Ascentis ES Cyano – A New “Extra Stable” Cyano Phase Shown as a Confirmatory Column for US EPA Method 8330

By: Carmen T. Santasania, Wendy Roe, Michael Buchanan, Reporter US Volume 28

Carmen T. Santasania, Wendy Roe and Michael Buchanan


Military installations and armament manufacturers have experienced closures in the last several years due to non-proliferation treaties and disarmament agreements. These closures have forced the military and environmental regulators to closely examine the explosive materials left behind in water, soil and sediment.

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US EPA Method 8330

The United States Environmental Protection Agency (US EPA) developed Method 8330 (1) for the trace analysis of explosive residues by HPLC with ultraviolet detection (LC-UV). This method is used to identify and determine levels of the explosives shown in Figure 1, and recommends the use of two columns for confirmatory analysis. For instrument calibration, the fourteen analytes are divided in two mixes, termed Mix A and Mix B, with 5-point calibration curves required for both. After calibration, the instrument software combines the retention time and response factor data from both mixes into a single file. The results of the subsequent analyses of sample extracts are then compared to this combined calibration file.

Figure 1. Explosive Analytes

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Primary (C18) and Confirmatory (Cyano) Columns

Figure 2 shows the analysis of these two mixes on an Ascentis C18 column. Using identical instrument conditions, both mixes were also analyzed on an Ascentis ES cyano column (chromatograms shown in Figure 3). These two columns contain different ligands with their own analyte interactions, resulting in a different selectivity for each column. This differing selectivity is necessary for confirmatory analysis.

Figure 2. Analysis of Explosives on Ascentis C18 using EPA Method 8330 (581325-U)

Figure 3. Analysis of Explosives on Ascentis ES Cyano using EPA Method 8330 (577307-U)

The choice of the Ascentis ES Cyano column to provide alternate selectivity for the explosives is based on π-π and/ or dipole-dipole interaction on a cyano phase (2). The π acidic solutes, such as the nitro-substituted aromatic explosives have been shown to have preferential retention for a cyano phase. (2,3,4). In addition, the method mobile phase of 50:50 water:methanol has been shown to enhance retention on a cyano phase (5).

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Analyte Identification and Quantitation

The results obtained from the primary (C18) column can be used for eight of the analytes, as indicated in Table 1. A number of co-elutions and partial co-elutions exist on the primary (C18) column, so it can be seen that an alternative selectivity column would be necessary to confirm the identity of each peak. As an illustration, Table 1 shows that the analyte pair nitrobenzene and tetryl will co-elute on the Ascentis C18, with retention times of 11.54 and 11.59 minutes, respectively. Retention times on the Ascentis ES Cyano are also listed in Table 1. Note the large differences in retention times for this analyte pair, 8.92 and 23.81 minutes, respectively. Another example, also shown in Table 1, are the partially co-eluting analyte pair 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene. When these analytes are run on the Ascentis ES Cyano phase, their retention times are nearly two minutes apart. Table 1 also shows the column that would be used when this column pair is used for quantitation.

Table 1. Peak IDs, Retention Times, and Quantitation Strategy

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Ascentis ES Cyano Column Stability

An important issue that has arisen in the past is the stability of a cyano phase. Figure 4 shows results of a stability test run under rigorous conditions. At 50° C, with a mobile phase containing of 0.1% TFA in 65% water and 35% acetonitrile, a test mixture of uracil, nitrobenzene and butyl paraben was injected every hour for several days. The leading competitive cyano column was included in this test as a control. The Y-axis shows the number of column void volumes pumped through the column during the test, with the X-axis showing retention (k). Note the 2.2% loss in k for the nitrobenzene on the competitor cyano phase versus the 0.78% loss on the Ascentis ES Cyano phase. Recall that the nitrobenzene is also one of the explosive compounds included in EPA Method 8330, discussed above. Additionally, butyl paraben shows a 3% loss of k on the competitor Cyano, while a negligible loss of 0.072% is seen on the Supelco Cyano phase. This data shows the stability of this new phase under extreme conditions of pH and temperature in LC analysis.

Figure 4. Stability of Ascentis ES Cyano vs. Competitor

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US EPA Method 8330 is used to determine explosive residues in water, soil and sediment. It specifies the use of two columns with alternative selectivity to confirm results. The data presented here shows that the Ascentis C18 and Ascentis ES Cyano are an ideal column pair for this analysis. Stability of the Ascentis ES Cyano is favorable when compared to the leading competitive cyano phase.

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  1. US EPA Method 8330A, “Nitroaromatics and Nitramines by High Performance Liquid Chromatography (HPLC)” Revision 1 (February 2007), obtained from the web site.
  2. K. Croes et al. J. Chromatogr. A 1098 (2005) 123.
  3. A. Tchapla, S. Heron, E. Lesellier, J. Chromatogr. A 656 (1993)81
  4. S. Heron, A. Tchapla, J. Chromatogr. A 725 (1996) 205.
  5. J. Horak, N.M. Maier, W. Lindner, J. Chromatogr. A 1045 (2004) 43.

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