PURPOSE :
This document describes the procedure for purchase, installation, storage,
performance evaluation, usage, cleaning and withdrawl of HPLC
columns.
II.
SCOPE :
Applicable to all HPLC columns used in the Quality Control department.
III.
RESPONSIBILITY :
It is the responsibility of the Quality Control personnel involved in the usage of
HPLC columns, to follow the procedure as mentioned in this Standard Operating
Procedure.
IV.
DOCUMENT REFERENCE :
SOPs : None
Forms : HPLC Column inwards register, Column performance work book &
tag.
V.
1.0
PROCEDURE :
PURCHASE OF COLUMNS :
Responsibility : Department
Head
1.1
Indent & receive the columns from Purchase department and verify whether correct
columns are received against to Specify the column make, brand name, dimensions
and particle size.
1.2
Check the column for physical damage which may have occurred during shipping.
2.0 INSTALLATION OF COLUMNS
Responsibility : Section Head
2.1
Retain safely the column performance certificate sent by the manufacturer.
2.2
Ensure that the column ends are securely closed.
2.3
Store the column in the designated area.
2.4
Test the column immediately to verify quality and allot individual
3.0 COLUMN PERFORMANCE EVALUATION
Responsibility : Section
Head/Analyst
3.1
Prior to column performance, flush the column with suitable solvent (compatible)
based on the solvent in which the column is shipped.
3.2
Read and follow the instructions such as temperature, pH, back pressure limits
mentioned for one particular column.
3.3 Test the column for performance by the following procedure :
3.3.1 For reverse phase bonded packings (C18, C8, Cyano, Amino etc.)
3.3.1.1 Uracil stock solution : Weigh accurately about 50 mg of Uracil in a
50 ml
volumetric flask. Dissolve in 25 ml mobile phase and dilute upto the mark with
the mobile phase.
3.3.1.2 Standard solution : Weigh accurately 40 mg of phenol and 60 mg of anisole
transfer
in a 50 ml volumetric flask, containing about 20 ml of mobile phase and dissolve.
Add 2 ml of uracil stock solution and dilute up to the mark.
3.3.1.3
Chromatographic conditions :
Mobile phase : Methanol : water (70 : 30)
Flow : 1.0 ml/minute
Wavelength : 254 nm
Injection volume : 20 µl
3.3.1.4
Procedure : Inject the standard solution 5 times. Calculate the column efficiency
(theoretical plates). Symmetry factor and capacity factor for the peaks of phenol
and anisole. Use the retention time of uracil as void volume for determination of
capacity factor. Relative standard deviation of peak area for 5 replicate injections
should not be more than 2.0% for phenol and anisole.
3.3.1.5
Acceptance criteria : Capacity factor
: For phenol : Not less than 0.30
For Anisole : Not less than 1.00
Theoretical plates :
For phenol : Not less than 2000
For Anisole : Not less than 2000
USP tailing : Not more than 2.0 (each for phenol and
anisole)
3.3.2
For Adsorption/Normal Phase columns
3.3.2.1 Preparation of the standard : Weigh about 200 mg each of acetophenone ,
80 mg of 2,4-dinitrotoluene and 200 mg of toluene in a 100 ml volumetric flask.
Dissolve in 25 ml of mobile phase and dilute upto the mark with mobile phase.
Dilute 3 mL of the above to 100 mL in a volumetric flask with mobile phase.
3.3.2.2
Chromatographic conditions
Mobile phase : Hexane : Ethyl acetate (98:2)
Flow : 1.0 ml/minute
Wavelength : 254 nm
Injection volume : 20 µl
Run time
: 20 Minutes for 250mm and above 250mm length columns.
10 Minutes for below 250mm length columns.
3.3.2.3 Procedure : Inject the standard solution 5 times. Calculate the column efficiency
(theoretical plates), symmetry factor and capacity factor for the peaks of
acetophenone and 2,6-dinitrotoluene. Use the retention time of toluene as void
volume for determination of capacity factor. Relative standard deviation of peak
area for 5 replicate injections should not be more than 2.0% for acetophenone and
2,4-dinitrotoluene.
