ANALYTICAL SEPARATION METHOD

CHM 510
EXPERIMENT 2:
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC): METHOD DEVELOPMENT

NAME : NUR HAZIMAH BINTI MAHAMAD POZI

PARTNER: SITI NORMALIA BINTI SULAIMAN
LECTURER:
GROUP: ASB2Ac
DATE OF EXPERIMENT: 9/10/2013
DATE OF SUBMISSION: 23/10/2013

(2012448048)
(2012237674)

TITLE
High Performance Liquid Chromatography (HPLC) Method Development

OBJECTIVE
To study development for optimizing a separation of a mixture of three compounds which are
standard mixtures of caffeine, phenatole and methyl benzoate using HPLC by varying the mobile
phase composition.

ABSTRACT
A further refinement to HPLC has been to vary the mobile phase composition during the
analysis; this is known as gradient elution. The gradient separates the analyte mixtures as a
function of the affinity of the analyte for the current mobile phase composition relative to the
stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid
extraction but in continuous, not step wise. In this experiment, using water / acetonitrile gradient,
the more hydrophobic components will elute when the mobile phase consist mostly of
acetonitrile which giving a relatively hydrophobic mobile phase. The more hydrophilic
compounds will elute under conditions of relatively low acetonitrile and high water. The choice
of solvent, addictives and gradient depend on the nature of the stationary phase and the analyte.
Often a series of tests are performed on the analyte and the number of trial runs may be
processed in order to find the HPLC method which gives the best separation of peaks.

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.

INSTRUMENTS
Liquid Chromatography (Agilent G1314A HPLC) equipped with UV detector,
5mm RP C18 column and 10 uL sample loop.
REAGENTS AND SOLVENTS
HPLC grade acetonitrile, deionized water, standard mixture of caffeine,
phenatole, and methyl benzoate (100 ppm).
SAMPLE
Standard mixture of caffeine, phenatole, and methyl benzoate (100 ppm)

ANALYTICAL PROCEDURE
1. Instrument set up
Detector wavelength
Flow rate
Mobile phase

: 254 nm
: 1.5ml/min
: acetonitrile: water (50: 50 v/v)

2. Effect of mobile phase on Liquid Chromatography separation

a. The instrument is initially set-up as in B then sample is injected.
b. The mobile phase composition is changed to (acetonitrile:water)
60:40 and 70:30 then the sample is injected.
c. The best composition is determined by comparing the resolution
of the chromatogram produce.
3. Identification of components in standard mixture
For identification of components in methyl esters mixture, each
compound was injected individually to identify the components
of the mixture using the optimized LC conditions.
4. Separation using gradient elution
Based on the separation above, a gradient elution separation was
performed to improve the efficiency of the column.

RESULT

Response
Factor

Area

Mobile Phase

Retention

Ratio

Time

(minute)
52985.5

145506.21

134896.63

529855

50% H2O:50%

ACN

145506

60% H2O:40%

21

ACN

134896

70% H2O:30%

63

ACN

0.538

0.621

0.667

Sample ID: Caffiene Standard (100 ppm)

Respond Factor (RF)

Peak Area
Sample Amount (ppm)

For Standard 1

5298550

52985.5

100

For Standard 2

14550621 =

145506.21

100

For Standard 3

13489663 =

134896.63

100

Respond
Factor

Area

Mobile Phase Ratio

Retention
Time

(minute)
180227.45

1802274
5

50% H2O : 50%

ACN

1.902

547649.01

5476490
1

60% H2O : 40%

ACN

3.114

1323032.63

1323032
63

70% H2O : 30%

ACN

6.483

Sample ID: Methylbenzoate Standard (100 ppm)

Respond Factor (RF)

Peak Area
Sample Amount (ppm)

For Standard 1

18022745 =

180227.45

100

For Standard 2

54764901 =

547649.01

100

For Standard 3

132303263 =

1323032.63

100

Respond

Area

Mobile Phase

Retention

Factor

Ratio

Time
(minute)

353914.45

1031182.33

1272972.99

353914

50% H2O : 50%

45

ACN

103118

60% H2O : 40%

233

ACN

127297

70% H2O : 30%

299

ACN

3.281

6.471

15.316

Sample ID: Phenatole Standard (100 ppm)

Respond Factor (RF)

Peak Area
Sample Amount (ppm)

For Standard 1

35391445 =

353914.45

100

For Standard 2

103118233 =

1031182.33

100

For Standard 3

127297299 =

1272972.99
100

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 is phenatole,
methylbenzoate and caffiene 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 coelution 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).

CONCLUSION
In this experiment, students would know how the concept and method development of
optimizing a separation of a standard compounds using High Performance Liquid
Chromatography (HPLC) by varying the mobile phase composition instead of other factors that
effected to the elution process occurred.

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