Size Exclusion Chromatography SEC/GPC

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Size Exclusion Chromatography SEC/GPC

Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC) and gel filtration chromatography (GFC), is a chromatographic process in which analytes are separated according to size and structure. It is used to characterise natural and synthetic polymers, biopolymers, proteins and nanoparticles.

The largest molecules are the first to leave the separation column. The smaller the molecules are, the better they can penetrate the pores of the stationary phase and the later they are eluted from the column (see illustration).

Subcategories of size exclusion chromatography

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TSKgel GMHHR-H(S), 300x7.8mm, 13 µm

TSH-17361

Product Type: SEC/GPC Columns Base Material: Styrene-divinylbenzene Delivery Time: approx. 4 days Column Type: Analytical Column Manufacturer: Tosoh Matrix: Polymer Internal Diameter: 7.8 mm Length: 300 mm Technology: HPLC

TSKgel GMHHR-H, 5 µm, 75-650 Å 300 mm x 7.8 mm

TSH-17360

TSKgel G7000HHR, 5 µm, >650 Å 300 mm x 7.8 mm

TSH-17359

TSKgel G6000HHR, 5 µm, >650 Å 300 mm x 7.8 mm

TSH-17358

TSKgel G5000HHR, 5 µm, 650 Å 300 mm x 7.8 mm

TSH-17357

TSKgel G4000HHR, 5 µm, 200 Å 300 mm x 7.8 mm

TSH-17356

TSKgel G3000HHR, 5 µm, 75 Å 300 mm x 7.8 mm

TSH-17355

TSKgel G2500HHR, 5 µm, 30 Å 300 mm x 7.8 mm

TSH-17354

TSKgel G2000HHR, 5 µm, 20 Å 300 mm x 7.8 mm

TSH-17353

TSKgel G1000HHR, 5 µm, 15 Å 300 mm x 7.8 mm

TSH-17352

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Technical Data

Basics

Stationary phases of the SEC

Either porous silica or polymer-based materials are used as stationary phases. In order to minimise adsorption or other undesirable interactions between the analyte and the stationary phase, some of these are modified or undergo special treatment during the manufacturing process. Polymer materials for the GFC are usually based on polymethacrylate or polyhydroxymethacylate. Styrene-divinylbenzene copolymer (PS-DVB) is often used for polymer-based GPC columns.

Mobile phases of SEC

Aqueous mobile phases are used in gel filtration chromatography (GFC). GFC is used as a bioseparation technique for the separation of proteins and other large biomolecules. Gel permeation chromatography (GPC), on the other hand, uses organic mobile phases to separate non-polar polymers (plastics).

SEC detectors

In principle, the same detectors can be used for size exclusion chromatography as in HPLC. In addition, light scattering and viscosity detectors are also used for polymer analysis in order to obtain specific molecular and structural information about the analyte. When SEC is combined with light scattering, viscometers and concentration detectors (known as triple detection), it can calculate absolute molecular weight, molecular size and intrinsic viscosity as well as provide information on macromolecular structure, conformation, aggregation and branching.

Reference materials for SEC

By using reference materials with known molecular size and distribution, the GPC system can be calibrated to allow precise analysis of molecular weights and molecular size distribution in a sample. Calibration standards are essential to regularly check the performance of the GPC system and ensure that results are consistent and reliable over time. These standards also help to optimise separation conditions and improve analytical accuracy, making them a critical component of high quality GPC analysis. MZ-Analysentechnhik GmbH offers you a wide range of different SEC reference materials from manufacturers such as Sigma Aldrich, Tosoh, Shodex or Waters.

Columns for the SEC/GPC

To select a suitable column, a few basic questions should be clarified first.

1. What is the polarity of the analyte and which solvents are suitable?

For very polar biomolecules, such as proteins or antibodies, purely aqueous buffers are often used as eluents. Silica gel has proven to be a suitable base material for this. For polar to medium polar polymers, aqueous or polar organic eluents or mixtures of both are used. The base material is often polymethacrylate or polyvinyl alcohol. For non-polar polymers for whose analysis purely organic eluents are used, styrene-divinylbenzene copolymers (PS/DVB) are usually used as stationary phases.

2. What is the molecular weight of my analyte?

The molecular weight of the analyte or its size in the solvent determines the pore size of the stationary phase. The absolute pore sizes range from about 25 to 30,000 Å, covering a molecular weight range of about 100 to > 20,000,000 Daltons. For samples with very different molecular weights, either several columns with different pore sizes can be connected in series or columns containing particles with different pore sizes can be used. These are often labelled "linear", "mixed gel" or "multipore gel".

In many cases, suitable pore sizes can be easily determined with the help of the usually very extensive calibration data provided by the manufacturers.

