EXPERIMENT 2

HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC): METHOD
DEVELOPMENT

NAME

: MOHD IQBAL BIN NORAZMAN

STUDENT ID

: 2015840192

GROUP

: AS2452s

PARTNERS NAME

: 1. AKMAL ARSYAD BIN MOHD. RAFFI

2. MUHAMMAD RAHIMI BIN ZAHURI
3. SYARAFUDDIN BIN MOHD SHAMSIBY

DATE OF EXPERIMENT :
DATE OF SUBMISSION

Introduction
High Performance Liquid Chromatography (HPLC) is the most widely used of all of the
analytical separation technique. This type of instrument is suitable for the separation of
nonvolatile compounds. There are four types of liquid chromatography procedure at which the
most common one is the partition chromatography that divides into two categories that which are
normal-phase chromatography and reverse-phase chromatography. For this experiment, reversephase is used at which stationary phase is non polar while the mobile phase is polar. This will
make the most polar component to be eluted first. In the liquid chromatography, it is more
complicated due to the interaction of the components of analyte with both the stationary phase
and the mobile phase. By that, increasing the mobile phase polarity will increases the retention
time of the components. The polarities of various analytes functional groups in increasing order
are as shown below:
Hydrocarbon < Ether < Ester < Ketones < Aldehydes < Amides < Amines < Alcohols
Water is the most polar compounds among the compounds containing any of the preceding
functional groups. In this experiment, 5 compounds which are caffeine, acetone, methyl
benzoate, phenatole, and phenanthrene are determined by varying the mobile phase composition
throughout the analysis. This is known as gradient elution technique. Gradient elution is a
condition at which the composition of the mobile phase is changed during the separation. Two or
more solvents that differ in polarity are employed. After sample introduction, the ratio of these
solvents is programmed to vary either continuously or in steps. The method was intended to
decrease the retention time for the later eluting components so that they can elute faster which
then produces narrower peak thus improves the peak shape. This experiment uses
water/acetonitrile gradient at which more hydrophobic components will elute when the mobile
phase consists of mostly acetonitrile which gives a relatively hydrophobic mobile phase. The
more hydrophilic compounds will elute under conditions of relatively low acetonitrile and high
water. The choice of solvents, addictives, and gradient is dependent of the nature of the
stationary phase and the analyte. A series of tests were done on the analyte and the number of
trial runs may be processed in order to find the method which gives the best separation of the
peaks. The objective of this experiment is to study the development for optimizing the separation
of mixture of five compounds which are standard mixtures of caffeine, acetone, methyl benzoate,

phenatole, and phenanthrene using HPLC by varying the mobile phase composition, identified
the individual components exist in the mixture and optimize the separation between each peaks
by using gradient elution method.

Method
Instrument used: Liquid chromatograph (Agilent G1314A HPLC) equipped diode array detector
(DAD) and 5m RP C18 column and 20L sample loop. Instrument set up by detector
wavelength at 254nm and flow rate was 1.5mL min -1 and mobile phase used was acetonitrile and
water. In the effect of mobile phase on HPLC separation, the instrument was set up with the
mobile phase ratio acetonitrile:water (50:50 v:v) and the sample was injected into the column.
The mobile phase composition was then changed to acetonitrile:water (70:30 v:v) and the sample
was injected into the column. The resolution for both the composition was calculated and
compared. The suitable composition of mobile phase of these compounds was identified. In
identification of components in the mixture, each individual compound was injected into the
column and the component of the mixture was identified using the selected HPLC conditions. In
separating by using gradient elution, the gradient elution method was performed in order to
improve the column efficiency. The suitable ratio of the mobile phase is set up and the sample
was injected into the column. The method was repeated until a suitable ratio of mobile phase was
identified and all of the peaks were separated well with short analysis of time.

