EXPERIMENT 2

High Performance Liquid

Chromatography (HPLC): Method
Development

NAME: DANG HUMAIRAH BT ANUAR

MATRIC NO: 2020989185

CLASS: RAS2453B

DATE OF SUBMISSION: 22/1/2021

LECTURER’S NAME: DR SHARIZAL HASAN

OBJECTIVE

To separate mixture of 5 compounds using High Performance Liquid Chromatography

(HPLC) by differed their mobile phase composition.

INTRODUCTION

High Performance Liquid Chromatography (HPLC) is one of the analytical techniques

used to separate and determine each of the component in a mixtureThis chromatography's
mobile phase is liquid, while the stationary phase is liquid, too. The separation is based on
variations in analyte polarity. The first elute analyte is the analyte that less interacts with the
stationary phase and the late elute analyte is the one that most interacts with the stationary
phase. In this experiment, reversed phase chromatography was used. The reverse phase is
where, while the stationary phase is nonpolar, the mobile phase is polar. The relationship
between the analyte and the stationary stage will be influenced by adjusting the polarity of
the mobile phase shift. The effectiveness of compound separation would also be affected.
Two methods may be used to vary the composition of the mobile phase. First, by isocratic
elution, in which the composition of the mobile process during the study remains the same.
Second, with the use of gradient elution. During separation, the composition of the mobile
process varies either continuously or in steps to distinguish analytes of different polarity
ranges.
REAGENTS AND SOLUTIONS
HPLC grade acetonitrile
Deionised water
Standard mixture of caffeine, acetone, ethyl benzoate, phenatole and phenanthrene (around
100 ppm)

INSTRUMENT
Liquid chromatograph (Agilent G1314A HPLC) equipped with diode array detector (DAD),
RPC 18 column and 2μL sample loop.

ANALYTICAL PROCEDURE
1. The instrument set up as below:
Detector wavelength: 254 nm
Flow rate: 1.5 mL/min
Mobile phase: acetonitrile:water

2. Effect of mobile phase on HPLC separation.

The mobile phase using polar solvent which acetonitrile and deionized water with the
ratio 50:50. The standard mixture was injected. Then, the composition of mobile
phase was changed to ratio 70:30.

3. Identification of components in the mixture

To investigate the component in the mixture using the selected high performance
liquid chromatography or HPLC specification, each compound was injected
individually in automatic injection.

4. Separation using gradient elution

Based on the separation, gradient elution separation was performed to improve the
efficiency of the column by enhance the ratio of the column.
RESULT
1. The consequences of the change composition of mobile phase on resolution by
isocratic elution:

2(t R 2−t R 1)
Rs = W 2 +W 2

Composition Peak no. Retention Base width of Resolution Average

of mobile time (min) peak (min) resolution
phase (ACN:
H 2O)
50:50 2-1 1.356, 1.135 0.1319, 0.1674 1.4768 12.8583
3-2 3.964, 1.356 0.2201, 0.1319 14.8182
4-3 6.887, 3.964 0.2767, 0.2210 11.7673
5-4 26.141, 6.887 1.3710, 0.2767 23.3708
70:30 2-1 1.234, 1.169 0.1069, 0.1435 0.5192 6.1078
3-2 2.089, 1.234 0.1247, 0.1069 7.3834
4-3 2.800, 2.089 0.1303, 0.1247 5.5765
5-4 6.377, 2.800 0.5229, 0.1303 10.9522

Average resolution of isocratic elution

12.8583+ 6.1078
= 2
= 9.4831
 2. The impact of the composition of the mobile phase by gradient solution.

Composition Peak no. Retention Base width of Resolution Average

of mobile time (min) peak (min) resolution
phase (ACN:
H 2O)
50:50 (0 min 2-1 1.278, 1.138 0.0991, 0.1540 1.1063 6.0542
– 1.8 min)
3-2 2,582, 1,278 0.1934, 0.0991 8.9162
70:30 (after
1.8 min – 8.0 4-3 3.487, 2.582 0.1521, 0.1934 11.7673
min)
5-4 5.403, 3.487 0.2758, 0.1934 23.3708
50:50 (0 min 2-1 1.278, 1.136 0.1013, 0.1598 1.0877 5.7411
– 1.8 min)
3-2 2.582, 1.278 0.2222, 0.1013 8.0618
70:30 (after
1.8 min – 3.0 4-3 3.489, 2.582 0.1507, 0.2222 4.8646
min)
5-4 5.403, 3.489 0.2770, 0.1507 8.9502
85:15 (3.0
min until 8.0
min)

Average resolution of gradient elution

6.0542+ 5.7411
=
2
= 5.8977

3. The retention time of the components that modify HPLC mode (70:30) ration of
(ACN: H 2 O )

Standard mixture of the Retention time on

components individual standard (min)
Caffeine 1.121
Acetone 1.332
Methyl benzoate 2.095
Phenatole 2.812
Phenanthrene 6.353
DISCUSSION

High performance liquid chromatography (HPLC) is process of chromatography used

to separate sample mixture into small compounds with mobile phase and stationary phase are
in the form of liquid. Method development HPLC is a process used to show the stability of
the sample for the future by adjusting the column temperature, volume of injection, sample
flow rate, and others. In this experiment, the analyte's polarity is the one that is being
changed. Analyte with low contact between the stationary phase will first elute and analyte
who stays most in the stationary phase will later elute. Analyte polarity is directly
proportional to the retention time. As the polarity of analytes increases, the retention time will
also increase. A narrow and sharp peak would have a lower retention time. polarity of the
analyte as determined by the composition of the mobile phase. The reverse process is used
depending on the experiment performed. Liquid chromatography is a reverse phase
chromatography method where the stationary phase is non-polar and the polar phase is
mobile. Reverse phase chromatography is used to distinguish molecules based on
hydrophobic interactions between the mobile phase solvent molecules and the stationary
phase-attached ligands.

