Evaluation of the Base Stability of Hydrophilic Interaction Chromatography Columns Packed with Silica or Ethylene-Bridged Hybrid Particles

Round 1

Reviewer 1 Report

To investigate the base stability of eight different HILIC columns, key mobile phase parameters including temperature, pH and concentration of the aqueous component were adjusted for one of the columns in the most effective (destructive) manner. All columns were tested with these optimized parameters, providing great insight into the relationship between physical properties and chemical stability.

The manuscript is well written, has a clear structure and was easy to follow. The determined efficiency losses were evaluated by using three columns of every type which is a great approach and sufficient to generate significant results. The tables and graphics, which are shown, are easily accessible to the reader and underline the effects described in the text.

The test conditions should be able to cause a 50 % efficiency loss after approximately 5 h of exposure to the basic solutions, which was achieved for the selected CORTECS HILIC column. However, there is no information provided why this specific column was chosen to optimize the conditions. Considering L77/L78 it would have been an interesting approach to figure out the limits columns that already show improved base stability.

Minor remarks:

·         Page 1, L11: First mention of “…hydrophilic interaction chromatography…”. The abbreviation HILIC is explained in L13 and should already be mentioned earlier.

·         Page 1, L39: “Si(OH)4” The “4” must be subscript.

·         Page 2, LL52-53: Please use the same number of digits when comparing two values.

·         Page 2, L86: “Millipore Milli-Q^TM system” (Manufacturer, town, state and country are missing)

·         Page 3, L98: ”…advanced electrochemistry meter from Thermo Fischer….” (Town, state, country are missing)  

·         Page 3, L106: use “water” instead of H₂O to be consistent.

·         Page 4, Table 1: What Information does the column “Curve” provide? It is not mentioned in the text.

·         Page 4, L169: Probably an ammonium hydroxide solution was used instead of the salt “ammonium hydroxide” 

·         Page 5, Figure 1: For the blue circles it looks like the last circle at/beyond 15 h is missing. In the description (LL191-192) “water” is used to describe the aqueous component. Actually “buffer” would be the more accurate word. (also: Page 6 L251)

·         Page 5, L 194: “…we observed large (100-400 %) increases…”. At what time could those effects be seen?

·         Page 10, L328: “than” --> “that”

Major remarks:

·         Page 9, Figure 5: In the y-axis the signals of the UV-measurement are presented. Please explain why there is a difference in peak height, when the only parameter changed is the stationary phase.

·         Page 10, Table 4: Number of replicates?

Comparing Table 4 and Table 3 (page 8): The order of the stationary phases is not immediately understandable. I would consider ordering all columns in the same manner or to provide a “sum” column in Table 4 where the average retention factor changes are summed up. 

Comparing Table 4 and Table 3 (page 8): Use “Column” or “Stationary Phase”.

·         For the figures: One of the central aspects of this work was to figure out the time when efficiency loss was 50%. However, on the y-axis the value of 50 % cannot be spotted immediately but must be guessed in between 45 and 60%. I recommend improving the scaling of the axis. 

·         The tailing factor is mentioned as one of three parameters that were calculated to determine column performance. Nevertheless, this parameter is hardly mentioned in the remaining manuscript.

·         Please provide some information why a CORTECS HILIC column was used for the optimization of measuring parameters.

Author Response

Regarding the reviewer remarks for manuscript separations-1761930, I herein provide my response. The reviewer remarks are in black type. Responses in red indicate that a change was made to the manuscript, while those in blue indicate that no change was made.

1. [Comments and Suggestions for Authors] The test conditions should be able to cause a 50% efficiency loss after approximately 5 h of exposure to the basic solutions, which was achieved for the selected CORTECS HILIC column. However, there is no information provided why this specific column was chosen to optimize the conditions. Considering L77/L78 it would have been an interesting approach to figure out the limits columns that already show improved base stability.

A sentence explaining why the CORTECS HILIC column was chosen has been added as new lines 136-137 (see point 8 below). We agree that that exploring the limits of the ethylene-bridged hybrid columns would be interesting, and are planning to carry out these experiments.

2. [Major remarks] Page 9, Figure 5: In the y-axis the signals of the UV measurement are presented. Please explain why there is a difference in peak height, when the only parameter changed is the stationary phase.

The differences in peak height are due to differences in the peak widths. This was confirmed by checking the peak areas, which are similar for the separations obtained using the different columns.

3. [Major remarks] Page 10, Table 4: Number of replicates?

The number of replicates (3) was added to the header of Table 4.

4. [Major remarks] Comparing Table 4 and Table 3 (page 8): The order of the stationary phases is not immediately understandable. I would consider ordering all columns in the same manner or to provide a “sum” column in Table 4 where the average retention factor changes are summed up.

A column titled “Overall Avg” was added to Table 4, to make it clear that the stationary phases are listed in order of increasing overall average. In Table 3 the stationary phases are listed in alphabetical order, and no change was made to this table.

5. [Major remarks] Comparing Table 4 and Table 3 (page 8): Use “Column” or “Stationary Phase”.

“Column” was changed to “Stationary Phase” in Table 4 for consistency with Table 3.

