University of Bahrain

College of Engineering
Department of Instrumentation and Control Engineering

High Performance Liquid

Chromatography (HPLC)

CHEMY 313
Analytical Chemistry

pg. 1
 Table of Contents
1. Aim.......................................................................................................3
2. Introduction..........................................................................................3
3. Equipment And Chemicals...................................................................4
4. Calculations.........................................................................................4
4.1 Raw Data...........................................................................................4
4.2 Substituting.....................................................................................5
5. Conclusion...........................................................................................5
6. References...........................................................................................6

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1. Aim
Separation and Identification of components of a mixture by High Performance
Liquid Chromatography (HPLC).

2. Introduction
High performance
liquid chromatography
is basically a highly
improved form of
column
chromatography. Instead
of a solvent being
allowed to drip through
a column under gravity,
it is forced through
under high pressures of
up to 400 atm.

It also allows using a very much smaller particle size for the column packing
material which gives a much greater surface area for interactions between the
stationary phase and the molecules flowing past it. This allows a much better
separation of the components of the mixture.
For a particular compound, the retention time will vary depending on: the
pressure used (because that affects the flow rate of the solvent),the nature of the
stationary phase (not only what material it is made of, but also particle size),the
exact composition of the solvent, the temperature of the column, That means that
conditions have to be carefully controlled if you are using retention times as a way
of identifying compounds.

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3. Equipment And Chemicals

 High performance liquid chromatography instrument) .

 Unknowns Samples .

4.

Calculations

4.1 Raw Data

Standard solution Graph

90
80
Concentratio f(x) = 0.67 x − 17.84
Height of
n of Caffeine 70
Curve
(ppm) 60
Height of Curve

50
50 17.037 40
30
100 46.910 20

150 84.296 10
0
40 60 80 100 120 140 160
Concentration of Caffeine (ppm)

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 Unknowns samples

Unknowns 1 Unknowns 2
Height of Curve 45.100 52.236

3.2 Substituting

Unknowns 1:
Height of Curve = 45.100

by straight line equation : H = 0.6726xC - 17.845

45.100 = 0.6726xC - 17.845

Concentration (C) =93.585 ppm
Unknowns 2 :
Height of Curve = 52.236

by straight line equation : H = 0.6726xC - 17.845

52.236= 0.6726xC - 17.845

Concentration (C) =104.194 ppm

5. Conclusion
In this experiment, we used the High Performance Liquid Chromatography
(HPLC) method experiment to measure the Concentration of Caffeine. We used
linear equation which is showing a linear relationship between Height of Curve
and concentration .by plotting the results ,the relation be shown easily.

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We can summary the experiment in few sentences,
• The HPLC has developed into a universally applicable method so that it
finds its use in almost all areas of chemistry, biochemistry, and pharmacy,
Analysis of drugs , Water purification, Analysis of synthetic polymers and
Separation and purification of enzymes or nucleic acids.
• Advantages of (HPLC) : Speed ,Efficiency and Accuracy .

6. References
1- Ppt slides , Mr. Sadiq Mayoof , Chemistry Department , University of
Bahrain
2- fundamentals of analytical chemistry 9E, Douglas A. Skoog , Donald M.
West, F.James Holler, Stanley R. Crouch .
3- YouTube video, Zeal’s pharmacy tutorials channel, Fluorescence
spectroscopy.

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