This is the 2nd part of our Chromatographic Maintenace series. Please read through…

Cyano column

The cyano column is also a column that can be used in both normal-phase and reversed-phase modes. The weakness of the cyano column under reversed-phase conditions is that it is very sensitive to moderately polar solvents, such as THF (tetrahydrofuran), EtOH (ethanol), etc. Therefore:

  1. These moderately polar solvents should be avoided during use, and should not be stored in such solvents;
  2. The cyano-column phase switching should be fully excessive, and then all the mobile phase and mixed standard are newly prepared to avoid the influence of the solvent. According to 100% methanol–100% acetonitrile–100% isopropanol–100% acetonitrile , each flushing for more than 40min (isopropanol has high viscosity and high pressure, pay attention to adjusting the flow rate within the appropriate range);
  3. Allow the mobile phase to equilibrate for a long enough time (initial injection if necessary), and when the baseline is stable, inject the sample in the order of empty injection–blank solvent–reference substance–test sample;

SCX/SAX

The cation/anion exchange (SCX/SAX) column should avoid high proportion of organic phase and high concentration of salt (>100mM), otherwise the bonded phase will easily fall off, or the column may be blocked by salt precipitation, and the column is not recommended to exist in the high proportion of organic phase. ; In addition, if the column is stored for a long time, it is easy to cause the bonded phase to fall off (it is generally recommended to store it in 10% methanol at 4°C, and then take it out and rinse it every other day to ensure that the bonded phase is in an active state).

  1. When there are problems such as the decrease of resolution and the drift of retention time, firstly rinse the column with 10% methanol (1-2 hours), rinse the salt and acid, and then reconstitute a new mobile phase to balance the chromatographic column. After fully equilibrating, inject the sample in the order of empty needle–blank solvent–reference substance–test substance;
  2. If washing with 10% methanol water is ineffective, use 100mmol/L NaClO4 solution (adjust pH 4.0)-10% methanol water for SCX column, and wash each solvent for more than 60min;
  3. If washing with 10% methanol water is ineffective, use 1M ammonium nitrate — 40% methanol — 100% isopropanol — water — 10% methanol water for the SAX column, and wash each solvent for more than 60 minutes;

Sugar-H/Sugar-Ca

Polymer-based chromatographic columns are tightly packed in the expanded form of the resin. If the sample is well prepared, most problems are related to the column bed and packing. During use, care should be taken to control the column temperature and flow rate to avoid damage to the column bed caused by temperature and pulses, and the maximum proportion of organic phase should not exceed 5%.

Sugar-H: When there are problems such as abnormal peak shape, decreased column efficiency, and decreased resolution, you can use:

  1. The acid solution with pH 2.5 is washed at low flow rate for 12 hours. The acid can be used sulfuric acid, hydrochloric acid, phosphoric acid, perchloric acid (avoid the use of strong oxidizing acids such as nitric acid), and the pH is adjusted to 2.5. When flushing, pay attention to setting the temperature to 80°C or 85°C and the flow rate to be 0.5mL/min. Try not to interfere with other cations in the water in which the acid solution is prepared;
  2. Equilibrate with mobile phase after rinsing, and then inject samples in the order of empty needle–blank solvent–reference substance–test substance;
  3. In daily use, pay attention to whether there is a temperature difference between the head end of the column and the tail end of the column, and try not to contain other cations in the water for preparing the mobile phase (especially pay attention to monovalent ions such as Na⁺ and K⁺);

Sugar-Ca: The problems of calcium cation columns are mostly caused by the loss of Ca²⁺ during use, so it is necessary to pay attention to replenish the lost Ca²⁺ in time (generally when the above problems occur, or after using 5L of mobile phase, you need to use Ca²⁺ solution reactivation):

  1. Rinse with 0.5g/L EDTA Ca (CAS: 23411-34-9) solution at low flow rate for 12 hours. When rinsing, pay attention to setting the temperature to 80°C or 85°C, and the flow rate is mostly 0.5mL/min. Try not to interfere with other metal cations in the water for preparing Ca²⁺ solution;
  2. Equilibrate with mobile phase after rinsing, and then inject samples in the order of empty needle–blank solvent–reference substance–test substance;
  3. In daily use, pay attention to whether there is a temperature difference between the head end and the tail end of the column, and try not to interfere with other metal cations in the water in which the mobile phase is prepared (especially alkali metal ions such as Na⁺ and Mg²⁺);

Zn Powder reduction column

The Zn powder reduction column should not touch water, and the Zn powder will be deactivated when exposed to water, so when encountering problems, the general treatment is as follows:

  1. 100% methanol rinse, 1.0 flow rate backwash for more than 3 hours;
  2. Equilibrate with mobile phase after rinsing, and then inject samples in the order of empty needle–blank solvent–reference substance–test substance;
  3. If the white substance is washed out, it may be that the packing is damaged, and the chromatographic column needs to be repurchased;
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