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).

Schematic function of the size exclusion mechanism

Subcategories of size exclusion chromatography

Products

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MZ-Gel SDplus 100 Å 5µm 300x8,0mm SEC / GPC Columns

MZ5015-300080

€1,169.00

Product Type: SEC / GPC Columns Length: 300 mm Internal Diameter: 8.0 mm Particle Size: 5 µm Pore Size: 100 Å Manufacturer: MZ-Analysentechnik GmbH Delivery Time: approx. 1-2 days Column Type: Analytical Column Base Material: Styrene-divinylbenzene Exclusion Limit: 5.000 (PS) Mode of Separation: SEC / Organic Size Exclusion Chromatography (GPC) Hardware Type: Ready-to-use Column Matrix: Polymer Particle Morphology: spherical, fully porous Technology: HPLC Upper Molecular Weight: 3.000 (PS)

MZ-Gel SDplus 500 Å 5µm 300x8,0mm SEC / GPC Columns

MZ5055-300080

€1,169.00

Product Type: SEC / GPC Columns Length: 300 mm Internal Diameter: 8.0 mm Particle Size: 5 µm Pore Size: 500 Å Manufacturer: MZ-Analysentechnik GmbH Delivery Time: approx. 1-2 days Column Type: Analytical Column Base Material: Styrene-divinylbenzene Exclusion Limit: 20.000 (PS) Mode of Separation: SEC / Organic Size Exclusion Chromatography (GPC) Hardware Type: Ready-to-use Column Matrix: Polymer Particle Morphology: spherical, fully porous Technology: HPLC Upper Molecular Weight: 20.000 (PS)

MZ-Gel SDplus 1.000 Å 5µm 300x8,0mm SEC / GPC Columns

MZ5305-300080

€1,169.00

Product Type: SEC / GPC Columns Length: 300 mm Internal Diameter: 8.0 mm Particle Size: 5 µm Pore Size: 1.000 Å (10e3 Å) Manufacturer: MZ-Analysentechnik GmbH Delivery Time: approx. 1-2 days Column Type: Analytical Column Base Material: Styrene-divinylbenzene Lower Molecular Weight: 1.000 (PS) Exclusion Limit: 70.000 (PS) Mode of Separation: SEC / Organic Size Exclusion Chromatography (GPC) Hardware Type: Ready-to-use Column Matrix: Polymer Particle Morphology: spherical, fully porous Technology: HPLC Upper Molecular Weight: 40.000 (PS)

MZ-Gel SDplus 10.000 Å 5µm 300x8,0mm SEC / GPC Columns

MZ5405-300080

€1,169.00

Product Type: SEC / GPC Columns Length: 300 mm Internal Diameter: 8.0 mm Particle Size: 5 µm Pore Size: 10.000 Å (10e4 Å) Manufacturer: MZ-Analysentechnik GmbH Delivery Time: approx. 1-2 days Column Type: Analytical Column Base Material: Styrene-divinylbenzene Lower Molecular Weight: 4.000 (PS) Exclusion Limit: 700.000 (PS) Mode of Separation: SEC / Organic Size Exclusion Chromatography (GPC) Hardware Type: Ready-to-use Column Matrix: Polymer Particle Morphology: spherical, fully porous Technology: HPLC Upper Molecular Weight: 500.000 (PS)

MZ-Gel SDplus 100 Å 10µm 50x8,0mm SEC / GPC Guard Columns

MZ5010-050080

€408.00

Product Type: SEC / GPC Guard Columns Length: 50 mm Internal Diameter: 8.0 mm Particle Size: 10 µm Pore Size: 100 Å Manufacturer: MZ-Analysentechnik GmbH Delivery Time: approx. 1-2 days Base Material: Styrene-divinylbenzene Exclusion Limit: 5.000 (PS) Mode of Separation: SEC / Organic Size Exclusion Chromatography (GPC) Hardware Type: Ready-to-use Column Matrix: Polymer Particle Morphology: spherical, fully porous Technology: HPLC Upper Molecular Weight: 3.000 (PS)

MZ-Gel SDplus 1.000 Å 10µm 300x8,0mm SEC / GPC Columns

MZ5300-300080

€913.00

Product Type: SEC / GPC Columns Length: 300 mm Internal Diameter: 8.0 mm Particle Size: 10 µm Pore Size: 1.000 Å (10e3 Å) Manufacturer: MZ-Analysentechnik GmbH Delivery Time: approx. 1-2 days Column Type: Analytical Column Base Material: Styrene-divinylbenzene Lower Molecular Weight: 1.000 (PS) Exclusion Limit: 70.000 (PS) Mode of Separation: SEC / Organic Size Exclusion Chromatography (GPC) Hardware Type: Ready-to-use Column Matrix: Polymer Particle Morphology: spherical, fully porous Technology: HPLC Upper Molecular Weight: 40.000 (PS)

