Analyzing Phthalates Using an Inert LC Column

Phthalates are a class of compounds commonly used as plasticizers and are found in a wide variety of products. These compounds can help increase the flexibility and lifetime of products and can also be used in many different personal care products and for industrial uses. These compounds are readily found in our environment and food sources due to leaching, and the potential for high exposure is likely.¹ In the US and EU a number of these compounds are being replaced due to their adverse health effects, toxicity, and bioaccumulative properties.^1,2 In this work, LC-MS/MS method development was explored for phthalic acid and six widely used phthalates.

Method Development

Phthalic acid and six phthalates were chosen as the analytes of interest (Figure 1).

Figure 1. Phthalic acid and six additional analytes of interest

Two of these compounds, DIBP and DBP, are structural isomers and require chromatographic separation. A Raptor ARC-18 column was chosen as the analytical column, and a method was developed using 0.1% formic acid in water and methanol. The results of these experiments are shown in Figure 2. 

Figure 2. Method using 0.1% formic acid in water and methanol mobile phases.

This methodology was able to retain phthalic acid and resolve the two structural isomers, but the peak shape for the last two eluting compounds, DINP and DIDP, was poor. Next, the acid modifier in the mobile phase was increased to determine if the peak shapes could be improved by decreasing the pH. The results when decreasing the pH and keeping all other analytical parameters the same are shown in Figure 3.

Figure 3. Chromatogram obtained using 0.5% formic acid in both mobile phases.

The results of these experiments show excellent peak shape for the last two eluting compounds, while still maintaining the resolution of the isomers and retention of phthalic acid.

The Effect of Inert Hardware

Inert column hardware can be useful in mitigating the effects of compounds that participate in non-specific binding to metal surfaces. Compounds capable of non-specific binding typically suffer from poor peak shape and/or reduced sensitivity that can be exacerbated by several factors including analytical conditions, matrix, pH, and temperature. A temporary solution to combat non-specific binding is system and on-column passivation. This can be achieved by a number of different ways and one such method includes the use of a passivation solution, such as Restek’s LC Passivation Solution (cat.# 32475), following the instructions for both LC system and LC column passivation found here. However, this is not a permanent solution, and both the system and column will require intermittent re-passivation. For a permanent solution to LC column passivation, inert column hardware can be used to eliminate non-specific binding of compounds to the column hardware. In the next step of method development, inert hardware was tested and compared to traditional column hardware. The results of this experiment showed improved sensitivity for a majority of the analytes when using inert hardware.  

Figure 4. Comparison of signal intensity on traditional column hardware versus inert hardware.

The inert hardware column showed a significant increase in sensitivity for almost all of the compounds tested. The results for all compounds analyzed using the inert column hardware and 0.5% formic acid can be see below:

Figure 5: Final method conditions for the analysis of phthalic acid and six phthalate compounds using Raptor Inert ARC-18 and 0.5% formic acid as mobile phase modifier.

Based on these results, it is evident that the use of inert hardware is critical to achieve the lowest levels of detection. Though phthalates can be challenging, several analytical parameters can be optimized to ensure that the best quality peak shape and sensitivity can be achieved. At Restek, achieving your analytical goals is our priority. Interested in trying one of our new INERT LC columns? Reach out to your local sales representative today!

1. US Environmental Protection Agency. https://www.epa.gov/sites/default/files/2015-09/documents/phthalates_actionplan_revised_2012-03-14.pdf. Accessed 5/21/2024.
2. European Union- LEX Access to European Union Law. Regulation - 143/2011 - EN - EUR-Lex (europa.eu). Accessed 5/21/2024