Care, Maintenance, and Troubleshooting of HPLC Columns

Page 1

Care, Maintenance, and

Troubleshooting of

HPLC Columns

Columns and Consumables

Edward Kim

Applications Engineer

January 17, 2008

Page 2

Goals for this presentation:

1. Introduce the most commonly observed column related

problems in HPLC.

2. Explore the reasons for these column problems.

3. Propose preventative maintenance and method

development/optimization approaches to minimize HPLC

column problems and increase column lifetimes.

Nano LC

Capillary LC

Analytical LC

Prep LC

Chip LC

Page 3

Troubleshooting in HPLC

20

00

15

00

10

00

5

0

5

1

0

1

5

2

0

2

5

Time

(min)

mA

U

Page 4

Major Areas of Column Problems -

Dramatic Changes in 3 Key Areas:

1. HPLC System Pressure

2. Chromatogram - Peak Shape

3. Chromatogram - Peak Retention/Selectivity

Page 5

Plugged inlet frit

Large pressure change Column contamination

Plugged packing

1. Pressure Issues

Column Observations

Potential Problems

Page 6

Determining the Cause and

Correcting High Back Pressure

• Check pressure with/without column - many

pressure problems are due to blockages

elsewhere in the system.

If Column pressure remains high:

• Rinse column (remove detector from flow path!)

– Eliminate column contamination and

plugged packing

– high molecular weight/adsorbed compounds

– precipitate from sample or buffer

• Back flush column – may clear plugged column

inlet frit

• Change column inlet frit (… or discard column)

Eliminate pressure issues – add a disposable 0.5 or 2 um

in-line filter to system.

Page 7

Pressure Problem I

Pressure Too

High

• Column inlet frit contaminated

• Frit in purge valve contaminated

• Column contaminated

• Blockage in a capillary, particularly

needle seat capillary

• Rotor in injection valve plugged

• Injection needle or needle seat

plugged

Pressure Measurement

Use this valve

to divide the

system

Page 8

Pressure Problem II

Pressure Too Low

• Solvent inlet frit plugged

• Leak in a capillary connection or

other part (pump seals)

• Wrong solvent or flow rate

• AIV (Active inlet valve) defective

• Multichannel Gradient valve

incorrectly proportioning

• Ball valve defective

• Column defective

(stationary phase)

Pressure Measurement

Solvent inlet

frits

Page 9

1100 and 1200 Pumps

Exploded View

Pistons

Plunger Housing

Seals

Piston Support Rings

Outlet Valve

Active Inlet Valve

Purge

Valve

Holding Screw

Pump Housing

Page 10

Pump Check Valves

Active Inlet Valve

(common to all)

Outlet Ball Valve

Iso/Quat Pump G1311-60012

Binary Pump G1312-60012

Purge

Valve

G1312-60009

2

5

6

7

Cartridge

New style

1

Old style

G1312-60010

5062-8568

5062-8562

1. Gold Washer 5001-3707 5. Gold Seal 5001-3707

2. Plastic cap 01018-21207 6. Cap(4pk) 5062-2485

3. Gold Seal 5001-3707 7. PTFE (5pk) 01018-22707

4. Cap(4pk) 5062-2485

Page 11

1. Mobile phase without buffer salts (water/organic)

2. 100% Organic (MeOH or ACN)

3. Is pressure back in normal range?

4. If not, discard column or consider more drastic conditions:

75% Acetonitrile:25% Isopropanol, then

5. 100% Isopropanol

6. 100% Methylene Chloride*

7. 100% Hexane*

Column Cleaning

Use at least 10 x V

m

of each solvent for analytical columns

Flush with stronger solvents than your mobile phase.

Make sure detector is taken out of flow path.

Reversed-Phase Solvent Choices

in Order of Increasing Strength

When using either Hexane or Methylene Chloride the column must be flushed

with Isopropanol before returning to your reversed-phase mobile phase.

