CONTENT: PAGE

INTRODUCTION 2

METHODOLOGY 3

RESULTS AND DISCUSSION 5

CONCLUSION 9

REFERENCES 9

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EXPERIMENT 6: High Performance Liquid Chromatography (HPLC):Method Development

1.0 INTRODUCTION

In recent years the technology used in chromatographic analysis has greatly improved.
Advances in this area have led to the common use of High Performance Liquid
Chromatography, a laboratory technique that allows for every efficient separation of small
amounts of the components of a mixture. The technique has essentially the same operational
basis as liquid chromatography, but HPLC allows for far better separation of the components
of a mixture. The column in an HPLC instrument contains tiny particles of only about 5 µm
diameter, ensuring a very large surface area to which molecules may adsorb. These particles
comprise the stationary phase of the chromatographic system. Because these tiny particles are
so tightly packed, the mobile phase solvent must be forced through the column under very high
pressure. A detector and a computer are connected to the HPLC instrument in order to signal
when eluents are coming off the column and fractions should be collected.

Identification of sample is carried out by comparison of their retention times. A known amount
of a known compound (a standard) is injected, the retention time is recorded and the width of
the peak can be calculated. A sample containing the same analyte is then injected under the
same experimental parameters. If the analyte is the same compound as the standard, then they
should give identical retention times. The ratio of peak areas should give the amounts of the
components present.

Reverse phase partition chromatography in HPLC employs a polar mobile phase and a non-
polar stationary phase. This is the most frequently used form of HPLC. Other modes of liquid
chromatography include normal phase partition, a non-polar mobile phase and polar stationary
phase.

The column used in this experiment consists of a layer of an alkane C-18 chemically bonded
to the surface of very small silica particles. The alkane stationary phase is non-polar, the silica
is the inert support material with high surface area and the mobile phase is a polar solvent
(water/acetonitrile)

The objective of the experiment is to optimise the separation of a mixture of five compounds
(caffeine, acetone, methyl benzoate, phenatole and phenanthrene) using HPLC by varying the
mobile phase composition.

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2.0 METHODOLOGY

a. Preparation of Solvents

a-i. Quality

Before experiment is carried out, solvent has passed through several process. This is
important because it can save vast amount of time spent troubleshooting spurious peaks and
baseline noise. All reagent and solvent must be at highest quality, where the impurities are
absent in it. Modern water purification instruments use this mechanism to produce water of
suitable quality in high volumes.

a-ii. Buffers

The buffers should be prepared freshly, to ensure that the pH of the buffer is not affected
by prolonged storage and present of microbial growth. The changes in pH and microbial growth
can affect chromatography

a-iii. Filtration

HPLC solvents should be filtered before use. This is to ensure any particulates are
removed that may cause blockages. After the filtration, the solvent is then stored in covered
reservoir, to prevent contamination with dust. By undergoing the filtration process, it will
benefit the HPLC system. Pump plungers, seals and check valve will perform better, and
lifetimes will be maximized.

a-iv. Degassing

Before pumping the mobile phase into the system, the solvent has been undergone
degassing process, to remove all dissolved gasses. Dissolved gas can be removed from solution
either bubbling with helium, sonication, or vacuum filtration. If the solvent is not degassed, the
air bubble can form in HPLC, which can cause system instability, and produce ghost peaks and
shorten the lifetime of the machine.

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b. Purging the System

The system has been purged before use. The purpose of the process is to remove any
air bubbles trapped in the system and to refresh the mobile phases that are inside the system.
In other words, it simply flushed all remaining solvent through the lines from a previous
analysis or wash and replaced with the new mobile phase.

c. Experimental

The samples were prepared by laboratory assistant, which are individual standards
(caffeine, acetone, methyl benzoate, phenatole, phenanthrene), and standard mixture (consist
of all 5 individual standards). Separation of each mixture in standard mixture was optimized
by varying the mobile phase composition by two methods:

c-i. Isocratic Elution

The standard mixture was automatically injected in HPLC system with ratio of 70:30
and 50:50 twice, for each ratio. The optimum ratio of mobile phase was identified by
calculating the Resolution (Rs). As the experiment was carried out, it shows that isocratic
elution has the optimum ratio. Thus, each individual standard was carried out by using isocratic
elution’s method.

c-ii. Gradient Elution

This technique was mostly chosen when sample contains components of wide range of
polarities. The gradient elution offers most complete separation of peaks in shorter time period
without loss of resolution in earlier peaks.

