Experiment 2

High Performance Liquid Chromatography(HPLC)

Method Development.
Name: Hamizan Bin Hashim
Matrix number: 2014960875
Group member:
1. Nur Liyana binti Zainal
2. Siti Shazwani binti Salim *
3. Siti Nur Inani binti Baharuddin
Group: AS2453D1
Lecturer name: Prof Madya Zuraidah Abdullah Munir
Date: 19.3.2015

Introduction:
In this experiment, high performance liquid chromatography (HPLC) with UV detector
was used to separate a mixture of five compounds(caffeine, acetone, methyl benzoate,
phenolate and phenanthrene).HPLC mode used was reversed phase as Mobile phase
acetonitrile:water (slightly polar) and stationary phase C-18 column(non-polar). The
mobile phase act carrier of sample solution as it travel into column until reach detector.
As sample(analyte) travel in stationary phase, there was an interaction between with
mobile phase and stationary phase. The rate of interaction may difference due to
polarities of analyte as like dissolve like concept.First eluted compound has strong
interaction with mobile phase compare to last eluted compound.HPLC has been
conducted with two different mode, that was isocratic elution and gradient elution. In
isocratic elution, composition of mobile phase remain constant until end of elution. On
the other hand, mobile phase in gradient elution mode programmed to vary either
continuously or by step. Gradient elution mode used to achieve best separation of mixture
compound with short elution time. Therefore, it work efficiently.

Figure 1: schematic diagram of High Performance Liquid Chromatography (HPLC).

Experimental:
a) Instrumental set-up
detector wavelength: 254nm
flow rate: 1.5mL min-1 .
mobile phase: acetonitrile:water
b) Effect of mobile phase on HPLC separation.
1. Mobile phase ration has been set (acetonitrile:water;50:50).
2. Sample was injected 20L into injected port and wait until the elution done.
3. The mobile phase composition was changed into (acetonitrile:water;70:30) and sample
has been injected.
4. The suitable mobile phase composition has been determined.
c) Identification of compound in the mixture.
1. The individual standard has been injected to identify the mixture component using
suitable HPLC condition.
d) Separation using gradient elution.
1. A gradient elution was performed in order to improve the efficiency of the column.
2. 2. 80:20 mobile composition was set on first 3minutes, then 75:25 mobile composition
was set for the next 4 minutes until end elution time.
3. The mixture was injected at this optimum condition.

Result and Discussion:

Mobile
phase
composition
Peaks

50:50 (ACN: Water)

Width (min)

Retention time(tR)
(min)
0.976

70:30(ACN:Water)
Width (min)

0.0568

Retention time(tR)
(min)
0.964

1.124

0.0613

1.0.65

0.0513

3.336

0.1225

1.758

0.0612

5.759

0.1758

2.350

0.0712

21.596

0.5026

5.217

0.1478

Table 1: Table of the data of the sample at isocratic elution condition

Sample calculation resolutions of the experiment:
RS = 2(tR2- tR1) / (W1+W2)
For example of the calculations for peaks 2 and 1 from table 2
= 2(1.124-0.976)/ (0.0568 + 0.0613)
= 2.51
Mobile
phase
composition

Resolution of 50:50 (ACN:

Water)

Resolution of
70:30(ACN:Water)

1-2

2.51

2.07

2-3

24.07

12.32

3-4

16.24

8.94

4-5

46.68

26.13

Peaks

Table 2: Resolution of 50:50 and 70:30 mobile phase composition.

0.0463

Standard mixture
Retention time (min)
Width (min)
Caffeine
0.962
0.0468
Acetone
1.068
0.0459
Methyl benzoate
1.763
0.0557
Phenatole
2.350
0.0718
Phenanthrene
5.238
0.1188
Table 3: Data of the standard mixture in the optimum conditions ACN: Water (70:30)

Peaks
Retention time (min)
Width (min)
1
0.972
0.0656
2
1.274
0.1080
3
1.453
0.0552
4
1.771
0.0599
5
3.332
0.1266
Table 4: Table for data of the sample in the gradient elution systems (80:20) ACN:Water
for three minutes and (75:25) ACN:Water for above three minutes.
Peaks

Resolution, Rs

1-2

3.86

2-3

0.52

3-4

5.52

4-5

16.74

Table 5: Resolution data in gradient elution.

Discussion:
In the HPLC experiment, composition of mobile phase is main factor that affect the
retention time of the analyte to be eluted through column. Strong eluent strength will
decrease the retention time of analytes. Strong eluent means, the composition of organic
used is higher than the water. As the data present, 70:30(ACN:H2O) eluted more faster
compare to 50:50(ACN:H2O) ratio. This show 70:30(ACN:H2O) ratio has strong eluent
strength. The separation quality is depanding on resolution(Rs) calculated between peaks.
If Rs value more than 1.5 it will produced good peak with baseline separation. Based on
results, caffeine and acetone has low Rs value but it is still separated.
Next, gradient elution was use to increase the efficiency of HPLC. Gradient elution
normally used in wide range polarities compound mixture. In this experiment, gradient
elution has reduce the elution time and produced good separation. It show, by using
gradient elution mode, separation become better and analysis time become shorter.

Conclusion:
At the end of the experiment, the suitable mobile phase composition used in isocratic
mode was determined that is 70:30(ACN:H20). Furthermore, efficient analysis has been
proceed through gradient elution mode 80:20(ACN:H20) at 0 - 3 minutes,
75:25(ACN:H20) 3 minutes to end of elution. Caffein and acetone become more separated
and Phenanthrene elute more faster, at 3.332 minute retention time.

Reference:
1.http://www.waters.com/waters/en_MY/How-Does-High-Performance-LiquidChromatography-Work%3F/nav.htm?cid=10049055&locale=en_MY
2. http://en.wikipedia.org/wiki/Reversed-phase_chromatography
3. http://www.phy.ohiou.edu/~small/c325/appendix3.pdf

4. Norashikin s., Ruziyati., Mardiana S. (2012), Analytical Separation Methods

Laboratory Guide (2nd edition).

Appendix: