Here you can read Pharmaceuticals Standard Operating Procedures (SOP), Guidelines,
Method of Analysis, Test Procedures, Instruments Calibrations and Validation.
HPLC Problems and Solutions | HPLC Troubleshooting Guide | Common error occurs during HPLC Analysis |
Topic covers below points:
Pre-Analysis Checks
Before “RUN”
Pre-Stabilization Checks
After “RUN”
Post-Analysis Checks
After Analysis
Mobile Phase – Solvent Grade
Mobile Phase – Use of buffer solutions
Mobile Phase – Premixing of Solvents
Waste Bottle Maintenance
COMMON PROBLEMS IN HPLC
Pump
Injector
Detector
Problems in Retention Time
Decreasing Retention Time:
Increasing Retention Time:
Treatment of Column
Useful Tips
Pre-Analysis Checks
Before “RUN”
Filter the Mobile Phase & Sample
Always filter the mobile phase & Sample with 0.45μ & 0.2 μ membrane filter respectively
Wait the mobile phase to reach room temperature
Because the temperature of recently mixed solution can vary greatly from the ambient temperature, verify that the mobile phase is at room temperature before analysis. Until the mobile phase reaches room temperature, there may be considerable baseline instability, especially drift. Bubble may also form in flow lines
Purge the flow lines
Ensure the flow lines are free from bubbles.
Bubble in flow lines may result in inaccurate concentrations and instability, causing changes in retention time and poor reproducibility
Replace mobile phase as necessary
Replacing mobile phase with a miscible solvent.
Replacing mobile phase with an immiscible solvent.
Replacing mobile phase if either old or new solvent is a buffer solution.
Replacing mobile phase with a miscible solvent
Pour some of the new mobile phase into a clean beaker.
Remove the solvent reservoir bottle, insert it into the beaker and rinse the filter well by shaking.
Purge the connected flow lines.
Insert the solvent reservoir filter into the new mobile phase reservoir bottle and rinse the flow lines.
Connect the column and rinse the flow lines.
Replacing mobile phase with an immiscible solvent
First replace the old mobile phase with an intermediate solution that is miscible to both the old and new mobile phases. Then replace the intermediate solution with the new mobile phase.
Follow the procedure for replacing the mobile phase with a miscible solvent for both the intermediate solution and new mobile phase.
Replacing mobile phase if either old or new solvent is a buffer solution
First replace the old mobile phase with distilled water as an intermediate solution. Then replace the water with the new mobile phase. This prevents the buffer solution salts from precipitating.
If this procedure is not followed, organic solvents, such as isopropyl alcohol, may precipitate buffer crystals or other residual material and cause the flow lines to clog.
Verify that the seal rinse bottle is prepared for use
If the mobile phase contains buffer solution or phosphoric acid, ensure that the rinse bottle has been filled so that the seal will be rinsed. If the mobile phase is distilled Water or contains an immiscible solvent, the rinse bottle should be empty.
Use distilled water to fill the rinse bottle.
Replace the water every two or three days to prevent bacterial growth
Verify that the autosampler rinse bottle is full
Use mobile phase as the rinse solution. If the mobile phase contains a salt, use the equivalent solution without the salt.
The rinse solution mixes with the mobile phase in the high pressure valve; this will ensure that no crystals form.
Purge the auto sampler measuring pump
If there is a large change in room temperature between uses, or if the instrument remains idle for several days, bubbles may form in the pump head, resulting in inconsistent injection volume. Purge the measuring pump to eliminate bubbles
Pre-Stabilization Checks
After “RUN”
Check for leaks
After the instrument starts running an analysis, verify that there are no leaks in the flow lines.
If a leak is discovered, either tighten the connection or replace the damaged part.
Check the pump pressure
The pump pressure varies with the entered pressure parameter and type of mobile phase used
Perform a baseline check
Verify that the baseline is stable.
Rinse the seal
If the mobile phase contains buffer solution or phosphoric acid, rinse the seal. Otherwise, salt or phosphoric acid may precipitate out at the rear seal surface where it can scratch the seal and plunger, shortening the life of these parts.
Rinse the seal with distilled water.
Note: Delivering Rinse Solution
If the rinse solution is not delivered when Pump is pressed, insert the injector syringe as shown in the figure, and aspirate the rinse solution into the syringe.
