Chromatography Forum

This is my first time posting here, so I don’t know exactly what information to give, but I would really appreciate some help with my LC-MS column (MS is ESI). I’m still relatively new to LC-MS, and I’ve been analyzing aqueous solutions of sucrose with the Phenomenex Luna 3um NH2 column, HILIC chromatography. My mobile phase has acetonitrile and water with 0.1% ammonium hydroxide. I understood that I was supposed to leave a trickle of mobile phase running through the column over night, so after I finish using the instrument I’ve been leaving it on 50% acetonitrile, 25% pure water, and 25% water with 0.1% ammonium hydroxide (to keep the pH constant). This has been working just fine until last week. All of a sudden, the pressure shot up, and after testing different parts of the instrument, I determined my column was almost completely blocked somehow. I only ran aqueous sucrose that day. I tried to run water and methanol through the column to get it to open up, and it has improved somewhat, but the pressure is still around 1500 psi with only 0.03 mL/min flow. It should be around 1500 psi with 0.2 mL/min. I’ve been filtering solvents and purging about every week. Should I keep running some type of solvent, or reverse the flow, or any other suggestions?

The Luna NH2 material is based on silica, and silica dissolves at a significant rate at a pH > 8. Possibly the high pH conferred by 0.1% NH4OH dissolved enough silica that some of the particles collapsed. The resulting small fragments would account for the increase in backpressure you describe. Where did you get these operating conditions?

The Luna NH2 material is based on silica, and silica dissolves at a significant rate at a pH > 8. Possibly the high pH conferred by 0.1% NH4OH dissolved enough silica that some of the particles collapsed. The resulting small fragments would account for the increase in backpressure you describe. Where did you get these operating conditions?

I was following a paper for sucrose separation. They actually put the 0.1% NH4OH in with both the acetonitrile and water but I only wanted a very small pH change.

Using 0.1% NH4OH in the mobile phase during analytical runs is one thing. Leaving it in the column constantly for days or weeks is something else. You now know that that's not innocuous.

Hi LCHelp,

Definitely ask for Phenomenex's 2020/2021 Chromatography Product Guide-- it holds a lot of information pertaining to not only the Luna series of columns, but their entire catalogue along with a variety of useful calculations, expected pressure values, recommended wash solutions, etc. As a preface, I do not work for Phenomenex, but most of my columns are from them and if you're switching between a few often, it can be helpful to have this book handy. As for your column, I completely agree with Andy. Luna columns, especially Luna Amino, are particularly susceptible to degradation at high pH values. Acids and bases actually do more damage when they're not under pressure (like a low-flow over-night). I would recommend that each time you use a mobile phase with any additives wash the column after you're finished with all of your samples. Then I would leave it in that mixture if you plan to keep a low-flow overnight. The operating conditions themselves are not bad, and this issue does not have to do with the addition of base to both aqueous and organic phases, but you never want to leave that base (or any additive) on the column overnight. This column is probably completely dead, and I'd wager that the USP you're basing this analysis off of went through columns much quicker than if they didn't have these additives. For some people that's not really a problem, but if you need columns to last then definitely stick to what the company recommends.

Hi LCHelp,

Definitely ask for Phenomenex's 2020/2021 Chromatography Product Guide-- it holds a lot of information pertaining to not only the Luna series of columns, but their entire catalogue along with a variety of useful calculations, expected pressure values, recommended wash solutions, etc. As a preface, I do not work for Phenomenex, but most of my columns are from them and if you're switching between a few often, it can be helpful to have this book handy. As for your column, I completely agree with Andy. Luna columns, especially Luna Amino, are particularly susceptible to degradation at high pH values. Acids and bases actually do more damage when they're not under pressure (like a low-flow over-night). I would recommend that each time you use a mobile phase with any additives wash the column after you're finished with all of your samples. Then I would leave it in that mixture if you plan to keep a low-flow overnight. The operating conditions themselves are not bad, and this issue does not have to do with the addition of base to both aqueous and organic phases, but you never want to leave that base (or any additive) on the column overnight. This column is probably completely dead, and I'd wager that the USP you're basing this analysis off of went through columns much quicker than if they didn't have these additives. For some people that's not really a problem, but if you need columns to last then definitely stick to what the company recommends.

