High Performance Liquid Chromatography

Performed By: Derek Hafstad

Partner: Jacob McClaskey
Date Performed: April 12, 2018
Date Submitted: May 3, 2018

Abstract:

High performance liquid chromatography was used to analyze a mixture of anisole,

benzaldehyde, and toluene. This was done to understand the effects of polarity, solvent strength, and

selectivity in reverse phase separations. The ideal composition of the mobile phase was determined to

be 70% methanol and 30% water. The peaks in the mixed standard were identified using spiked

solutions. It was determined that benzaldehyde, anisole, and toluene had retention times of 11:48,

18.38, and 34.63 minutes, respectively. Reverse phase HPLC separation resulting in well resolved peaks

for these compounds with a resolution of 3.01 between the benzaldehyde and anisole peaks and 4.05

between the anisole and toluene peaks. Both of which are above 1.5, so they are considered well

resolved.
Introduction:

High Performance Liquid Chromatography (HPLC) is a form of liquid chromatography that was

created during the 1960s and 1970s. This technique separates a mixture into its individual components

in order to identify and quantify them. (Thammana, 2016) A mobile phase is pumped through a

stationary phase column to achieve separation. Each component has a different retention time due to

their different affinity for the column and mobile phase. (Thammana, 2016) HPLC has the advantage

over gas chromatography of being able to analyze nonvolatile compounds. (Sundaram)

A schematic diagram for a HPLC system is shown in Figure 1. Solvent reservoirs contains the

mobile phase which may be a mixture of polar and nonpolar solvents. A pump forces the mobile phase

out of the solvent reservoirs at high pressure to produce reproducible and constant amounts at a

constant flow rate through a stationary phase. (Sundaram) Microliter volume samples are injected using

a loop injector. (Thammana, 2016) The sample in the mobile phase is then passed through an HPLC

column containing a stationary phase with particle sizes of 3 um to 10 um.(Thammana, 2016) A UV,

fluorescence, or mass spectrometer detector then gives a signal response proportional to

concentrations as each analyte elutes from the column.(Thammana, 2016)

Figure 1: Schematic diagram of a HPLC system (Czaplicki, 2013)

 From a chromatogram, you can determine the half width or the width at the bottom of the

peak, which are related by the equations: (Hallock-Waters)

Eq.1 and 2

W is the width at the base in minutes. tr is the retention time of the peak in minutes. W1/2 is the width of

the peak at half height in minutes. From the width at the base of the peak, the resolution can be

determined through the equation: (Hallock-Waters)

Rs = 2[(tr)B-(tr)A]
WA + WB Eq. 3

tr is retention time in minutes and W is the width in minutes at the base. Well separated peaks will have

a resolution greater than 1.5. (Hallock-Waters)

This experiment involved using reverse phase high performance liquid chromatography to

determine an optimal mobile phase composition to separate a mixture of anisole, benzaldehyde, and

toluene.

Experimental Procedure:

Preparation of Solutions Solutions of 10000 ppm anisole, toluene, and benzaldehyde standards

were prepared by adding 100.0 uL of pure anisole, toluene, and benzaldehyde to 10.0 mL volumetric

flasks and diluting to the mark with HPLC-grade methanol. One milliliter of each of these standards was

added to separate 10.0 mL volumetric flask and filled to the mark with HPLC-grade methanol to create

1000 ppm anisole, toluene, and benzaldehyde standards. Two hundred and fifty microliters of the 1000
ppm standards were added to 10.0 mL volumetric flasks and filled to the mark with HPLC-grade

methanol to prepare 25.0 ppm anisole, toluene, and benzaldehyde solutions. Two hundred and fifty

microliters of each 1000 ppm anisole, toluene and benzaldehyde were added to the same 10.0 mL

volumetric flask and diluted to the mark with HPLC-grade methanol to create a 25.0 ppm mixed

standard. All solutions were filtered through a 0.45 um PTFE syringe filter.

Analysis of Solutions Solutions were analyzed on a Dionex UltiMate 3000 HPLC system. An

Acclaim 120 4.6mm x 250mm C18 column with a particle size of 5 um and pore size of 120 Å was used

for the separation. The mobile phase flow rate was 0.500 mL/min and column temperature of 25

degrees Celsius. The injection volume was 25.00 uL. Absorbance was measured at 254 nm and 280 nm.

Results/Discussion:

The standards were first analyzed over several trials while changing the polarity of the mobile

phase. The first trial started with a 70% methanol and 30% water mobile phase and proceeding trials

were performed while increasing the polar component, H2O, in 5% increments, and decreasing the non-

polar component, CH3OH, by 5%. These trials show the effect of polarity of the mobile phase on the

retention of the components. The best solvent mixture was determined to be 70% methanol and 30%

water due to the sharp peak resolutions and the short analysis times. Increasing the polar component,

H2O, by 5% resulted in the most retained component eluting at a time longer than total analysis time.

When the mobile phase was 35% methanol and 65% water, the elution carried over to the next trial.
Figure 2: An example of a chromatogram

The individual peaks were identified by analyzing the mixed standard as well as standards spiked

with benzaldehyde, toluene, and anisole separately. The mixed standard contained peaks at 11.48

minutes, 18.38 minutes, and 34.63 minutes. The solution with a benzaldehyde spike showed a peak at

16.88 minutes. The solution spiked with toluene showed a peak at 39.29 minutes, while the solution

with an anisole spike had a peak at a retention time of 21.79. From these, it was determined that the

peak at a retention time of 11.48 minutes in the mixed solution was benzaldehyde. The peak at a

retention time of 18.38 minutes was determined to be anisole, and the peak at a retention time of 34.63

minutes was toluene. The resolutions between the benzaldehyde and anisole peaks was determined to

be 3.01, and the resolution between the anisole and toluene peaks were determined to be 4.05. Since

both resolutions are above 1.5, the peaks are well resolved.

The column used for this experiment was a reverse phase HPLC column, and it produced well

defined peaks. Other columns can result in different retention times. Another column that is useful for

hydrophobic compounds are mixed-mode HPLC columns.(HPLC) If the peaks are not well resolved, a

change in column or flow rate could result in more resolved peaks. Changing the composition of the

polar or non-polar components of the mobile phase will also increase peak resolution.
Conclusions:

The ideal composition of the mobile phase for the separation of anisole, benzaldehyde, and

toluene on a non-polar C18 column was determined to be 70% methanol and 30% water. The peaks in

the mixed standard were identified using spiked solutions. It was determined that the peak at a

retention time of 11.48 minutes in the mixed solution was benzaldehyde. The peak at a retention time

of 18.38 minutes was determined to be anisole, and the peak at a retention time of 34.63 minutes was

toluene. The resolutions between the benzaldehyde and anisole peaks was determined to be 3.01, and

the resolution between the anisole and toluene peaks were determined to be 4.05. Since both

resolutions are above 1.5, the peaks are well resolved. It shows that a reverse phase HPLC column

creates well resolved peaks for the aromatic compounds anisole, benzaldehyde, and toluene.
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Hallock-Waters, Dr. Kristen A. 4/11/2018 Introduction to Chromatographic Separations 2, Blackboard,

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