Good chromatographic practices

GOOD CHROMATOGRAPHIC
PRACTICES

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Introduction
Chromatography is a physical method of separation in which the
components to be separated are distributed between two
phases
one of which is stationary (stationary phase) while the other
(the mobile phase) moves through it in a definite direction.
The chromatographic process occurs due to differences in the
distribution constant of the individual sample components.


Contents
∗ Mobile phase
∗ Stationary Phase
∗ Solvent Management system
∗ Sample Management system
∗ Routine startup
∗ Pre analysis checks
∗ Sample set sequence
∗ Bracketing standards
∗ System failures & Incidents
∗ Integration & Re-integration
∗ Processing
∗ Empower
∗ Reporting
∗ Post analysis check

Mobile Phase
Water used for HPLC
∗ Always use Purified water / Milli Q water as both inorganic
and organic contaminants are removed.
∗ Do not use RO water / de-ionsed water for HPLC.
∗ Due to existence of impurity, deionized water shows higher
absorption.


Mobile Phase
Water used for HPLC
∗ Ghost peak will be appeared in gradient elution if water is of
bad quality!


Mobile Phase
Solvents used for HPLC
∗ Always use HPLC/ Chromatographic grade solvents.
∗ Do not use analytical grade solvents.


Mobile Phase
Solvents used for HPLC –
Precautions for using THF
∗ Analytical grade THF contains butylated hydroxytoluene
(BHT) as an oxidation inhibitor. As BHT has strong UV
absorption, it is not added for HPLC grade THF.
∗ As such, HPLC grade THF must be stored in cool and dark
places to reduce oxidation to form peroxides. Accumulation
of large amount of organic peroxides may lead to
explosions.


Mobile Phase
Solvents used for HPLC –
Precautions for using Chloroform
∗ Analytical grade chloroform contains 3% ethanol to prevent
generation of phosgene, a highly toxic gas.
However, ethanol, like water, may deactivate silica gel used
in normal phase separation and thus lead to unstable
retention times.
∗ HPLC Grade chloroform therefore does not contain ethanol
as a stabiliser.
∗ Once seal is opened, it should be stored in dark and cool
places to reduce the possibility of generation of phosgene.


Mobile Phase
Buffers used for HPLC
∗ All the solutions should be clear, homogenous & free from
particulate matter.
∗ Buffer solutions must be filtered through 0.45µm filter or as
specified in the method by using Solvent Filtration Kit .


Mobile Phase
∗ Do not keep mobile phase under the suction for more than 4
to 5 minutes as the composition of mobile phase change
especially if highly volatile solvents are used.


Mobile Phase
Buffers used for HPLC
∗ Buffer solutions must not be left in system to avoid
crystalisation.
∗
∗
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effect on pump -- damage plunger and seal
effect on column -- creation of column voids
effect on flow line -- corrosion of stainless steel lines

∗ Possible bacterial growth especially phosphate buffers -good medium for bacterial and fungus growth.
∗ Ideally, solutions should be prepared fresh everyday.


Mobile Phase
Premixing of solvents
∗ For isocratic systems, different solvents are premixed. They
are to be measured separately for accuracy in measuring.
E.g. To prepare 1000 mL of 50:50 mixture of
water/methanol,
Measure 500 mL of water, transfer into a bottle
and 500 mL of methanol, transfer into same bottle
then mix them together & sonicate for about 15 minutes.


Mobile Phase
Material of the container
∗ Use glass bottle
∗ Use Plastic bottle for high sensitivity inorganic analysis.
Always cover the container
∗ To prevent evaporation of solvents
∗ To prevent dust enter the mobile phase
∗ To reduce the vapors in the room
Do not cover the mobile phase bottle with parafilm. The
leachable from the parafilm may contaminate the analysis.


Mobile Phase
∗ Do not expose the bottle to direct sunlight/wind.
∗ Always keep reservoir above solvent delivery system
∗ Always use a suction filter
∗ Prevent particulates from reaching pumps
∗ Clean the suction filter by sonicating in isopropanol or 1N nitric
acid and water.


Mobile Phase

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Label all the mobile phase and Solvents bottles.
Write all the details on the label clearly.
Use the mobile phase before expiry period.
Establish the validity of the Mobile Phase.


