Abstract

Reversed phase High performance was conducting in order to identify the unknown and
separate the aliphatic substituted benzene compounds. The mobile phase polarity was
optimised which generated suitable conditions to separate these compounds and enough UV
detection to identity the unknown. The mobile phase that was used was made of Methanol
and water which allowed it to be polar. The stationary was packed with alkyl groups which
caused it to be nonpolar. It was then noticed that polar components eluted faster than nonpolar components due to the fact that the polar components interacts with the polar mobile
phase and the nonpolar interacts with the nonpolar stationary phase .Chromatograms showed
each components retention time and show which components eluted faster on the basis of
polarity .The order was: uracil, benzene, toluene and ethylbenzene. The 70% Mobile phase
acts as the optimum mobile phase as a result of resolution (evenly spaced peaks and peak
heights) of its chromatogram. The calculation of the resolution was 14.30 for the
70%MeOH.The unknown was then identified by using the retention times .It displayed a
similar retention to that of benzene and therefore is identified as benzene. It can be concluded
that an increase when the mobile phase undergoes an increase in polarity it is caused by
increasing the concentration of methanol in the solution and this causes the retention time to
increase. Therefore if the mobile phase polarity increases, elution increase. To calculate the
efficiency the column, the theoretical plate number and theoretical plate heights must be
calculated. An average efficiency is discovered between 80% MeOH and 90%MeOH

Introduction

HPLC is a useful technique which can separate, identify and quantify components in a
mixture. It is relevantly useful when the samples or compounds are thermally unstable and
have high molecular weights. Reverse phase HPLC focuses on separation and purification
with regards to analytic and preparation application. When molecules are hydrophobic in
nature, they qualify to by separated using reversed phase chromatography that gives optimal
recovery and resolution. The mechanism however proceeds by binding interaction between
the stationary phases also known as they immobile hydrophobic ligand and the solute which
is present in the mobile phase1. Initially the mobile phase binding conditions are aqueous
which shows that both the solute and ligand molecules are surrounded by water. The area that
is hydrophobic can be minimised which is exposed to the solvent when the solute and ligand
bind to one another. This allows for the water to decrease which increases the entropy of the
system which adds an advantage to the hydrophobic moieties1.
The samplesathataareaofainterestaareainjectedaintoatheaHPLCaandaUValightaisaallowed
pass through the sample 2.The amount of light that is absorbed by each component produces
spectral bands on the chromatogram, which identifies the compounds. Benzenes which are
aliphatic substituted compounds are commonly used due to its electron cloud of the ring
acting as a chromophore. This allows for UV absorption. Eluting of an analyte will therefore
depend on the functional groups that are attached to the benzene ring2. When the
chromatogram is produced it displays a graph, depicting the response detected by the amount
of UV absorbed and retention time.

The components of an HPLC include a column ,pumps ,injectors ,detectors and an oven .The
column used in this practical is the c18 column which has a high surface area and phase
stability for separation . It also has high non-polar retentivity and gives symmetrical peak
shapes. It contains the stationary phase3. The pumps cause variations in flow rates of the
mobile phase that effect elution timeaofasampleacomponentsaandaresultainaerrors.
Pumpsaprovideaconstantaflowaofamobile phaseatoatheacolumnaunderaconstantapressure3.
Injectors are used in order to allow a constant amount which enter the mobile phase. The
column oven is used to keep the temperature constant in order for the eluting components to
not have varying retention times1. This is done by circulating the air. Detectors are used to
give signals by responding to the components separated by the column2.

HPLC starts by pumping the liquid phase constantly to a column that contains the stationary
phase .The sample is first injected into the carrier gas which pushes it into the column .When
the sample reaches the column the components of the sample are retained due to the chemical
interactions between the analyte molecules and the stationary phase2. Based on the conditions
of operation, the mobile phase elutes the components of the sample. Techniques of detection
are put into place and the components are then quantified2.

