Part 2: Optimizing EPA Method 610
Guest Author: Dr. Jeff Layne
Welcome back! In the last article, we looked at the EPA method for the analysis of polynuclear aromatic hydrocarbons—EPA Method 8310—and presented data on Phenomenex’s new core-shell based LC column for PAH analysis – Kinetex® 3.5 µm PAH. I won’t go over the background again, but if you are interested, please go back and check it out.
EPA Method 8310 consists of a mixture of 18 PAHs, but it is not the only method used for PAH analysis. Another method often referred to is EPA Method 610, which is a mixture of 16 PAHs, some of which are distinct from EPA 8310. A comparison of the analyte lists for EPA 610 and EPA 8310 is shown in Table I below.
Table I. Analyte lists for EPA Method 610 and EPA Method 8310
As you might expect, with the simpler analyte list of EPA Method 610 compared to EPA Method 8310, we should be able to have an easier time separating our target PAHs, and should see shorter analysis times. But one of my goals is to demonstrate the performance improvements that are achievable when core-shell technology is applied to PAH analysis. Let’s start by looking at EPA Method 610 separated using a standard full-porous LC column.
Figure 1 is a representative chromatogram obtained using a fully-porous column marketed specifically for PAH analysis (Vendor X 5 µm, 100 x 4.6mm) and the gradient and flow rate were reproduced from the manufacturer’s product marketing literature. Using said column and running conditions, you can see that all 16 analytes in the EPA Method 610 are baseline resolved, with a total cycle time of about 30 minutes with re-equilibration. Under these conditions, the lowest resolution is obtained between peaks 14 and 15, Dibenzo[a,h]anthracene and Benzo[ghi]perylene, with an Rs of 2.43.
Figure 1. Separation of the 16 PAHs in EPA Method 610 mixture using a fully-porous 100 x 4.6mm column packed with conventional fully-porous 5 µm media, using running conditions (gradient profile and flow rate) recommended by the manufacturer. App. ID. 24599.
When run using the identical running conditions, the core-shell Kinetex 3.5 µm PAH, 100 x 4.6mm column has a total cycle time of about 25 minutes (with re-equilibration), which is a few minutes shorter than the fully porous 100 x 4.6mm column (Figure 2). However, under these running conditions, peaks 14 and 15, Dibenzo[a,h]anthracene and Benzo[ghi]perylene, are not completely baseline resolved on the Kinetex 3.5 µm PAH column (Rs = 1.45). This may be due to differences in the surface area of the core-shell Kinetex media compared to the fully porous media.
The crucial point to note here is that, although columns packed with core-shell media will almost always provide greater efficiency than columns packed with fully porous media, it is not always possible to use the same gradient profile and get the appropriate selectivity required to separate all target analytes. This is especially true when running extremely complex samples mixtures such as these PAHs.
Figure 2. Analysis of the same 16 PAHs in EPA Method 610 performed using a 100 x 4.6mm column packed with core-shell Kinetex 3.5 µm PAH media, using the identical running conditions as Figure 1. Under these conditions, peaks 14 and 15 (indicated by the large arrow) have an Rs value of 1.45 on the Kinetex 3.5 µm PAH column. App. ID. 24600.
Thus, to compensate for this, we need to simply make an adjustment to the gradient profile to improve the resolution between those two peaks, and can also use a shorter, faster gradient to dramatically decrease our overall cycle time (Figure 3). In this case, we are using a simple linear gradient going from 40 to 100% B over 8 minutes. Under these new conditions, the Kinetex 3.5 µm PAH 100 x 4.6mm column provides full baseline resolution of all 16 PAHs in the EPA Method 610 mixture in a total cycle time of about 10 minutes. That cycle time is less than half the time the comparable separation takes when using the fully porous 5µm 100 x 4.6mm column, and our minimum resolution value is 2.57 between peaks 3 and 4 (acenaphthene and fluorene).
Figure 3. Improved analysis of the EPA Method 610 PAH mixture using a 100 x 4.6mm column packed with Kinetex 3.5um PAH media under adjusted gradient conditions. App. ID. 24601.
Increasing column efficiency leads to narrower peaks that provide improved resolution and increased peak height response. The increase in peak height can be very useful in improving method LOD and LOQ values. As an example, consider the first peak in the series, naphthalene. Using the original fully porous 100 x 4.6mm under the column manufacturer’s conditions, the peak height response for naphthalene is 13.9 mAU (from Figure 1). Now, when we compare that to the same sample injected using the Kinetex 3.5 µm PAH 100 x 4.6mm column under our optimized gradient profile (from Figure 3), the peak height for naphthalene is increased to 55.0 mAU, almost a four-fold increase in peak height response. The overlaid peaks for naphthalene obtained using the two columns is shown in Figure 4 to clearly illustrate the improvement in peak height response obtained when using the Kinetex 3.5 µm PAH column.
Figure 4. Comparison of the peak height response for naphthalene obtained using the fully porous 100 x 4.6mm (red trace) with the same sample analyzed using the 100 x 4.6mm column packed with Kinetex 3.5um PAH media (blue trace). App. ID. 24602.
The new Kinetex 3.5um PAH brings the core-shell efficiency advantage to the field of PAH analysis. The ultra-high efficiency of the core-shell morphology can provide the analyst with the ability to dramatically improve their resolving power, sensitivity, and productivity. In this case, baseline resolution of the 16 component PAH mixture specified in EPA Method 610 is achieved with a total cycle time of less than 10 minutes, compared to about 30 minutes using a typical fully porous alternative column. In addition, single peak height response is increase by almost four-fold, allowing analysts to achieve greater LOD and LOQ values in a fraction of the time compared to a traditional fully porous HPLC column.
For more information on Kinetex columns and your PAH media analysis, contact our nearly 24/7 Technical Experts!