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HPLC troubleshooting
Troubleshooting
There is no standard troubleshooting procedure.

General Pattern:
 Locate the problem by ranking possible causes.
 Verify the presence of the most probable cause.
 If present – fix the problem, otherwise verify the existence of
the next possible cause.

First try to distinguish

System problem or Method problem

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HPLC System Components

•Pump

•Injector/ Autosampler

•Column

•Detector

•Data System/Integrator

Problems Can be related to all components in the system

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Method vs. System Troubleshooting
System Method
Parameters Parameters
• Flow stability • Flow rate
• Backpressure • Eluent composition
• Clogging • pH &pH modifier (type)
• Detector problems • Injection volume
• Injection suitability • Temperature
• Gradient profile

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 System Parameters
 Simple preliminary verification of system setup can save time.

Solvent Degasser Pump Autosampler Column Detector

Bottle fill-in Flush if Backpressur Vial fill-in Column type Wavelength
solvent e Flow connections connections
Inlet filter
change >15 stability cross-
date mL Check-valves contamination

Critical connections. Minimize tubing length

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Categories of Column and System Problems

I. Pressure

II. Peak shape

III. Retention

IV. Detection
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 I. Pressure Issues
Column observations Potential Problems

High pressure - Plugged frit

- Column contamination
- Plugged packing
Low Pressure - Leak
- Flow Incorrect

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Determining the Cause and Correcting
High Back Pressure
Many pressure problems are due to blockages in the system.

If Column pressure is high:

• Back flush column – Clear “dirty” frit surface
• Wash column – Eliminate column contamination
and plugged packing
➢– high molecular weight/adsorbed compounds
➢– precipitate from sample or buffer

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Column Cleaning
Flush with stronger solvents than your mobile phase
Use at least 25 mL of each solvent for analytical columns

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*

* When using either Hexane or Methylene Chloride the column must be flushed with
Isopropanol before returning to your reversed-phase mobile phase.
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Prevention Techniques for column problems
Use column protection
➢In-line filters

➢Guard columns

• Filter samples

• Filter buffered mobile phases

• Sample clean-up (i.e. SPE)

• Appropriate column flushing

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II. Peak Shape Issue
What Are Common Peak Shape Issues?

1. Split peaks

2. Peak tailing

3. Broad peaks
• Many peak shape issues are also combinations - i.e. broad and
tailing or tailing with increased retention

•Symptoms do not necessarily affect all peaks in the chromatogram

•Each of these problems can have multiple causes

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Peak Splitting Caused By Disrupted Sample Path

•Flow Path Disrupted by Void

•Sample Allowed to Follow Different Paths
Through Column
•Poorly Packed Bed Settles in Use
•High pH Dissolves Silica

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Split Peaks from Column Contamination

•Column: StableBond SB-C8, 4.6 x 150 mm, 5 μm 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. Chlorpheniramine

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Split Peaks from Injection Solvent Effects
Sample diluent:
1- 50/50 MeOH/Water 50/50 Buffer/MeOH
2 - 80/20 MeOH/Water Buffer: 20 mM Citrate, pH=4.6
3 - 90/10 MeOH/Water
4 - 95/5 MeOH/Water
5 - 100 MeOH

1
2
3
4
5
Incompatible solvents may cause sample precipitation and column clogging
Different eluent pH and composition may cause peak splitting

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Peak Tailing, Broadeningand Loss of Efficiency

May be caused by:

➢Column “secondary interactions”

➢Column contamination

➢Column aging

➢Column loading

➢Extra-column effects

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 Peak Tailing
Identifying Column “Secondary Interactions”
Column: Alkyl-C8, 4.6 x 150 mm, 5μm 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. 5. Phenteramine

10 mM TEA
No TEA USP TF (5%)
USPTF(5%)
1. 1.19
1. 1.29
2. 1.18
2. 1.91
3. 1.20
3. 1.63
4. 1.26
4. 2.35
5. 1.14
5. 1.57

Tip: Mobile phase modifier (TEA) competes with Sample for surface ion exchange sites
at mid-range pH values

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 Low pH Minimizes “Secondary Interactions”
for Amines
Column: Alkyl-C8, 4.6 x 150 mm, 5μm 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

pH 3.0
pH 7.0 USP TF (5% )
USP TF (5% ) 4. 1.33
4. 2.35

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

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 Peak Tailing - Column Contamination

Trick: Reverse Column and Run Sample –If Improved, Possible Cleaning Will Help -No
improvement-Column Damaged and Needs to be Replaced

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 Peak Shape: Fronting Peaks

Symmetry < 0.9

Causes:
Column Overload
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Column Overloading

1 ml

5
ml

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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.
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 Unknown “Phantom” Peaks
Column: Extend-C18, 4.6 x 150 mm, 5 μm 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

