Chromatography is one of the essential techniques used for the separation, identification, purification and quantification of components from a heterogenous solution. The instruments behind these techniques are often considered the “work horse” of the lab so keeping your system up and running is a must if you want your experiments to work. Although these instruments can seem daunting with all of the tubing and valves, there are simple and straightforward maintenance tips involving sample preparation, buffers, columns and the overall system to keep your experiments humming along.
Sample preparation
It all starts with your sample and as they say, "garbage in garbage out". Sample and mobile phase filtration are simple, not to mention economical, maintenance practices that can help to extend the life of consumable parts and decrease wear and tear on the system. Generally, filtering samples through a 0.45 or 0.2 μm filter is sufficient to remove particulates. If your sample does not flow easily through one of these filters, try a 0.8 μm filter. The larger filter pore size works well for proteins with visible particulates. Be sure to select a low protein binding membrane such as polyvinylidene fluoride (PVDF) or polyethersulfone (PES) as nylon filters often bind protein resulting in a loss of yield.
Buffer preparation
One of the easiest tasks to perform to keep your system running smoothly is to keep your buffers and solvents free of particulates. There’s nothing worse than having something happen to your run when you could have prevented it – like a clogged column or line due to a dirty buffer. Similar to filtering your samples before running them on your system, filtering your buffers and solvents helps keep your system free of clogging. Try these few tips for buffer preparation to help maintain your system.
- Use HPLC-grade materials and water for your buffers. This will help to lessen unwanted particulates in your buffers.
- Filter the buffer after it has been prepared. Although the buffer may be chemically pure, dust, particulate matter from a bottle-cap, or precipitation of salts may be present by the time the buffer is made. For best results, use a 0.5 µm filter prior to use.
- Degas your buffers. Degassing and/or vacuum filtering buffers prior to use with your system minimizes air and particulates in your mobile phase.
- Occasionally inspect buffer bottles/bags for signs of bacterial growth. Solutions that appear cloudy upon shaking or stirring should be discarded. Treatment with a bacteriostatic agent such as 0.02% sodium azide will prolong solution storage. For best results, make solutions fresh, as needed.
- Set an expiration date for discarding unused buffer. Although you may be able to get longer stability, many laboratories specify one-week expiration dating on dilute buffers.
Column maintenance
Columns can be expensive, as with any investment in the lab, care should be taken when handling, using and storing your columns. By following the simple tips below, you can be sure to extend the life of your column.
- Use a column guard when appropriate. Use of an inline pre-column filter or guard (20 μm) between your inject valve and column is an effective way to increase column lifetime.
- Replace worn frits and O-rings in manually packed columns, a small accessory part like this can derail an important experiment.
- Store columns correctly. Bacteria thrive in the neutral environment of buffers. Prevent bacterial growth by running HPLC-grade water through the column and then place columns in bacteriostatic solutions, such as those containing 20% ethanol, and in the refrigerator at 4oC to 8oC until you are ready to use them again.
Tubing maintenance
There are several factors to consider when deciding on the tubing for your application. If the right tubing is used and maintained, this part of the system will last for many runs.
- Use the best material that is chemically compatible with your mobile phase. Poly ether ether ketone (PEEK) polymer tubing stands up to most operating conditions and is typically used in the high-pressure areas of the system. Use polytetrafluoroethylene (PTFE) tubing in parts of the system exposed to high protein concentrations such as inject and outlet lines. PTFE tubing limits protein sticking and is clear enabling you to see buffers and identify air bubbles within the buffer lines.
- Tubing size is another factor. In most cases, you want to choose small-diameter tubing (commonly 0.15 to 0.75 mm) to reduce volume within the system but be aware of possible increased back-pressure with smaller tubing. The tubing on your system should be short enough to minimize dead volume but long enough to provide flexibility in component positioning.
- Rinse lines with HPLC-grade water at the end of each day to clean away the buffers used as they can precipitate salts and clog lines. This is especially important when working at low temperature where salts can precipitate.
System maintenance
Your hardware also requires regular maintenance. Below are additional general tips for maintaining your system.
- Periodically clean sample inlet lines and loops with 1N NaOH solution followed by HPLC-grade water or neutralizing buffer to eliminate sample buildup in the tubing.
- Replace your piston washing solution weekly with fresh 20% ethanol or isopropyl alcohol. At minimum, regularly inspect for fluid level depletion or bacterial growth and replace or supplement as needed.
- Periodically replace PEEK tubing that comes in regular contact with sample, especially lines connected to your fraction collector. The tubing surface is exposed to large volumes of protein-rich solutions that can start to clump and eventually clog the line.
- Store all system lines in fresh 20% ethanol when not in use to avoid bacterial growth. Periodically check system tubing for cracks and bends. Tubing bends can restrict flow and increase system backpressure.
- For best results, occasionally sanitize your entire system with 1 N NaOH to clear the flow path from pump to fraction collector.
Modern chromatography systems like Bio-Rad’s NGC system, utilize automated processes that run user-defined cleaning methods for the system. This along with the easy access and high utility inlet values provide users with a convenient and effective flushing of the system so it is ready for subsequent runs. Establishing a routine maintenance schedule utilizing these tips should keep your chromatography system up and running for years to come.
Image: Courtesy of Bio-Rad
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