Mobile Phase
∗ Changing Over of Mobile Phase.

Do not keep the channel tubing directly in another
mobile phase.


Mobile Phase
Do not keep the channel tubing directly
from buffer solution to aqueous organic
solvent.

Do not keep the channel tubing
directly from aqueous organic solvent
to Non aqueous organic solvent.


Mobile Phase
∗ Before changing to totally organic phase in the system, the
whole system should be flushed with water/organic (1:1)
mixture to remove the buffers used.
∗ Otherwise, crystallisation may occur when the organics
come into contact with leftover buffers in system.


Mobile Phase
Degassing
∗ Degas the Mobile phase before use & use online degasser
while running the HPLC.


Mobile Phase
Degassing
∗ Do not use Online degasser while using THF in the Mobile
phase as it may degrade the Teflon tube which is connected
to vacuum chamber.


Stationary Phase
Precautions to be followed
∗ Avoid pressure shocks on the column.
∗ Pressure shocks lead to channeling in the column, which
results in peak splitting in corresponding chromatogram.
∗ Always keep both ends of the column closed, after usage.
∗ Keep the columns in the designated column cabinets after
use.
∗ Use the pH range of 2 to 8 or follow the manufacturers
instructions.


Stationary Phase
Equilibration of Column
∗ The amount of mobile phase which should be flushed
through a column before it is ready to use is usually
expressed in terms of the column volume i.e. the amount of
mobile phase required to fill the column.
∗ When a column has just installed on a reverse phase HPLC
system then it will typically require 10 to 20 column volumes.
(with few exceptions like methods which include on pairing
agents & chiral methods).


Stationary Phase
∗ This column volume is more correctly called as void volume
(Vm).
∗ This is the volume of the HPLC column that s not taken up
by the stationary phase. This is typically approximately 70%
of the total column volume.
∗ Methods to calculate Vm
∗ Volume of a cylinder V = π r2 L
∗ By injecting an unretained solute to obtain t0
Void volume, Vm = F x t0
Where F is flow rate expressed n mL/min


Stationary Phase


Stationary Phase
Storage of HPLC Columns

∗

∗

Do not store HPLC columns in buffers. A buffer may precipitate inside
the column, resulting in plugged hits and packing material.
A buffer may also encourage bacterial growth, which can plug both the
column frit and packing material. This is more likely to occur with
mobile phases which have close to or 100% buffer.
Bacteria may also affect your analytes, and organic products from the
dead bacteria may cause "ghost peaks" in chromatograms.
Do not store HPLC columns in solvents that degrade easily
tetrahydrofuran (THF), triethylamine (TEA), trifluoroacetic acid (TFA).
Unstabilized THF can form peroxides which may degrade the column.
TEA and TFA are more likely to become contaminated from the lab
environment and should be refrigerated during storage. Contamination
may change the chromatography or change the column.


Stationary Phase
Storage of HPLC Columns
∗ For short term storage, i.e. over night, columns can be stored in
the eluent used in last analysis.
∗ For middle term storage, i.e. 2 days or over the weekend, columns
should be flushed with pure water to prevent microbial growth.
∗ For long term storage, silica based columns should be stored in an
aprotic solvent.
∗ The water content should not be higher than 50%. The best
storing solvent is Acetonitrile.
∗ Make sure that all buffers are washed out of the column before
flushing with Acetonitrile. Buffer salts are mainly not soluble in
Acetonitrile and can block the capillaries and the column.

Log book
∗ Record the column usage in Column usage log books /
sheets of respective column.
∗ Make entry of relevant details in HPLC log book before using
the system.


Solvent Management System


∗ To prevent damage to the Separations Module, be sure to use
needle wash and plunger seal wash solutions that are miscible
with the mobile phase you are using.
∗ Use separate solutions and containers for plunger seal wash
and needle wash. Because the functions of these solutions
differ, the use of one solution for both functions may
compromise the effectiveness of either needle washing or
plunger seal washing.


