Waters Gel Permeation Chromatography (GPC)

1

Gel Permeation  
 

Chromatography (GPC)

[ GPC Capabilities ]

2

In 1964 John C. Moore, of the Dow Chemical Company, published his work on the preparation of Gel Permeation Chromatography 
(GPC) and changed how scientists studied synthetic polymers and macromolecules. Shortly thereafter Waters Corporation licensed 
the technology from Dow to produce the first commercially-available gel permeation chromatograph, the GPC-100. With dedicated, 
purpose-built instrumentation combined with the innovations from the Dow Chemical Company it was possible for GPC to provide 
critical information to scientists that was difficult to obtain by other methods. 

For over 40 years, Waters has continued to refine the instrumentation, packing materials, and technology to improve GPC and SEC 
analysis. T hese innovations allow size-exclusion techniques to expand outside of the original polymer analysis to include 
applications for separating small and large molecules from interfering matrices, such as those found in foods, pharmaceutical 
preparations, and natural products.

As a market leader in GPC analysis, Waters provides you with the highest quality GPC products and expert applications  
support. As a primary manufacturer of chromatographic instrumentation and consumables, all our facilities follow strict ISO, FDA 
and cGMP guidelines. This is your assurance that Waters will continue to provide you with solutions that will be at the forefront of 
separation science.

Gel Permeation Chromatography (GPC)

Table of Contents

Organic Soluble Polymers
Standards for Non-Aqueous Calibration ..........................................................4
GPC Columns for Non-Aqueous Samples .........................................................6

Water Soluble Polymers and Small Molecules
Standards for Aqueous Calibration................................................................14
SEC Columns for Aqueous Samples ...............................................................16
SEC Columns for Protein Analysis and Characterization....... .......................18

Autosampler Vials
LC/GC Certified Vials ..................................................................................... 20
TruView LCMS Certified Vials ........................................................................ 20
Choosing the Right Vial and Septum for Your Application ............................21
Vial Closures Guide ........................................................................................21

Tips and Frequently Asked Questions
Solvent Considerations ................................................................................. 24
Frequently Asked Questions ......................................................................... 26

3

Gel Permeation Chromatography (GPC)

4

Reference Materials for Non-Aqueous Samples

We understand that accurate and reliable data is only achieved using a properly calibrated system. By providing you with well-characterized 
polymer standards and reference materials we help you to focus on results and maintain your productivity. T he polymers used in our 
reference materials have been specifically manufactured to provide known molecular weight data for a wide range of analysis. W hether 
your choice is for an individual standard or a cocktail mix, you can count on the traceability of our performance-based reference materials.

Non-Aqueous GPC Standards Guide

Reference materials for 
the analysis of organic 
soluble polymers.

Effective Molecular Weight Range

Effective Molecular Weight Range

101

102

103

104

105

106

107

101

102

103

104

105

106

107

Polystyrene ReadyCal 

Polybutadiene

Polyisoprene

Polymethylmethacrylate

Polymethylmethacrylate

ReadyCal Standards

A ReadyCal kit allows you to quickly and accurately prepare a multi-point calibration curve without the need to weigh chemicals. Each vial contains a 
polymer mix that spans a molecular weight range to provide baseline resolution of each component. Simply add solvent directly to the vial and mix.

Description*

Part No.

Polystyrene ReadyCal Standards 4 mL Kit 

WAT058930

A complete kit of ready-to-use polystyrene calibration standards. Kit contains thirty 4 mL autosampler vials which contain four polystyrene standards per vial. There are three separate  
molecular weight ranges in each kit, ten units of each range. Range is from 400 to 2,000,000 Da

Polystyrene ReadyCal Standards 2 mL Kit 

WAT058931

A complete kit of ready-to-use polystyrene calibration standards. Kit contains thirty 2 mL autosampler vials which contain four polystyrene standards per vial. There are three separate 
molecular weight ranges in each kit, ten units of each range. Range is from 400 to 2,000,000 Da

[ORGANIC SOLUBLE POLYMERS ]

Standards for Non-Aqueous Calibration

*Values listed are approximate molecular weights.

5

Polymer Specific Calibration Standards

Tailored specifically for different types of polymer analysis, these conveniently prepared calibration standards provide the analyst a quick 
and reliable reference to known molecular weight ranges. Polymer type and MW ranges are specified in the product guide below.

Description*

Part No.

Polybutadiene Standards Kit 

WAT035709

0.5 g/vial polybutadiene at each molecular weight: 1000, 3000, 7000, 10,000, 30,000, 70,000, 100,000, 300,000, 700,000, 1,000,000

Polyisoprene Standards Kit 

WAT035708

0.5 g/vial polyisoprene at each molecular weight: 1000, 3000, 10,000, 30,000, 70,000, 100,000, 300,000, 500,000, 1,000,000, 3,000,000

Polymethylmethacrylate Low MW Standards Kit

WAT035707

0.5 g/vial polymethylmethacrylate at each molecular weight: 1000, 1700, 2500, 3500, 5000, 7000, 10,000, 13,000, 20,000, 30,000

Polymethylmethacrylate Mid MW Standards Kit

WAT035706

0.5 g/vial polymethylmethacrylate at each molecular weight: 2400, 9500, 31,000, 52,000, 100,000, 170,000, 270,000, 490,000, 730,000, 1,000,000

Individual MW Reference Materials

In many cases a single calibration standard is used to verify a molecular weight component in a sample mixture or extend the range of an 
existing calibration solution. T hese individual component standards make molecular weight identification simple and straightforward.

Description*

Part No.

Polystyrene Standard 400 

WAT011590

10 g/vial polystyrene, 400 MW

Polystyrene Standard 530 

WAT011592

10 g/vial polystyrene, 530 MW

Polystyrene Standard 950 

WAT011594

10 g/vial polystyrene, 950 MW

Polystyrene Standard 2,800 

WAT011596

5 g/vial polystyrene, 2,800 MW

Polystyrene Standard 6,400 

WAT011598

5 g/vial polystyrene, 6,400 MW

Polystyrene Standard 10,100 

WAT011600

5 g/vial polystyrene, 10,100 MW

Polystyrene Standard 17,000 

WAT011602

5 g/vial polystyrene, 17,000 MW

Polystyrene Standard 43,000 

WAT011604

5 g/vial polystyrene, 43,000 MW

Polystyrene Standard 110,000 

WAT011606

5 g/vial polystyrene, 110,000 MW

Polystyrene Standard 180,000 

WAT011608

5 g/vial polystyrene, 180,000 MW

Description*

Part No.

Polystyrene Standard 430,000 

WAT011612

5 g/vial polystyrene, 430,000 MW

Polystyrene Standard 780,000 

WAT011614

5 g/vial polystyrene, 780,000 MW

Polystyrene Standard 1,300,000 

WAT011616

1 g/vial polystyrene, 1,300,000 MW

Polystyrene Standard 2,800,000 

WAT011618

1 g/vial polystyrene, 2,800,000 MW

Polystyrene Standard 3,600,000 

WAT011620

1 g/vial polystyrene, 3,600,000 MW

Polystyrene Standard 4,300,000 

WAT011622

1 g/vial polystyrene, 4,300,000 MW

Polystyrene Standard 5,200,000 

WAT011624

1 g/vial polystyrene, 5,200,000 MW

Polystyrene Standard 6,200,000 

WAT011626

1 g/vial polystyrene, 6,200,000 MW

Polystyrene Standard 8,400,000 

WAT011628

1 g/vial polystyrene, 8,400,000 MW

Polystyrene Standard 20,000,000

WAT011630

1 g/vial polystyrene, 20,000,000 MW

*Values listed are approximate molecular weights.

*Values listed are approximate molecular weights.

