not separate the mixture well. This is because the polarity of the mobile phase will affect the elution time of the analyte or compound to be separated. Less polar characteristic of the mobile phase will reduce the elution time of the nonpolar analyte because decreasing the polarity of mobile phase will increase the solvent strength and the eluent strength. Therefore, the compounds will elute so much faster and the retention time will decrease as separation becomes more efficient. Thus, the objective has been determined which ratio of mobile phase is suitable for the separation of these compounds, by using mobile phase ratio of ACN: H2O 70:30 in order to analyze each compound that exists in the standard mixture. Gradient elution is used for compound to be separated more efficiently by changing the mobile phase composition in the duration of analysis gradually or continuously. The purpose is to move strongly retained components of the mixture faster, but least retained component well resolved. Firstly, start with low solvent strength (ACN: H2O) 50:50 which is high polarity of mobile phase to separate least retained compound. Then, gradually increase the solvent strength to ACN: H2O 70:30 so that the strongly retained compound elutes faster. This elution mode will decrease the retention time for the analytes to be eluted from the column while producing more efficient separation. This in turn result in lower resolution values as compared to the resolution values when using isocratic elution. Although from the calculations showed that the resolution values for both Program 1 and 2 have exceed 5.0 with highest resolution that is below 9.0, but it shows that gradient elution produce a more efficient separation as compared to isocratic elution because the resolution values for isocratic elution is very large and can exceed 10.0 for late eluting peaks. In part C where is the identification of individual components for all five compounds occur by using the composition of mobile phase of ACN: H2O 70:30 isocratic system by comparing the retention times of compounds in the individual standard chromatogram. The retention time for acetone, caffeine, methyl benzoate, phenanthrene and phenetole in the individual standard chromatogram are 1.332 min, 1.121 min, 2.095 min, 6.353 min and 2.812 min respectively. Based on these retention times, the comparison is made by observing the retention times in the ACN: H2O 70:30 isocratic system obtained. By this technique, caffeine

eluted at the 1.121 minute, as shown in the chromatogram of the individual injection of the compound. Then, the 2nd eluting compound is acetone at 1.332 minute, also shown in the chromatogram of the individual injection of the compound. The 3rd eluting compound would be methyl benzoate, at 2.095 minute, shown at the chromatogram of the individual injection of the compound. Phenetole is the 4th eluting compound, at the 2.812 minute, as shown in the chromatogram of the individual injection of the compound and the last and 5th eluting compound is phenantrene, at 6.353 minute, as shown in the chromatogram of the individual injection of the compound. Upon observation, it was confirmed that the compounds with retention times of 1.169 minute, 1.234 minute, 2.089 minute, 2.800 minute and 6.377 minute represent as caffeine, acetone, methyl benzoate, phenetole and phenanthrene respectively. Since the mobile phase is polar and the stationary phase is non-polar, it will retain non-polar compounds and less retain polar compounds. By that, the most polar compound is caffeine, followed by acetone, methyl benzoate, phenatole and the least polar or non-polar compound is phanantrene.

CONCLUSION The objective of the experiment was determined whereas the optimized mobile phase composition for the separation of the mixture is 70:30 (ACN: H2O). The higher composition of organic solvents will increase the solvent strength which will shorten the analysis time. Hence, the first peak corresponds to caffeine and is followed by acetone, methyl benzoate, phenatole and phenanthrene peak.

REFERENCES Chemistry

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