6. [Major remarks] For the figures: One of the central aspects of this work was to figure out the time when efficiency loss was 50%. However, on the y-axis the value of 50% cannot be spotted immediately but must be guessed in between 45 and 60%. I recommend improving the scaling of the axes.

The y-axis scales were changed in Figures 1-4 to allow the 50% points to be easily located. In addition, horizontal lines at 50% efficiency loss were added.

7. [Major remarks] The tailing factor is mentioned as one of three parameters that were calculated to determine column performance. Nevertheless, this parameter is hardly mentioned in the remaining manuscript.

As stated in the original manuscript (L198-199), the information provided by the changes in tailing factors is redundant with the efficiency trends. For this reason, we didn’t comment further on the changes in the tailing factors. No change was made.

8. [Major remarks] Please provide some information why a CORTECS HILIC column was used for the optimization of measuring parameters.

A sentence was added at L136-137 in the revised manuscript to explain why CORTECS HILIC columns were used when studying the effects of the water concentration, buffer pH and temperature: "CORTECS HILIC columns were chosen because they showed significant changes after < 15 h of contact with the challenge mobile phase." 

9. [Minor remarks] Page 1, L11: First mention of “…hydrophilic interaction chromatography…”. The abbreviation HILIC is explained in L13 and should already be mentioned earlier.

The abbreviation HILIC was moved to the first mention of “hydrophilic interaction chromatography” in L11.

10. [Minor remarks] Page 1, L39: “Si(OH)4” The “4” must be subscript.

This change has been made.

11. [Minor remarks] Page 2, LL52-53: Please use the same number of digits when comparing two values.

In L53, “50.5” was changed to “50.50”.

12. [Minor remarks] Page 2, L86: “Millipore Milli-Q™ system” (Manufacturer, town, state and country are missing)

The town, state and country have been added (L86). The manufacturer (Millipore) was already given.

13. [Minor remarks] Page 3, L98: “…advanced electrochemistry meter from Thermo Fischer…”” (Town, state, country are missing)

The town, state and country have been added (L98).

14. [Minor remarks] Page 3, L106: use “water” instead of H₂O to be consistent.

This change has been made (line 107 in revised manuscript).

15. [Minor remarks] Page 4, Table 1: What information does the column “Curve” provide? It is not mentioned in the text.

A footnote was added to Table 4 explaining what “Curve” designates, and what the values 11 and 6 signify: "Curve indicates the shape of the change of the solvent composition for each gradient segment. Curve 11 is a step change at the end of the segment and curve 6 is a linear change during the segment."

16. [Minor remarks] Page 4, L169: Probably an ammonium hydroxide solution was used instead of the salt “ammonium hydroxide”

In L174 of the revised manuscript “ammonium hydroxide” was changed to “an ammonium hydroxide solution”.

17. [Minor remarks] Page 5, Figure 1: For the blue circles it looks like the last circle at/beyond 15 h is missing. In the description (LL191-192) “water” is used to describe the aqueous component. Actually “buffer” would be the more accurate word. (also: Page 6 L251)

The x-axis span in Figure 1 was increased to show the last data point. The captions for Figures 1-4 were revised to use “buffer” in place of “water” (L197-198, 238, 259 and 284 in the revised manuscript).

18. [Minor remarks] Page 5, L 194: “…we observed large (100-400 %) increases…”. At what time could those effects be seen?

A phrase was added (L202-203 in the revised manuscript) to indicate when the increases were seen: "Accompanying the decreases in efficiency, we observed large (100 - 400%) increases in the tailing factors of the three test compounds that occurred at the times of the steepest drops in efficiency. 

19. [Minor remarks] Page 10, L 328: “than” --> “that”

This change has been made in L339 in the revised manuscript.

Reviewer 2 Report

Comments on the manuscript Separations-1761930 “Evaluation of the Base Stability of Hydrophilic Interaction Chromatography Columns Packed with Silica or Ethylene-Bridged Hybrid Particles” by T. H. Walter, B. A. Alden, and K. Berthelette.

The authors studied stability of several commercial HILIC stationary phases under basic pH conditions. The influence of pH, water amount, and temperature was evaluated on CORTECS columns in detail. Then, eighth silica and ethylene-bridged hybrid sorbents were stressed and the effect of challenge solution on the individual sorbents compared. The work is important and interesting and it fits the scope of the journal. I have two minor comments to the manuscript.     

Table 1 and the gradient program: First, it is unclear why the times are specified with two decimal places precision. For instance, the challenge solution was applied for 20.37 min exactly; here I do not understand why for such specific time. This is unclear, strange, and unexplained. Second, concerning the column “Curve”, the authors should explain, at least in the legend of the table, the meaning of the numbers. The readers are not obliged to be familiar with the system of numbering of the various gradient profiles used by Waters.

Author Response

The gradient times were calculated based on specific numbers of column volumes for the gradient segments. To indicate this, a phrase was added in L128 of the revised manuscript: "A challenge solution of varying composition was then passed through the column at 0.4 mL/min for 20.57 min (75 column volumes), ...".

Regarding explaining the meaning of “Curve” and the meaning of the numbers in Table 1, a footnote was added to Table 1: "Curve indicates the shape of the change of the solvent composition for each gradient segment. Curve 11 is a step change at the end of the segment and curve 6 is a linear change during the segment."