6

GPC Columns for Non-Aqueous Samples

A GPC column is selected based on the goals of the separation, which often ranges from one of maximum speed for screening to that of 
maximum resolution for determining product quality control. Each analysis provides unique challenges for separation. By providing you 
with a comprehensive selection of GPC columns, you can be certain that the column or column bank that you choose will be compatible with temperature, 
solvent, and polymer type.

T he following charts may be used to quickly compare the molecular weight ranges for the specified columns. By connecting two or more 
columns in series, the effective molecular weight range can be extended to provide coverage for more complex sample analysis.

101

102

103

104

105

106

107

108

Effective Molecular Weight Range

Effective Molecular Weight Range

UHDPE
UH-Polystyrene
UH-Isoprene
UH-PMMA

Ultra-High Molecular Weight 
Waters Styragel HMW Series
20 µm particle size
Ambient to 150 oC

Styragel HMW 7

Styragel HMW 6E

Styragel HMW 2

Applications:

ABS
Acetyl
Acrylics
EVA
Nylon
PEEK
PET/PBT

Mid-Range Molecular Weight
Waters Styragel HT Series
10 µm particle size
Ambient to 150 oC

Styragel HT 6E

Styragel HT 6

Styragel HT 5

Styragel HT 4

Styragel HT 3  

Styragel HT 2

Applications:

Additives
Epoxy
Oligomers
Phenalics
Unsaturated Polyester
Urea/Formaldehyde
Polyethylene Glycol
Ethanolamines
Melamine Resin

Styragel HR 5E

Styragel HR 4E

Styragel HR 6

Styragel HR 5

Styragel HR 4

Styragel HR 3

Styragel HR 2

Styragel HR 1

Styragel HR 0.5

Low Molecular Weight 
Waters Styragel HR Series
5 µm particle size
Ambient to 80 oC

Applications:

101

102

103

104

105

106

107

108

Styragel Columns Selection Guide

HMW—High Molecular Weight

HT—High Temperature

HR—High Resolution

[ORGANIC SOLUBLE POLYMERS ]

7

High Temperature
Waters HSPgel HT Series
5 µm particle size
Ambient to 180 °C

Room Temperature
Waters HSPgel RT Series
3 µm particle size
Ambient to 80 °C

High Resolution
Waters HSPgel HR Series
3 µm particle size
Ambient to 80 °C

101

102

103

104

105

106

107

108

Effective Molecular Weight Range

Effective Molecular Weight Range

101

102

103

104

105

106

107

108

HSPgel HT MB-H 

HSPgel HT 1.0 

HSPgel HT 2.0 

HSPgel HT 2.5 

HSPgel HT 3.0 

HSPgel HT 4.0 

HSPgel HT 5.0 

HSPgel HT 6.0 

HSPgel HT 7.0 

HSPgel HT MB-L 

HSPgel HT MB-L/M 

HSPgel HT MB-M 

HSPgel RT 2.0 

HSPgel RT 2.5 

HSPgel RT 3.0 

HSPgel RT 4.0 

HSPgel RT 5.0 

HSPgel RT 6.0 

HSPgel RT MB-L 

HSPgel RT MB-L/M 

HSPgel RT MB-M 

HSPgel RT MB-H 

HSPgel HR 1.0 

HSPgel HR 2.0 

HSPgel HR 2.5 

HSPgel HR 3.0 

HSPgel HR 4.0 

HSPgel HR MB-L 

HSPgel HR MB-M 

HSPgel RT 1.0 

HSPgel Columns Selection Guide*

*MW ranges for HR and RT are based on polystyrene chain lengths. 

HR—High Resolution
RT—Room Temperature

HT—High Temperature
MB—Mixed Bed

L—Low MW Range
M—Medium MW Range

L/M—Low/Medium MW Range
H—High MW Range

8

Styragel Columns for Polymer Characterization

Styragel HR (High-Resolution) Columns

Designed particularly for low molecular weight samples, the Waters Styragel® HR Columns are ideal for the analysis of oligomers, epoxies, 
and polymer additives where high resolution is critical. Packed with rigid 5-µm particles, these columns deliver unrivaled resolution and 
efficiency in the low-to-mid molecular weight region. 

Calibration Curves for the Waters Styragel HR Series 
of High-Resolution Columns

Styragel HR

Mo

lecu

lar We

ig

ht

12 min

10

8

6

4

102

103

104

105

106

107

Styragel HR 0.5
Styragel HR 1
Styragel HR 2
Styragel HR 3

Styragel HR 4
Styragel HR 5
Styragel HR 6
Styragel HR 4E
Styragel HR 5E

Elution Volume (mL)

Sample: Polystyrene
Mobile Phase: 

THF

Flow Rate: 

1 mL/min

45 min

40

35

30

25

20

45 min

40

35

30

25

20

Column Bank: 

Styragel HR 0.5, 1, 2, and 3

Part Numbers:   WAT044231, WAT044234,   
 

WAT044237, WAT044222

Mobile Phase: 

THF

Column Temp.:  35 °C
Flow Rate: 

1 mL/min

Sample: 

Polystyrene standards: 

0.5K, 5.05K, 49.8K

Conventional Styragel HR Columns 
7.8 x 300 mm

Column Bank: 

Styragel HR 0.5, 1, 2, and 3

Part Numbers:   WAT045835, WAT045850,   
 

WAT045865, WAT045880

Mobile Phase: 

THF

Column Temp.:  35 °C
Flow Rate: 

0.35 mL/min

Sample: 

Polystyrene standard mix  

0.5K, 5.05K, 49.8K

Solvent-Efficient Styragel HR Columns 
4.6 x 300 mm

Styragel HR Columns for Unrivaled Resolution 
of Low Molecular Weight Samples

Styragel HR Columns (4.6 x 300 mm)

T he 4.6 x 300 mm solvent-efficient Styragel Columns offer the same high resolution performance as our conventional 7.8 x 300 mm 
Styragel Columns, with the added advantage of reducing solvent consumption by two-thirds. 

Column

Effective  

MW Range

Part No.  

THF

Part No.  

DMF

Part No.  

Toluene

Styragel HR 0.5

0–1,000

WAT045835

WAT045840

WAT045830

Styragel HR 1

100–5,000

WAT045850

WAT045855

WAT045845

Styragel HR 2

500–20,000

WAT045865

WAT045870

WAT045860

Styragel HR 3

500–30,000

WAT045880

WAT045885

WAT045875

Styragel HR 4

5,000–600,000

WAT045895

WAT045900

WAT045890

Styragel HR 4E

50–100,000

WAT045805

WAT045810

WAT045800

Styragel HR 5E

2,000–4,000,000

WAT045820

WAT045825

WAT045815

Styragel HR Columns (7.8 x 300 mm)

Column

Effective  

MW Range

Part No.  

THF

Part No.  

DMF

Part No.  

Toluene

Styragel HR 0.5

0–1,000

WAT044231

WAT044232

WAT044230

Styragel HR 1

100–5,000

WAT044234

WAT044235

WAT044233

Styragel HR 2

500–20,000

WAT044237

WAT044238

WAT044236

Styragel HR 3

500–30,000

WAT044222

WAT044223

WAT044221

Styragel HR 4

5,000–600,000

WAT044225

WAT044226

WAT044224

Styragel HR 4E

50–100,000

WAT044240

WAT044241

WAT044239

Styragel HR 5

50,000–4,000,000

WAT054460

WAT054466

WAT054464

Styragel HR 5E

2,000–4,000,000

WAT044228

WAT044229

WAT044227

Styragel HR 6

200,000–10,000,000

WAT054468

WAT054474

WAT054470

Styragel Guard Column 4.6 x 30 mm

—

WAT054405

WAT054415

WAT054410

[ORGANIC SOLUBLE POLYMERS ]

9

Styragel HT (High-Temperature) Columns

T he Styragel HT Columns can be used with aggressive solvents at high temperatures without sacrificing resolution or column lifetime. 
Packed with rigid 10-µm particles, they have a typical plate count greater than 10,000 plates per column. T hese columns are extremely 
durable due to a narrow particle size distribution that results in a very stable column bed. Suitable for both ambient and high-temperature 
analysis, the Styragel HT Columns offer excellent resolution of polymers in the mid-to-high molecular weight range.