Post-Analysis Checks
After Analysis
Rinse the column
If the instrument will remain idle for some time, rinse the column with storage solution and detach it from the instrument.
The cleaning and storage procedure varies by column, so refer to the documentation provided with the column or contact the manufacturer.
Clean the flow line
If a buffer solution is used, rinse the contact areas with water.
If the instrument is to remain idle for some time, rinse the column, and detach it from the instrument. Connect the inlet and outlet column ports with a coupling, then rinse the flow lines. To prevent the growth of mold or bacteria, rinse the flow lines with an alcohol, such as isopropyl alcohol or methanol, and leave the alcohol in the system.
Also disconnect and rinse the solvent reservoir filter and UV-VIS detector outlet tubing. Store them in a beaker of rinse solution to prevent them from drying out.
Mobile Phase – Solvent Grade
If the solvent has background absorption,
linear range will be smaller, noise will be larger
baseline stability especially during gradient runs will be poor.
Water grade
Pure water
Distilled water
Deionized water
Due to existence of impurity, deionized water shows higher absorption.
The use of pure water systems (such as those supplied by Millipore or Barnstead) is ideal as both inorganic and organic contaminants are removed.
Modern pure water systems give TOC values as indication of total organic content in water.
Ghost peak will appear in gradient elution if water is of bad quality
Difference of analytical and HPLC grade solvent
Mobile Phase – Use of buffer solutions
Buffer solutions must be filtered before using.
Buffer solutions must be filtered before use.
Buffer solutions must not be left in system to avoid crystallisation.
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.
E.g. To prepare 1000ml of 50:50 mixture of water/methanol,
Measure out 500ml of water and 500ml of methanol separately, and then mix them together.
Measure 500ml of water, and top up to 1000ml mark of measuring cylinder/volumetric flask with methanol.
Measure 500ml of methanol and top up to 1000ml mark of measuring cylinder/volumetric flask with water.
All three methods will give different compositions. Whichever method selected must be used consistently for repeatable results.
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.
COMMON PROBLEMS IN HPLC
Pump
Leaks:
POSSIBLE CAUSE
SOLUTION
Leakage from the pump head.
Replace the pump seal; Check the piston for scratches and if necessary replace.
Decreasing Pressure:
POSSIBLE CAUSE
SOLUTION
Insufficient flow from pump
Loosen cap on mobile phase reservoir
Leak in lines from pump to column
Tighten or replace fittings; tighten rotor in injection valve
Leaking pump check valve or seal
Replace or clean check valve; replace pump seals
Suction filter clogged
Sonicate or replace
Increasing Pressure:
POSSIBLE CAUSE
SOLUTION
Blocked flow lines
Systematically disconnect components from pump to column end to find blockage; replace or clean blocked component
Particulate buildup at head of column
Filter sample; use 0.45μ filter; disconnect and back flush column; replace line filter
Fluctuating Pressure:
POSSIBLE CAUSE
SOLUTION
Bubble in pump head
Suck up bubbles through the drain tubing with a syringe.
Leaking pump seals, flow lines or faulty check valve.
Replace pump seals, tight the flow line connection; Sonicate check valves or replace.
Old Solvents remains in the pump chamber
Purge or increase the flow rate.
High Back Pressure:
POSSIBLE CAUSE
SOLUTION
Column Blocked
Improve sample cleanup; use guard column; reverse flush column with strong solvent to dissolve blockage.
Microbial growth on column.
Store column in at least 25% organic solvent without buffer.
Mobile phase viscosity too high
Use lower viscosity solvents or high temperature
Plugged frit in line filter or guard column
Replace frit or guard column
Plugged inlet frit
Replace end fitting or frit assembly
Salt precipitation (especially in reversed phase chromatography with high conc. of organic solvent in mobile phase)
Ensure mobile phase compatibility with buffer conc.
Injector
Unstable Pressure during Sample injecting:
POSSIBLE CAUSE
SOLUTION
Valve Clogged
Clean the valve or replace rotor seal.
Leaks:
POSSIBLE CAUSE
SOLUTION
Needle seal has deteriorated
Replace the needle seal
Valve Rotor or Stator has deteriorated
Replace the rotor or stator seal
Area Reproducibility:
POSSIBLE CAUSE
SOLUTION
Auto sampler was not rinsed
Check the level of rinsing solvent and ensure that it is being rinsed
Column has deteriorated
Check the column performance under known analysis condition or replace the column.
