# FAMEs blog part 4: Struggling with using hydrogen for AOCS methods Ce 1j-07 or Ce 1h-05?

16 Dec 2018

Recently I came across customers’ issue with AOCS method Ce 1h-05 used with hydrogen as a carrier gas and I’ve decided to look more closely into what conditions are suggested for this analysis. While at it, I looked also into conditions of methods Ce 1j-07 (AOCS) and AOAC 996.06. What I’ve noticed at first glance is that these methods are written with helium in mind, however, even when using helium, the flow suggested in the method might not provide the best resolution. The method's parameters are summarized below:

Table 1: Summary of method parameters

What I highlighted in the table are the suggested linear velocities. As the color-coding suggests, 3 out of 5 linear velocities are completely outside of the optimal range. The optimal linear velocity (according to van Deemter) is ~ 25 cm/sec for He and ~ 40-50 for H_{2}. This means that we can speed up the analysis and gain efficiency!

To best show the differences, I’ve decided to use the method translator and EZGC. For the analytes list, I choose to use only the critical separations for the individual methods and then the full food FAMEs list with the addition of available C18:1 *cis/trans *FAMEs.

Let’s start with the AOCS Ce 1h-05, the isothermal method. Using the original parameters (H_{2} or He @ 1mL/min) the time of analysis for the full set of FAMEs is approximately 105 and 144 minutes, respectively. Unsurprisingly, using H_{2} at 1 mL/min leads to worst resolution. A simple increase of flows to 1.75 and 1.4 mL/min (H_{2} and He, respectively), which is on the lower side of the recommended linear velocity, decreases the time to approx. 75 and 109 minutes, respectively, with small improvements to the resolution. Increasing the flow even further (up to 3.5 and 2.5 mL/min, respectively), does not decrease the resolution and cuts the time of analysis by 50% (H_{2}) and 40% (He).

The second AOCS method (Ce 1j-07) has a temperature gradient which improves the elution of high boiling peaks, such as C22:6, with a 65-minute runtime with both H_{2} at 1 mL/min and He at 2 mL/min. Using He with this flow places it squarely into the recommended linear velocity. Resolution criteria (Table 1, Figure 1) are met, and there is no need for adjustment.

Figure 1: Critical resolutions of method AOCS Ce 1j-07 under recommended conditions for helium

The situation is not as clear-cut with hydrogen as the carrier gas. While the flow of 1 mL/min meets the resolution criteria and provides fairly similar retention times, the width of peaks can be almost doubled compared to the optimal flow of 2.5 mL/min (Figure 2).

Figure 2: Critical resolutions of method AOCS Ce 1j-07 under original (A) and optimal (B) conditions for hydrogen

Running the analysis at the optimal flow also cuts down the retention time of the last peak by 10 minutes, however, if we take advantage of method translator and translate the method from helium to hydrogen (Figure 3), we can shave off an additional 10 minutes.

Figure 3: translation of AOCS Ce 1j-07 method from helium to hydrogen

The last example addresses AOAC method 996.06. While this method doesn’t specify the hydrogen flow (Table 1), it features low helium flow (0.75 mL/min) which is outside of the helium recommended range. Let’s start with optimizing the He flow. The method has specific critical separations (Table 1), however, these are met using the lower flow and while the resolution improves with higher flow, the difference is small (Figure 4). What improves significantly is (again) the overall analysis time and the peak width.

Figure 4: Critical resolutions of method AOAC 996.06 under original (A) and optimal (B) conditions for helium. Optimal conditions decrease run times by nearly 15 minutes.

Because the separation criteria are met, we can use the method translator to help speed up the method with a higher flow (Figure 5 A) and subsequently use the improved method to translate to hydrogen (Figure 5 B). The translation helps cut down the analysis time by almost 15 minutes (optimal He flow) and by almost 30 minutes when using the hydrogen.

Figure 3: translation of AOAC 996.06 method to higher helium flow (A) and from helium to hydrogen (B)

To conclude, using optimal gas flows with method translation does not decrease the resolution (quite contrary!) and it significantly improves the peak width and analysis time.