Chm 510 Experiment 2

CHM 510 ANALYTICAL SEPARATION METHODS EXPERIMENT 2 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): METHOD DEVELOPMENT NAME: NABILAH BINTI ABD RAHMAN STUDENT ID: 2015484718 LAB PARTNERS: 1. ANIZA BINTI ABDULLAH (2015827038) 2. NIK NURFARAHAIN RIFHAN BINTI NIK AZMAN (2015896328) GROUP: AS2453D1 LECTERUR’S NAME: DR. MARDIANA BINTI SAAID DATE PERFORMED: 30TH MARCH 2016 DATE OF SUBMISSION: 27TH MAY 2016

TITLE OF EXPERIMENT Experiment 2 – High Performance Liquid Chromatography (HPLC): Method Development

OBJECTIVE To optimize the separation of mixture of 5 compounds (caffeine, acetone, methyl

benzoate,

phenatole,

phenantrene)

using

HPLC

method

development by varying the mobile phase composition.

INTRODUCTION Chromatography is a technique to separate mixtures of substances into their components on the basis of their molecular structure and molecular composition. This involves a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Sample components that display stronger interactions with the stationary phase will move more slowly through the column than components with weaker interactions. This difference in rates causes the separation of various components. Chromatographic separations can be carried out using a variety of stationary phases, including immobilized silica on glass plates (thin-layer chromatography), volatile gases (gas chromatography), paper (paper chromatography) and liquids (liquid chromatography). High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. All

chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation.

Normal Phase HPLC method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these. Polar samples are thus retained on the polar surface of the column packing longer than less polar materials. In

Reverse

Phase

HPLC

the

stationary

phase

is

nonpolar

(hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic interactions hence the more nonpolar the material is, the longer it will be retained. In Size-exclusion HPLC the column is filled with material having precisely controlled pore sizes, and the particles are separated according to its their molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. Ion-Exchange HPLC’s stationary phase has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.

EXPERIMENTAL PROCEDURE 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: The standard mixture was injected into the HPLC by using mobile phase composition of acetonitrile:water (50:50 v:v). The mobile phase ratio of acetonitrile:water was then changed for the second injection to 70:30. The resolution of the two chromatograms were calculated and compared to determine the best composition for that analysis.

c.

Identification of components in the mixture: Each of the standard compounds (caffeine, methyl benzoate, phenatole and phenantrene) was injected individually with the optimized HPLC conditions to be compared with the retention time of the standard mixture.

d.

Separation using gradient elution: Gradient elution separation was performed based on the separation by using isocratic elution to improve the efficiency of the column.

EXPERIMENTAL RESULTS, DATA AND CALCULATIONS A.

Effect Of Mobile Phase On HPLC Separation Isocratic Elution of Standard Mixture

Mobile Phase Ratio (Compositi on) of ACN:water

50:50

70:30

Peaks

Retentio n Time of Peaks (min)

Base Peak Width of Peaks (min)

Area of Peaks (mAU*s)

Peak 1

0.975

0.0637

Peak 2

1.118

0.0593

Peak 3

3.253

0.1115

Peak 4

5.582

0.1563

Peak 5

20.672

0.5183

1.17225e 4 484.1832 9 5215.045 41 4499.707 03 1.30105e 5

Peak 1

0.956

0.0528

8957.484 9

Resolutio n of 2 peaks (peak 2 & peak 3)

25

(Average from injection 1 and injection 2)

Peak 2

1.056

0.0503

Peak 3 Peak 4

1.728 2.296

0.0602 0.0716

Peak 5

5.053

0.2088

1708.031 56 4607.312 4266.629 15 4.51238e 4

12.16

Calculation of resolution of 2 peaks: 1.

Mobile Phase Ratio (Composition) of ACN:water ; (50:50) Rs(2,3) = 2( Rt,3- Rt,2 ) (Wb,2 + Wb,3) Rs(2,3) = 2( 3.253 - 1.118 ) (0.0593 + 0.1115) = 25 #

2.

Mobile Phase Ratio (Composition) of ACN:water ; (70:30) Rs(2,3) = 2( Rt,3- Rt,2 ) (Wb,2 + Wb,3) Rs(2,3) = 2( 1.728 - 1.056 ) (0.0503 + 0.0602) = 12.16 # The resolution for both mobile phase compositions of the standard

mixture is above 1.5, which is and ideal separation or complete separation between peaks. But the elution time for the mobile phase composition of 50:50 has a longer elution time for the last or late eluting compound, the

mobile phase composition of 70:30 ACN:H 20 is chosen. In other words, the mobile phase composition of 70:30 ACN:H20 has shorter analysis time.

