GC-MS Determination of Bisphenol A in Water

READING (FROM HARRIS TEXT):

• • 116-119 (Internal Standards)

  • 597-598 (Transmission Quadrupole Mass Spectrometer)

  • 642-644 (What Is Chromatography?)

  • 668-671 (The Separation Process in Gas Chromatography)

  • 676-679 (Temperature and Pressure Programming; Carrier Gas)

  • 687-688 (Gas Chromatography-Mass Spectrometry)

  • 826-828 (Solid-Phase Extraction)

OBJECTIVES:

• • To determine the BPA concentration in unknown water samples using SPE with GC-MS

  • To better understand gas chromatography-mass spectrometry (GC-MS)

  • To become familiar with solid-phase extraction (SPE)

  • To understand the need for derivatization in GC-MS analysis

  • To understand the use of an internal standard in a quantitative determination

This experiment will be an introduction to quantitative analysis using gas chromatography/mass spectrometry (GC-MS). The GC-MS is one of the most powerful tools available for the analysis of complex organic and biochemical mixtures. It is simple to use and provides both qualitative (what?) and quantitative (how much?) information.

As the name implies, GC-MS is a combination of two instruments. A gas chromatograph (GC) separates the components of a mixture, and a mass spectrometer (MS) helps to identify these components. The diagram below shows the basic design of the GC-MS instrument. 

As with all chromatographic techniques, GC separations are based on compounds in a mixture being partitioned between two phases – one mobile and one stationary.  The sample mixture is injected into an oven where it is vaporized. The mixture is then swept up by a stream of helium gas (the mobile phase) and carried to the coiled GC column. This column is a 30-meter long thin tube made of fused silica (SiO2), the inner walls of which are coated with a film of liquid polymer (the stationary phase). As the mixture of compounds moves through the column, the individual components separate from each other as some compounds spend more time in the stationary phase and others more time in the mobile phase. This partitioning between phases is typically based on the relative polarity of the compound versus the polarity of the stationary phase. The more polar the compound, the more time it will spend in a polar stationary phase, and the less time it will spend in a non-polar stationary phase. Also, small compounds tend to move through the column faster than large compounds, just as you would expect.

The GC column is housed in a programmable oven, and we can also use temperature to change the amount of time it takes a compound to move through the column. The warmer the column, the less time a compound will spend in the stationary phase. Oftentimes, we can speed up a separation by slowly warming the column after the sample has been injected. This is called temperature programming. We start at a relatively cool temperature to allow the compounds to interact with the stationary phase and separate from each other. Then we increase the temperature of the column to move the separated components through more quickly.

As the individual compounds emerge (elute) from the GC column, they are carried to the mass spectrometer. As shown in the figure below, the mass spectrometer consists of three distinct regions: ionizer, mass analyzer, and detector. Compounds enter the ionizer and encounter a beam of electrons emitted from a filament. The electrons bombard the sample, breaking the molecules into fragments, and turning these fragments into ions. The fragmented ions then encounter the quadrupole mass analyzer. The quadrupole consists of four parallel metal rods to which both a dc (direct current) voltage and an oscillating radiofrequency voltage are applied simultaneously. This causes ions entering the poles to move in an oscillating path.  If an ion has the correct mass-to-charge ratio (m/z), it will follow a stable (resonant) path between the rods. Ions without the correct m/z, however, will follow a non-resonant path and collide with the rods. By very quickly changing the values of the voltages applied to the rods, an entire mass range spanning several hundred mass units can be scanned in milliseconds. Resonant ions emerge from the quadrupole and hit the face of the detector where they eject electrons from the detector surface. These ejected electrons are carried through the detector, continuing to collide with the detector walls, creating even more ejected electrons and resulting in a measurable electrical current (i).

The mass spectrometer can be operated in different modes. In the total ion current (TIC) mode, all m/z values within a given range are monitored following injection of the sample into the GC. The chromatogram is simply a plot of the TIC versus time. Sometimes, however, it is better to simply monitor one or more selected m/z values. The chromatogram is then just a plot of a single m/z versus time. This mode is called selective ion monitoring (SIM) and is especially effective if two compounds with different m/z values are not separated and come off the column together. We’ll be using the SIM mode in our experiment (more on that below).

Below is a picture of our GC-MS instrument. The column length is 30 m, with a diameter of only 0.25 mm. The thickness of the stationary phase on the inner wall of the column is 0.25 µm and consists of a film of polydimethylsiloxane (PDMS) in which 5% of the methyl groups in the polymer have been replaced with diphenyl groups. This is typically referred to as 5% diphenyl-PDMS.

BISPHENOL-A (BPA):

Bisphenol-A (BPA) is a monomer used to make epoxy resins and polycarbonate plastics since it enhances the strength and durability of the material. Now classified as an endocrine-disrupting chemical (EDC) that mimics the role of estrogen in the body, Found in a wide variety of products such as food packaging, baby bottles, and medical devices, . While most human exposure to BPA comes through food, breakdown of this compound and mishandling of its waste has caused it to leak into water sources. There is still much to be learned about the risks associated with BPA, but the Minnesota Department of Health (MDH) has developed a guidance value of 20 ppb, meaning that someone drinking water at or below this level would have little to no health risk.

SOLID-PHASE EXTRACTION (SPE):

Many potentially harmful pollutants in the environment are present at levels to low to detect with common instrumentation. We say the concentration is below the limit of detection. So we must find some way to concentrate the pollutant to bring it above the limit of quantitation for the instrument. One way to do this is through solid-phase extraction (SPE) which adsorbs the species of interest on a solid support when the sample is passed through a tube containing this support.  Solid-phase extraction can be use to concentrate an analyte and/or to remove other species from the sample mixture that might interfere with detection. The steps in a typical SPE procedure are shown in the picture below. The SPE cartridge is first conditioned with solvent, then the sample is passed through which "loads" the analyte onto the solid support particles. (Note that the interferents are not retained and pass through to waste.) The loaded column is then washed in the third step before the analyte is "eluted" and collected with a small volume of solvent in the final step.

Since BPA is a non-polar aromatic compound, a solid support with a non-polar coating is typically used to retain BPA. The most common non-polar coating is octyldecyl (C18) chains bonded to solid silica (SiO2) particles. The hydrophobic interactions of BPA with the C18 chains allow BPA to stick to the gel bedding which is then desorbed and eluted with a small volume of less polar solvent such as methanol. Hence, a large volume of dilute sample can be passed through the SPE and efficiently concentrated into a small elution volume. 

The picture below shows the SPE apparatus we'll be using.  The SPE cartridges (three in the image) are inserted into the top of a manifold which is connected to a vacuum pump. When the vacuum pump is turned on, the valves below the tubes control the flow rate of solvent through the cartridge.

INTERNAL STANDARDS:

Since we are interested in determining the amount of BPA 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 BPA, and then inject them in to the GC-MS. We would then measure the area of the BPA peak and construct a calibration curve by plotting peak height versus BPA concentration.

There is one serious problem with this simple approach, however. We shouldn't assume that ALL the BPA is being retained on the SPE cartridge, or that all the SPE that is retained is washed off my the desorption solvent. If we were to run the same sample over and over, we would invariably end up with very different BPA peak areas for each run.  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 BPA and an internal standard.  Both compounds are retained by the SPE cartridge.  While the absolute amount of each compound adsorbed may differ from run to run, the ratio of the signals from each compound should be a constant.  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 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 GC-MS, it is common to use an isotopically-labeled version of the analyte.  So in our analysis, our internal standard will be bisphenol A-d16.  As shown in the figure below, all the hydrogen atoms in normal BPA are replaced with deuterium atoms.  This gives a molecular weight increase of 16 amu.  Except for this mass difference these two molecules should behave identically, showing identical adsorption in the SPE cartridge.  Furthermore, there is virtually no chance of finding the isotopically-labeled form present in the sample matrix.

METHOD OF STANDARD ADDITIONS:

In this experiment we will also be making use of the method of standard additions for calibration of the instrument. Again, calibration is usually performed by running a series of pure standard solutions of known concentration through the instrument (BPA standards in our case) and generating a calibration curve of signal versus concentration. Then we run our sample through the instrument and determine the concentration of our analyte by matching our sample signal to the calibration curve generated with the standards.  Ideally, our standard solutions should be similar to our sample solution since other components of the sample solution (the matrix) can interfere with our analyte signal (a matrix effect). Many times, however, it is difficult to prepare standard solutions with a matrix similar to that of our sample.  Instead, we can add known amounts of standard directly to the sample solution. In this way the standard is put in the same matrix environment as the analyte in the sample. We then are interested in the increase in signal due to the addition of the standard. Typically we add several different amounts of standard solution to our sample and then generate a curve of signal versus the added analyte concentration. Our original analyte concentration in the sample can then be determined by calculating the x-intercept of the resulting curve. Study the standard additions plot below until you understand why we can say the concentration of analyte in our original sample is 18 ppm.  

DERIVATIZATION:

In a GC analysis it is important that the analyte is quickly and efficiently vaporized to the gas phase in the GC injector. The polarity introduced by the phenol groups on BPA make efficient vaporization challenging. Therefore we must "derivatize" the molecule to make it more volatile. This is accomplished by added a "derivatizing agent" which in our case is bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMS) and pyridine. As shown below, the product of this reaction is bisphenol-A-di-TMS which readily loses a methyl group in the ionizer of the mass spectrometer to give a species with m/z 357 that becomes the largest peak in our mass spectrum. Similarly, the internal standard is derivatized to give a product with a mass of 386 amu that loses a -CD3 group to give a base peak with m/z 386. 

Since both forms of BPA have identical chemical and physical properties, it should make sense that they will not be separated well by a GC column. They may “co-elute” (emerge from the column together).  So why isn’t this a problem?  This is where the beauty of the MS detector comes in.  We’ll use the SIM mode of detection described above.  We’ll monitor mass 357 for BPA and mass 368 for the deuterated form.  So even though they’ll come off the column at the same time, the MS will allow us to obtain distinct signals for each.

REFERENCE:

Mead, Ralph N.; Seaton, Pamela J. J. Chem. Educ. 2011 88 1130-1132.

MDH; Eh; Esa; Hra. Bisphenol A in Drinking Water. https://www.health.state.mn.us/communities/environment/risk/docs/guidance/gw/bpainfosheet.pdf