CH251 Instrumental

Analysis
Determination of the concentration of methyl paraben and
propyl paraben using high-performance liquid
chromatography (HPLC)
Dunie Navarro, James Yu


10/15/12
Prof. Ruben Savizky

Abstract
Two calibration curves from standard solutions of methyl paraben and propyl paraben were
generated from high-performance liquid chromatography analysis. Solutions of 20ppm, 40ppm, 60ppm
and 100ppm of the compounds were submitted for identification of methyl and propyl parabens and
construction of the calibration plot. Chromatographic measurements were recorded using a Waters 1525
Binary HPLC Pump and Waters 2487 Dual Absorbance Detector. The chromatographic analysis was
performed using a reverse-phase HPLC. The mobile phase was (75:25) CH
3
OH/H
2
O. The flow rate used
was to 0.75 mL/min with an injection volume of 20 L. The quantitative determination of the parabens
was performed at = 254nm. The retention times of methyl and propyl parabens were 2.74 and 4.00 min,
respectively. The separation factor for the two compounds was = 1.46. Unknown #1069 was submitted
for HPLC analysis and reported concentrations of 56 6.0 and 74 8.0 ppm for methyl and propyl
parabens, respectively.
Introduction
High performance liquid chromatography (HPLC) is an essential analytical tool for separating
and quantifying components in complex liquid mixtures. HPLC has several advantages over other
methods, and is consequently a widely used technique for research. HPLC is applied in many different
fields including energy industries, food, cosmetics, pharmaceuticals and environmental conservation.
Liquid chromatography consists of multiple separation modes such as adsorption
chromatography, reversed-phase chromatography, liquid-liquid partition chromatography,
chromatography with chemically bonded phases, ion-exchange chromatography, ion-pair
chromatography, ion chromatography, size-exclusion chromatography, and affinity chromatography. [1]
This experiment is concerned with reversed-phase chromatography. In reversed-phase chromatography, a
relatively non-polar compound with a high specific surface area is used as the stationary phase. The
mobile phase is relatively polar (water to dioxanes). [2] The different extents to which a variety of
molecules in the mixture are adsorbed in the stationary phase provide the separation effect.
Information obtained from a chromatographic process is presented by a chromatogram, which are
measurements of the concentration of the sample compounds. The chromatograms (see Appendix) are
series of patterns (normal Gaussian curves) of compounds generated by a detector, which senses the
change in concentration of the individual compound at the end of the column as a function of the time (or
volume of the mobile phase). Additionally, it provides information about separated components in the
mixture, peak area, height and retention times in the column.
Using a chromatogram, two kinds of analysis can be carried, qualitative and quantitative. For
qualitative purposes, the retention time of a component can be recorded since it is constant under identical
chromatographic conditions. A peak can be identified by injecting the relevant substance and comparing
the retention time to standards. In the case of quantitative analysis, the area of a peak is proportional to
the amount of a compound injected. Using peak areas from compounds prepared at known concentrations,
a calibration graph can be prepared to find the amount of a component in a solution.
The aim of the present experiment is to determine the concentration of two compounds, methyl
and propyl parabens, in an unknown solution. [3] Parabens are generally used as preservatives by
pharmaceutical and cosmetic industries. The chemical structures of the compounds are presented below.
OH
O
CH
3
O
OH
O
CH
3
O

methyl paraben propyl paraben
To determine the concentration of each of the compounds in an unknown from a calibration curve
of standard solutions the following formula is used
Concentration of the analyte =

(1)
where A is the peak area of the analyte in unknown solution, I is the y-intercept of the calibration curve
and m is the slope of the calibration curve.
The time between sample injection and recording of the peak maximum is known as retention
time, t
R
. Two compounds can be separated if they have different retention times. Net retention times are
given by
t

R
= t
R
t
o
(2)
where t
o
is an identical retention time representing the mobile phase retention time.
Two components in a mixture cannot be separated unless they have a relative retention, , greater
than 1. Relative retention is a measure of the chromatographic systems potential for separating the two
compounds. Relative retention or separating factor is given by
=

(3)
where t
R2
and t
R1
are the respective retention times of the components.














