SOP for Linearity Check of High Performance Liquid Chromatograph (HPLC)
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
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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
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6.1 SOP - Standard Operating Procedure
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.
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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