Calibration Curves for the Waters Styragel HT 
Series of High-Temperature Columns

Styragel HMW

Mo

lecu

lar We

ig

ht

12 min

10

11

7

8

9

4

5

6

102

103

104

105

106

107

108

109

Elution Volume (mL)

Styragel HT 2
Styragel HT 3
Styragel HT 4

Styragel HT 5
Styragel HT 6
Styragel HT 6E

Sample: Polystyrene
Mobile Phase: 

THF

Flow Rate: 

1 mL/min

Styragel HT Columns Deliver Superior Performance — 
Even at High Temperatures

Branching

Cumulative Distribution

Distribution

3

4

5

6

Log (MW)

% 

MW

100

80

60

40

20

0

Column: 

Styragel HT 6E

Part Number:  

WAT044218

Mobile Phase: 

TCB

Column Temp.: 

140 °C

Sample: LDPE

Column

Effective  

MW Range

Part No. 

THF

Part No. 

DMF

Part No. 

Toluene

Styragel HT 3

500–30,000

WAT045920

WAT045925

WAT045915

Styragel HT 4

5,000–600,000

WAT045935

WAT045940

WAT045930

Styragel HT 5

50,000–4,000,000

WAT045950

WAT045955

WAT045945

Styragel HT 6

200,000–10,000,000

WAT045965

WAT045970

WAT045960

Styragel HT 6E

5,000–10,000,000

WAT045980

WAT045985

WAT045975

Styragel HT Columns (7.8 x 300 mm)

Column

Effective

MW Range

Part No.

THF

Part No.

DMF

Part No.

Toluene

Styragel HT 2

100–10,000

WAT054475

WAT054480

WAT054476

Styragel HT 3

500–30,000

WAT044207

WAT044208

WAT044206

Styragel HT 4

5,000–600,000

WAT044210

WAT044211

WAT044209

Styragel HT 5

50,000–4,000,000

WAT044213

WAT044214

WAT044212

Styragel HT 6

200,000–10,000,000

WAT044216

WAT044217

WAT044215

Styragel HT 6E

5,000–10,000,000

WAT044219

WAT044220

WAT044218

Styragel Guard Column 4.6 x 30 mm

—

WAT054405

WAT054415

WAT054410

Styragel HT Columns (4.6 x 300 mm)

The same high performance as our conventional Styragel HT Columns with the added advantage of reducing your solvent consumption 
by two-thirds.

10

Calibration Curves for Waters Styragel HMW 
Series of High-Molecular Weight Columns

Styragel HMW (High-Molecular Weight) Columns

T he Styragel HMW Columns were specifically designed for the analysis of ultra-high molecular weight polymers susceptible to shearing. 
Combining high-porosity 10-µm frits and 20-µm particles, the Styragel HMW Columns minimize polymer shear effects. T hese state-of-the-art 
columns can be used at ambient or elevated temperatures, and exhibit excellent column lifetime.

Styragel HMW

Styragel HMW 2

Elution Volume (mL)

Styragel HMW 6E
Styragel HMW 7

Mo

lecu

lar We

ig

ht

12 min

10

11

7

8

9

4

5

6

102

103

104

105

106

107

108

109

Sample: Polystyrene
Mobile Phase:  THF
Flow Rate: 

1 mL/min

Styragel HMW Columns are Optimized for Analysis of Shear-Sensitive, Ultra-High Molecular Weight Polymers

0.0000

0.1000

0.1500

0.2000

0.2500

0.3000

0.3500

0.4000

0.4500

0.5000

0.5500

10.0000

20.0000

30.0000

Retention Time (min)

De

tector Re

sp

onse

40.0000 min

PS Mix 10.2M, 1.075M, 95K, 13K, & 1.68K

0.0000

0.1000

0.1500

0.2000

0.2500

0.3000

0.3500

0.4000

0.4500

0.5000

0.5500

10.0000

20.0000

30.0000

Retention Time (min)

De

tector Re

sp

onse

40.0000 

PS Mix 14.4M, 3.84M, 330K, 34.5K, & 3.25K

0.0000

0.1000

0.1500

0.2000

0.2500

0.3000

0.3500

0.4000

0.4500

0.5000

0.5500

10.0000

20.0000

30.0000

Retention Time (min)

De

tector Re

sp

onse

40.0000 min

PS Mix 10.2M, 1.075M, 95K, 13K, & 1.68K

0.0000

0.1000

0.1500

0.2000

0.2500

0.3000

0.3500

0.4000

0.4500

0.5000

0.5500

10.0000

20.0000

30.0000

Retention Time (min)

De

tector Re

sp

onse

40.0000 

PS Mix 14.4M, 3.84M, 330K, 34.5K, & 3.25K

Column Bank: 

2 Styragel HMW 7 and 2 Styragel HMW 6E

Part Numbers: 

WAT044200 & WAT044203

Column Dimensions:   

7.8 x 300 mm

Polystyrene Stds: 

14.4M, 3.84M, 330K, 34.5K, 3.25K

Flow Rate: 

1 mL/min

Column Temp.: 

145 °C

Solvent: 

 TCB

Column Bank: 

2 Styragel HMW 7 and 2 Styragel HMW 6E

Part Numbers: 

WAT044200 & WAT044203

Column Dimensions:   

7.8 x 300 mm

Polystyrene Stds: 

10.2M, 1.075M, 95K, 13K, 1.68K

Flow Rate: 

1 mL/min

Column Temp.: 

145 °C

Solvent: 

 TCB

Styragel HMW Columns (7.8 x 300 mm)

Styragel HMW Columns (4.6 x 300 mm) 

T he same high performance as our conventional Styragel HMW  
Columns with the added advantage of reducing your solvent  
consumption by two-thirds.

Column

Effective  

MW Range

 Part No. 

THF

Part No. 

DMF

Part No.  

Toluene

Styragel HMW 2

100–10,000

WAT054488

WAT054494

WAT054490

Styragel HMW 7

500,000–1 x 108

WAT044201

WAT044202

WAT044200

Styragel HMW 6E

5,000–1 x 107

WAT044204

WAT044205

WAT044203

Styragel Guard 
Column 4.6 x 30 mm

—

WAT054405

WAT054415

WAT054410

Column

Effective  

MW Range

 Part No.

THF

Part No.

DMF

Part No.  

Toluene

Styragel HMW 7

500,000–1 x 108

WAT046805

WAT046810

WAT046800

Styragel HMW 6E

5,000–1 x 107

WAT046820

WAT046825

WAT046815

*System dead volume must be minimized for maximum column performance.

[ORGANIC SOLUBLE POLYMERS ]

11

Ultrastyragel Columns

Ultrastyragel™ Preparative Columns provide high-efficiency GPC separations for compound isolation and sample clean-up.  Closely related to 
Styragel GPC Columns, the family of Ultrastyragel Columns provide a two- to three-fold increase in efficiency (plates/meter) that improves 
separation speed and reduces solvent consumption for preparative isolation. Separations that once required several smaller Styragel Columns 
can be performed on a single, more efficient Ultrasytragel Preparative Column.

Ultrastyragel Columns (19 x 300 mm)

For high resolution preparative applications, these columns are available in toluene or THF.

Pore Size

Effective  

MW Range

Flow Rate  

(mL/min)

Part No.  

Toluene

Part No. 

