How can analyte protectants and matrix help improve peak shapes?

In my last blog, I presented a new technique called low pressure gas chromatography (LPGC, Figure 1). Just to recap, the LPGC system consists of a relatively short analytical column (10 – 15 m) with large ID and thick film (e.g. 0.53 mm and 1.0 µm, respectively) which is restricted with a narrow guard column (e.g. 5 m x 0.18 mm). The restrictor (guard column) allows for maintaining head pressure on the inlet, while the analytical column is under near-vacuum pressure.

Figure 1: LPGC schematics


During my analysis, I’ve run into issues with early eluting peaks. At the initial 80 °C starting temperature the first two compounds, methamidophos and dichlorvos, showed up as distorted, split peaks. I’ve found that the optimal initial temperature was 70 °C, but the question remains: what if the sample is in the matrix? Or what if we use analyte protectants?
I’ve decided to investigate so I compared the original (solvent) analysis of the QuEChERS Performance Mix (#31152 ) to the analysis of added analyte protectant (0.1 mg/mL shikimic acid) and finally compared those to the celery matrix (Figure 2).

Figure 2: Comparison of pesticide residues’ runs with no matrix or analyte protectant (black trace), celery matrix (red trace) and with analyte protectant (green trace)


Figure 2 shows that the matrix (red trace) distorts the peak shape even further at both tested temperatures. Celery isn’t a very “dirty” matrix (after using dSPE), therefore, it doesn’t act as an analyte protectant for these compounds. On the other hand, the analyte protectant (green trace) helps significantly with the peak shape at both 80°C and 70 °C. At 80 °C, the effect is more pronounced while at 70 °C it helps reduce the tail of methamidophos and narrows the dichlorvos peak.
In conclusion, the analyte protectant (shikimic acid) can help with the peak shape at the original temperature, however, it is still preferable to lower the initial temperature to achieve a good solvent trapping.

4 Responses to “How can analyte protectants and matrix help improve peak shapes?”

  1. Tim Strutt says:

    Hi Jana,
    In your previous blog, Yuk discussed polarity mismatch between solvent and stationary phase. I think that another reason for your peak splitting is because of an incomplete solvent-trapping effect. Koni Grob’s original work shows that solvent trapping normally requires an initial oven temperature of 20-25C lower than the solvent BP and a retention gap big enough to trap all of the solvent – I think Koni discussed this with me (at Riva in the 1980s?), and kindly gave me a copy of his book, after I talked to Carlo Erba when had a similar problem. We saw from your previous work that when the initial temperature is dropped to 60C the peak shape for Dichlorvos (the 2nd eluter) improves as expected, but the splitting of Methamidophos becomes worse. This could be because some of the solvent has escaped from the empty retention gap and is condensing onto the first part of the Rxi-5ms causing the volatile analyte to condense in both places, so you have two sets of conditions. The first has your analyte safely trapped onto the ACN in the empty column as expected; and the second has analyte dispersed between pools of solvent and the stationary phase – Which as Yuk suggests is a classic polarity mismatch problem. This peak split would be worse than normal because when the ACN evaporates the analyte had been condensed into two locations. You may be able to improve things by using a longer length of (0.2 or 0.25mm?) restrictor to allow all of the ACN to condense in the empty retention gap and still keep the same inlet pressure, but it would probably increase your run time. Your use of analyte protectant implies that the Methamidophos “sticks” to it in the injector and retention gap giving a single unretained location when the ACN evaporates.

  2. Jana Rousova says:

    Hi Tim,
    Thank you for the comment! I agree that the peak splitting is mostly due to incomplete solvent trapping. However, I’m intrigued by your suggestion to extend the retention gap. In our case, we used a 5 m x 0.18 mm guard column, which I assumed would serve good enough. Would you suggest increasing the length to 10 or 15 m? Using a larger diameter guard column is no desirable because can hinder the near-vacuum conditions of the analytical column.

  3. Tim Strutt says:

    Hi Jana,

    I agree that making the restriction capillary/retention gap wider may compromise the inherent speed of the method, and could cause the inlet pressure to drop so that air is pulled into the column, but if we make a couple of assumptions…

    The classic retention gap work was often done with alkane solvents. ACN would give about 2.5 x the vapour volume of hexane at the same pressure. Assuming that the column head pressure is about 5-7psi, the BP of ACN would be about 91-95C at the start of the retention gap, and will drop to the “normal” 82C when the pressure has dropped to one atmosphere somewhere along the retention gap or column (That’s also why you get a small solvent trapping effect with DCM [BP 40C] in the standard EPA 8270E method with a 30m 0.25 column and a 40C initial oven temp: The 5psi head-pressure increases the solvent BP to ~48C). We want all of the ACN to condense in the retention gap – If the initial column temp is sufficiently low to rapidly condense the solvent, we can assume that it will, if there is “enough room” (Retention gaps for split-less injections are usually “a good thing”, if you can ensure that the joint doesn’t leak!).

    For a 1uL injection of hexane on a 0.32ID column, a typical retention gap is ~3-5m (say 4m) long. The internal surface area of of the gap is proportional to its diameter (Pi*d) so a 0.18 gap would need to be about ~(0.32/0.18)*4= ~7m long to contain about the same amount of solvent. If we increase the ID of the gap to 0.2mm, the EZGC indicates that the column length needs to roughly double to give a similar pressure drop. That suggests using a 10m length giving ~(0.2/0.18)*(10/5)= 2.2 the total surface area, or more than twice the trapping potential. If we increase the ID to 0.25 to give us more capacity, the length goes up to ~20m – Which is impractical, but would give give about 7 times the trapping capacity! So a good place to start might be to use a 0.18 restrictor but increase the length to 7m (Raising the head pressure to give the same linear flow should still be OK). I’m not sure how well a 10m 0.2id restrictor would work, but it might be worth trying.

  4. jaap de zeeuw says:

    Length of Retention gap needed for focusing in splitless injection depend on many parameters. Amount injected, (+ injection speed) vapor pressure of solvent, volumetric flow, temperature of the oven, pressure, diameter and length of the retention gap and its polarity. Note that in the analytical column we have a reduced pressure and the chances that condensation takes place, is minimal.
    When I worked on the “restriction – liner ” concept, we saw that we only could do splitless injections when the column was kept at low temperature which also helped the focusing by retention.
    It may be another interesting study..

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