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Good Chromatography Practices - SOP & Guideline - Pharma Beginners
Good Chromatography Practices - SOP & Guideline - Pharma Beginners
Good Chromatography Practices - SOP & Guideline - Pharma Beginners
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H O M E PA G E # HPLC
HPLC
Increase/ decrease the mobile phase flow rate stepwise and slowly.
Ensure that the date and time of data acquisition appear on each
chromatogram and the chromatograms are compiled in succession.
While going through change over from reverse phase to normal phase
and normal phase to reverse phase follow the changeover steps.
Never mix the aqueous phase and organic phase portion in measuring
cylinder as it can give erroneous composition.
Column shall be washed pre and post running of the sample set.
If the mobile phase contains a buffer solution, first calculate the quantity
of the mobile phase required for complete analysis (including quantity
:
of the mobile phase required for complete analysis (including quantity
required for dilution) and prepare the buffer solution as per the
procedure.
Set the pH (If required) and do not use concentrated acid/alkali directly
for pH adjustment,
The filter also can be done at the time of the final mobile phase
composition, if applicable.
Add to the stoppered bottle containing a buffer solution and mix well.
It may alter the composition while sonication ensures that the bottle cap
is loosened to avoid the pressure built up.
If the mobile phase is a normal phase then rinse the filtration assembly
and collection vessel with the water-miscible component of the mobile
phase before filtration followed by the mobile phase.
Physically check the column intactness and then Connect the column
on HPLC.
Saturate the column with the mobile phase for about 30 minutes or
more until the baseline gets stabilized.
:
more until the baseline gets stabilized.
Vial No.
Injection volume.
Before starting the sequence, the reviewer or section head shall ensure
that the sequence (sample set) parameter and respective instrument
method parameters are as per STP/GTP.
There shall not be any trial injection run to check the suitability of the
system (except System Suitability run defined in respective STP/GTP).
Peak shape,
:
Peak shape,
Retention time,
Resolution,
Asymmetry,
After satisfactory system suitability run (if applicable) inject blank (i.e.
diluents, mobile phase, etc.) standard solution, check the system
suitability parameters, and if it meets then start sequence.
Make entry of previously used column in the column usage log with
the reason of discontinuation and keep for washing.
Make an entry in the instrument usage logbook with the reason for
discontinuation of the mobile phase.
System suitability check is a must for every new mobile phase and
column.
Directions Call
Each of the items in the following list is the maximum variation that can
be considered unless otherwise directed in the ATP.
The following are the general criteria, which provide the extent of
:
The following are the general criteria, which provide the extent of
allowable variation to get the system suitability.
The pH of the aqueous buffer used in the mobile phase preparation can
be adjusted to within ±2 pH units.
(Ex.: If the specified concentration is 1.0% then the allowable limit for
adjustment is 0.90 % – 1.10 %).
Flow rate: When column dimensions have been modified, the flow rate
can be adjusted using
Where,
F1: Flow indicated in the monograph in ml/min,
F2: Adjusted flow rate, in ml/min,
L1: Length of column indicated in the monograph,
L2: Length of column used,
d: Column inner diameter of the column indicated in the monograph
D: Internal diameter of the column used
Additionally, the flow rate can be adjusted ± 50 %.
Note: If the column temperature and sample temperature are not mentioned
in the ATP/STP, or it is mentioned to keep it “Ambient”, then in both cases
the column temperature shall be set to 25°C and sample temperature to
15°C.
:
15°C.
Directions Call
Detector wavelength:
Injection volume:
Therefore, the mobile phase ratio may be adjusted only within the range
of 40:60 to 60:40.
Therefore the second component may be adjusted only within the range
of 25% to 45% absolute.
The duplicate standard shall be applied for the Assay test (irrespective
of sample category).
The first standard (Initial standard) shall be injected as per the above
schedules, while the second standard shall be injected in duplicate.
log the Lab Incident/Event as per the current version of SOP – Lab
Incident, investigate and repeat the analysis by preparing the standard
and establish the similarity factor prior to sample injection.
:
and establish the similarity factor prior to sample injection.
After every defined sample injections or after every test (Club analysis
or individual analysis) standard preparation in single shall be injected
(Called as Bracketing standard preparation).
After testing the sample (A) preparations (6th and 7th ) and one
bracketing standard preparation injection (8th ) shall be injected.
:
Then further sample (B) preparation injections ( 9th and 10th ) followed
by bracketing standard (11th ).
Standard Avg. Area = Avg. Area of std (2nd, 3rd, 4th 5thand 8th).
Standard Avg. Area = Avg. Area of std (3rd,4th,5th, 8th, and 11th ).
The analyst shall log the Lab Incident (as per the current version of
SOP for Lab Incident) and investigate the reason for failure, adopt the
strategy as follows.
Note1: Chromatograms shall be processed within one working day from the
completion of the sequence. If exceeds, Log the Lab incident, Investigate /
Justify and then process the acquired data.
:
Note 2: No single chromatogram shall remain unprocessed irrespective of
Blank, Placebo, System check, standard, sample, etc.