Page 12

• Use at least 50 mL of each

solvent

• 50% Methanol : 50% Chloroform

• 100% Ethyl Acetate

Column Cleaning

Normal Phase Solvent Choices

in Order of Increasing Strength

Page 13

Preventing Back Pressure Problems:

In-Line Devices

Mobile Phase

From Pump

Pre-Column

Injector

Guard

Column

Analytical

Column

To Detector

Filter and Guard Column Act on Sample

Pre-Column Acts on Mobile Phase

Filter

Page 14

Preventing Column Back Pressure Problems

1. Filter mobile phase:

- filter non-HPLC grade solvents

- filter buffer solutions

- Install an in-line filter between auto-sampler and column

(removes pump seal debris, ALS rotor debris, and sample

particulates). Use 2 um frit for 3.5 um columns, use 0.5 um frit for

1.8 um columns.

2. Filter all samples and standards

3. Perform sample clean-up (i.e. SPE, LLE) on dirty samples.

4. Appropriate column flushing – flush buffers from entire system at

end of day with water/organic mobile phase.

Page 15

2. Peak Shape Issues in HPLC

• Split peaks

• Peak tailing

• Broad peaks

• Poor efficiency (low N)

• Inconsistent response

• Many peak shape issues are also combinations - i.e. broad

and tailing or tailing with increased retention

Page 16

Split Peaks

Can be caused by:

• Column contamination

• Partially plugged frit

• Column void (gap in packing bed)

• Injection solvent effects

Page 17

0 5 10 15

1

3

4

2

Time (min)

0 5 10 15

1

3

4

2

Time (min)

0 5 10 15

1

3

4

2

Time (min)

Split Peaks

Column Contamination

Column: StableBond SB-C8, 4.6 x 150 mm, 5 μm Mobile Phase: 60% 25 mM Na

2

HPO

4

, pH 3.0 : 40% MeOH Flow Rate: 1.0 mL/min

Temperature: 35°C Detection: UV 254 nm Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP 3. Unknown 4. Chlorpheniramine

Injection 1 Injection 30

Injection 1

After Column Wash

with 100% ACN

• Column washing eliminates the peak splitting, which resulted from a contaminant on the column.

Page 18

Split Peaks

Injection Solvent Effects

Column: StableBond SB-C8, 4.6 x 150 mm, 5 μm Mobile Phase: 82% H

2

O : 18% ACN

Injection Volume: 30 μL Sample: 1. Caffeine 2. Salicylamide

A. Injection Solvent

100% Acetonitrile

B. Injection Solvent

Mobile Phase

• Injecting in a solvent stronger than the mobile phase can cause

peak shape problems, such as peak splitting or broadening.

• Note: earlier peaks (low k) most affected

0 10

Time (min)

1

2

0 10

Time (min)

1

2

Page 19

• Void Volume in Column

• Partially Blocked Frit

• Only One-Peak a Doublet- Coeluting Components

• Early (low k) peaks most affected

Normal

Doublet

Peaks

Void Volume in Column

Peak Shape Problems - Doublets

Page 20

Determining the Cause of Split Peaks

1. Complex sample matrix or many samples analyzed - likely

column contamination or partially plugged column frit.

2. Mobile phase pH > 7 - likely column void due to silica

dissolution (unless specialty column used, Zorbax Extend-

C18 stable to pH 11)

3. Injection solvent stronger than mobile phase - likely split

and broad peaks, shape dependent on injection volume and

k value.

Page 21

Peak Tailing, Broadening

and Loss of Efficiency (N, plates)

May be caused by:

1. Column “secondary interactions”

2. Column packing voids

3. Column contamination

4. Column aging

5. Column loading

6. Extra-column effects

Page 22

Peak Tailing

Column “Secondary Interactions”

• Peak tailing of amine analytes eliminated with mobile phase

modifier (TEA, triethylamine ) at pH 7

Column: Alkyl-C8, 4.6 x 150 mm, 5μm Mobile Phase: 85% 25 mM Na

2

HPO

4

pH 7.0 : 15% ACN Flow Rate: 1.0 mL/min

Temperature: 35°C Sample: 1. Phenylpropanolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine

No TEA

USP TF (5%)

1. 1.29

2. 1.91

3. 1.63

4. 2.35

5. 1.57

10 mM TEA

USP TF (5%)

1. 1.19

2. 1.18

3. 1.20

4. 1.26

5. 1.14

T

Ime (min)

Time (min)