THF

100 Å

50–1,500

4–10

WAT025866

WAT025859

500 Å

100–10,000

4–10

WAT025867

WAT025860

1000 Å

200–30,000

4–10

WAT025868

WAT025861

10,000 Å

5,000–600,000

4–10

WAT025869

WAT025862

100,000 Å

50,000–4,000,000

4–10

WAT025870

WAT025863

1,000,000 Å

200,000–10,000,000

4–10

WAT025871

WAT025864

Linear 

2,000–4,000,000

4–10

WAT025872

WAT025865

12

HSPgel Columns for High-Speed GPC Analysis

Waters HSPgel™ Column offering for high-speed GPC analysis,  
provides accurate and precise molecular weight determination,  
increased sample throughput, and greatly reduced solvent  
consumption and disposal. Waters offers a series of 6.0 x 150 mm 
high-speed GPC columns. 



  HSPgel HR series for high resolution, room temperature GPC



  HSPgel RT series for routine room temperature GPC



  HSPgel HT series for high temperature GPC

T he HSPgel HR series is designed for high resolution, room  
temperature, organic polymer GPC. T hese columns are packed  
in THF and can be converted once to toluene, methylene chloride, 
or chloroform. 

Column

Solvent

Particle  

Size

MW Range

Part No.

High-Temperature GPC

HSPgel HT 1.0

THF

5 µm

100–1,000 

186001761

HSPgel HT 2.0

THF

5 µm

500–10,000 

186001762

HSPgel HT 2.5

THF

5 µm

1,000–20,000 

186001763

HSPgel HT 3.0

THF

5 µm

2,000–60,000 

186001764

HSPgel HT 4.0

THF

5 µm

10,000–400,000 

186001765

HSPgel HT 5.0

THF

5 µm

25,000–4,000,000 

186001766

HSPgel HT 6.0

THF

5 µm

50,000–10,000,000 

186001767

HSPgel HT 7.0

THF

5 µm

100,000–15,000,000

186001768

HSPgel HT MB-L

THF

5 µm

100–1,000 

186001769

HSPgel HT MB-L/M

THF

5 µm

500–400,000 

186001770

HSPgel HT MB-M

THF

5 µm

1,000–4,000,000 

186001771

HSPgel HT MB-H

THF

5 µm

5,000–10,000,000

186001772

HSPgel HT 1.0

ODCB

5 µm

100–1,000 

186001773

HSPgel HT 2.0

ODCB

5 µm

500–10,000 

186001774

HSPgel HT 2.5

ODCB

5 µm

1,000–20,000 

186001775

HSPgel HT 3.0

ODCB

5 µm

2,000–60,000 

186001776

HSPgel HT 4.0

ODCB

5 µm

10,000–400,000 

186001777

HSPgel HT 5.0

ODCB

5 µm

25,000–4,000,000 

186001778

HSPgel HT 6.0

ODCB

5 µm

50,000–10,000,000 

186001779

HSPgel HT 7.0

ODCB

5 µm

100,000–15,000,000

186001780

HSPgel HT MB-L

ODCB

5 µm

100–1,000 

186001781

HSPgel HT MB-L/M

ODCB

5 µm

500–400,000 

186001782

HSPgel HT MB-M

ODCB

5 µm

1,000–4,000,000 

186001783

HSPgel HT MB-H

ODCB

5 µm

5,000–10,000,000

186001784

Column

Solvent

Particle  

Size

MW Range

Part No.

Ultra-High Resolution GPC*

HSPgel HR 1.0

THF

3 µm

100–1,000 

186001741

HSPgel HR 2.0

THF

3 µm

500–10,000 

186001742

HSPgel HR 2.5

THF

3 µm

1,000–20,000 

186001743

HSPgel HR 3.0

THF

3 µm

2,000–60,000 

186001744

HSPgel HR 4.0

THF

3 µm

10,000–400,000 

186001745

HSPgel HR MB-L 

THF

3 µm

500–700,000 

186001746

HSPgel HR MB-M

THF

3, 5 µm

1,000–4,000,000 

186001747

Column

Solvent

Particle  

Size

MW Range

Part No.

Room-Temperature GPC*

HSPgel RT 1.0

THF

3 µm

100–1,000 

186001749

HSPgel RT 2.0

THF

3 µm

500–10,000 

186001750

HSPgel RT 2.5

THF

3 µm

1,000–20,000 

186001751

HSPgel RT 3.0

THF

3 µm

2,000–60,000 

186001752

HSPgel RT 4.0

THF

3 µm

10,000–400,000 

186001753

HSPgel RT 5.0

THF

3 µm

25,000–4,000,000 

186001754

HSPgel RT 6.0

THF

5 µm

50,000–10,000,000 

186001755

HSPgel RT MB-L

THF

3 µm

100–10,000 

186001757

HSPgel RT MB-L/M

THF

3 µm

500–400,000 

186001758

HSPgel RT MB-M

THF

3 µm

1,000–4,000,000 

186001759

HSPgel RT MB-H

THF

3, 5 µm

5,000–10,000,000

186001760

T he HSPgel RT series are designed for room temperature, routine 
work of organic polymer GPC. T hese come packed in THF and can 
be converted multiple times from THF to toluene, chloroform, 
methylene chloride, DMF, DMSO, etc. 

T he HSPgel HT series are designed for room temperature to high 
temperature (180 °C) organic GPC. T he columns come shipped in 
either THF or ODCB. T he ODCB packed column should be used for 
direct conversion to TCB. T hese columns can withstand multiple 
solvent switches.

*MW ranges for HR and RT are based on polystyrene chain lengths

HR—High Resolution
RT—Room Temperature

HT—High Temperature
MB—Mixed Bed

L—Low MW Range
M—Medium MW Range

L/M—Low/Medium MW Range
H—High MW Range

[ORGANIC SOLUBLE POLYMERS ]

MV

- 

10.

0. 

10.

20.

30.

40.

50.

60.

70.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0 min

Column: HSPgel MB-M, 6.0 mm x 15 cm
Flow Rate:  0.6 mL/min
Injection Volume: 5 µL

PS Standards

3,840,000

2,890,000

1,260,000

775,000

422,000

186,000

42,800

16,700

5,570

2,980

890

474

High Speed GPC of Polystyrene Standards

13

Shodex GPC Columns

Waters is proud to distribute Shodex™ GPC Columns and accessories. 
For over 25 years, Shodex GPC Columns have been used successfully 
by scientists worldwide.  The following selection of highly-reproducible 
GPC columns contains styrene divinylbenzene resins.

K-800 Series (8 x 300 mm)

Ultra-high-efficiency columns designed for high-resolution performance.  
They are available in THF, DMF, or chloroform.

Type

Polystyrene Exclusion Limit

Part No.

HFIP-803

70,000

WAT035605

HFIP-806M (linear)

40,000,000

WAT035611

HFP-LG Pre-column (8 x 50 mm)

—

WAT035612

Type

Polystyrene Exclusion Limit

Part No.

KF-800 (THF)

KF-801

1500

WAT030697

KF-802

5000

WAT030698

KF-802.5

20,000

WAT030699

KF-803

70,000

WAT034100

KF-804

400,000

WAT034101

KF-805

4,000,000

WAT034102

KF-807

200,000,000

WAT034104

KF-806M (linear)

40,000,000

WAT034105

KF-G Pre-column (4.6 x 10 mm)

—

WAT034106

K-800 (Chloroform)

K-802.5

20,000

WAT034109

K-803

70,000

WAT034110

K-804

400,000

WAT034111

K-805

4,000,000

WAT034112

K-G Pre-column (4.6 x 10 mm)

—

WAT035524

KD-800 (DMF)

KD-801

2500

WAT034116

KD-802

5000

WAT034117

KD-802.5

20,000

WAT034118

KD-803

70,000

WAT034119

KD-804

400,000

WAT034120

KD-806 

40,000,000

WAT034122

KD-807

200,000,000

WAT034123

KD-806M (linear)

40,000,000

WAT034124

KD-G Pre-column (4.6 x 10 mm)

—

WAT034125

KS-800

KS-801

1000

WAT034276

KS-802

10,000

WAT034277

KS-804

400,000

WAT034279

KS-800 Pre-column (4.6 x 10 mm)

—

WAT034282

HFIP-800 Series (8 x 300 mm)

T hese columns have the same high efficiency as the K-series 
columns but are available in HFIP.

