High Performance Liquid Chromatography (HPLC). Cleaning SOP



1.0  OBJECTIVE:
To lay down the procedure for cleaning of High Performance Liquid Chromatography (HPLC).
2.0  SCOPE:
This shall be applicable to the High Performance Liquid Chromatography (HPLC) installed in Quality Control instrument Laboratory.
3.0  RESPONSIBILITY:
3.1  Technical Assistant/ Helper.
3.2  Q.C Analyst.
4.0  ACCOUNTABILITY:
4.1  Manager-Quality Control.
5.0  MATERIAL AND EQUIPMENT:
5.1  High Performance Liquid Chromatography (HPLC).
5.2  Tissue paper.
5.3  Clean dry cloth.
5.4  Purified water.
6.0  PROCEDURE:
6.1  For system cleaning:
6.1.1        After completion of analysis, clean the HPLC as follow.
6.1.2        Transfer the used glassware for washing & keep aside test tube stands lying near the HPLC system. Clean the surrounding area with tissue paper.
6.1.3        Clean the syringe with repeated rinses of purified water.
6.1.4        Clean the outer surface of injector by flushing the purified water with syringe forcefully near the injector hole and joints of loops. Clean it with tissue paper.
6.1.5        Clean the inner surface injector and loops by injecting purified water into loop with syringe three to four times.
For normal phase system, use non-polar solvents like acetonitrile, methylene chloride, n-hexane, & tetrahydrofuran for cleaning.
6.1.6        Clean the plunger seal by injecting purified water three to four times with syringe.
6.1.7        Remove the column and replace with joints for the cleaning of tubings & flush with 6N nitric acid, purified water and then with methanol.
6.1.8        Remove the air in suction pipe by purging the line and flushing with purified water for some time.
6.2  Clean the suction filter by sonicating into nitric acid for 10 to 15 minutes and then wash it with purified water.
6.3  Replace the joints with column and wash the column first with purified water and then with methanol.
6.4  Switch “OFF” the pump, detector and integrator.
6.5  Clean the outer surface of instrument with dry clean cloth.
6.6  Remove any dirt or spot with Dettol.
6.7  Dry it with tissue paper.
6.8  Washing of HPLC column after use:
6.8.1        For C18, C8, C6, Phenyl, CN, Amine, Silica column:
6.8.1.1  Wash the column with the solvent which is use in individual product mobile phase preparation. E.g. suppose purified water is use in mobile phase preparation then wash the column with purified water.
6.8.1.2  Allow the minimum 30.0ml washing medium to flow through the column.
6.8.1.3  Allow the purified water to flow through the column at the rate of 1ml/min for 30 minutes. Check that the pressure does not exceed 400 bars.
6.8.1.4  Change the mobile phase from water to HPLC grade 80% Acetonitrile.
6.8.1.5  Set the instrument to flow rate 1 ml/min and wash the column for 30 minutes.
6.8.1.6  Ensure that the column contains 80% Acetonitrile.
6.8.1.7  Disconnect the column from the HPLC unit and store in an appropriate place.
6.8.2        For protein pack column:
6.8.2.1  Wash the column with minimum 30.0ml of 2.5M acetic acid or flush 1:10 solution of isopropyl alcohol & purified water.
6.8.2.2  Allow mobile phase to flow through the column at the rate of 1 ml/min for 30 minutes. Check that pressure does not exceed 400 bars.
6.8.2.3  Ensure that the column contains mobile phase.
6.8.2.4  Disconnect the column from the HPLC unit and store in an appropriate place.
6.8.3        For L17 column:
6.8.3.1  Change the mobile phase to a mixture of 0.01N H2SO4: ACN (85:15 v/v) and flush at flow rate of 0.6 ml/min minimum for 2 hrs.
6.8.3.2  Take care that flow rate should not exceed 0.6 ml/min.
6.8.3.3  Change the mobile phase from (H2SO4: ACN) to purified water. Flush the column with purified water at flow rate of 0.6 ml/min, for 2 hours.
6.8.3.4  Ensure that the column contains purified water.
6.8.3.5  Disconnect the column from the H.P.L.C unit and store in an allocated place.
6.8.3.6  Affix the “CLEANED” label and fill the “Cleaning Equipment Log Book”.
6.9  Frequency:
6.9.1        General cleaning must be daily.
6.9.2        Syringe, loop and injector plunger seal cleaning must be after completion of each analysis.
6.9.3        Flow cell, pipeline, suction filter cleaning when problem arise.
6.9.4        Keep the record of cleaning.
7.0  REVISION LOG:
Revision No.
Effective Date
Reason
00

New SOP
8.0  REFERENCES:
8.1  Not Applicable.
9.0  ANNEXURES:
Annexure 1: Cleaning record of High Performance Liquid Chromatography (HPLC) (Cleaning Equipment Log Book).

Annexure: 1
(Cleaning Equipment Log Book)
Cleaning record of High Performance Liquid Chromatography (HPLC).
CLEANING RECORD OF UV/VIS SPECTROPHOTOMETER
Date
Cleaned by
Checked by
Remarks 





















































































10.0    ABBREVIATIONS:
Abbreviation
Expanded Form
SOP
Standard operating procedure
&
And
No.
Number  
Ltd.
Limited
Q.C
Quality control
QCEC
Quality control equipment cleaning
HPLC
High Performance Liquid Chromatography
N
Normal
Min
Minutes
ml/min
Milliliter per minute
Hrs.
Hours
ml
Milliliter
%
Percentage
CN
Cyanide
M
Molar
v/v
Volume by volume


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