Resolution between two peaks depends upon three things:
1. proper retention factor (k) – a function of the mobile phase strength),
2. correct selectivity (α)– the chemical interaction provided by the stationary phase and mobile phase),
3. enough efficiency (N)– a mechanical function of the column and packing dimensions).
Possibility of mass overload
use a higher capacity stationary phase (i.e. increased % carbon or pore size),
use a column with an increased diameter,
decrease the absolute sample amount or volume injected
Operate at a lower pH
As silanol groups are acidic, secondary interactions can be minimised by performing the chromatographic separation at a lower pH — thereby ensuring the full protonation of such ionisable residual silanol groups.
Work at high pH when analyzing basic compounds
From a practical point of view, it is usually only possible to use pH to suppress acid ionisation, as base ionisation typically requires pH values of greater than 8, at which pH silica dissolution can occur.
The silica surface being protected from dissolution by the use of bi- and tridentate ligands.
These ligands are bidentate and are bridged using proprietary chemistry, prior to their application to the stationary phase.
Their bridged structure affords steric protection against hydrolysis of the silica surface.
Use a highly deactivated (‘end-capped’)column
End-capping is a process whereby residual silanol groups are treated to convert them to substantially less polar surface functional groups. This has the effect of reducing the amount of secondary interaction they can potentially have with polar analyte molecules.
However, due to steric hindrance factors end-capping only reduces approximately 50% of the number of unreacted residual silanol groups. Consequently, a “fully end-capped” column is not quite as it may seem!
Chemical reagents typically used in the end-capping process include;
Trimethyl chlorosilane (TMCS)
Hexamethyl disilazane (HMDS)
Possibility of column bed deformation
If a void is suspected, reverse the column, disconnect it from the detector, and wash in 100% strong solvent (at least 10 column volumes).
The blockage of column frits can be avoided though the regular replacement of solvents filers, use of in-line filters, and guard columns.
Use a sample clean-up procedure
Solid Phase Extraction (SPE) can be used to remove any interfering contaminants.
Broad Asymmetrical Peaks
If all peaks are broad, the column efficiency (N) may be too low. Try
smaller particle diameter
optimal flow rate.
If only one peak in a mixture shows poor precision, possible causes could be wrong pH (analyte is only partially ionized)Try
If retention factor (k) is too low (<2), the peak may be overlapping with other peaks. Decrease the strength of mobile phase.
short time variation of the baseline from a straight line.
caused by electric signal fluctuations, lamp instability, temperature fluctuations and other factors.
Noise usually has much higher frequency than actual chromatographic peak.
Noise is measured “peak-to-peak”: i.e., the distance from the top of one such small peak to the bottom of the next.
Noise limits detector sensitivity. A practical limit for qualitative purposes is a 3 x signal-to-noise ratio, but quantitative detection limit better be chosen as 10x signal-to-noise ratio.
long-term noise and is defined as a change in the baseline position. It is usually measured for a specified time, e.g., 1/2 hour or one hour. Drift usually associated to the detector heat-up in the first hour after power-on.
drift is mainly caused by changes of temperature, or solvent programming and temperature effects on the detector.