Abstract

The aim of this experiment was to use reverse phase mode HPLC to identify and unknown
compound and to optimize the chromatographic conditions for the separation of a series of aromatic
hydrocarbons namely; uracil benzene toluene and ethylbenzene by optimising the mobile phase!
This was done by obtaining the chromatograms for standards of each component and matching the
retention times with the unknown the mobile phase polarity was altered and a mixture containing
all of the standards was analysed "ualitatively! The mobile phases used contained #$ %$ &$ and
'$( methanol! The compounds eluted in order of decreasing polarity the order of elution was as
follows) uracil benzene toluene ethylbenzene! The retention times and plate heights decreased as
the amount of methanol in the mobile phase was increased whereas the number of theoretical plates
and the resolution between the peaks increased! The mobile phase with the optimum condition
between the retention times and the peak resolution was found to be the one containing &$(
methanol therefore the unknown compound was in*ected in this mobile phase and the retention time
was +!+,- minutes which matched with that of uracil!
Introduction
High performance li"uid chromatography .HPLC/ is a type of li"uid chromatography that makes
use of a high pressure pump to transport the mobile phase through the column .0oel +$$$/!
Partition chromatography is the type of HPLC used in this experiment this method separates
components based on their polarity and reverse phase mode or normal phase mode can be used for
analysis .Cazes +$$-/! 1n reverse phase the stationary phase is non polar and the mobile phase is
polar therefore the more polar components will elute first since the separation is based on each of
the components affinity for the mobile phase .0oel +$$$/! 2ormal phase mode is the opposite of
reverse phase!
HPLC can be used for both "uantitative and "ualitative analysis and the shape separation and
retention times of the peaks is very important! The time that it takes for each component to elute
from the column is called the retention time and it can be used for "ualitative analysis by matching
the retention times of unknown samples to those standards .0oel +$$$/!
The separation that occurs in chromatography can be explained using the plate theory and the rate
theory!
The plate theory is based on how simple distillation occurs and the column efficiency can be
estimated by calculating the number of theoretical plates! 3ach plate is a representation of a region
in the column where the analyte reaches e"uilibrium between the mobile and stationary phase
therefore an increase in the number of plate means that the peaks will be sharper which is good for
analysis .Pillay +$4,/! The retention times and base widths of ad*acent peaks can be used to
measure the degree of separation of two peaks which is called the resolution a resolution that is
greater than 4 usually means that the peaks are well separated and can be used for an accurate
"uantification! 5or the analyte components to be accurately "uantified the peaks need to be narrow
well resolved and symmetrical .Pillay +$4,/!
The method of separation can be optimized by changing the mobile phase polarity in order to get a
good balance between the resolution of the peaks and the time it takes for each analyte to elute!
1n this experiment the separation of substituted aliphatic benzenes is investigated therefore a
6787is detector is suitable because the benzene ring behaves as a chromophore! This is due to the
delocalisation of electrons and the con*ugated bonds which means that it can absorb 67 light
.2ueman +$$-/! The detected signal is recorded in the form of sharp narrow peaks which are
distinct for each component in the mixture!
Methods
9tandards of uracil benzene toluene and ethylbenzene were in*ected into a high performance li"uid
chromatograph with a mobile phase that contained %$( methanol and :$( water! ; +$ <L sample
of each standard was in*ected! ;fter each standard had been analyzed a +$ <L sample of a mixture
that contained all the standard components was then in*ected into the instrument!
=ixtures of methanol and water were accurately prepared into bottles these mixtures contained #$
&$ and '$( methanol! These methanol and water mixtures were used as the mobile phase for the
analysis of a mixture that contained all the standard components! The mixture of the standard
components was in*ected into the instrument for each of the different mobile phases each new
mobile phase was left to e"uilibrate for % minutes!
The instrument used was a Perkin 3lmer 9eries +$$ HPLC with a 6787is detector! ; 2ucleosil 4$$
.+-$ mm x , mm/ column packed with - <m silica particles bonded with C
4&
.octadecylsilane/ was
used! The flow rate of the mobile phase was $!%- mL8 min!

