hplc column troubleshooting - agilent€¦ · 4 soc 6 c 12-oc j.c. 268(1983) 1 j.c. 111(1975) 149...

HPLC Column Troubleshooting

Gulf Coast Conference 2014

John Palmer

Pump

Injector/Autosampler

Column

Detector

Data System/Integrator

HPLC System Components

Problems Can Be Related to All Components in the System

There Can Be Many Causes Start Asking Questions That Lead to The Cause

•1st Did System Suitability or the Sample Fail?

•2nd Review Method for Compliance - Is The Procedure Being Followed Properly?

- Are Instrument Settings Correct?

•3rd Ask More Questions! - When Did the System Last Function Properly?

- Has Anything Been Changed?

•4th Review ALL parameters! - The Obvious Is Not Always the Cause

- Was There More Than One Change?

Categories of Column and System Problems

A. Pressure

B. Peak shape

C. Retention

Column Observations Potential Problems

High pressure - Plugged frit

- Column contamination

- Plugged packing

Low Pressure - Leak

- Flow Incorrect

Pressure Issues

Determining the Cause and Correcting High Back Pressure

• Check pressure with/without column

• Remove Guard

• Remove Column

• If Column pressure is high:

• Back flush column – Clear “dirty” frit surface • • Wash column – high molecular weight/adsorbed compounds – precipitate from sample or buffer

• Replace Column

Change frit – Clear plugged frit PREVENT THIS!

Peak Splitting Caused By Disrupted Sample Path

Split or Double Peaks

Normal Double

Peaks

Tip: Similar Effect Can be Caused by Partially Plugged Frit

•Flow Path Disrupted by Void

•Sample Allowed to Follow Different Paths

Through Column

•Poorly Packed Bed Settles in Use

•High pH Dissolves Silica

Split Peaks from Injection Solvent Effects

September 28, 2014Month

##, 200X

Group/Presentation Title

Agilent Restricted

Column: StableBond SB-C8, 4.6 x 150 mm, 5 mm Mobile Phase: 82% H2O : 18% ACN

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

A. Injection Solvent

100% Acetonitrile

B. Injection Solvent

Mobile Phase

Tip: Injecting in a solvent stronger than the mobile phase can cause peak shape

problems such as peak splitting or broadening

Trick: Keep Organic Concentration in Sample Solvent < Mobile Phase

0 10Time (min)

1

2

0 10Time (min)

1

2

Split Peaks from Column Contamination

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)

Column: StableBond SB-C8, 4.6 x 150 mm, 5 mm Mobile Phase: 60% 25 mM Na2HPO4, 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

Tip: Column washing eliminates the peak splitting, which resulted from a contaminant on the column

How could this be prevented? (Guard Column, SPE clean up of samples, Periodic column wash)

Peak Tailing - Column Contamination

0.0 2.5 5.0

2

41

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)

Column: StableBond SB-C8, 4.6 x 250 mm, 5mm Mobile Phase: 20% H2O : 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

Tip: Quick Test to Determine if Column is Dirty or Damaged

Trick: Reverse Column and Run Sample –If Improved, Possible Cleaning Will Help

- No improvement - Column Damaged and Needs to be Replaced

Column Cleaning

Use at least 25 mL of each solvent for long analytical columns

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% Acetonitrile

• 75% Acetonitrile:25%

Isopropanol

• 100% Isopropanol

• 100% Methylene Chloride*

• 100% Hexane*

Must Reverse

to

Re-Equilibrate

This Is Time Consuming

Often Performed Offline

Changing a Frit May Not Be a Good Idea May not be possible with new generation columns

May damage high performance columns

Column

Inlet Frit

Compression

Ferrule

Column Body

Female End Fitting

Male End Fitting

Wear gloves

Do not allow bed to dry

Do not touch the column -

body heat will extrude packing

Do not overtighten

Tip: Prevention is a Much Better Idea!

The Trick: Prevention Techniques - A Better Choice!

• Filter samples

• Filter buffered mobile phases

• Use column protection

- In-line filters

- Guard columns

• Sample clean-up (i.e. SPE)

• Appropriate column flushing

Easy

Not As Easy

Inexpensive Filters Prevent Column Frit Plugging

Regenerated Cellulose (RC) Recommended •Universal hydrophilic membrane, compatible

with most solvents - aqueous and organic

•High purity, extremely low extractables and

binding

•More Uniform Surface

•Different than Other Cellulose Filters!!