Sample flow line is clogged
Verify the sample is completely dissolve, Clean the clogged flow line or replace.
Valve rotor or stator has deteriorated
Replace the rotor or stator seal
Ghost Peak:
POSSIBLE CAUSE
SOLUTION
Contamination in column
Flush column to remove contamination; use HPLC grade solvent
Elution of analytes retained from previous injection
Flush column with strong solvents at end of run; end gradient at higher solvent concentration
Unknown interferences in sample
Use sample cleanup or prefractionation before injection
Detector
Leak:
POSSIBLE CAUSE
SOLUTION
Blocked column outlet (leak in flowcell)
Replace outlet tube or change gasket in flowcell.
Noise:
POSSIBLE CAUSE
SOLUTION
Bubble flowing or trapped in the flow cell
Apply pressure to the flow cell outlet, use MeOH to flush flow cell
Cell window is strained
Cell rinsing.
D2 Lamp Degradation
Replace a new D2 Lamp
Strained lens and gasket
Replace with a new one
Mobile phase pulsation
Eliminate pulsation from pump by dampener
Drift:
POSSIBLE CAUSE
SOLUTION
Cell Window is strained
Cell Rinsing
Impurities in mobile phase
Use fresh, covered mobile phase
Unit is in the presence of strong air current
Change the location of the instrument or place it in a protective environment.
Problems in Retention Time
POSSIBLE CAUSE
SOLUTION
Contamination Buildup
Flush the column occasionally with suitable solvents
Improper flow or pressure
Check the leaks in flow lines; check the check valves
Equilibration time insufficient for gradient run or changes in isocratic mobile phase
Condition the column
Inconsistent on-line mobile phase mixing
Ensure the gradient system is delivering a constant composition
Selective evaporation of mobile phase component
Cover solvent reservoirs; Prepare fresh mobile phase
Varying column temperature
Thermostat or insulate column; ensure laboratory temperature is constant.
Decreasing Retention Time:
POSSIBLE CAUSE
SOLUTION
Column overloaded with sample
Decrease sample amount or use large diameter column
Increase flow rate
Check the reset the pump flow rate
Varying column temperature
Thermostat or insulate column; ensure laboratory temperature is constant
Increasing Retention Time:
POSSIBLE CAUSE
SOLUTION
Decreasing flow rate
Check and reset the flow rate. Check for leaking pump seals and other leak in system.
Changing mobile phase composition
Cover solvent reservoirs; ensure that gradient system is delivering correct composition
Varying column temperature
Thermostat or insulate column; ensure laboratory temperature is constant
Treatment of Column
For new columns, it is best to condition under low flow rate (0.2 – 0.3 ml/min) for about 30 mins before operating under analytical flow rate. It applies for columns that have not been used for long time.
During shutdown, it is best to gradually reduce the flow rate so as not to subject column to sudden pressure drop.
When using buffer solutions, remember to flush with water before changing to organic solvents to prevent crystallization.
It is best to filter mobile phase and sample before use to reduce clogging by particulates in column frit.
Sample is best dissolved in mobile phase or if not possible then as clse to mobile phase composition as possible. This reduce chances that sample may participate in column bed in mixing with mobile phase.
Flush with stronger solvent after daily use. For Eg. ODS columns may flushed with Acetonitrile anger use daily to remove strong absorbed materials from column.
Prevention is by filtering both sample and mobile phase through 0.45um. Use 0.2 um membrane filter if it is very dirty.
Normal flushing with solvents may dissolve and remove some of these particles but in cases where the particulates are insoluble, flushing the column in reverse may help.
Replace filter frit if it cannot be washed. Be careful not to disturb column bed or performance may drop and in some cased, the stainless steel frits are fused to the columns and these cannot be replaced.
As HPLC columns are pressure-packed, subjecting them to pressure shock such as dropping the column or opening of drain valve during operation many result in formation of voids.
Useful Tips
Good habit
Log book – record pressure and mobile phase
Read manual / instruction
If any problem happens
Observe the instrument carefully – look, smell, and feel
Check the simple things first
Remember the last thing that happen before the problem started