Plunger Seal Wash
∗ Seal wash is used to rinse the backs of the piston seals in the
pump of a HPLC system.
∗ The piston is the part of the pump which moves back and
forward in the pump head drawing in and pushing out the
mobile phase solvent with each movement. Because it is a
moving part, a seal around this piston is required to prevent
the liquid leaking out the back of the pump.


Plunger Seal Wash
∗ Over a period of time small amounts of mobile phase
solvents seep through the seal to the back of the pump
head.
∗ If these solvents contain buffers then the salts may
precipitate out forming deposits which can shorten the life
of the seal.
∗ Seal wash is used when the mobile phase contains buffers.
The wash flushes the back of the piston seals removing any
deposits and maximising the lifetime of the seal.


Seal wash - Composition
∗ It follows that the composition of the seal wash should be
aqueous to dissolve buffers.
∗ A small amount of organic solvent is added to prevent
bacteria growth and also to reduce the surface tension of
the water (this helps the wash solvent cling to surfaces).
∗ Typical seal wash composition is 80% water and 20% organic
solvent.
∗ The organic solvent may be methanol, acetonitrile or
isopropyl alcohol (IPA)


Prime the solvent management system by:
∗ Dry prime option when the solvent lines are dry
Opens the fluidic path (from the selected solvent reservoir
to the prime/vent valve) to replace air with solvent, then
performs a prime.
∗ Wet prime option when you want to change between
miscible solvents.


Sample Management System


Sample Management System
Purge the sample management system whenever
∗ Prime the solvent management system
∗ Change solvents
∗ See bubbles in the syringe
∗ Start using the Separations Module at the beginning of each day.
To purge the sample management system:
∗ In the Main screen, press Menu/Status to display the Status screen.
∗ Enter the appropriate solvent composition in the Composition
fields.
∗ Press Direct Functions to display the Direct Functions menu.
∗ Select Purge Injector, then press Enter.

Needle wash
∗ The needle-wash pump flushes the needle in the sample
management system, preventing carryover of sample between
injections.
∗ The needle-wash also extends the life of the injector seals by
removing buffered mobile phase and sample from the needle.
∗ The needle in the HPLC system is used to introduce the sample into
the mobile phase so that it can be separated on the HPLC column.
∗ The needle wash is used to clean the needle after an injection.
∗ Use “Extended needle wash” from the instrument method if the
carry over is expected, especially during the analysis of Related
substances.

Needle wash
∗ The composition of the needle wash needs to be matched
to the sample since this is what you want to clean off the
needle.
∗ Typically a composition of needle wash which matches the
proportions of aqueous and organic solvents in the mobile
phase will be appropriate.


Adjust seal pack
Adjust the seal pack whenever:
∗ Start up the Separations Module for the first time. The
Separations Module does not perform injections or compression
checks until the seal pack has been adjusted.
∗ A “Compression Check Failed” error appears on the screen.
∗ A “Missing Restrictor” alarm appears during a diagnostic
procedure.
∗ Change the seal pack.
∗ Rebuild the seal pack.
Before you adjust the seal pack, purge the sample management
system to ensure that there is no air in the syringe. Air in the
system may cause Alarm Seal Geometry or Alarm Missing
Restrictor dialog boxes to (erroneously) appear.

Sample Treatment
∗ Always filter sample with 0.2μm or 0.45μm filters before
injection.
∗ If the sample contains a lot of solid, centrifuge it, and the
filter the solution.


Sample Treatment
∗ Check that the filter type you are using is compatible with
the sample solvent.
∗ Extractable components from the filter may show up as
ghost peaks in a run. Incompatible filter types, which may
partially dissolve in your solvent, introduce material into a
column, which can cause plugging.
∗ Use the filters which are mentioned in the validated method


Injector Maintenance
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∗
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∗

Purge flow line at least once a day.
Change purge liquid frequently.
Never use salt solutions as purge liquid.
Use septums for sample vial recommended by manufacturer
only.
∗ Septums should not be reused.
∗ Do not fill vials to the brim.


Waste Bottle Maintenance
∗ Remember to empty waste bottles regularly.
∗ Do not mix concentrated nitric acid with organic solvents.
∗ Do not immerse the drain tubing in the waste liquid as this
may create back pressure on the flow cell.