0.0 2.5 5.0

5

4

3

2

1

5

4

3

2

1

0.0 2.5 5.0

Page 23

Peak Tailing

Column “Secondary Interactions”

Column: Alkyl-C8, 4.6 x 150 mm, 5μm Mobile Phase: 85% 25 mM Na

2

HPO

4

: 15% ACN Flow Rate: 1.0 mL/min

Temperature: 35°C Sample: 1. Phenylpropanolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine

pH 3.0

USP TF (5%)

4. 1.33

pH 7.0

USP TF (5%)

4. 2.35

• Reducing the mobile phase pH reduces interactions with silanols that

cause peak tailing. No TEA modifier required.

Time (min)

0.0 2.5 5.0

5

4

3

2

1

5

4

3

2

1

Time (min)

0.0 2.5 5.0

Page 24

0.0 2.5 5.0

2

4

1

3

Time (min)

2

4

1

3

0.0 2.5 5.0

Time (min)

2

41

3

0.0 2.5 5.0

Time (min)

Peak Tailing

Column Contamination

Column: StableBond SB-C8, 4.6 x 250 mm, 5μm Mobile Phase: 20% H

2

O : 80% MeOH Flow Rate: 1.0 mL/min

Temperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 2. Phenol 3. 4-Chloronitrobenzene 4. Toluene

Plates TF

1. 7629 2.08

2. 12043 1.64

3. 13727 1.69

4 13355 1.32

Plates TF

1. 7906 1.43

2. 12443 1.21

3. 17999 1.19

4 17098 1.25

Plates TF

1. 7448 1.06

2. 12237 1.21

3. 15366 1.11

4 19067 1.17

QC test forward

direction

QC test reverse direction

QC test after cleaning

100% IPA, 35°C

Page 25

Analytical vs. Preparative Scale HPLC. Non-linear

Adsorption Isotherms, or Overload Conditions:

k’

W

Amount injected

Analytical Prep

Retention

Peak Width

Group/Presentation Title

Agilent Restricted

October 28, 2008Month ##,

200X

Page 26

Peak Tailing/Broadening

Sample Load Effects

Columns: 4.6 x 150 mm, 5μm Mobile Phase: 40% 25 mM Na

2

HPO

4

pH 7.0 : 60% ACN Flow Rate: 1.5 mL/min

Temperature: 40°C Sample: 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine

Broadening

Competitive C8

Plates

A.

B.

C.

D.

High Load

x10

Low Load

CD

1. 850 5941

2. 815 7842

3. 2776 6231

4. 2539 8359

5. 2735 10022

6. 5189 10725

Tailing

Eclipse XDB-C8

USP TF (5%)

AB

1. 1.60 1.70

2. 2.00 1.90

3. 1.56 1.56

4. 2.13 1.70

5. 2.15 1.86

6. 1.25 1.25

0 5 10

Time (min)

0 5 10

Time (min)

0 5

Time (min)

0 5

Time (min)

Page 27

Peak Broadening, Splitting

Column Void

• Multiple peak shape changes can be caused by the same column

problem. In this case a void resulted from silica dissolved at high pH.

Mobile Phase: 50%ACN: 50% Water : 0.2% TEA

(~ pH 11)

After 30 injections

Initial

Page 28

Peak Tailing

Injector Seal Failure

Column: Bonus-RP, 4.6 x 75 mm, 3.5 μm Mobile Phase: 30% H

2

O : 70% MeOH Flow Rate: 1.0 mL/min

Temperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 2. Phenol 3. N,N-Dimethylaniline

Plates USP TF (5%)

1. 2235 1.72

2. 3491 1.48

3. 5432 1.15

Plates USP TF (5%)

1. 3670 1.45

2. 10457 1.09

3. 10085 1.00

Before

After replacing rotor seal

and isolation seal

• Overdue instrument maintenance can sometimes cause peak shape problems.

