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Chromatography !

Chromatography

Chromatography is a key technique used to separate, identify, and analyze chemical mixtures. It is widely used in pharmaceuticals, environmental analysis, forensic science, and food safety. The method works by passing a sample mixture through a stationary phase while being carried by a mobile phase. Different components in the mixture travel at different speeds, leading to separation.

Principles of Chromatography

Chromatography relies on different interactions between molecules and the stationary and mobile phases. The main mechanisms include:

Adsorption Chromatography : Molecules adhere to the surface of the stationary phase.
Partition Chromatography : Separation occurs based on solubility in the mobile and stationary phases.
Ion Exchange Chromatography : Charged molecules are separated based on electrostatic attraction.
Size Exclusion Chromatography (SEC) : Molecules are separated based on size.

Affinity Chromatography : Uses specific binding between molecules, such as antibody-antigen interactions.

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Key Components of a Chromatography System

A typical chromatography system consists of :

Mobile Phase: The fluid that carries the sample.
Stationary Phase : The solid or liquid phase where separation occurs.
Column or Plate : Holds the stationary phase and facilitates separation.
Detector : Identifies the separated components (e.g., UV-Vis, fluorescence, mass spectrometry (MS)).
Data System : Records chromatograms for analysis.

Types of Chromatography

I. Liquid Chromatography (LC)

Liquid Chromatography (LC) is a widely used analytical technique that separates compounds in a mixture based on their interaction with a stationary phase and a liquid mobile phase. It is employed in various industries, including pharmaceuticals, environmental analysis, biotechnology, and food safety, due to its high sensitivity, precision, and adaptability.

Principle of Liquid Chromatography

LC operates based on the differential partitioning of analytes between the mobile phase (liquid) and the stationary phase (solid or liquid-coated surface). The separation occurs as the sample components travel at different rates due to their varying affinities toward the stationary phase. The key factors influencing LC separation include :

-Polarity of analytes and phases : Determines retention and separation efficiency.

-Column packing material : Affects selectivity and efficiency.

-Flow rate of the mobile phase : Impacts resolution and analysis time.

-Detection method : Enhances sensitivity and identification accuracy.

Types of Liquid Chromatography

Liquid Chromatography has multiple variations, each tailored for specific applications :

1. High-Performance Liquid Chromatography (HPLC)

HPLC is the most common LC technique, using high pressure to push the mobile phase through a tightly packed column. It provides excellent resolution, speed, and reproducibility.

Normal-Phase HPLC (NP-HPLC) : Uses a polar stationary phase (e.g., silica) and a nonpolar mobile phase. Ideal for separating polar compounds.
Reverse-Phase HPLC (RP-HPLC) : Employs a nonpolar stationary phase (e.g., C18) with a polar mobile phase (e.g., water-methanol). Preferred for separating hydrophobic compounds.
Gradient HPLC : The composition of the mobile phase changes over time, improving separation for complex mixtures.
Isocratic HPLC : Uses a constant mobile phase composition, suitable for simpler separations.

2. Ultra-High-Performance Liquid Chromatography (UHPLC)

UHPLC is an advanced version of HPLC that operates at even higher pressures (>15,000 psi), allowing for faster analysis, improved resolution, and reduced solvent consumption.

3. Size Exclusion Chromatography (SEC)

Also known as Gel Filtration Chromatography (GFC), SEC separates molecules based on size. Larger molecules elute faster as they do not penetrate the porous stationary phase, while smaller molecules take longer to pass through.

4. Ion Exchange Chromatography (IEC)

IEC separates charged molecules based on their interaction with oppositely charged stationary phase materials. It is widely used for proteins, peptides, and ionic compounds.

Cation Exchange Chromatography : Retains and separates positively charged ions.
Anion Exchange Chromatography : Separates negatively charged ions.

5. Affinity Chromatography

This technique exploits highly specific interactions between biomolecules (e.g., enzyme-substrate, antigen-antibody, receptor-ligand). It is commonly used for protein purification and biomarker detection.

6. Hydrophilic Interaction Liquid Chromatography (HILIC)

HILIC is useful for separating highly polar compounds. It operates similarly to normal-phase HPLC but with an aqueous-organic mobile phase.

7. Chiral Liquid Chromatography

Chiral chromatography is designed to separate enantiomers of chiral molecules using a chiral stationary phase. It is crucial in pharmaceutical research to differentiate between drug isomers.

Detection Methods in LC

Various detectors are used in LC to identify and quantify separated compounds :

UV-Vis Detector (UV-Vis) : Measures absorbance at specific wavelengths.
Fluorescence Detector (FLD) : Enhances sensitivity for fluorescent compounds.
Mass Spectrometry (LC-MS) : Provides structural identification of molecules.
Refractive Index Detector (RID) : Useful for compounds without UV absorbance.

Conductivity Detector : Commonly used in ion chromatography.

II. Gas Chromatography (GC)

Gas Chromatography (GC) is an analytical technique used to separate, identify, and quantify volatile compounds in a mixture. It is widely applied in chemistry, biology, forensics, environmental analysis, and industrial quality control.

Principle of Gas Chromatography

GC operates by vaporizing a sample and carrying it through a column using an inert gas (mobile phase). The components separate based on their interaction with the column's stationary phase and their volatility. The separated components reach a detector, which records their retention time and concentration.