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3.0 RESULTS AND DISCUSSION

To find Rs,

2[𝑇𝑅2−𝑇𝑅1]
Rs= 𝑊𝑎+𝑊𝑏

RESULTS:

a. STANDARD MIXTURE

a-i. Isocratic Elution (ratio70:30)

Compound Retention Width (min) Area (%) Resolution(Rs)

Time (TR)
1 1.712 0.1528 22.3004 1.373(compound 1 and 2)
2 1.923 0.1546 12.7850 8.209(compound 2 and 3)
3 3.186 0.1531 7.9499 6.925(compound 3 and 4)
4 4.285 0.1643 4.8983 22.368(compound 4 and 5)
5 9.519 0.3037 51.6816 -

a-ii. Isocratic Elution (Ratio 50:50)

Compound Retention Width (min) Area (%) Resolution(Rs)

Time (TR)
1 1.172 0.1296 20.0667 1.509(compound 1 and 2)
2 1.370 0.1329 10.3852 17.007(compound 2 and 3)
3 3.984 0.1745 7.1307 14.857(compound 3 and 4)
4 6.887 0.2163 4.0042 44.355(compound 4 and 5)
5 25.638 0.6292 57.8741 -

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a-iii. Gradient Elution

Compound Retention Width(min) Area(%) Resolution(Rs)

Time(TR)
1st 2nd 1st 2nd 1st 2nd 1st 2nd
1 1.173 1.173 0.1295 0.1289 27.3015 24.9488 1.533 1.523
2 1.371 1.372 0.1289 0.1324 14.1143 12.8562 16.785 16.99
3 3.778 3.846 0.1579 0.1588 9.5133 8.7506 13.382 14.067
4 5.899 6.191 0.1591 0.1746 5.3227 4.8867 35.852 46.457
5 11.413 14.393 0.1485 0.1785 42.374 47.3587 - -

b. INDIVIDUAL STANDARD

b-i. Isocratic Elution (70:30)

(Each of the samples has been run individually using isocratic elution method)

Compound Retention Time (TR) Width (min) Area (%)

Caffeine 1.146 0.1415 100.0
Acetone 1.234 0.1643 100.0
Methyl Benzoate 1.830 0.1351 100.0
Phenatole 2.346 0.1410 100.0
Phenanthrene 4.812 0.2815 100.0

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DISCUSSION:

The purpose of the experiment is to optimize the separation of a mixture of five

compounds, which are caffeine, acetone, methyl benzoate, phenatole, and phenanthrene. Using
HPLC by varying the mobile phase composition.

HPLC is one of the most powerful instruments for analytical chemistry, where it can
separate, identify and quantitate the compound that are present in any sample that can be
dissolve in liquid. There are lots of application for the instruments such as pharmaceuticals,
food, cosmetics, environmental matrices, forensic samples and industrial chemicals. The basic
component of HPLC are solvents (mobile phase), degassing system, pump, injector, column
and detector.

To optimize the separation of the mixture, it can be either by changing the mobile phase
ratio or composition (to get an optimum separation), adjusting the temperature or using the best
detector, which is diode array detector (DAD). The advantage of this detector is that it can set
up different wavelength. This detector was applied in the HPLC which has been used during
the experiment was carried out.

There are two kinds of HPLC, which are Normal phase HPLC or Reverse phase HPLC.
Normal phase is effective for separation of structural isomers. Mobile phase is a polar while
the stationary phase is a non-polar. Revers phase can be a wide range of application. It is
effective for separation of homologs. Mobile phase is a non-polar while the stationary phase is
a polar. In reverse phase, the retention time of HPLC will increase as it decreased in polarity
of particular species. The compounds should be eluted at shorter time, which is better. HPLC
that used during experiment was Reverse Phase HPLC.

Through the experiment that has been conducted, there are two methods that was
used, which are Isocratic Elution and Gradient Elution method.

Isocratic elution was used when the compound has either one substance or has narrow
range of polarities. If the composition of mobile phase is constant throughout the experiment,
it is said to be an isocratic elution method.

