HPLC Determination of Phthalates in Juice

READING (FROM HARRIS TEXT):

• • 116-110 (Internal Standards)

  • 642-644 (What is Chromatography?)

  • 703-710 (The Chromatographic Process)

  • 712-715 (Isocratic and Gradient Elution)

  • 719-720 (Solvents)

  • 721-724 (Pumps and Injection Valves, Spectrophotometric Detectors)

  • 824 (dispersive liquid-liquid microextraction)

OBJECTIVES:

• • To become familiar with the practice of high-performance liquid chromatography (HPLC).

  • To extract phthalate esters from a juice sample and measure their concentration using HPLC.

  • To be introduced to deep eutectic solvents (DESs) and dispersive liquid-liquid microextraction (DSSME) as tools for "green analytical chemistry."

Phthalates

Phthalate esters, or phthalates, are a family of compounds commonly used as plasticizers to make plastics more flexible and durable. They are found in a number of consumer products (e.g., PVC and vinyl products, personal care products like cosmetics and fragrances, food packaging, children's toys) and are known to leach out of these products into the environment over time. Concerns about their impact on health and safety have arisen as phthalates are known to be endocrine disruptors, meaning they interfere with hormone production and function and have been linked to reproductive issues, developmental problems, and other health effects.

Phthalates are diesters of phthalic acid (1,2-benzenedicarboxylic acid), which gives them the characteristic structure shown below. The specific properties of each phthalate are related to the alkyl chains (R and R') which also determine the name of the compound. For example, dibutyl phthalate (DBP) has two butyl chains, benzyl butyl phthalate (BBP) has one benzyl and one butyl chain, and bis(2-ethylhexyl) phthalate (DEHP) has two 2-ethylhexyl chains. The structures of all the phthalates we'll be measuring are shown below.

HPLC

Chromatographic techniques are widely used in all areas of science because they allow the analyst to both separate and quantify the components of a mixture. The principle on which chromatographic methods are based is simple. Compounds in a mixture are separated from each other based on their preferences for one of two different solvents (or phases) in contact with each other. For example if a polar solvent and a nonpolar solvent are brought into contact, polar molecules will prefer to be in the polar solvent, while nonpolar molecules prefer to be in the nonpolar solvent. In a chromatography experiment one of these solvents is stationary (the stationary phase), while the other solvent (the mobile phase) flows over it. A small volume of sample mixture is injected into the flowing mobile phase and is carried to a column which contains the stationary phase. As the components of the mixture pass through the column they are separated from each other based on their different affinities for the mobile and stationary phases. High-performance liquid chromatography (HPLC) utilizes a liquid mobile phase flowing through a small-diameter column packed with small particles coated with the stationary phase. 

A diagram of the basic components of an HPLC system is shown in the figure above. A piston-based pump continuously delivers liquid mobile phase at a steady flow rate from the mobile phase reservoir through the system. A very small volume of sample (usually a few microliters) is injected with a syringe into the flowing mobile phase which carries the sample to the column. In the column the various components of the sample mixture are separated from each other into bands. These bands “elute” from the column and move toward the detector. Several different types of HPLC detectors are available, but the most common simply focuses light of a particular wavelength through the flowing solution and measures the absorbance of the solution. When a species with an absorbance different from the mobile phase passes the through the detector, a peak is generated. The result is a plot of detector signal (absorbance) vs. time.  This plot is called a chromatogram. The image below shows a chromatogram resulting from the separation of five compounds detected in an aqueous sample over a 9.5-min separation measured from the time of injection (t = 0). (Note: the y-axis units are mAU which stands for milli-absorbance units).

The chromatographic column you will use in this experiment is a reversed-phase column, meaning that the mobile phase is polar and the stationary phase is nonpolar (this is the reverse of older methods which are referred to as normal phase). The most common reversed-phase columns are packed with small, porous silica particles that have hydrocarbon chains eighteen carbon atoms long chemically bonded to their surfaces. (We refer to this as a C18 column.) The components of a mixture separate based on differences in their polarity, with more polar compounds spending more time in the polar mobile phase and eluting earlier, and less polar compounds spending more time in the stationary phase and eluting later.

The mobile phase in our experiment is a mixture of water and acetonitrile (CH3CN, sometimes abbreviated ACN). If the relative amounts of each stay constant over the course of the separation, it is called isocratic elution. Many times, however, we need to change the relative amounts of each to get a better or faster separation. This is called gradient elution, which we'll be using in our experiment. A typical gradient elution separation begins with a mostly aqueous mobile phase (either pure water or an aqueous buffer if pH control is required), and then gradually increases the amount of organic solvent (usually methanol or acetonitrile). Our separation will begin with a 50:50 mix of water and acetonitrile, then gradually increase the organic fraction until we end with 100% acetonitrile.

For our experiments we will be using our Agilent 1260 HPLC system shown in the picture above. On top you’ll notice there are multiple mobile phase containers. One will hold our water and another will hold HPLC-grade acetonitrile. These bottles are connected to the pump at the bottom of the tower. Through the instrument software we'll tell the pump to deliver the solvents in the ratio we need for this separation. 

