GOOD CHROMATOGRAPHIC
PRACTICES
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.
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
Contents
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.
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.
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.
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
Mobile Phase
Buffers used for HPLC
Buffer solutions must not be left in system to avoid crystalisation.
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
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
Mobile Phase
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.
Mobile Phase
Changing Over of 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
Changing Over of 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.
Mobile Phase
Degassing Degas the Mobile phase before use & use online degasser while running the HPLC.
Mobile Phase
Degassing
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.
Stationary Phase
Equilibration of Column
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
Equilibration of Column
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.
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.
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.
Solvent Management System
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
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.
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.
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.
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
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.
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
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
Routine start up
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.
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
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 System over pressure
Communication error
Failure of system suitability Peak splitting
Lost prime error
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.
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
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,
Integration
From the sample set, process the complete set.
Integration
Integrate all the injections including set failures.
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.
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