HPLC Troubleshooting: Spinco Biotech PVT LTD

HPLC
Troubleshooting
A.NARENDER
Asst.Service Manager
Spinco Biotech Pvt Ltd.

Daily Maintenance
Pre-analysis checks (1)
• Filter mobile phase

• Wait for mobile phase to reach room temperature
• Purge all the flow lines with mobile phase
• Replace mobile phases as necessary
• If buffer is used as mobile phase, wash the back of plunger seal
• If autosampler is used, verify that the autosampler rinse bottle is full
• Purge the autosampler rinse solution
• Check for leaks
• Check the pump pressure
• Check the oven temperature
• Perform a baseline check
2
Daily Maintenance
Post-analysis checks
• Wash the column
• If the column is not going to be used for long time, take off it from
the system after washing, and cap both ends of the column, store it
in cool and dark place
• Clean the flow line or the rest of the instrument, as necessary
• Store the solvent reservoir filters in rinse solvent to prevent them

from drying out
3
Outline
- Troubleshooting Process
- Categories of Problems
Pressure Issues
Baseline Issues
Peak Shape Issues
Retention Issues
- Troubleshooting Examples
4
Troubleshooting Process
Gather the facts – not theories.

Compare the performance obtained to the expected performance.
List possible causes.
Check the simplest things first – it’s easier.
Work through the possible causes in a step-by-step manner
checking the outcome from any changes made.
As a last resort – get help from elsewhere, for example your
instrument supplier help desk or your local technical support
department.
5
Potential Sources of Problems
– Mobile phase
– Injector
– Pump
– Sample
– Column and guard column
– Detectors
– Tubing and connectors
– etc.
• Analysts should simplify the problem source

– chemistry or mechanical?
6
Troubleshooting Guide
Main Troubles with HPLC

1. Pressure 2. Unstable Baseline
Low pressure Drift/noisy
Pressure fluctuation Spikes
High pressure Sawtooth
Cyclic
No change
3. Distorted Peak Shape 4. Retention Time

Broading Changing Retention
Tailing
Leading
Splitting
No peaks
7
Pressure – Low pressure
Leaking in pumps, check

valves, seals or connectors
Pump cavitation
No pumping
8
Pressure Fluctuation in System
Possible causes :
1. Air bubbles in pump head
2. Leaks in system
3. Check valves are dirty
4. Plunger seal is damaged
5. Plunger is spoilt.
Remedy:
1. Purge the pump
2. Check for leaks in all the connections, especially new connection.
3. Clean check valves in ultrasonicator
4. Replace plunger seal
5. Replace plunger
9
High Pressure in System
Possible Causes Too much work ?
Clogging in
• Detector cell
• Column
• Pump
Too much nagging ?
• Mixer
• Piping
Medical problem ?
10
Locate the point of the pressure increases
Column or system?
Check the system methodically:

• Disconnect column from detector, if the pressure problem goes
way, the problem is clogging of detector cell or piping to and from
flow cell.
• If pressure persist, disconnect guard column and main column. If
pressure becomes normal, clean/replace main column.
• If pressure persist, remove guard column. If pressure is normal,
replace guard column.
• Continue in this manner from auto-injector (if present) to mixer and
finally to pump until find out the high pressure spot.
11
High Pressure in System
Possible reason:
Clogging in system. It may occur in any part of system.
Remedy :
If clogging in piping, it may be possible to flush out using
IPA or nitric acid at higher flow rate. In this case, do not
connect column or detector.
Clean or replace in-line filter.
12
High Pressure in Column
Possible reason:
Column clogging/failure.
Accumulation of particulates and/or adsorbed substances on column
Remedy :
1.Clean guard column and column.
2.Replace filter frit on column if possible.
3.Flush the column reversely or repair the column (This is recommended
only as a last resort).
Precaution:
In these cases, it is always recommended to use guard column
whenever
possible. The guard columns are cheaper and can be easily replaced
when clogging or adsorption occurs.
13
Precautions for Column
• Buffer solution must be filtered

• by 0.2 or 0.45 µm membrane filter.
• Sample must be filtered

• by 0.2 or 0.45 µm membrane filter.
• Be sure do not jump from buffer to pure organic or from organic to

buffer. This can lead to buffer precipitation, plugging and pressure
problems.
• Always use a washout, intermediate solvent.
14
Solution (1)
Wash the column with suitable solvent
Open the column end, carefully remove the

filter/frit with the column in an upright
position, and sonicate it.
Change the filter/frit.
Wash the column reversely at low flow rate.
15
Column Cleaning
• Washing the column will remove non-polars.
• Buffered mobile phase:

must wash out the buffer with the same mobile phase minus
the buffer first
• Aqueous organic solvent

