Identifying the Ubiquitous Phthalate Esters in the Environment Using HPLC, Photodiode Array Detection, and environment using HPLC, photodiode array detection, and confirmation by GC-MS

5.15.1 Background and Summary of Method

The most commonly found organic contaminant in landfills and hazardous waste sites has proved to be the homologous series of aliphatic esters of phthalic acid. This author has found phthalate esters in almost every Superfund waste site sample GC-MS report that he reviewed during the mid-1980s while consulting for an environmental testing laboratory in New York State.

The molecular structures for two representative phthalate esters are drawn below. Dimethyl phthalate (DMP) and bis(2-ethylhexyl) phthalate (Bis) illustrate one example of a lower- molecular-weight phthalate ester versus a higher-molecular-weight ester. DMP and the higher homologs, diethyl phthalate (DEP), di-n-propyl (DPP), and di-n-phthalate (DBP), are the focus of this exercise.

The photodiode array UV absorption detector provides both spectral peak matching and, if desired, peak purity determinations. This is nicely illustrated below. A peak is identified in the first drawing and its UV absorption spectrum can be matched against a library of UV absorption spectra. Note that the UV absorption spectrum from the peak at or near a retention time tR = 39 min in the HPLC chromatogram is retrieved from a stored library file. The UV spectrum for the peak and that for a reference standard are compared. The second drawing demonstrates how overlays of UV absorption spectra use three points across the chromatographically resolved

HPLC and are used together with an algorithm to calculate a purity match. Note the difference between the overlaid UV absorption spectra for the impure vs. the pure peak. You will not be using the peak purity algorithm in this exercise. Analytical Method Development Using HPLC

Analytical method development in HPLC usually involves changing the composition of the mobile phase until the desired degree of separation of the targeted organic compounds has been achieved. One starts with a mobile phase that has a high solvent strength and moves downward in solvent strength to where a satisfactory resolution can be achieved. Recall the key relationship for chromatographic resolution:

A useful illustration of the effects of selectivity, plate count, and capacity factor is shown below:

HPLC chromatogram (A) shows a partial separation of two organic compounds, e.g., DMP from DEP. This degree of resolution, Ry could be improved by changing k N, or a. In (B), k' is increased, which changes the retention times and shows a slight improvement in Rr Increasing N significantly increases as shown in (C); the greatest increase in Rs is obtained by increasing a, as shown in (D). Refer to the Suggested Readings at the end of this experiment or an appropriate textbook chapter or monograph on HPLC to enlarge on these concepts. GC-MS Using a Quadrupole Mass Spectrometer

In a manner similar to obtaining specific U V absorption spectra for chromatographically separated peaks, as in HPLC-PDA, GC-MS also provides important identification of organic compounds first separated by gas chromatography. The mass spectrometer that you will use consists of four rods arranged to form parallel sides of a rectangle, as shown below. The beam from the ion source is directed through the quadrupole section, as shown below.

The quadrupole rods are excited with a large DC voltage superimposed on a radio frequency (RF) voltage. This creates a three-dimensional, time-varying field in the quadrupole. An ion traveling through this field follows an oscillatory’ path. By controlling the ratio of RF to DC voltage, ions are selected according to their mass-to-charge ratio (m/z). Continuously sweeping the RF/DC ratio will bring different m/z, ratios across the detector. An oversimplified sketch of a single quadrupole MS appears below:

5.15.2 Of What Value is This Experiment?

The goal of this experiment is to provide an opportunity for students to engage in analytical method development by identifying an unknown phthalate ester provided to them. This is an example of qualitative analysis. The reference standard solution consists of a mixture of the four phthalate esters: dimethylphthalate (DMP), diethylphthalate (DEP), di-n-propylphthalate (DnPP) and di-n-butylphthalate (DnBP). Molecular structures for these four phthalate esters are shown below:

Each group will be given an unknown that contains one or more of these phthalate esters. A major objective would be to use available instrumentation to identify the unknown phthalate ester! Students will have available to them an HPLC in the reversed-phase mode (RP-HPLC) and also access to the department’s gas chromatograph-mass spectrometer (GC-MS) system.

