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How To Calculate System Suitability in Chromatography: Resolution
How To Calculate System Suitability in Chromatography: Resolution
How To Calculate System Suitability in Chromatography: Resolution
Chromatography
How to calculate System Suitability in Chromatography
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
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.
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.
Assymetry factor
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
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=
Precision
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%.
Theoretical plates
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.
or
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
2.6.4 Oxidative Hydrolysis- To 10 ml of above methanolic stock solutions tertiary mixture and
pharmaceutical formulation, 10 ml of 3 %v/v H 2O2 was added separately.These mixtures were
refluxed separately for 1 hour at 80C on oil bath. The forced degradation in oxidative media was
performed in the dark in order to exclude possible photo-degradation. The degradation samples
were then cooled to room temperature. Suitable aliquots of resultant degradation samples were
taken and subjected to analysis after suitable dilutions with mobile phase.
2.6.6Dry Heat Degradation- For dry heat degradation, tertiary mixture and pharmaceutical
formulation were placed in oven at 80C for 24 hours under dry heat condition in the dark and
then cooled to room temperature. Degradation samples were subjected to analysis after suitable
dilutions with mobile phase.
2.6.7 Wet Heat Degradation- The above stock solutions of tertiary mixture and
pharmaceutical formulation were refluxed separately for 4 hours at 80C on oil bath for wet heat
degradation. The degradation samples were then cooled to room temperature. Suitable aliquot of
resultant degradation samples were taken and subjected to analysis after suitable dilutions with
mobile phase.