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.
6.8.2.4
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
|
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
|