Students must first optimize the separation of the esters using RP-HPLC, record and store the ultraviolet absorption spectra of the separated esters, and compare the spectrum of the unknown against the stored UV spectra. In addition, staff will be available to conduct the necessary GC- MS determination of the unknown. A hard copy of the chromatogram and mass spectrum will be provided so that the student will have additional confirmatory data from which to make a successful identification of the unknown phthalate ester.

5.15.3 Experimental

High-performance liquid chromatograph with ultraviolet absorption photodiode array detection (HPLC-PDA) set up for reversed-phase liquid chromatographic separations.

Capillary gas chromatograph-mass spectrometer incorporating a quadrupole mass-selective detector (C-GC-MS). This instrument should be available for students to use or to drop off their samples at a location outside of the instrumental teaching laboratory location. Preparation of Chemical Reagents

Note: All reagents used in this analytical method contain hazardous chemicals. Wear appropriate eye protection, gloves, and protective attire. Use of concentrated acids and bases should be done in the fume hood.

  • Accessories to Be Used with the HPLC per Student or Group
  • 1 HPLC syringe. This syringe incorporates a blunt end; use of a beveled-end GC syringe would damage inner seals to the Rheodyne HPLC injector.
  • 1 Four-component phthalate ester standard. Check the label for concentration values.
  • 1 Unknown sample that contains one or more phthalate esters. Be sure to record the code for the unknown assigned.
  • Procedure

Unlike other lab exercises, no methods have been developed for this exercise. Consult with your lab instructor regarding the details for developing a general strategy. You will be introduced to Turboscan®, software that will allow you to store and retrieve UV absorption spectra.

First, find the mobile-phase solvent strength that optimizes the separation of the four phthalate esters. Second, retrieve the UV absorption spectrum for each of the four and build a library. Third, inject the unknown sample and retrieve its UV spectrum. Fourth, make arrangements with the staff to get your unknown analyzed using GC-MS.

5.15.4 For the Report

Include your unknown phthalate ester identification code along with the necessary laboratory data and interpretation of results to support your conclusions.

Please address the following in your report:

  • 1. Compare the similarities and differences for the homologous series of phthalate esters on both UV absorption spectra and mass spectra from your data.
  • 2. Explain what you would have to do if you achieved the optimum resolution and suddenly ran out of acetonitrile! Assume that you have only methanol available in the lab. Would you use the same mobile-phase composition in this case?
  • 3. This exercise introduces you to the quadrupole mass filter. Briefly describe how the mass spectrum is obtained, and if you so desire, attempt to provide a brief mass spectral interpretation. You may want to review a text that discusses the principles and practice of GC-MS.
  • 5.15.5 Suggested Readings

To develop this experiment, the author consulted the following resources:

  • • Snyder L., J. Kirkland. Introduction to Modern Liquid Chromatography. 2nd ed. New York: John Wiley & Sons, 1979.
  • • Snyder L., J. Glajch, J. Kirkland. Practical HPLC Method Development. New York: John Wiley & Sons, 1988.
  • • Snyder L., J. Kirkland, J. Glajch. Practical HPLC Method Development. 2nd ed. New York: John Wiley & Sons, Inc. 1997.

• Sawyer D., W. Heineman, J. Beebe. Chemistry Experiments for Instrumental Methods, New York: John Wiley & Sons, 1984, pp. 344-360.

To see how the LC-MS-MS determinative technique has significantly impacted the practice of TEQA, refer to two recent contributions:

  • • Shrestha P. et al. Current Trends in Mass Spectrometry> October, 2019, pp. 7-15. The authors compared LC-MS-MS quantitation results obtained between APPI vs. ESI interface with respect to wastewater analysis for various pharmaceuticals.
  • • Parry E., T. Anumol. Quantitative analysis of per fluoro alkyl substances (PFAS) in drinking water using liquid chromatography tandem mass spectrometry. Current Trends in Mass Spectrometry October, 2019 pp. 21-24.

Current Trends in Mass Spectrometry is published (as of this writing) by MultiMedia Healthcare LLC that also publishes monthly issues of LC-GC North America, LC-GC Europe and Spectroscopy. Interested readers who qualify can receive the print and e-versions without cost. Issue archives are also available online.

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