What is Hplc
HPLC or also known as High-Performance Liquid Chromatography as full form, is a widely used sophisticated instrument used to separate a mixture of components. When a mixture of particles moves through a tightly packed column at high pressure, the particles move through it at different rates. This is due to attraction of sample to the particles in the column. Say if in a mixture of two compounds A and B, if A is more attracted to the column then it will move through it slowly. Compare at this flow rate and column if compound B is less attracted to column then the compound B will be leaving the column at faster rate than compound A, this causes compound B to reach first to the detector and compound A to reach slow at detector. In the detector these compounds will be analyzed (will create peaks) and because they reached the detector at different times there will be two peaks.
The peaks that formed in a graph known as Chromatogram, this method provides a accurate and fast result. HPLC is one of most advanced versions of chromatography, therefore let’s first learn about Chromatography
What is Chromatography?
Chromatography is a technique used to separate a mixture of compounds into its individual compounds. It consists of two phases stationary phase and mobile phase; the stationary phase is stable and does not move from its position while the mobile phase moves through the stationary phase. Mobile phase helps in the movement of samples through the stationary phase.
Types of Chromatography
Paper Chromatography – uses paper as the stationary phase .
Thin-Layer Chromatography (TLC) – uses coated glass or plastic plate.
Column Chromatography – uses vertical glass columns packed with solid adsorbent.
HPLC – High-pressure method for precise and fast separation of components.
GC – Separates volatile compounds through gas as the mobile phase.
Ion Exchange Chromatography – Separates ions based on their charge.
Affinity Chromatography – Uses specific binding interactions.
Size-Exclusion Chromatography – Separates molecules by size.
Flash Chromatography – fastest version using compressed gas.
Supercritical Fluid Chromatography – Uses supercritical CO₂ as the mobile phase.
Reverse-Phase Chromatography – Stationary phase is non-polar, mobile phase is polar.
Normal-Phase Chromatography – Stationary phase is polar, mobile phase is non-polar.
2D Chromatography – Combines two chromatographic methods for improved separation.
Chiral Chromatography – Separates optical isomers using a chiral stationary phase.
Planar Chromatography – by using a flat surface like TLC or paper as the stationary phase.
Micellar Electrokinetic Chromatography – through surfactants in capillary electrophoresis to separate neutral molecules.
Counter-Current Chromatography – two immiscible liquid phases without a solid stationary phase.
Hydrophobic Interaction Chromatography – Separates based on hydrophobicity.
Simulated Moving Bed Chromatography – continuous method for industrial-scale separations
HPLC Instrumentation
Hplc is a sophisticated instrument and consists of many parts which work together to create a constant flow and pressure. This system also consists of a very sensitive detector therefore even a slight change in pressure composition of solvent and flow rate complete reaction peak will get disturbed. Let’s go in depth about these main components which help run this sophisticated instrument.
The key components include
HPLC Pump
This delivers the mobile phase from the mobile phase reservoir which is normally placed above the hplc system. These pumps create a very high pressure and constant flow. Again in the pump we can find many other equipment like pulse dampeners, check valves, and degassers. Which helps in creating a good flow.
Any fluctuation in the flow rate can affect the retention time and peak shape in the chromatogram, this part is in a constant evolution which in each version performs better and longer.
There are many types of hplc pump which include
Reciprocating Piston Pump-Uses a piston moving back and forth to deliver precise volumes of solvent at high pressure.
Syringe Pump-Delivers solvent by pushing it out of a syringe barrel.
Displacement Pump-Maintains a constant volume by displacing solvent with a mechanical Component
Constant Pressure Pump-Applies a steady pressure to the mobile phase rather than controlling flow rate directly.
Dual-Piston (or Binary) Pump– Uses two pistons working alternately to reduce pulsation and ensure a smoother flow.
HPLC Injector
Injector is the component which is responsible for introducing the sample into the flow of the mobile phase. This sample through the mobile phase then reaches the column for separation. It also functions to deliver a precise volume of sample(in microliter).A poorly functioning injector can lead to sample loss, poor peak shape, or inconsistent results.
These injectors must be able to withstand high pressure and sample loss or contamination must be minimal.
Types of injectors
Manual Injector
The user injects the sample manually using a syringe into a fixed loop.This is commonly used in basic or educational setup, also using this requires skill and time.