Thank you. Is there anything I could possibly do? Getting another column is not an option as far as I know. I didn't know a trace amount of base would be a problem

Sorry, but you're going to have to get another column anyway or else you won't be performing this analysis. Once a column has become blocked because of silica collapse, the increase in backpressure is irreversible.

I just checked the pH of the solution and it is neutral, not even above 7. Could it still be causing silica collapse at that pH?

Hi LCHelp,

PLEASE READ THE ENTIRE POST BEFORE MAKING A DECISION, I DISCOVERED A LOT REGARDING THESE COLUMNS AS I DID MORE RESEARCH which is displayed later on in the post!

According to the booklet, the Luna Amino (NH2) column actually has a stability from pH 1.5-11.0! So I was completely incorrect about the pH stability, and rather I was thinking about their cyano columns that are non-stable at higher than neutral pH. Here is an excerpt from their booklet that I found quite interesting: "Column life for most amino columns can be problematic as the amino bonding easy strips off the silica". Immediately after they imply their column is more rugged than other amino columns-- I can't say anything on this front as I don't use this phase regularly. However, I do believe that this disproves the pH theory, but it does not rule out the fact that much of your mobile phase was running at very low pressure with a potentially harmful additive. From many of the vendors I talk to, they recommend to never leave your column in a buffered mobile phase (include bases and acids) and that the pressure is what keeps many of them from degrading in this environment. I'm not sure of the exact reasoning--hopefully someone will pop in and answer this for the both of us, but this could be enough to eventually destroy a column. What I can do is recommend a cleaning procedure directly from the booklet for this column!
Below is their recommended cleaning procedure for Silica-based columns:
-Flow Rates should be 1/5-1/2 of typical flow-rate
-To estimate column volume: V=(pi)r^2*L
V is column volume in mL, r is radius in cm and L is length in cm.
"Reverse Phase Columns (C18,C12...NH2)
Rinse with 10 column volumes each of:
95% Water/5% Acetonitrile
(for buffer removal) (potentially precipitated sugars/salts in your case?)
THF (100%)
95% Acetonitrile/5% Water
Mobile Phase"

^ This is the general procedure for reverse-phase cleaning
NOTE: Reverse the column's flow direction prior to the cleaning!!!! If there is a build-up of some particulate on the end of your column (rather than phase collapse) this could dislodge that blockage and send it down-stream to the waste (disconnect your detector/anything down-stream from column during this exercise).
Another interesting note regarding amino columns that I read is:
"Stationary Phase considerations:
Maintain pH between 2.0-8.0
Use Guard Columns
AVOID ALDEHYDES AND KETONES WITH AMINO COLUMNS"
Since you are working with sugars you're probably dealing with aldoses and ketoses, but this column should be designed for the analysis of sugars so I'm not sure why this comment pops up so late in the booklet. I would probably contact someone from Phenomenex to elaborate on this portion.

Here is what they say regarding column storage (sort of an alternative to having it low-flow over-night every night would be store it in an inert mobile phase and then wash and condition the next morning in your desired mobile phase):
-Column storage conditions affect column lifetime
-NEVER store columns containing buffers or ion-pairing reagents (even though you're not using a buffer, the ammonia hydroxide or whatever base your were using is included in this statement due to the potential of precipitation on the column).
-Flush with at least five column volumes of mobile phase without buffer to remove buffers or salts.

OH GOODNESS I found ANOTHER exception for the Luna Amino column (this thing must be really special: "Exception: (this is under the column cleaning procedure and applies directly to the use reverse-phase amino column usage)
Recommended for cleaning Luna Amino when used in reverse-phase mode:
1. Wash with at LEAST 30 columns volumes of sodium hydroxide pH 11.0
2. Flush with at least 30 column volumes of water (HPLC grade)
3. Re-equillibrate to mobile phase conditions."

