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FPLC
Troubleshooting Guide

Basics. Prevention. Safety tools.

Identification and problem solving.

think LC. think KNAUER.

FPLC Troubleshooting Guide

BASICS
• Identify and resolve your FPLC problems by • Change only one thing at a time.
checking for obvious explanations. • Know your system: perform a standard
• Use a logical sequence of steps to isolate separation, if anything goes wrong repeat
possible causes. the method and compare before and after.

A1 A2 bypass 5x
B1 B2
multi
injection 6x
valve waste
detection

UV Con
column outlet
pump A pump B 1 2 selection valve fraction
valve 3 collector
sample
automation
pump
air
sensor

Typical flow scheme of a FPLC system and points of optimization​

1 multi
injection
valve
INJECTION WITH A SAMPLE LOOP

The flow rate of the sample entering the loop creates a parabolic flow profile in the
loop. To fill the loop completely, a larger volume needs to be loaded. For small loops
a 3-fold overfill is recommended. For larger sample volumes less overfill is needed.

Flow during loop filling

Partial loop fill

− Low reproducibility
+ Low sample loss

Full loop fill

+ High reproducibility
− High sample loss

Flow during loop emptying

TIP Load the sample into the sample loop. Always leave the syringe in the injection port until
the sample has been injected onto the column and the valve is switched back into the
load position to avoid sample loss due to siphoning.

2
 bypass 5x

2
6x waste
detection
PEAK BROADENING
UV Con
column outlet
pB selection valve
Peak
valvebroadening isthe effect that a product
fractionis not staying concentrated, but is
collector
sample diffusing while passing through the system. This effect can impair the resolution of
automation
pump your separation. One can avoid peak broadening by shortening the tubing length
air
sensor of the overall system and reducing inner tubing diameter. But keep in mind, that
smaller inner tubing diameters are also increasing the system pressure.

Smaller ID – Capillary between

higher back column outlet
pressure! and UV detector
mAU
should be as short
as possible!

volume

mm Colour Dead volume Recommended Article Max. pressure

ID coding (µl/m) flow rate No. (bar)

0.13 Red striped 13 Up to 0.25 ml/min A2522 420

0.25 Blue striped 49 0.25–2 ml/min A2524 385

0.50 Orange striped 196 2–10 ml/min A2525 240

0.75 Green striped 442 10–50 ml/min A2526 240

3
detection

UV Con FRACTION DELAY VOLUME / DEAD VOLUME

fraction
collector
The fraction delay volume of a system is the volume between the UV detector flow
cell and the fraction collector. It is important to know this volume for collecting peaks
accurately. The software will take this volume into account and start fractionation
in a delayed manner accordingly.

Peak is detected Peak reaches the

fraction collector
Fraction delay =
dead volume/time

TIP Determine the delay volume by calculating the volume of the capillaries and adding the
volume of the flow cells of the detectors.

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FPLC Troubleshooting Guide

PREVENTION
Always degas your buffer by applying for example a vacuum or by sonication. Additionally filter
your buffer. Ideally this can be combined by using a vacuum filtration device.

Buffers used at room temperature should be • Filter your sample

renewed on a daily basis. Alternatively consider • Refresh buffers on a regular basis
the use of a preservative to increase life time of • Never leave system overnight on high salt buffer
your buffer (e. g. adding 0,02 % NaN₃). • Regular clean up routine
• Check system performance with system test
Another option is to run the pumps over night • Clean columns according to manufacturers
with low flow rates. recommendation on a regular basis

Storage of your system Overnight Over the weekend Longer then a week

Buffer high salt

(e. g. 0.5–1 M NaCl)

Buffer low salt

(e. g. 150 mM NaCl)

Water

20 % Ethanol

1. START UP ROUTINE 2. SYSTEM CHECK UP

• Switch on detector/system • Check for leakage
• Degass and filter buffers • Check for stable pressure
• Temper buffers • Check for stable UV signal
• Purge pump • Exchange tubes in fraction collector
• Wash system • Check waste level
• Equilibrate column • Check buffer levels
• Prepare sample • Check or renew back piston rinsing
solution

4. SHUT DOWN ROUTINE 3. CLEANING OF THE SYSTEM

• Flush system with suitable solution for storage • Remove pH electrode from the flow path
• Flush injection port and sample loop • Flush system with 50 ml water
• Rinse fraction collector valve • Flush system with 50 ml 0.5–1M NaOH
• Optional: switch of detector lamp • Flush with a minimum of 50 ml water
• Rinse pH electrode and store in storage solution • Store in 20 % Ethanol
• Never switch of system at 4°C!

4
SAFETY TOOLS
AIR SENSOR
Air sensors are very useful tools to protect columns from damage by air and supports automatic
sample loading.