B.

Identification Of Each Component In The Mixture Mobile phase composition of ACN:water is 70:30

1.

Caffeine

2nd Injection 2.

Retention time in standard (min) 1.056

Retention time in Mixture (min) 1.057

Retention time in standard (min) 1.728

Retention time in Mixture (min) 1.729

Retention time in standard (min) 2.296

Retention time in Mixture (min) 2.270

Area (mAU*s) 8916.14160

Area (mAU*s) 501.43103

Methyl benzoate

1st Injection 4.

Retention time in Mixture (min) 0.962

Acetone

2nd Injection 3.

Retention time in standard (min) 0.956

Area (mAU*s) 4969.65430

Phenetole

1st Injection

Area (mAU*s) 1912.42883

5.

Phenantrene

1st Injection

C.

Retention time in standard (min) 5.053

Retention time in Mixture (min) 4.227

Area (mAU*s) 3546.25146

Separation Using Gradient Elution Gradient Elution of Standard Mixture

1.

1st injection of standard mixture for gradient elution 0 min : (70:30) 0 – 2 min : (75:25) 2 – 3 min : (80:20)

3 – 5 min : (80:20) Mobile Phase Ratio (Compositi on) of ACN:water

50:50

Peaks

Retentio n Time of Peaks (min)

Base Peak Width of Peaks (min)

Area of Peaks (mAU*s)

Peak 1

0.973

0.0540

Peak 2

1.120

0.0529

Peak 3

2.144

0.0513

Peak 4

2.798

0.0627

Peak 5

4.992

0.1496

8783.035 16 1196.049 93 3055.491 46 3080.991 21 2.74360e 4

Calculation of resolution of 2 peaks Rs(2,3) = 2( Rt,3- Rt,2 ) (Wb,2 + Wb,3) Rs(2,3) = 2( 2.144 - 1.120 ) (0.0529 + 0.0513) = 20 #

2.

2nd injection of standard mixture for gradient elution 0 min : (70:30) 0 – 2 min : (70:30) 2 – 3 min : (80:20)

Resolutio n of 2 peaks (peak 2 & peak 3)

20

3 – 4 min : (80:20) Mobile Phase Ratio (Compositi on) of ACN:water

50:50

Peaks

Retentio n Time of Peaks (min)

Base Peak Width of Peaks (min)

Area of Peaks (mAU*s)

Peak 1

0.977

0.0622

Peak 2

1.123

0.0548

Peak 3

2.117

0.0552

Peak 4

2.695

0.0632

Peak 5

4.710

0.1408

1.11516e 4 1712.522 58 4358.705 57 4370.255 86 3.07807e 4

Calculation of resolution of 2 peaks Rs(2,3) = 2( Rt,3- Rt,2 ) (Wb,2 + Wb,3) Rs(2,3) = 2( 2.117 - 1.123 ) (0.0548 + 0.0552) = 18 #

3.

3rd injection of standard mixture for gradient elution

Resolutio n of 2 peaks (peak 2 & peak 3)

18

0 min : (70:30) 0 – 1.5 min : (80:20) 1.5 – 4 min : (80:20) Mobile Phase Ratio (Compositi on) of ACN:water

50:50

Peaks

Retentio n Time of Peaks (min)

Base Peak Width of Peaks (min)

Area of Peaks (mAU*s)

Peak 1

0.970

0.0658

Peak 2

1.116

0.0550

Peak 3

2.070

0.0533

Peak 4

2.553

0.0588

Peak 5

4.220

0.1414

1.16349e 4 1840.950 56 4705.826 17 4711.999 02 3.04860e 4

Calculation of resolution of 2 peaks Rs(2,3) = 2( Rt,3- Rt,2 ) (Wb,2 + Wb,3) Rs(2,3) = 2( 2.070 - 1.116 ) (0.0533 + 0.0550) = 18 #

Resolutio n of 2 peaks (peak 2 & peak 3)

18

4.

4th injection of standard mixture for gradient elution 0 min : (70:30) 0 – 1 min : (80:20) 1 – 3.5 min : (80:20)

Mobile Phase Ratio (Compositi on) of ACN:water

50:50

Peaks

Retentio n Time of Peaks (min)

Base Peak Width of Peaks (min)

Area of Peaks (mAU*s)

Peak 1

0.970

0.0562

Peak 2

1.116

0.0514

Peak 3

2.037

0.0500

Peak 4

2.469

0.0529

Peak 5

4.116

0.1522

9628.161 13 1393.817 38 3498.078 61 3560.180 18 2.80843e 4

Calculation of resolution of 2 peaks Rs(2,3) = 2( Rt,3- Rt,2 ) (Wb,2 + Wb,3) Rs(2,3) = 2( 2.037 - 1.116 ) (0.0500 + 0.0514) = 18 #

