How to calculate System Suitability in

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

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

Resolution of Chromatographic Peaks

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.

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

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=

T should be less than or equal to 2 to satisfy the system suitability requirement.

Precision

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%.
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.

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

or

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

2.6.1 Acidic Hydrolysis- To 10 ml of above methanolic stock solutions of tertiary mixture and
pharmaceutical formulation, 10 ml of 1 M HCl, was added separately. These mixtures were
refluxed separately for 1 hour at 80C on oil bath. The forced degradation in acid 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 neutralized for assay after suitable dilutions with mobile phase.

2.6.3 Alkaline Hydrolysis- To 10 ml of above methanolic stock solutions of tertiary mixture

and pharmaceutical formulation, 10 ml of 0.5 M NaOH, was added separately. These mixtures
were refluxed separately for 1 hour at 80C on oil bath. The forced degradation in alkaline 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, neutralized and subjected to analysis after suitable dilutions with mobile
phase.

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.

2.6.5 Neutral Hydrolysis- To 10 ml of above methanolic stock solutions of tertiary mixture

and pharmaceutical formulation, 10 ml of water was added separately. These mixtures were
refluxed separately for 6 hours at 80C on oil bath. The forced degradation in neutral media was
performed in the dark in order to exclude possible degradation effect of light. 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.

2.6.8 Photochemical Degradation- The photochemical stability of drug was studied by

exposing stock solutions of tertiary mixture and formulation in direct sunlight for 3 days (from
09-00 am to 05-00 pm) on wooden plank and kept on terrace. Degradation samples were
subjected to analysis after suitable dilutions with mobile phase.