High Performance Liquid Chromatography (HPLC) and calculation of chromatogram

 

Title: Standard operating procedure for performance on High Performance Liquid Chromatography (HPLC) and calculation of chromatogram.

 

1.0                 OBJECTIVE:

To lay down the procedure for analysis on High Performance Liquid Chromatography (HPLC).

2.0                 SCOPE:

This shall be applicable on installed High Performance Liquid Chromatography (HPLC) of Quality Control Instrument Lab.

3.0                 RESPONSIBILITY:

3.1  Technical Assistant.

3.2  Q.C Analyst.

4.0                 ACCOUNTABILITY:

4.1  Manager-Quality Control.

4.2  Head of the Q.C Department.

5.0                 MATERIAL AND EQUIPMENT:

5.1  High Performance Liquid Chromatography (HPLC).

5.2  Reference solution.

5.3  Sample solution.

6.0                 PROCEDURE:

6.1  Firstly prepare the test solution, reference solution and mobile phase according to the requirements.

6.2  The solutions must be free from solid particles.

6.3  Prepare the apparatus.

6.4  The mobile phase solvent mixtures must be deaerated prior to use either by boiling or by applying a partial vacuum to the solvent reservoir.

6.5  Equilibrate the column with the prescribed mobile phase, flow rate and at temperature specified until a suitable baseline is achieved.

6.6  Test solution of the mixture to be separated is now introduced into the mobile phase with the help of an injector just before entering the separating column.

6.7  As the eluate leaves the column it enters a detector, where it is continuously monitored at the specified λ.

6.8  The electrical signal obtained from detector is amplified and routes to recorder which record the developed chromatogram.

6.9  Calculate the percentage content of substance using the declared content of reference standard.

6.10    Calibration frequency:

6.10.1               Monthly and after maintenance.

7.0                 REVISION LOG:

Revision No.	Effective Date	Reason
00		New SOP

8.0                 REFERENCES:

8.1  Not Applicable.

9.0                 ANNEXURES:

Annexure 1: Observations and calculations of HPLC method.

 

Annexure: 1

Observations and calculations of HPLC method

Analysis on HPLC Instrument: ___________________                                           Date: _________________ Model: ___________________ Column size: Length= θ= Stationary phase:   Temperature:   Mobile phase:   Flow rate:   Injection size:   Detector:   Wavelength: λ=   Sample solution: _______________________ Reference standard solution: ______________ Impurities: ____________________________ (calculate each component calculation separately) OBSERVATIONS: Attach chromatogram.           CALCULATIONS: 1.   Retention time:                                                                                n= no. of peak Retention time of unretained peak (tm)= _____________ No. of peaks Retention time of peak of interest (tr)n Height of peak of interest (h)n Width of peak of interest (w)n Area of peak of interest A=1/2(h x w)                                         2.   Retention volume: Flow rate= _______________ml/min. No. of peaks Retention time of peak of interest (tr)n Retention volume = retention time x flow rate                         3.   Retention factor: Retention time of unretained peak (tm)= _____________ No. of peaks Retention time of peak of interest (tr)n Retention factor of a component k= (tr-tm)/tm                         4.   Separation factor (α): No. of peaks Retention factor of a component (kn) Relative retention of two adjacent peaks α = k2/k1                         5.   Resolution: Retention time of unretained peak (tm)= _____________ No. of peaks Retention time of peak of interest (tr)n Width of peak of interest (w)n Resolution Rs = 2 (tr2-tr1)         (w1-w2)                                 6.   Efficiency: No. of peaks or components Retention time of peak of interest (tr)n Width of peak of interest (w)n Efficiency (No. of theoretical plates) N= 16 (tr/w)2                                   7.   Height equivalent to a theoretical plate (HETP): Length of column = ________________________ No. of peaks or components No. of theoretical plates (N) Height equivalent to a theoretical plate HETP = L/N                         8.   Symmetry factor (tailing factor): No. of peaks or components Distance from the peak max. to leading edge of the peak (f) Width w Symmetry factor At 5% At 10% As = w5%        2f As = w10%        2f                                                 9.   Response factor & Relative response factor: Conc. (mg/ml)= ___________________ No. of peak Peak area Response factor = (peak area/conc.) Relative response factor = (response factor of impurity/response factor of API)                                 10.                                                                                       Relative standard deviation (%RSD): Use formula of relative standard deviation where it is required i.e., 11.                                                                                       Percentage of content: Percentage content = (rU/rS) x (CS/CU) x 100. rU= peak response of substance from the sample solution. rS= peak response of substance from the standard solution. CS= concentration of substance in the standard solution (mg/mL). CU= concentration of substance in the sample solution (mg/mL).     RESULTS: ________________________________________________________________________________________________________________________________________________