Good laboratory practices include proper use of any HPLC column including the Cogent TYPE-C, silica hydride columns. On this page you will learn how to condition the TYPE-C™ columns, how to store them long term and How To develop good methods for each TYPE-C™ phase.
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Defining Aqueous Normal Phase, Reversed Phase & Normal Phase
Cogent TYPE-C™ Silica HPLC columns can be used in:
- Reversed Phase = RP
- Aqueous Normal Phase = ANP
- Normal Phase = ONP
To avoid confusion it is best to define ANP and ONP as separate techniques, but they are in reality different ends of the same technique; hexane/ethyl acetate as a mobile phase or 60+% Acetonitrile/water (for ionic compounds at appropriate pH) are both Normal Phase systems that work well on Cogent TYPE-C™ Columns.
- Normal Phase: Retention times decrease as most polar solvent % increases
- Reversed Phase: Retention times increase as most polar solvent % increases
How to Condition All TYPE-C™ Columns for Use
First Things First
- Before installing your column, purge the solvent lines of any previous mobile phase unless it is identical to the one you will be using.
- Purge the injection port to remove any residual compounds from previous analyses.
- Attach the column to your instrument following good laboratory practices.
- Condition the column with 50:50 mixture of organic solvent and water including any additives that will be used in your method for 30 min.
- Equilibrate the column for 15 min at the starting conditions for your method.
- For RP, start at 50:50 organic/water in the mobile phase. To increase retention - add more water. Proceed with method development the same way as with any other hydrophobic column (for example C18).
- For ANP, start with 50:50 organic/water in the mobile phase. To increase retention - add more organic. For method development tips for each phase, see below.
- For method development tips for each phase, see below.
- Before removing the column, wait until there is no pressure in the lines or the column or it could damage the column. Then fill it with 90:10 organic/water mobile phase for storage.
- Click HERE for complete Storage Instructions.
- An increase in organic solvent concentration will increase retention of the polar analyte in an exponential way to the point that you may not see it in the chromatogram. If any peak doesn’t change RT with higher % organic IT IS NOT your polar analyte.
- To make sure that you are detecting a polar analyte use a simple ANP gradient (0.00 min 90%B, 6 min, 20%B, hold 2 min, 10 min 90%B). Always run the solvent in which you prepared your analyte as a blank to see gradient end peak.
- When you determine the RT of the analyte in a gradient run, you might go back to designing an isocratic run by determining the percentage of organic solvent from the position of the peak in the gradient run.
TROUBLESHOOTING TIPS FOR ALL TYPE-C™ COLUMNS
Retention, resolution and peak shape problems are seldom due to the column, especially a new recently installed one. Here are some typical issues that are often blamed on the column:
- Make sure you have thoroughly purged the solvent lines of your instrument and injector.
- Before making the first run of the day, allow the column to equilibrate for 15 min under the mobile phase conditions to be used in the analysis.
Poor Peak Shape can be caused by:
- Fronting; column overload or sample matrix effects (use guard column)
- Peak Broadening: too large of an injection volume, long retention time (make a mobile phase using a stronger solvent or use a gradient), too large detector cell for optical systems, fittings and tubing have too much volume (us hardware that has the smallest dead volume and use tubing with the smallest diameter), sample diluent too strong (match sample solvent to mobile phase).
- Peak Splitting: plugged frits (replace, backflush column, use a guard column or an inline filter), sample over load, the sample solvent is incompatible with the mobile phase (use sample solvents as close to the mobile phase as possible), injector valve malfunction (service or replace).
- Peak Tailing: usually caused by secondary interactions. Try changing the mobile phase pH, use an additive such as TFA or EDTA, use or change the gradient.
- Noisy Baselines can be caused by bubbles in the solvent lines (check degasser system), detector problems (lamp or phototube in an optical detector or a dirty source in MS as an example), contamination from the sample, poor solvents or injector system, unstable pressure due to a pump problem such as valves or seals.
- Retention Time changes can be due to pressure fluctuations (pump not working properly), insufficient buffer concentration, some component of the mobile phase has evaporated, are the solvent bottles not sealed tightly, column overload (sample volume or concentration too high), column contamination from previous samples or matrix (flush column with strong solvent), equilibration time insufficient after gradient run (before subsequent injections, ensure that pressure has stabilized), temperature effects (thermostat column if possible).
- Check to see if the wrong method loaded or method is corrupted in your instrument.
Notes on Using TYPE-C™ HPLC Columns in Normal Phase modes
When using Normal Phase (ANP & ONP) HPLC, you will likely observe the retention time change or the relative selectivity change with adjustments of the organic modifier of + or – 1% or with temperature changes of + or – 1° Celsius.
Temperature changes between 10° and 50° Celsius have been shown to change the elution order of antidepressant probes, therefore it is important to precisely control the temperature of any column used in Normal Phase Separation Methods. Similarly a minor change in percentage from 8 to 10 % Aqueous content can significantly increase Retention and Resolution (Relative Selectivity) of some compounds.
It is critical to accurately control the concentration of solvents A to B in Normal Phase modes and therefore makes it impractical to develop methods in fine detail when using 100 % A + 100% B and using a conventional HPLC pump gradient mixing (high or low pressure gradient formation).
It is recommended that the approximate percent concentration of solvents are found by this method (mixing pumps), but final percent concentrations are defined either by isocratic v/v mixing (note as organic solvents are not additive on volume when mixing, separately measure out each solvent; add, shake and de-gas, the ultimate volume of mix is irrelevant) or change solvent A to a mixture of solvents A + B ( say 20% A in 80% B ) to be called C, thus 10% solvent A becomes a 50:50 mixture of solvents B and C and 5% A, a mix of 75:25 B + C.
Isocratic premixing and accurate temperature control with a forced air circulation oven/chiller is always preferred in ANP or ONP for reproducible and robust TYPE-C™ column methods.
Start up Guides - "How To Use TYPE-C™ Columns" - Links
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