Further Evaluation of the Base Stability of Hydrophilic Interaction Chromatography Columns Packed with Silica or Ethylene-Bridged Hybrid Particles
Round 1
Reviewer 1 Report
The authors present a work focused on studying the influence of different parameters causing the loss of base stability in HILIC columns packed with silica or ethylene bridget hybrid particles. My opinion about of this article is positive, I read this upgrade of the previous publication with considerable interest because studies of this sort can help all analytical chemists who, daily or for the first time, approach HILIC column-based applications. The paper is well written, although I found it quite similar to the previous one.
The paper lacks in more clarification regarding the choice to use 70 °C as temperature of the mobile phase, since that is not a temperature at which anyone works in HPLC. Wouldn't it have been more appropriate to stop at a lower temperatures (for example 40 degress?), since the tests of several temperatures were done in the previous publication. I also did not understand how this temperature of the mobile phase is maintained: is only the column in the column compartment of the HPLC heated? Please clarify it in the text.
Adding chromatograms comparing the various stationary phases tested is also very interesting. This can be a more straightforward way to see the trend of the base stability loss as time passes.
After these modifications/clarifications the paper can be published.
Author Response
- Regarding the comment: "The paper lacks in more clarification regarding the choice to use 70 degrees C as temperature of the mobile phase, since that is not a temperature at which anyone works in HPLC. Wouldn't it have been more appropriate to stop at a lower temperature (for example 40 degrees?), since the tests of several temperatures were done in the previous publication." We chose 70 degrees C for these evaluations because we wanted to shorten the time to column failure, to allow for a sufficient number of tests to be completed in the time available for this study. Our goal is stated in lines 64 - 66, and the experiments described in our previous publication (reference 29) show that the time to failure increases as the temperature decreases. If we had used a lower temperature (e.g. 40 degrees C), a considerably longer time would have been needed to complete the evaluations. Regarding usage of temperature in the 60 - 80 degree range in HPLC, this is actually not uncommon, particularly for separations of biopolymers like proteins, peptides, oligosaccharides and nucleic acids. A recent review of HILIC separations of biopharmaceutical drugs (T. Ikegami, J. Sep. Sci. 2019, 42, 130-213) shows that temperatures in this range have been reported in a number of publications. Other articles (Z. Hao et al, J. Sep. Sci. 2008, 31, 1449-1464; Y. Guo et al, Chromatographia 2007, 66, 223-229) have highlighted the use of elevated temperatures to adjust the selectivity of HILIC separations. No change has been made.
- Regarding the comment "I also did not understand how this temperature of the mobile phase is maintained: is only the column in the column compartment of the HPLC heated? Please clarify it in the text." The temperature was controlled using an ACQUITY Column Manager, which not only heats the column, but also the mobile phase, using an active pre-heater. The use of the ACQUITY Column Manager was added in line 84.
- Regarding the comment "Adding chromatograms comparing the various stationary phases tested is also interesting. This can be a more straightforward way to see the trend of the base stability loss as time passes." We added four representative chromatograms showing the changes that occurred after carrying out the stability evaluations. This is now Figure 2, and the subsequent figures were renumbered accordingly.
Reviewer 2 Report
The work is very good. It should certainly be published in Separations. Its topic is very timely and will find interest among scientists working with HILIC. I have only one request: please add exemplary chromatograms for one compound and several columns at the beginning of the study and after tests conducted during the course of the study.
Author Response
Regarding the comment "please add exemplary chromatograms for one compound and several columns at the beginning of the study and after tests conducted during the course of the study." We added chromatograms showing representative separations for two columns before and after the stability evaluations (lines 182-191). This is now Figure 2, and the subsequent figures were renumbered accordingly.