In-line Filters Easy to Use and replace

Frits Available in 0.2,0.5 and 2.0µ Porosity

Much Less expensive than a Column

Easier and Faster to Replace than a Column Frit

Mini-UniPrep Filtering Vials

•Pre-assembled filtration devices for removing particulate matter from samples.

•Single disposable unit can replace the combination of syringe filters, syringes, auto-sampler vials, transfer containers, septa and caps.

•Quick, environmentally conservative way to filter samples prior to HPLC analysis.

Manufactured by Whatman, a division of GE Healthcare

Separation Conditions and the Effect on Retention*

16

Flow Rate +/- 1% +/- 1% Tr

Temp +/- 1 deg C +/- 1 to 2% Tr

%Organic +/- 1% +/- 5 to 10% Tr

pH +/- 0.01% +/- 0 to 1% Tr

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

Mobile Phase: Aqueous Component Experimental Variables That Impact Resolution

Column

Mobile Phase

• Aqueous Component - Importance of Buffers

- Considerations for Buffer Selection

- Buffer pH

- Buffer Concentration

• Organic Component

Sample

Instrument

Gradient Separations

September 28, 2014

CI0126C

17

Your opportunity to improve

robustness and ruggedness

Mobile Phase pH and pH Buffers Why Are These So Important in HPLC?

•pH Effects Ionization

- Silica Surface of Column

- Sample Components of Interest

• Buffers

- Resist Changes in pH and Maintain Retention

- Improve Peak Shape for Ionizable Compounds

• Effects Column Life

- Low pH strips Bonded Phase

- High pH Dissolves Silica

Why Worry About pH? pH, pKa and Weak Bases

At pH 9 – the sample exists as protonated and unprotonated diphenhydramine

in a ratio of 1:1. Peak shape can be poor.

At pH 10 – 91% of the sample exists as unprotonated diphenhydramine.

At pH 8 – 91% of the sample exists as protonated diphenhydramine.

Ka = [R3N][H+]

[R3NH+]

Ka = 1 x 10-9

pKa = 9

R3NH+ R3N + H+

CHOCH CH N2 2

CH3

CH3

+

HCH

3

CH3

CHOCH CH N2 2 + H+

Why Worry About pH? pH, pKa and Weak Acids

At pH 4.2 – the sample exists as benzoic acid and the benzoate ion in a ratio

of 1:1. Peak shape can be poor

At pH 5.2 – 91% of the sample exists as the benzoate ion. RP retention decreases.

At pH 3.2 – 91% of the sample exists as benzoic acid. RP retention increases.

RCOOH RCOO- + H+

Ka = 6.4 x 10-5

pKa = 4.2

Ka = [RCOO-][H+]

[RCOOH]

+ H+

COOH COO _

pH vs. Selectivity for Acids and Bases

5

SCD

+

+

2 +

1

1.5

1.0

0.5

0.0

-0.5

log

k«

3 4 5 6 7 8 pH

A B C

2

4

5

3

3 5 7 9

ELUENT pH

40

30

20

10

10

Ret

entio

n

+++

3

17,12 - OC

UDC

SOC4

6

C12-OC

J.C. 268(1983) 1

J.C. 111(1975) 149

Column: Nucleosil-C18

Mobile Phase: 45% ACN/55% phosphate buffer

Sample: Bile Acids

Column: mBondapak-C18

Mobile Phase: 60% 25 mM phosphate buffer

40% Methanol

1. Salicylic acid

2. Phenobarbital

3. Phenacetin

4. Nicotine

5. Methamphetamine

• Retention and selectivity can change dramatically when pH is changed.

Why Use Buffered Mobile Phases?

September 28, 2014

CI0126C

22

A = pH 7.0

water A = pH 7.0, 25 mM phosphate

buffer

Column: ZORBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm, 3.5 µm Mobile Phase: 44% A : 56% methanol

Flow Rate: 1.0 mL/min Temperature: 25°C Detection: UV 250 nm

Sample: 1. ketoprofen 2. ethyl paraben 3. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol 7. ibuprofen

• Buffered mobile phases enhance retention, resolution, and peak shape.