Auto-injector (Autosampler)
Fully automated this injector can select and inject samples from vials in sequence. Users just need to fill in the sequence provided by the software. They are useful when a large number of sample testing is required and is used almost all labs
HPLC Column
They are tightly packed tube filled with stationary phase particles, where the separation of sample components occurs, through this when the sample passes due to its mixture of compounds having different affinity interaction to the column they move at different rates.
It is one of most important part, that’s why proper care to column in terms of storage, in sample and mobile phase preparation is done any mishandling can cause damage to column or poor peak formation
Basic Structure of an HPLC Column
Material-made of stainless steel to withstand high pressure
Length-Commonly ranges from 50 mm to 250 mm.
Internal Diameter-Varies from 2.1 mm to 4.6 mm for analytical columns, and up to 50 mm or more for preparative columns.
Packed with: Stationary phase particles
Stationary Phase Types
Reverse-Phase (RP)-Stationary phase is non-polar (e.g., C18 or C8 bonded silica) and mobile phase is polar in nature like (water with methanol or acetonitrile).in this the Non-polar compounds interact more and eluate later
Normal-Phase-Stationary phase is polar (like unmodified silica) and Mobile phase is non-polar (e.g., hexane).in this Polar analytes stick longer and elute slowly.
Ion Exchange- In this the Stationary phase contains charged groups and used to separate the Ions
Size-Exclusion (Gel Filtration)-In this Stationary phase the separation happens based on size therefore Large molecules come out first; small ones get delayed.
Chiral Columns-Designed to separate enantiomers (mirror-image molecules).
Column Maintenance & Care
Column Maintenance is important to extend the life of the column for this it is important to always filter the sample to avoid the clogging and by using the guard columns to protect the column.
Store the the column properly which includes flushing with appropriate solvent after use and during working it is important to avoid the pressure spikes to prevent damage to packed beds
Types of Columns
C18 Column (Octadecylsilane), C8 Column, C4 Column, Phenyl Column, Pentafluorophenyl (PFP) Column, Cyano Column (CN), Amino Column (NH₂), Silica Column, Hydrophilic Interaction Liquid Chromatography (HILIC) Column, Hydrophobic Interaction Chromatography (HIC) Column, Ion Exchange Column (including Strong Anion Exchange – SAX, Weak Anion Exchange – WAX, Strong Cation Exchange – SCX, Weak Cation Exchange – WCX), Size‑Exclusion (Gel Filtration / Gel Permeation) Column, Chiral Column (e.g. polysaccharide‑ or protein‑based), Affinity Column, Monolithic Column (silica‑ or polymer‑based), Porous Graphitic Carbon (PGC) Column, Superficially Porous (Fused‑Core) Column, Polymer‑based Reversed‑Phase Column (e.g. polyvinyl alcohol resin), Mixed‑Mode (Multimodal) Column.
HPLC Detector
After the separation of the sample mixture the individual analytes reach the detector at a different rate. Detectors are responsible for identifying and quantifying the compounds as they elute (exit) from the column.The detector’s signal forms the chromatogram, which displays the peaks corresponding to different compounds based on their retention time
How Does a Detector Work in hplc
When the compound passes through the detector, the detector can sense the change in the physical properties (such as UV absorption, refractive index, fluorescence, etc.). Common methods for light detection include the light bean passing through the cell which is a part of mobile phase and sample flow after column through this flow cell when the components flow they causes the change in light beam wavelength this difference in the wavelength is detected and changed into electronic signal which is then displayed as peak on a graph which is known as chromatogram
Common Types of Detectors in HPLC
UV-Visible (UV-Vis) Detector-Measures how much UV or visible light is absorbed by the sample
Fluorescence Detector-Extremely sensitive, used for compounds that naturally fluoresce or can be made to fluoresce.
Mass Spectrometric Detector (LC-MS)-Offers very high sensitivity and specificity. Identifies compounds based on mass-to-charge ratio. Ideal for complex mixtures or unknowns.
Photodiode Array (PDA/DAD) – Captures a full spectrum at once (typically 190–800 nm), ideal for peak purity
Conductivity Detector – Detects ionic species by measuring the electrical conductivity of the eluent; common in ion chromatography.
Light Scattering Detector (LSD) – Includes multi-angle light scattering (MALS), ideal for determining molar mass of polymers and proteins.