1. columns for organic mobile phases (GPC)

Manufacturer	Column lines	Characteristics
MZ-Analysentechnik GmbH	MZ-Gel SDplus(LS)	Base material: PS/DVB Particle sizes: 3, 5 & 10 µm Pore sizes: 50 - 106 Å & linear Further features: LS suitable for light scattering detection
MZ-Analysentechnik GmbH	MZ-SuperFG	Base material: silica gel Particle sizes: 7µm Pore sizes: 100 - 4,000 Å & linear Further features: For fluorinated solvents
Shodex™	K-800 Series KD-800 Series KF-800 Series	Base material: PS/DVB Particle sizes: 6 - 18 µm Pore sizes: 50 - 20,000 Å & "mixed-gel" Other features: For efficient GPC separations
Shodex™	KF-600 Series KF-400HQ Series HK-400 Series	Base material: PS/DVB Particle sizes: 3 - 18 µm Pore sizes: 50 - 20,000 Å & "mixed-gel" Other features: For fast and ultra-fast applications
Shodex™	LF Series	Base material: PS/DVB Particle sizes: 6 µm Pore sizes: Multi-pore gels Other features: Very large linear calibration range
Shodex™	HT and UT Series	For high temperature applications up to 210 °C
Shodex™	HFIP Series	For HFIP as eluent
Tosoh Bioscience™	TSKgel Alpha TSKgel SuperAW	Base material: Highly cross-linked polymethacrylate Particle sizes: 4 - 13 µm Pore sizes: 25 - >1,000 Å & "mixed-gel" Further features: For polar and non-polar eluents
Tosoh Bioscience™	TSKgel H Series	Base material: PS/DVB Particle sizes: 5 & 10 µm Pore sizes: 15 - >1,000 Å & "multi-pore" Further features: High temperature GPC up to 220°C
Sepax™	Mono GPC	Base material: PS/DVB Particle sizes: 5 & 10 µm Pore sizes: 100 - 1,000 Å & linear Other features: High eluent compatibility
Macherey-Nagel™	Nucleogel GPC	Base material: PS/DVB Particle sizes: 5 & 10 µm Pore sizes: 50 -¹⁰⁴ Å Further features: Wide application range
Waters™	Styragel™ Ultrastyragel™	Base material: PS/DVB Particle sizes: 5, 10 & 15-20 µm Pore sizes: 50 -¹⁰⁷ Å & linear Further features: HT for high temperature GPC
SMT™	SMTgel	Base material: silica gel Particle sizes: 5 & 10 µm Pore sizes: 100 - 2000 Å & linear Other features: Various surface modifications available

2. columns for aqueous mobile phases (GFC) and aqueous/organic mobile phases

Manufacturer	Column lines	Characteristics
Tosoh Bioscienace	TSKgel SW Series	Base material: silica gel Particle sizes: 2 - 17 µm Pore sizes: 125 - 450 Å & "mixed-gel" Other features: Industry standard for protein analysis
Tosoh Bioscience	TSKgel PW Series	Base material: polymethacrylate Particle sizes: 3 - 17 µm Pore sizes: 125 - >1,000 Å & "multi-pore" Other features: Wide application range for polar polymers
Shodex™	K-800 Series KD-803 Series KF-400 Series	Base material: Silica gel Particle sizes: 3 - 7 µm Pore sizes: 400 - 2,000 Å Other features: For protein analyses
Shodex™	SB-800HQ Series	Base material: Polyhydroxymethacrylate Particle sizes: 8 - 35 µm Pore sizes: 100 - 30,000 Å & "mixed-gel" Further features: For the analysis of polar polymers
Shodex™	LB-800 Series	Base material: Polyhydroxymethacrylate Particle sizes: 6 - 13 µm Pore sizes: 800 - 15,000 Å Other features: For light scattering detection
Shodex™	GF-HQ	Base material: Polyvinyl alcohol Particle sizes: 5 & 9 µm Pore sizes: 180 - 10,000 Å Further features: For aqueous as well as organic eluents
Sepax™	Unix Zenix SRT SRT-10	Base material: silica gel Particle sizes: 1.8 - 10 µm Pore sizes: 80 - 2,000 Å Other features: For proteins, mAb's, nucleic acids, virus, etc.
Sepax™	Unix-C Zenix-C SRT-C SRT-10C	Base material: silica gel Particle sizes: 1.8 - 10 µm Pore sizes: 80 - 2,000 Å Other features: For membrane proteins, derivatised mAb's, etc.
Mitsubishi Chem. Corp.™	MCI GEL™ CQP-Series	Base material: Hydrophilic polymer Particle sizes: 10 µm Pore sizes: 120 - 600 Å Other features: For polar polymers, such as oligosaccharides or PEG
Supelco	Discovery^® BIO GFC	Base material: silica gel Particle sizes: 5 µm Pore sizes: 100 - 2,000 Å Other features: For the analysis of biomolecules
Waters™	Ultrahydrogel™	Base material: Polyhydroxymethacrylate Particle sizes: 6 - 12 µm Pore sizes: 120 - >2,000 Å & linear Other features: For polar polymers

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