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Tittle: High Performance Liquid Chromatography (HPLC) Method Development

1.0 Objective: 1) To separate compound into a single component and separate the mixture. 2) To study the change of mobile phase ratio and polarity and its effect or interaction to Sample, stationary phase and efficiency of separation.

2.0 Abstract: High performance liquid chromatography (HPLC) is an analytical instrument for classifying and quantifying components in complex fluid mixtures. By selecting the appropriate equipment (i.e. Column and detector), this method is applicable to samples with components running from small organic and inorganic molecules and ions to polymers and proteins with high molecular weights. The chromatographic process was started by throwing in a sample solution into the mobile phase through the injector port.This experiment was run by isocratic elution improved by gradient elution and type of component tested by individual test.Five type of component was used which are Caffeine, Acetone, Phenatole, Phenanthrene and Methyl Benzene. How good this separation was measured by resolution, separation peak and rentation time. Type of instrument used is Agilent Technologies 1200 siries, Mobile phase Acetonitrile (ACN), Water (H2O), Column ZORBAX SB-C184.6 x 150 mm, 5 m and flow rate at 2.0mL/min.

3.0 Procedure: A. Instrument set up 1. The HPLC needs to analytialize 20 minutes before it started to run. When the system and the methods are ready a blank run should be performed to test the system and verify that it is clean from interferences 2. Setting HPLC parameters (according to ------)

Figure 3.1: HPLC set up picture was taken from Successful Operation of HPLC System.

B. Solutions:

Provided by lab assistant: Mobile phase - Acetonitrile (ACN) , Water (H2O) Standard samples Caffeine, Acetone, Phenatole, Phenanthrene and Methyl Benzene.

Once the data have been collected, use the View menu to see the report containing the chromatograms at each monitored wavelength and the integrated peak areas, etc. From here report were printed as well as view and print full spectrum at any peak.

C. Instrument

Figure 3.2: High Performance Liquid Chromatography (picture from Successful Operation of HPLC System.)

4.0 RESULT: Table 4.1:Average resolution run at ratio 50(ACN):50(WATER) Peak Rs 1,2 Rs 3,2 Rs 4,3 Rs 5,4 Resolution 2.21 20.89 17.1 31.66

Table 4.2:Average resolution run at ratio 70 (ACN): 30 (WATER) Peak Rs 1,2 Rs 3,2 Rs 4,3 Rs 5,4 Table 4.3: Gradient Elution result Peak Rs 1,2 Rs 3,2 Rs 4,3 Rs 5,4 Resolution 1.74 22.85 6.64 18.74 Resolution 1.64 10.54 7.93 21.04

5.0 DISCUSSION: Chromatography is a technique for separating mixtures into their individual components so that they can be identified and measured. In liquid chromatography (LC), a moving liquid (the mobile phase) carries the sample across a stationary phase (the solid support found within an LC column). The sample components separate based on their differing affinity with the stationary phase. LC is suited for analyzing nonvolatile and thermally fragile molecules including such high molecular weight compounds as proteins. The goal of any HPLC experiment is to achieve the desired separation in the shortest possible time and with better resolution at range 1.50-2.50. To design a chromatographic separation system we create competition for the various compounds contained in the sample by choosing a mobile phase and a stationary phase with different polarities. Then, compounds in the sample that are similar in polarity to the stationary phase will be delayed because they are more strongly attracted to the particles. Compounds whose polarities are similar to that of the mobile phase will be preferentially attracted to it and move faster. In this experiment we use Acetonitrile (ACN) and water (H20) as mobile phase, Column ZORBAX SB-C184.6 x 150 mm, 5 m and flow rate at 2.0mL/min. The first step was used are isocratic elution where the composition of mobile phase remains constant at 50 (ACN): 50 (H20) during the separation. The standard mixture of five components was injected at the injected port, the chromatogram showed average 1st peak at 0.77 minutes and the last peak at 11.95 minutes, chromatogram showed also do not have good resolution between peak it showed in table 4.1 Next the ratio of mobile phase was adjusted to 70 (ACN): 30 (H2O), According toRonald E. Majors, LC Columns Technical Support rentation time and resolution of peak can be improving by adjusting the ratio of mobile phase, however the result showed from this adjusting show a shorter retention time compared before. At the average 1st component showed a peak at 0.739min and the 5th component showed a peak at 3.864 min and the resolution showed improving it can be seen in table 4.2 However, the peak hasn't shown a good peak separation, the wider the peaks, the poorer the separation. Now to be treated by rate theory, the gradient elution method was used, in a gradient work the solvent strength is increased with time during the chromatographic run. According to Agilent technologies, gradient elution is the solvent composition of the mobile phase is changed with time during separation. The two components of mobile phase are ACN and H2O, H2O is the weak solvent which allows the solute to elute only slowly and ACN is the strong solvent which rapidly elutes the solutes from the column, organic solvent of the mobile phase is to reduce the retarding strength of aqueous component. From this method component 1 and 2 is more like to

