How to calculate System Suitability in Chromatography

System Suitability Testing in Chromatographic Analysis

How to calculate System Suitability in Chromatography

In my earlier post on generation of authentic chromatographic data I had emphasized the need for evaluation of system suitability before proceeding with analysis. 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

Resolution of Chromatographic Peaks

 

 R_S=\frac {tR_B - tR_A}{0.5 (W_A + W_B) }

Where tR_B and tR_A are retention times of peaks A and B

Peak widths W_A and W_B are obtained from the intersection of tangents with baseline

Resolution is considered complete if it equals or exceeds 1.5

Asymmetry or Tailing factor (A_s)

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

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 =  \frac {a+b}{2a}

 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

N =16{[\frac{(t_R)}{W}}]^2 Where W is the peak at base

or

N = 5.54{[\frac{(t_R)}{W\frac{1}{2}}}]^2

Where  W_1_/_2 is peak width at half height where

 t_R is retention time and  W_1_/_2 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

k' = \frac{t_r - t_m}{t_m}

Higher the value of k’ greater is the retention of a compound on a column

Ideally k’ should be greater than 2.0

About Dr. Deepak Bhanot

Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise beginning from sales and product support of analytical instruments. After completing his graduation and post graduation from Delhi University and IIT Delhi he went on to Loughborough University of Technology, UK for doctorate research in analytical chemistry. His mission is to develop training programs on analytical techniques and share his experiences with broad spectrum of users ranging from professionals engaged in analytical development and research as well as young enthusiasts fresh from academics who wish to embark upon a career in analytical industry.

Comments

  1. what do you mean by the factor in resolution formula 0.5 and in theatrical plates formula 16 or 5.54. how it was introduced in formula and what is use of that. i mean from where it came. please sir i am new for this profession.

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