exp 1 hplc

TITLE: High Performance Liquid Chromatography (HPLC): Method Development OBJECTIVE: To study development for optimizing a separation of a mixture of 5 compounds which are caffeine, acetone, methyl benzoate, phenotate and phenanthrene using HPLC by varying the mobile phase composition. ABSTRACT A further refinement to HPLC method has been developed and validated for the determination of 5 compounds which are caffeine, acetone, methyl benzoate, phenotate and phenanthrene by varying the mobile phase composition during the analysis; this is known as gradient elution. Gradient elution is where the mobile phase compostion is change with time during the separation. The method was intended to decrease the retention of the later-eluting components so that they elute faster, giving a narrower (& taller) peaks for most components and improves the peak shape for tailed peaks. In Optimum resolution was achieved by gradient elution on an analytical column with the mobile phase consisting of a acetonitrile:water (20:80 v:v at a flow rate of 2.0 mL/min. The retention times of caffeine, acetone, methyl benzoate, phenotate and phenanthrene were about 0.783, 0.864, 2.049, 2.434 and 3.717 min, respectively. Data acquisition was carried out using a photo diode array detector in the wavelength 254 nm. Extraction of chromatograms was carried out by timed wavelength. Data obtained in these studies indicated that the method was suitable for the intended purpose.

PROCEDURE

A.INSTRUMENT SET UP

Detector wavelength: 254nm

Flow rate: 2.00 mL min-1

Mobile phase: acetonitrile: water

B. EFFECT OF MOBILE PHASE ON HPLC SPERATION

The instrument was set up with mobile phase ratio of acetonitrile:water (50:50 v:v) and the sample is injected into the column.

This ratio is repeated for three times to verify the results.

After that, the mobile phase composition is change to acetonitrile:water (70:30 v:v) and the sample is injected into the column.

This ratio is also repeated for three times to verify the results.

Then, the resolution for both composition is calculated and compared .

The suitable composition of mobile phase of these copmounds is identified.

C. IDENTIFICATION OF COMPONENTS MIXTURE

Each individually compound is injected into the column and the components of the mixture is identifed by using selected HPLC conditions.

This step is repeated for two times to verify the results.

D. SEPERATION USING GRADIENT ELUTION

Based from the seperation above, the gradient elution seperation is performed to improved the column efficiency.

The suitable ratio of mobile phase is set up and the sample is injected into the column.

This method is repeated until the sitable ratio of mobile phase is identified and all peak is seperated nicely and short resist time.

RESULT All the chromatogram for this experiment has been analyzed and it is attached behind the lab report. The resolution for isocratic elution for mobile phase composition ACN:H2O (50:50 v:v) is tabulated in Table 1.1, 1.2, 1.3 and for mobile phase composition ACN:H2O (70:30 v:v) is tabulated in Table 2.1, 2.2 and 2.3 . While, the average resolution of both mobile phase compositions is tabulated in Table 3. Lastly, resolution for gradient elution is tabulated in Table 4. Resolution (Isocratic elution) Mobile phase composition: ACN:H2O (50:50 v:v) Peak

Calculation

Resolution, Rs

1-2

=2.912

2-3

= 22.618

3-4

= 15.776

4-5

= 37.441 Table 1.1: Resolutions run 1

Peak

Calculation

Resolution, Rs

1-2

= 2.161

2-3

= 23.273

3-4

=17.011

4-5

= 39.873 Table 1.2: Resolutions run 2

Peak

Calculation

Resolution, Rs

1-2

= 2.286

2-3

= 25.493

3-4

=18.525

4-5

= 42.599 Table 1.3: Resolutions run 3

Mobile phase composition: ACN:H2O (70:30 v:v) Peak

Calculation

Resolution, Rs

1-2

= 1.558

2-3

= 9.396

3-4

= 6.878

4-5

= 17.040 Table 2.1: Resolutions run 1

Peak

Calculation

Resolution, Rs

1-2

= 1.799

2-3

= 12.213

3-4

= 9.492

4-5

= 28.463 Table 2.2: Resolutions run 2

Peak

Calculation

Resolution, Rs

1-2

= 1.571

2-3

= 10.050

3-4

= 7.425

4-5

= 17.656 Table 23: Resolutions run 3

Mobile phase composition

Average Resolution, Rs

ACN:H2O (50:50 v:v)

