Skip to main content

HPLC Mobile Phase: Types, Preparation, Isocratic vs Gradient Explained

Mobile Phase in HPLC – Types, Preparation & Role in Separation 💧

In High-Performance Liquid Chromatography (HPLC), the mobile phase is the liquid that moves through the column and carries the sample with it. Choosing the right mobile phase is critical for good separation, sharp peaks, and accurate results. In this post, you'll learn about the types of mobile phases used in HPLC, how to prepare them, and their impact on the analysis.

What is the Mobile Phase in HPLC?

The mobile phase is the solvent or mixture of solvents that flows through the HPLC column. It interacts with the stationary phase and the sample, helping separate the compounds based on polarity, charge, or size.

Types of Mobile Phases 📌

Mobile phases are generally divided into two types:

1. Aqueous Phase (Water-Based) 💦

  • Usually includes water with a buffer (e.g., phosphate, acetate)
  • Used in: Reverse-Phase HPLC
  • pH control: Important for ionizable compounds

2. Organic Phase (Solvent-Based) 🧪

  • Common solvents: Methanol, Acetonitrile, Ethanol
  • Used in: Both RP-HPLC and NP-HPLC
  • Acetonitrile gives better peak shape; methanol is more economical

Mobile Phase in Reverse-Phase HPLC (RP-HPLC)

In RP-HPLC, the mobile phase is polar, and the stationary phase is non-polar. This means polar compounds move faster, while non-polar compounds are retained longer.

  • Example: Water + Acetonitrile (60:40), pH adjusted with phosphate buffer

Mobile Phase in Normal-Phase HPLC (NP-HPLC)

In NP-HPLC, the mobile phase is non-polar (like hexane), and the stationary phase is polar (like silica). This setup is used for separating polar analytes.

  • Example: Hexane + Ethyl Acetate (80:20)

Mobile Phase Preparation 🧫

Proper preparation ensures reproducible results and longer column life.

  1. Use HPLC-grade solvents: Free from UV-active impurities
  2. Filter (0.45 or 0.22 µm): To remove particles
  3. Degas: By sonication or vacuum to remove air bubbles
  4. pH Adjustment: Use dilute acids or bases (e.g., orthophosphoric acid, triethylamine)

Isocratic vs Gradient Elution 💡

The way the mobile phase is delivered during the run affects separation:

Isocratic Elution

  • The mobile phase composition stays the same throughout the run
  • Best for simple mixtures with closely related compounds
  • Example: Water:Acetonitrile (70:30) for paracetamol assay

Gradient Elution

  • The mobile phase composition changes gradually during the run
  • Used for complex samples or compounds with wide polarity range
  • Improves resolution, especially for early and late eluting peaks

Example Gradient Program ⚙️

Below is a common gradient method setup:

  • Initial: 10% Acetonitrile / 90% Buffer
  • 5 min: 30% Acetonitrile / 70% Buffer
  • 10 min: 60% Acetonitrile / 40% Buffer
  • 15 min: 90% Acetonitrile / 10% Buffer
  • Hold 2 min at 90% ACN, then re-equilibrate

This helps elute both polar and non-polar compounds in one run.

Impact of Mobile Phase on Results 🔬

  • Peak shape: Depends on solvent strength and pH
  • Retention time: Affected by polarity and flow rate
  • Resolution: Optimized by changing mobile phase composition

Summary ✅

  • Mobile phase plays a key role in separation efficiency
  • Reverse-phase uses water-organic mixtures; normal-phase uses non-polar solvents
  • Use isocratic for simple compounds, gradient for complex samples
  • Always filter, degas, and pH-adjust your mobile phase

Keywords 🔑

HPLC mobile phase, reverse phase mobile phase, mobile phase preparation, isocratic elution, gradient elution, gradient program HPLC, pharmaceutical HPLC, mobile phase selection

Written by Nirmal Patel | o2h.space

Labels:

Comments

Post a Comment

Popular posts from this blog

What is HPLC ? A Beginner’s Guide to High-Performance Liquid Chromatography

What is HPLC ? A Simple Introduction for Beginners High-Performance Liquid Chromatography (HPLC) is a powerful analytical technique used to separate, identify, and quantify the components in a mixture. It is widely applied in pharmaceutical, environmental, food, and chemical industries for quality control and research. In the pharmaceutical industry, HPLC is essential for ensuring the safety and efficacy of drugs by analyzing the purity, potency, and stability of active pharmaceutical ingredients (APIs) and formulations. Why is HPLC Important? Even minor variations in drug composition can lead to serious health risks. HPLC provides precise and reliable data that help manufacturers comply with regulatory standards and deliver safe medications to patients. It is also used for: Detecting impurities and degradation products Quantifying drug release rates in formulations Monitoring stability under different environmental conditions Basic Principle: HPLC separates c...
Post a Comment
Read more

Types of HPLC – Explained with Examples

Types of HPLC – Explained with Examples 🔬 High-Performance Liquid Chromatography ( HPLC ) has different types depending on how the separation is done. Each type is designed to handle specific compounds and sample properties . In this post, you’ll learn the main types of HPLC used in the pharmaceutical industry and how they work. 1. Reverse-Phase HPLC (RP-HPLC) 🔄 Reverse-phase HPLC is the most commonly used type in pharma labs. In this method, the stationary phase is non-polar (hydrophobic), and the mobile phase is polar (water-based solvents). Used for: Non-polar or moderately polar compounds Example: Testing drug substances like paracetamol, ibuprofen, or vitamins 2. Normal-Phase HPLC (NP-HPLC) ⚖️ This method uses a polar stationary phase (like silica) and a non-polar mobile phase (like hexane). It separates polar compounds better than RP-HPLC. Used for: Separation of isomers, sugars, amino acids Example: Identification of sm...
Post a Comment
Read more

HPLC Columns Explained: Types, How to Choose & Maintain

Understanding HPLC Columns: Types, Selection & Maintenance 🔬 The HPLC column is the most critical part of the chromatography system. It holds the stationary phase and is responsible for separating the compounds in your sample. The choice of column directly impacts the quality, speed, and reproducibility of your analysis. What is an HPLC Column? An HPLC column is a stainless steel tube packed with tiny particles called the stationary phase . When the liquid mobile phase pushes the sample through the column, interactions between the analytes and stationary phase cause them to separate and elute at different times. Types of HPLC Columns Columns are categorized based on the type of stationary phase they contain: Reverse-Phase Columns (RP): These are the most widely used columns in pharmaceutical analysis. They use non-polar stationary phases, commonly octadecyl (C18) or octyl (C8) bonded silica. Reverse-phase columns are excellent for separating non-polar to moderately ...
Post a Comment
Read more