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
phase usually cannot compete successfully for the sample components; the retention time becomes shorter for practical application. When the situation where the polarity of the analyte and the stationary phase are too alike and totally different from the mobile phase, thus the retention time becomes too long. Due to theories of mobile phase and stationary phase interaction at any given set of sample component are impacted, and at best, we can only narrower the choice of the stationary has to a general type. Hence, because of this choice, we then can perform a series of set trial and error experiment until a satisfactory separation is identified. During this experiment, a High Performance liquid chromatography (HPLC) Agillent G1311A equipped with DA detector, 5 mm reverse phase C18 column and 10 microliter sample loop was used. At flow rate 2.0 ml/min and detector wavelength at 254nm, the mobile phase ratio was set up at 50% acetonitrile and 50% water at the beginning at the experiment in other to analyze and observe the effect of mobile phase composition on the chromatography separation. After the standard mixture is injected, the process is run and the peak obtained is analyzed. The process is repeated 3 times to get ideal result. Next, the resolution of the process is identified by calculating using formula below:
Where t is the retention time and W is the peak width. After performing calculation for resolution as shown in Table 1.1, 1.2, and 1.3 we get average resolution of 2.213.
Next, with the same requirement of flow rate and detector wavelength, the mobile phase composition was changed to 70% acetonitrile and 30% water. The same steps have been done and the process is also repeated for 3 times to get ideal result. After performing calculation for resolution as shown in Table 2.1, 2.2, and 2.3 we get average resolution of 1.643. Resolution describes the ability of a column to separate the peaks of interest, and so the higher the resolution, the easier it is to achieve baseline separation between two peaks. Resolution takes into consideration efficiency, selectivity and retention. It is useful to relate the resolution to the number of plates in the column, the selectivity factor and the retention factors of the two solutes:
One can improve resolution by improving any one of these parameters. To obtain high resolution, the three terms must be maximized. An increase in N, the number of theoretical plates, by lengthening the column leads to an increase in retention time and increased band broadening which may not be desirable. Instead, to increase the number of plates, the height equivalent to a theoretical plate can be reduced by reducing the size of the stationary phase particles. It is often found that by controlling the capacity factor, k', separations can be greatly improved. This can be achieved by changing the composition of the mobile phase (in Liquid Chromatography).
The selectivity factor, α, can also be manipulated to improve separations. When α is close to unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable time. A value of 1 is the minimum for a measurable separation to occur and to allow adequate quantitation. A value of 0.6 is required to discern a valley between two equal-height peaks. Values of 1.7 or greater generally are desirable for rugged methods. A value of 1.5 is considered to be a baseline separation and ensures the most accurate quantitative result. By comparing the average resolution for both mobile phase compositions, we can see that with mobile phase ratio of 70% acetonitrile and 30% water gives the best resolution which is 1.64. It is indicate that the resolution is good and efficiency of separation is increase. Meanwhile, for the mobile phase ratio of 50% acetonitrile and 50% water, the resolution is 2.21 shows that it has good resolution, but the efficiency of the separation is very weak. After that, the components in standard mixture is identified when each of the component is injected individually by using mobile phase compositions of 70% acetonitrile and 30% water as the best composition baseline separation. The compound in the standard mixture is identified by comparing the retention time of standard mixture with the retention time of individual compound. From the result obtained, peak 1 indicate compound of caffeine, peak 2 is acetone, peak 3 is methyl benzoate, peak 4 is phenatole and the last peak is phenantrene. Other than that, the rate theory of chromatography / Van Deemter equation is related in HPLC broadening peak. Below are Van Deemeter equations: H = A + B/v + Cv
