METHOD

1. The computer was turned on and logged in.

2. The pump and detector was turned on.
3. Instrument Online icon was clicked on the desktop.
4. Pump and detector were turned on by choosing System On under the Instrument
Menu. (Wait until diagram turn to green/system ready)
5. Solvent and waste bottle were checked.
The level of the mobile phase bottle was checked before starting any analysis.
i. The solvent bottles was checked and the samples was make sure that there is
enough solvent to run. (Click on the bottle icon Solvent bottles filling OK)
ii. The volume levels in the program were adjusted, if the solvent bottles were
filled.
Enter new volumes Click OK.
iii. The waste bottle on the floor was checked. The waste bottle was changed if full.

6. The method was opened

a) The line was flushed before analysis (loosen the pump-anticlockwise, pressure 1 bar)
File Load Method CHM260_A.m
b) Sample analysis (tighten the pump-clockwise, pressure will increase up to 400 bar)
7. Select Run Control Sample Info Enter Subdirectory Enter Sample Name
(Refer diagram)
8. Sample injection:
a) 10 uL sample was took by using syringe
b) Before injection: position was LOADED (Diagram 1)
c) Syringe was inserted, the sample was injected into the sample injection port (Diagram 2)
d) Turned to INJECT position (Diagram 3). The analysis will run by system.
9. The column was flushed before the HPLC system was turned off.
i. Line A was removed from mobile phase solvent bottle (acetonitrile:water) and was
rinsed thoroughly with DI water. This will avoid contaminating the solution.
ii. Line A was placed in 100percent acetonitrile solvent bottle.
iii. Method was opened:
File load method CHM260_FLUSH.m OK
10. The result was saved and printed:
Report Print File name Save
11. The pump was make sure to turn off once the samples have run. The software was
closed out and the instrument was turned off.
CALCULATION

Rt std caffeine = 100 3five26.09790

=0.0284

(Rt 100 plus) 0.0284 = X 410.3676five

= 11.6380 ppm

(Rt coke) 0.0284 = X 410.3676five

= 76.3114 ppm
DISCUSSION
In this experiment, HPLC is used to determine the concentration of standard caffeine,
100plus and coke in a soft drink sample. Although gas chromatography (GC) has a higher
sensitivity and speed, it cannot be utilized in this case as both compounds are not sufficiently
volatile and may degrade at high temperatures.

Caffeine has the chemical name of 1,3,7-trimethylxanthine and a molecular formula of

C8H 10N4O2. It is a psychoactive stimulant that is usually added into soft drinks as an additive,
it contributes a bitter taste as well as induces a stimulating effect. Caffeine, being colourless,
can be detected within the UV absorption range of 210nm and 275nm. Thus in this experiment,
a wavelength of 2FIVE4nm was used to measure the elution of caffeine using the photodiode
array detector conducting a UV/VIS scan.

The retention time of sugars from the chromatogram obtained for standard caffeine is
1.(FIVE)17 while for 100 plus is 1.five five five and for coke is 1.five12 which is the average for
each sample are 1.FIVE17, 1.FIVE FIVE FIVE and 1.five12. The example of calculations for
glucose standard dilution :

Rt std caffeine = 100 3five26.09790

=0.0284

(Rt 100 plus) 0.0284 = X 410.3676five

= 11.6380 ppm

(Rt coke) 0.0284 = X 410.3676five

= 76.3114 ppm

The peak area for the standard caffeine is 3five26.09790 while the peak height is
4five4.38470. The peak area for 100 plus is 410.3676five and the peak height is 48.62five10
while the peak area and peak height for coke is 2687.02271 and 40.41five9.
 Error might not have happened in this experiment but the precaution steps must have in
this experiment. The first is do not close the cap of the bottle tightly during boiling the samples
in the water bath to avoid from the presence air bubbles. Next, according to safety data,
caffeine is a skin irritant. So, upon the exposure it should wash away with water to avoid
irritation. Lastly, the HPLC eluent, with 60% methanol and 40% water, is a flammable mixture
and an irritant. Any residue should be placed in the waste container and spillages should be
washed away with plenty of water to avoid from flammable and irritation.

REFERENCES
1. http://chromsci.oxfordjournals.org/content/38/6/259.full.pdf
2. https://books.google.com/books?isbn=1461306817
3. www.art-xy.com/2009/11/lab-report-on-high-performance-liquid.html