Laura Moriarty is Web Marketing Manager in the Protein Function Division at Bio-Rad Laboratories.
In our 60 years’ experience supporting chromatography applications, we at Bio-Rad Laboratories have learned that a chromatography system can be the workhorse of your lab. Your system needs to work day and night to keep up with purification demands. It needs to perform a wide variety of protocols, all with minimal downtime. Here are some easy-to-implement tips to ensure your system keeps up with your lab’s requirements.
Tubing tips
When it comes to tubing, the smaller the better. The tubing on your chromatography system should be short enough to minimize dead volume but long enough to provide flexibility in component positioning. In most cases, you want to choose a smaller-diameter tubing—commonly 0.15 to 0.75 mm in diameter—to reduce volume within the system. However, the trade-off is that the smaller the diameter, the higher the back pressure.
Typically PEEK® (polyetheretherketone) tubing is used in the high-pressure areas of the system. In the parts of the system exposed to high protein concentrations, for example, the inject and outlet lines, and in some cases in the buffer lines, use PTFE (polytetrafluoroethylene) instead. (PTFE tubing is clear and thus allows you to see buffers and identify air bubbles within the buffer lines.) And remember to clean your buffer lines with purified water at the end of each day. This is especially important when working at low temperatures, as salts can precipitate.
Column tips
Columns may be expensive, so you should always degas and/or vacuum filter buffers prior to use with your system to minimize air and particulates in your mobile phase. Air entering the system also may be detected prior to entering a column by using an air sensor.
For analytical purifications, in which the column may be extremely expensive, such as Aminex columns, you might consider using a guard column. This smaller guard column is used in-line, before the main column, and is filled with similar material as the main column (in some cases with a larger bead size). This column collects any junk in your sample, keeping it off your more expensive analytical column.
Keeping buffers fresh
There’s an old saying: Garbage in, garbage out. If your buffers are bad, so too will be your results. So make sure your buffers are always ready for use. Regularly inspect buffer bottles/bags for signs of bacterial growth, and if solutions appear cloudy upon shaking or stirring, discard them. For best results, make solutions fresh, as needed. You also may try a bacteriostatic agent, such as 0.02% sodium azide, for prolonged solution storage.
Clean instrument tips
Your instrument hardware also requires regular maintenance. We recommend replacing your piston washing solution weekly with fresh 20% ethanol or isopropyl alcohol. At a minimum, regularly inspect solutions for fluid-level depletion or bacterial growth and supplement or replace as needed.
Periodically clean your sample inlet lines and sample loops with 1N NaOH solution, followed by a thorough rinse with water or neutralizing buffer, to eliminate sample buildup in the tubing.
Also, replace PEEK tubing that comes in contact with your sample on a regular basis, especially lines connected to your fraction collector. This 20 cm to 30 cm of tubing needs to be clean or replaced often, as the surfaces are exposed to large volumes of protein-rich solutions. Regular system maintenance may not clean these lines, as typically those solutions are directed to waste instead. As a result, they can become sites of cross contamination or bacterial growth. To prevent this, store all system lines in fresh 20% ethanol when not in use. We recommend that this be done at the end of each work day
Conclusion
Ultimately, your chromatographic separation will only be as good as the hardware on which it runs. These simple tips should keep your chromatography system running around the clock and for years to come.
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