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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