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 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.


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


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.


  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.

  2. daud bangash says:

    how to calculate lower detection limit for hplc

    • Dr. Saurabh Arora says:

      Hi Daud, LOD is calculated by signal to noise ratios or from the slope and intercept of linearity studies. S/N is usually the preference.

  3. What are the factors responsible for the Relative Standard Deviation to exceed the limit?

    • Dr. Saurabh Arora says:

      Dear Sunil A host of factors can increase the RSD like injection volume, flow rates, sample prepration, temperature variation, evaporation of the stock solutions, gas quality variation You need to ID the one responsible by isolation and then solve it.

  4. what is difference between resolution and usp resolution in empower

  5. Why 5 or 6 replicate injection used in sst

  6. Why we don’t used 10 injection in sst instead of 5 or 6?

    • Hi Meena,
      If you can achieve the required precision in 5-6 injections why waste your time in making 10 injections. There is no guarantee that you will get improvements in 10, 15 or even 20 injections over your results so 6 is the optimum number .

  7. What is RsD calculation in system suitability

  8. How to get sharp peak in phenomenox column?what s washing column procedure

  9. Hi sir this srikanth
    In system suitability parameters calculation
    Formulas in resolution0.5,railing factory 2,
    Teritacal plates 16…numbers is used could be explain sir……

  10. Why Assay specification is broad I.e 95% to 105%. But purity specification is NLT 99. 0%. What is the main reason

    • Dr. Saurabh Arora says:

      Hi Mahesh, the assay is for formulations where there are other ingredients and also the fact that on storage the assay might come down depending on stability. Thus the broader range.

  11. Hello Dr. Deepak,
    Is there any videos available to learn integration of peaks.

  12. hello sir,
    what is the system suitability requirement for bracketing standard and how many injections we should give .Because some companys are running sst 6 injections,check standard 2 injections and Two point claiberation 2 injections.And after 6 Injections of assay samples again they are giving two injections of two point it really needed.

    • Dr. Saurabh Arora says:

      This is usually based on the internal STP which is established during the method validation.

  13. Suresh Singaram says:

    hi sir, in my Assay analysis for Acarbose tablets,pre injection of resolution got exactly 1.5(limit NLT 1.5)but in main sequence got 1.42, why, can you explain the reason

  14. Sir tell me about ideal peak

  15. How we will set oven temperatur in gc

  16. How we will caleculate s/n in gc,
    Plese explain which is exat rt to caleculate SN

    • S/n ratio is defined as the lowest concentration at which the signal is 3 times the average noise level.There is no particular Rt defined for measuring it. You can use a reference compound whose Rt can be established through repeated runs

  17. Hello sir,
    Why we use Acetonitrile, and Methanol in preparation of HPLC mobile phase, why we not used remaining solvents and what is the reason

    • Solvent composition decides the overall polarity of the mobile phase. As the sample may contain more than one compounds of different polarities a mixture is generally used. The selected solvents should be fully miscible. Acetonitrile and Methanol are fully miscible with water as in caes of most aqueous phases.

  18. Hello sir,
    What are the units of vaccume, why some products are analysed in a vaccum oven.

    • Units of vacuum are torr. However, I have not come across any application which requires a vacuum to be maintained in the column oven. In any case since the fittings are airtight column oven pressure should not play any role in GC separations.

  19. Hi sir good morning
    Why we are not identify unknown peacks with there boiling point

    • Peaks are usually identified by retention times under the set of operating conditions using standard mixtures.The boiling points of compounds depend on purity. As purity can vary with sample matrix basing your judgement on boiling points can be misleading.

  20. How we will set split ratio

  21. suresh gajula says:

    What thing are keep in mind when we are integrate the chromotogram

  22. suresh gajula says:

    In lab online courses are only Hplc,Aas,safety…..?
    I want to join Gc direct classes

  23. suresh gajula says:

    Which operating conditions are show sequencel order rt based on there boiling point

    • It was difficult to make sense from the question but if I have understood it correctly you mean what operating conditions are needed to get retention time in a sequence matching the boiling points of components.From basic physics you will agree that the components will vaporize at different temperatures depending on their boiling points. As the column oven temperature increases the components begin to evaporate in a sequence of increasing boiling points.

  24. suresh gajula says:

    Good morning sìr
    What is defference between known and uknown impurity

  25. suresh gajula says:

    Please tell me sir types of sample and types of Gc columns

    • Samples constitute any matrix which contain compounds of your interest whose identity and amount needs to be determined. These can be synthetic mixtures, or specimens of pharmaceuticals, foods, environmental samples, forensic residues,etc.GC columns are of different types depending on their dimensions and packing material. You are advised to register for the free course on GC which will give you clarity on fundamental concepts.

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