System suitability in HPLC Analysis


System suitability is to prove that system is working perfectly before the analysis on HPLC, GC, TOC analyser or any other system. It is required to done before every sample analysis. HPLC, short for High-performance liquid chromatography is a technique used for separating the components in a mixture.

HPLC chromatographic technique is used in pharmaceutical industries for analysis. System suitability testing limits are acceptance criteria that must met before starting the analysis.

There are some System suitability parameters which can be used to check the system before starting the sample analysis are listed below.

1.    Retention time

2.    Resolution

3.    Repeatability

4.    Plate Count

5.    Tailing Factor

6.    Signal-to-noise ratio

7.    Pressure

Retention Time:

In liquid chromatography and gas chromatography, the retention time, tR, is defined as the time elapsed between the injection of the sample and the appearance of the maximum peak response of the eluted sample zone. tR may be used as a parameter for identification. Chromatographic retention times are characteristic of the compounds they represent but are not unique. Coincidence of retention times of a sample and a reference substance can be used as a partial criterion in construction of an identity profile but may not be sufficient on its own to establish identity. Absolute retention times of a given compound may vary from one chromatogram to the next.

 Resolution (Rs):

Resolution is a 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 components to characterize their separation.

Resolution is calculated by following formula,

RS = 2(tR2 − tR1) / (W1 + W2)

Where, tR2 and tR1 are retention times of two compounds, W2 and W1 are the corresponding widths at the bases of the peaks obtained by extrapolating the relatively straight sides of the peaks to the baseline.

Repeatability/Precision:

Replicate injections of a standard preparation are used to demonstrate the system performance when it gets exposed to some specified column usage, environment, and plumbing conditions. Data from five or six replicate injections are used if requirement of relative standard deviation is less than 2%.

Plate Count/ Column Efficiency:

The Column Efficiency is measured by following formula,

N = 16(tR/W)2

Where tR is the retention time of the substance, and W is the peak width at its base, obtained by extrapolating the relatively straight sides of the peak to the baseline. The value of N depends upon the substance being chromatographed as well as the operating conditions, such as the flow rate and temperature of the mobile phase or carrier gas, the quality of the packing, the uniformity of the packing within the column, and, for capillary columns, the thickness of the stationary phase film and the internal diameter and length of the column.

Tailing Factor/Symmetry Factor:

Tailing Factor is calculated by following formula,

AS = W0.05/2f

where W0.05 is the width of the peak at 5% height and f is the distance from the peak maximum to the leading edge of the peak, the distance being measured at a point 5% of the peak height from the baseline.

Signal-to-noise ratio:

This parameter is used for the lower-end calculation of the performance of the system. 

Noise: It is measured between two specific lines that bracket the baseline. 

Signal: It is measured starting from the baseline’s middle and ending to the peak’s top.

Once calculating both these factors, the ratio can be measured by dividing the signal value by the noise value. With this, generally, the noise value has to be reduced using one of the following methods:

  • Signal Averaging
  • Reagent and Solvent Purity
  • Column Flushing and Sample Clean-Up
  • Temperature Control
  • Additional Pulse Damping and Mixing

The signal-to-noise ratio (S/N) is a useful system suitability parameter. The S/N is calculated as follows:

S/N = 2H/h

where H is the height of the peak measured from the peak apex to a baseline extrapolated over a distance ≥5 times the peak width at its half-height; and h is the difference between the largest and smallest noise values observed over a distance ≥5 times the width at the half-height of the peak and, if possible, situated equally around the peak of interest

System Pressure:

System suitability tests must be performed under controlled pressure limit. Monitor the pressure variation throughout the analysis.

Conclusion: The above mentioned system suitability parameters are not must. These parameters and acceptance criteria are performed during the method validation and fixed based on the method development outcome results. But system suitability should meet the acceptance criteria before starting the sample analysis.

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