Frequently asked questions (FAQs)

HPLC columns

C18 packing specifications

See C18 ProteCol HPLC columns product page.

What are the pressure limits of the 150 µm trap columns?

It’s 800 psi + the pressure generated by the analytical column.

What is the loading capacity for ProteCol trap columns?

The amount is very much dependent on the size of the molecule for two reasons. First, a given surface area can only accommodate a certain number of molecules but also because smaller molecules can penetrate into a larger percentage of pores than large molecules. The 300 Å packing material has a specific surface area of 100 m²/g. The 300 μm x 10 mm trap column contains ~0.37 mg of packing material which means there is 0.036 m² surface area available for peptide/protein binding. Assuming that about 50% can be covered gives a capacity for angiotensin II (a small peptide) 86 nmol but for hSA (human serum albumin - a much larger protein) about 600 pmol. This does not take into account that hSA cannot penetrate all the pores in the system.

How do I store my column?

Storage conditions are dependent on the duration of the storage:

  • Overnight – no specific precautions are necessary unless the column contains solvent with an extreme pH.
  • Over the weekend – store the column in salt-free mobile phase.
  • A week or longer – rinse the column with pure water to wash out all traces of buffer salts and then flush the column for more than the equivalent of 10 column volumes with 100% acetonitrile or methanol. Use blank end-nuts to seal the column and store it in a cool place.
  • To avoid accidental damage to your column, we recommend storing it in the packaging it came in.
What is the maximum recommended back pressure for the ProteCol columns?

Back pressure is dependent on column length. For columns 150 mm and 250 mm in length, the recommended maximum pressure drop is 6000 psi or 400 bar. For 100 mm columns the recommended pressure is 4000 psi (275 bar) and for 50mm columns it is 2000 psi (140 bar).

Will I get identical results using an "alternative" column from Trajan?

In general, no, as columns can differ in a number of ways. For the most common stationary phase, C18, there are more than 400 different types of columns on the market. They can differ physically (particles size, pore size and their distributions) and chemically (purity of the silica matrix, bonding density, monomeric or polymeric modification, end-capping and specialty modifications such as polar embedded).

Within a group (polymeric A vs polymeric B or polar embedded A vs polar embedded B) compatibilities are more likely than between those groups. Furthermore, the sample composition will have an impact on how easy it is to transfer methods between column A to column B. “Less complex samples with mainly hydrocarbon/aromatic compounds will behave more similarly on a variety of columns than samples with a number of reactive centers such as bases, carboxylic acids, phosphates etc.

Many deviations in the retention time can be reversed by adjusting the mobile phase conditions slightly.

How long do your columns last?

The biggest threat to an HPLC column is particulate matter in the mobile phase. Particulates can originate from a broken stationary phase (fines), undissolved samples or buffer components or microbial growth in the mobile phase. These small particles can get lodged between the stationary phase particles and cause an increase in backpressure and, by means of nonspecific interactions, a shift in retention time.

When using clean samples, a filtered mobile phase, moderate temperatures and pHs and avoiding sudden pressure changes, a column can last for several thousand injections. Running a column for extended periods of time at or above the previously mentioned limits can shorten the life time of the column. Having dirty samples (such as blood, saliva, cell or plant extracts) can reduce the column lifetime to only 150 to 200 injections.

Having incomplete elutions can also lead to a build-up of sample components on the column which will compete with the C18 chains for interaction and lead to a shift in retention time.

Some precautions that can extend the life of your column:

  • Filter mobile phases.
  • Do not use aqueous mobile phases for more than seven days to avoid microbial growth.
  • Do not store the column in aqueous solutions.
  • Filter or spin samples to remove particulate matter.
  • Wash the column frequently with high organic solvents.
  • Use guard columns.
What temperature range can I use ProteCol columns?

We recommend a temperature limit of 60°C.

Can I use stainless steel nuts and ferrules with ProteCol columns and guard columns?

Yes, as long as you make the original connection into the ProteCol column. We do not recommend using pre-swaged fittings and tubing as the geometries of various column vendors vary. Having the wrong length of capillary protruding from the ferrule or having the wrong ferrule geometry can lead to leaking connections, bad peak shapes due to void volumes or even irreversible damage to the column.

To avoid complications with the column connections, we recommend the use of PEEK fingertight fittings.

What pH range can I use ProteCol columns?

It is recommended you maintain a pH range of 1.0 – 8.0 for the C18 G, Q and P phases and a pH range of 1.0 – 11 for the C18 H phase at room temperature.

With the C18 H phase a pH of 12 can be used successfully for up to 500 column injections.

The pH range narrows at elevated temperatures as hot acids and bases are more aggressive.

Why use metal-free chromatography?

Many pharmaceutical active substances are rich in oxygen and can therefore interact with metal (Fe). Tests have shown the N-hydroxypyridine-2-on – the chelating part of the anti-fungal cyclopirox molecule – is a powerful probe for metal activity.

In one experiment, a PEEK lined ProteCol-P C18 Q105 coated column was compared to a stainless steel ProteCol C18 Q105 column where the connection tubing also varied between stainless steel and PEEKsil (PEEK coated fused silica tubing).

As shown, peak tailing was minimized with the reduction of metal in the flow path resulting in increased peak height and hence, sensitivity.

A second experiment was conducted with tetracycline antibiotics. Tetracycline and related drugs have a number of potential chelating groups aligned on one side of the molecule and are known to form metal complexes (dietary calcium and iron render the antibiotic ineffective).

The chromatogram of tetracycline and its major degradation product demonstrates the improved peak sensitivity using the PEEK lined ProteCol-P C18 Q105 column format. Note the peak broadening on the base of the peak run through the stainless steel column.

The inset shows the tetracycline molecule depicting the three potential chelating groups.

More information

Also see: Product literature