Chemistry 421 Advanced Instrumental Analysis - Spring Semester 2010

Lab 6: High Performance Liquid Chromatography (HPLC) Required time: 3 Lab periods

I.

Overview

Chromatographic techniques are used to separate the components of a mixture before detection and analysis, and can be used to determine the composition of a mixture and the quantity of individual chemical compounds within a mixture. An example would be to determine how much caffeine there is in a cup of coffee. High Performance Liquid Chromatography (HPLC) is an analytical technique for the separation and determination of organic and inorganic solutes in samples. The technique is particularly applicable to biological, pharmaceutical, food, environmental, and industrial analyses. Chromatographic techniques provide both qualitative and quantitative information. The quality of the analysis is determined by how well separated the analytes are and how efficient the separation process is. In order to obtain as much analytical information as possible from a chromatographic separation, it is important to understand the theory and practice of operation.

Figure 1: Illustration of the basic chromatographic process. All chromatographic separations utilize the same basic approach. The sample (a mixture of the analytes and other compounds, including a solvent) is introduced into a flowing stream called the mobile phase. The mobile phase carries the sample through a column that contains a second phase, the stationary phase, which is fixed in place. The analytes partition between the mobile and stationary phases, and those analytes with stronger attraction to the stationary phase 20

Chemistry 421 Advanced Instrumental Analysis - Spring Semester 2010

take longer to travel through the column. A detector that responds to the analytes is placed at the downstream end of the column. The detector monitors the concentration of the analytes as they elute from the column, providing a chromatogram. This process is illustrated in Figure 1. Further information on chromatography can be found in Chapter 26 in Skoog, and from Harris, Chapter 23. The various forms of chromatography are generally classified by the nature of the mobile phase. Gas chromatography utilizes a gas as the mobile phase, while liquid chromatography utilizes a liquid mobile phase. Reversed-phase chromatography, which is the most popular form of HPLC and is the mode that will be employed in this lab, utilizes a non-polar stationary phase and a polar mobile phase. Other modes of liquid chromatography include normal phase (polar stationary phase), ion exchange, and size exclusion chromatography. Further information on liquid chromatography can be found in Chapter 28 of Skoog, from Chapter 25 in Harris.

Figure 2: Schematic of an HPLC system. HPLC instruments consist of a pump, injector, column, detector, and data analysis and control system (Figure 2). The pump is used to force the liquid mobile phase through the column and past the detector. The injector is used to introduce the sample into the flowing stream. The analytes in the sample are (hopefully) separated as they travel through the column, and are then detected by the detector. All of the components are controlled by a computer, which also collects, stores and analyzes the signal from the detector. In HPLC, the column is packed with very small particles (3-10 m), and high pressures are required to pump the solvent through the system. Thus the pump and all plumbing are designed to generate and withstand high pressure (up to 6000 psi). The injector must also be engineered to allow the sample to be introduced into a flowing stream at very high pressures. The detector on the system to be used in this exercise is a UV/Vis diode array absorbance detector. The system to be used also has two solvent

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Chemistry 421 Advanced Instrumental Analysis - Spring Semester 2010

reservoirs, and is equipped with an electronically controlled metering system to control the composition of the mixed solvent. A typical chromatogram is illustrated in Figure 3. Important data that can be obtained from the chromatogram are the retention times (tr), peak widths (w), and peak areas. Retention times can be used to identify compounds, as each compound will (hopefully) have a different affinity for the stationary phase and a different retention time. Peak widths determine the quality, or efficiency, of a separation. Narrow peaks are advantageous because this allows the separation of compounds with similar retention times. The peak area can be used to determine the quantity or concentration of a given analyte in the mixture. Important figures of merit for a chromatographic separation are the retention factor (k), the selectivity (), the resolution between peaks (Rs), and the plate number or efficiency (N):
t t k= r 0 ; t0 k = 2 ; k1 t r 2 t r1 ; Rs = (w1 + w2 ) / 2

tr t N = 16 r = 5.545 w w 1/ 2

t0 Area, A

solvent

w
Analytes

Figure 3: Typical chromatogram.

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Chemistry 421 Advanced Instrumental Analysis - Spring Semester 2010

In reversed-phase HPLC, the stationary phase is lipophylic (hydrophobic or nonpolar), and the mobile phase is a mixed aqueous/organic solvent. The more lipophylic, nonpolar or hydrophobic an analyte is, the stronger is its interaction with the stationary phase. Thus, solutes generally elute from the column with the most polar first and the most nonpolar last. This is illustrated in Figure 4. Additionally, the affinity of all solutes for the stationary phase will be affected by the composition of the mobile phase. As the fraction of organic solvent (acetonitrile in the current exercise) is increased and the fraction of water is decreased, the affinity of all compounds for the stationary phase will be reduced and the retention times will become shorter. A gradient of solvent composition with increasing fraction of organic solvent can be used to elute strongly retained solutes.

Figure 4: Expected elution order in reversed-phase HPLC.

