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Knauer Webinar 12-01-24
Solve your problems – KNAUER (U)HPLC Troubleshooting II
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Knauer Webinar 12-01-24
Solve your problems – KNAUER (U)HPLC Troubleshooting II
Dr. Silvia Marten
Head of Applications and Columns Dep.
Rene Borstel
Applications and Columns Dep.
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Knauer Webinar 12-01-24
Troubleshooting alocation
System or Column ?
mobile phase
pump problems
injector problems
detector problems
columns problems
Problems can be everywhere in your
HPLC system!
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Knauer Webinar 12-01-24
Troubleshooting categories
major categories pressure abnormalities
leaks
problems with chromatogram
other problems
Report in your HPLC system recordbook !
injector problems
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Knauer Webinar 12-01-24
Troubleshooting categories
Problem indicators
Retention tR
Pressure
Baseline
Peak shape
broad peaks
split peaks
peak tailing
Peak area
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Knauer Webinar 12-01-24
broad peaks Cause change in mobile phase
flow rate to low
leak between column
and detector
extra column effects:
- column overload
- cell volume to large
- tubing between column
and detector to long
Solutions
check make up of mobile
phase
adjust flow rate;
use higher temperatures
check lose fittings
- inject smaller volumes
- dilute sample
- use smaller flow cell
- use 0.25 or 0.1 mm ID
tubings
Troubleshooting: tips and tricks
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Knauer Webinar 12-01-24
Troubleshooting: tips and tricks
tailing Cause buffer concentration to
low
guard column
contaminated
column contaminated
void at column inlet
column temperature too
low
Solutions
increase concentration
(10 mM – 50 mM)
replace guard column
replace or reequilibrate
column
replace column
increase temperature
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Knauer Webinar 12-01-24
Troubleshooting: tips and tricks
broad peaks
mixing
chamber
Avoid poorly made HPLC connections !
leakage
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Knauer Webinar 12-01-24
Troubleshooting: tips and tricks
Fittings used in HPLC
1 Fitting only for one port !
stainless steel
high pressure sealing
(re-useable)
PEEK (black) P3860
high pressure sealing
frequent connection
changing
Dynaseal
< 400 bar
re-useable
fingertight
(U)HPLC (U)HPLC 0.5 mm ODHPLC
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Knauer Webinar 12-01-24
Troubleshooting; tips and tricks
split peaks
regular sample
problem sample
Cause column/precolumn
contaminated
sample solvent
incompatible with mobile
phase
void at column inlet
partially blocked frit
broken column bed
Solutions
replace column/
precolumn
dilute sample in mobile
phase
replace or repack top of
column
replace frit
replace column
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Knauer Webinar 12-01-24
Troubleshooting: tips and tricks
frontingCause interference in sample
shoulder
column overload
incompatible sample
solvent
Solutions
Check column performance
with standard
increase efficiency
try another column type
inject smaller volumes
inject lower concentration
adjust sample solvent
www. chromservice.cz
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Knauer Webinar 12-01-24
Minutes
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
-20
0
20
40
60
80
100
120
140
160
180System optimization:
minimize tubing length
smallest inner diameter
low flow cell volume
poor HPLC connections
Minutes
0.575 0.600 0.625 0.650 0.675 0.700 0.725 0.750 0.775 0.800
0.825 0.850 0.875 0.900 0.925
0
20
40
60
80
100
120
140
160
Troubleshooting: tips and tricks
extra column dispersion
Avoid system dead volume !
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Knauer Webinar 12-01-24Minutes
0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0
mA
U
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
green: UHPLCblue: HPLC plus
1
2
3
4
5
Troubleshooting: tips and tricks
Peak 5: Phenetol
tR [min]0,982 0,979
Area
725858 729245
Plates
1567 5437
Optimization of resolution
Avoid system dead volume !
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Knauer Webinar 12-01-24
HPLC Troubleshooting Guide
available as .pdf
www.knauer.net
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Knauer Webinar 12-01-24
Troubleshooting rules
1. Failure in system or sample?
2. Check method parameter: are instrument settings correct?
4. Step by step changings: modify or replace only one thing!
3. Anything be changed in method or system/column?
How to fix the problem?
Instrument and method logbook can help you !
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Knauer Webinar 12-01-24
Troubleshooting rules
Prevention (main rules)
3. periodical replacement of inline filter
1. periodical replacement of pump seals
4. filtration of mobile phase and samples
6. avoid pH > 8 / < 2
Realize routine preventive maintenainance !
5. use guard/saturator columns
2. periodical replacement of detector lamp
7. flush buffer from the HPLC system
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Knauer Webinar 12-01-24
Summary
Troubleshooting
1. systematic troubleshooting (step by step)
2. report troubleshooting / maintenance
3. avoid poor HPLC connections
4. periodical maintenance
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Knauer Webinar 12-01-24
1. Operators and service Manuals for instruments
2. experienced other people in the lab
3. Manufacturer of instruments or columns
4. KNAUER technical hotline: [email protected]
5. Seminars, workshops and trainings: www.knauer.net
6. Troubleshooting LC Systems
by J.W. Dolan and L.R. Snyder, (Humana Press, 1989)
7. LC-GC Magazine (http://www.chromatographyonline.com/)
Advanstar Publishing
8. KNAUER HPLC Troubleshooting Guide www.knauer.net
Troubleshooting
Additional help / References
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Knauer Webinar 12-01-24
Troubleshooting
Further questions and answers via mail
[email protected] or
[email protected]
Questions ?
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Knauer Webinar 12-01-24
Column Troubleshooting
Webinar in 2012
26.06.2012