Column

Solvent

Dimension

Part No.

Envirogel GPC Cleanup

Methylene Chloride

19 x 150 mm

WAT036555

Envirogel GPC Cleanup

Cyclohexane/Ethyl Acetate

19 x 150 mm

186001915

Envirogel GPC Cleanup

Methylene Chloride

19 x 300 mm

WAT036554

Envirogel GPC Cleanup

Cyclohexane/Ethyl Acetate

19 x 300 mm

186001916

Envirogel GPC Guard

Methylene Chloride

4.6 x 30 mm

186001913

Envirogel GPC Guard

Cyclohexane/Ethyl Acetate

4.6 x 30 mm

186001914

Envirogel High-Resolution GPC Cleanup Columns

The Envirogel™ High-Efficiency GPC Cleanup Columns are specifically 
designed to remove low volatility, high-molecular-weight interferences, 
such as lipids and natural resins, from environmental samples as 
specified in EPA Method 3640A. In the past, the cleanup procedure 
for environmental samples was performed on low-efficiency 
GPC columns based on packing particle diameters of 37–75 µm 
(200–400 mesh) Bio-Beads® S-X resins. T he high-efficiency 
Envirogel GPC Cleanup Columns increase the speed of this process 
while simultaneously reducing solvent consumption.

For optimum capacity and resolution, a 150 mm column is used 
in series with the 300 mm column. T he use of both the 150 mm 
column and the 300 mm column provides maximum loading 
capacity while the 300 mm column provides maximum throughput 
and reduction in solvent consumption when used alone.

Column Optimization

1

2 3

4

5

Collect

Column: 

Envirogel GPC,  

19 x 300 mm and  

19 x 150 mm

Part Numbers:   WAT036555, WAT036554
Sample: 

2000 µL

Solvent: 

Methylene chloride

Flow Rate: 

5 mL/min

Detection: 

UV @ 254 nm, 1.5 AUFS

Compounds:
1.  Corn oil, 62.5 mg/mL
2.  Bis(2-Ethylhexyl) Phthalate, 2.5 mg/mL
3.  Methoxychlor, 0.5 mg/mL
4.  Perylene, 0.05 mg/mL
5.  Sulfur, 0.2 mg/mL

14

[ WATER SOLUBLE POLYMERS AND SMALL MOLECULES ]

Reference Materials for Aqueous Samples
Reliable SEC results depend on the quality of the reference materials used for the molecular weight calibration. Waters SEC calibration 
standards are precisely formulated to provide you with accurate molecular weight reference materials that are conveniently packaged to 
minimize errors in SEC calibration methods. Our fully traceable aqueous-based polymer reference kits simplify routine calibration procedures 
that improve your workflow and increase your productivity.

This chart may be used to determine the appropriate component standard and corresponding molecular weight range. This information can be used in conjunction 
with the full range of aqueous SEC standards listed on page 15.

Aqueous SEC Standards Guide

Standards for the 
analysis of anionic, 
cationic, and 
neutral polymers

Effective Molecular Weight Range

101

102

103

104

105

106

107

108

Effective Molecular Weight Range

101

102

103

104

105

106

107

108

Polyacrylate 

Pullulan 

Dextran 

Polyethylene Oxide 

Polyethylene Glycol 

Standards for Aqueous Calibration

15

Full-Range Calibration Standards for SEC

T hese conveniently prepared and prepackaged standards provide you with an accurate calibration range for molecular weight determination 
of common water soluble polymers. T he kits contain a series of well-characterized standards of the specified polymer type and include 
certificates that list component ranges and concentrations. 

Description*

Part No.

Polyacrylic Acid Standards Kit

WAT035714

250 mg/vial polyacrylic acid at each molecular weight: 1000, 3000, 7000, 15,000, 30,000, 70,000, 100,000, 300,000, 700,000 and 1,000,000

Polyethylene Glycol Standards Kit

WAT035711

1.0 g/vial polyethylene glycol at each molecular weight: 100, 200, 400, 600, 1000, 1500, 4300, 7000, 13,000 and 22,000

 Polyethylene Oxide Kit

WAT011574

500 mg/vial polyethylene oxide at each molecular weight: 24,000, 40,000, 79,000, 160,000, 340,000, 570,000, and 850,000

Dextrans Standard

WAT054392

500 mg/vial dextrans at each molecular weight: 5000, 12,000, 24,000, 48,000, 148,000, 273,000, 410,000 and 750,000

 Pullulan Kit

WAT034207

200 mg/vial pullulan at each molecular weight: 5000, 10,000, 20,000, 50,000, 100,000, 200,000, 400,000, and 800,000

Individual Calibration Standards for SEC

In many cases a single calibration standard is used to verify a molecular weight component in a sample mixture. T hese individual component 
standards make molecular weight identification simple and straightforward. Package quantity: 0.5 g.

Description*

Part No.

Polyethylene Oxide Standard 24,000 

WAT011574

Polyethylene oxide, 24,000 MW

Polyethylene Oxide Standard 40,000 

WAT011576

Polyethylene oxide, 40,000 MW

Polyethylene Oxide Standard 79,000 

WAT011578

Polyethylene oxide, 79,000 MW

Polyethylene Oxide Standard 160,000 

WAT011580

Polyethylene oxide, 160,000 MW

Polyethylene Oxide Standard 340,000 

WAT011582

Polyethylene oxide, 340,000 MW

Polyethylene Oxide Standard 570,000 

WAT011584

Polyethylene oxide, 570,000 MW

Polyethylene Oxide Standard 850,000 

WAT011586

Polyethylene oxide, 850,000 MW

*Values listed are approximate molecular weights.

*Values listed are approximate molecular weights.

16

SEC Columns for Aqueous Samples

Size Exclusion Chromatography (SEC) and Gel Filtration Chromatography (GFC) are synonymous techniques that are used to separate 
macromolecules in aqueous environments based on their hydrodynamic volume. Waters SEC columns allow scientists to efficiently separate 
cationic, anionic and non-ionic macromolecules under a wide range of physical, chemical and biological environments. W hether you are 
choosing a column bank for maximum molecular weight resolution or selecting a column for quick screening, you can count on the stability, 
lifetime and performance of a Waters Ultrahydrogel™ SEC Column or HSPgel SEC Column.

This chart compares the molecular weight ranges for the specified columns. By connecting two or more columns in series, the effective molecular weight range 
can be extended to provide coverage for more complex sample analysis.

