In the research and development of new drugs, ion chromatography technology is widely used in drug ingredient analysis, content detection and impurity control. Ion chromatography technology makes up for the shortcomings of conventional chromatography technology in drug research and development, providing reliable technical support for drug quality control and drug safety evaluation. Through ion chromatography technology, drug developers can better understand the ingredients and properties of drugs, which can help optimize drug formulas and improve production processes, thereby improving the efficacy and safety of drugs.
Recently, in Medicilon Cloud Lecture Hall CMC special live broadcast, we received enthusiastic feedback and questions about the interpretation of ion chromatography. This article has compiled the questions in the live broadcast and answers from Jian Xu, Senior Team Leader of Medicilon’s Process Analysis Department, hoping to help reduce distress and confusion in the research and development.
Q：Anion mode, cation mode, what are the common eluents? What are their differences?
A：Anion and cation detection usually uses an ion exchange column. When eluting anions, we mainly use eluents such as potassium hydroxide, sodium hydroxide, sodium carbonate and sodium bicarbonate. In cationic systems, for example, sodium ions and methane ions require the use of acidic eluents. The more commonly used acids are methylsulfonic acid and nitric acid. These two acids are commonly used in cationic eluents. Therefore, when selecting an eluent, you need to choose an alkaline or acidic eluent according to the specific situation. For acid detection, an alkaline eluent is used; for cation detection, an acidic eluent is used.
In addition, the requirements for the eluent are also important, and a higher level of eluent needs to be selected to ensure accuracy. When manually preparing the cation eluent, you need to pay attention to using mass spectrometry grade or superior grade pure nitric acid or methanesulfonic acid to prepare the eluent. Otherwise, it may cause high detection background contamination.
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Q：Is the relationship between the different retention of ion valence states and temperature due to the effect of reaction rate? Is the retention of halide ions related to the ionic radius?
A：Yes, the reaction rate may be affected by temperature, which affects ion retention. The part about the halogen ion radius is not shown in my PPT due to limited space. During the live broadcast, I listed some classic cases to discuss the influencing factors of ion retention. The larger the ionic radius, the more significant the retention effect. Through the case in the figure below, take fluoride ions, chloride ions, bromide ions and iodide ions as examples. These ions all have the same charge, but their ionic radii gradually increase. Observing the chart, we can find that the larger the ionic radius, the stronger the retention effect under the same chromatographic conditions.
Q：Will sample X- not pass through the cation membrane and combine with H+?
A：In a cation exchange membrane, only positively charged ions are allowed to pass. These negative ions cannot pass through the cationic semipermeable membrane, so they will not pass through the semipermeable membrane and combine with hydrogen ions. Only positively charged hydrogen ions pass through the cation exchange membrane and then combine with X-, such as chloride ions, bromide ions or nitrate ions to form the corresponding acid.
Q：Can chiral separation be separated?
A：The effect of chiral separation is not ideal. Although we considered trying to separate some compounds with chiral structures by ion chromatography, the separation was poor. Therefore, other methods of detection will be chosen, such as CAD detectors, which can use chiral separation in reverse-phase mode or normal-phase separation mode with CAD as the detector.
Q：Have you ever encountered components with large molecular weights such as polyethylene glycol that cannot be eluted during ion chromatography detection, resulting in reduced column performance? How to solve this problem?
A：In the experiment, you can use the chromatography regeneration function. Our system allows the organic phase to exist. If you are in eluent generator mode, you can disconnect and short it to the column pump outlet and then connect it directly to the detector, avoiding the need to connect the suppressor and eluent generator. Then you can use organic solvents for flushing, such as using 50% acetonitrile aqueous solution to regenerate. The organic phase is used to clean the column because polyethylene glycol may be present inside, preventing elution. It should be noted that after cleaning, the chromatographic column must be stored in the eluent system. For example, when using AS chromatography columns, A series chromatography columns, and anion chromatography columns, store them in at least 5 mmol sodium hydroxide and potassium hydroxide systems, and avoid storing them in water or other organic phase systems. In addition, the decrease in column efficiency may also be due to the residual compounds. If there is a problem that cannot be eluted, you can elute with an organic item, such as 50% or 30% acetonitrile, after the sequence is completed.
Q：How to choose the mobile phase here?
A：In routine experiments, anionic eluents are often used in published literature and methods, most commonly potassium hydroxide or sodium hydroxide. These two types of eluents can solve at least 90% of anion detection. For the cation part, methylsulfonic acid is commonly used, which is currently the most common eluent in cation detection. Methanesulfonic acid is one of the most commonly used eluents when testing potassium, sodium, calcium and magnesium ions, and ammonium ions.
Q：How to inject undissolved samples?
A：For samples that are not easily soluble, a suitable diluent needs to be selected to dissolve the sample. In that case, you can choose organic reagents, such as dimethyl sulfoxide (DMSO), methanol or acetonitrile. First dissolve the sample with organic reagents, then add water or eluent to dilute it. Normally, conventional ions will no longer be wrapped or adsorbed by the compound after the first dissolution, and can usually be detected, thus not affecting the recovery rate and the accuracy of the detection amount. The main thing to note is that generally the amount of organic reagents added should not exceed 5%. Although it is okay to exceed 5%, the amount of organic reagent added is relatively large. When a sample is injected in a pure aqueous phase system, the sample may precipitate instantly, causing the risk of clogging the injection needle, the six-way valve, the chromatographic column and even the detector. So it is best not to exceed 5%, we usually use 1% or 2% organic phase. If it is more useful, more observation is needed to see if there will be a large amount of precipitation after the organic reagent is added.
Q：What ratio can be used to have how many conflicts? Will it damage the column?
A：According to the literature, it is usually okay to use 50% acetonitrile for rinsing, but be careful not to leave it in the acetonitrile. After rinsing, it should be transferred to water phase and then stored in eluent. For example, if you are using an AS series column and you need to rinse the column after the measurement, you can use 50% or 30% acetonitrile for a certain period of time. Generally, 20 times the column volume can be considered clean. Then, you can use pure water for transition, and then transition to the eluent, such as potassium hydroxide or sodium hydroxide.
Q：The life of the cation suppressor is relatively short. How should we pay attention to it during use?
A：The suppressor is most afraid of dry burning and sample adsorption. The main reason is that the semipermeable membrane in the suppressor is relatively fragile. The semipermeable membrane will be irreversibly damaged under conditions of high pressure, dry burning and excessive sample adsorption. It is recommended to use external circulating water mode and do not use detector waste liquid as suppressor water; if the suppressor is not used for a long time, rinse it once a week.
Q：What solution should be used for short-term and long-term storage of suppressors?
A：Short-term ones can be stored in eluent and ultrapure water. Pay attention to distinguish the flow paths and rinse them once a week. It is not recommended to store it for a long time. If the suppressor dries up, it may cause damage. If it has been stored for a long time without weekly maintenance, you can use the manufacturer’s suppressor regeneration method. Regenerate the suppressor before use. Do not use it directly connected to the instrument.
