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Column Care andColumn Care and
MaintenanceMaintenance
Nicola JonesHichrom Limited
Column Problems
Lifetime 63 %
Pressure 23 %
Reproducibility 18%
Performance 12%
LC/GC Survey
Column Problems
♦ Severe peak tailing
♦ High back pressure
♦ Lower efficiency
♦ Split peaks
Symptoms
0.0 2.5 5.0 7.5 10.0Time (min)
The First Step
♦ The performance either with the
manufacturers conditions or your
own standard conditions
♦ Solvent compatibility
♦ Storage recommendations
A new column:
CHECK
HPLC Columns
♦ Columns are the consumable part of
an HPLC system
♦ They do not last forever!
♦ Select the right column for the job
♦ Consider all operational variables
mobile phase, pH, temperature
Outline
♦ Simple preventative measures
♦ Column lifetime
♦ Pressure
♦ Performance
♦ Reproducibility
♦ Conclusion
Simple Preventative Measures
DETECTOR
Injection valve
Guardcolumn
Analytical column
DataSystem
Solventreservoir
PUMP
Inletfilter
Inlinefilter
Pre column
Sample Purification
Solvent Filtration
Simple Preventative Measures
♦ Use of high quality solvents – HPLC grade
♦ Removal of particulates- solvent filtration
- in-line filter
- sample purification
- RP to NP
- use of buffered mobile phase
♦ Implementation of washing protocols
Outline
♦ Simple preventative measures
♦ Column lifetime
♦ Pressure
♦ Performance
♦ Reproducibility
♦ Conclusion
Low pH– ligand cleavage– accelerated with T
OH
OH
HO
OH
OH
OHHO
HOHO
HOHO
Column Lifetime
High pH– silica dissolves– effected by T, mM buffer & type, mobile phase strength
Column Lifetime
High pH
Mobile phase : 50%H2O, / 50%MeCN / 0.2% TEA, pH 11
Initial analysis
After 30 injections
column voided
Column Lifetime
♦ Restricted pH range 2 < pH < 7
♦ Temperature < 50ºC
♦ Use of guard column
Is it possible to work outside these “ideal” conditions?
The “ideal” conditions
YES
Column Lifetime
Low pH stability
Components; 1: Uracil, 2:Dimethyl phthalate, 3: Toluene, 4: Biphenyl, 5:Phenanthrene
Conditions:0.1% TFA in H2O – MeCN (50:50)pH 1.81.0 ml / min, 22oC150x4.6mm
Column Lifetime
High pH stability
Components; 1: Uracil, 2:Dimethyl phthalate, 3: Toluene, 4: Biphenyl, 5:Phenanthrene
Conditions:25mM pyrrolidine in H2O - MeCN (50:50)pH 11.01.0 ml / min, 22oC
Column Lifetime
High Temperature Application – 80oC
Camurus, Malmo Sweden
Sample: Cyclosporin A & UColumn: Ace C18 5um 250x3.0mm
Mobile phase: H2O / MeCN / MTBE / phosphoric acid (460:510:30:1)Flow rate: 0.8 ml / minTemperature: 80oCInjection Volumne: 20ulDetection: 210nm
Column Lifetime
112 hours
3 hours
300 hours
381 hours
Column Lifetime
High Temperature Application
Retention vs Analysis Time
0
10
20
30
0 200 400 600 800 1000
Analysis Time (hours)
Ret
enti
on
(m
inu
tes)
CsU
CsA
Column Lifetime
High Temperature Application
Time (hrs) CsU CsA CsU CsA3 25.86 27.56 5790 75478 25.43 27.06 5308 767541 26.16 27.93 5615 769658 25.5 27.16 5539 7565112 26.12 27.86 5566 7624145 26.18 27.91 5499 7528212 25.37 27.02 5450 7501240 25.29 26.91 5600 7635300 24.7 26.32 5519 7491352 24.74 26.32 5453 7443381 24.86 26.53 5470 7327509 25.02 26.61 5195 6892592 24.39 25.97 5237 6976660 25.11 26.76 5194 6796750 25.68 27.29 4704 6081790 26.16 27.9 4832 6146
Retention (min) Plate count (N)
Column Lifetime
Flush with 100% H2O for 24 hours, 1.0ml/min
High Aqueous Conditions
Conditions: MeCN/50mM KH2PO4, pH3.5 (7:93)Components; 1: Uracil, 2:Dimethyl phthalate, 3: Toluene, 4: Biphenyl
0 1 2 3 4 5 6 7 8 9 10
Time - Minutes
0
100
200
300
400
500
600
700
Re
sp
on
se
- M
illiV
olts