Procedure
Unknown #1069 and samples of methyl paraben (C
8
H
8
O
3
) and propyl paraben (C
10
H
12
O
3
) were
obtained from the stockroom. Using a digital weight balance, 0.0102 g of C
8
H
8
O
3
and 0.0101 g of
C
10
H
12
O
3
were weighed to prepare two solutions of 100ppm from each compound. A 25:75 parts solution
of HPLC grade water (H
2
O) and HPLC grade methanol (CH
3
OH) was prepared in a 500mL volumetric
flask using a 100mL volumetric cylinder and a 10mL pipette to measure volume. The parabens samples
were each dissolved separately using the stock solution in two 100mL volumetric flasks. The solutions
were mixed thoroughly. The parabens solutions and the unknown were carried to the HPLC station.
HPLC Conditions
Chromatography was performed using a Waters 1525 Binary HPLC Pump and Waters 2487 Dual
Absorbance Detector. The chromatographic analysis was performed using a reverse-phase HPLC. The
mobile phase was (75:25) CH
3
OH/H
2
O. The flow rate was adjusted to 0.75 mL/min and an injection
volume of 20 L was required. The quantitative determination of the parabens was performed at =
254nm by running the UV detector in single wavelength mode. In the Breeze software the run time was
set to 10 min to record each sample peaks.
Quantitative determination of products
A 100ppm methyl paraben injection was loaded by injecting 20 L of the sample in the HPLC
port after clicking Injection in the software. The peaks were recorded in the computers interface and the
peaks area was computed using the software integrator. The same procedure was followed for the
100ppm propyl paraben and the unknown sample. Using the peaks area of the unknown, standard
solutions were prepared to cover the range of the peaks area in order to construct a calibration curve.
Two standard solutions of 20:20 ppm and 40:40 ppm of methyl paraben and propyl paraben were
prepared from the 100 ppm solutions by mixing with appropriate volumes in 5 mL volumetric flasks.
Samples of 60 ppm of each paraben were additionally prepared separately in 5mL volumetric flasks. Each
of the standard solutions was analyzed by the HPLC; their peaks area was computed and recorded. The
calibration plots were constructed by plotting the peak areas of methyl paraben and propyl paraben versus
their concentrations.




Results and Discussion
Peaks area values found using the HPLC integrator are presented in Table 1 for methyl paraben
and propyl paraben at 20ppm, 60ppm and 100ppm concentrations. Peaks area is computed in contrast to
peaks height because it results in better quantitative approximations of the concentration in the sample.
Table 1 Peaks area for methyl paraben and propyl paraben

() ( ) ( )
20 2425728 2239674
60 8855926 8593502
100 14865864 15122648

The data above was plotted and calibration curves were obtained in order to calculate the concentration of
the components in the unknown solution. A calibration curve for methyl paraben is plotted in Fig 1 below
resulting in almost a perfect fit of the data. The equation of the calibration curve can be stated as P
A
=
155502C 614263, where P
A
is the peak area and C is the concentration.
Fig 1 Calibration curve of methyl paraben

y = 155502x - 614263
R = 0.9996
0
2
4
6
8
10
12
14
16
0 20 40 60 80 100 120
P
e
a
k

A
r
e
a

(

s
)


M
i
l
l
i
o
n
s

Concentration (ppm)
Calibration Curve of Methyl Paraben
Analogously, a calibration curve was computed for propyl paraben from the peaks area and
concentrations. The curve represents a good fit of the data for the component and is plotted in Fig 2.
Similarly, the calibration curve can be stated as P
A
=161037C 1000000 where P
A
is the peak area and
C is the concentration of the component.
Fig 2 Calibration curve of propyl paraben


Unknown #1069 was analyzed using the calibration curves and respective peak areas for methyl
paraben and propyl paraben. The calibration curves were isolated to calculate concentrations from peaks
area. The results of the concentrations from each component in the unknown are presented in Table 2
below.
Table 2 Concentrations in Unknown #1069
Unknown Analysis
( ) ()
8066299 56 6.0
10872416 74 8.0


y = 161037x - 1E+06
R = 0.9999
0
2
4
6
8
10
12
14
16
0 20 40 60 80 100 120
P
e
a
k

A
r
e
a

(

s
)


M
i
l
l
i
o
n
s

Concentration (ppm)
Calibration Curve of Propyl Paraben
Retention times for methyl paraben and propyl paraben were recorded as a mean to characterize
the variation of the HPLC conditions. The peaks of the samples in the unknown were identified by
comparing its peaks to the solutions of known concentrations. The retention times for methyl paraben and
propyl paraben are presented in Table 3.
Table 3 Retention time of compounds in solution samples
Retention time (min)


2.74 4.00

2.74 3.99
2.72 3.99
2.75 4.02
2.75 4.01
2.75 4.01
2.75 4.00
2.74 4.00
0.01 0.01
0.02 0.01

The results above confirm that variations in the HPLC conditions are reduced to a minimum and barely
affect retention times for compounds. The peaks of the unknown are easily identified from the data
presented, confirming the presence of methyl paraben and propyl paraben in the unknown solution. The
relative retention or separation factor for the two compounds is = 1.46, indicating a successful
separation of the compounds.
Error Discussion
The results from R
2
reported a good fit for the calibration curves of methyl paraben and propyl
paraben. However the uncertainty in the concentration calculated from peaks area was assessed by
calculating the uncertainty in the fit of the line. The correlation coefficients were in average 0.99. Each of
the concentrations calculated carry an uncertainty of
C
= 5 ppm and
C
= 7 ppm from the fit procedure
for methyl paraben and propyl paraben respectively. The errors were supported due to the evaluation of
presence of remaining solution in the HPLC column from the previous injection every time a run was
done. The measurements for the 40 ppm concentrated solutions were discarded after being considered
outliers in the data, affected by a previous injection and low peak areas.
The use of volumetric glassware and pipets provide another source of error. From a 100mL
volumetric flask, a tolerance of 0.08 mL is present while for a 500mL a tolerance of 0.20 mL is
reported. At small concentrations in the range of 20-100ppm, volumetric uncertainties become important.
Additionally, weighing of the compounds powder in the digital weight balances carries an error of
0.0001 g for each of the samples. The propagation of error from these uncertainties was calculated in the
appendix providing an uncertainty in concentration of 1 ppm for methyl paraben and propyl paraben.
The final uncertainty on the concentration is found by considering the influence of volumetric
glassware and line fit. The reported uncertainty was 6ppm and 8ppm for the concentrations from
calibration curve procedure for methyl paraben and propyl paraben.