Columns for the 
analysis of anionic, 
cationic, and 
neutral polymers

Effective Molecular Weight Range

Ultrahydrogel DP

Ultrahydrogel 120

Ultrahydrogel 250

Ultrahydrogel 500

Ultrahydrogel 1000

Ultrahydrogel 2000

Ultrahydrogel Linear

101

102

103

104

105

106

107

108

Effective Molecular Weight Range

101

102

103

104

105

106

107

108

Columns for the 
analysis of anionic, 
cationic, and 
neutral polymers

HSPgel AQ MB-H  

HSPgel AQ 6.0  

HSPgel AQ 5.0  

HSPgel AQ 4.0  

HSPgel AQ 3.0 

HSPgel AQ 2.5  

Higher Speed

Maximum Resolution

Aqueous SEC Column Selection Guide

[ WATER SOLUBLE POLYMERS AND SMALL MOLECULES ]

17

Ultrahydrogel Columns

Packed with hydroxylated polymethacrylate-based gel, Waters 
Ultrahydrogel SEC Columns are ideal for the analysis of aqueous-
soluble samples, such as oligomers; oligosaccharides; polysaccharides; 
and cationic, anionic, and amphoteric polymers. Measuring 7.8 x 300 mm, 
these high-resolution columns offer many advantages over conventional 
aqueous SEC columns, such as:



  Wide-pH range (2-12)



  Compatibility with high concentrations of organic solvents  

(up to 20% organic, 50% organic if the mobile phase is  
introduced by gradient)



  Greater flexibility for the mobile phase



  Minimal non-size-exclusion effects

Ultrahydrogel Columns Calibration Curves

106

105

104

103

102

4

6

8

10

12

14 min

Mo

lecu

lar We

ig

ht

A B C

F

D

E

A Ultrahydrogel 120
B Ultrahydrogel 250
C Ultrahydrogel 500
D Ultrahydrogel 1000

E Ultrahydrogel 2000
F Ultrahydrogel Linear

Column Dimensions:   

7.8 x 300 mm

Part Numbers:  

WAT011525, WAT011530, WAT011535

Sample: 

PEG and PEO standards

Mobile Phase: 

Distilled water

Flow Rate: 

1 mL/min

Gelatin Sample

30 min

25

20

15

mV

MW = 7500K

MW = 90K

MW = 250K

MW = 480K

Column: 

Ultrahydrogel 250, 500, and 1000

Part Numbers:  

WAT011525, WAT011530, WAT011535

Temperature: 

80 °C

Eluent: 

Water, pH 7, phosphate buffer

Flow Rate: 

1 mL/min

Detection: 

Waters 410 Differential Refractometer

Ultrahydrogel Columns (7.8 x 300 mm)*

Column

Pore Size

Exclusion Limit

Part No.

Ultrahydrogel 120

120 Å

5000

WAT011520

Ultrahydrogel 250

250 Å

80,000

WAT011525

Ultrahydrogel 500

500 Å

400,000

WAT011530

Ultrahydrogel 1000

1000 Å

1,000,000

WAT011535

Ultrahydrogel 2000

2000 Å

7,000,000

WAT011540

Ultrahydrogel Linear

Blend

7,000,000

WAT011545

Ultrahydrogel DP*

 120 Å

5000

WAT011550

Ultrahydrogel Guard Column

N/A

N/A 

WAT011565

Ultrahydrogel Guard Column DP*

N/A

N/A

WAT011570

*DP = Degree of Polymerization, choice of column when working with glucose oligomers.

HSPGel Columns

Waters HSPgel SEC Columns are optimized for high-speed polymer analysis in aqueous solution. HSPgel Columns will reduce solvent 
consumption, increase throughput and provide accurate molecular weight data for any room-temperature analysis. T he column dimensions 
are 6.0 x 150 mm.

Aqueous GPC**

Solvent

Particle Size

MW Range

Part No.

HSPgel AQ 2.5

Water

4 µm

500–2,000 

186001785

HSPgel AQ 3.0

Water

4 µm

1,000–60,000 

186001786

HSPgel AQ 4.0

Water

6 µm

10,000–400,000 

186001787

HSPgel AQ 5.0

Water

7 µm

50,000–4,000,000 

186001788

HSPgel AQ 6.0

Water

9 µm

100,000–10,000,000

186001789

HSPgel AQ MB-H

Water

9 µm

500–10,000,000 

186001790

**Exclusion limits for AQ series extrapolated from highest MW PEO standard (~900,000).

HSPgel Columns for High-Speed GPC Analysis

18

SEC Columns for Protein Analysis and Characterization

BioSuite Size-Exclusion HPLC Columns

BioSuite™ Ultra-High Resolution (UHR), High Resolution (HR), and Standard Size-Exclusion Columns contain silica-based sorbents that 
are stable from pH 2.5–7.5. As indicated in the calibration curve tables, the exclusion limit of the BioSuite SEC packings is determined 
by the pore size of the silica-base material. T he particle size of the SEC packing media, as well as column length, is an important 
parameter that determines the separation efficiency. T he BioSuite UHR Columns (4-µm particle size) provide maximum separation effi-
ciency, followed by BioSuite HR Columns (5- and 8-µm particle sizes) and BioSuite Standard SEC Columns (10-, 12- and 17-µm particle 
sizes). To maximize column life of analytical (4.6- or 7.8-mm ID) or preparative (21.5-mm ID) SEC columns, use of BioSuite Guard Columns 

[ WATER SOLUBLE POLYMERS AND SMALL MOLECULES ]

Protein Calibration Curves for BioSuite 
Ultra-High Resolution (UHR) SEC Columns

Sample: 

Thyroglobulin (MW 670,000 Da), Gamma globulin (MW 155,000 Da), Boviine  

serum albumin (66,330 Da), Beta lactoglobulin (MW18,400 Da), Lysozyme  

(14,300 Da), Cytochrome C (12,400 Da), Triglycine (189 Da)

Columns:  

BioSuite 250, UHR SEC, 4.6 x 300 mm, 4 μm

BioSuite 125, UHR SEC, 4.6 x 300 mm, 4 μm 

Part Numbers:  

186002162 (BioSuite 250) & 186002161 (BioSuite 125)

Eluent: 

0.15 M sodium phosphate, pH 6.8

Flow Rate:  

0.35 mL/min

Column Temp.: 

25 °C

Detection:  

UV @ 220 nm

Column

Globular Protein  

MW Range

Branched  

Dextrans

Linear  

PEG/PEO

BioSuite 125

5,000–150,000

1,000–30,000

500–15,000

BioSuite 

250 

10,000–500,000

2,000–70,000

1,000–35,000

Protein Calibration Curves for BioSuite 
High Resolution (HR) SEC Columns

Elution Volume  (mL)

10

6

8

12

102

103

104

105

106

Molecular Weight (Da)

BioSuite 125, 5 m HR SEC

BioSuite 250, 5 m HR SEC

BioSuite 450, 8 m HR SEC

Column

Globular Protein 

MW Range

Branched  

Dextrans

Linear  

PEG/PEO

BioSuite 125

5,000–150,000 

1,000–30,000

500–15,000

BioSuite 250 

10,000–500,000 

2,000–70,000

1,000–35,000

Sample:  

Thyroglobulin (MW 670,000 Da), IgG (MW 156,000 Da), BSA (66,330 Da), Ovalbumin  

(MW 43,000 Da), Peroxidase (40,200 Da), Beta lactoglobulin (MW 18,400 Da),  

Myoglobin (MW 16,900 Da), Ribonuclease A (MW 13,700 Da), Cytochrome C (12,400 Da),  

Glycine tetramer (246 Da)

Columns:  

BioSuite 450, HR SEC, 7.8 x 300 mm, 8 μm 

BioSuite 250, HR SEC, 7.8 x 300 mm, 5 μm 

BioSuite 125, HR SEC, 7.8 x 300 mm, 5 μm 

Part Numbers:   186002166 (BioSuite 450), 186002165 (Biosuite 250), & 186002164 (BioSuite 125)
Eluent: 

0.1 M sodium phosphate, pH 7.0 containing 0.3 M sodium chloride

Flow Rate:  

1.0 mL/min

Column Temp.:  25 °C
Detection:  

UV @ 220 nm

Protein Calibration Curves for BioSuite Standard SEC Columns

Molecular Weight (Da)

Elution Volume (mL)

106

105

103

104

20

25

10

15

BioSuite 125, 10 m SEC
BioSuite 250, 10 m SEC
BioSuite 450, 13 m SEC

Sample:  

Thyroglobulin (MW 670,000 Da), IgG (MW 156,000 Da), BSA (66,330 Da), Ovalbumin (MW 43,000 Da), Peroxidase (40,200 Da),  

Beta lactoglobulin (MW18,400 Da), Myoglobin (MW 16,900 Da), Ribonuclease A (MW 13,700 Da), Cytochrome C (12,400 Da),  

Glycine tetramer (246 Da)

Columns:  