0.0 0.5 1.0 1.5

Time (min)

2

1

3

0.0 0.5 1.0 1.5 2.0

Time (min)

2

1

3

Page 29

Peak Tailing

Extra-Column Volume

Column: StableBond SB-C18, 4.6 x 30 mm, 3.5 μm Mobile Phase: 85% H

2

O with 0.1% TFA : 15% ACN Flow Rate: 1.0 mL/min

Temperature: 35°C Sample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine acetic acid 3. Asp-phe 4. Aspartame

10 μL extra-column

volume

50 μL extra-column

volume (tubing)

Time (min)

0.0 0.5 1.0 1.5 2.0

4

3

2

1

Time (min)

0.0 0.5 1.0 1.5 2.0

4

3

2

1

Page 30

Determining the Cause of Peak Tailing

• Evaluate mobile phase effects - alter mobile phase pH

and additives to eliminate secondary interactions

• Evaluate column choice - try column with high purity

silica or different bonding technology

• Reduce sample load – volume injection and

concentration

• Eliminate extra-column effects – tubing, fittings,

Uv cell

• Flush column and check for aging/void

Page 31

Reproducibility

Typically,

• Area and Peak Height problems together point to the autosampler system

• Area and Retention Time problems together point to the pump

Peak retention time precision:

⇒ with oven: <

0.3%

⇒ without oven: <

0.7%

Peak area precision: <

1.5%

Page 32

Problems with Reproducibility – Peak Areas

Peak Areas not

Reproducible

Rotor Seal

Seal

Pump

Column

With peak height

• Rotor seal cross-port leak or injection valve not tight

• Piston seal of metering unit leaking

• Needle partially blocked

With retention time

• Variable pump flow rate

Other

• Capillary from injector to detector not tight

• Detector equilibration problems

Page 33

3. Retention Issues

• Retention time changes (t

r

)

• Retention factor changes (k’)

• Selectivity changes (a)

Page 34

Retention time t

R

, Retention factor k’, and

Selectivity factor α

Retention factor k’ = (t

R

-t

0

)/t

0

Selectivity factor α

α = k

2

/k

1

Page 35

Changes in Retention (k) -

Same Column, Over Time

May be caused by:

1. Column aging

2. Column contamination

3. Insufficient column equilibration

4. Poor column/mobile phase combination

5. Change in mobile phase

6. Change in flow rate

7. Change in column temperature

8. Other instrument issues

Page 36

0 20 30

Time (min)

0 10

Time (min)

Mobile Phase Change

Causes Change in Retention

Fresh TFA Added to

Mobile Phase

60% MeOH: 40% 0.1%TFA

• Volatile TFA evaporated/degassed from mobile phase.

Replacing it solved problem.

• Chromatography is from a protein binding study and

peak shape as expected.

Page 37

Separation Conditions That

Cause Changes in Retention*

Flow Rate ± 1% ± 1% t

r

Temp ± 1° C ± 1 to 2% t

r

%Organic ± 1% ± 5 to 10% t

r

pH ± 0.01% ± 0 to 1% t

r

*excerpted from “Troubleshooting HPLC Systems”, J. W. Dolan and L. R. Snyder, p 442.

Page 38

Determining the Cause of Retention

Changes

Same Column

1. Determine k’, a, and t

r

for suspect peaks

2. Wash column

3. Test new column - note lot number

4. Review column equilibration procedures

5. Make up fresh mobile phase and test

6. Check instrument performance

Page 39

Change in Retention/Selectivity

Column-to-Column

1. Different column histories (aging)

2. Insufficient/inconsistent equilibration

3. Poor column/mobile phase combination

4. Change in mobile phase

5. Change in flow rate

6. Other instrument issues

7. Slight changes in column bed volume (t

r

only)

Page 40

Example Change in Retention/Selectivity

Mobile Phase Variation

Column-to-Column

“I have experimented with our mobile phase, opening new bottles of all

mobile phase components. When I use all fresh ingredients, the problem

ceases to exist, and I have narrowed the problem to either a bad bottle of

TEA or phosphoric acid. Our problem has been solved.”

Column 1

Column 2 - Fresh

mobile phase

Column 2

0 4 6

Time (min)

0 2 3 4 5 6 7

Time (min)

0 4 6

Time (min)

Page 41

Minimize Change in Retention/Selectivity

Lot-to-Lot

1. All causes of column-to-column change*

2. Method ruggedness (buffers/ionic strength)

3. pH sensitivity (sample/column interactions)

*All causes of column-to-column change should be considered first,

especially when only one column from a lot has been tested.

