Testing the Performance of an Ion Chromatography Column

Ion chromatography column performance test Ion column chromatography is used to separate cations and anions. Ion separation using ion exchange resin (In this laboratory, an anion exchange resin is used as a high molecular weight polymer.) The time required for a particular ion to pass through a column depends on the affinity of the ion. In this laboratory, the mobile phase of anionic sodium bicarbonate drives out ions from the column.

For ion exchange chromatography, the use of charge helps to isolate rubisco. For ion exchange chromatography, a DEAE cellulose fast flow column was used. The column was equilibrated by adding 30 mL buffer A (10 mM Tris, pH 0, 3 mM EDTA). The dialyzed sample was centrifuged at 1,000 rpm for 3 minutes on a benchtop centrifuge and 1 ml of P1 and P2 samples were transferred to an Eppendorf tube and labeled. P1 was diluted 1: 50 by adding 5,000 μL Buffer A and 100 μL P1. 3 ml of diluted P1 was added to one column and 3 ml of undiluted P2 was added to the other column. Store eluate. 10 ml of low salt buffer (buffer A + 50 mM NaCl) was added to each of the two columns. The eluate was collected in a 2 mL fraction in a cuvette and the highest eluate was stored for spectral analysis. The OD of low salt flow of P1 and P2 at 280 nm was measured. Collect flows that pass through 1-2 mL fractions in the cuvette

Chromatographic focusing is a special form of ion exchange chromatography requiring resins containing charged groups with high buffering capacity. The column is equilibrated and loaded with protein at a predetermined starting pH. The column is then eluted with a buffer different from the pH of the starting buffer. In this way, a pH gradient is formed in the column as the column elutes buffer drops to set up the ion exchanger. For example, in order to produce a decreasing pH gradient (pH 9-6) the ion exchanger must be at a higher pH than the eluent. For that reason, it is necessary to use an anion exchanger as ion exchange requires a basic buffer set. The column was first equilibrated at pH 9 and the pH of the eluent was set to pH 6. Among them, the most acidic one is combined with the basic group on the anion exchanger. In this way. The pH of successive points in the column gradually decreases