0 1 2 3 4 5Time (min)

1

2

3

4

6

7

5

0 1 2 3 4

1, 4, 6, 7

2

3

5

Time (min)

Considerations For Buffer Selection

Buffer Type

• Inorganic vs. organic buffers—choice can affect resolution, column lifetime and MS compatibility

Buffer pH

• Select buffer based on desired pH and optimum buffer pH range.

• Measure pH of buffer solution before mixing with organic modifier.

• Compare resolution at desired pH ± 0.1–0.2 pH units. Buffer Concentration and Ionic Strength

• Start at 20 – 25 mM.

• Prepare buffer according to accepted procedures.

• Avoid overshoot and readjustment when setting pH.

• Compare resolution at desired buffer concentration ± 5–10 mM.

September 28, 2014

CI0126C

23

Changes in Buffer Concentration Can Affect Retention, Peak Width and Peak Shape

September 28, 2014

CI0126C

24

USP Tf

(5%)

1. 1.62

2. 1.65

3. 1.63

4. 1.77

5. 1.83

6. 1.12

0.0 0.02.5 2.55.0 5.0

6

6

5

54

4 3

3

2

2

1

1

7.5

Time (min) Time (min)

10 mM Phosphate 25 mM Phosphate

Column: ZORBAX Eclipse XDB-C8, 4.6 x 150 mm, 5 µm

Mobile Phase: 40% phosphate buffer (pH 7.0) : 60% ACN Flow Rate: 1.5 mL/min Temperature:

40°C

Sample: Tricyclic Antidepressants, 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6.

Trimipramine

USP Tf

(5%)

1. 1.41

2. 1.50

3. 1.33

4. 1.39

5. 1.36

6. 1.00

Effect of pH on Peak Shape at or Near the Sample pKa

0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10

Time (min) Time (min)

Column: ZORBAX SB-C8 4.6 x 150 mm, 5 mm Mobile Phase: 40% 5 mM KH2PO4: 60% ACN

Flow Rate: 1.0 mL/min. Temperature: RT

pH 4.4 pH 3.0

Ibuprofen

pKa = 4.4

CH3CHCOOH

CH2CH(CH3)2

• Inconsistent and tailing peaks may occur when operating close to an analyte

pKa and should be avoided.

Lot-to-Lot Selectivity Change Related to pH Choice


##, 200X


Agilent Restricted

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

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

• pH 3.0 shows no selectivity change from lot-to-lot

• Indication of poorly controlled ionization

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

Test for pH Robustness

September 28, 2014

CI0126C

27

1 2

3

4

6 7

5

0 1 2 3 4 5 6

Time (min)

0 1 2 3 4 5 6

1 2

3

4

6

7

5

Time (min)

pH 7.00

pH 7.25

Column: ZORBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm, 3.5 µm

Mobile Phase: 44% 25 mM phosphate, pH 7.00 : 56% methanol Flow Rate: 1.0 mL/min Temperature: 25°C

Detection: UV 250 nm

Sample: 1. ketoprofen 2. ethyl paraben 3. Hydrocortisone 4. fenoprofen 5. propyl paraben 6. Propranolol 7.

ibuprofen

• The resolution of ionizable compounds can change

markedly with pH changes—even as small as 0.05–

0.25 pH units.

Don’t Forget - Match Column to pH of Mobile Phase for Maximum Column Lifetime low pH and high temperature (pH 0.8, 90°C)

000023P1.PPT

Purge Solvent:

50% methanol/water with

1.0% TFA

Solute: Toluene

Kirkland, J.J. and J.W. Henderson, Journal of Chromatographic Science, 32 (1994) 473-480.

Don’t Forget - Match Column to pH of Mobile Phase for Maximum Column Lifetime High pH and Room Temperature (pH 11 RT)

Tip: Use Columns Designed for chosen pH

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

(~ pH 11)

After 30 injections

Initial

Mobile Phase: Organic Component

Column

Mobile Phase • Aqueous Component

• Organic Component

- % Organic Modifier

Sample

Instrument


September 28, 2014

CI0126C

30

Even a Small Change in % Organic Modifier Can Change Resolution

September 28, 2014

CI0126C

31

12

34

5

6

0 1 2 3 4

Time (min)

0 1 2 3 4

12

34

5

6

Time (min)

1 2

34

5

6

0 1 2 3Time (min)

4

• Verify that resolution doesn’t change

significantly around desired

conditions (for example, %B ± 1–

2%).

