CH251 Instrumental Analysis

Determination of the concentration of methyl paraben and propyl paraben using high-performance liquid chromatography (HPLC)

Dunie Navarro, James Yu

10/15/12Prof. 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 λ Absorbance Detector. The chromatographic analysis was

performed using a reverse-phase HPLC. The mobile phase was (75:25) CH3OH/H2O. The flow rate used

was to 0.75 mL/min with an injection volume of 20 μL. The quantitative determination of the parabens

was performed at λ = 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 α = 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.

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 = A−I

m

(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, tR. Two compounds can be separated if they have different retention times. Net retention times are

given by

t’R = tR – to (2)

where to 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, α, greater

than 1. Relative retention is a measure of the chromatographic system’s potential for separating the two

compounds. Relative retention or separating factor is given by

α = tR2−t o

tR1−t o (3)

where tR2 and tR1 are the respective retention times of the components.

Procedure

Unknown #1069 and samples of methyl paraben (C8H8O3) and propyl paraben (C10H12O3) were

obtained from the stockroom. Using a digital weight balance, 0.0102 g of C8H8O3 and 0.0101 g of

C10H12O3 were weighed to prepare two solutions of 100ppm from each compound. A 25:75 parts solution

of HPLC grade water (H2O) and HPLC grade methanol (CH3OH) 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

λ Absorbance Detector. The chromatographic analysis was performed using a reverse-phase HPLC. The

mobile phase was (75:25) CH3OH/H2O. The flow rate was adjusted to 0.75 mL/min and an injection

volume of 20 μL was required. The quantitative determination of the parabens was performed at λ =

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 μL of the sample in the HPLC

port after clicking Injection in the software. The peaks were recorded in the computer’s 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

Methyl paraben Propyl ParabenConcentration ( ppm) Peak Area(μV · s) Peak Area (μV · s)

20 2425728 223967460 8855926 8593502100 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 PA = 155502·C – 614263, where PA is the peak area and C is the concentration.

Fig 1 Calibration curve of methyl paraben

10 20 30 40 50 60 70 80 90 100 1100

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

f(x) = 155501.7 x − 614262.666666666R² = 0.99961972356155

Calibration Curve of Methyl Paraben

Concentration (ppm)

Peak

Are

a (μ

V · s

)

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 PA = 161037·C – 1000000 where PA is the peak area and C is the concentration of the component.

Fig 2 Calibration curve of propyl paraben

10 20 30 40 50 60 70 80 90 100 1100

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

f(x) = 161037.175 x − 1010289.16666667R² = 0.999938273352303

Calibration Curve of Propyl Paraben

Concentration (ppm)

Peak

Are

a (μ

V · s

)

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 AnalysisComponent Peak Area(μV · s) Concentration( ppm)

Methyl paraben 8066299 56 ± 6.0Propyl Paraben 10872416 74 ± 8.0

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)Methyl paraben Propyl Paraben

2.74 4.002.74 3.99

Unknown 2.72 3.992.75 4.022.75 4.012.75 4.012.75 4.00

Mean 2.74 4.00Standard Deviation 0.01 0.01

Deviation of Unknown 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 α = 1.46, indicating a successful

separation of the compounds.

Error Discussion

The results from R2 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 σC = 5 ppm and σC = 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 H2O and CH3OH. The required weight to prepare the solutions was calculated as follows:

mg of solute= required concentration( ppm)106 ×volume of flask

100 ppm106 ×100 mL=0.01 gof solute required

¿¿

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 PA = 155502·C – 614263, where PA is the peak area and C is the concentration. To solve for concentration, C is isolated as C = (PA + 614263)/155502:

C of methyl paraben∈unknown solution=Peak area of m. p∈unknown+614263155502

C of methyl paraben∈unknown solution=8066299+614263155502

=56 ppm

¿¿

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 PA = 161037·C – 1000000 where PA is the peak area and C is the concentration of the component. To compute the concentration, C is isolated as C = (PA + 1000000)/161037:

C of methyl paraben∈unknown solution=Peak area of p . p∈unknown+1000000161037

C of methyl paraben∈unknown solution=10872416+1000000161037

=74 ppm

¿¿

4. Calculations of the separation factor of the mixture

Relative retention is a measure of the chromatographic system’s potential for separating the two compounds. Relative retention or separating factor is calculated by

α=tR2−to

tR1−to=4.00−0

2.74−0=1.46

Appendix II - Error Propagation

1¿ Uncertainty∈the concentrationof methyl paraben∈unknown solution

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 y is normally distributed about its true value A+B xi. [4]

σ y=√ 1N−2∑ ( y i−A−B x i)

2

A represents the y-intercept of the best-fit line, and B 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, N=3 , A=−614262 , B=155502:

σ y=√ 13−2

{¿¿

σ y=171570

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

2¿ Uncertainty∈the concentrationof propyl paraben∈unknown solution

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 y is normally distributed about its true value A+B xi. [5]

σ y=√ 1N−2∑ ( y i−A−B x i)

2

A represents the y-intercept of the best-fit line, and B 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, N=3 , A=−1000000 , B=161037:

σ y=√ 13−2

{¿¿

σ y=73754

The peak area values have an uncertainty of 73754 calculated from concentration measurements. Using the equation for the calibration curve, the uncertainty, σx, on the concentration is found be σx = 7 ppm.

3¿ Uncertainty∈the concentrationof 100 ppm standard solutions

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 δVvf = 0.08 mL while the tolerance in the weight balance is δw = 0.0001g.

To confirm the concentration needed the formula below is used from the measured weight [5]:

C= 106

V vf (ml)× w of solute (g )

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

δCC

=√( δ V vf

V f) 2+( δw

w )2

δC100 ppm

=√( 0.008 ml100 ml ) 2+(0.0001 g

0.01 ) 2

δC=1.0 ppm