Ace AQ
0 1 2 3 4 5 6 7 8 9 10
Time - Minutes
0
100
200
300
400
500
600
700
Re
sp
on
se
- M
illiV
olts
C18
0 1 2 3 4 5 6 7 8 9 10
Time - Minutes
0
100
200
300
400
500
600
700
Re
sp
on
se
- M
illiV
olts
Ace AQ
0 1 2 3 4 5 6 7 8 9 10
Time - Minutes
0
50
100
150
200
250
300
350
400
450
Re
sp
on
se
- M
illiV
olts
C18
Outline
♦ Simple preventative measures
♦ Column lifetime
♦ Pressure
♦ Performance
♦ Reproducibility
♦ Conclusion
Pressure
♦ Avoid pressure shocks
♦ Increase flow gradually upon equilibration
♦ Turn valve quickly
♦ Blocked frit, line
♦ Buffer precipitation
♦ Column contamination
Potential Problems
Good Practice – primarily associated with irregular silicas
Pressure Problems
♦ Check the pressure with & without the column
Determining the cause
♦ Wash the column
♦ Back flush the column
If the column pressure is high
If the system pressure is high
♦ Change in-line filter
♦ Flush the system
Column Cleaning
Flush with stronger solvents than your mobile phase
Reversed phase solvent choices in order of increasing strength
♦ Mobile phase without buffer salts
♦ 100% methanol
♦ 100% MeCN
♦ 75%MeCN / 25% IPA
♦ 100% IPA
♦ 100% methylene chloride
♦ 100% hexane
Column Cleaning
Column Contamination
Mobile phase : 20% H2O, / 80% MeOH1: Uracil, 2: Phenol, 3: 4-Chloronitrobenzene, 4: Toluene
0.0 2.5 5.0
2
41
3
Time (min)
QC analysis
2
41
3
0.0 2.5 5.0Time (min)
QC analysis after cleaning 100% IPA, 35 oC
USP Tailing Factor Component 4: 1.17
USP Tailing Factor Component 4: 1.32
Summary
To help prevent back pressure problems
♦ Use column protection
- guard cartridges
- in-line filters
♦ Filter samples
♦ Sample clean up
♦ Appropriate column flushing
♦ Filter buffered mobile phases
♦ Flush the system regularly
Outline
♦ Simple preventative measures
♦ Column lifetime
♦ Pressure
♦ Performance
♦ Reproducibility
♦ Conclusion
Performance
♦ Peak tailing, broadening
♦ Loss of efficiency
Deterioration in performance
Can be caused by
♦ Column secondary interactions
♦ Column void
♦ Column contamination
♦ Column loading
♦ System dead volume
♦ Solvent effects
Si
Si
SiSiSi
Least Acidic Most Acidic
The Silica Surface
O
Si
OO Si
Si
O
O
OH
OHO
O
SiSi O
O Si
Si
HO
SiO
O
O Si
HO
O
OSi
O
SiO
O SiO
SiO
O
OH
O Si
Si
O
O
O
O
Si
O
OHO
Si
OOH
SISiSi
OHOH
OHOH
GeminalGeminal
HO
FreeFree
SiSiHOHO
MM++ChelatedChelated
SiSi
OHOH OHOH
SiSi
AssociatedAssociated
SiSiSiSi
OHOHOHOH
Least Acidic Most Acidic
Silica Bonding :Monomeric
+Si O H
Silica surface
C l Si
C H3
C H3
Monofunctional bonding reagent
C H
OSi Si
3
C H3 Bonded silica
Single point attachment
Reproducible bonding technique
Surface Chemistry
Silica Bonding :Endcapping
♦ remove residual silanols
♦ improve the chromatography of basic compounds
X S i
C H3
C H3
C H3
S i OH
S i O S i
C H3
C H3
S i O S i
C H3
C H3
S i O S i C H3
C H3
C H3
S i OH
S i O
S i OH
S i
C H3
C H3
S i O S i
C H3
C H3
+
Surface Chemistry
Performance
Column Secondary Interactions
Mobile phase : 85% 25mM Na2HPO4 / 15% MeCN1: Phenylpropanolamine, 2: Ephedrine, 3: Amphetamine, 4: Methamphetamine, 5: Phenteramine
TIme (min) Time (min)0.0 2.5 5.0
54
32
1
54
3
2
1
0.0 2.5 5.0
No TEA 10mM TEA
USP Tailing FactorComponent 5: 1.14
USP Tailing Factor Component 5: 1.57
Time (min)
0.0 2.5 5.0
54
32
1
54
32
1
Time (min)
0.0 2.5 5.0
Performance
Column Secondary Interactions
Mobile phase : 85% 25mM Na2HPO4 / 15% MeCN1: Phenylpropanolamine, 2: Ephedrine, 3: Amphetamine, 4: Methamphetamine, 5: Phenteramine
pH 3.0 pH 7.0
USP Tailing Factor Component 4: 2.35
USP Tailing Factor Component 4: 1.33