BioSuite 450, SEC, 7.5 x 300 mm, 13 μm; BioSuite 250, SEC, 7.5 x 300 mm, 13 μm; BioSuite 125, SEC, 7.5 x 300 mm, 10 μm 

Part Numbers:   186002172 (BioSuite 450), 186002170 (BioSuite 250) & 186002168 (BioSuite 125)
Eluent: 

0.1 M sodium phosphate, pH 7.0 containing 0.3 M sodium chloride

Flow Rate:  

1.0 mL/min

Column Temp.:  25 °C
Detection:  

UV @ 220 nm

Column

Globular Protein  

MW Range

Branched 

Dextrans

Linear  

PEG/PEO

BioSuite 125

5,000–150,000 

1,000–30,000

500–15,000

BioSuite 250 

10,000–500,000 

2,000–70,000

1,000–35,000

BioSuite 450

20,000–1,000,000 

4,000–500,000

2,000–250,000

6

100

1,000

10,000

100,000

1,000,000

8

10

12 min

MW

BioSuite 125, 4 µm UHR SEC

BioSuite 250, 4 µm UHR SEC

19

Description

Matrix

Diameter 

Width

Diameter 

Length

Column 
Volume

Suggested Volume Load for Maximum  

Multicomponent Resolution*

Multicomponent 

Resolution*

Part No.

BioSuite 125, 4 µm UHR SEC

Silica

4.6 mm

300 mm

4.98 mL

Less than 8 mg/mL

Less than 40 µL

186002161

BioSuite 250, 4 µm UHR SEC

Silica

4.6 mm

300 mm

4.98 mL

Less than 8 mg/mL

Less than 80 µL

186002162

BioSuite UHR Guard SEC

Silica

4.6 mm

35 mm

—

 —

 —

186002163

BioSuite 125, 5 µm HR SEC 

Silica

7.8 mm

300 mm

14.33 mL

Less than 8 mg/mL

Less than 200 µL

186002164

BioSuite 250, 5 µm HR SEC 

Silica

7.8 mm

300 mm

14.33 mL

Less than 8 mg/mL

Less than 200 µL

186002165

BioSuite 450, 8 µm HR SEC 

Silica

7.8 mm

300 mm

14.33 mL

Less than 8 mg/mL

 Less than 200 µL

186002166

BioSuite HR Guard SEC 

Silica

6 mm

40 mm

—

 —

—

186002167

BioSuite 125, 10 µm SEC 

Silica

7.5 mm

300 mm

13.25 mL

Less than 8 mg/mL

 Less than 200 µL

186002168

BioSuite 125, 13 µm SEC 

Silica

21.5 mm

300 mm

108.9 mL

Less than 8 mg/mL

 Less than 1.6 mL

186002169

BioSuite 250, 10 µm SEC 

Silica

7.5 mm

300 mm

13.25 mL

Less than 8 mg/mL

 Less than 200 µL

186002170

BioSuite 250, 13 µm SEC 

Silica

21.5 mm

300 mm

108.9 mL

Less than 8 mg/mL

 Less than 1.6 mL

186002171

BioSuite 450, 13 µm SEC 

Silica

7.5 mm

300 mm

13.25 mL

Less than 8 mg/mL

 Less than 200 µL

186002172

BioSuite 450, 17 µm SEC 

Silica

21.5 mm

300 mm

108.9 mL

Less than 8 mg/mL

 Less than 1.6 mL

186002173

BioSuite Guard SEC 

Silica

7.5 mm

75 mm

— 

—

—

186002174 

BioSuite Guard SEC 

Silica

21.5 mm

75 mm

— 

—

—

186002175 

* Using a BSA protein standard in a 50 mM phosphate buffer containing salt (either 0.1 M NaCl or 0.1 M Na

2SO4) eluent. Useful protein mass loads will vary depending upon separation eluent, complexity of sample, and on the  

type of proteins contained in mixture. In general, maximum component resolution is obtained by injecting the smallest possible volume of a dilute protein solution. 

* Note: Operating flow rates for BioSuite Ultra-High Resolution (UHR) SEC Columns (4.6-mm ID) are from 0.1–0.4 mL/min. Use of an HPLC system (e.g. Waters Alliance HPLC System) capable of operating at these flows is essential 

for optimal UHR SEC Column performance.

Protein-Pak and Shodex Size-Exclusion  
HPLC Columns
Waters offers two families of packings for size-exclusion chro-
matography. Protein-Pak™ packings are based on a 10 µm 
diol-bonded silica and are available in a selection of pore sizes 
and column configurations. In addition, Waters offers a series of 
Shodex 7 µm high-resolution, gel-filtration packings.

T he Protein-Pak size-exclusion columns can be expected to 
resolve proteins that differ in molecular weight by a factor of 
two and to distinguish proteins differing by as little as 15% in 
molecular weight. T he degree of resolution is more dependent 
on the sample mass and volume than the interaction between 
the sample and the stationary phase. Ideally, there should be 
no interaction between the stationary phase and the sample 
molecules. Secondary interactions are most often ionic and can, 
therefore, be reduced by increasing the ionic strength of the mo-
bile phase. Typical, salt concentrations range to 0.2–0.5 M NaCl. 
It may also be useful in some cases to consider adding 10–20% 
methanol to eliminate hydrophobic and other hydrogen-bonding 

Standard Protein Mix on KW-803 Column

25 min

1

2

3

4 5

6

7

8

This gel-filtration separation of protein standards demonstrates the ability  
to separate proteins in a wide range of molecular weights in minutes for high 
sensitivity analysis or protein isolation up to the milligram scale.

Column: 

Protein KW-803

Part Number:    WAT035946 
Eluent: 

25 mM sodium phosphate pH 6.8

Flow Rate: 

0.72 mL/min

Detection: 

UV @ 280 nm

Compounds:
1.  Blue dextran
2.  Ferritin
3.  Aldolase
4.  Bovine serum albumin
5.  Ovalbumin
6.  Chymotrypsinogen
7.  Cytochrome c
8.  Cytidine

Protein-Pak Columns and Packings

Steel Column

Dimension

MW Range

Part No.

Protein-Pak 60

7.8 x 300 mm

1,000–20,000

WAT085250

Protein-Pak 125

7.8 x 300 mm

2,000–80,000

WAT084601

Protein-Pak 300SW

7.5 x 300 mm

10,000–300,000

WAT080013

Protein-Pak 125 Sentry Guard Column 3.9 x 20 mm, 2/pkg (requires holder)

186000926

Sentry Universal Guard Column Holder

WAT046910

Protein-Pak 200SW

8 x 300 mm

500–60,000

WAT011786

Protein-Pak 300SW

8 x 300 mm

10,000–300,000

WAT011787

Inquire for additional offerings, including prep.

Shodex Size-Exclusion Columns

Column

Particle 

Size

Dimension

MW Range

Part No.

Protein KW-802.5

7 µm

8 x 300 mm

100–50,000

WAT035943

Protein KW-803

7 µm

8 x 300 mm 

100–150,000

WAT035946

Protein KW-804

7 µm

8 x 300 mm 

500–600,000

WAT036613

Protein-Pak 125 Sentry Guard Column 3.9 x 20 mm, 2/pkg (requires holder)

186000926

Sentry Universal Guard Column Holder

WAT046910

BioSuite Columns

20

Waters is a leading manufacturer of analytical instrumentation and consumable products. We understand the importance of autosampler 
vials for the performance of analytical instrumentation. T here are many factors to consider in selecting the proper vial:



  Needle design



  Autosampler tray design



  Chemical compatibility



 Cleanliness



  Optic and robotic specifications



 Volatility



  Sample volume

At Waters, we take all of these factors into consideration in the design, manufacture, and delivery of our vials and accessories. Unlike 
our competition, who offer Type I, 33-expansion glass in North America and Type I, 51-expansion glass in Europe or Japan, Waters 
single source manufacturing produces Type I, 33-expansion glass, the lowest free ion glass available, for worldwide distribution.