Pre-analysis checks
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Filter mobile phase
Sonicate the Mobile phase.
Wait for mobile phase to reach room temperature
Purge all the flow lines with mobile phase
Replace mobile phases as necessary
If the solvent tubing is not completely filled with mobile
phase, purge the flow line
∗ After periods of disuse or changes in ambient temperature,
purge the flow line


Routine start up
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Dry prime
Place the solvent reservoirs
Switch-on the degas
Wet prime
Purge injector
Equilibrating the system
Conditioning column
Check for leaks
Check the pump pressure
Perform a baseline check


Empower
∗ Use your own log in ID for any software.
∗ Don’t share your password to any body.


Sample set
∗ Prepare all the solutions viz. blank, system suitability,
working standard & Sample solutions as per the method.
∗ Equilibriate the column before sample set.
∗ Inject the Bracketing standard to ensure the system
condition during the sample run.
∗ Define the no. of samples to be injected for bracketing and
calculation for bracketing standard in the SOP.


Sample set
∗ For linearity include the amounts during the preparation of sample
set method


System failures
∗ If HPLC / GC system fails during analysis due to
∗
∗
∗
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System over pressure
Communication error
Failure of system suitability
Peak splitting
Lost prime error
Bracketing standard failure


System failures
∗ Define the procedure to record Lab incidents.
∗ Record the incident as per defined procedure.
∗ Process all the injections including the disregarded sets
and enclose with the Raw data.
∗ Investigate the root cause for the sample set failure.
∗ Rectify the problem, document the reason for failure.
∗ Repeat complete sample set of injections in case of 1
injection failure of Assay / dissolution / UOD.


Definitions
∗ A chromatogram is a series of detector responses, sampled
uniformly across a length of time. The elution of a compound
results in a characteristic chromatographic peak profile.
∗ Integration is the process of calculating an area that is
bounded in part or in whole by a curved line. The goal of
chromatographic peak integration is to obtain retention
times, heights, and areas of these peaks.
∗ Peak integration uses two key algorithms: one that detects
peaks and one that determines their baselines. Once the peak
apex and baseline are known, the retention time (RT), height,
and area can be calculated.

Processing
∗ Processing is the manipulation of data to determine the
identities and/or amounts of separated components. It most
often involves integrating chromatographic peaks to calibrate
standards and generate a calibration curve, and to quantitate
the source components.
∗ Processing methods define how Empower detects,
integrates, calibrates, and quantitates unprocessed, raw data
from a 2D channel or a 2D-derived channel.
∗ In Review, you start with unprocessed data acquired from a
known standard (Channels). You then create a multipoint
calibration curve by using a range of standard concentrations.
Adding a processing method to a method set allows the
software to process raw data while it is being acquired.

Integration
∗ Do not integrate any peak manually.
∗ Integrate all the sample sets batch wise.
∗ Always use same processing method for processing of
blank, standard & sample chromatograms in case of Assay
& dissolution tests.
∗ Verify the processing parameters like
∗
∗
∗
∗

threshold,
width,
system suitability,
Peak names etc.

∗ Save the processing method

Integration
∗ From the sample set, process the complete set.


Integration
∗ Integrate all the injections including set failures.
∗ For System suitability injection for which Tailing factor,
Resolution or Theoretical plates are to be checked,
consider only the first injection of the sequence. (Do not
consider this criteria for average of all the standard
injections unless otherwise specified.)


Re-integration
∗ Do not re-integrate the chromatograms without
documenting.
∗ Document reason for reintegration.


Post analysis check
To remove buffered mobile phase from the fluidic path of the
Separations Module:
∗ Replace the buffered mobile phase with Milli-Q water and wet
prime the system for 10 minutes at 3 mL/min.
∗ Flush the system with Milli Q water & solvent after use to prevent
salt deposition & precipitation of crystals & microbial growth.
∗ Perform three injector purge cycles to ensure that the sample
loop is clean.
∗ Replace the plunger seal-wash solvent with a fresh solution of
80% water: 20% methanol or a miscible solvent, and prime the
plunger seal-wash pump.
∗ Replace the needle-wash solvent with a solution of 90% methanol:
10% water or a miscible solvent, and perform a needle-wash
prime.

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Good chromatographic practices

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