Results
Table 4) The retention times for each of the standard components in when using a mobile phase with
%$( methanol!
Name of Standard Retention time, t
R
/ minutes
6racil +!+%&
>enzene :!':4
Toluene ,!'&4
3thylbenzene #!:+&
Table +) The retention times of each of the components in the mixture as the amount of methanol in
the mobile phase was changed!
Component % MeOH in mobile phase
6 ! " #
6racil +!+#4 +!+&4 +!+:- +!+,4
>enzene -!#:, ,!$+4 :!4#& +!%$&
Toluene &!,-, -!$,: :!#&: +!'+#
ethylbenzene 4+!,,- #!::4 ,!+44 :!4$-
Table :) The widths at the base of each of the component peaks in the mixture as the ( of methanol
was changed in the mobile phase!
Component % MeOH in mobile phase
6 ! " #
>enzene 4!$4 $!#- $!,+ $!:+
Toluene 4!4 $!&, $!,: $!+4
3thylbenzene 4!- $!'4 $!-- $!:-
Calculation for the theoretical number of plates and the plate height based on the benzene peak of
the mixture when using a mobile phase containing #$( methanol!
2 ? 4#.t
@
+
8 A
+
/
? 4#.-!#:,
+
min8 4!$4
+
min/
? ,'%!&#
H ? L82
? +-$ mm8 ,'%!&#
? $!-$+ mm
Calculation for the resolution between the benzene and toluene peaks when using a mobile phase
containing &$( methanol!
@
9
? .+Bt
@
8 w
;
Cw
>
/
? +.:!#&: D :!4#&/8 $!,: C $!,+
? 4!+4
Table ,) The number of theoretical plates calculated for each component in the mixture as the
percentage of methanol in the mobile phase was changed!
Component % MeOH in mobile phase
6 ! " #
>enzene ,'%!&# #4+!: '4$!:+ 44,-!&+
Toluene ',-!$# -%#!#' 44%:!%& :4$#!+
3thylbenzene 44$4!:- %%,!,: ':%!'4 4+-'!+,
Table -) The plate heights calculated for each component in the mixture as the percentage of
methanol in the mobile phase was changed!
Component % MeOH in mobile phase
6 ! " #
>enzene $!-$+ $!,$& $!+%- $!+4&
Toluene $!+#- $!,:, $!+4: $!$&4
3thylbenzene $!+%% $!:+- $!+#% $!4''
Table #) The resolution between the benzene and toluene peaks as the percentage of methanol was
changed in the mobile phase!
% Methanol in mobile phase Resolution, R
S
#$ +!#%
%$ 4!:%
&$ 4!+4
'$ $!&+
Table %) The resolution between the toluene and ethylbenzene peaks as the percentage of methanol
was changed in the mobile phase!
% Methanol in mobile phase Resolution, R
S
#$ :!$%
%$ 4!,%
&$ 4!+,
'$ $!-4
The mobile phase with &$( methanol was determined to give the optimum chromatographic
conditions therefore the unknown sample was in*ected into this mobile phase!
The retention time of the unknown was found to be +!+,- minutes!
5igure 4) Eraph of the retention times of benzene as the percentage of methanol in the mobile phase
was changed
60 65 70 75 80 85 90
0
1
2
3
4
5
6
% methanol in mobile phase
R
e
t
e
n
t
i
o
n

t
i
m
e
/

m
i
n
5igure +) Eraph of the retention times of toluene as the percentage of methanol in the mobile phase
was changed
5igure :) Eraph of the retention times of ethylbenzene as the percentage of methanol in the mobile
phase was changed
60 65 70 75 80 85 90
0
1
2
3
4
5
6
7
8
9
% methanol in mobile phase
R
e
t
e
n
t
i
o
n

t
i
m
e
/

m
i
n
60 65 70 75 80 85 90
0
2
4
6
8
10
12
14
% methanol in mobile phase
R
e
t
e
n
t
i
o
n