In this experiment, gradient elution mode is used to separate the mixture with large
polarity and adjust the polarity of the mobile phase composition continuously or in step
during the study. The eluent intensity will increase during the separation by adjusting the
composition of the mobile step and the analysis period will also decrease. It will create more
effective and good resolution of the separation. High eluent power is where the amount of
organic solvent used is greater than the amount of water used in the ratio. For the experiment,
the composition that have strong eluent is 70:30 (ACN: H 2 O ) compared to other composition
50:50 (ACN: H 2 O ). In composition 70:30, the analytes come out faster than the one with
composition 50:50. The peak has better separation when it has ideal resolution 1.5. The peak
that has resolution more than 1.5 will has better separation but the space between the peaks
much longer. Resolution less than 1.5 produce inadequate separation between peak or the
peak has already overlapped to each other. Both average resolutions of isocratic elution and
gradient elution is more than 1.5 which are 9.4831 and 5.8977.
 So, it concludes that the peaks have greater separation for the analyte to be eluted, but
have longer retention time. But the average gradient elution resolution is lower than the
isocratic solution. The gradient elution resolution is probably much greater than isocratic
elution. The qualitative analysis was performed by comparing the peaks in the mixture with
the peaks of the regular compound to classify the components in the mixture. The first peak is
the one that elutes first with shorter analyzing time. Thus, the first peak is caffeine, followed
by other compounds such as acetone, methyl benzoate, phenatole, and phenanthrene

CONCLUSION
High performance liquid chromatography (HPLC) is chromatography technique that
used pressure instead of gravity to separate mixture into small components but it has to
develop method to prove the stability of mixture for further used. In this experiment, polarity
of analyte has been changed. Polarity indicates to composition of mixture. Based on the
result, the most suitable composition of mixture is 70:30 (ACN: H 2 O ). When the polarity of
organic solvent increases the strength eluent also increase and the retention time will
decrease. The first peak is corresponding to caffeine followed by other compound which are
acetone, methyl benzoate, phenatole and phenanthrene peak.
REFERENCES
Analytical Laboratories Applications GC with Electron Capture Detector (GC-ECD). (n.d.). Retrieved
from AIR PRODUCTS : http://www.airproducts.com.my/industries/Analytical-
Laboratories/analytical-lab-applications/product-list/gc-with-electron-capture-detector-gc-
ecd-analytical-laboratories.aspx?
itemId=2ED69212C574443C9354860ABEFCFE2B#:~:text=Gas%20Chromatography
%20%E2%80%9

Solid Phase Extraction (SPE). (2020, June 9). Retrieved from Chem.LibreTexts:
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(An
alytical_Chemistry)/Analytical_Sciences_Digital_Library/Active_Learning/Contextual_Modul
es/Sample_Preparation/03_Solid-Phase_Extraction

Solid-Phase Extraction. (n.d.). Retrieved from ScienceDirect:

https://www.sciencedirect.com/topics/chemistry/solid-phase-extraction#:~:text=The
%20basic%20principle%20of%20SPE,greater%20affinity%20for%20the%20analytes.

What is Solid-Phase Extraction (SPE)? (n.d.). Retrieved from Waters:

https://www.waters.com/waters/en_US/Goals-and-Benefits-of-SPE/nav.htm?
cid=10083495&locale=en_US
APPENDIX

Figure 1 STANDARD MIXTURE gradient elution (injection 1)

Figure 2 STANDARD MIXTURE gradient elution (injection 2)
Gradient Program Time (min) %ACN:%Water

0 – 1.8 50:50

1.8 – 8.0 70:30

Figure 3 STANDARD MIXTURE ISOCRATIC ELUTION 70%: 30% ACN: Water
Figure 4 STANDARD ELUTION Individual standard CAFFEINE at 70%: 30% ACN: Water
Figure 5 STANDARD ELUTION Individual standard ACETONE at 70%: 30% ACN: Water
Figure 6 STANDARD ELUTION Individual standard METHYL BENZOATE
at 
70% ACN: 30% Water

Figure 7 STANDARD ELUTION Individual standard PHENATOLE at 

70% ACN: 30% Water
Figure 8 STANDARD ELUTION Individual standard PHENANTHRENE at 
70% ACN: 30% Water
Figure 9 STANDARD MIXTURE ISOCRATIC ELUTION, 50% ACN: 50% Water
Pea Retation time Width
k

4 6.887 0.2767

5 26.141 1.3710