Optimizing Mass on Column to Balance Sensitivity Requirements and Calibration Range with Split Injection

This is a continuation of the EPA Method 8270 blog series started in January of 2016. Previous posts: 1, 2, 3, 4, and 5.

We’ve been focusing on the advantages of split injection analysis, while highlighting the weaknesses of splitless injection. This blog is going to revisit the topic of column overload in more detail – focusing on optimizing a split injection method to maximize sensitivity while maintaining and extended calibration range. This is especially critical if you are migrating your method to a 20 m x 0.15 mm x 0.15 µm or 20 m x 0.18 mm x 0.18 µm column for fast analysis with the new GC Acclerator.

Typical semivolatiles calibrations on a 30 m x 0.25 mm x 0.25 µm 5-type column range from 0.5 µg/mL or 1.0 µg/mL to over 100 µg/mL (many analysts target 160 or 200 µg/mL). However, this column dimension (as well as the 0.32 mm ID format) will usually show signs of peak overload with less than 10 ng of any individual component on column. Isobars that elute close together quickly become coelutions as mass on column increases. Figure 1 highlights the most popular example of this, the benzo[b]fluoranthene and benzo[k]fluoranthene isomeric pair. The three highest concentration calibration standards (120, 80, and 40 µg/mL) do not meet the 50% valley resolution criteria under splitless conditions [technically, the resolution criteria are only evaluated at the midpoint used for the CCV evaluation, but they are good indicators of accurate integration potential]. The extreme peak fronting resulting from column overload makes it impossible to accurately integrate and generate a linear calibration including these points. Additionally, the peak apex of benzo[b]fluoranthene shifts more than 0.2 minutes (12 seconds) across the calibration standards, another symptom of severe column overload.

Figure 1 – Benzo Fluoranthene isomer resolution ( B and K ) at increasing mass on column

The concentration-dependent retention time shift requires wide windows in the data processing software, increasing the likelihood of identification errors when the automated integrator processes the data. This results in more time required for manual data review and integration [which can be a headache for those of you manually recording before and after chromatograms in compliance with your manual integration policies] with an elevated risk of error.

Under split conditions, the isomers meet resolution criteria (50% valley) in each of the 9 calibration standards, and the peak apices shift by at most 0.04 minutes (2.4 seconds) from 0.1 µg/mL to 120 µg/mL, indicating only minor peak overload at the high end of the calibration range. Figure 2 shows a comparison of splitless and split benzo fluoranthene isomer chromatography over the same calibration range.

Figure 2 – Comparison of benzo fluoranthene isomer retention time variation as injection concentration increases using splitless (top) and split 10:1 (bottom) injection techniques

The minimal shift of concentration-dependent retention times allows for a much more narrow integration window and a greater confidence in peak IDs. This is important because compound concentration isn’t the only factor that can cause retention time shifts. Complex sample matrices with an excess of co-extracted material can have the same effect on splitless injection by greatly broadening the initial sample band at the head of the column. Split injection minimizes this by transferring a fraction of the injection to the head of the column.

Figure 2 is a good illustration of how column overload is managed by split injection – but it introduces a new problem that needs to be dealt with: detector overload. Initially, when collecting the splitless injection data, we dropped the gain factor to 0.3 (from the default of 1.0), which reduces the voltage applied to the multiplier below the tune optimized level. This reduces instrument sensitivity, preventing detector overload at the high end of the calibration curve (120 ng on column). For the 10:1 split injection, we increased the gain factor to 3.0 (adding almost 250 volts to the tune optimized level) to make sure sensitivity wasn’t an issue at the low end of the calibration because the split injection delivers 1/11th of the sample to the analytical column. This was a gross over-correction, causing compounds with strong molecular ion responses (such as PAHs) to overload the detector at concentrations as low as 40 µg/mL. Through trial and error, we determined that the best balance between low-end sensitivity and high-end overload occurred at a gain factor of 0.8 (see Figure 3). This appears to be instrument dependent, as we operate a similarly configured 5977a with an optimized gain factor of 1.0 and see similar performance.

Figure 3 – Comparison of benzo fluoranthene isomer peak shapes demonstrating the effect of reducing the gain factor on detector overload.

Once you have established your linear mass on column range, you can adjust standard concentrations and the split ratio (or injection volume) to maximize your calibration range while maintaining sensitivity to meet method required LOQs.

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