EXPERIMENT 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

METHOD DEVELOPMENT

OBJECTIVE
I. To identify the components of the mixture using the selected HPLC conditions.

ABSTRACT
The chromatography process begins by injecting a sample solution into the mobile
phase through the injector port. As a sample solution in the mobile phase flows through a
column, the compounds of the solution will migrate according to the interactions of the
components with the stationary phase. The chemical interactions of the mobile phase and
sample solution, as well as with the stationary phase, determine the degree of migration and
separation of components contained in the sample. For example, the components that have
stronger interactions with the mobile phase than with the stationary phase will elute faster
from the column and have shorter retention time compared to those components which have
stronger interactions with the stationary phase. The type and composition of the mobile phase
is one of the influence in separation of the components. Therefore, the mobile phase can be
altered in order to manipulate the interactions of the sample components and the stationary
phase. In isocratic elution, components are eluted using constant mobile phase composition.
In gradient elution, different components are eluted by changing the mobile phase
composition as the separations proceeds. By increasing the strength of the mobile phase will
subsequently results in elution of retained components.

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.
In summary, polarities for solute, mobile phase and stationary phase must be carefully
blended if good partition chromatography separation are to be realized in a reasonable time.
Unfortunately, theories of mobile phase and stationary phase interaction with any given set of
sample component are impacted, and at best, we can only narrow the choice of stationary
phase to a general type. Having made this choice, we then perform a series of set trial and
error experiment in which chromatogram are obtained with various mobile phase until a
satisfactory separation is realized. If resolution of the entire component of a mixture proves to
be impossible, different types of column may have to be chosen.
ANALYTICAL PROCEDURE

a. Instrument set up (may vary depending on instrument):

Detector wavelength : 254 nm
Flow rate : 1.5 mL/min
Mobile phase : acetonitrile : water

b. Effect of mobile phase on HPLC separation

i. The instrument is set to use a mobile phase ratio of acetonitrile:water (50:50)
and the sample is injected.
ii. Then, the mobile phase is changed the mobile phase composition to 70:30.
The suitable composition of mobile phase for the separation of the compound
is determined.

c. Identification of components in the mixture

Each compound is injected individually and the components of the mixture is
identified by using HPLC conditions.

d. Separation using gradient elution

The gradient elution is performed based on the separation to improve the efficiency of
the column.

RESULT
FOR ISOCRATIC ELUTION;
Response Factor Area Mobile Phase Ratio Retention Time
(minute)
 79.3762 7937.625 1.128
28.1650 2816.501 1.273
26.7675 2676.751 2.591
25.3343 2533.430 50% H2O : 50% ACN 3.834
678.492 67849.20 10.392
Sample ID: Standard mixture (100 ppm)
Respond Factor (RF) = Peak Area
Sample Amount (ppm)
For Peak 1 = 7937.625 = 79.3762
100
For Peak 2 = 2816.501 = 28.1650
100
For Peak 3 = 2676.751 = 26.7675
100
For Peak 4 = 2533.430 = 25.3343
100
For Peak 5 = 67849.20 = 678.492
100

Respond Factor Area Mobile Phase Ratio Retention

Time (minute)
72.1883 7218.8344 1.066
25.6509 2565.0927 1.179
24.6158 2461.5817 1.969
22.0825 2208.2568 70% H2O : 30% ACN 2.644
457.5050 45750.500 5.923
Sample ID: Standard mixture (100 ppm)

Respond Factor (RF) = Peak Area

Sample Amount (ppm)

For Peak 1 = 7218.8344 = 72.1883

100
For Peak 2 = 2565.0927 = 25.6509
100
For Peak 3 = 2461.5817 = 24.6158
 100
For Peak 4 = 2208.2568 = 22.0825
100
For Peak 5 = 45750.500 = 457.5050
100

FOR GRADIENT ELUTION: i) First injection

Respond Factor Area Mobile Phase Ratio Retention Time
(minute)
78.6675 7866.7539 1.138
26.9818 2698.1826 1.274
24.1705 2417.0522 2.359
22.4718 2247.1877 70% H2O : 30% ACN 3.041
228.0344 22803.440 4.712
Sample ID: Standard mixture (100 ppm)
Respond Factor (RF) = Peak Area
Sample Amount (ppm)
For Peak 1 = 7866.7539 = 78.6675
100
For Peak 2 = 2698.1826 = 26.9818
100
For Peak 3 = 2417.0522 = 24.1705
100
For Peak 4 = 2247.1877 = 22.4718
100
For Peak 5 = 22803.440 = 228.0344
100

ii) Second injection

Respond Factor Area Mobile Phase Ratio Retention Time

(minute)
74.2430 7424.3032 1.108
25.6960 2569.6088 1.243
22.1251 2212.5109 2.228
21.5843 2158.4335 70% H2O : 30% ACN 2.719
244.3564 24435.640 4.086
Sample ID: Standard mixture (100 ppm)
Respond Factor (RF) = Peak Area
Sample Amount (ppm)
For Peak 1 = 7424.3032 = 74.2430
100
For Peak 2 = 2569.6088 = 25.6960
100
For Peak 3 = 2212.5109 = 22.1251
100
For Peak 4 = 2158.4335 = 21.5843
100
For Peak 5 = 24435.640 = 244.3564
100

Sample ID Respond Factor Area Mobile Phase Ratio Retention

(100ppm) Time (minute)
Caffeine 60.0627 6006.2705 1.072
Acetone 47.9063 4790.6377 1.197
Phenanthrene 31.9643 3196.4394 2.719
Methyl Benzoate 111.771 11177.100 70% H2O :30% ACN 2.014
Phenatole 187.127 18712.700 6.141

Respond Factor (RF) = Peak Area

Sample Amount (ppm)

For Caffeine = 6006.2705 = 60.0627

100
For Acetone = 4790.6377 = 47.9063
100
For Phenanthrene = 3196.4394 = 31.9643
100
For Methyl Benzoate = 11177.100 = 111.771
100
For Phenatole = 18712.700 = 187.127
100

DISCUSSION
During this experiment, a High Performance Liquid Chromatography (HPLC) Agilent
G1314A equipped with UV detector, 5 m Reverse Phase C18 column and 20 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 is
standard mixture, caffeine, acetone, phenanthrene, methyl benzoate, and phenatole were
injected, the ratio was changed to 70%:30% respectively on the same mobile phase. Based on
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 the result shows all the peaks from the injection process to the sample loop were
not separated well. In a reversed-phase separation, the strength of eluent 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.
Another reason, 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).
CONCLUSION
The components is identified as well as their peak retention time.

REFERENCES
1. Skoog, Holler and Nierman, 5th Edition. Principles of Instrumental Analysis.
Thomson Learning 1998
2. Skoog, D.A., West, D.M, Holler, F.J. 7th Edition, Fundamental of Analytical
Chemistry
3. Saim, N., Tajuddin, R., & Saaid, M. (2014). Analytical separation methods
laboratory guide. Selangor: UiTM Press.
 (EXPERIMENT 2)
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC):
METHOD DEVELOPMENT
NAME: NURUL HAZIQAH BINTI HASAN
STUDENT ID: 2016666772
PARTNERS NAMES: 1. NUR AININA BINTI MOHAMED AINI
2. NURUL JUNAIDAH BINTI TERMIZI
3. RAHAYU BINTI ABDUL RAHMAN
DATE OF SUBMISSION: 09/06/2017