Analysts shall ensure the peak shape and system suitability parameters
for all chromatograms in a sequence (i.e.Up to the last chromatogram)
before the set up of integration parameters.
Set the integration parameters like width, threshold, peak area, peak
height, scale are selected appropriately for proper peak marking and
detection and inhibit all others peaks except the principal peak in all
tests except for related substance test and degradation product,
Chromatography purity (but not limited to).
Blank-Diluent,
Placebo,
Principal peak,
Unknown impurity,
The custom report shall cover the following information but not limited,
which is for information and can be modified as per the specific need.
Test performed
Data Path
Injection volume
Vial no.
Column number / ID
WaveLength.
Date of acquisition
Date processed
:
Acquired by /Analyst
Instrument ID
Instrument method ID
Processing method ID
The peak table in the custom report shall cover the following data (But
not Limited to), however other data as per requirement.
Peak Name
Retention time
Area / Height
Area% / Height%
Integration type
The integration parameters shall set in such a way that the peak of at
least half of the disregard limit must be integrated, and shall be
documented in the Chromatogram checklist.
:
The scale of chromatograms shall be set properly so that the peak
shape of all interesting peaks and their integration can be seen clearly.
Where,
Where,
Where impurity standard is not injected and LOQ and RF are mentioned
in GTP/Protocol.
L x A x RF
C
Where,
Put the “ √ ” mark against the applicable point and ‘NA’ against the not
applicable points.
Put all chromatograms together and attach them with the relevant Batch
No./ A.R.No. Document.
Take the print out of all generated chromatograms put the canceled on
each chromatogram with proper justification and attaches with the
relevant document.
Unprocessed chromatograms,
Single injections,
Which are not part of the original sequences on a weekly basis and
Document the review observations as per the current version of SOP for
Analytical Data Review.
Any of the above events are observed, log the lab incident as per the
current version of the SOP-Lab Incident.
In the end of the sample sequence a different method for “D2 lamp off
and flow rate change to 0.2ml/min (or suitable)” shall be submitted to
keep the system stabilize in the mobile phase.
To prepare the specimen chromatogram and ensure its usage the below
procedure to be followed.
Peak shape
Retention time
:
Baseline (Baseline pattern is very important particularly in HPLC
gradient analysis)
Scale of chromatogram
Additional peak
Blank (diluent),
Resolution solution,
Standard solution,
Placebo solution,
File all the “ Specimen chromatograms” product and test wise for the
comparison of all future analyses.
During raw data review, the reviewer shall verify the chromatograms
against the “specimen chromatogram”, in case of any abnormality
observed.
Cancellation of Chromatograms :
After proper washing of the previously used column remove the column
from the column compartment and connect the Union.
:
from the column compartment and connect the Union.
Take 100 % HPLC water in Mobile phase reservoirs, and execute the
dry prime 5 minutes individually for each channel (line) at flow rate
5ml/minutes during the dry prime outlet valve shall be opened.
Execute the wet prime 3 minutes individually for each channel (line) at
flow rate 5ml/minutes during the dry prime outlet valve shall be opened.
Step 5:
Flush the HPLC system for 20 minutes by gradually increase the flow
1ml/minute to 5ml/minute, 25% flow from each channel / Pump (line).
Step 6 :
:
Repeat wet prime, needle wash, seal wash, and injector wash as per
procedure mentioned above in steps 2, 3, and 4 by using 100% HPLC
grade methanol.
Step 7 :
Repeat wet prime, needle wash, seal wash, and injector wash as per
procedure mentioned above in step 2, 3, and 4 by using 100 %HPLC
grade IPA.
Step 8 :
Use 100% IPA or other non-polar solvents as per GTP or template for
seal wash or needle wash during the analysis.
After proper washing of the previously used column remove the column
from the column compartment and connect the Union.
Keep all tubing reservoirs in the bottle containing HPLC water /solvent
(degassed) and proceed following steps.
Execute the wet prime 3 minutes individually for each channel (line) at
flow rate 5ml/minutes during the dry prime outlet valve shall be opened.
Step 5:
Flush the HPLC system for 20 minutes by gradually increase the flow
1ml/minute to 5ml/minute, 25% flow from each channel (line).
Step 6:
Repeat wet prime, needle wash, seal wash, and injector wash as per
procedure mentioned above in step 2, 3, and 4 by using 100% HPLC
grade methanol.
Step 7:
Repeat wet prime, needle wash, seal wash, and injector wash as per
procedure mentioned above in step 2, 3, and 4 by using 100 %HPLC
grade water.
:
grade water.
Step 8:
Flush the HPLC system for 20 minutes by gradually increase the flow
rate 1 ml/min to 5 ml/minutes, 25% flow from each channel (line), (In
case of the Quaternary pump).
Product / Material :
…………………………………………………………………………………………
B. No. / A.R. No. : 1. ………………….. 2. ………………..3. …………………. 4.
…………………
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Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an
ultimate pharmaceutical blogging platform. Email: pharmabeginers@gmail.com
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