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 <ether <ester
<ketones <aldehyde <amide <amines <alcohols. Water is more polar compounds
than compounds containing any of the functional group.
When in choosing a column partition chromatography separation, the
polarity of the stationary phase is matched roughly with analytes, while the mobile
phase is considerably in different polarity is then used for elution. The stationary

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, , can also be manipulated to improve separations.

When 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 20L.

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