How to Make HPLC Columns last Longer?

How long an HPLC or UHPLC column should last?

Column lifetime depends on how they are used and how well they are maintained. A C18 or aqueous C18 column generally lasts the longest, while something like an amino phase usually has the shortest lifetime.

While the relative cost of high-performance liquid chromatography (HPLC) columns has been reduced over time, extending column lifetime remains an important consideration for most laboratories. The following tips should help to protect your columns and significantly extend the useful lifetime of most phases.

1. Always read the manufacturer’s literature with respect to the recommended pressure, eluent pH, and temperature operating ranges for the column, and stick to these ranges.
2. Avoid mechanical shock of the column bed (dropping the column) and ramp the pressure and flow slowly (1 mL/min/min is ideal) each time eluent flow is initiated.
3. If columns have dried out, initiate the flow very slowly (0.1 mL/min/min) using an eluent containing at least 50% acetonitrile.
4. If samples are likely to contain particulate matter, choose a qood quality inline filter with the appropriate mesh size; 0.45 µm for traditional columns and 0.2 µm for UHPLC columns is typical
5. Columns should be properly washed after each use.
6. If column or frit contamination is suspected as a result of peak splitting or loss of efficiency, it is possible to reverse the direction of the column for back flushing purposes, and the column washing procedure mentioned above is a good “recipe” for this purpose.

Here are the most common LC column killers:

Particulates

These usually come from the unfiltered sample matrixes, but can also come from unfiltered mobile phases.

ALSO READ: SOP for HPLC Column Maintenance and Washing Procedure

Reactive material in samples 

The most obvious of these is derivatizing agent, but can also come from a sample matrix. Unless you have detailed info of your sample matrix, there are likely to be components that are unexpected and uncharacterized. This can even be present in matrices that appear to be fairly clean, such as urine.

Harsh pH conditions:

The suggested operating range for our columns is 2.5 – 8.0. A pH that is too low may remove the bonded phase from the silica, while a pH that is too high will start to hydrolyze the silica itself.

Incompatible mobile phase or flow conditions

Be aware that only certain phases are designed to be used with more than 95% water in the mobile phase. The bonded phase in a traditional C18 column will collapse under these conditions. (You should be using an Aqueous C18 column if this is a possibility.) This is one example of this type of column damage.

So… to help your columns live long and prosper, you should do the following:

• Make sure your flow rate and pressure are appropriate for your column dimensions and particle size. Here is a useful table from our current catalog:

If you have recently reduced your column ID, please ensure that you reduce the flow rate accordingly. As far as pressure goes, generally, it’s best to stay below a

backpressure of 5000 psi for HPLC and below 12000 psi for UHPLC (particle size below 3 um).

ALSO READ: Column Restoration Procedures

Pre-filter all samples prior to LC injection, either by using a syringe filter or Thomson Single Step filter vial.

If the sample volume is too low for either option above, make sure you use a cap frit with your guard cartridge.

If doing UHPLC, you will need to use one of our inline filters instead, either the Ultra-Shield UHPLC Precolumn Filter or the Ultra UHPLC Inline Filter.

• Use a guard cartridge for HPLC to minimize damage from reactive and adsorptive material. If you’ve had prior issues with short column lifetime, a longer cartridge might help.

The 20 mm length should be used for any samples potentially containing reactive or highly adsorbed components. 

• Pre-filter mobile phases if they contain buffer salts or any modifiers that come from a weigh-out of solid material. Also, make sure you are using solvents that are HPLC grade or better. 

ALSO READ: General HPLC Column Care

• If using buffer in your mobile phase, don’t make drastic changes from high to low aqueous content while pumping your column. If your intent is to switch to 100% organic solvent, make sure you flush out the buffer first using a mix that is at least 50% water for at least 7 column volumes. Failure to do this may result in salts falling out of the solution, clogging frits and increasing back pressure.
• Store your column in the appropriate solvent. When storing for longer term intervals (more than 2 days), it is always best to consult the column handling instructions for a given column. Most reverse-phase columns (including C18, aqueous C18 and IBD) are best stored in a mixture of methanol or acetonitrile and no more than 50% water. Some of our phases, such as the biphenyl, PFP Propyl and Amino are better stored in 100% acetonitrile.

Avoid incompatible mobile phases. watch for the materials that are known to be reactive to specific phases. This includes: 

1. Aldehydes and ketones with amino phase (this includes acetone!)
2. Sulfur-containing compounds with PFP propyl phase
3. Significant percentages and/or inconsistent amounts of water with bare silica

• Check the pH of your sample (and mobile phase if acids or bases are used). Most analysts use a pH meter, If you are using a meter, make sure it is calibrated properly and frequently. 
• Keep it clean. If normally running a gradient for reverse phase LC, give it an occasional flush with the mobile phase that contains slightly more water than your starting conditions and no buffer salts, not to exceed 90% water for traditional C18 phases. Likewise, make sure you occasionally flush with a mobile phase that contains slightly more organic than you usually use. For example, if your gradient goes up to 90% organic, flush it with 95% organic occasionally. About 7 column volumes for each of these, done once a week or so should do the trick for this. (You will probably be doing some of this anyway prior to storage, but it doesn’t hurt to mention it!) You may find that one of these two procedures is more beneficial than the other, depending on what is in your sample that tends to settle out or be absorbed. You can alter your maintenance as needed according to this. You might consider programming a flush cycle (with a gradient that holds the concentration at the highest and lowest points) at the end of each day or at regular intervals if it seems to help. 

ALSO READ: HPLC Column Care and Maintenance