Wash with Acetonitrile or methanol for at ~20 column
volumes.
16
Column Cleaning
For reversed phase column
• 1. Wash with mobile phase without buffer salts

• 2. Wash with methanol or acetonitrile
17
Solution (2)
only ~1 cm might be polluted
Last resort:
Remove the contaminated part and
repack with clean packing material.
Always use the same size and type of
material used originally in the column.
column
18
Baseline Troubleshooting
Noisy baseline Cyclic
Spikes
Sawtooth
Drift
Positive & negative peaks
19
Unstable Baseline
en? Why is it
not flat?
Contaminated column
Detector cell is dirty
System is contaminated
Weak detector lamp
Leaks
Unstable pumping
Mobile phase is not degassed properly
Air bubbles in pumps/detector
Electronic noise
Temperature is fluctuating
Wavelength is too low
20 More……
Spike Baseline
Bubbles in flow cell

• Apply back pressure to the flow cell
• Degas mobile phase
• Clean flow cell with IPA
Pump pulsation
• Check pump function
• Install damper
21
Baseline - Sawtooth
• Bubbles
• Mixing problem
• Plugged lines
• Electrical problems
22
Baseline - Cyclic
•Temperature fluctuations
•Mixing problem
•Mobile phase was not degassed
•Pump problem
23
Baseline – no change
• Faulty circuits
– replace any faulty parts
• Lamp is off
– set lamp parameter to turn on lamp power
• Range setting is not correct
– enter an appropriate range value
24
Peak Shape Issues
• Peak broading
• Peak tailing
• Peak fronting
• Peak splitting
• Other asymmetry peaks
• No Peaks
Many peak shape issues are combined
25
Peak Broading
Sample overload
Large extra system volume
Retention time is too long
Poor column efficiency
26
Peak Tailing
The flow-line contains extra dead volume

Sample overloading
Silica-based column degraded at high pH or high
temperature
27
Peak Fronting
Sample overloading
Channeling in column
28
Peak Splitting
Voids in the column caused by crystallization

or by pressure shock
Coeluting peaks or interfering compounds
Column overload
Incorrect storage of column leading to drying
up of packing
29
Distorted Peak Shape
Column overload
Incomplete endcapping
Coeluting peaks
Incorrect solvent for sample
Contaminated column or guard column
30
No Peaks
Sample is too diluted

Injection volume is too small
Detector lamp is off
Leaks
Pump is not pumping
Injector is damaged
Zoom setting of chromatogram is not correct
Column retains all compounds
Incorrect mobile phase/sample
31
Changing Retention Time
• Contamination was built up

• Equilibration time is insufficient
• Inconsistent mobile phase preparation
• Inconsistent on-line mobile phase mixing
• Selective evaporation of mobile phase component
• Unstable column temperature
• Leakage
• Unstable pumping
32
Tips
Good habits
Log book – record pressure and mobile phase
Wash the column after sample analysis
Read manual/instruction
If any problem happens

Observe the instrument carefully – look, smell, and feel
Check the simple things first
The hardware is not easy to be broken
33
Thank You!
34

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HPLC

Spinco Biotech Pvt Ltd.

• Filter mobile phase

• Wash the column

• Clean the flow line or the rest of the instrument, as necessary

• Store the solvent reservoir filters in rinse solvent to prevent them

Peak Shape Issues

Gather the facts – not theories.

• Analysts should simplify the problem source

Main Troubles with HPLC

3. Distorted Peak Shape 4. Retention Time

Leaking in pumps, check

Possible Causes Too much work ?

Check the system methodically:

• Buffer solution must be filtered

• Sample must be filtered

• Be sure do not jump from buffer to pure organic or from organic to

• Always use a washout, intermediate solvent.

Wash the column with suitable solvent

Open the column end, carefully remove the

Change the filter/frit.

Wash the column reversely at low flow rate.

• Washing the column will remove non-polars.

• Buffered mobile phase:

• Aqueous organic solvent

For reversed phase column

• 1. Wash with mobile phase without buffer salts

only ~1 cm might be polluted

Noisy baseline Cyclic

Bubbles in flow cell

Peak Shape Issues

Many peak shape issues are combined

The flow-line contains extra dead volume

Voids in the column caused by crystallization

Sample is too diluted

Changing Retention Time

• Contamination was built up

If any problem happens

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