59% MeOH

57.5% MeOH

56% MeOH

Column: ZORBAX Rapid Resolution Eclipse

XDB-C8

4.6 x 75 mm, 3.5 µm

Mobile Phase: A: 25 mM phosphate, pH

7.00 (10 mM TEA)

B: methanol (10 mM TEA)

Flow Rate: 1.0 mL/min

Temperature: 25°C controlled

Injection: 5 mL

Detection: 275 nm

Sample: 1. ketoprofen 2. ethyl paraben 3.

hydrocortisone

4. fenoprofen 5. propyl paraben 6.

propranolol

Rs (5,6) =

2.5

Rs (5,6) =

1.7

Rs (5,6) =

1.2

The Sample Experimental Variables That Impact Resolution

Column

Mobile Phase

Sample

• Injection volume

• Sample solvent

strength

Instrument


September 28, 2014

CI0126C

32

Injection Volume and Sample Solvent Strength

Injection Volume

• Lack of ruggedness typically seen

– when Vinj is increased to improve signal-to-noise (S/N) ratio, or,

– when column size is decreased.

• Use minimum Vinj for required repeatability and limit of detection.

• Compare resolution, peak shape and repeatability at 0.2X, 1X and 2–5X Vinj.

Sample Solvent Strength

• Match % organic modifier in mobile phase (or weaker).

• If stronger sample solvent needed (solubility, stability), keep Vinj to

minimum.

• Compare resolution, peak shape and width at desired solvent strength ±50% relative.

September 28, 2014

CI0126C

33

Test For Injection Volume Robustness

September 28, 2014

CI0126C

34

0 1 2 3 4 5

0 1 2 3 4 5

1 2

3

4 5

T im e (m in)

6 7

1 2

3

45

6

7

1

2

3

45

6

7

0 1 2 3 4 5

1 µL

5 µL

Column: ZORBAX Rapid Resolution Eclipse XDB-C8 4.6 x 75 mm, 3.5 µm

Mobile Phase: 44% 25 mM phosphate,

pH 7.00

56% methanol

Flow Rate: 1.0 mL/min

Temperature: 25°C


Sample: 1. ketoprofen 2. ethyl paraben 3. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol 7. ibuprofen

Rs(6,7) =

2.5

Rs(6,7) =

2.1

Rs(6,7) =

1.6

10 µL

• Varying injection volume can sometimes reveal lack of robustness for resolution and peak shape.

Peak Tailing, Broadening and Loss of Efficiency

May be caused by:

• Column “secondary

interactions”

• Column contamination

• Column aging

• Column loading

• Extra-column effects

Peak Shape: Tailing Peaks

Normal Tailing

Normal Tailing

Symmetry > 1.2

All Peaks Tail:

Extra-Column Effects.

Build up of Contamination on Column

Inlet.

Bad Column.

Causes

Some Peaks Tail:

Secondary - Retention Effects.

Residual Silanol Interactions.

Small Peak Eluting on Tail of Larger Peak.

Peak Tailing

Identifying Column “Secondary Interactions”

Tip: Mobile phase modifier (TEA) competes with Sample for surface ion exchange

sites at mid-range pH values

Column: Alkyl-C8, 4.6 x 150 mm, 5mm Mobile Phase: 85% 25 mM Na2HPO4 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

TIme (min) Time (min)

0.0 2.5 5.0

54

32

1

5

4

3

2

1

0.0 2.5 5.0

Peak Tailing

Low pH Minimizes “Secondary Interactions” for Amines

Column: Alkyl-C8, 4.6 x 150 mm, 5mm Mobile Phase: 85% 25 mM Na2HPO4 : 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

Tip: Reducing mobile phase pH reduces interactions with silanols and peak tailing.

Time (min)

0.0 2.5 5.0

54

32

1

5

4

32

1

Time (min)

0.0 2.5 5.0

Peak Tailing

High pH Eliminates “Secondary Interactions” for Amines

Peak Shape and Retention of this sample of basic compounds improves at high pH where column has high IEX activity. Why?