MZ-Gel SDplus 10.000 Å 10µm 300x8,0mm SEC / GPC Columns

MZ5400-300080

€913.00

Product Type: SEC / GPC Columns Length: 300 mm Internal Diameter: 8.0 mm Particle Size: 10 µm Pore Size: 10.000 Å (10e4 Å) Manufacturer: MZ-Analysentechnik GmbH Delivery Time: approx. 1-2 days Column Type: Analytical Column Base Material: Styrene-divinylbenzene Lower Molecular Weight: 4.000 (PS) Exclusion Limit: 700.000 (PS) Mode of Separation: SEC / Organic Size Exclusion Chromatography (GPC) Hardware Type: Ready-to-use Column Matrix: Polymer Particle Morphology: spherical, fully porous Technology: HPLC Upper Molecular Weight: 500.000 (PS)

MZ-Gel SDplus linear 5µm 300x8,0mm SEC / GPC Columns

MZ5005-300080

€1,270.00

Product Type: SEC / GPC Columns Length: 300 mm Internal Diameter: 8.0 mm Particle Size: 5 µm Pore Size: linear Manufacturer: MZ-Analysentechnik GmbH Delivery Time: approx. 1-2 days Column Type: Analytical Column Base Material: Styrene-divinylbenzene Lower Molecular Weight: 1.000 (PS) Exclusion Limit: 2.000.000 (PS) Mode of Separation: SEC / Organic Size Exclusion Chromatography (GPC) Hardware Type: Ready-to-use Column Matrix: Polymer Particle Morphology: spherical, fully porous Technology: HPLC Upper Molecular Weight: 1.000.000 (PS)

MZ-Gel SDplus 50 Å 10µm 300x8,0mm SEC / GPC Columns

MZ5530-300080

€913.00

Product Type: SEC / GPC Columns Length: 300 mm Internal Diameter: 8.0 mm Particle Size: 10 µm Pore Size: 50 Å Manufacturer: MZ-Analysentechnik GmbH Delivery Time: approx. 1-2 days Column Type: Analytical Column Base Material: Styrene-divinylbenzene Exclusion Limit: 3.000 (PS) Mode of Separation: SEC / Organic Size Exclusion Chromatography (GPC) Hardware Type: Ready-to-use Column Matrix: Polymer Particle Morphology: spherical, fully porous Technology: HPLC Upper Molecular Weight: 2.000 (PS)

MZ-Gel SDplus 100 Å 10µm 300x8,0mm SEC / GPC Columns

MZ5010-300080

€913.00

Product Type: SEC / GPC Columns Length: 300 mm Internal Diameter: 8.0 mm Particle Size: 10 µm Pore Size: 100 Å Manufacturer: MZ-Analysentechnik GmbH Delivery Time: approx. 1-2 days Column Type: Analytical Column Base Material: Styrene-divinylbenzene Exclusion Limit: 5.000 (PS) Mode of Separation: SEC / Organic Size Exclusion Chromatography (GPC) Hardware Type: Ready-to-use Column Matrix: Polymer Particle Morphology: spherical, fully porous Technology: HPLC Upper Molecular Weight: 3.000 (PS)

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Technical Data

Basics

Stationary phases in SEC

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

Mobile phases in SEC

In gel filtration chromatography (GFC), aqueous mobile phases are used. GFC serves as a bio-separation technique for the separation of proteins and other large biomolecules. Gel permeation chromatography (GPC), on the other hand, is carried out using organic mobile phases to separate non-polar polymers (plastics).

Detectors in SEC

In principle, the same detectors as those used in HPLC can be employed for size-exclusion chromatography. In addition, light-scattering and viscosity detectors are also used in polymer analysis 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 the 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 enable precise analysis of molecular weights and molecular size distribution in a sample. Calibration standards are essential for regularly checking the performance of the GPC system and ensuring that results remain consistent and reliable over time. These standards also assist in optimising separation conditions and improving analytical accuracy, making them a critical component of high-quality GPC analyses. MZ-Analysentechnik GmbH offers a wide range of different SEC reference materials from manufacturers such as Sigma Aldrich, Tosoh, Shodex and Waters.

Columns for the SEC/GPC

Before selecting a suitable column, a number of fundamental questions should first be clarified.

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

For highly 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 purpose. For polar to moderately 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 the analysis of which purely organic eluents are used, styrene-divinylbenzene copolymers (PS/DVB) are usually employed 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 approximately 25 to 30,000 Å, covering a molecular weight range of approximately 100 to > 20,000,000 daltons. For samples with widely varying molecular masses, either several columns with different pore sizes can be connected in series, or columns containing particles of different pore sizes can be used. These are often labelled ‘linear’, ‘mixed-gel’ or ‘multipore-gel’.

In many cases, suitable pore widths can be determined without difficulty using the manufacturers’ calibration data, which is usually very comprehensive.

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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