HPLC Chromatography troubleshooting

HPLC Chromatography

High-Performance Liquid Chromatography (HPLC) has revolutionized the field of analytical chemistry, offering a powerful and versatile tool for separating, identifying, and quantifying compounds in complex mixtures.  High-Performance Liquid Chromatography has become an indispensable tool in various scientific disciplines, contributing significantly to advancements in research, quality control, and diagnostics. Its versatility, sensitivity, and ability to handle complex samples make it a cornerstone in the analytical arsenal of scientists and researchers worldwide. As technology continues to evolve, so too will the capabilities and applications of HPLC, ensuring its continued prominence in the realm of analytical chemistry.

Applications of HPLC:

1. Pharmaceutical Analysis: HPLC plays a crucial role in pharmaceutical research and quality control, enabling the analysis of drug formulations, impurities, and degradation products.

2. Environmental Monitoring: HPLC is employed for the detection and quantification of environmental pollutants, such as pesticides, herbicides, and heavy metals in air, water, and soil samples.

3. Food and Beverage Industry: HPLC is utilized for the analysis of food and beverage products, ensuring the safety and quality of consumables by detecting contaminants and additives.

4. Clinical Diagnostics: In clinical laboratories, HPLC is used to analyze biological samples for the presence of drugs, metabolites, and other biomolecules, aiding in disease diagnosis and monitoring.

5. Biochemical Research: HPLC is a valuable tool in biochemistry for separating and analyzing complex mixtures of proteins, peptides, and nucleic acids.

HPLC Chromatography 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.

Two Types  of HPLC troubleshooting System problems or Method problems

HPLC System Components

•Injector/ Autosampler
•Data System/Integrator

Problems Can be related to all components in the system

Method vs. System Troubleshooting

System Parameters
• Flow stability
• Backpressure
• Clogging
• Detector problems
• Injection suitability

Method Parameters
• Flow rate
• Eluent composition
• pH &pH modifier (type)
• Injection volume
• Temperature
• Gradient profile

System Parameters

Simple preliminary verification of system setup can save time.

Solvent Degasser Pump Autosampler Column Detector
Bottle fill-in Inlet filter date Flush if solvent change >15 mL Backpressures  Flow stability Check-valves Vial fill-in connections cross[1]contamination Column type connections Wavelength

Critical connections. Minimize tubing length

Categories of Column and System Problems

I. Pressure
II. Peak shape
III. Retention
IV. Detection

I. Pressure Issues

Column observations Potential Problems
High pressure – Plugged frit

 – Column contamination

– Plugged packing

Low Pressure – Leak – Flow Incorrect

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

Column Cleaning

Flush with stronger solvents than your mobile phase
Reversed-Phase Solvent Choices in Order to Increasing Strength
➢Mobile phase without buffer salts
➢100% Methanol
➢100% Acetonitrile
➢75% Acetonitrile:25% Isopropanol
➢100% Isopropanol
➢100% Methylene Chloride*
➢100% Hexane*
Use at least 25 mL of each solvent for analytical columns

* When using either Hexane or Methylene Chloride the column must be flushed with Isopropanol before returning to your reversed-phase mobile phase.

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

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

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

HPLC troubleshooting

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

HPLC troubleshooting

HPLC troubleshooting

Peak Tailing, Broadeningand Loss of Efficiency

May be caused by:
➢Column “secondary interactions”
➢Column contamination
➢Column aging
➢Column loading
➢Extra-column effects

HPLC troubleshooting

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

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

HPLC troubleshootingHPLC troubleshooting

HPLC troubleshooting

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.

HPLC troubleshooting

III. Changes in Retention Time
Changes in Retention Can Be Chemical or physical and May be caused by:
➢Column aging
➢Column contamination
➢Insufficient equilibration
➢Poor column/mobile phase combination
➢Change in the mobile phase
➢Change in flow rate

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

Dependencies of Analyte Retention on the pH of the Mobile Phase

Ionization in general decreases hydrophobicity causing a decrease of HPLC retention.

HPLC troubleshooting

HPLC troubleshooting

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 is most stable between pH’s 2 – 8.
Don’t Forget – Match Column to pH of mobile phase for maximum column lifetime

IV. Detection Issues

Recognize Where the Problem Originates
➢ Is it a consequence of technique?
➢ Is It expected due to the use of certain mobile phase components?
➢ Can it be corrected by adjusting detector parameters?

Drifting Baselines

➢ Detector (UV) not set at absorbance maximum but at the slope of the curve
➢ Gradient Elution
➢ Temperature Unstable (Refractive Index Detector)
➢ Contamination in Mobile Phase
➢ Mobile Phase Not in Equilibrium with Column

Baseline Noise
➢ Mobile phase contaminated, deteriorated, or prepared from low-quality materials
➢ Mobile phase solvents are immiscible
➢ Air trapped in the system
➢ Air bubbles in a detector
➢ Detector cell contaminated (even small amounts of contaminants can cause noise)
➢ Weak detector lamp

HPLC troubleshooting

HPLC troubleshooting

➢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

HPLC Chromatography

About Abha Maurya

Ms. Abha Maurya is the Author and founder of pharmaceutical guidance, he is a pharmaceutical Professional from India having more than 18 years of rich experience in pharmaceutical field. During his career, he work in quality assurance department with multinational company’s i.e Zydus Cadila Ltd, Unichem Laboratories Ltd, Indoco remedies Ltd, Panacea Biotec Ltd, Nectar life Science Ltd. During his experience, he face may regulatory Audit i.e. USFDA, MHRA, ANVISA, MCC, TGA, EU –GMP, WHO –Geneva, ISO 9001-2008 and many ROW Regularities Audit i.e.Uganda,Kenya, Tanzania, Zimbabwe. He is currently leading a regulatory pharmaceutical company as a head Quality. You can join him by Email, Facebook, Google+, Twitter and YouTube

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