Using an air sensor for column protection. Using an air sensor for automated sample
injection.

air sensor air sensor

system sample
pump pump

Upon air detection, various functions can be When the end of the sample is detected, the
programmed. The run can be paused or stopped software allows the run to start or continue
to prevent air from entering the system. automatically.

BACK PRESSURE REGULATOR

A back pressure regulator (BPR) applies a constant
pressure to the system. It prevents the formation
of air bubbles due to the pressure drop after the
column which disturbs the detector signal.

back waste
detection pressure
regulator

UV Con
pH
fraction
collector
Place BPR after Place BPR
the UV detector before the
and conductivity pressure FPLC Tutorial:
monitor. sensitive pH Back pressure
electrode. regulator

www.knauer.net/fplc 5
FPLC Troubleshooting Guide

PRESSURE MEASUREMENT

Several FPLC columns and resins are very pressure sensitive, consequently pressure control is very
important. The pressure before and after the column is monitored and the software calculates
automatically the pressure difference over the column resin. If the pressure differential value (DP)
exceeds the pressure limit, the pump stops or pauses.

UV

System Post column

pressure pressure p2
(pressure
sensor of Pre column
pressure p1 Delta pressure p1–p2
system pump)
Pressure limit Pressure limit protects
protects the the column bed
column hardware

PRESSURE MEASUREMENT AND BACK PRESSURE REGULATOR

The additional pressure behind the column affects only the column hardware. The column bed is
not affected.

Without With
back back
pressure pump pressure pump

p1 = 3 bar p1 = 5 bar

Column Column
hardware hardware
∆p = 3 bar ∆p = 3 bar
pressure 3 bar pressure 5 bar
(3.0 + 0.0) (3.0 + 2.0)

p2 = 0 bar p2 = 2 bar
Back pressure
regulator adjusted
to 2 bar

6
FILTER

Injection of samples via the system pumps is possible. Always filter samples before injection. When
using the pump P 6.1L for injection of sample the filter located in the pressure sensor must be
removed to avoid blockage. Insert the adapter, which you can find in the accessory kit of the pump.
The adapter has no filter effect. Use an inline filter for column protection which you can simply install
in front of your column.

If using the pump P 6.1L for sample application, Instead use an inline filter for
replace the filter cartridge by dummy cartridge. column protection.

Filter

FPLC Tutorial: FPLC Tutorial: FPLC Tutorial:

Pressure sensor Exchange of inline Inline filter
filter frit

www.knauer.net/fplc 7
FPLC Troubleshooting Guide

IDENTIFICATION AND
PROBLEM SOLVING
Problem Cause Solution
System pressure to high 1. Column clogged 1.+2. Clean the system and/or column with
2. Filter of pressure sensor Ethanol or 0.5–1M NaOH
clogged when applying 2. Exchange filter of the pressure sensor by
sample via the major pump dummy or use a sample pump
3. ID of tubing to small 3. Use capillaries with ID according to the
recommended flow rate
4. Viscositiy of buffer or
sample to high (especially 4. Dilute and/or filter sample and decrease
working at 4°C) the flow rate

Pressure fluctuation 1. Air bubbles in buffer 1. Degas buffer or/and use degasser in the
2. Air bubbles in pump head system. Adjust temperature of the buffer
3. Positioning of buffer bottles 2. Check for prefilled inlets and purge pump
Pressure signal

with 50 % of maximum flow rate

4. Blocked check valves
3. Remove persistent air bubbles by flushing
Volume the system with 20 % isopropanol
4. Place bottles on top or at the same heigth
of the system/pump
5. Renew check valves
UV signal fluctuation 1. Dirty flow cell 1. Clean the flow cell according to manual
2. Air in flow cell 2. Flush flow cell and use back pressure
Detector signal

regulator

Volume

Loss of signal/No peaks 1. Detector off 1. Switch on detector

loss of signal 2. Leakage 2. Check for leakage and tighten capillary
3. No flow connection or renew capillary
4. Problem during injection 3. Check the purge valve, check the flow
rate at the outlet with a graduated vessel,
no signal
exchange check valve
4. Check position of injection valve

Retention time delay/shift 1. Leakage 1. Check for leakage and thighten capillary
2. Gradient mixing not precise connection or renew capillary
regular
3. Column problem 2. Check the flow rate of each pump head,
Check buffer composition. Purge the
4. Pump/flow problem
system with 100 % A or B and higher flow
rates. Renew and adjust the solvents
problem
3. Clean column, Check for column
performance, if mandatory renew
4. Check the flow rate at the outlet with a
graduated vessel, exchange check valve
tR
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