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 Gradient elution method is a method that varying mobile phase composition throughout
the experiment, gradually and slowly. Gradient elution method was used to maintaining its
peak resolution and it is used to compound that has wide range of polarities. The gradient
elution offers most complete separation of peaks in shorter time period without loss of
resolution in earlier peaks.

For standard mixtures, it was carried out by using both method, which are isocratic
elution and gradient elution. Isocratic elution was carried out using two different ratios, which
are 70:30 and 50:50. Based on the observation of the peaks, the resolution each of the
separations are above 1. As Rs>1, then it shows a good separation. Therefore, gradient elution
is the best method for standard mixtures because of the Rs>1.5, which is an optimum
separation.

For individual standards, it was carried out by using isocratic elution with the ratio of
70:30. This is because it is the optimum ratio for separation, where it only takes 10minutes to
be well separated compared to 50:50 that consumed up to 25minutes of separations. The mobile
phase that has been used was Acetonitrile and Distilled water. Each of the compound has been
injected twice, to get an accurate result. Both ratio has its own pros and cons, where 70:30 has
smaller Rs compare to 50:50 but with shorter time analysis, while 50:50 has bigger Rs, that is
above 1.5 but with a longer analysis time.

As we compare the result of standard mixture and individual mixture based on their
retention times, it shows that caffeine has the first peak, second peak is acetone, third peak is
methyl benzoate, fourth peak is phenatole and the last peak is phenanthrene. In other words,
compound 1 represents caffeine, compound 2 represents acetone, compound 3 represents
methyl benzoate, compound 4 represents phenatole, compound 5 represents phenanthrene.

The precautions during the experiment are the mobile phase should undergoes several
processes, to prevent any dissolve gas or contaminant presence in the mobile phase, which can
clog the column of the HPLC, produce ghost peaks, thus shorten the system’s lifetime. The
system must be purged before using it. Glove and lab coat should be worn, to prevent any
contact with unwanted chemicals in the laboratory.

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4.0 CONCLUSION

The conclusion of the experiment is to use gradient elution method for the compound
that has wide range of polarities, for example standard mixture. This is because for the peaks
to be well separated. Plus the Rs of standard mixture for gradient elution method is above
1.5,which is an optimum separation of the peaks.

For individual standard, isocratic elution with ratio of 70:30 (ACN:H2O) has an
optimum separation, with shorter time of analysis compare to ratio of 50:50, where it takes up
to 25minutes of separation. Rs for ratio of 70:30 is above 1,which is good separation while
50:50 is above 1.5, which is an optimum separation and take longer time to separate.

5.0 REFERENCES

(Ghiotti & Boccuzzi, 1987; Ishii, Tsuboi, Sakane, Yamashita, & Breedlove, 2009; Kalmus &
Harper, 1915; Kumar, Kumar, & Dass, 2013; Nitschké, Ertl, & Küppers, 1981)

Ghiotti, G., & Boccuzzi, F. (1987). Chemical and Physical Properties of Copper-Based
Catalysts for CO Shift Reaction and Methanol Synthesis. Catalysis Reviews, 29(2–3),
151–182. https://doi.org/10.1080/01614948708078069

Ishii, T., Tsuboi, S., Sakane, G., Yamashita, M., & Breedlove, B. K. (2009). Universal
spectrochemical series of six-coordinate octahedral metal complexes for modifying the
ligand field splitting. Dalton Trans., 0(4), 680–687. https://doi.org/10.1039/B810590A

Kalmus, H. T., & Harper, C. (1915). Physical Properties of the Metal Cobalt. Journal of
Industrial & Engineering Chemistry, 7(1), 6–17. https://doi.org/10.1021/ie50073a004

Kumar, D., Kumar, A., & Dass, D. (2013). Syntheses and Characterization of the Coordination
Compounds of N-(2-hydroxymethylphenyl)-C-(3′-carboxy-2′-
hydroxyphenyl)thiazolidin-4-one. International Journal of Inorganic Chemistry, 2013, 1–
6. https://doi.org/10.1155/2013/524179

Nitschké, F., Ertl, G., & Küppers, J. (1981). Coordination chemistry of metal surfaces:
Chemisorption of PF 3. The Journal of Chemical Physics, 74(10), 5911–5921.
https://doi.org/10.1063/1.440909

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