t
i
m
e
/

m
i
n
5igure ,) Eraph comparing the retention times of each component in the mixture as the percentage
of methanol in the mobile phase was changed!
5igure -) Eraph of the calculated number of theoretical plates based on the benzene peak in the
mixture as the percentage of methanol in the mobile phase was changed!
60 65 70 75 80 85 90
0
2
4
6
8
10
12
14
Benzene
Toluene
Ethlbenzene
% methanol in mobile phase
R
e
t
e
n
t
i
o
n

t
i
m
e
/

m
i
n
60 65 70 75 80 85 90
0
200
400
600
800
1000
1200
1400
% methanol in mobile phase
!
o
"

o
#

t
h
e
o
$
e
t
i
%
a
l

p
l
a
t
e
s
5igure #) Eraph of the calculated number of theoretical plates based on the toluene peak in the
mixture as the percentage of methanol in the mobile phase was changed!
5igure %) Eraph of the calculated number of theoretical plates based on the ethylbenzene peak in the
mixture as the percentage of methanol in the mobile phase was changed!
60 65 70 75 80 85 90
0
500
1000
1500
2000
2500
3000
3500
% methanol in mobile phase
!
o
"

o
#

t
h
e
o
$
e
t
i
%
a
l

p
l
a
t
e
s
60 65 70 75 80 85 90
700
800
900
1000
1100
1200
1300
1400
% methanol in mobile phase
!
o
"

o
#

t
h
e
o
$
e
t
i
%
a
l

p
l
a
t
e
s
5igure &) Eraph of the calculated height of each theoretical plate based on the benzene peak in the
mixture as the percentage of methanol in the mobile phase was changed!
5igure ') Eraph of the calculated height of each theoretical plate based on the toluene peak in the
mixture as the percentage of methanol in the mobile phase was changed!
60 65 70 75 80 85 90
0
0"1
0"2
0"3
0"4
0"5
% methanol in mobile phase
&
l
a
t
e

h
e
i
'
h
t
/

m
m
60 65 70 75 80 85 90
0
0"1
0"2
0"3
0"4
0"5
% methanol in mobile phase
&
l
a
t
e

h
e
i
'
h
t
/

m
m
5igure 4$) Eraph of the calculated height of each theoretical plate based on the ethylbenzene peak
as the percentage of methanol in the mobile phase was changed!
$iscussion
The purpose of this experiment was to use reverse phase mode HPLC to identify an unknown
sample and to further investigate the changes in the chromatographic conditions when the
composition of the mobile phase was changed! This was done by comparing the retention times of
the unknown sample to the retention times of the standard samples of uracil benzene toluene and
ethylbenzene!
5or the "ualitative analysis of the chromatographic peaks the polarity of the mobile phase was
changed by changing the water to methanol ratio and the mixture was in*ected and analysed for
each mobile phase! >ecause reverse phase mode was used the polarity of the stationary phase is
non polar which means that the less polar components will have a higher affinity for the stationary
phase compared to the mobile phase and therefore take longer to elute .0oel +$$$/! >enzene and its
derivatives are fairly non polar therefore they will interact more with the non polar stationary phase
.2ueman +$$-/ the components will elute in order of decreasing polarity which means that uracil
will elute first since it is the most polar followed by benzene toluene and finally ethylbenzene! The
polarity of toluene and ethylbenzene is decreased due to the increasing branched substituents
.2ueman +$$-/!
9ince water is more polar than methanol increasing the percentage of methanol in the mobile phase
will decrease the polarity of the mobile phase therefore the mobile phase with #$( methanol is
more polar than the mobile phase with '$( methanol! ;s it can be seen in figure 4 + and : the
retention times for each of the mixture components decreased as the percentage of methanol in the
mobile phase was increased! This is because the components will have an increased affinity for the
less polar mobile phase and will interact less with the column meaning that they will elute faster!
However a fair balance between the retention times and the separation of the peaks needs to be
maintained for accurate analysis!
60 65 70 75 80 85 90
0
0"05
0"1
0"15
0"2
0"25
0"3
0"35
% methanol in mobile phase
&
l
a
t
e

