Properties of organic acids

Organic acids refer to some acidic organic compounds. The most common organic acids are carboxylic acids, the acidity of which originates from the carboxyl group (-COOH). Sulfonic acid (-SO3H), sulfinic acid (RSOOH), sulfuric acid (RCOSH), etc. also belong to organic acids. Organic acids are mostly soluble in water or ethanol, but insoluble in other organic solvents.

Cause of tailing

The retention and separation ability of some organic acids on the C18 column is very weak, so the ionic force needs to be used to separate the compounds, but the mobile phase system of liquid mass spectrometry cannot add non-volatile salts and volatile ion-pair reagents. (such as trifluoroacetic acid, etc.) will also reduce the mass spectral response and cannot meet the quantitative limit requirements, so the zwitterion column HILIC-Amphion II has become a good choice.

Figure 1. L-glutamic acid tailing peak

However, in the test, it was found that the target would have serious tailing phenomenon, and it was still very serious after adjusting the pH. After removing the chromatographic column and using two-pass, it was found that the target 4 (L-glutamic acid) was more than other targets. Some retention time, and the tailing is still serious, it is inferred that the liquid phase system may be contaminated (the mobile phase system of the previous test project contains trifluoroacetic acid, which may cause residues), resulting in serious tailing.

Figure 2. Four kinds of organic acids are connected to the two-way peaks


Step 1

Remove column and replace with union.

Step 2

Remove all mobile phase bottles, replace with 0.1% formic acid aqueous solution, and rinse at a flow rate of 0.3mL/min for 2h.

Step 3

Replace 0.1% formic acid aqueous solution with 0.1% ammonia aqueous solution, rinse at 0.3mL/min flow rate for 2h.

Step 4

Replace the 0.1% ammonia solution with an aqueous solution, and rinse at a flow rate of 0.3 mL/min for 2h.

Step 5

Change the aqueous solution to methanol solution, wash at 0.3mL/min for 2h.

Note: During cleaning, the liquid phase is not connected to the mass spectrometer. If there are other sources of contamination in the liquid phase system and the cleaning effect is still unsatisfactory according to the above, you can rinse with isopropanol after methanol cleaning, and then replace the system with methanol. isopropanol solution.

Figure 3. L-glutamic acid peaking situation after liquid phase system cleaning

Discussion: In most cases, when we find peak tailing, we think more about the problem of the liquid phase method or the chromatographic column, and rarely eliminate the contamination problem in the liquid phase system. I hope this case can help you in your experience. When the peak is tailed and the cause cannot be identified, provide an idea.

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