SOP for Linearity Check of High Performance Liquid Chromatography (HPLC)
Standard operating procedure to check performance of the high performance liquid chromatograph (HPLC) for accurate results.

 1.0 Objective
To check the performance of the equipment for reliable and accurate results.

2.0 Scope
This procedure is applicable to check the performance of                                                                             the high-performance liquid chromatography.

3.0 Responsibility
3.1 Doing: Technical Assistant/Executive
3.2 Checking: Executive /Manager


4.0 Accountability
Head of the Department

5.0 Procedure
5.1 Operate the instrument as per the respective standard operating procedure of HPLC.
5.4 Instrument Performance ( Linearity of Results)
      Frequency: Two months.
5.4.1 Prepare the mobile phase consisting of 70% Methanol: 30 % water.
5.4.1 Stock solution preparation: Prepare a mixture of 0.2 % Benzene and 0.2 % Toluene in methanol.
5.4.2 Make dilution in the following manner.
5.4.1 Take 5 ml, 10 ml, 15 ml, 20 ml of stock solution respectively in 25 ml of volumetric solution                and makeup with methanol.
5.4.3 Set flow rate at 1.0 ml/min.
5.4.4 Allow the system to be saturated with mobile phase for at least 15 minutes before injecting the           different solutions.
5.4.5 Record the area and retention time of both benzene and toluene as per Annexure - I.
5.4.6 Calculate the Linearity for both Benzene and Toluene respectively.

6.0 ABBREVIATION
6.1 SOP - Standard Operating Procedure

ANNEXURE-I
Q. C. Department
LINEARITY CHECK REPORT OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPH

PERFORMANCE DATE
DATE OF LAST PERFORMANCE DONE
NEXT DUE FOR PERFORMANCE



INSTRUMENT DETAILS
INSTRUMENT NAME
INSTRUMENT MAKE
INSTRUMENT IDENTIFICATION NO.



OBSERVATION    
1) Instrument performance ( Linearity of results)
Stock solution preparation :
Mixture of 0.2 % Benzene and 0.2 % Toluene in Methanol.
Solution 1) : 5.0 ml of Stock solution                   25 ml with methanol.
Solution 2) : 10.0 ml of Stock solution                 25 ml with methanol.
Solution 3) : 15.0 ml of Stock solution                 25 ml with methanol.
Solution 4) : 20.0 ml of Stock solution                 25 ml with methanol.
Solution 5) : Direct stock solution.

HPLC Parameter :
Column                  : ODS (25 cm X 4.6 mm, 5 μ)
Mobile phase         : Methanol : Water
                                            70 : 30
Flow rate                : 1.0ml/min.
Wave length           : At 254 nm
Solution No.
Conc.
Inj. No.
Bin No.
Area of Benzene
Area of Toluene
1
0.04%
1





2



2
0.08%
1





2



3
0.12%
1





2



4
0.16%
1





2



5
0.20%
1





2



Linearity : (Limit NLT 99.95%)