0 5

1

2,3

4

5

Time (min)

7

6

0 5 10

1

2

3

4

Time (min)

6

5

7

Column: ZORBAX Extend-C18, 4.6 x 150 mm, 5 m m Mobile Phase: See Below Flow Rate: 1.0 mL/min Temperature: RT


Sample: 1. Maleate 2. Scopolamine 3. Pseudoephedrine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. Diphenhydramine

pH 7

30% 20 mM Na2HPO4

70% MeOH

pH 11

30% 20 mM TEA

70% MeOH

tR = 8.5 tR = 11.4

40

Peak Shape: Broad Peaks

All Peaks Broadened:

• Loss of Column Efficiency.

• Column Void.

• Large Injection Volume.

Some Peaks Broadened:

• Late Elution from Previous Sample

(Ghost Peak).

- High Molecular Weight.

- Sample - Protein or Polymer.

Unknown “Phantom” Peaks

Time (min)

3

1

2

0 5 10 15

Column: Extend-C18, 4.6 x 150 mm, 5 mm Mobile Phase: 40% 10 mM TEA, pH 11 : 60% MeOH Flow Rate: 1.0 mL/min

Temperature: R.T. Detection: UV 254 Sample: 1. Maleate 2. Pseudoephedrine 3. Chlorpheniramine

Plates

1. 5922

2. 9879

3. 779

Tip: The extremely low plates for moderately retained peaks are an indication of a

very late eluting peak from a preceding run.

Time (min)

1

0 5 10

Sample 1: Chlorpheniramine maleate

Peak 1: maleate

Sample 2 : Chlorpheniramine

maleate

and Pseudoephedrine

Peak 1: maleate

Peak 2: pseudoephedrine

Peak 3: chlorpheniramine (from 1st

injection)

“Phantom” peak from

first injection

Changes in Retention Can Be Chemical or Physical

May be caused by:

• Column aging

• Column contamination

• Insufficient equilibration

• Poor column/mobile phase combination

• Change in mobile phase

• Change in flow rate

• Different Gradient Delay Volumes

Column Aging/Equilibration Causes Retention/Selectivity Changes


##, 200X


Agilent Restricted

Column 1 - After Cleaning

with 1% H3PO4

/Equilibration

• The primary analyte was sensitive to mobile phase aging/

conditioning of the column

• The peak shape was a secondary issue (metal chelating

compound) resolved by “de-activating” the active metal

contamination

Column 1 - Next Day Column 1 - Initial

0 3 5 9 12 15Time (min)

2

1

0 3 5 9 12 15Time (min)

2

1

Column Temperature Adequate Temperature Control is Essential

Laboratory temperatures can vary by ±5°C or more. Column temperature changes affect resolution and repeatability. Useful tool for changing selectivity, retention and efficiency when developing separations. Important parameter to control during method development and validation. Compare resolution, peak width and peak shape at desired temperature ±5°C.

September 28, 2014

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44

Small Temperature Changes Can Cause Dramatic Changes in Resolution

September 28, 2014

CI0126C

45

12

34 6

5

0 0.5 1 1.5 2 2.5 3 3.5

Time (min)

0 0.5 1 1.5 2 2.5 3 3.5

12

34 6

5

Time (min)

0 0.5 1 1.5 2 2.5 3 3.5

1 2

34 6

5

Time (min)

Column: ZORBAX Rapid Resolution Eclipse XDB-C8 4.6 x 75 mm, 3.5 µm

Mobile Phase: Isocratic, 28%B : 72%A

A: 5/95 methanol/pH 7.00 buffer 25 mM, 10 mM TEA

B: 80/20 methanol/pH 7.00 buffer 25 mM, 10 mM TEA

Flow Rate: 1.0 mL/min.

Temperature: See Figure

Injection: 5 mL

Detection: 275 nm

Sample: 1. ketoprofen 2. ethyl paraben 3. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol

20°C

25°C

30°C

• Column temperature

control will produce the

most consistent

results.