The picture shows that our system uses an autosampler instead of a manual injector. (There actually is a manual injector seen to the right of the tower in the picture, but we won't use it here.) The autosampler will allow us to run many samples in a row automatically. Underneath the autosampler you can see the the column. (The column is housed in a heater that we use for precise temperature control. In the picture the heater door is open so we can see the column inside, but this will be closed during our experiments.)  Below the column oven is the detector, which is an absorbance detector as described above. This particular model actually allows us to monitor multiple wavelengths in the UV and/or visible region simultaneously. (In fact, we have a photodiode array detector that can measure the entire spectrum all at once.) 

GREEN ANALYTICAL CHEMISTRY

Green analytical chemistry is a movement within the analytical community that focuses on four goals: 

1. elimination or reduction of the use of chemical substances

2. minimization of energy consumption

3. proper management of analytical waste

4. increased safety for the operator

Deep eutectic solvents

Deep eutectic solvents (DESs) are a relatively recent discovery in the chemical world that are now finding their way into a number of green analytical methods - largely because they have proven to be an effective substitute for traditional organic solvents in a variety of applications. There are different types of DESs, but the most common are mixtures of two compounds, one a hydrogen bond donor (HBD) and one a hydrogen bond acceptor (HBA),  with the mixture having a lower melting than either of the original components. The reaction diagram below shows the DES we will be using in our experiment that forms when mixing phenol (the HBD) and choline chloride (the HBA) in a 2:1 molar ratio. The hydrogen bonding between the phenols and the chloride weakens both the ionic bond in choline chloride and the hydrogen bonding interactions between the phenols, resulting in a DES product with a melting point significantly lower than the two reactants. So in this case, you should start with two solids at room temperature and see a liquid form when you mix them together!

Dispersive liquid-liquid microextraction

Liquid-liquid solvent extraction is a very common method for removing target organic compounds from a sample solution. A sample in water, for instance, is mixed with a smaller volume of an immiscible organic liquid (e.g., chloroform, hexane, dichloromethane) and the target compounds become concentrated in the organic phase. The drawback, of course, is the sometimes large amount of organic solvent waste that is generated. This is where DESs have been shown to be an effective green replacement, as we will see in our experiment.

Another green alternative to traditional liquid-liquid extraction is dispersive liquid-liquid microextraction (DLLME). In this technique, a small volume of extraction solvent is added to the aqueous sample and a larger volume of a disperser solvent that is miscible in both phases is added. The disperser creates a cloudy emulsion that maximizes the interaction between the sample and the extraction solvent. The mixture is centrifuged to separate the layers and the target organic analytes concentrated in the extraction solvent. 

The experiment we'll be conducting is based on a recent journal article by Santa-Mayor and coworkers. In our experiment, we will extract phthalates from water using DLLME, but using a choline chloride/phenol DES as the extraction solvent and tetrahydrofuran (THF) as the dispersive (emulsifying) agent. We will remove the DES and inject it into the HPLC to separate and ultimately quantify the extracted phthalates in a sample of juice that has been intentionally spiked with phthalates.

INTERNAL STANDARDS

Since we are interested in determining the amount of phthalates in our sample, we’ll have to generate a calibration curve for our data. The simplest way to do this is to prepare a number of solutions with known concentrations of phthalates, and then inject them in to the HPLC. We would then measure the area of the phthalate peak and construct a calibration curve by plotting peak area versus phthalate concentration.

There is one serious problem with this simple approach, however. We shouldn't assume that ALL the phthalates are being retained in each part of the experiment. In an experiment like this, it is very easy to lose some sample at various points in the procedure. We overcome this problem by adding an internal standard to our solutions.  An internal standard is a substance we add to the solution in a known amount that gives us a consistent reference for comparing different standard and sample injections. Now imagine doing multiple runs with a solution containing a phthalate and an internal standard. Both compounds are extracted by the DES. While the absolute amount of each compound extracted may differ from run to run, the ratio of the signals from each compound should be a constant. If any sample is lost at some point in the experiment, we assume we are losing the same relative amount of both analyte and internal standard, so their concentration ratio should stay the same. So when we prepare our solutions, we will add a constant amount of internal standard to all the solutions.  The calibration curve is then simply a plot of Sanalyte/SIS versus the concentration of the analyte, where Sanalyte and SIS are the signals (peak areas) from the analyte and internal standard, respectively.  Similarly, we will calculate the same ratio for our sample solution and fit it to our calibration curve.

It can be difficult to identify a good internal standard for a given analysis. Ideally, the internal standard should give a signal comparable in magnitude to the signal from the analyte.  It must also not be present in the sample matrix. In phthalate determinations it is common to use benzyl benzoate (shown below), an ester of benzyl alcohol and benzoic acid that has structural similarities to phthalates, but that should not be found in our samples.

REFERENCES

V.L McDevitt, A. Rodriguez, and K.R. Williams, J. Chem Ed., 1998, 75, 625-629.

A. Gałuszka, Z. Migaszewski, and J. Namieśnik, TrAC Trends in Analytical Chemistry, 50, 2013, 78-84.

W. Guo, Y. Hou, S. Ren, S. Tian, and W. Wu, Journal of Chemical & Engineering Data 58, 2013, 866-872.

Á. Santana-Mayor, B. Socas-Rodríguez, R. Rodríguez-Ramos, and M. Rodríguez-Delgado, Food Chemistry, 312, 2020, 125798.

A. Shishov, A. Gorbunov, L. Moskvin, and A. Bulatov, Journal of Molecular Liquids, 301, 2020, 112380.