Mobile Phase in HPLC – Types, Preparation & Role in Separation π§ 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 π Mobile phases are generally divided into two types: 1. Aqueous Phase (Water-Based) π¦ 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-Base...
HPLC Detectors – Types and Their Applications in Pharma π After a sample is separated by the HPLC column, it needs to be detected and measured. This is where HPLC detectors come in. Detectors help us know what compound is coming out of the column and how much of it is present. Different detectors are used depending on the chemical nature of the compound. 1. UV-Visible Detector (UV/Vis & DAD) π This is the most commonly used detector in pharmaceutical analysis. It measures how much UV or visible light a compound absorbs. Single Wavelength UV: Measures absorbance at one fixed wavelength (e.g., 254 nm). DAD – Diode Array Detector: Records absorbance across a range of wavelengths. It helps in identifying peak purity and overlapping compounds. Best for: UV-absorbing compounds like drugs with aromatic rings or conjugated systems. Example: Paracetamol, aspirin, ibuprofen, antihistamines 2. Fluorescence Detector ✨ This detector measures the light emitted (fluor...