HPLC (High-Performance Liquid Chromatography)

HPLC (High-Performance Liquid Chromatography)

HPLC (High-Performance Liquid Chromatography) columns are the core component of an HPLC system, where the actual separation of sample components occurs. They are packed with stationary phase materials that interact with the sample mixture to separate its components based on differences in their chemical or physical properties.

Types of HPLC Columns:

1. Reverse Phase (RP-HPLC) – Most common; uses non-polar stationary phases (e.g., C18, C8) and polar mobile phases (e.g., water, methanol, acetonitrile).
2. Normal Phase (NP-HPLC) – Uses polar stationary phases (e.g., silica) and non-polar mobile phases (e.g., hexane, chloroform).
3. Ion Exchange (IEX-HPLC) – Separates charged molecules using ionic interactions with a charged stationary phase.
4. Size Exclusion (SEC-HPLC) – Separates molecules based on size using porous stationary phases.
5. Chiral Columns – Used for separating enantiomers (optical isomers).

Important point to have  to select  HPLC Column:

• Column length and diameter (affect separation efficiency and run time)
• Particle size of the stationary phase (smaller particles provide better resolution but higher pressure)
• Pore size (important for large molecules like proteins)
• Stationary phase chemistry (determines selectivity and retention)

HPLC columns are  based on the separation mechanism used. Here are the main types:

1. Reverse Phase HPLC (RP-HPLC) Columns

• Highly used type.
• Stationary Phase: Non-polar (e.g., C18, C8, Phenyl, C4).
• Mobile Phase: Polar (e.g., water, methanol, acetonitrile).
• Used For: Separation of non-polar to moderately polar compounds (e.g., pharmaceuticals, biomolecules, food analysis).

2. Normal Phase HPLC (Columns

• Opposite of reverse phase chromatography.
• Stationary Phase: Polar (e.g., silica, cyano, amino).
• Mobile Phase: Non-polar (e.g., hexane, chloroform).
• Used For: Separation of polar compounds (e.g., sugars, lipids, natural products).

3. Ion Exchange HPLC Columns

• Separates charged molecules based on their ionic interactions.
• Stationary Phase: Charged resin (either cationic or anionic).
• Cation Exchange: Negatively charged stationary phase for separating positively charged molecules.
• Anion Exchange: Positively charged stationary phase for separating negatively charged molecules.
• Used For: Separation of proteins, amino acids, nucleotides, and other charged biomolecules.

4. Size Exclusion HPLC Columns

• Separates molecules based on size.
• Stationary Phase: Porous beads (e.g., silica, polymer).
• Mobile Phase: Typically aqueous or organic solvents.
• Used For: Analysis of polymers, proteins, and large biomolecules.

5. Chiral HPLC Columns

• Separates enantiomers (optical isomers).
• Stationary Phase: Chiral selectors like cyclodextrins or polysaccharides.
• Mobile Phase: Depends on application (normal or reverse phase).
• Used For: Pharmaceutical and drug analysis (enantiomeric purity).

6. Hydrophilic Interaction Liquid Chromatography (HILIC) Columns

• Hybrid between normal and reverse phase for highly polar compounds.
• Stationary Phase: Polar (e.g., silica, amide, cyano).
• Mobile Phase: Water-miscible organic solvents (e.g., acetonitrile with a small amount of water).
• Used For: Analysis of sugars, metabolites, polar drugs, peptides.

7. Affinity Chromatography Columns

• Based on specific binding interactions (e.g., antigen-antibody, enzyme-substrate).
• Stationary Phase: Ligand-functionalized material (e.g., protein A, lectins).
• Mobile Phase: Buffer solutions.
• Used For: Protein purification, antibody analysis, biomolecule separations.

We are dealing with different (makes) of HPLC columns, each offering a variety of stationary phases, particle sizes, and column dimensions for different applications.

1. Agilent :

• ZORBAX (Eclipse Plus, SB-C18, Bonus-RP) – Widely used for pharmaceutical and biotech applications.
• Poroshell 120 – Core-shell technology for high efficiency.

2. Waters :

• XBridge – High pH stability, ideal for method development.

• XBridge C18 → Highly durable C18 column, ideal for pharmaceutical and biotech applications.
• XBridge C8 → Provides shorter retention for hydrophobic compounds.
• XBridge Phenyl → Suitable for separating aromatic and polar compounds.
• XBridge Shield RP18 → Designed for improved peak shape and retention of polar analytes.

