-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.
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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.
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.
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.
- 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.
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.
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.
- 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.
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.
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.
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.
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.
2.Procedure:
- Inject the sample into the SEC column.
- Allow molecules to separate as they pass through the porous beads.
- Collect fractions containing different-sized molecules.
- Analyze using absorbance or mass spectrometry.