Results and Calculations

Resolution (Isocratic Elution)
Mobile phase composition: ACN:H2O (50:50 v:v)
Peak

Calculation

Resolution, Rs

1-2

2( 1.1260.978)
=
0.0551+0.0681

2.40

2-3

2( 3.2511.126)
=
0.0551+0.1068

26.30

3-4

2( 5.5523.251)
=
0.1548+0.1068

17.59

4-5

2( 20.5855.552)
=
0.5191+0.1548

44.61

Table 1.1 Resolution Run 1

Mobile phase composition: ACN:H2O (70:30 v:v)
Peak

Calculation

Resolution, Rs

1-2

2( 1.0570.957)
=
0.0591+0.0520

1.80

2-3

2( 1.7361.057)
=
0.0650+0.0520

11.61

3-4

2(2.3111.736)
=
0.0650+ 0.0837

7.73

4-5

2( 5.1732.311)
=
0.2342+ 0.0837

18.00

Table 2.1 Resolution Run 1

Peak

Calculation

Resolution, Rs

1-2

2( 1.0590.959)
=
0.0496+0.0558

1.90

2-3

2( 1.7401.059)
=
0.0600+0.0496

12.43

3-4

2( 2.3151.740)
=
0.0753+0.0600

8.50

4-5

2( 5.1662.315)
=
0.2192+0.0600

20.42

Table 2.2 Resolution Run 2

Component

Retention Time

Width

Caffeine

0.960

0.0614

Acetone

1.059

0.0513

Methyl Benzoate

1.739

0.0652

Phenatole

2.315

0.0779

Phenanthrene

5.175

0.1307

Table 3.0 Retention time for individual component

Resolution (Gradient Elution)
Peak

Calculation

Resolution, Rs

1-2

2( 1.1280.981)
=
0.0714+0.0540

2.34

2-3

2( 2.4171.128)
=
0.0531+0.0540

24.07

3-4

2( 2.7992.417)
=
0.0550+0.0531

7.07

4-5

2( 4.0162.799)
=
0.1448+ 0.0550

12.18

Table 5.0 Resolution for Gradient Elution

Discussion

Based the experiment, we injected the sample into the injector port that have sample loop. This is
to ensure that the injector port hold the fixed volume which is 20 L. So that, it will not be
overloaded. During injection, the injector port at load mode then we rotate to inject mode for
injector to introduce sample to column. The isocratic elution was done first by use the
composition of mobile phase 50:50 (ACN:H2O). All the peaks were well separated, but the
analysis time was too long which is more than 20 minutes. After that, isocratic elution was done
by using 70:30(ACN:H2O) composition. The analysis time getting shorter which is 5 minutes
but the peaks not well separated. So, in order to get high quality chromatogram, gradient elution
was done. After several times doing gradient elution (try an error), the highest quality of
separation that we got is by using 40:60(ACN:H2O) from 0 minute to 1.20 minutes then change
the composition to 85:15(ACN:H2O) from 1.20 minutes until 4.6 minutes. The resolution was
calculated and recorded in result part. This result actually not a good result but we do so many
times and still got the same result. So, we pick the best and this occurred maybe due to
degradation of the column that effect our result. Then, we continue by injecting individual
standard and identified by comparing with the sample chromatogram. The result that we got is
Caffeine, Acetone, Methyl Benzoate, Phenatole, and Phenanthrene, at 0.960, 1.059, 1.739, 2.315,
and 5.175 respectively. From sample chromatogram, Caffeine, Acetone, Methyl Benzoate,
Phenatole, and Phenanthrene are at 0.957, 1.057, 1.736, 2.311, and 5.173 respectively. Caffeine
elute faster among others which mean Caffeine is polar than others. The chromatogram that we
got have many ghost peak, even we have rinsed the syringe. So, we conclude that, maybe that
ghost peak is from mobile phase signal or the impurities that already present in the injector port.

Conclusion

Gradient elution help in getting the optimize separation. The gradient elution condition that we
got is by using 40:60(ACN:H2O) from 0 minute to 1.20 minutes then change the composition to
85:15(ACN:H2O) from 1.20 minutes until 4.6 minutes. The individual standard has been
compared with sample chromatogram and has been identified.

References

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http://www.chemguide.co.uk/analysis/chromatography/hplc.html
(n.d.). Retrieved from High performance liquid chromatography:
http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatograp
hy/High_performance_liquid_chromatography
Saim, N., Tajuddin, R., & Saaid, M. (2014). Experiment 1 Gas Chromatography(GC):
Optimization of Flow Rate and Column Temperature. In ANALYTICAL SEPERATION
METHODS LABORATORY GUIDE 2nd edition (pp. 1-2). Selangor: UiTM Press.
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