Changes in Elution Order between Phenols and Phenyl Acetates

In order to determine contamination levels for phenols in water, the French normalized method, NF EN 12673, recommends an in-situ derivatization with acetic anhydride, followed by extraction with hexanes. The derivatization process (diagrammed in Figure 1) converts the phenolic compounds into phenyl acetates, which perform much better on GC/MS columns such as the Rxi-5Sil, due to the protective acetic acid ester in place of the acid proton. The acetic acid ester changes the polarity of the phenols, which creates some interesting changes in the elution order for some of the target analytes.

Table 1: The 19 chlorophenol compounds determined using NF EN 12673.

A total of 41 phenolic compounds were analyzed individually by adding 50 µL of acetic anhydride to 1 mL of the concentrated compound in methanol. This reaction allows for both the phenol compound and phenyl acetate to exist in one mixture since the excess methanol will out-compete the phenol and react with most of the acetic anhydride. Each compound (together with its derivatized analog) was analyzed individually by GC/MS on the Rxi-5Sil MS column to determine retention times.

 

Figure 1: Nucleophilic acyl substitution reaction used in NF EN 12673 to derivatize phenol compounds.

For 36 of the 41 compounds, the phenol eluted earlier than its phenyl acetate counterpart, indicating that the phenyl acetate group increased the compound’s affinity for the stationary phase (Figure 2). The remaining 5 compounds had a specific substitution pattern that decreased the relative retention time of the derivatized compound. The pattern places a highly electrophilic compound such as a chlorine or nitro group in the para and/or meta positions. This substitution pattern decreases the compound’s stationary phase affinity, causing the phenyl acetate to elute before the phenol (Figure 3).

Figure 2: 2,4,5-trichlorophenyl acetate (left) and 2,4,5-trichlorophenol (right) are an example of a pair of compounds that does not see an elution order shift.

Figure 3: 3,4,5-trichlorophenol (left) and 3,4,5-trichlorophenyl acetate (right) are an example of a pair of compounds with the substitution pattern that sees an elution order shift.

 

 

Figure 4: (From left to right) 3,4-dichlorophenyl acetate, 2,4,-dichlorophenyl acetate, and 3,5-dichlorophenyl acetate. The orange line divides the molecules into meta/para substitution and ortho-substition zones. Disubstution that is limited to the m/p zone results in an elution order change.

Table 2: All 41 phenolic compounds determined using the NF EN 12673 derivatization method. The compounds in red indicate the five compounds which undergo the discussed change in elution order. The compounds in green are the compounds which exhibit changes in co-elution compared to non-derivatized compounds.