System suitability tests (SST) ensure that chromatography systems are operating as expected prior to sample analysis. Key parameters evaluated include column efficiency, resolution, asymmetry, retention time, capacity factor, and precision. SST solutions contain analytes of interest and closely eluting components at known levels. Acceptance criteria are established during validation. If an SST fails, the analysis is stopped and the system diagnosed before re-running the SST. SSTs help reduce unnecessary retesting of samples by identifying potential issues early.

1. System suitability test for HPLC
Dr. Vrushali Tambe
PES Modern College of Pharmacy (for ladies),
Moshi

2. • System suitability tests are based on the concept that the
equipment, electronics, analytical operations, and samples to be
analyzed constitute an integral system that can be evaluated as
such.
• System suitability test parameters to be established for a particular
procedure depend on the type of procedure being evaluated.
• Ensures that both methodology and instrumentation are
performing within expectations prior to the analysis of the test
samples
• Used to verify column efficiency and repeatability of
chromatographic system and electronics
• The performance of any chromatographic system may continuously
change during their regular use, which can affect the reliability of
the analytical results.
Its criteria should be established during the method validation
The produced data by the system should be reliable.

3. • Recommended by ICH and USP
• Used during assay, impurity determination, dissolution testing
and bioassay
• Performed before and throughout the regulated assays (eg. 5
SSS injections before and one SSS injection every 10 assays)
whenever instrument malfunctions.
• Assessed with any significant change in equipment or in a critical
reagent
• It represents minimum acceptance performance
The operation parameters of the whole chromatographic system
can be checked with properly selected SST mixtures. These
mixtures are used to establish characteristic chromatographic
parameters

4. Constitution of System suitability solution
At least the major analyte of interest and, ideally, a
closely eluted component or components that could be
found in actual samples at known levels
Analyte should be sensitive to operating conditions and
should indicate slight deterioration of the system
Standard solution can be used
Single component calibration standard is not adequate
for SST as system separation capability is not tested
SST solution should consist of main component to be
tested and related impurities critical to separate from API

5. Typically, the SST involves performance parameters, such as
• Number of Theoretical plates/ Column efficiency
• Resolution
• Asymmetry/ Tailing factor
• Retention time/ Relative retention time/ Retention Volume
• Detection limit
• Selectivity
• Capacity factor
Most of the performance parameters are calculated
automatically by data evaluation software of sophisticated
chromatography equipment
The system is then only declared suitable if the responses are
within given limits.

6. Resolution (Rs)
Measure for the ratio of the distance of two adjacent peak maxima and their
widths. For complex sample mixtures Rs should be determined for the critical
pairs of component to characterize their separation.
𝑅𝑠 =
2 (𝑡𝑅2 − 𝑡𝑅1)
𝑊𝑏1 + 𝑊𝑏2
• where, tR2 and tR1 are the retention times of closely eluted compounds,
and
• Wb1 and Wb2 are their peak widths at the base.

7. Resolution-
• Function of column efficiency
• Measure of resolving power of system
• NLT 2
• Most closely eluting species

8. Column efficiency
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
• N=16 (tR/w)2 Where W is the peak at base
• Theoretical plates should not fall below 2000

10. Precision
• Replicate injections of a standard preparation
are used to ascertain if requirements of
precision are met
• Data from five replicate injections are used
Relative standard deviation should be less
than 1%.

11. Asymmetry or Tailing factor (As)
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 occurrence 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
T should be less than or equal to 2 to satisfy
the system suitability requirement.

12. Asymmetry is most sensitive indicator of system contamination

13. Retention factor (k’)/ Capacity factor
• 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
• k′=tr–tm/tm
• tr: retention time of analyte
• tm: retention time of unretained species
• Higher the value of k’ greater is the retention of a compound on a
column
• Ideally k’ should be greater than 2.0
• It tells about how much interaction analyte have along with
stationary phase

14. Retention time: The resolution should be fairly constant.
Retention volume:
It is volume of mobile phase required for elution of compound
Vr= tR * F
Relative retention time (RRT)
is the ratio of the retention time of analyte peak relative to that
of another used as a reference obtained under identical
conditions.
Pressure: The suitability testing must be carried out within set
pressure limits. This is to ensure that wearing of system
components is reduced.
Signal-to-Noise Ratio: S/N ratio is a measure of the system's
performance at the lower end.

16. For dissolution or release profile test methods
using an external standard method, k', T and %
RSD are minimum recommended system
suitability tests.
For acceptance, release, stability, or impurities
degradation methods using external or internal
standards k’, T, Rs, and %RSD are recommended
as minimum system suitability testing
parameters

17. When SST fails, if possible the assay sequence
should be stopped immediately
Analyst diagnose the system problem, make
necessary adjustments and repairs
Perform SST again
If SST passes further analysis can be done

18. Reasons for SST failure
• Poor precision of autosampler
• Aged column
• Pump problem
• Mobile phase preparation error
• Air bubbles
• SST is early warning to reduce sample retesting

19. Thank You…