Procedure
Table 1: Showing the details of the HPLC instruments and its components
Instrument used was the HPLC which contained a UV/VIS detector. The column was a
Nucloesil 100 that contained 5 m of silica particles bonded with C18. The flow rate of the
sample was 0.75 mL/min. The detection range is 210 nm. The mobile phase contains water
and methanol.
Establishing Retention Time (tR) of individual components
The standard solutions (uracil, benzene, toluene and ethyl benzene) were prepared for us
.They contained approximately 10mg/mL of mobile phase solvent. An unknown was also
prepared which contained all the standards .The 70 % mobile phase was prepared by the ratio
of 70:30 which tells us there is 70 percent methanol and 30 percent water.500mL were
prepared of this solution. The standards were then added into vials and put into certain
positions on the tray which was inserted into the machine , where they could be automatically
injected, removing the risk of further contamination. 10 l was injected each time.
Chromatograms were created for each sample.
Optimisation of mobile phase polarity
500mL solutions of 60%, 80% and 90% were also prepared, which allowed for variance in its
polarity. A mixture solution containing all solutions was then injected for each mobile
phase .Approximately 7 minutes was used to switch between mobile phases. Chromatograms
were obtained for each mobile phase.

Identification of unknown10 L was injected automatically injected which used the 70%
methanol solution as it is the optimal percentage methanol mobile phase. Chromatogram
were received fir the unknown and with that its retention times were compared to that of the
standard which can be used to indicate what the unknown is.

Results
The data tabulated below was obtained from the appendix which contains the chromatograms
for each analysis.

Table 2: Showing the retention time for each compound using the 70% mobile phase.
Components

Retention time /min

Uracil

2.54

Benzene

6.89

Toluene

10.18

Ethylbenzene

14.67

Unknown

6.88

Table 3: Showing the retention times of the components in a mixture using different mobile
phases.
% Methanol
Component

60

70

80

90

Uracil (tR/min)

1.60

2.54

2.48

2.48

Benzene (tR/min)

2.52

6.89

5.59

3.84

Toluene (tR/min)

11.05

10.18

7.37

4.45

Ethylbenzene (tR/min)

19.22

6.88

9.35

5.01

3
2.5

f(x) = 0.01x + 1.35

R = 0.49

Retention time/min 1.5

1
0.5
0
55

60

65

70

75

80

85

90

95

%MeOH

Figure 1: A graph showing retention time /min versus %MeOH for Uracil

8
7
6
5

Retention Time/min

f(x) = 0.03x + 2.76

R = 0.03

4
3
2
1
0
55

60

65

70

75

80

85

% MeOH

Figure 2: A graph showing retention time/min versus % MeOH for Benzene

90

95

12
f(x) = - 0.23x + 25.29
R = 0.95

10
8

Retention time/min

6
4
2
0
55

60

65

70

75

80

85

90

95

90

95

%MeOH

Figure 3: A graph showing Retention time /min versus %MeOH for Toluene

25

20
f(x) = - 0.48x + 48.1
R = 1

15

Retention time/min
10

0
55

60

65

70

75

80

85

% MeOH

Figure 4: A graph showing retention time/min versus % MeOH for Ethylbenzene

Sample Calculation of Number of Theoretical plates (N) using benzene (70% methanol)
N=16 (tR) 2/w2
= 16(6.89)2/ (15.6509/60)2
=11163.04

Table 4: Showing the calculated number of theoretical plates (N) for each mixture component

Figure 5: Graph of %MeOH against the number of theoretical plates for Uracil

Figure
6:
Graph
of %MeOH against the number of theoretical plates for Benzene

Figure 7: Graph of %MeOH against the number of theoretical plates for Toluene

Figure 8: Graph of %MeOH against the number of theoretical plates for Ethlybenzene

Showing an example of calculation of theoretical plate height (H) :

(Using benzene with 70% MeOH)

H=L/N

=250mm/18265.01

=0.01387
Table 5: Showing the calculated theoretical plates height (H) for each mixture component
%MeOH