In this exercise, the separation of three parabens and a phthalate will be optimized. Parabens are common preservatives used in cosmetics, and phthalates are common plasticizers used to give polymeric materials greater flexibility. Phthalates are partly responsible for the socalled new car smell. The structures of the compounds to be separated are given in Figure 5.
A
OH

OPr OPr

R O O

Figure 5: Structures of A. Parabens (R=Me, Et, Pr) and B. diethyl phthalate (replace Pr with Et).

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Chemistry 421 Advanced Instrumental Analysis - Spring Semester 2010

II.

Objectives

Your objectives are to learn the basics of liquid chromatography and to learn how to operate the Hewlett Packard 1090 HPLC. The separation of four compounds will be optimized by both isocratic and gradient elution methods. The compounds will be identified using your knowledge of reversed phase HPLC and the diode array detector. Various figures of merit will be calculated for the separations. The HPLC will then be used to perform a qualitative and semi-quantitative analysis of an unknown.
III. Procedure

A.

Using the UV-VIS, obtain a UV spectrum of 5-10 ppm ethyl paraben and diethyl phthalate in 40% acetonitrile/60% water. Be sure to use 40/60 acetonitrile/water for the blank. Note differences in the spectra, and record the best wavelengths for the analysis of parabens (they have very similar spectra) and phthalate. Use the peak maxima as the choice for the analytical wavelengths (2 wavelengths) in the method below. Starting the HPLC 1. Make sure the instrument is turned on, and that the air and He tanks are turned on. Open the valve on the back of the instrument and purge the solvent reservoirs with helium for several minutes. Start the Chemstation software. Once the software has started, and the solvents have been purged with He, load the first method that you will use, and turn the instrument on. From the pump control window, set the flow to 0.1 mL/min and wait about 30 minutes for the lamp to warm up. When the lamp is warmed up, the baseline on the plot should stop drifting. You will use the manual inject module on the front of the instrument. I will show you how to inject sample. Several methods have been stored in the system with names such as CH342_##, where the ## indicates the percentage of acetonitrile that the method uses. To conduct an experiment, load the appropriate method and edit it. Be sure to confirm parameters such as the flow rate (1.0 mL/min), percent B (acetonitrile), wavelengths of detection (choose 2), and stop time. If you have not changed the %B from that indicated in the file name, you can save it under that name. If you do change the %B, save the method under a different name. The injection volume should be between 5 and 10 L.

B.

2. 3.

C.

Develop a method

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Chemistry 421 Advanced Instrumental Analysis - Spring Semester 2010

1.

Make separate injections of the blank and the mixed standard using 80% acetonitrile as the mobile phase and a run time of about 5 minutes. Put in the lab directory. Any differences between the two chromatograms are due to analytes in the sample. Since there are four compounds, the optimized separation should have 4 peaks in addition to those observed for the blank. If this initial separation does not have four analyte peaks, it is most likely because the concentration of acetonitrile is too high. Perform additional separations using 20% and 60% acetonitrile. Repeat the injections and note any differences in the chromatograms. Print and save the chromatograms (in ASCII text files) at both selected wavelengths. Include a copy of the chromatograms and the conditions in your report. There will most likely be a long period between the first three peaks and the last peak. This is classic situation (the general elution problem) that calls for a gradient elution method. Load the CH342_grd method and edit it. Make some educated guesses about what the initial and final fractions of acetonitrile should be. Keep in mind that a) the initial and final fractions should be as close as possible to facilitate re-equilibration before the next injection and b) the rate of programming should not exceed 10%/min. Be sure to program a return to the initial condition and a post time for the column to re-equilibrate before the next injection. Optimize the separation by trial and error. Include a copy of the optimized separation and the conditions used in your report.

2. 3.

4.

D.

Run the mixed standard once and the unknown at least 3 times using the optimized gradient method from above. Record the retention times of all four peaks and the retention times and peak areas of the unknown and corresponding standard. Use the spectra from the HPLC detector to identify the phthalate. Remember to turn off the pump and the lamp and close the air and He tank valves.

E.

IV.

Results and Discussion

A.

Include the paraben and phthalate spectra recorded on the UV-VIS and the chromatograms collected in the preceding procedure. In a table, report the separation conditions using both isocratic and gradient elution. Using reversed phase retention theory and the spectral information for the parabens and phthalates, identify each of the four peaks in the mixed standard.

B.

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Chemistry 421 Advanced Instrumental Analysis - Spring Semester 2010

C.

Calculate and report the retention factors for methyl, ethyl and propyl parabens, and calculate the selectivity between each pair for the best isocratic separation and the optimized gradient separation. Discuss the similarities or differences in the selectivity values between the two separations. Calculate and report the resolution between methyl and ethyl paraben with the optimized gradient elution. Calculate and report the average efficiency (N) for each peak for the optimized separation. Discuss any significant differences. Comment on the gradient separation. Discuss the advantages and disadvantages of the approach, keeping such factors as sensitivity, baseline stability, and total analysis time in mind. Also comment on the width of the last peak using the isocratic and gradient methods. Report the identity of your unknown and report an estimate of its concentration with an estimate of the error. To estimate the concentration, use a one point calibration curve (peak area versus concentration) and assume a zero intercept. Homework assignment: Chapter 26: 2, 6, 8, 11, 12, 13, 14, 22 Chapter 28: 4, 13, 19.

D. E. F.

G.

H.

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