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HPLC is most widely used in all of the analytical separation method. It is suitable for
separating non-volatile species or thermally fragile ones. Partition chromatography is the
most widely used of all the four types of liquid chromatography procedure. It is divide into
two; reversed phase and normal phase. For this analysis, we used reversed phase
chromatography. In reversed phase chromatography, the stationary phase is non-polar and
mobile phase is relatively polar. The most polar component will elute first, and increasing the
mobile phase polarity increases the elution time. Method development tend to be more
complex in liquid chromatography because the sample components interact with both the
stationary phase and mobile phase.
Successful chromatography with interactive mobile phase requires a proper balance
of intermolecular forces among the three active participants in the separation process; the
solute, the stationary phase and the mobile phase. These intermolecular forces are describe
quantitatively in term of the relative polarity of three reactants. The polarities of various
analytes functional groups in increasing order are; hydrocarbon <ether <ester <ketone
<aldehyde <amide <amine <alcohol. Water is more polar compound than compounds
containing any of the preceding functional groups. Often in choosing a column for a partition
chromatography separation, the polarity of the stationary phase is matched roughly with that
of the analytes; a mobile phase of considerably different polarity is then used for elution.
This procedure is generally more successful than one in which the polarities of the
solute and mobile phase are matched but different from that of the stationary phase. Here, the
stationary phase often cannot compete successfully for the sample components; the retention
times become too short for practical application. At the other extreme, of course, is the
situation where the polarity of the solute and stationary phase are too much alike and totally
different from that of the mobile phase. Here, the retention time becomes inordinately long.
The type and composition of the mobile phase is one of the variables influencing the
separation. Therefore, the mobile phase can be altered in order to manipulate the interactions
of the sample components and the stationary phase. In isocratic elution, components are
eluted using constant mobile phase composition. In gradient elution, different components are
eluted by changing the mobile phase composition as the separation proceeds. By increasing
the strength of the mobile phase will subsequently results in elution of retained components.
Each components elute from the column as a narrow band (or peak), detected and
displayed as a chromatogram on a computer screen. Detection of the eluting components can
be either selective or universal, depending on the detector used. To collect, store and analyse
the chromatographic data, computers, integrators, and other data processing equipment are
frequently used. The objective of this experiment is to optimize a separation of caffeine,
methyl benzoate, phenatole and phenanthrene using HPLC by varying the mobile phase
composition.
CHEMICALS
1) Individual standard (caffeine, acetone, methyl benzoate, phenatole, phenanthrene)
2) Standard mixture ( consist of all 5 individual standard)
3) Mobile phase (acetonitrile (ACN), deionized water)
PROCEDURE
A) ISOCRATIC ELUTION
ACN H2O
50 50
70 30
Standard mixture was injected 1x for mobile phase of ratio 50:50 and 2x for mobile
phase of ratio 70:30. The optimum ration of mobile phase was determined by calculating the
Resolution (Rs). The best ratio was chosen and then 1x injection was applied for each
individual standard for identification.
B) GRADIENT ELUTION
Our own method was developed based on our finding and observation during the
isocratic elution method. Our own method was set up by varying more than one mobile phase
composition where it should be two or more different polarity of solvents employed in the
same injection. 2x injection were performed on the same succeeded method once the own
method was set up successfully. The result was discussed and the ways to improve the
efficiency of the column were observed.
ISOCRATIC ELUTION
The retention time of compound in standard mixture at optimized HPLC condition (70:30) of
(ACN:H2O)
Retention time in Retention time in Retention time in
Standard compound standard (min) mixture (min) of mixture (min) of
first injection second injection
Caffeine 1.157 1.155 1.155
Methyl benzoate 2.144 2.140 2.136
Phenatole 2.884 2.876 2.869
Phenantherene 6.381 6.398 6.374
Acetone 1.295 1.284 1.291
DISCUSSION
During this experiment, High Performance Liquid Chromatography (HPLC) Agilent
G1314A equipped with UV detector, 5mm reverse phase C18 column and 10µl sample loop
was used. At flow rate 1.5 ml/min and detector wavelength 254 nm, the mobile phase ratio
(v/v) was set at 50:50 at the beginning in order to analyse and observe the effect of mobile
phase on LC separation. After all the standard samples which are phenatole, methyl benzoate,
caffeine, phenantherene and acetone are injected, the ratio was changed to 70:30 respectively
on the same mobile phase. By the actual procedure, from this experiment we need to identify
the components contained in the standard mixture by using the optimized LC conditions
getting from the above ratio of the mobile phase as well as we should perform a gradient
elution separation to improve the efficiency of the column.
Composition of the mobile phase is strongly affected by the retention time of the
analytes to be eluted throughout the column. Since it is a reversed phase partition liquid
chromatography, the stationary phase is a polar while the mobile phase liquid is non-polar.
Strong eluent strength tends to decrease the retention time of the analytes. Strong eluent
strength is where the composition of the organic solvent used in the ratio is higher than the
water. In comparison between 50:50 (ACN:H2O) and 70:30 (ACN:H2O), 70:30 ratio has
higher eluent strength so that the analytes come out earlier than ratio of 50:50. The separation
quality is depending on the resolution between peaks that ideally 1.5. Resolution that is
higher than 1.5 will produce good separation between peaks but needs longer retention time
for the last analyte to be eluted. Low resolution than the ideal value of 1.5 produces
inadequate separation between peaks or in other words, the peaks is overlap between them.
Gradient elution mode is used when there are wide polarities of compounds to be
separated. Gradient elution changes the polarity of mobile phase composition throughout
analysis either continuously or in step during the separation. Gradient elution mode will
decrease the retention time for the analytes to be eluted throughout the column. The gradient
elution will give better separation, high efficiency and good resolution in separating the
mixture with wide polarities. Based on the result obtained, there is an overlapping between
peak 1 and 2. To give a better separation, we have tried various ratio of the same mobile
phase. 30:70 at 0 minute, 75:25 at 1.50 minutes and 85:15 at 2.50 minutes were chosen as it
gives a better separation than before even though the two peaks are not totally separated.
The qualitative analysis was done to identify the components in the mixture by
comparing the peaks in the mixture with the peaks of the standard compound. For gradient
elution, we have choose certain ratio to separate peak 1 and 2. 30:70 at 0 minute, 75:25 at
1.50 minutes and 85:15 at 2.50 minutes were chosen as it gives a better separation. Caffeine
is said to be eluted first and indicates the first peak followed by methyl benzoate, phenatole
and phenanthrene.
CONCLUSION
The optimized mobile phase composition for the separation of the mixture is
70:30(ACN:H2O). Higher composition of organic solvent will increase the solvent strength
that will shorten the analysis time. The first peak is corresponds to caffeine, secondly is
methyl benzoate, phenatole peak and lastly is corresponds to phenanthrene peak.
REFERENCES