How to calculate System Suitability in Chromatography

How to calculate System Suitability in Chromatography
System Suitability Testing in Chromatographic Analysis
How to calculate System Suitability in Chromatography

High-Performance Liquid Chromatography (HPLC) technique is applied in various places to separate out a mixture’s components. Through this process, the liquid’s effectiveness is examined by passing it over an absorbent material.

One of the major applications of this technique is in the pharmaceutical industry, where experts research and test different components. System suitability testing is a part of this procedure.

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.

But let’s first understand the concept of system suitability testing

System Suitability Test (SST)

This testing is used for examining a liquid chromatographic system’s specs. That is why it is crucial to opt only for an appropriate method for the calculations. On the other hand, an acceptance criterion is also set, called the SST limits. It is vital to meet these limits before analyzing any sample for your purpose.

Now, let’s move on to the parameters that have to be checked under this testing.

Resolution

The resolution in HPLC is a measure of the quality of separation between two chromatographic peaks. It plays a crucial role in checking the feasibility of this critical separation according to the given circumstances.

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

Almost all peaks show a bit degree of tailing. That is why the resolution is considered complete only if it equals or exceeds 1.5. Another important factor to consider here is that the equation can not be used if the peaks are not resolved at the level of baseline. But this is not something to worry about because it is virtually impossible for the peaks to overlap at the bottom. Even the peak baseline width measurement is not feasible. 

In all other cases, this resolution formula in chromatography provides a much efficient outcome.

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  the tailing factor formula is expressed as

T = [Latex] \frac {a+b}{2a}[/latex]

 T should be less than or equal to 2 to satisfy the system suitability requirement.

The tailing factor in HPLC is also known as the symmetry factor.

Precision

Replicate injections of a standard preparation are used to ascertain if requirements of precision are met. This is used to demonstrate the system performance when it gets exposed to some specified column usage, environment, and plumbing conditions. 

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%.

Signal To Noise Ratio (S/N)

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

Theoretical Plates: Column Efficiency

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. That is why it is always preferred to keep the plate number high for any provided column. The number of plates can be calculated by:

\(
N =16{[\frac{(t_R)}{W}}]^2\) 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

It is recommended that Theoretical plates should not fall below 2000

The benefit of opting for this method is that the number of theoretical plates can be calculated even if the resolution is poor, and the two consecutive peaks are not entirely differentiable. It becomes possible due to the use of half-height in the formula. However, the valley that lies between these peaks must be lower than their half-heights. Only then can the number be calculated. 

The method is not much utilized in the manual calculations. Instead, it is mostly preferred for automatic verifications done on data systems. In the case of calculating the theoretical plate number, the following formula can be used:

Plate per meter = Number of theoretical plates in one column x 100 /HPLC column length in cm

A USP method for theoretical plate calculation can be used for other cases. 

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. This is one of those parameters that can be identified without any hassle. 

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

You can easily calculate all the factors using these equations. However, you must remember that the final results can show extreme deflections based on the type of analysis you are performing and the analytical conditions around it. Plus, these tests are not only done at the beginning. You need to conduct them periodically to ensure that the system performance remains intact. The values may change with time because of the continuous use of the system.

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  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.

    1. 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.

    1. 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.

      1. In Empower software,there is two resolutions-one is Resolution and another one is USP Resolution.what is the difference between them.

    1. You need to make replicate injections to achieve a high degree of reproducibility. After making a single injection you will not be sure about the accuracy of volume injected.

    1. 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 .

      1. There is a concept of 6 sigma and 3 sigma where in we need minimum 3-points for the agreement
        i mean to see that your system is precised enough for the analysis u need to meet the criteria of RSD either with 3-points or with 5-6 points

  2. 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……

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

    1. 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.

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

  5. 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 caliberation.is it really needed.

  6. 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

    1. 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

  7. What is the difference between Drug Purity and Drug Potency?

    Please explain

  8. 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

    1. 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.

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

    1. 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.

    1. 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.

    1. In manual operation you have to get the best fit with baseline. However, the softwares have auto fit capability so this is taken care on its own.

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

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

    1. 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.

    1. The answer is simple. Known impurities are those whose identity is known before the analysis and unknown are those whose identity is unknown.

    1. 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.

    1. Analytical validation of a new method is important as it verifies that the method adopted will provide you reliable analysis. For more information you may browse through our articles on the topic which were published on 7th Feb 2017.

  12. Hi there, You have done an excellent job. I will definitely digg it and personally suggest to my
    friends. I’m sure they’ll be benefited from this site.

  13. Thank you very much sir. I have indeed gain alot from your knowledge. may God increase u in knowledge,meanwhile i want to learn more on hplc

    1. gister for our free course on HPLC through the site. Click on About us on top menu and then click on Free courses in the drop down menu.You can select the HPLC free course and register for it by providing your name and mail ID in the dialog box.On submission you will get a link in your mailbox . Click on th link and your registration is complete. You will start receiving the successive modules after a gap of 3-4 days each.

    1. A Gaussian peak is a perfect bell shaped peak without any distortions. It rises above a perfect horizontal baseline and drops back to the same baseline after reaching the maximum height.

    1. Accuracy relates to closeness of observed value to the true value whereas precision relates to closeness of observed values to a value which may or may not be a true value.

  14. Good morning sir..!!
    What is the effect on our assay or RS if talling factor will be out of limit… Why be use limit of talling..

    Ex:- our talling factor limit 1.5… And after calculations it will be found 1.8, Does it effect on our assay or rs?

    1. Tailing factor will affect the area under the peak.In case you are getting tailing beyond the limit value obviously your quantitative result.

  15. Good morning Sir
    What is the difference between checking standard and bracketing standard?
    And what is acceptance criteria for both?

    1. Assay gives you the amount of active ingredient present in a pharmaceutical formulation whereas RS or related substances give you amounts of impurities present. In HPlC chromatogram the main peak is the active ingredient ant the other small peaks represent the related substances.

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  17. what is the void volume role in the empower calculation. and can you give clear definition of void volume

  18. Fill your response that both compounds 1 and 2 satisfy ICH guidliness for asymmetric factor and tailing factor. If your answer is yes or no than will provide the reason.

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