1) What Is a System Suitability Test (SST)? Explain the Important SST Parameters in HPLC and GC.
A System Suitability Test (SST) is performed to verify that the chromatographic system is functioning properly and is suitable for the intended analytical procedure.
It evaluates the performance of the complete chromatographic system under the specified method conditions, including the instrument, injector, column, detector, mobile phase or carrier gas, and data-processing system.
The commonly evaluated SST parameters include:
- Precision
- Retention factor (Capacity factor)
- Selectivity factor
- Resolution
- Theoretical plate count
- Tailing factor or symmetry factor
- Signal-to-noise ratio
- Peak-to-valley ratio
Note: The applicable SST parameters and acceptance criteria must be specified in the analytical method or applicable pharmacopoeial requirements. All parameters are not mandatory for every method.
2) WHAT IS PRECISION? EXPLAIN?
Precision is the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under specified conditions.
In chromatographic system suitability testing, precision is commonly evaluated by injecting a standard solution repeatedly and calculating the percentage relative standard deviation (%RSD) of the peak areas or responses.
%RSD = Standard Deviation*100/Mean.
For Assay by HPLC, the maximum permitted relative standard deviation does not exceed the appropriate value given in the below table as per USP & EP.
Note:
1.B (percent) means upper limit of your assay -100.
That is If your assay limit is 98%-102%, B (percent) is 102%-100% = 2.0
That implies your %RSD should not more than 0.85% for 6 injections and 0.73 for 5 injections.
2.This requirement does not apply to tests for related substances.
3) WHAT IS CAPACITY FACTOR or RETENTION FACTOR?
It is the ratio of the adjusted retention volume (or time) to the hold-up or Void volume (or time). (or) It is the migration rate of analyte on a column (or) It is a measure of time of sample component resides in the stationary phase relative to the time it resides in the mobile phase.
Simply it is measure of where the peak of interest located with respect to void volume (retention volume of unretained compound).
Capacity factor (K¹) = VR¹/Vm = tR¹/ tm = (tR-tm)/tm
VR¹= Retention volume of analyte.
VM=Retention volume of unretained compound
tR=Retention time of analyte.
tm or to=Retention time of unretained compound
Note:
1. Generally the ideal valve of K¹ is in between 2 to 5. But the acceptable valve is in between 1 to 20 .
4) WHAT IS SELECTIVITY FACTOR or RELATIVE RETENTION? EXPLAIN?
The selectivity factor (α) describes the relative retention of two components and indicates the ability of a chromatographic system to distinguish between them.
It is calculated as the ratio of the retention factor of the later-eluting compound to that of the earlier-eluting compound.
Selectivity factor (a ) = K2/K1 = (tR2-tm)/ (tR1-tm)
tR2=Retention time of our analyte
tR1=Retention time of reference compound.
Note:
1. As per USP the selectivity factor should be always greater than 1.
5) WHAT IS RESOLUTION? EXPLAIN?
Resolution (Rs) is a measure of the separation between two adjacent chromatographic peaks.
It is calculated using the difference between their retention times and their peak widths.
As per USP:
Resolution(R) = 2 (tR2-tR1)/ (w1+w2)
tR2&tR1 are retention times of two components.
w1&w2 are corresponding peak widths at base.
As per EP:
Resolution(R) = 1.18 (tR2-tR1)/ (wh1+wh2)
tR2&tR1 are retention times of two components
wh1&wh2 are corresponding peak widths at half height.
Note:
1. Base line resolution achieved at R=1.5. But more than two is desirable.
6) WHAT IS THEORITICAL PLATE? HOW NUMBER OF THEORITICAL PLATES EFFECT THE COLUMN EFFICIENCY? EXPLAIN?
The theoretical plate is a hypothetical concept used to describe the efficiency of a chromatographic column.
The number of theoretical plates (N) indicates column efficiency. A higher plate number generally represents lower peak broadening and better column efficiency under comparable conditions.
As per USP:
Number of theoretical plates (N) = 16 (tR/W) 2
tR=Retention time of analyte
W=Peak width at base
As per EP:
Number of theoretical plates (N) = 5.54 (tR/Wh) 2
tR=Retention time of analyte
Wh =Peak width at half height.
7) WHAT IS SYMMETRY FACTOR or TAILING FACTOR? EXPLAIN?
An ideal chromatographic peak is approximately symmetrical and follows a Gaussian distribution.
Deviations from peak symmetry are commonly described as:
- Peak fronting: The front side of the peak is broader.
- Peak tailing: The rear side of the peak is broader.
The tailing or symmetry factor describes the shape of a chromatographic peak.
As per USP & EP:
Tailing factor (As) = w 0.05/2d
w 0.05=Width of peak height at one-twentieth of peak height
d=Distance between the perpendicular dropped from the peak maxima and the leading edge of the peak at one-twentieth of the peak height
Note:
1. As per EP, In related substance test or assay the symmetry factor should be in between 0.8 to 1.5 unless otherwise specified prescribed.
8) WHAT IS SIGNAL TO NOISE (S/N) RATIO? EXPLAIN?
The signal-to-noise ratio compares the analyte response with the background detector noise.
It is particularly important in trace-level analysis, such as related-substances testing, where low analyte concentrations must be detected and quantified reliably.
Signal to noise(S/N) ratio is calculated from following equation
S/N = 2H/h
Where
H= Height of concerned peak measured from the peak apex to the base extrapolated over a distance ≥5 times the peak width at its half height.
h= Difference between the largest and smallest noise valves observed over a distance ≥5 times width at half height of the peak.
9) WHAT IS PEAK TO VALLEY (p/v) RATIO EXPLAIN?
The peak-to-valley ratio is used as a system suitability parameter in certain related-substances tests when two specified peaks are not completely separated. It evaluates the degree of separation between a minor peak and an adjacent major peak.
p/v = Hp/Hv Where
Hp= Height above the extrapolated baseline of the minor peak.
Hv= Height above the extrapolated baseline at the lowest point of the curve separating the minor and major peaks.
