Laboratory Report

ANALYTICAL INSTRUMENTATION
(INST-2135)

Experiment 6: HPLC (High Performance Liquid Chromatography)

Submitted to: Bita
Submitted by: Ishani Patel
Lab Partner: Hiral Joshi, Harshul Patani

Date of performance: 03/31/14, 04/07/14

Date of submission: 04/14/14

OBJECTIVE
Experiment-1
In this experiment, peaks of the mixture of three different compounds were observed by
injecting the sample to HPLC. These peaks were identified by comparing peak area
depending on the retention time of the individual peaks with that of the mixture.
Experiment-2
In this experiment, Standard solution of caffeine was prepared and an unknown sample
(coke) was injected to HPLC to obtain their peak area. From the calibration curve of
standard solution, concentration of caffeine in an unknown sample can be determined.
INTRODUCTION
HPLC is widely used in quantitative separation of various components in a mixture
because of its sensitivity. It is also used for determination of non-volatile and thermally
fragile components.
In HPLC, sample is injected through injection port. Approximately 25 L of the sample is
automatically injected to pump, which pushes the solvent (mobile phase) through the
column (stationary phase) to a detector for separation, identification and quantitative
determination of components. The mobile phase here, is a solvent, which carries a sample
to be detected over a column (stationary phase). The solution flows over the stationary
phase, which retains the components resulting in each component reaching the detector at
different time. All the compounds have different retention time, so they are separated and
identified depending on the retention time and area.
INSTRUMENTALCOMPONENTS
Injection port
Sample is injected to the injection port. Approximately 25 L of the sample is

automatically injected to the pump.

HPLC Pump
It is used to pump the sample. The solution is pumped to column to retain the sample by
the stationary phase and for further detection of the components being separated. Types
of Pumps used for HPLC analysis are Reciprocating Piston Pump, Syringe type pump,
Constant pressure Pump.

Column
It has a layer stationary phase to retain the sample and separate the components.
Detectors
Various detectors are used in GC depending on its selectivity. Specific detectors are used
for specific compounds.
Experiment 1
OBJECTIVE
To identify the components present in a prepared mixture by comparing it with the peak
area of the individual components depending on the retention time.
INSTRUMENTAL CONDITIONS
Methanol : water 70 : 30
Wavelength: 254 nm
Flow: 1.00
Manually injected: 40 60 L
Automatically injected: 25 L
Range: 1.0000
RSP: 0.02
REAGENTS: Toluene, ethyl benzene, Propyl benzene, methanol
PROCEDURE
Is followed as per Analytical Instrumentation Laboratory Manual INST 2135 Durham
College. Pg 5 9
OBSERVATION
Sample

Retention time

Peak area

Peak 1

Peak 2

Peak 1

Peak 2

Mixture

4.733

6.666

18034.0340

17327.9210

Toluene

4.900

53160.3240

Ethyl benzene

6.966

18563.5680

Experiment2
OBJECTIVE
Todetermineconcentrationofcaffeineinanunknownsamplefromthecalibrationcurve
producedfromthepeakareaofthestandardcaffeinesolutionsusingHPLC.
INSTRUMENTAL CONDITIONS
Methanol : water 70 : 30
Wavelength: 238 nm
Flow: 1.00
Manually injected: 40 60 L
Automatically injected: 25 L
Range: 1.0000
RSP: 0.02
REAGENTS: Toluene, ethyl benzene, Propyl benzene, methanol
PROCEDURE
Is followed as per Analytical Instrumentation Laboratory Manual INST 2135 Durham
College. Pg 5 9
OBSERVATION
Concentration (mg)

Retention time

Peak area

1.533

1553.5460

10

1.500

2342.7150

15

1.483

3911.5660

Unknown (coke)

1.500

357.5210

Peak area vs Concentration of caffeine

5000
P
e 4000
a
k 3000
2000
a
r 1000
e
0
a

f(x) = 235.8x + 244.59

R = 0.96

10

12

14

16

Concentration (mg)

Fromthegraph
Y=235.8x+244.59
PeakareaY=357.5210
X = 0.47 mg
5ml of unknown sample (coke) contains 0.47 mg caffeine
So, Coca cola bottle (590 ml) contains ? mg caffeine
590mlx0.47mgcaffeine=55.46mginoriginalsample(590mlcocacola)
5ml
% error = theoretical value actual value x 100 = 56 mg 55.46 mg x 100
actual value
56 mg
= 0.98 %
DISCUSSION
Inthisexperimentweidentifiedindividualcomponentspresentinthemixtureby
comparingtheretentiontimeoftheindividualcomponentswiththatofthemixture.
Compoundsthathavestrongerinteractionwiththestationaryphase(column)areretained
forlongertime(moreretentiontime)takingtimetoreachthedetectorlikepropyl
benzene.Peakofpropylbenzenewasnotobservedinthegraphbecausethetimeinserted
tothesoftwaretorunthesamplewas10minutes.So,becauseofthehumanerrorpeak
wasnotdetectedandthegraphofindividualpeakofpropylbenzenewasnotobtained
duetoprintingerror.Theothertwopeaks,Tolueneandethylbenzenewasidentifiedby
matchingtheretentiontimeofthemixturewiththatoftheindividualpeak.So,thethird
peakisoughttobepropylbenzene.Thecompoundthatisstronglyattractedtomobile

phaseisretainedforlesstime(toluene)andisdetectedfaster.Methanol:wateristhe
mobilephasehavinghighestaffinityforthetoluene>ethylbenzene>propylbenzene.
Stationaryphaseinthisexperimentisthecolumn.Isocraticelutionwasusedinthis
experimenti.econstantmobilephasecompositionwasused.Thecompoundswereeasily
identifiedduetoitsgoodresolution.
Concentrationofcaffeineinanunknownsample(coke)wasdeterminedfromthe
calibrationcurveobtainedbyinjectingstandardcaffeinetoHPLC.Calibrationcurveof
peakareavsconcentrationofcaffeinewasplotted.
CONCLUSION
Thecomponentspresentinagivenmixturewereidentifiedbymatchingtherepeakswith
theindividualcomponentsdependingonthereretentiontime.
Theconcentrationofcaffeineinanunknownsamplecokewasfoundtobe55.46mgin
590ml.Theamountofcaffeinefoundonthelabelwas56mgin590ml.The%errorwas
0.98%.
REFERENCE
1. Analytical Instrumentation Laboratory Manual INST 2135 Durham College. Pg 5
-9
2. Caffeineinformer retrieved from http://www.caffeineinformer.com/caffeinecontent/coca-cola-classic