• XSelect – High selectivity columns for complex separations.

• XSelect CSH C18 → Charged surface hybrid for superior peak shape in LC-MS applications.

• XSelect Phenyl-Hexyl → Ideal for aromatic compounds.

• Symmetry – Traditional HPLC columns with excellent reproducibility.

• Symmetry C18 → Traditional C18 column for pharmaceutical analysis.
• Symmetry Shield RP18 → Improved retention of polar compounds.

•  
• SunFire – High-performance reverse-phase columns.
• SunFire C18 → General-purpose C18 column with excellent peak shapes.
• SunFire C8 → Shorter retention for hydrophobic compounds.

3. Thermo Fisher Scientific

• Accucore – Core-shell technology for high efficiency.( Accucore C18 – General-purpose reverse-phase column)
• Hypersil GOLD – High-resolution, high-efficiency columns.( Hypersil GOLD C18 – Ideal for pharmaceutical and environmental analysis.)
• Syncronis – Ultra-high-performance columns for LC-MS applications.( Syncronis C18 – High-resolution column for method development.)
• BetaBasic – Good for a broad range of compounds.( BetaBasic C8 – Used for peptides and pharmaceutical compounds.)

4. Phenomenex

• Luna – High-purity silica for RP, NP, and HILIC applications.
• Kinetex – Core-shell technology for ultra-fast separations.
• Synergi – Unique selectivity for polar and non-polar compounds.
• Gemini – High pH stability for method flexibility.

5. Shimadzu

• Shim-pack GIST – Universal RP columns with high reproducibility.

6. Merck (Sigma-Aldrich, Supelco)

• Ascentis Express – Fused-core technology for high-speed separations.
• Supelcosil – Traditional silica-based columns for RP and NP applications.
• Discovery – Broad range of stationary phases for diverse applications.

7. YMC

• YMC-Triart – Hybrid silica-based columns for a wide pH range.
• YMC-Pack Pro C18 – High-performance C18 columns.

8. Daicel Chiral Technologies

• CHIRALCEL – Polysaccharide-based chiral columns.

• CHIRALCEL OD-H → Covered with  Cellulose with hexane as the mobile phase.
• CHIRALCEL OJ-H → Similar to OD-H but with a different selective Phase.

• CHIRALPAK – Used in pharmaceutical enantiomer separation.

• CHIRALPAK AD-H → Coated Amylose for normal-phase chiral separations.
• CHIRALPAK AS-H → Excellent for separating alcohols and amines.

• CHIRALPAK IA, IB, IC, ID → chiral selectors for greater method flexibility.

9. GL Sciences

• Inertsil – Popular for reproducible RP-HPLC separations.

1. Inertsil(High-Purity Silica-Based Columns)

• Inertsil ODS-3 → Popular C18 column for pharmaceutical and environmental analysis.
• Inertsil ODS-4 → Improved version of ODS-3 with enhanced performance and durability.
• Inertsil SIL-100A → Normal-phase silica column for non-polar compounds.
• Inertsil NH2 (Amino) → Used for carbohydrate analysis.
• Inertsil Phenyl → Suitable for aromatic compound separations.
• InertSustain – High stability and efficiency.
• InertSustain C18 → Universal C18 column with high reproducibility.
• InertSustain AQ-C18 → Designed for highly aqueous mobile phases.

GC Column?

A Gas Chromatography (GC) column is a key component in Gas Chromatography systems, responsible for separating different chemical components in a sample based on their volatility and interaction with the stationary phase.

GC columns are typically long, narrow tubes made of fused silica or stainless steel, coated internally with a stationary phase. The sample is vaporized and carried through the column by an inert gas (carrier gas like helium, nitrogen, or hydrogen), where separation occurs before reaching the detector.

Types of GC Columns

GC columns are broadly classified into two main types:

1. Packed Columns (Traditional, Less Common)

• Shorter columns (typically 1–5 meters in length).
• Made of stainless steel or glass and packed with a solid support coated with a stationary phase.
• Used for analyzing gases and volatile compounds.
• Example stationary phases: Porapak, Molecular Sieve, Carbopack.

