Group Members: Chereem Beaufort 1102067

Felicia Reeves 1104148

Mikkel Green 1102269
Lab - #8-Experiment 10
Date: July 9, 2014
Title: Determination of Caffeine in Drugs and Beverages
Aim: To determine the Caffeine content in tea using HPLC and Standards

Abstract: In this lab exercise, the determination of caffeine in tea bag was analyzed
using the high performance liquid chromatography. Chromatograph of different concentrations
of standards were prepared and used to determine the caffeine present in tea. For this reason, the
peak of the sample and the standard peak are proportional to the concentration of the caffeine
present in the tea sample analysed. The concentration of the caffeine present in tea is ----
Introduction: High-performance liquid chromatography (HPLC) is also known as high-pressure
liquid chromatography. It is an instrumental system based on chromatography that is commonly
used in forensic science. HPLC is used in drug analysis, toxicology, explosives analysis, ink
analysis, fibers, and plastics. In this experiment HPLC is used to analyze the caffeine present in
tea.
HPLC is based on selective partitioning of the molecules of interest (in this case caffeine) in the
given sample between two different phases, the mobile and stationary phases. The mobile phase
in this case is a solvent or solvent mix that flows under high pressure over beads coated with the
solid stationary phase. The mobile phase has the purpose of carrying the solutes through the
stationary phase along with the help of a pump. It is also seen to be used to adjust the
chromatographic separation and retention. the ultimate purpose of the mobile phase however is
to dissolve the components of the sample to be separated thus a non-polar sample is used a non-
polar mobile phase is used and if a polar sample is used a polar mobile phase is used as likes
dissolve likes and thus the application of the reverse or normal mood.
The purpose of the stationary phase is to act as a surface area, a constant one on which the
components of the mobile phase (that of the dissolved mixture) can be separated based on their
affinity for this phase.
While traveling through the column, different components in the sample partition selectively
between the mobile phase and the stationary phase will move at different rate, thus, separating
the components according to the size and shape of the molecules. Those that interact more with
the stationary phase will lag behind those molecules that partition preferentially with the mobile
phase. As a result, the sample introduced at the front of the column will emerge in separate bands
called peaks, with the bands emerging first being the components that interacted least with the
stationary phase and as a result moved quicker through the column. The components that emerge
last will be the ones that interacted most with the stationary phase and thus moved the slowest
through the column. A detector is placed at the end of the column to identify and record the
components that elute. This represent the area of the peak of interest in comparison to the other
peaks eluded first and is proportional to the concentration of the component of interest in the
sample (caffeine). The caffeine peaks found in each sample could have compared with the
prepared standard. This is because identical peaks will have identical retention time. Retention
time is the time taken for a particular compound to travel through the column to the detector,
therefore can be measure from the time at which the sample is injected to the point at which the
displays shows a maximum peak height for that compound. This provides a pure or nearly pure
sample of the component of interest. This technique is sometimes referred to as preparative
chromatography.
Resolution is the most important concept in HPLC chromatography is resolution (R), which is
dependent on all the factors involved. R is calculated as the difference in retention time of two
analytes divided by the average width of the two peaks at the baseline.
Resolution (R) = (RTpeak 1 - RTpeak 2)
1/2 (widthpeak 1 + widthpeak 2)

The equation above shows that developing a method with good resolution and a reasonable run
time is a balancing act between selectivity and efficiency. The most effective way to improve
resolution is by changing the stationary and mobile phases.

Selectivity factor is the ratio of the capacity factors of two peaks. Selectivity factor is a
measurement of the difference in interactions of two analytes with the mobile and stationary
phases, and therefore the difference in retention times. As with capacity, the selectivity of a
column is an effect of the column packing material and the eluents used.

Selectivity = k1
k2

The Capacity factor (k) is the ratio of a compounds elution volume, or retention time, relative to
the time of elution of an unretained compound. It is a measure of how much time a compound
spends in the stationary phase versus the mobile phase. Peaks of interest should have a k>2 for
optimum resolution. The capacity factor is generally independent of the equipment and is a
function of the choice of column and mobile phase.
Capacity factor (k) = (RT analyte RT unretained)
(RT unretained)
Height Equivalent of a Theoretical Plate (HETP) also known Separation efficiency (N) is a
measure of the sharpness of peaks eluting from a specific column, and is described as the number
of theoretical plates in a column. It is calculated from the retention time and the width of the
peak at peak height. In general shorter columns decrease analysis time, but do not increase the
system efficiency (N). Often, increasing column temperature will increase the efficiency of
separation.
Separation efficiency (N) = RT
( Width
half height
)


Results:
Mass of caffeine used: 1.0002g in 1L
Wavelength- 250nm
150mm column
20l sample analyzed
Flow rate- 2ml per minute
Method isocratic
Ratio- water 55: methanol 45
Column type-C
18
reverse phase chromatography
4.6 150 diameter agilent column
TABLE SHOWING THE STANDARD CONCENTRATION AND VOLUME PIPETTED
[Stock]= 1000ppm
[Standard]/ppm Vol of flask(ml) Dilution factor Vol pipette (ml)
20 50 50 1
40 50 25 2
60 50 16.67 3
80 50 12.5 4
100 50 10 5

TABLE SHOWING CONCENTRATIONS OF CAFFEINE INSAMPLE AND HPLC
READING
Conc(ppm) HPLC reading for area (mAUHS)
0 0
20 466.14032
40 907.61682
60 1356.71387
80 1827.13965
100 2237.80688


Discussion: In this experiment High Performance Liquid Chromatography (HPLC) was used to
determine the caffeine content in tea. HPLC is a type of chromatography that employs a liquid
mobile phase and a very finely divided stationary phase. In order to obtain satisfactory flow
rates, the liquid was pressurized to several hundred or more pounds per square inch. In liquid
chromatography, the mobile phase was a liquid solvent containing the sample (tea) as a mixture
of the solutes. The components were separated according to the size and shape of the molecules.
As each set of molecules elutes from the column, a detector recognizes it and recorded a peak.
The sources of error in this experiment could have been both quantitative and qualitative.
Quantitative errors might include:
There could have been false calibration
Poor integration due to wrong base line adjustment or too low peak start
In Incomplete separation, the peaks may overlap because of non optimized selectivity or
insufficient separation power.
This can result if the detector is working outside the detector working range
Qualitative errors might include:
The separation power and selectivity can be changed because the column temperature
usually has a stronger influence in liquid chromatography than believed. This results in the
separation of important substance pairs and is a source of error
Poor separation power
Certain substances from the samples can be adsorbed strongly in and on the stationary
phase
The selectivity changes of the stationary phase can be due to highly sorptive impurities in
the mobile phase this can result in a source of error
Conclusion: The experiment was successful and the aim achieved which was to determine the
content of caffeine in tea using HPLC. The concentration of caffeine in tea was found to be






Reference:
Clark,J. (2007) High Performance Liquid Chromatography-HPLC. Retrieved from
http://www.chemguide.co.uk/analysis/chromatography/hplc.html