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- Posts: 9
- Joined: Sat Mar 01, 2025 12:40 am
Hello,
I am trying to bring a version of an Agilent underivatized amino acid method in-house: https://www.agilent.com/cs/library/appl ... gilent.pdf
This uses an Agilent HILIC-Z column, and the mobile phases are (A) 100% water + 0.1% formic acid + 10 mM ammonium formate, and (B) 90% acetonitrile 10% water + 0.1% formic acid + 10 mM ammonium formate. The gradient starts at 100% B, ramps down to 20% B, then re-equilibrates at 100% B. The Q1 and Q3 masses I'm using are 147.1, and 130.1/84.1.
So far, all I've done is (1) condition the column per Agilent's instructions, and (2) test the handful of amino acids we happened to have in-house. These are not analytical standards; they're generally 98+ or 99+% grade. I made a concentrated aqueous solution, then diluted into MPB from 1 ppb up to 2.5 ppm, following the Agilent paper. My calibration curves all look nice, with one exception: I get a large and constant integrated area for lysine (regardless of concentration), the peak shape isn't very Gaussian, and the peak even shows up with a zero-volume injection.
I've tried disconnecting the column and pumping through a union directly into the MS. When I did this, I saw no peak when injecting a blank, but I did see a peak when injecting a standard. So I am pretty sure that I have somehow contaminated my column with lysine, even though I've only ever injected a small number of relatively dilute standards.
I should also mention: the lysine I used was rather old. It was yellow and the top layer of powder had hardened.
I have tried cleaning the column overnight with 50-50 acetonitrile-water + 0.1% formic acid + 200 mM ammonium formate. I followed this with an extended 80% aqueous flush, then re-equilibrated with full organic. This did not help. The first blank after this procedure showed about 2-3X as much lysine signal as before (and even worse peak shape), but then the second and third blanks looked exactly like the ones before cleaning. Same shape, same area.
Is this actually lysine/lysine byproducts contamination? Did I maybe fail to properly condition the column? Is there something else / more aggressive I can do to clean the column that's still safe for HILIC-Z? How high on aqueous content can I safely go with this column? I would appreciate any help at all. Thank you!
I am trying to bring a version of an Agilent underivatized amino acid method in-house: https://www.agilent.com/cs/library/appl ... gilent.pdf
This uses an Agilent HILIC-Z column, and the mobile phases are (A) 100% water + 0.1% formic acid + 10 mM ammonium formate, and (B) 90% acetonitrile 10% water + 0.1% formic acid + 10 mM ammonium formate. The gradient starts at 100% B, ramps down to 20% B, then re-equilibrates at 100% B. The Q1 and Q3 masses I'm using are 147.1, and 130.1/84.1.
So far, all I've done is (1) condition the column per Agilent's instructions, and (2) test the handful of amino acids we happened to have in-house. These are not analytical standards; they're generally 98+ or 99+% grade. I made a concentrated aqueous solution, then diluted into MPB from 1 ppb up to 2.5 ppm, following the Agilent paper. My calibration curves all look nice, with one exception: I get a large and constant integrated area for lysine (regardless of concentration), the peak shape isn't very Gaussian, and the peak even shows up with a zero-volume injection.
I've tried disconnecting the column and pumping through a union directly into the MS. When I did this, I saw no peak when injecting a blank, but I did see a peak when injecting a standard. So I am pretty sure that I have somehow contaminated my column with lysine, even though I've only ever injected a small number of relatively dilute standards.
I should also mention: the lysine I used was rather old. It was yellow and the top layer of powder had hardened.
I have tried cleaning the column overnight with 50-50 acetonitrile-water + 0.1% formic acid + 200 mM ammonium formate. I followed this with an extended 80% aqueous flush, then re-equilibrated with full organic. This did not help. The first blank after this procedure showed about 2-3X as much lysine signal as before (and even worse peak shape), but then the second and third blanks looked exactly like the ones before cleaning. Same shape, same area.
Is this actually lysine/lysine byproducts contamination? Did I maybe fail to properly condition the column? Is there something else / more aggressive I can do to clean the column that's still safe for HILIC-Z? How high on aqueous content can I safely go with this column? I would appreciate any help at all. Thank you!
