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

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

Related: Relative Response Factor Calculation in HPLC

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%)

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