Resolutio n of 2 peaks (peak 2 & peak 3)

18

DISCUSSION High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). A moving carrier gas stream of helium or nitrogen carries the sample. HPLC has the ability to separate, and identify compounds that are present in any sample that can be dissolved in a liquid in trace concentrations as low as parts per trillion. Because of this versatility, HPLC is used in a variety of industrial

and

scientific

applications,

such

as

pharmaceutical,

environmental, forensics, and chemicals. Sample retention time will vary depending on the interaction between the stationary phase, the molecules being analyzed, and the solvent, or solvents used. As the sample passes through the column it interacts between the two phases at different rate, primarily due to different polarities in the analytes. Analytes that have the least amount of interaction with the stationary phase or the most amount of interaction with the mobile phase will exit the column faster. In this experiment, is to optimize the separation of mixture of 5 compounds (caffeine, acetone, methyl benzoate, phenatole, 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 were first done for mobile phase composition of ACN:H 2O 70:30 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 first step of the experiment is to find the suitable mobile phase composition. A mobile phase composition of ACN:H 2O of 50:50 and 70:30 were done. The resolution for both mobile phase compositions of the standard mixture is above 1.5; 50:50 mobile phase compositions has a resolution of 25 and 70:30 mobile phase composition has a resolution of 12 which are an ideal separation or complete separation between peaks. But the elution time for the mobile phase composition of 50:50 has a longer elution time for the last or late eluting compound, at the 20 th minute but the elution time of last eluting compound for the mobile phase composition of 70:30 ACN:H20 is at the 5th minute, so this mobile phase ratio is chosen since it is more suitable. In other words, the mobile phase composition of 70:30 ACN:H20 has shorter analysis time. The second step is to inject the compounds individually (caffeine, acetone, methyl benzoate, phenatole, phenantrene) to identify the components in the mixture using the selected HPLC conditions (CAN:H 2O 70:30). If the composition of the mobile phase remains constant throughout the HPLC separation, the separation is deemed an isocratic elution. By this technique, we know and found out that caffeine eluted at the 0.9th minute, as shown in the chromatogram of the individual injection of the compound. Then, the 2nd eluting compound is acetone at 1st minute, also shown in the chromatogram of the chromatogram of the individual injection of the compound. The 3 rd eluting compound would be methyl benzoate, at 1.7th minute, shown at the chromatogram of the individual injection of the compound. Phenetole is the 4th eluting compound, at the 2nd minute, as shown in the chromatogram of the individual injection of the compound and the last and 5 th eluting compound is phenantrene, at 5th minute, as shown in the chromatogram of the individual injection of the

compound. 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. Often the only way to elute all of the compounds in the sample in a reasonable amount of time, while still maintaining peak resolution, is to change the ratio of polar to non-polar compounds in the mobile phase during the sample run. Known as gradient chromatography, this is the technique of choice when a sample contains components of a wide range of polarities. This is the third and last step in this experiment. For a reverse phase gradient, the solvent starts out relatively polar and slowly becomes more non-polar. The gradient elution offers the most complete separation of the peaks, without taking an inordinate amount of time. A sample containing compounds of a wide range of polarities can be separated by a gradient elution in a shorter time period without a loss of resolution in the earlier peaks or excessive broadening of later peaks. However, gradient elution requires more complex and expensive equipment and it is more difficult to maintain a constant flow rate while there are constant changes in mobile phase composition. Gradient elution, especially at high speeds, brings out the limitations of lower quality experimental apparatus, making the results obtained less reproducible in equipment already prone to variation. If the flow rate or mobile phase composition fluctuates, the results will not be reproducible. In this experiment, gradient elution was done to improve the efficiency of the column. This is done so that the late eluting compounds will elute faster, and the results shown in the results and data section showed that the 4 th injection for gradient elution is most suitable and the best. Since the response factor is above 1.5 (18) it is still deemed an efficient and complete separation. So, the gradient elution for the 4 th injection is set at 0 min (70:30), 0 – 1 min (80:20) and 1 – 3.5 min (80:20).

Conclusion The most polar compound is caffeine (0.9 th minute), followed by acetone (1st minute), methyl benzoate (1.7th minute), phenatole (2nd minute) and the least polar or non-polar compound is phanantrene (5 th minute) and a mobile phase composition of ACN:H2O is 70:30. References 1.

http://laboratoryinfo.com/hplc/

2.

http://www.chemguide.co.uk/analysis/chromatography/hplc.html