CASE STUDIES

Example: Change in Retention/Selectivity


##, 200X


Agilent Restricted

Unintended Mobile Phase Variation

Tip: The Source of the Problem is Often Not the Obvious Change

“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 6Time (min)

0 2 3 4 5 6 7

Time (min)

0 4 6Time (min)

Case History: Problem – Selectivity Does Not Appear the Same from Column-to-Column

Details:

• 3 Columns with the Same Bonded Phase were used

• They were the same dimensions, just different particle sizes (and therefore

different lots of material)

• They were tested on the same day on the same instrument and with the

same mobile phase

• Isocratic elution; binary (two channel) pump generated mobile phase

Problem:

• The selectivity was different on each of the columns

48

49

Selectivity between particle sizes of Eclipse Plus C18, 4.6 x 50 mm, using same test solutes

John W. Henderson JrGroup/Presentation

Title

Agilent Restricted Month ##, 200X

min 0 1 2 3 4 5

mAU

-5

0

5

10

15

20

25

DAD1 B, Sig=280,16 Ref=360,20 (F:\EPC18SELECTIVITY\ANALINE000015.D)

0.2

93

0.3

60

0.7

12

0.9

61

1.2

29

1.6

35

3.9

07

min 0 1 2 3 4 5

mAU

-5

0

5

10

15

20

25

DAD1 B, Sig=280,16 Ref=360,20 (F:\EPC18SELECTIVITY\3P5MICRON200001.D)

0.0

17

0.3

00

0.3

69

0.7

25

0.9

96

1.2

90

1.7

22

4.1

98

min 0 1 2 3 4 5

mAU

-5

0

5

10

15

20

25

DAD1 B, Sig=280,16 Ref=360,20 (F:\EPC18SELECTIVITY\ANALINE000019.D)

0.2

99

0.7

84

1.0

71

1.3

89

1.9

00

4.8

97

2 3

5 1 4

6

5 um, UXE01033, NEP0652003

3.5 um, UXF01309, B08111

1.8 um, UXG03882, B08021

m.p.: A: water, B: acetonitrile (60:40 A:B)

Flow : 1.5 mL/min

Temp: 25 C

Detection: UV 280nm,16, ref=360,20

Flow cell: 6mm, 5uL

Data rate: 0.2s

Inj. Vol 2 uL

N=3800

N=7100

N=9900

tr k alpha

uracil 0.3 0.00

aniline 0.78 1.60 #DIV/0!

o-toluidine 1.07 2.57 1.60

4-chloroaniline 1.39 3.63 1.42

2-naphthylamine 1.9 5.33 1.47

3,3'-dichlorobenzidine 4.9 15.33 2.88

tr k alpha

uracil 0.3 0.00


toluidine 1 2.33 1.63

chloroaniline 1.29 3.30 1.41

naphthylamine 1.72 4.73 1.43

dichlorobenzidine 4.2 13.00 2.75

tr k alpha

uracil 0.29 0.00


toluidine 0.96 2.31 1.60

chloroaniline 1.23 3.24 1.40

naphthylamine 1.63 4.62 1.43

dichlorobenzidine 3.91 12.48 2.70

What Questions Should We Ask and What Should We Try to diagnose problem?

Compare more columns – see if they are different

Try another LC

Premix the mobile phase

50

51

One channel premixed mobile phase shows similar α whereas previous two channels doesn’t show similar α!


Title


0 1 2 3 4

mAU

-5

0

5

10

15

20

DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\ONECHANNEL00002.D)

0.2

49

0

.28

8

0.3

52

0.6

96

0.9

46

1.2

14

1.6

15

3.8

79


0.2

88

0.3

53

0.6

95

0.9

44

1.2

13

1.6

12

3.8

73

5 um, UXE01033, NEP0652003

min 0 1 2 3 4 5 6

mAU

-5

0

5

10

15

20

25

30


0.2

90

0.3

51

0.4

17

0.7

14

0.8

48

0.9

71

1.2

41

1.6

68

4.0

73


0.2

87

0.3

48

0.7

13

0.9

70

1.2

41

1.6

66

4.0

72

1.8 um, UXG03882, B08021

one channel tr k alpha

uracil 0.29 0.00


o-toluidine 0.94 2.24 1.63




one channel tr k alpha

uracil 0.29 0.00


o-toluidine 0.97 2.34 1.62




m.p.: A: water, acetonitrile (60:40 v/v)

Flow : 1.5 mL/min

Temp: 25 C


Flow cell: 6mm, 5uL

Data rate: 0.2s

Inj. Vol 2 uL

46 bar

246 bar

52

Repeat experiment of two channel isocratic mobile phase confirms different selectivity probably due to wrong composition of mobile phase being delivered