2.21

ACN:H2O (70:30 v:v)

1.64

Table 3: average resolution of both mobile phase compositions Resolution (Gradient elution) Peak

Calculation

Resolution, Rs

1-2

= 1.744

2-3

= 22.854

3-4

= 6.644

4-5

=18.744 Table 4: Resolutions for gradient elution

DISCUSSIONS HPLC is a technique for separation, identification and quantification of components in a mixture. It is especially suitable for compounds which are not easily volatilized, thermally unstable and have high molecular weights. High performance liquid chromatography is basically a highly improved form of column 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. It also allows us to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture. The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive. According to…………. for the column and the solvent, there are two variants in use in HPLC depending on the relative polarity of the solvent and the stationary phase which is normal phase HPLC and reversed phase HPLC. Normal phase HPLC is essentially just the same as in thin layer chromatography or column chromatography. Although it is described as normal, it isn't the most commonly used form of HPLC. The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for example. A typical column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm. Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column.

Next, for the reversed phase HPLC, in this case, the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface typically with either 8 or 18 carbon atoms in them. A polar solvent is used for example, a mixture of water and an alcohol such as methanol. Hence, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent. Non polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column. That means, now it is the polar molecules that will travel through the column more quickly. The liquid phase is pumped at a constant rate to the column packed with the stationary phase. Before entering the column the analysis sample is injected into the carrier stream. On reaching the column the sample components are selectively retained on the basis of physic chemical interactions between the analyte molecules and the stationary phase. The mobile phase moving at a steady rate elutes the components based on the operating conditions. Detection techniques are employed for detection and quantification of the eluted components. The HPLC schematic diagrams are shown in Figure 1 and HPLC machine is shown in Figure 2. In HPLC system, there are about eight important components.

Some of the significance and role of each component part of the HPLC system is discussed.

Figure 1: HPLC schematic diagram

Figure 2: HPLC machine First of all, the mobile phase. Mobile phase serves to transport the sample to the system. Essential criteria of mobile phase are inertness to the sample components. Pure solvents or buffer combinations are commonly used. The mobile phase should be free of particulate impurities and degassed before use.

Next is mobile phase reservoir. These are inert containers for mobile phase storage and transport. Generally transparent glass bottles are used so that so as to facilitate visual inspection of mobile phase level inside the container. Stainless steel particulate filters are provided inside for removal of particulate impurities in the mobile phase if any. Other than that, variations in flow rates of the mobile phase effect elution time of sample components and result in errors. Pumps function in providing constant flow of mobile phase to the column under constant pressure and to pressurize the liquid mobile phase Next is the injector. Injectors are used to provide constant volume injection of sample into the mobile phase stream. Inertness and reproducibility of injection are necessary to maintain high level of accuracy. Column contains the bed of stationary phase. It is a vital component and should be maintained properly as per supplier instructions for getting reproducibility separation efficiency run after run. Other than column, the column oven is also important part. The variation of temperature during the analytical run can result in changes of retention time of the separated eluting components. A column oven maintains constant column temperature using air circulation. This ensures a constant flow rate of the mobile phase through the column A detector gives specific response for the components separated by the column and also provides the required sensitivity. It has to be independent of any changes in mobile phase composition. It is function to detect the presence of components as they exit the column and lastly the recorder. The recorders function to record the detector signal.

Modern HPLC systems are computer based and software controls operational parameters such as mobile phase composition, temperature, flow rate, injection volume and sequence and also acquisition and treatment of output. Those are the main parts of a basic HPLC system more specialized equipment might also have solvent selection valves, vacuum degasser, auto samplers, column switchers, pre or post column derivatization and fraction collectors. As we mention earlier, there are two variants which is normal phase chromatography and reversed phase chromatography. For this analysis, we used reversed phase chromatography. In reversed phase chromatography, the stationary phase is non polar and the mobile phase is relative polar. The most polar component will elute first, and increasing the mobile phase polarity increase the elution time. Hence, because of the sample components interact with both the stationary phase and the mobile phase the method development tends to be more complex in liquid chromatography Interactive mobile phase requires proper equalization intermolecular forces among the three members in the separation process which is the solute, the mobile phase and the stationary phase in other to get successful chromatography. These intermolecular forces are describes in term of the relative polarity of the reactants. The polarities of various analytes functional groups are hydrocarbon