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

Where and are retention times of peaks A and B

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

**Resolution is considered complete if it equals or exceeds 1.5**

#### Asymmetry or Tailing factor ()

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.

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 =

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

Where W is the peak at base

or

Where is peak width at half height where

is retention time and 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

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

**Ideally k’ should be greater than 2.0**

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.

how to calculate lower detection limit for hplc

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.

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

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.

what is difference between resolution and usp resolution in empower

Suresh your question is not clear. Can you elaborate it.

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

the formula is different, you can check in the help file for exact details.

Why 5 or 6 replicate injection used in sst

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.

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 .

What is RsD calculation in system suitability

Hi Meena, it is Relative standard deviation the formula is standard deviation divided by mean times 100.

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

Hi meena, the washing procedure is different for different column phases. What are you using?

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……

Hi Srikanth

Your question is not understood . Please clarify what you wish to know

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

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.

Hello Dr. Deepak,

Is there any videos available to learn integration of peaks.

Hello Neena,

Yes videos on software control of instrument in our paid certificate course covers this aspect.

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.

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

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

This could be due to sample degradation, are you using a refrigerated auto sampler?