Title


min 0 1 2 3 4 5 6

mAU

-5

0

5

10

15

20

DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL00005.D) DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL00006.D)

min 0 1 2 3 4 5 6

mAU

-5

0

5

10

15

20

25

DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL00002.D)

0.3

00

0.3

68

0

.38

7

0.7

81

1.0

75

1.4

01

1.9

18

4.9

91

DAD1 B, Sig=280,16 Ref=360,20 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL00003.D)

0.3

00

0.3

67

0

.38

7

0.7

82

1.0

74

1.4

00

1.9

16

4.9

85

5 um, UXE01033, NEP0652003

1.8 um, UXG03882, B08021

45 bar

238bar

m.p.: A: water, B: acetonitrile (60:40 A:B)

Flow : 1.5 mL/min

Temp: 25 C


Flow cell: 6mm, 5uL

Data rate: 0.2s

Inj. Vol 2 uL

two channel (2nd exp) tr k alpha

uracil 0.29 0.00


o-toluidine 0.98 2.38 1.60




two channel (2nd exp) tr k alpha

uracil 0.3 0.00


o-toluidine 1.07 2.57 1.60




Typical Case History: Lack of Efficiency on Poroshell 300

53

Agilent Publication Customer Results

Looked Bad – What Did We Do?

1. Customer needed to understand that the column used – 2.1

x 75mm, Poroshell 300 column---is a low volume column, and

therefore, low column volume guidelines should be followed

2. What about the instrument and sample ?

3. Turns out that instrument was quaternary pump 1100, not set

up appropriately for low volume use (difficult to optimize

completely)

4. Customer provided with details on how to achieve a better

low volume set-up, based on 1100-RRHT Kit documents

5. Customer now achieving acceptable results

54

Conclusions

• High pressure (prevention better than the cure)

• Undesirable peak shape

• Changes in retention/selectivity

Often these problems are not associated with the column and

may be caused by instrument and chemistry issues.

•pH of mobile Phase

•Instrument Connections

•Detector Settings

•Metal Contamination

Start With the Correct Questions

•Find the Answers

•The Answers will Lead to Solutions

HPLC column problems are evident as

APPENDIX

Important Buffer Systems

September 28, 2014

CI0126C

57

Buffer Selection

Buffer Selection

Buffer pKa pH Range UV Cutoff (A > 0.5)

Trifluoroacetic acid <<2 (0.5) 1.5-2.5 210 nm (0.1%)

KH2PO4/phosphoric acid 2.12 1.1-3.1 <200 nm (0.1%)

tri-K-Citrate/hydrochloric acid 1 3.06 2.1-4.1 230 nm (10 mM)

Potassium formate/formic acid 3.8 2.8-4.8 210 nm (10 mM)

tri-K-Citrate /hydrochloric acid 2 4.7 3.7-5.7 230 nm (10 mM)

Potassium acetate/acetic acid 4.8 3.8-5.8 210 nm (10 mM)

tri-K-Citrate /hydrochloric acid 3 5.4 4.4-6.4 230 nm (10 mM)

Ammonium formate 3.8

9.2

2.8-4.8

8.2-10.2 (50 mM)

Bis-tris propane·HCl/Bis-tris propane 6.8 5.8-7.8 215 nm (10 mM)

Ammonium acetate 4.8

9.2

3.8-5.8

8.2-10.2 (50 mM)

KH2PO4/K2HPO4 7.21 6.2-8.2 <200 nm (0.1%)

Tris·HCl/Tris 8.3 7.3-9.3 205 nm (10 mM)

Bis-tris propane·HCl/Bis-tris propane 9.0 8.0-10.0 225 nm (10 mM)

Ammonium hydroxide/ammonia 9.2 8.2-10.2 200 nm (10 mM)

Borate (H3BO3/Na2B4O7·10 H2O) 9.24 8.2-10.2

Glycine·HCl/glycine 9.8 8.8-10.8

1-methylpiperidine·HCl/1-methylpiperidine 10.1 9.1-11.1 215 nm (10 mM)

Diethylamine·HCl/diethylamine 10.5 9.5-11.5

Triethylamine·HCl/triethylamine 11.0 10.0-12.0 <200 nm (10 mM)

Pyrollidine·HCl/pyrollidine 11.3 10.3-12.3

Adapted from Practical HPLC Method Development, 2nd Edition, Snyder, L.R., Kirkland, J.J. and

Glajch, J.L., page 299.

