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Improved Liquid Chromatography for Anionic Compounds

A liquid chromatography column, liquid chromatography analysis technology, applied in separation methods, chemical instruments and methods, material separation, etc.

Active Publication Date: 2021-10-19
AGILENT TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, mass spectrometers cannot be used in positive ion mode when the mobile phase contains EDTA due to its high ionization efficiency in positive ion mode analysis, thus limiting the method to negative ion mode analysis using mass spectrometers

Method used

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  • Improved Liquid Chromatography for Anionic Compounds
  • Improved Liquid Chromatography for Anionic Compounds
  • Improved Liquid Chromatography for Anionic Compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] EDTA as a mobile phase additive

[0085]As discussed in the background, there are reports in the literature of the use of EDTA to improve the performance of LCs. See Pesek et al. 2011 and Myint et al. 2009. To investigate this, several experiments were further performed. Organic acids (succinic acid, malic acid, citric acid / isocitric acid, α-keto Samples (15 ng) of glutaric acid (α-KG), L-glutamine, and glutamic acid (Sigma Aldrich, St. Louis, MO)). The connecting capillary was PEEK lined and a 1290 binary pump was coupled to a 6545Q-TOF instrument (Agilent Technologies, Inc., Santa Clara, CA). Solvent A consisted of 10 mM ammonium acetate (pH 9.0) in water. Solvent B consisted of 10 mM ammonium acetate (pH 9.0) in 90:10 acetonitrile / water. For each experiment, the flow rate was 0.25 ml / min and a sample volume of 3 μl was injected onto the column. After loading the sample solution, the column was conditioned with 90% solvent B for 2 min, and then the gradient was ...

Embodiment 2

[0088] Pyrophosphoric acid as mobile phase additive

[0089] Samples (15 ng) were analyzed on a HILIC column using a mobile phase containing 10 mM ammonium acetate, pH 9.0, with or without 5 μM pyrophosphate in the eluent. The connecting capillary was PEEK lined and the 1290 binary pump was coupled to the 6545Q-TOF instrument. The result is as Figure 3A-3B shown. Phosphorylated compounds and organic acids showed better peak shape when 5 μM pyrophosphate was added to the mobile phase. Surprisingly, the ion suppression effect observed with the addition of EDTA was not observed when pyrophosphoric acid was used as an additive.

Embodiment 3

[0091] Methylene diphosphate as mobile phase additive

[0092] Samples (15 ng) were analyzed on a HILIC column using a mobile phase containing 10 mM ammonium acetate (pH 9.0) with and without 5 μΜ alkylene diphosphate in the eluent. The connecting capillary was PEEK lined and the 1290 binary pump was coupled to the 6545Q-TOF instrument. The result is as Figures 4A-4B shown. Phosphorylated compounds and organic acids showed better peak shape when 5 μM of alkylene diphosphate was added to the mobile phase. Surprisingly, the ion suppression effect observed when EDTA was added was not observed here. What's more, alkylene diphosphoric acid gave better results than pyrophosphoric acid and thus worked as a better mobile phase additive.

[0093] Analysis of 15 ng sample using a second LC system, on a HILIC column, in stainless steel (SSTL) hardware, using 10 mM ammonium acetate, pH 9.0, with or without 5 μM ADP as mobile phase solvent . All connecting capillaries are SSTL excep...

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Abstract

The present disclosure generally relates to improved liquid chromatography for anionic compounds, which is particularly useful in conjunction with mass spectrometry. The method includes adding an additive to the mobile phase carrying the sample. Mobile phase additives are effective in improving the peak shape of target analytes in the acquired data and / or eliminating or at least reducing ion suppression. The method is particularly suitable for anionic compounds, such as phosphorylated compounds.

Description

technical field [0001] The present disclosure generally relates to improved liquid chromatography. In particular, the disclosure relates to improved methods for isolating and analyzing compounds generally using liquid chromatography-mass spectrometry (LC-MS) systems. The method is particularly suitable for anionic compounds, such as phosphorylated compounds. Background technique [0002] In a liquid chromatography (LC) system, a mobile phase consisting of one or more solvents is driven at high system pressure through a separation unit, usually provided in the form of a chromatography column. In high performance LC (HPLC) systems and ultra high performance LC (UHPLC) systems, the system pressure can be as high as, for example, about 1200 bar. The column contains a stationary phase, which in LC is usually provided as a packed bed of particles such as silica beads. Particles are formulated and / or functionalized to separate different analytes or components (eg, chemical compo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
CPCG01N30/02G01N30/34G01N30/7233B01D15/305B01D15/325B01D15/34B01D15/424G01N2030/8831G01N30/16G01N30/88G01N30/06G01N2030/027G01N2030/065G01N2030/884
Inventor 乔迪·J·荷斯奥洪峰·尹奥斯卡·G·波特
Owner AGILENT TECH INC