Evaluate:

Page 42

0 2 4 6 8 10 12 14 16 18

Time (min)

2-Base

3

4-Base

1

0 2 4 6 8 10 12 14 16 18

Time (min)

2

3

4

1

Lot-to-Lot Selectivity Change - pH

• pH 4.5 shows selectivity change from lot-to-lot for basic compounds

• pH 3.0 shows no selectivity change from lot-to-lot, indicating silanol

sensitivity at pH 4.5

• Evaluate several pH levels to establish most robust choice of pH

pH 4.5 - Lot 1

pH 3.0 - Lot 1

pH 4.5 - Lot 2

pH 3.0 - Lot 2

0 2 4 6 8 10 12 14 16 18

Time (min)

2-Base

3

4-Base

1

0 2 4 6 8 10 12 14 16 18

Time (min)

2

3

4

1

Page 43

Problems with Reproducibility – Peak Areas

Peak Areas not

Reproducible

Rotor Seal

Seal

Pump

Column

With peak height

• Rotor seal cross-port leak or injection valve not tight

• Piston seal of metering unit leaking

• Needle partially blocked

With retention time

• Variable pump flow rate

Other

• Capillary from injector to detector not tight

• Detector equilibration problems

Page 44

Problems with Reproducibiliy – Retention Time

Retention Times not Reproducible

• Pump Problems

–Mobile phase composition problems

–Valves AIV, ball valve defective

–Flow rate problems

•Column Oven Problems

–Temperature fluctuations

•Other

–Column equilibration

–Column deterioration

Page 45

Autosampler

Principle of Operation

From pump

To column

Metering device

Rheodyne 7750

Sampling

unit

To waste

Vial gripper

Standard loop volume300µl

Total delay volume 300µl + Vinj

Minimal (bypass) delay volume 6.2ul

O 4-port rotor seal

Widest dynamic injection range:

0.1 µl-1.5 ml (w/addt'l hardware)

Page 46

Evaluate Retention Changes

Lot-to-Lot

1. Eliminate causes of column-to-column

selectivity change

2. Re-evaluate method ruggedness - modify method

3. Determine pH sensitivity - modify method

4. Classify selectivity changes

5. Contact manufacturer for assistance

*

Agilent Column Support: 800-227-9770, option 4, option 2 (LC columns)

Page 47

1. High pressure

2. Undesirable peak shape

3. Changes in retention/selectivity

These problems are not always

associated with the column and may

be caused by instrument and

experimental condition issues.

Conclusions

HPLC column problems are evident as:

Page 48

The End – Thank You!

Agilent LC Column Tech Support: 800-227-9770 #4, #2 Email:

[email protected]

Page 49

Separation Fundamentals

Agilent Restricted

December 11, 2007

Agilent LC Columns and Agilent J&W GC

Columns Scientific Technical Support

800-227-9770 (phone: US & Canada)

*

302-993-5304 (phone)

For LC columns

Select option 4, then option 2

For GC Columns

* Select option 4, then option 1.

www.agilent.com/chem

Page 50

Separation Fundamentals

Agilent Restricted

December 11, 2007

Looking for More Information on Agilent’s LC Systems

and Software?

Agilent offers a full range of LC training courses including hands-on

courses with the latest 1200 series equipment including Rapid

Resolution, and additional

1100 series courses.

Each course includes a course manual for future reference and a

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are taught by industry experts.

Call 800.227.9770, Option 5 or visit

www.agilent.com/chem/education

to register today!

Page 51

Separation Fundamentals

Agilent Restricted

December 11, 2007

New On Demand Webinar:

Utilizing Sub

-

Two Micron Particles to Optimize

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A Four Part Workshop on Managing Chemistry and Pressure for Fast

er

and More Efficient HPLC Separations

Be sure to register after today

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Page 52

Separation Fundamentals

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Upcoming LC e-Seminars

New Technology for HPLC Environmental Assay

January 22, 2008 – 1:00 pm EST

A Look at Column Choices- Series 3

February 13, 2008 – 1:00 pm EST

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March 18, 2008 – 2:00 pm EST