How do you scale ion exchange chromatography?
According to the theory, a successful scale-up is accomplished by keeping the residence time of the fluid flowing through the column constant [7], [8], [9], [10]. This means that the volumetric flow, gradient length and load are scaled to the column volume.
How is the chromatographic column efficiency measured?
Column efficiency, indicated as the number of theoretical plates per column, is calculated as N = 5.54 (tR / w0.5)2 where tR is the retention time of the analyte of interest and w0.5 the width of the peak at half height.
What is bed height in chromatography?
Chromatography Purification The bed heights are usually from 10 to 30 cm regardless of column diameter. The quantity of resin required is based on the product titer. Resin loading is typically in the range of 5–20 g/L depending on the protein and the resin composition.
How do you fill a chromatography column?
There are two common ways you can fill a chromatography column:
- Dry Packing. For this method, add your dry solid phase to the column and pass equilibria buffer or starting solvent to saturate the solid.
- Wet Packing. Here, you mix both liquid and solid outside of the column and pour it into the column.
How do you elute in ion exchange chromatography?
Retained proteins are eluted from the column by applying a modified buffer. Elution is most commonly achieved by gradually increasing ionic strength of the buffer via salt gradient, and proteins are eluted in order of increasing their net charges.
How can ion exchange chromatography be improved?
The easiest way to improve the resolution from an IEX run is to modify the running conditions. Different pH will affect the charged surface on the protein, which affects the resolution. A smaller amount of sample will improve the resolution: typically use up to 30% of the complete capacity to maintain good resolution.
How do you increase the efficiency of a column?
Decreasing particle size thus is a useful method for improving column efficiency and providing better separations. changing to particles that are half as big, while keeping the column length the same, will double the performance, but increase the required pressure by a factor of four.
What is a good resolution value in chromatography?
Scientists consider a resolution of 1.0 or higher to represent an adequate separation. Measure the widths of two adjacent peaks in the chromatogram by noting where the x-axis values are at the base of each peak. The x-axis represents retention time, usually measured in seconds.
How do you calculate column bed height?
Fill any two fields in a column and hit the Convert button. The empty field is calculated using the formula: Bed volume (L) = Bed height (cm) * Column crossectional area (cm2) / 1000.
How do you speed up column chromatography?
Add solvent to the top of the pipette and allow it to drain. A pipette bulb can be attached to the top and squeezed at this point to speed solvent flow and help the silica pack more densely. Add your compound and elute as usual. Use the pipette bulb to increase the pressure and flow rate.
What elutes faster in column chromatography?
In column chromatography, molecules reversibly adsorb to the stationary phase as they flow through the column, thereby slowing their progress. Compounds that interact weakly with the stationary phase are faster to exit the column, or elute. Compounds that interact strongly with the stationary phase are slower to elute.