Main Components of a Gas Chromatograph

1.Carrier Gas (Mobile Phase)

Typically helium, nitrogen, or hydrogen.

Transports the vaporized sample through the column.

2.Injection System

Introduces the liquid or gaseous sample into the system.
Common methods : Split, splitless, on-column.

3.Column & Oven

Column : Contains the stationary phase (coated on the inner wall of a capillary column or packed column).
Oven : Controls the temperature, which affects separation efficiency.

4.Detector

Detects separated compounds and converts them into an electrical signal.

Common detectors :

-Flame Ionization Detector (FID) – For hydrocarbons.

-Thermal Conductivity Detector (TCD) – For general applications.

-Electron Capture Detector (ECD) – For halogenated compounds.

-Mass Spectrometer (MS) – Provides molecular identification.

5.Data System

Collects, processes, and interprets the chromatogram output.

Types of Columns in GC

Capillary Columns :

-Thin fused silica tube with a stationary phase coating

-Higher efficiency, better resolution

Packed Columns :

-Packed with solid particles coated with the stationary phase

-Used for larger sample volumes but lower resolution

III. Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC) is a simple, rapid, and cost-effective separation technique used for identifying and analyzing small amounts of compounds in a mixture. It is widely used in chemistry, pharmaceuticals, forensics, and food analysis.

Thin Layer Chromatography (TLC)

TLC is based on the differential migration of compounds on a thin layer of stationary phase (silica gel, alumina, or cellulose) under the influence of a mobile phase (solvent). The separation occurs due to differences in adsorption and solubility of the analytes in the stationary and mobile phases.

1.Stationary Phase

-Thin layer of silica gel (most common), alumina, or cellulose coated on a plate (glass, aluminum, or plastic)

-Provides a surface for analyte separation.

2.Mobile Phase (Solvent System)

-A liquid solvent or mixture of solvents.

-Moves up the plate by capillary action, carrying the analytes.

3.Sample Application

-The sample is dissolved in a suitable solvent and spotted near the bottom of the TLC plate using a capillary tube or micropipette.

4.Development Chamber

-A sealed container holding the mobile phase.

-Ensures proper solvent movement and saturation for consistent separation.

5.Detection System

-UV Light (Short/Long Wave) : Fluorescent compounds or pre-treated TLC plates.

-Chemical Sprays : Ninhydrin (amino acids), Iodine vapors (organic compounds), Dragendorff’s reagent (alkaloids).

-Derivatization : Chemical modification to enhance visibility.

IV. Paper Chromatography

Paper Chromatography is a simple separation technique used to analyze and identify mixtures of compounds, particularly for qualitative analysis. It is based on capillary action, where the mobile phase (solvent) moves through a stationary phase (filter paper), separating components based on their solubility and affinity.

Paper Chromatography

1.Principle :

The stationary phase: Special-grade filter paper (cellulose)
The mobile phase: A solvent or a mixture of solvents
The separation occurs due to partitioning between the mobile phase and the stationary phase. More soluble components travel further up the paper.

2.Procedure :

Spot the sample onto the base of the chromatography paper.
Place the paper in a solvent-filled chamber, ensuring the sample is above the solvent level.
Allow the solvent to travel up the paper by capillary action.
Remove, dry, and analyze the chromatogram.

V. Affinity Chromatography

Affinity Chromatography is a highly selective method used to purify biomolecules (proteins, enzymes, antibodies) by exploiting specific binding interactions between a target molecule and a ligand attached to a stationary phase.Learn how to quickly set up and start using our services with our step-by-step onboarding process.

Affinity Chromatography

1.Principle :

The stationary phase: A ligand covalently attached to a solid support (agarose, cellulose, or magnetic beads).

The mobile phase: A buffer solution to wash unbound substances.

The target molecule binds to the ligand, while other components are washed away.

The target molecule is eluted using a competing ligand or by altering pH/salt conditions.

2.Procedure :

Load the sample onto the affinity column.

Wash unbound substances using a buffer.

Elute the bound molecules using an elution buffer.

Collect and analyze the purified biomolecule.

VI. Ion Exchange Chromatography (IEC)

Ion Exchange Chromatography (IEC) separates molecules based on their charge by using charged stationary phases that attract oppositely charged solutes. It is widely used in protein purification, water treatment, and biochemical research.

Ion Exchange Chromatography (IEC)

1.Principle:

Cation Exchange Chromatography (CEX) : The stationary phase carries a negative charge and retains positively charged molecules.
Anion Exchange Chromatography (AEX) : The stationary phase carries a positive charge and retains negatively charged molecules.
The separation is influenced by pH and ionic strength of the mobile phase.

2.Procedure:

Load the sample into the ion-exchange column.
Wash weakly bound molecules with a buffer.
Elute bound molecules by gradually increasing the salt concentration or adjusting pH.
Collect and analyze the separated molecules.

VII. Size Exclusion Chromatography (SEC)

Size Exclusion Chromatography (SEC), also known as Gel Filtration Chromatography, separates molecules based on size by passing them through a porous stationary phase. Larger molecules elute faster because they bypass small pores, while smaller molecules take longer as they diffuse into the porous matrix.

Size Exclusion Chromatography (SEC)

1. Principle :

The stationary phase : A column filled with porous beads (dextran, agarose, or silica).
The mobile phase : A buffer or solvent that carries the sample.
Large molecules travel through faster, while smaller ones are delayed by entering the pores.