Troubleshooting Reversed Phase Chromatography (RPC)

Ghosting

Poor-quality eluent components can cause a phenomenon referred to as “ghosting”. Trace levels of organic impurities bind to the medium, concentrating during equilibration and sample application. When elution begins, these contaminants appear in the chromatogram as unknown, or “ghost” peaks. The size of a ghost peak will usually depend on the equilibration time and the level of organic impurities in the eluent.

Ghosting may also be caused by incomplete elution of molecules in a previous run. Run a blank gradient, with no sample, as a check, especially if subsequent runs are to be performed with highsensitivity detection.

Baseline drift: balancing eluents

During a typical run the baseline can progressively increase or decrease in an approximately linear fashion as the proportion of eluent B increases. This phenomenon may originate from an ion-pairing agent (or strong acid component) or an organic modifier that absorbs significantly at the detection wavelength. The background absorbance caused by eluent components is corrected for during column equilibration. As the proportion of organic component increases so the absorbance properties change.

Compensate for a drifting baseline by using different concentrations of UV-absorbing ion-pairing agents (or buffer acids) in eluent A and B and thereby balancing the “concentrations” with respect to UV-absorption properties to give an approximately straight baseline. Because of batch-to-batch variations in the absorption properties of eluent components and other differences between the conditions in different runs, it is not practical to give specific recommendations. The following example can assist to illustrate the principle: gradients from TFA in water to TFA in acetonitrile will usually require that the concentration of TFA in acetonitrile is 10–30% lower than in water. The balanced concentrations of UV-absorbing components should then be determined empirically. The difference in concentration of ion-pairing agent between the two eluents is generally not large enough to adversely affect the separation. A typical example would be to use 0.065% TFA in eluent A and 0.05% TFA in eluent B.

Situation	Cause	Remedy
Reduced or no flow through the column.	Outlet closed or pumps not working.	Open outlet. Check pumps for signs of leakage.
	Blocked filter, end-piece, adaptor, tubing or pre-column.	Remove and clean or replace if possible. Always filter samples and eluents before use.
Reduced flow through column	Precipitation in the column.	Follow cleaning procedures.
Back pressure increases during a run or during successive runs.	Bed is compressed.	Adjust eluents to maintain sample solubility.
Sample does not elute during gradient elution.	pH caused precipitation.	Adjust pH to avoid precipitation.
	Final concentration of organic modifier too low.	Increase concentration of organic modifier in gradient or in eluent B.
	Eluting power of organic modifier too weak.	Change to a less hydrophobic RPC medium or change organic modifier.
Sample elutes before gradient elution begins.	Sample components not sufficiently hydrophobic.	Add or increase concentration of ion pairing agent or use an organic modifier with less eluting power or change to a more hydrophobic RPC medium.
	pH unsuitable.	Adjust pH to enhance binding.
	Impurities bound to the column.	Clean and re-equilibrate column.
	Concentration of organic modifier in initial eluent too high.	Decrease organic modifier concentration.
	Column not equilibrated properly.	Re-equilibrate column.
	pH unsuitable.	Adjust pH to enhance binding.
Leading or very rounded peaks in chromatogram.	Column overloaded.	Decrease sample load and repeat.
	Column contaminated.	Clean using recommended procedures.
Peaks are tailing	Column poorly packed. Sample has precipitated on column.	Repack or use a prepacked column.
	Sample has precipitated on column.	Clean column, replace top filter or precolumn if possible.
Peaks are tailing or have a leading edge.	Column packing compressed.	Check column efficiency (see Appendix 2)
Peaks too small.	Sample absorbs light.	If appropriate, check absorbance range on monitor. If satisfactory, use a different wavelength. Check UV cut offs of eluent components.
	Different assay conditions have been used before and after the chromatographic step.	Use same assay conditions for all assays.
	Excessive peak broadening.	Check column efficiency. Repack if necessary. Adjust eluent to suppress ionization of silanois or replace pre-column. Replace column if necessary
Resolution less than expected.	Large mixing spaces at top of or after column.	Top up surface of medium if possible. Reduce all post-column volumes.
	Sub-optimal elution conditions e.g., gradient too steep, flow rate too high.	Alter elution conditions: use shallower or plateau gradient, reduce flow rate.
	Column poorly packed.	Check column efficiency (see Appendix 2). Repack if possible. Use prepacked columns.
	Column overloaded	Clean and re-equilibrate column. Decrease sample load.
	Lipoproteins or protein aggregates have precipitated.	Clean and re-equilibrate column. Adjust eluent to maintain solubility.
	Column aging	Adjust eluent to improve ion suppression or replace pre-column.
	Mixed-mode retention due to surface silanols.	Lower pH to suppress silanols or replace column.
	Sample not filtered properly.	Clean the column, filter the sample and repeat.
Samples do not bind or elute as expected.	Sample has not been filtered.	Prepare fresh sample
	Column equilibration incomplete.	Repeat or prolong the equilibration step until baseline is stable.
	Lipoprotein or protein aggregates have precipitated.	Clean and re-equilibrate column.
	Incorrect eluent conditions compared to previous runs (possibly due to evaporation).	Check conditions required. Prepare fresh solutions.
Medium/beads appears in eluent.	Column operated at too high pressure.	Do not exceed recommended operating pressure for medium or column
Low recovery of activity, but normal recovery of mass.	Sample may be denatured or inactive in the eluent.	Determine the pH and organic solvent stability of the sample. Reduce separation time to limit exposure to organic conditions or change to a less powerful organic modifier or use a less hydrophobic RPC medium.
	Enzyme separated from co-factor or similar.	Test by pooling aliquots from the fractions and repeating the assay.
Sample yield lower than expected.	Sample may have been degraded by proteases or nucleases.	Add inhibitors or minimize separation time.
	Absorption to filter during sample preparation.	Use another type of filter.
	Sample precipitated.	Decrease sample load or change eluent conditions.
	Basic sample components bound to medium by ionic interaction.	Increase pH or add/adjust ion-pairing agent concentration.
More activity recovered than was applied to the column	Different assay conditions have been used before and after the chromatography step.	Use same assay conditions for all assays.
	Removal of inhibitors during separation.
Air bubbles in the bed.	Eluents not properly degassed.	Degas eluents thoroughly.
	Column packed or stored at cool temperature and then warmed up.	Remove small bubbles by passing degassed eluent through the column. Take special care if eluents are used after storage in a fridge or cold-room. Do not allow column to warm up due to sunshine or heating system. Repack column, if possible.
Negative peaks.	Refractive index effects.	Ensure sample is dissolved in initial eluent.
Unexpected peaks or spikes in chromatogram. Peaks appear on blank gradients.	Impurities in the eluent.	Use high-quality HPLC grade reagents.
Peaks appear on blank gradients	Incomplete elution of previous sample.	Wash the column according to recommended methods.
Spikes in chromatogram.	Air bubble trapped in UV low cell	Always use degassed eluents. Ensure the flow restrictor has an appropriate pressure range. Rinse the chromatography system with 100% methanol.
UV baseline rises with gradient	Eluents A and B absorb differently at the same wavelength	Balance eluent components or use a different wavelength.
	Impurities in the eluent.	Use high-quality HPLC grade reagents.
Retention time of a component increases over time. Peak width increases over time.	Mixed-mode retention due to surface silanols.	Lower pH suppress silanols or replace column.
Excessive baseline noise.	UV absorption by components in eluent.	Monitor at different wavelength or reduce concentration of UV absorbing component (usually the ion-pairing agent) or change organic modifiers if this is absorbing.