Practical Guide to Manual Loading of Industrial Column Chromatography
Suzhou Vdo Biotech Co.,Ltd
Suzhou Vdo Biotech Co., Ltd. is a specializes in production of biotechnology microspheres.
I. Column Loading
a. Determine the column volume and bed height based on the internal diameter and height of the column (filling volume should not exceed 70% of the maximum column volume).
b. Calculate the amount of chromatography resin required based on the column volume (compression ratio for the resin should be approximately 1.1-1.15). Accurately weigh a specified quantity of clean chromatography resin and wash it with injection-grade water. The purpose of washing is to remove any residual substances from the chromatography resin. Residues of 20% ethanol can affect the settling of the packing material and the column loading process.
a. Clean the chromatography column system, ensuring that the inner and outer walls of the column and the tubing are free of visible particles. Ensure that the column is level.
b. Connect all tubing of the chromatography column, fill the column with injection-grade water, close all outlet lines, pressurize to 0.5 MPa for 15 minutes, with pressure drop below 0.01 MPa.
a. Prepare a suspension of resin by adding a suitable amount of clean resin to injection-grade water, with a solid-liquid ratio of approximately 1:1.5. Stir and sonicate for about 3 minutes, or if no sonication equipment is available, extend the stirring time as much as possible to ensure uniform dispersion of the packing material.
b. Gradually pour the re-suspended resin into the column in one continuous motion (avoid introducing air bubbles). A peristaltic pump can be used for this step. Allow the resin to settle completely and record the settling level.
a. Attach the column head to the chromatography column, purge the tubing of air, and slowly rotate the column head to the marked level or compression ratio position based on the previous step's marking (pay attention to changes in column pressure difference; if the pressure difference exceeds 0.3 MPa, pause, as this indicates the final position of the column bed height).
b. Adjust the system pump to a low flow rate of injection-grade water (about 30 cm/h) and wash the column for approximately 1.0 column volume (CV).
II. Column Efficiency Assessment
After the column is loaded, to evaluate its separation performance, conduct a column efficiency assessment immediately. The performance of the packed column is typically assessed using parameters such as theoretical plates per meter (PPM) and asymmetry factor (As). A higher PPM value indicates better separation capability, while As values closer to 1.0 indicate better symmetry.
III. Common Problems and Solutions During Column Loading
If column leakage is observed when operating the chromatography system after column loading, first inspect the source of the leak. If it is in the tubing, it may indicate tubing damage and require replacement.
Check if the column valves and seals are tightened. If any of these issues are present, tighten them as necessary. Another possible cause of leakage is aging or loss of elasticity in the column head seals, in which case, contact the column manufacturer for replacement parts.
The appearance of cracks or non-uniform color in the chromatography resin loaded into the column may result from the introduction of external air bubbles into the column. If the column is needed urgently, you can assess the column efficiency. If PPM and As are within acceptable ranges, you can continue to use the column without affecting production. Otherwise, you may need to reload the column.
Sometimes, a loaded column may exhibit widened peak shapes and reduced resolution after multiple uses. This can affect the purity and yield of the target compounds, particularly when collecting fine fractions. In such cases, check whether the sample injection volume during column loading is too large (for group separation, ≤30%; for fine separation, ≤4.0%). Additionally, inspect the column for signs of extended use, gel clumping, or dry packing. If any of these issues are present, perform maintenance and cleaning of the chromatography resin before reloading the column (see the general cleaning procedure in the following section).
Insufficient pre-treatment of purified samples, high turbidity, high viscosity, and high total protein concentration can also affect resolution and purification results during column chromatography.
Extended use of the chromatography resin or neglecting to clean the column after each purification batch may lead to discoloration of the chromatography resin. It is recommended to clean and disinfect the resin after each purification batch using 1 M sodium hydroxide for general packing materials.
Non-compliance with PPM standards is a common issue after column loading. This can occur due to the introduction of air bubbles during column loading, inadequate mixing of the chromatography resin, fragmentation of the resin, or excessively high flow rates during column packing (recommended flow rate is 30 cm/h). If there are issues with the chromatography resin itself, consider replacing it. If problems occurred during column loading, follow the methods mentioned above to reload the column.
After loading the column, if you observe forward peaks (As < 1.0, as shown in Figure 1), it may indicate that the chromatography resin is compressed too tightly during column loading. In such cases, if PPM is within specifications, you can slightly raise the column head using backpressure and retest the column efficiency. If you observe tailing peaks during column efficiency testing (As > 1.0, as shown in Figure 2), it may indicate that the chromatography resin is compressed too tightly during column loading. In this case, if PPM is within specifications, you can lower the column head slightly and retest the column efficiency.
After loading the column, if you experience abnormal backpressure or if the column pressure difference is higher than usual when using the same solvent and flow rate, it may indicate that the chromatography resin requires cleaning and maintenance. In such cases, you can use a low flow rate to clean the resin with 2 M sodium chloride or 1 M sodium hydroxide. Note that Ni Focurose FF (IDA) and Ni Focurose FF (IMAC) should be stripped of nickel before cleaning with sodium hydroxide.
Additionally, blockages or fragmentation of the chromatography resin, blockages in the chromatography system tubing, injection heads, or online filters can also lead to excessive backpressure during the chromatography process. The solution is to replace the packing material and corresponding components as needed.
In summary, assuming the chromatographic resin is in good condition, there are three key points in the industrial chromatography column packing process. First, ensure the uniform dispersion of the chromatographic resin into the chromatography column. Second, strive to avoid the formation of bubbles during the packing process. Third, maintain control over the compression ratio during column packing. If these three points are well-managed, the column efficiency of the chromatography column will not be significantly compromised.