Upon further investigation it seems that amino problems kind of suck all-around when it comes to longevity. Here's anecdotal evidence on Research Gate regarding the life-span of these columns: https://www.researchgate.net/post/Rapid ... PLC-column
Anyway, I hope that second cleaning method is useful and can restore your column. I hope it is just some minor blockage and not phase degradation/collapse! Good luck!

In my opinion there is no way that silica-based amino column would be stable at pH 12, may be for a day or two. I am still puzzled why people are using amoinopropyl column for HILIC since there are a much better and more robust solutions for HILIC. In a lot of cases the peak shape is not that good and stability even at pH below 7 is an issue. It is good for column manufacturers since people are buying more columns, but it is not good at all for customers. I am column manufacturer, and I would be hesitant to commercialize something which is not stable and robust.

Here's another puzzle about this application, Vlad. In HILIC, the alpha- and beta- anomers of reducing sugars are separated. The result is a pattern of two peaks connected by a continuum. Raising the pH above 7.5 or so accelerates the rate of mutarotation, causing the peaks to collapse into a singlet. Normally this can be accomplished by including 0.1% triethylamine or something similar in the mobile phase. I speculate that that's the function of the HN4OH in the Phenomenex instructions here. Using a column material with an amine immobilized on the surface creates a locally high pH there and accomplishes the same thing. What puzzles me about this Phenomenex column method is the combination of an amine column material plus the mobile phase with the elevated pH. One of them's redundant.

Here's another puzzle about this application, Vlad. In HILIC, the alpha- and beta- anomers of reducing sugars are separated. The result is a pattern of two peaks connected by a continuum. Raising the pH above 7.5 or so accelerates the rate of mutarotation, causing the peaks to collapse into a singlet. Normally this can be accomplished by including 0.1% triethylamine or something similar in the mobile phase. I speculate that that's the function of the HN4OH in the Phenomenex instructions here. Using a column material with an amine immobilized on the surface creates a locally high pH there and accomplishes the same thing. What puzzles me about this Phenomenex column method is the combination of an amine column material plus the mobile phase with the elevated pH. One of them's redundant.

We developed a few methods when we used a low pH to collapse two sugar peaks. It is usually done with TFA. I agree with your assessment on column/mobile phase. I would expect HILIC to be much weaker at high and very low pH, which can be compensated by using more ACN.

[quote="TylerSmith123"]Hi LCHelp...


Thank you for all this information on cleaning the column! I am running HILIC, not reverse phase, but I did find some cleaning instructions for this column for HILIC.

I have had a problem with my peaks splitting slightly which might be related to the anomers separating. The NH4OH helped with that to a degree. I am less familiar with HILIC chromatography than with other types, so I'm figuring this out as I go. If anyone has any ideas about any of these issues, or more suggestions for cleaning the column, I would appreciate hearing them!

Hi LC,

Sorry about the confusion! The standard column cleaning procedure from Phenomenex on the HILIC is 10 column volumes of 95% Water and 5% Acetonitrile followed with 95% 100mM Ammonium acetate pH 5.8 with 5% acetonitrile. After that re-run in the initial 95% water, 5% acetonitrile followed by your mobile phase. I'm much less familiar with HILIC, but I agree with a lot of the points Vlad and Andy brought up. I'd be very surprised even in an analytical setting the robustness of the amino columns at high and low pHs. Either way, I anticipate that if you're using a mobile phase at either of those extremes, column lifetime will be significantly reduced. However, with a buffered mobile phase that is barely over seven I don't believe it would impact your performance in a similar fashion (unless it's being left on overnight). This may be a shot in the dark but could this collapse be due to a sudden shift in pressure on the column which could potentially damage the silica packing?

Hi everyone,

Thanks to those who have shared diagnostic steps and practical fixes.

For anyone dealing with a clogged LC-MS column, I wanted to share an article that covers common causes, troubleshooting strategies, and a few prevention tactics:

LC and LC-MS Column Clogging: Common Causes and Proven Prevention Strategies

I hope it’s helpful!