HPLC Column Companion, Section 6: Injection-to-Injection Reproducibility

Retention and Resolution Variability
If peak retention

(t_r) and resolution (R_s) are not reproducible from injection-to-injection, or even day-to-day, the accuracy and precision of the HPLC method may not be acceptable. Poor reproducibility is usually caused by either changes related to chromatographic conditions or variability in column performance (see Table 6-1).

Identifying and Correcting Variability in Column Performance
Reproducibility problems

caused by variability in column performance usually show up as continuous changes in retention time and/or loss in peak symmetry. Identifying and correcting the cause of column variability can be difficult, however, a systematic approach to taking corrective action will usually be successful. Table 6-2 lists the causes of poor injection-to-injection reproducibility that are due to variability in column performance along with identifying symptoms and recommended corrective actions.

Table 6-2 Correcting Variability in Retention

Causes of Poor Reproducibility	Symptom	Corrective Action

Inadequate equilibration	Systematic decrease or increase in t_r Poor peak shape	Allow more time for column equilibration. (Prior to injection, purge with at least 15 column volumes of mobile phase, i.e. 35 mLs for a 25 x 0.46 cm column) If the chromatographic conditions call for a weak mobile phase, purge the column initially with a stronger mobile phase. This assists in solvating the bonded phase consistently.
Column fouling	Continuous decrease in t_r, usually correlated with sample injection Poor peak shape Increasing pressure (occasionally)	Clean the column by reverse flushing with strong solvent Replace column if cleaning does not work. In either event, consider using a guard column to protect the analytical column
Loss of bonded phase	Continuous change, usually decrease, in t_r Poor peak shape	Replace the column when it no longer meets the separation requirements, i.e., suitability test. Use stabilized bonded phases when operating at low pH (see 2-2)
Column overload	Decrease in t_r that correlates with higher sample mass injected or change in sample matrix Tailing peaks	Inject diluted sample Choose a column having a larger inner diameter

This general troubleshooting information for injection-to-injection reproducibility problems applies to isocratic and gradient HPLC methods; but gradient methods require some additional troubleshooting hints.

Retention Time Variability in Gradient Separations
Gradient separations

have many advantages for the analysis of complex samples with components that vary widely in polarity. During a gradient separation, the column and analytes are subjected to a constant change in mobile phase conditions. This makes troubleshooting column or system problems more difficult. For example, the problem of gradient elution retention time irreproducibility, varying retention times from injection-to-injection, has several unique causes.

CAUSE: Incomplete column equilibration

SOLUTION: Make sure the column is equilibrated

A standard 4.6 x 250 mm column has an internal column volume of approximately 2.5 mL and needs at least 10 to 15 column volumes to completely equilibrate.Your gradient equilibration time may need to be adjusted to match this volume according to your flow rate. Duplicate injections after equilibration will verify adequate equilibration.
Column equilibration is easier when both the organic and aqueous/buffer components of a mobile phase contain the same concentration of mobile phase additives. For instance, TFA is often added to a mobile phase to improve peak shape, especially for peptide separations, and should be added to both the organic and aqueous mobile phase components.

CAUSE: Highly retained material "fouls" the column

Not all sample material may be completely eluted from a column if the final gradient composition is not strong enough. For example, the analysis of a sample in a complex matrix may require a gradient that starts at 10% methanol and ends at 60%, but it may require 75%-100% organic to elute all matrix components. Figure 6-1 shows peaks eluting late in a blank gradient run of 0-1000% methanol. This example demonstrates that the gradient conditions of 0-60% methanol are not sufficient to remove all matrix components. If the organic composition is not increased beyond 75%, matrix components will build up on this column and the result may be changes in retention time of eluting sample peaks. Noneluted material can be removed by extending both the gradient % organic and gradient time proportionately or washing the column with 100% methanol after a to-be-determined number of sample injections.

Figure 6-1
Blank Gradient Run Showing Retained Matrix Components

No injection
0-100% methanol in 50 min
2 mL/min

SOLUTION: Back-flush the column with a strong solvent

If the column has become heavily contaminated, first back flush the column. Disconnect the column from the detector, turn it in the opposite flow direction and Bush out all buffers with an aqueous/ organic mobile phase. Then, switch to 100% organic. Acetonitrile is a good organic solvent to start with, since it is stronger than methanol in a reversed-phase system and it may elute additional strongly retained components. If this flush is not sufficient, isopropanol is another good solvent choice. Isopropanol is more effective at solubilizing many proteins, peptides, and fats and is a stronger solvent than acetonitrile or THF. Unlike THF, isopropanol is compatible with PEEK tubing.


Section 6 Injection-to-Injection Reproducibility