h
e
i
'
h
t
/

m
m
The number of theoretical plates represents the regions in the column where the analyte reaches an
e"uilibrium between the stationary and mobile phase .Pillay +$4,/! These can be calculated from
the retention times and the widths of the base of the chromatographic peaks! The number of plates
for each component increased as the percentage of methanol in the mobile phase was increased!
This is further confirmed by the chromatographic peaks as they become narrower and sharper for
the mobile phase with '$( methanol! The height of each peak was calculated from the number of
theoretical plates and from figure & ' and 4$ it can be seen that the plate heights reached an
optimum when the mobile phase with %$( methanol was used and thereafter decreased! The
smaller plate heights are preferred because it means the column will have an increased number of
plates which will result in sharper and more symmetrical peaks!
The resolution is a measure of how well two ad*acent peaks are separated .Pillay +$4,/ and as
percentage of methanol in the mobile phase was increased the resolution decreased as seen in
figure & ' and 4$! This is because when the polarity is reduced the components with similar
polarity will elute too fast and very closely which results in an overlap of the peaks! Ahen peaks are
overlapping the base of each peak cannot be separated which results in poor accuracy if it is used
for "uantification! ; resolution of 4 or less generally means that the peaks are not well separated
.Pillay +$4,/! 5rom the results obtained the resolution between the toluene and ethylbenzene peak
was calculated to be :!$% for the mobile phase with #$( methanol! ;lthough this means the peak is
well resolved and there is good separation between the peaks the time taken for the component to
elute was 4+!,,- minutes which is very long! The peaks obtained were also broad compared to the
other mobile phases this broadening is because the analyte spends more time in the column and
becomes more diffuse!
The resolution between toluene and ethylbenzene for the mobile phase with '$( methanol was
calculated to be $!-4 which is very low the peaks obtained from the chromatogram were
overlapping because the was minimal interaction between the analyte and the mobile phase which
means e"uilibrium between the two phases cannot not be reached! ;lthough all the retention times
for the peaks of this chromatogram were less that - minutes the peaks are not good to use for
analysis! The mobile phase which showed a fair balance between the elution rate and the peak
separation was when &$( methanol was used the resolution between the peaks was greater than 4
which means the separation is good! The retention times were not very long ethylbenzene eluted at
,!+44 minutes which is very good compared to 4+!,,- and #!::4 minutes for the #$ and %$(
methanol mobile phases respectively!
Therefore the unknown was in*ected into the &$( methanol mobile phase and the retention time
was found to be +!+,4 minutes! Ahen compared to the retention times of the mixture at &$(
methanol which were +!+:- :!4#& :!#&: and ,!+44 minutes for uracil benzene toluene and
ethylbenzene respectively; it can be concluded that the unknown compound was uracil because of
the similarity in retention times!
Conclusion
The column efficiency and can be increased by reducing the polarity of the mobile phase for reverse
phase mode HPLC! 1ncreasing the percentage of methanol in the mobile phase resulted in a
decrease in the retention times of the components and the mobile phase that provided the optimum
conditions was that consisting of &$( methanol! The unknown sample was identified to be uracil by
matching the retention times of the peaks from the chromatogram!
References
4! 0oel F! +$$$ G@eversed Phase HPLCG in HPLC: Practical and Industrial Applications pp!
4#$H4#4!
+! Cazes! 0! +$$- GHigh Performance Li"uid ChromatographyG in Ewing's Analytical I
nstrumentation Handbook, pp! &$H&- =arcel Iekker 2ew Jork!
:! Pillay L! +$4, Chem !" Instrumental Analysis: Chromatography, lecture notes, pp! #H'!
,! 2euman @! C! +$$- G9ubstituent 3ffectsG in #rganic Chemistry, pp! :$H:4!
Khttp)88web!chem!ucsb!edu8Lneuman8orgchembyneuman8M ;ccessed on + =ay +$4,!