3.3.2.4 Acceptance criteria :
For Below 250 mm length columns
Capacity factor : For acetophenone : Not less than 0.15
For 2,4 dinitrotoluene : Not less than 0.30
Theoretical plates : Not less than 1000 (Each for acetophenone and 2,4
dinitrotoluene)
USP tailing : Not more than 2.0 (Each for acetophenone and 2,4 dinitrotoluene)
For 250 mm and above 250mm length columns
Capacity factor : For acetophenone : Not less than 0.40
For 2,4 dinitrotoluene : Not less than 2.0
Theoretical plates : Not less than 1000 (Each for acetophenone and 2,4
dinitrotoluene)
USP tailing : Not more than 2.0 (Each for acetophenone and 2,4 dinitrotoluene).
3.4
After completion of the performance test, attach a tag to the column indicating the
column number, date of performance check
and signature.
3.5
Update the index of column to include the new column.
3.6
If the column fails to meet the acceptance criteria, then return the column to the
supplier and arrange for replacement.
3.7
After meeting the requirements of column performance allot the ID no as given
below
3.8
QCD/LC/xxx , xxx is the serial number.
3.9
Dedicate the columns individually for finished product and stability testing of each
product (where ever possible) and label the same on the label.
4.0
USAGE OF COLUMNS
Responsibility : Analyst
4.1
Handle the column with extreme care. Never allow the column to experience
mechanical shocks or vibrations.
4.2
Select the column by referring to the HPLC column list and as indicated in the
specification.
4.3
Allow the column to stabilize on the system under the set parameters of the
instrument and the mobile phase.
4.4
given
Perform the analysis on the column as recommended in the ‘Tests and Methods’
under specification.
4.5
On completion of analysis, flush and clean the column with the relevant solvents as
recommended in the section on cleaning and care of HPLC columns.
4.6
Replace back the column to the designated place .
5.0
CLEANING OF COLUMNS
Responsibility : Analyst
5.1
Always use filtered and degassed mobile phase/ solvents.
5.2
Before change over of a mobile phase, make sure that solvents used for column
flushing
are miscible with the mobile phase.
5.3
Equilibrate the column for about 30 minutes at the flow rate as specified in the
individual specification. If ion-pairing agents or additives are used, flush the
column for about 1 hour at the specified flow rate for complete equilibration.
5.4
After completion of analysis, flush the column with the appropriate solvents (as
specified in the individual column log). If the mobile phase contains buffer, flush
the column first with sufficient quantity of water and then with an organic solvent,
used for mobile phase preparation.
5.5
For longer storage, store the column in 100% methanol and be sure that the end
plugs are firmly in place. Never let the column dry out.
5.6
Storage conditions for HPLC columns :
Column type
Reverse phase
Storage solvent
100% methanol
(C18, C8, C4, C2, C1, Phenyl)
Normal phase
(Silica, CN, NH2, Diol, Alumina)
Isopropanol or n-hexane
Ion exchange
(SAX, SCX)
Size exclusion
(Diol)
5.7
Methanol
(Flush the column with 50 ml water
prior to storage solvent)
0.05% sodium azide in water or in
10% methanol
While changing the mobile phase, gradually increase the flow rate of the new
mobile
phase in increments from 0.1 ml/minute upwards.
5.8
A shift in retention or resolution is an indication of insufficient flushing of the
column. Flush with a organic solvent which may be a part of the mobile phase of
the product for which the column is dedicated.
5.9
If buffers like citrate or ammonium carbonate are used in the mobile phase, ensure
that the HPLC columns which can withstand the conditions of analysis or the
specified HPLC columns as given in the specification are used. Other HPLC
columns may get deactivated in such conditions (condition this point while
selecting a column for particular applications).
6.0 WITH DRAWAL OF COLUMNS
6.1
Responsibility : Department Head/Section Head
Withdraw the column from usage in case of any of the following instances:
6.1.1 Physical damage
6.1.2 Failure to comply system suitability requirements as per specification .( Note: When
failure to the system suitability requirements as per specification, evaluate once
again the column performance and take the decision of withdrawl )
6.1.3
Failure to comply with performance check requirements.
6.1.4 Indicate the withdrawal on the column inwards register and update the column
index.