Separation Ruggedness Buffer Preparation

1. Dissolve salt in organic-free water in 1- or 2-L beaker. Use appropriate volume to leave room for pH

adjustment solution. Equilibrate solution to room temperature for maximum accuracy.

2. Calibrate pH meter. Use 2-level calibration and bracket desired pH. Use appropriate audit solution to

monitor statistical control (for example, potassium hydrogen tartrate, saturated solution, pH = 3.56).

3. Adjust salt solution to desired pH. Minimize amount of time electrode spends in buffer solution

(contamination). Avoid overshoot and readjustment (ionic strength differences can arise).

4. Transfer pH-adjusted buffer solution quantitatively to volumetric flask, dilute to volume, and mix.

5. Filter through 0.45 µm filter. Discard first 50 – 100 mL filtrate. Rinse solvent reservoir with small

volume of filtrate and discard. Fill reservoir with remaining filtrate or prepare premix with organic

modifier.

– Agilent Solvent Filtration Kit, 250-mL reservoir, 1000-mL flask, p/n 3150-0577

– Nylon filter membranes, 47 mm, 0.45 µm pore size, p/n 9301-0895 (not for proteins!)

September 28, 2014

CI0126C

58

Using Buffers Successfully Initial Column and System Equilibration

In an appropriate vessel, test highest % organic/buffer ratio to verify

that buffer will not precipitate. With stirring, add organic to buffer

first, not vice versa.

Equilibrate column with, in order:

• 100% organic modifier (if brand new)

• mobile phase minus buffer

• buffered mobile phase containing highest % organic modifier (gradient high end)

• buffered mobile phase containing lowest % organic modifier (gradient low end).

Inject standard or sample several times until RTs stable, or for

gradient methods, precede former with 1 or 2 blank gradients.

September 28, 2014

CI0126C

59

Using Buffers Successfully Shutdown State and Instrument Flushing

Next day use—using same buffers

• Pump mobile phase very slowly (for example, 0.01 – 0.1mL/min). When flushing column or for longer term column storage • Flush with 20/80 organic/water, then 80/20 organic/water or 100%

organic.

Instrument flushing

• Replace column with capillary tubing. Leave disconnected from detector.

• Flush pumps with water, then connect capillary tubing to detector. • Inject water 2-3 times at maximum injection volume setting. • Flush all pumps with 100% organic for long term storage.

September 28, 2014

CI0126C

60

Importance of pH and Buffers A Practical Example

•Why the Sample Dictates Use

•What Happens When Buffer Used Effectively

•What Happens When Buffer Ignored or Used Improperly

Importance of pH and Buffers - A Practical Example Optimized Isocratic Conditions for Cardiac Drugs

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Time (min)

5

4

32

1

0 1 2 3 4 5 6Time (min)

5

4

32

1Column: StableBond SB-C18, 4.6 x 150 mm, 5 mm Mobile Phase: 45% 25 mM NaH2PO4, pH 3.0 55% MeOH Flow Rate: 2.0 mL/min.

Temperature: 35°C


Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine

5. Flunarizine

I Don’t Have Time to Make Buffers or Adjust pH …

0 5 10 15 20 25

Time (min)

Column: StableBond SB-C18 4.6 x 150 mm, 5 mm

Mobile Phase: A: 20% H2O B: 80% MeOH

Flow Rate: 1.0 mL/min.

Temperature: 35°C

UV Detection: 254 nm

Sample: Cardiac Drugs

• Buffers are critical to good retention and peak shape in many separations.

Even at very high % MeOH Most Components

Strongly Retained with Poor peak Shape Due to

IEX at Surface

What If You Work Outside the Buffer Range?

0 5 10 15 20 25

5

1

2

34

Time (min)

Columns: StableBond SB-C18

4.6 x 150 mm, 5 mm

Mobile Phase:A: 30% 25 mM NaH2PO4, pH 4.8 unbuffered B: 70% MeOH Flow Rate: 1.0 mL/min.

Temperature: 35°C

UV Detection: 254 nm

Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine

Unsuitable Peak Shape

hplc column troubleshooting - agilent€¦ · 4 soc 6 c 12-oc j.c. 268(1983) 1 j.c. 111(1975) 149...

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