Experiment 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): METHOD

DEVELOPMENT
Date of Experiment: 20/4/2016
Group: AS245 3D1
Lecturer:

DR MARDIANA SAAID

PROF ZURAIDAH ABDULLAH MUNIR

Name: AMAR SAFWAN BIN MOHD ALI HANAPIAH

Student ID: 2015272222
Group members:

SALMA IZATI BINTI SINAR MASHURI (2015839778)

NURUL SHAZREENA BINTI ZULKAFLY (2015258246)

Experiment 2: High Performance Liquid Chromatography (HPLC): Method Development

Objective
To optimize a separation of a mixture of 5 compounds using HPLC by varying the mobile phase
composition

Introduction
High performance liquid chromatography is the most widely used of all of the analytical
separation technique. Its suitable for separating nonvolatile species or thermally fragile ones.
Partition chromatography is the most widely used of all the four types of liquid chromatography
procedure.

It

divides

into

two;

normal-phase

chromatography

and

reverse-phase

chromatography.
For this analysis we used reversed phase chromatography. In reverse-phase chromatography, the
stationary phase is non-polar and the mobile phase is relatively polar. The most polar component
will elute first, and increasing the mobile phase polarity increase the elution time. Method
development tends to be more complex in liquid chromatography because the sample
components interact with both the stationary phase and the 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 mobile phase, and
the stationary phase. These intermolecular forces are described qualitatively in term of the
relative polarity of three reactants. The polarities of various analytes functional groups in
increasing order are: hydrocarbon <ether <ester < ketones < aldehyde < amides < amines <
alcohols. Water is more polar compounds than compounds containing any of the preceding
functional groups.
Often in choosing a column for a partition chromatographic 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; retention time becomes 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 times becomes
inordinately long.

Procedure
1. The instrument wavelength is set up to 245 nm. The flow rate is set 1.5 mL min -1. While,
the mobile phase is used acetonitrile and water.
2. The instrument is set to use a mobile phase ratio acetonitrile: water (50:50 v: v) and the
sample is injected. Then, the mobile phase composition is changed to 70:30. And the best
composition of mobile phase is chosen.
3. Each component is injected individually to identify the component mixture using the
selected HPLC conditions.
4. Based on the separation, a gradient elution separation is performed to improve the
efficiency of the column.

Result

Effect of the variation of composition of mobile phase on resolution (isocratic elution):

Composition of
mobile

phase Injection

(ACN:H2O)
50:50
70:30

1
1
2

Retention time of
peak 1 and 2 (min)
1.000,1.150
0.960,1.503
0.956,1.049

Base peak width

of peak 1 and 2 Resolution
(min)
0.0933,0.0891
0.0840,0.0755
0.0876,0.0744

1.64
1.17
1.15

Effect of the gradient elution program: (ACN: H2O)

Composition
mobile
(ACN:H2O)

of Base

peak

phase width of peak 1

and 2 (min)

0.00min 70:30

Retention
time of peak
1

and

Resolution

(min)
0.958,1.103

0-2.5min 75:25
0.0660,0.0510

2.48

2.5-5min 80:20

0 min 70:30
0-1 min 80:20

Ra
t Rbt

Rs =
2

1.001,1.146
0.0641,0.0528

2.48

Average
Resolution
1.64
1.16

The retention time of compound in standard mixture at optimized HPLC condition (70/30) of
(ACN:H2O):
Retention time
Standard compound

in
(min)

Caffeine
Methyl benzoate
Phenatole
Phenantherene
Acetone

0.959
1.728
2.297
5.079
1.049

standard

Retention time Retention

in

mixture time

(min)
injection
0.960
1.731
2.302
5.070
1.053

in

1st mixture (min)

2nd injection
0.956
1.734
2.314
5.144
1.049

Discussion
During this experiment, a High Performance Liquid Chromatography (HPLC) Agilent
G1314A equipped with UV detector, 5 mm Reverse Phase C18 column and 10 l sample loop
was used. At flow rate 1.5 ml / min and detector wavelength at 254 nm, the mobile phase ratio
(v/v) was set at 50% water and 50% acetonitrile at the beginning in order to analyze and observe
the effect of mobile phase on LC separation. After all the standard samples which are phenatole,
methylbenzoate, caffeine, phenantherene, and acetone were injected, the ratio was changed to
60%:40% and 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. 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, increasing amounts of water are added to acetonitrile to create a continuous
gradient.
But what was happened is all the peaks from the injection process to the sample loop
were not separated well. In a reversed-phase separation, eluent strength decreases as the solvent
becomes more polar. Acetonitrile has high eluent strength, and all compounds are eluted rapidly.
All the peaks are observed overlapping. From the result of chromatogram and area calculation,
we can see that the Response Factor for all the standards injected is almost same. It was so
difficult to determine the resolution of the peaks since the peaks got overlap because the mixture
is in high concentration. As we know, the quantitative analysis in separation method depends
upon direct relationship between the area under a peak or peak height in the chromatogram and
the amount of the compound corresponding to that peak in the analyzed sample. Therefore, each
peak should be totally resolved from any neighboring peaks. A co-elution or other anomalies
such as tailing or fronting will distort or obscure the beginning and ending points of the peak.

There are some factors that contribute to all the problems stated above. The sample must
be degassing properly. Sometime when the pressure was not consistent, there must be any air
bubble in the mobile phase that fluctuant the instrument. Therefore the instrument should be
purge to let the pressure stable. Mobile phase that is too cooled also effect the pressure. The
254nm is the most suitable wavelength because give us very nice and sharp peak. The flow rate
or velocity of the mobile phase is very essential in HPLC (according to the Van Deemter
Equation).

To identify the components in the mixture by comparing the peaks in mixture with the
peaks of standard compound the qualitive analysis was done in the experiment. There is caffeine
indicated for 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).
The higher composition of organic solvent the more increase the solvent strength that will
shorten the analysis time.
The first peak is corresponds to caffeine, secondly isacetone. Thirdly, methyl benzoate, next is
phenatole peak and lastly is corresponds to phenanthrene peak.

Reference
1. High Performance Liquid Chromatography, Liquid Chromatography. Linde Group. 2016.
Retrieved on
http://hiq.lindegas.com/en/analytical_methods/liquid_chromatography/high_performance_liquid_chrom
atography.html
2. Liquid Chromatography. Chapter 28. Principles of Instrumental Analysis. 6 th edition.
Holler, Skoog & Crouch. 2007. Brooks/Cole Cengage Learning.
3. Skoog, D.A., West, D.M, Holler, F.J. 7th Edition, Fundamental of Analytical Chemistry