Performance
XTerra MS C18Ace 5 C18
Symmetry 5 C18 Waters Spherisorb 5 ODS2
N 0.1 = 63,400 pl/mAs = 1.13
Column Secondary Interactions pH 6.0
Mobile phase : 80% MeOH / 20% 25mM KH2PO41: Norephedrine, 2: Nortriptyline, 3: Toluene, 4: Imipramine, 5: Amitriptyline
N 0.1 = 21,800 pl/mAs = 1.83
N 0.1 = 100 pl/mAs = 5.93
N 0.1 = 24,400 pl/mAs = 1.33
Performance
Column Void
Column: Partisil 10um SAX, 250x4.6mmEvaluation Conditions: 5mM KH2PO4, 1.0ml/min 1: CMP, 2: UMP
Initial performance
Av N = 24,000 pl/m
Performance after 100 injections
Av N = 3,200 pl/m
Performance
Column Contamination
100% IPA100% MeOH
100% Ethyl acetate
New Column
Column Returned from Customer
Column After Flushing
Lichrospher 60A silica, 5um, 250x4.0mm, Heptane/ethyl acetate, (90:10) 1: butylbenzene, 2: methyl benzoate, 3: nitrobenzene
Performance
System Dead Volume
Time (min)0.0 0.5 1.0 1.5 2.0
4
32
1
Time (min)0.0 0.5 1.0 1.5 2.0
4
32
1
10µl loop 50µl loop
Mobile phase : 85%H2O, 0.1% TFA / 15%MeCN, 1.0ml.min1: Phenylalanine, 2: 5-benzyl-3,6-dioxo-2-piperazine acetic acid, 3:Asp-Phe, 4:Aspartame
Performance
Solvent Effects
Injection volume: 30ulMobile phase: 82% H2O / 18% MeCN1: Caffeine, 2: Salicylamide
Injection solvent 100% MeCN
12
Injection solvent mobile phase
1
2
Performance
Solvent Effects
Mobile phase : MeOH / H2O, 0.1% TFA, (60:40)
Fresh TFA added to mobile phase
Current mobile phase
Performance
Temperature variation
Column: Inertsil ODS 3V 250x4.6mm, 5umMobile phase : 20% H2O / 80% MeOHComponents: 1: Uracil, 2: Dimethyl phthalate, 3: Toluene, 4: Biphenyl, 5: Phenanthrene
AmbientTemp
40 oC
♦ Performance increased
Peak 5: 57600 to 70000 pl/m
♦ Pressure reduced
1340 psi to 1050 psi
♦ Run time reduced20 min to 17.5 min
Summary
♦ Minimise system dead volume
♦ Match the guard column to the analyticalcolumn
♦ Keep the column clean
- pre-treat samples
- replace guard cartridge periodically
- implement washing protocol
To help gain optimum performance from your column
Outline
♦ Simple preventative measures
♦ Column lifetime
♦ Pressure
♦ Performance
♦ Reproducibility
♦ Conclusion
EARLY CONVENTIONAL CONVENTIONAL
Reproducibility
PARTIALLY VALIDATED FULLY VALIDATED
Reproducibility
CONVENTIONAL BASE DEACTIVATED
Reproducibility
1.51.5
22
2.52.5
33
3.53.5
44
22 44 66 88 1010
Column NumberColumn Number
kk’’
+ 4 %
1.51.5
22
2.52.5
3.53.5
44
22 44 66 88 1010
Column NumberColumn Number
kk’’
± 50%
33 + 10%+ 10%
Column Reproducibility
Column Reproducibility
Batch Validation
Single Chromatographic Validation Test
Batch Validation
Multiple Chromatographic Validation - Test 1
Batch Validation
Multiple Chromatographic Validation - Test 2
c
Batch Validation
Multiple Chromatographic Validation - Test 3
Ace C18Ace C18 3.703.70Hichrom C18Hichrom C18 4.924.92Symmetry C18Symmetry C18 5.99 5.99 Luna C18(2)Luna C18(2) 7.047.04HypurityHypurity C18 C18 9.869.86
Validated Materials
YMC Pro C18, Waters XTerra & Inertsil ODS 3V are alsovalidated but the variation in hydrophobicity is not specified
k’(neutral molecule) ± (%)
NB: All data taken from manufacturers validation certificates
Summary
♦ Good column to column reproducibility
♦ Good batch to batch reproducibility
♦ Increased testing by manufactures
♦ Use of validated columns provides long term
reproducibility
♦ Simple measures can be adopted to prolong column
lifetime
♦ Regular performance tests to monitor any deterioration
♦ Select the column most suitable for your application
♦ Consider all operational variables
- the column is not always the problem
Conclusion