Uracil

Benzene

Toluene

Ethylbenzene

60

0.06823

0.1459

0.01331

0.01054

70

0.01617

0.01387

0.01067

0.009319

80

0.3081

0.4802

0.03312

0.008035

90

0.06780

0.01192

0.009674

0.008949

Figure 9: Graph of %MeOH versus theoretical plate height of Uracil

Figure 10: Graph of %MeOH versus theoretical plate height of Benzene

Figure 11: Graph of %MeOH versus theoretical plate height of Toluene

Figure 12: Graph of %MeOH versus theoretical plate height of Ethylbenzene

Sample Calculation showing the Resolution for each mobile phase

This was done using toluene and ethylbenzene in 70 %MeOH

Rs= 2(tR2-tR1) WA+WB

=2(14.74-10.25) (16.086860)+(21.598260)
=14.30

Discussion
The chromatograms attached under the appendix depict the retention time peaks for each pure
standard compound at 70%MeOH and for the analytes at 60%, 70%, 80% and 90%MeOH.
Acknowledging the differences of each compound structurally, using the chromatograms of
the pure standard samples, it was easy to identify the components of each mixture. The least
polar compound was ethylbenzene and the most polar compound was Uracil. Due to polarity
we are able to tell that uracil eluted first, and then benzene, toluene and finally ethylbenzene.
In the 60% chromatogram the peaks depict wide spaces between them .The peaks for uracil
and benzene are recorded more to the left and seem to sharp and narrow peaks. Benzene and
ethylbenzene have very lengthy peaks as compared to uracil and toluene which are smaller in
height. In the 70 % MeOH, it shows much more evenly spaced out peaks. Uracils peak
seems to be widening whereas the other three compounds peaks are approximately medium
width. In the 80% MeOH, the peaks seem to have shifted to the right and it is also noticed
that there are less spaces between these peaks. The tallest peak in this chromatogram seems to
be toluene whilst uracil and ethyl benzene have approximately average peak heights. Benzene
has the smallest peak height .The peaks discovered in the 90% MeOH are also to the right.
Uracil shows a broad peak whist the other three are narrow.
In table 2, it is noticed that uracil has the shortest retention time of 2.54min during the
evaluation of the 70% MeOH mobile phase. Ethylbenzene had the longest retention time of
14.67 min. A short retention time is directly proportional to polarity and thus the more it
reacts with a polar mobile phase and results in the fastest elution. The cause of this scenario is
lone pairs of electrons present on the oxygen atoms of the uracil compound. It can now be
said that because ethylbenzene showed the longest retention time, it is the non-polar and has
an interaction with the nonpolar stationary phase which allows it to elute the slowest. The
ethyl group which is on the benzene ring is non polar and contains no lone pair electron
which causes the slowest elution. The retention time of the unknown was 6.88mins which is
relatively similar to that of benzenes which was 6.89 mins thus we can assume the unknown
to be benzene.
By variation of the phases by polarity we can optimize the mobile phases. As mentioned, a
60%, 70%, 80% and 90% MeOH solution was prepared. In table 3, the retention times whilst
using these different mobile phases were recorded. Graphs of % MeOH were plotted against
retention times (figures 1-4). In figures 1 and 2, due to the negative gradient of the trend line
we say they are inversely proportional. In figure 3 and 4, it can be said that they are directly
proportionally due to the positive gradient. A decrease in the %MeOH can cause a decrease in
polarity of mobile phase and that elutes slower.

The pi cloud of a benzene ring causes UV absorption in al compounds .This is due to the
delocalization of these pi electrons. The oxygen present in uracil also acts as an absorbing
factor.
Due to how fast uracil elutes and does not stay in the column, it is commonly used in HPLC
techniques and to calibrate the instrument .This is to ensure the instrument function optimally
and gives accurate results during runs. Another reason why uracil may be used for calibration,
is due it responding to may wavelengths.
The number of theoretical plate number (N) were calculated and tabulated in table 4.This was
done for each mobile phase. The results were obtained using graphs (figure 4-5). Figure 5
shows a positive trend line while figures 6, 7 and 8 show a negative trend line. For uracil as
the %meOH increases the number of theoretical plates increase, where as in 6, 7 and 8, it
decreases. Benzene seemed to have shown the lowest value of theoretical plates (520.65) in
the 70% whilst ethylbenzene showed the highest (31112.50) in the 80%
The theoretical plate height was then calculated for each mobile phase. The results were then
plotted on graphs as %MeOH against H (Theoretical plate height)(Figures 9-12) .figures 9 ,
10 and 11 relay negative trend lines whilst figure 12 has a positive trend line. For 9, 10 and
11, if %MeOH increases then H decreases. Benzene shows the highest value of 0.4802 in
80% MeOH and ethyl benzene showed the lowest of 0.009319 in 70%MeOH.
The above results of theoretical plate numbers and heights define the efficiency of the column
with respect to the mobile phase. The higher the value, the more efficient the column is
.Benzene had a low theoretical plate number yet a high plate height but it was the opposite
trend for ethyl benzene. The columns efficiency was found at a higher %MeOH in regards to
number of theoretical plates, but lower %MeOH with regards to plate heights.
In order to calculate whether the mobile phase was optimal, the resolution is calculated. As a
resulted it was calculated to be 14.30.This is suggested to be the optimal mobile phase. No
major shifts or widths were noticed.