water compared to ACN because from the first ratio 50(water):50(CAN) first and second component separate well compared when changing ratio to 30(water):70(CAN) it not separated well, for this method changing of ratio of mobile phase was changed at certain time for first two minute ratio 80(water):20(ACN) was used and for the rest ratio 20(water):80(ACN) result showed show improving in retention time, separation and resolution, this method showed improved in rentation time and resolution from the beginning and now the 5th component out at rentation time 3.717 min with, better resolution compared before and not broadening peak. In addition, from the individual test we can determine the compound of each peak, from the individual test first peak is caffeine, followed by acetone, methyl benzoate, prenatal and phenantrene. There a few precautionary steps that should be considered before running this experiment, firstly Solvent Delivery System, before any analysis by HPLC the solvents for the mobile phase are prepared. All types of solvents should be filtered before any use (or verify that they were sold already filtered), to prevent clogging of the column. Filtration needs to be performed with a special solvent filtration system that should exist in every analytical lab that uses HPLC. Next, Programming of the mobile phase composition, Mixing between different solvents after the preparation of the solutions and solvents a protocol is decided, i.e., the composition of the mobile phase during the run. For this purpose, it there is a need to mix various solvents. Then elimination of gasses in the solvents, degassing of solvents is removing all air in them, and it is imperative in most HPLC methods. There are several ways to degases solvents the most common one currently is by using in-line vacuum degasser. The other method is by sparging helium in the reservoir. The vacuum degasser contains vacuum chambers, one for every reservoir, where the solvents enter, effuse all the gasses from them instantly, and proceed degassed. Next, purging the System when the solvents are ready for work the system is purged with the degassed solvents to wash the tubing all the way to the columns connection. This is done to drive out previous solvents and or air from the system. Preparation of the Column, this is the stage where the column can be connected to the system. It is important not to use excessive force to tighten and or release the column connections. Lastly, the Detector, after connecting the column into the detector it is time to prepare it for the analysis. It is important to remember that the detector is the eyes of the system. The baseline is actually the detector's response to the mobile phase flowing out of the column constantly, even before the injection and following it. It can serve as an indication of the status of the column or the solvents. If the column is polluted or the solvents are not appropriate for HPLC work the baseline is not stable. If the solvents are not appropriate for HPLC they can contain contaminations from the procedure of their production step, then that it is impossible to apply them for gradients due to adsorption and release of the contaminations all the time while the composing of the fluid phase is varied.

CONCLUSION:

In consideration of the numerous sources of error, this experiment has been a success to separate the standard mixture and to improve the resolution and shorter the rentation time.The individual component also had been identified caffeine, followed by acetone, methyl benzoate, penatole and phenantrene.

REFERENCES 1. Levin, D. S. (1997). Successful Operation of HPLC System. Retrieved March 17.2014, from chm510/HPLC.Resources-Main.htm. 2. D.C.Harris, Exploring Chemical Analysis, 3rd ed, W.H. Freeman & Co. 2004 3. Majors, R. E. (2011,March).Agilent In Liquid Chromatography. Retrieved April 15,2014, from http://agilenttechnologies.com .