In HPLC the B term can be neglected due to diffusion is 100 times less in liquids than in the gas. While the C term is the largest contribution to H. Consider the following mobile phase mass transfer coefficient: CMv = (fM(k’)dp2/DM)v Where dp is particle diameter of packing and DM is mobile phase diffusion coefficient. CMv is less if dp is smaller hence greater surface area or the solute diffusion coefficient in the mobile phase, DM is larger. Next, a gradient elution separation method is performed in other to improve te efficiency of the column. Meaning that, isocratic elution is performed with a single solvent or constant solvent mixture. If one solvent does not provide sufficiently rapid elution of all components, then gradient elution can be used. In this case, by increasing amount of water was added to acetonotrile to create a continuous gradient. In this experiment, using acetonitrile and water gradient, the more hydrophobic components will elute when the mobile phase consist mostly of acetonitrile which giving a relatively hydrophobic mobile phase. The more hydrophilic compounds will elute under conditions of relatively low acetontrile and high water. Often a series of test are performed on the analyte and the number of the trial runs has been done in other to find the HPLC method which gives the best separations peak. From the gradient elution method, the average resolution obtained is 1.74 and it is indicate as good resolution and efficiency of separation is high. From the chromatogram obtain from both method which are isocratic elution method and gradient elution method, there are some differences can be obtain from the separation condition. In isocratic elution method, peak width increases with retention time linearly according to the equation for N, the number of theoretical
plates. This leads to late eluting and peaks get a little bit flat and broad. In isocratic elution method, the retention times of caffeine, acetone, methyl benzoate, phenotate and phenanthrene were about 0.739, 0.812, 1.318, 1.763 and 3.864 min. Meanwhile, the gradient elution method decreases the retention of the later eluting components so that they elute faster, giving narrower, taller and sharp peaks for most components. 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. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height which where the peak look sharp which is important in trace analysis. Thus, the gradient elution method gave a shorter overall analysis with similar resolution of the critical pair compared to isocratic elution without sacrificing repeatability in retention time, peak area and peak height. From the result, it shows that almost the entire peak is well separated from the neighbor compound. But what was happened is only the peak 1 and the peak 2 is not well separated. Peak 1 indicates the caffeine compound and the peak 2 indicate acetone compound. The mobile phase composition used is 20% acetonitrile and 80% water. It shows that, just a bit separation has occurred. As we know, the quantitative analysis in separation method depends upon direct relationship between the area under a peak or height in the chromatogram and the amount of compound corresponding to that peak in the analyzed sample. Therefore, each peak should be totally resolved from any neighboring peaks. There are some factors leads to problem stated above. The mobile phase must be degassed properly. It is because mobile phases entrap air from the
atmosphere and this trapped air gets released as small bubbles under high pressures encountered during the HPLC analysis. Such bubbles can lead to noise in detector response or hinder flow of mobile phase through columns. In order to overcome such problems degassing of mobile phase becomes essential. It is require a very clean & pure HPLC grade solvent to prevent column degradation with impurities & to minimize detector background signals from contaminants (usually UV transparent). The gas that interference the HPLC analysis, such as N2, O2, and CO2. The gas bubbles create difficulties with pumps destabilize pressure, columns bad separation & detectors. Therefore we need to degasse the mobile phase in other to get good analysis. There are few methods in degassing the mobile phase. Vacuum devices (vacuum applied to headspace above solvent). Ultra sonication (high frequency vibration drives gasses out of solvent) Heating (decreases solubility of gases). He sparging. Sparging is a process in which dissolved gases are swept out of a solvent. There are some step is carefully taken when doing the experiment. When injecting the sample into the loop, the sample volume is no more than volume indicated on a loop. Besides, sample injection only with flat end needle to prevent damage to the injection port. Other than that, the syringe is washed at least 5 times with washing solution and wash 3 times with sample before the sample is inejcted to the column to get desire peak and no contiminants. When filling the syringe with the sample, the maximum value should be 20μL.
CONCLUSION From the experiment, the concept and method development of optimizing a separation of a standard compounds using hplc by varying the mobile phase composition is studied and determined. REFERENCES 1. Skoog, Hooler and Nierman, 5th Editon. Principles of Instrumental Analysis. Thomson Learning 1998. 2. Nor’ ashikin, Ruziyati and Mardiana, 2nd Edition. Analytical Separation Methods Laboratory Guide