Conclusion
In conclusion it can be said that HPLC method can be used efficiently ro separate and
identify compounds. The retention time of a compound that is eluting decrease with an
increasing %MeOH. It can be said that the number of plates decreased as %MeOH increased
for benzene and uracil, but was the opposite for toluene and ethylbenzene .As theoretical
plate number increased it was due to the increase in %MeOH. With regards to theoretical
plate height and uracil and benzene, it is observed that it increases as %MeOH increaases,but
decreased with Increasing %MeOH with toluene and ethylbenzene.70% MeOH was found to
be the optimium mobile phase as its peaks were evenly spaced out. This was also suugested
by the resolution calculation which resulted in 14.30. The unknown compound had a similar
retention time to benzene and therefore is identified as benzene.Other functionally groups
were also identified by its retention time which concludes that the most polar elute first due to
its interaction with the polar mobile phase.

References

1.high Performance Liquid Chromatography HPLC ,date accessed : 12/03/2016

http://lab-training.com/landing/free-hplc-training-programme-5/

2.Reverse phase chromatography - principles and methods, Amersham bioscience, pages

5-13 ,date accessed :9/03/2016
http://wolfson.huji.ac.il/purification/PDF/ReversePhase/AmershamRPCManual.pdf

3..Acentix C18 HPLC columns ,Date accessed :09/03/2016

http://www.sigmaaldrich.com/analytical-chromatography/hplc/columns/ascentis-hplccolumns/c18.html

Aim: The practical is aimed at obtaining the amount of manganese in steel by the standard
addition method of spectrophotometry and to analyse manganese by Atomic absorption
spectrophotometry (AAS).

Principle:
Chemicals of every nature transmits absorbs or reflects light at a certain wavelength.
Spectrophotometry uses a spectrophotometer to measure how much of light gets absorbed by
the sample. The basic operation of this instrument is that it allows a beam of light to pass
through a sample and a photodetector records its intensity of the light. The light contains a
stream of photons. The sample absorbs the photon as its being passed through it which causes
a reduction of the photons which is equivalent to reducing the intensity of light.
There are two parts to the spectrophotometer, it consists of a spectrophotometer that produces
the light of any wavelength and a photometer which measures the lights intensity. When this
light passes through sample it causes a relationship between the concentration of the solute
and the light intensity. This is referred to as Beers Law. The range of the wavelength of the
light can be classified into two different types. UV-visible spectrophotometer uses light of the
UV range and visible range of EM radiation spectrum. Theres also IR spectrophotometer
which uses light over the infrared range.
The spectrophotometer is made up of many different components such as a lamp, a
photodetector, and a monochromator. .A lamp which can be deuterium or tungsten
(depending on the wavelength) acts as the light source .The lights enters the diffraction
grating which behaves like a prism and acquires components of the wavelengths by
separating the light. A specific wavelength reaches the slit in order to exist only by rotating
the grating .The light is then allowed to interact with the sample. The detector measures how
much of the sample gets absorbed and how much gets transmitted. Transmittance refers to the
amount of light that passes completely through the sample and makes it to the detector .The
detector then converts this to the read out.

Atomic absorption spectroscopy is a quantitative analysis of a chemical species by measuring

the absorption of the radiation by the free atoms in its gaseous state. AAS can measure the
concentration in minute amounts such as parts per billion. The basis of this technique is
wavelengths of light that are specified for certain elements. When a sample is analysed, it
uses light from the element and passes it through the sample. The sample is converted to its
ground state that contains free atoms in its certain vapour state. The radiation is passed
through the gaseous sample from the atoms in its excited state. The element in the sample

then absorbs the radiation given off .The greater number of atoms there is, the more absorbed
radiation there is.

References
1. Date accessed: 09/03/2016
http://www.chm.davidson.edu/vce/spectrophotometry/Spectrophotometry.html

2. Date accessed: 12/03/2016

http://chemwiki.ucdavis.edu/Core/Physical_Chemistry/Spectroscopy/Electronic_Spectroscop
y/Electronic_Spectroscopy%3A_Application

3. Date accessed: 09/03/2016

.http://faculty.rmu.edu/~short/chem3550/chem3550-references/RSC-AA-Leaflet.pdf