Popular GC Column Manufacturers

• Agilent Technologies → DB Series (DB-1, DB-5, DB-WAX)
• Restek → Rtx Series (Rtx-1, Rtx-5, Rtx-WAX)

SGE is a well-known manufacturer of GC columns and accessories. SGE specializes in high-performance capillary columns for various gas chromatography (GC) applications.

SGE’s BP Series includes various stationary phases for different applications:

• BP1 (100% Dimethylpolysiloxane, Non-Polar)
• Similar to Agilent DB-1 or Restek Rtx-1.
• Used for hydrocarbons, essential oils, and non-polar compounds.
• Applications: Petroleum, environmental, and food analysis.
• BP 5 (5% Phenyl + 95% Dimethylpolysiloxane, Slightly Polar)
• Similar to Agilent DB-5 or Restek Rtx-5.
• General-purpose column for pesticides, aromatics, and drugs.
• Applications: Forensic, pharmaceutical, and environmental analysis.
• BP10 (10% Phenyl + 90% Dimethylpolysiloxane, Medium Polar)
• Enhanced selectivity for aromatic hydrocarbons and volatile organics.
• Applications: Flavors, fragrances, and industrial chemicals.
• BPX50 (50% Phenyl + 50% Dimethylpolysiloxane, Polar)
• Similar to Agilent DB-17 or Restek Rtx-17.
• Good for separating complex mixtures of alcohols, esters, and ketones.

• Macherey-Nagel (MN) HPLC Columns
• Macherey-Nagel (MN) is a leading manufacturer of HPLC columns, known for its NUCLEOSIL® and NUCLEODUR® These columns are widely used in pharmaceutical, environmental, food, and clinical applications.

• 
  One of the first spherical silica HPLC columns developed.
• Available in different particle sizes (3 µm, 5 µm, 7 µm, and 10 µm).
• Used in normal-phase, reversed-phase, ion-exchange, and size-exclusion chromatography (SEC).
• Popular NUCLEOSIL® Phases:
• NUCLEOSIL 100-5 C18 → General-purpose C18 column for pharmaceuticals.
• NUCLEOSIL 100-5 C8 → For shorter retention of hydrophobic compounds.
• NUCLEOSIL 100-5 CN → Cyanopropyl phase for polar interactions.
• NUCLEOSIL 100-5 NH2 → Aminopropyl for sugar analysis.

1. NUCLEODUR® (High-Purity Silica for Better Performance)

• Advanced, ultra-pure silica for higher efficiency and resolution.
• Available in reversed-phase (RP), normal-phase (NP), and HILIC modes.
• Compatible with UHPLC and high-speed separations.

• NUCLEODUR C18 Gravity → High-purity C18 for pharmaceutical and biotech separations.
• NUCLEODUR C18 Pyramid → Unique C18 with steric selectivity, ideal for large molecules.
• NUCLEODUR C18 ec → Economical C18 for routine analysis.
• NUCLEODUR Sphinx RP → Polar-modified C18 for better retention of polar compounds.
• NUCLEODUR HILIC → Hydrophilic interaction chromatography (HILIC) for polar analytes.

Shodex is a leading manufacturer of HPLC and) columns, known for high-performance polymer-based columns. They specialize in size-exclusion chromatography (SEC), sugar analysis, ion chromatography (IC), and reversed-phase (RP) separations.

Popular Shodex  Columns:

• Asahipak GF Series → Aqueous SEC for proteins & biopolymers.

• PROTEIN KW Series → SEC columns for antibodies (mAbs), peptides, and proteins.
• OHpak SB Series → SEC columns for polysaccharides and water-soluble polymers.

Hichrom HPLC Columns

Hichrom is a UK-based manufacturer of high-performance HPLC columns, now part of Avantor (VWR). Hichrom produces a wide range of silica-based and polymer-based columns,

1. Hichrom HPLC Column Series
2. Hichrom Reversed-Phase (RP) Columns

• Designed for non-polar and semi-polar compounds in pharmaceutical, environmental, and food analysis.
• Available in C18, C8, Phenyl, and CN phases.

Popular RP Columns:

• Hichrom C18 → General-purpose C18 column, high efficiency.
• Hichrom C8 → Shorter retention for hydrophobic compounds.
• Hichrom Phenyl → Better selectivity for aromatic compounds.
• Hichrom CN (Cyanopropyl) → Suitable for polar & non-polar analytes.