Conclusions
Determination of the concentration of methyl and propyl parabens in an unknown solution was
carried by HPLC analysis. A calibration curve for each compound was constructed from standard
solutions of increasing concentrations. The equation describing the calibration curve was used to solve for
concentrations from recorded peaks area. Unknown #1069 was analyzed using HPLC and reported
concentrations of 56 6.0 and 74 8.0 ppm for methyl and propyl parabens, respectively. This
experiment introduced the principles of HPLC for the determination of solute concentrations in organic
and inorganic solutions. Additionally, successful compounds separation was accomplished at low
retention times confirming the advantages of HPLC to separate a mixture of liquids.











References

[1] Meyer, Veronika R. Practical High-Performance Liquid Chromatography. New York: John Wiley &
Sons, Inc, 1994.
[2] Skoog, Douglas A, F. James Holler and Stanley R. Crouch. Principles of Instrumental Analysis.
California: Thomson Brooks/Cole, 2007.
[3] Newmark, Andrea. "High Performance Liquid Chromatography" n.d. Cooper Union Moodle. 8
October 2012 <https://moodle.cooper.edu>.
[4] Taylor, John R. An Introduction to Error Analysis . Sausalito, CA: University Science Books, 1997.
[5] Skoog, Douglas A., Donald M. West and James F. Holler. Fundamentals of Analytical Chemistry.
Orlando: Harcourt, Inc, 1997.












Acknowledgments
The authors would like to thank Prof. Ruben Savizky for teaching them how to use the HPLC
instrumentation and software, and Victoria Heinz and the stockroom staff for providing the chemicals and
glassware used during the experiment.















Appendix I- Sample Calculations
1. Calculations of input concentrations into spectrophotometer.
Two standard solutions of 100ppm of methyl paraben and propyl paraben were prepared from the
compounds powder and diluted in 25:75 parts of H
2
O and CH
3
OH. The required weight to prepare the
solutions was calculated as follows:

()

2. Calculations of the concentration of methyl paraben in unknown solution
The calibration curve obtained from plotting the standard solutions peaks area versus concentration
allowed to calculate the concentration of methyl paraben in the unknown solution. The equation is given
by P
A
=155502C 614263, where P
A
is the peak area and C is the concentration. To solve for
concentration, C is isolated as C = (P
A
+ 614263)/155502:

3. Calculations of the concentration of propyl paraben in unknown solution
The concentration of propyl paraben in the unknown solution was computed from the peak area versus
concentration calibration graph. The equation is given by P
A
=161037C 1000000 where P
A
is the peak
area and C is the concentration of the component. To compute the concentration, C is isolated as C = (P
A

+ 1000000)/161037:

4. Calculations of the separation factor of the mixture
Relative retention is a measure of the chromatographic systems potential for separating the two
compounds. Relative retention or separating factor is calculated by

Appendix - Error Propagation
)
To determine the uncertainty associated with the random error given in the measurements of
concentrations from peaks area, the formula provided below can be used; it is assumed that each value of
is normally distributed about its true value

. [4]

represents the - of the best-fit line, and represents the slope of the best fit line. The
uncertainty as a result of random error can therefore be determined by substituting the data into the above
expression. For the HPLC measurements of methyl paraben, :

*,( ()-

,( ()-

The peak area values have an uncertainty of 171570 calculated from concentration measurements. Using
the equation for the calibration curve, the uncertainty,
x
, on the concentration is found be
x
= 5 ppm.

)
To determine the uncertainty associated with the random error given in the measurements of
concentrations from peaks area, the formula provided below can be used; it is assumed that each value of
is normally distributed about its true value

. [5]

represents the - of the best-fit line, and represents the slope of the best fit line. The
uncertainty as a result of random error can therefore be determined by substituting the data into the above
expression. For the HPLC measurements of propyl paraben, :

*,( ()-

,( ()-

The peak area values have an uncertainty of 73754 calculated from concentration measurements. Using
the equation for the calibration curve, the uncertainty,
x
, on the concentration is found be
x
= 7 ppm.
)
To determine the uncertainty associated with the 100mL volumetric flask and the weight balance, the
expression to calculate concentration from measured weights is implemented.
The tolerance in the 100mL volumetric flask is given by V
vf
= 0.08 mL while the tolerance in the weight
balance is w = 0.0001g.
To confirm the concentration needed the formula below is used from the measured weight [5]:

()
()

From the uncertainties above and the formula for concentration, the uncertainty in concentration would be