In my earlier post on generation of authentic chromatographic data I had emphasized the need for evaluation of system suitability before proceeding with analysis. Some factors contributing to system suitability failures in HPLC were discussed. The current post introduces you to system suitability parameters and their acceptance limits.
Resolution is a measure of the separation between two chromatographic peaks.
Well resolved peaks are basic requirement in both qualitative and quantitative estimations. Separation between closely spaced peaks is governed by affinity for the stationary phase.
Co-eluting compounds can be resolved by:
- Change of mobile phase polarity
- Increase of column length
- Reducing particle size of stationary phase
Where and are retention times of peaks A and B
Peak widths and are obtained from the intersection of tangents with baseline
Resolution is considered complete if it equals or exceeds 1.5
Asymmetry or Tailing factor ()
An ideal chromatographic peak should be of symmetrical Gaussian shape but due to various factors the shape often deviates. Peak tailing is the commonly observed peak deformation. It is mainly due to occurence of more than one mechanism of analyte retention. Tailing can be reduced by changing mobile phase pH or end-capping of stationary phase.
where A and B are peak widths at 10% of the height for leading and tailing ends of the peak
Ideal peak has As =1 but values in the range 0.9 – 1.1 are acceptable
Tailing becomes apparent when asymmetry factor As equals to or exceeds 1.2
As per USP definition the tailing is considered as the ratio of the widths a and b at 5% of peak height and is expressed as
T should be less than or equal to 2 to satisfy the system suitability requirement.
Replicate injections of a standard preparation are used to ascertain if requirements of precision are met
Data from five replicate injections are used if requirement of relative standard deviation is less than 2%. Data from six replicate injections are used if the requirement of relative standard deviation is more than 2%.
The plate theory concept assumes that the chromatographic column comprises a large number of imaginary separation layers called theoretical plates. Equilibrium of the sample takes place between the stationary and the mobile phase in these imaginary plates. The analyte moves down the column by transfer of equilibriated mobile phase from one plate to the next.
Column efficiency is expressed in terms of theoretical plates(N).High resolution means greater number of plates in a given length of column
Where W is the peak at base
Where is peak width at half height where
is retention time and is the peak width at half height
Theoretical plates should not fall below 2000
Retention factor (k’)
Retention factor (k’) or partition ratio or capacity factor is the relation of time spent by a compound in stationary phase to the time it spends in the mobile phase.
k’ is a unitless quantity
Higher the value of k’ greater is the retention of a compound on a column
Ideally k’ should be greater than 2.0