1. Hichrom Normal-Phase (NP) Columns

• Used for lipids, steroids, and non-polar organics.

Popular NP Columns:

• Hichrom Si (Silica) → Unmodified silica for classic normal-phase separations.
• Hichrom NH2 (Amino) → Suitable for sugar analysis and polar compounds.

1. Hichrom Ion-Exchange (IEX) Columns

• Used for proteins, amino acids, and charged molecules.

Popular IEX Columns:

• Hichrom SAX (Strong Anion Exchange) → For nucleotides and organic acids.
• Hichrom SCX (Strong Cation Exchange) → For amino acids and peptides.

Cosmosil HPLC Columns (Nacalai Tesque, Japan)

Cosmosil is a well-known HPLC column brand from Nacalai Tesque (Japan), specializing in silica-based and polymer-based stationary phases. They offer a wide variety of columns for reversed-phase (RP), normal-phase (NP), size-exclusion (SEC), and chiral separations.

Cosmosil HPLC Columns WE offers

  Cosmosil 5C18-AR-II → General-purpose C18 column with excellent peak symmetry.

  Cosmosil 5C18-MS-II → C18 with high loading capacity, ideal for pharmaceuticals.

  Cosmosil 5C18-PAQ → C18 for polar compounds (100% aqueous mobile phases).

  Cosmosil C8-MS-II → Shorter retention for hydrophobic compounds.

D2 (Deuterium) Lamp

A D2 Lamp (Deuterium Lamp) is a high-intensity light source used in HPLC detectors, UV-Vis spectrophotometers, and other analytical instruments. It emits a continuous UV spectrum (190-400 nm), making it ideal for detecting compounds that absorb UV light.

1. Key Features of a D2 Lamp

✔ Wavelength Range: 190 - 400 nm (UV region)
✔ Stable Light Intensity: Provides low noise and high stability for accurate measurements.
✔ Long Lifespan: Typically lasts 1,000 to 2,000 hours, depending on usage.
✔ Used in: HPLC UV-Vis Detectors, UV-Vis Spectrophotometers, TOC Analyzers, and Colorimeters.

We Offers Below D2 Lamp

• Hamamatsu (Japan) → High-quality, long-life D2 lamps.
• Agilent (USA) → Standard in Agilent HPLC UV detectors.
• Waters (USA) → Used in Waters HPLC-UV detectors.
• Shimadzu (Japan) →Shimadzu lamps for Shimadzu instruments.

Consumable Spare Parts for HPLC & GC Instruments

HPLC (High-Performance Liquid Chromatography) and GC (Gas Chromatography) systems require regular maintenance to ensure accurate and reliable performance. Below are the most common consumable spare parts used in these systems.

1. HPLC Consumables & Spare Parts
2. Detectors (UV, PDA, RID)

• D2 Lamp (Deuterium Lamp) → UV/Vis detectors (lifespan: 1,000-2,000 hours).
• Tungsten-Halogen Lamp → Visible light range (400-900 nm).
• Flow Cell (Quartz or PEEK) → Ensures proper light transmission and detection.

1. HPLC Pump & Fluidic System

• Pump Seals / Piston Seals → Prevents leaks and maintains pressure.
• Pump Pistons (Sapphire or Ceramic) → Ensures precise solvent flow.
• Check Valves → Controls solvent movement and prevents backflow.

1. HPLC Columns & Fittings

• HPLC Columns → Regularly replaced due to wear or contamination.
• Column Guard Filters → Protects main column from debris.
• Ferrules & Nuts (Stainless Steel / PEEK) → Secure tubing connections.
• PEEK Tubing & Stainless-Steel Tubing → Solvent-resistant liquid flow paths.

Syringe Filters in HPLC & Sample Preparation

A syringe filter is a disposable, membrane-based filter used to remove particulates, bacteria, and contaminants from liquid samples before HPLC, GC, and other analytical applications. They help protect columns, injectors, and detectors from clogging and contamination.

1. Key Features of Syringe Filters

✔ Membrane Pore Sizes: 0.2 µm (for sterilization), 0.45 µm (for HPLC sample filtration).
✔ Filter Diameters: Common sizes 4 mm, 13 mm, 25 mm, 30 mm.