Sample a: Chlorpheniramine maleate Plates

Peak 1: Chlorpheniramine maleate 1. 5922
2. 9879
3. 779
Sample b : Chlorpheniramine
Maleate and Pseudoephedrine
Peak 1: Chlorpheniramine maleate
Peak 2: pseudoephedrine
Peak 3: chlorpheniramine (from 1st
injection)

Tip: The extremely low plates for moderately retained peaks are an indication of a very
late eluting peak from a preceding run.
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III. Changes in Retention Time
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

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III. Changes in Retention Time
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 24

III. Changes in Retention Time
Dependencies of Analyte Retention on the pH of the Mobile
Phase

RNH3+ RCOOH
RCOO- Base RNH2 Acid
pH=2.3 Base pH=2.3
Acid
pH=6.0 pH=6.0
Absorbance

5 10 15
Retention Time (min.)
Ionization in general decreases hydrophobicity causing a decrease of HPLC
retention.
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III. Changes in Retention Time
Effect of pH on Acidic Analyte Retention
HPLC Conditions: M obile phase
Column – Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm 30% Acetonitrile
Flow rate – 1.0 mL/min 70% 20mM Na2HPO4 buffer; variable pH
Detection – UV 220nm acidic modifier = perchloric acid
Injection volume – 1mL

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III. Changes in Retention Time
Effect of pH on Basic Analyte Retention
Chromatographic Conditions
Column: 15 cm x 0.46 cm Zorbax Eclipse XDB-C18 Eluent: 90% Aqueous / 10% MeCN
Buffer: 10 mM Na 2 HPO4 •7H2 O + xH3 PO4 Flow rate: 1 ml/min
Temp: 25o C

A : P y rid in e , pK a
= 5.17
B : 2 -P ic o lin e , p K = 5 .9 6
a

C : 2 , 4 L u t id in e , p K = 6 .7 4
a

D : 4 - e t h y lp yr id in e p K = 5 .8 7
a

E : P h e n y l e th y l a m i n e p K a
=9 .83
F : 2 , 3 d im e t h y l a n i l i n e p K = 4 .7
a

B
C

D
F

pH = 8.04 E

C
B
A

D F

E
pH =7 .04

B C

D F
E

pH =6 .03

B + C C

A D
E
F

p H = 5 .0 8

B + C

A
E
D
F
pH = 4.04
A + B
C

E
p H = 3.0 5 F

C
B

A D

E F
pH = 2.04

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Importance of pH and Buffers
➢ pH is an effective tool for adjustment of selectivity and retention

➢ pH can be used to optimize the resolution

➢ Reversed phase packaging are most stable between pH’s 2 - 8.

➢ Don’t Forget - Match Column to pH of mobile phase for maximum

column lifetime.

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IV. Detection Issues

Recognize Where the Problem Originates

➢ Is it a consequence of technique?

➢ Is It expected due to use of certain mobile phase components?

➢ Can it be corrected by adjusting detector parameters?

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Drifting Baselines

➢ Detector (UV) not set at absorbance maximum

but at slope of curve

➢ Gradient Elution

➢ Temperature Unstable (Refractive Index Detector)

➢ Contamination in Mobile Phase

➢ Mobile Phase Not in Equilibrium with Column

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Baseline Noise

➢ Mobile phase contaminated, deteriorated, or prepared

from low-quality materials

➢ Mobile phase solvents immiscible

➢ Air trapped in system

➢ Air bubbles in detector

➢ Detector cell contaminated (evensmall amounts of

contaminants can cause noise)

➢ Weak detector lamp

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Chromatographic Results with “Wrong” Lamp at 214 nm
Wavelength

OEM Lamp

Lamp from Generic Source

Tip: Could also be a symptom of aging lamp

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Expanded View of Chromatographic ResultsGeneric Source
Lamp at 214 nm Wavelength
Peak 1 S/N = 150
Peak 2 S/N = 400
OEM Lamp Peak 3 S/N = 300

OEM Lamp

Peak 1 S/N = 15
Peak 2 S/N = 50
Peak 3 S/N = 50

Lamp from Generic Source

Tip: Poor S/N makes it difficult to detect low level impurities

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Conclusions
HPLC column problems are evident as
▪ 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 34
 ‫َّ‬ ‫َ‬ ‫ْ‬ ‫َّ‬ ‫ْ ه‬
‫ّللا الرحم ِن الر ِح ِيم‬
‫ِبس ِم ِ‬

‫وح ِم ْن أَْم ِر َرِّب َوَما‬

‫وح قُِل الُّر ُ‬ ‫ك َع ِن ُّ‬
‫الر ِ‬ ‫َويَ ْسأَلُونَ َ‬
‫ل‬ ‫ي‬‫ِ‬
‫أُوتِيتُم ِّمن الْعِْلِم إِالَّ قَل ا‬

‫‪35‬‬