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 compound’s 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 ½ 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

20µl sample analyzed

Flow rate- 2ml per minute

Method isocratic

Ratio- water 55: methanol 45

Column type-C18 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 50 140 50 25 260 50 16.67 380 50 12.5 4100 50 10 5

TABLE SHOWING CONCENTRATIONS OF CAFFEINE INSAMPLE AND HPLC READING

Conc(ppm) HPLC reading for area (mAUHS)0 020 466.1403240 907.6168260 1356.7138780 1827.13965100 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