LC/GC Certified Vials

LC/GC Certified Vials are tested by HPLC using UV detection. T he HPLC test was developed to look for trace levels of chemicals used in the 
manufacturing and packaging process. T hese chemicals include lubricants, surfactants, antistatic, and antioxidants from packaging. 
T he tests are run on each batch of vials, after they have been packaged for several days, to ensure cleanliness. An additional head-
space GC test is done to look for proper curing of the silicone septa.

TruView LCMS Certified Vials

TruView™ LCMS Certified Vials include the stringent dimensional tolerances and UV and MS cleanliness tests required of the LC/
GC and LCMS Certified Vials lines. T he additional product attribute of TruView vials is the glass surface exhibits low polar analyte 
adsorption. T he vials are manufactured under tightly controlled process conditions (patent pending) that limit the concentration of free 
ions on glass surface. Low levels of free ions on the surface of glass can cause analyte adsorption. Waters TruView LCMS Certified Vials 
are tested for high recovery of analyte at 1 ng/mL concentration using UPLC/MS/MS (MRM) and yield little adsorption. T hese vials 
exhibit the lowest adsorption of autosampler vials in the market.

[ AUTOSAMPLER VIALS ]

21

Choosing the Right Vial and Septum for Your Application

There are three decisions you need to make when choosing the correct vial for your application: the septum, the closure, and the vial itself. 
Read through the selection options below to determine the proper combination for your application. For your convenience, Waters  
offers many of these choices as combination packs. T he vial, cap, and septum come pre-packaged as packs of 100 for ease and  
convenience in ordering. 

PTFE 



  Recommended for single injection applications  



  Ideal for use in MS applications  



  Excellent solvent resistance and chemical compatibility  



  Does not reseal upon puncturing  



  Not recommended for long-term sample storage

PTFE/Silicone 



  Demonstrates excellent resealing characteristics  



  PTFE chemical resistance until punctured, then the septum will have the chemical compatibility of silicone  



  Working temperature range from -40 ˚C to 200 ˚C

Vial Closures Guide

Vials are available in three closure types: crimp, snap, and screw cap. Each closure has its advantages and disadvantages.

Crimp caps squeeze the septum between the rim of the glass vial and the crimped aluminum cap. T his forms an excellent seal prevent-
ing  
evaporation. T he septum stays seated during piercing by the autosampler needle. T he crimp cap vial requires crimping tools to carry out 
the 
sealing process. For few samples, manual crimper tools are the best choice. For large numbers of samples, automated crimpers are avail-
able.

Snap caps are an extension of the crimp cap system of sealing. A plastic cap is stretched over the rim of the vial to form a seal by squeez-
ing the septum between the glass and the stretched plastic cap. T he plastic cap creates tension when trying to return to its original 
size. T his tension forms the seal between glass, cap and septum. Plastic snap caps do not require any tools to assemble.

Snap caps are not as effective a seal as other closures. 



  If the fit of the cap is very tight, the cap is hard to apply and may be subject to crack. 



  If the fit is too loose, the seal is poor and the septum may dislodge.

LectraBond™ Screw Caps are available through Waters. T his screw cap has a PTFE/silicone septum bonded to the polyethylene cap, using 
a non-solvent bonding process. T his bonding technology is designed to keep the septum/cap together during shipment and assembly 
onto vials. T he bond will aid in preventing dislodging of the septum during use, but the primary sealing mechanism is the mechanical 
force applied by tightening the cap to the vial.

Cap tightening is the mechanism that forms the seal and holds the septum in place during needle insertion. T here is no need to over-tight-
en the cap, as it can compromise the seal and lead to dislodging. T he septum starts to cup or indent when you begin to over-tighten.

Cap Design

Strength Design 

Comment

Crimp 

Excellent seal 

Requires tools

Snap

Moderate seal

Fast, no tools, some cap cracking

Screw 

Excellent seal 

Universal

22

Clear

Amber

Max Recovery

Amber Max

Total Recovery

TruView LCMS Certified Combination Packs

Part No.

Part No.

Part No.

Part No.

Part No.

Vial, Cap, and Silicone/PTFE Septum

186005660CV

186005667CV

186005668CV

186005664CV

186005669CV

LC/GC Certified Combination Packs

Bonded Silicone/PTFE Septum

186000272C

186000846C

186000326C

186003885C

186000384C

Bonded Pre-Slit Silicone/PTFE Septum

186000307C

186000847C

186000327C

186003886C

186000385C

Injectable Volumes Alliance 2690/2695

Max

1100 µL

1100 µL

—

—

950 µL

Residual

750 µL

750 µL

—

—

9 µL

Injectable Volumes Alliance 2790/2795/2707

Max

1700 µL

1700 µL

1500 µL

1500 µL

—

Residual

170 µL 

170 µL

22 µL

22 µL

—

Inserts

150 µL with Poly Spring

WAT094171(DV)1

WAT094171(DV)1

—

—

—

Max Volume Injection/Max Residual Volume

144 µL/6 µL

144 µL/6 µL

—

—

—

Compatible Systems

Alliance 2690/2695







Alliance 2790/2795/2707









All items come in quantities of 100, unless otherwise noted. 

Screw Cap 12 x 32 mm Vials for Alliance Systems

This table highlights the most commonly used vials for GPC analysis. For a complete listing of Waters vial products refer to the Waters Quality Parts, Chromatography 
Columns and Supplies Catalog, www.waters.com/catalog.

[ AUTOSAMPLER VIALS ]

23

4 mL Screw Cap

10 mL Screw Neck

Components

Part No.

Part No.

Vial

186000840

186001420

Black Screw Cap

600000162

186001421

PTFE Septum

 WAT0727141

186001422

1

 Item contains 144 vials.

GPC 2000 Vials

4 mL Screw Neck

Amber Screw Neck

Total Recovery

15 x 45 mm Vials

Combination Packs

Part No.

Part No.

Part No.

Vial, Cap, and LectraBond PTFE/Silicone Septum

186000838C

186001133C

186002629C

Vial, Cap, and LectraBond Pre-Slit PTFE/Silicone Septum

186000839C

186001134C

186002630C

Max Volume Injection/Max Residual Volume

2400 µL/1600 µL

2400 µL/1600 µL

3000 µL/40 µL

Insert

Part No.

Part No.

Part No.

250 µL Glass Insert2

WAT072704(DV)1

WAT072704(DV)1

—

Max Volume Injection/Max Residual Volume

244 µL/6 µL

244 µL/6 µL

—

Springs for LVI, 100/pk

WAT072708

WAT072708

—

2

 Inserts require springs (Part No. WAT072708).

Vials for Waters Breeze with 717 Autosampler

These tables highlight the most commonly used vials for GPC analysis. For a complete listing of Waters vial products refer to the Waters Quality Parts, Chromatography 
Columns and Supplies Catalog, www.waters.com/catalog.

24

Solvent Considerations

One of the most important decisions for an analyst is finding a suitable solvent to dissolve the polymer for analysis. T his may sound trivial, 
but remember that GPC is a separation technique based on the size of the polymer in solution. Polymer chains will open up to a certain relaxed 
conformation in solution, and the solvent chosen will determine what this size will be. Many polymers are soluble at room temperature in various 
solvents, but in some cases (especially for highly crystalline polymers) high temperature is required for dissolution. T he following is a guide for 
both aqueous and non-aqueous soluble polymers.

Aqueous SEC Solvent Selection Guide

Polymer

Class

Eluent

Polyethylene oxide
Polyethylene glycol
Polysaccharides, Pullulans 
Dextrans 
Celluloses (water soluble)
Polyvinyl alcohol 
Polyacrylamide

0.10 M Sodium nitrate

Neutral

Polyacrylic acid 
Polyalginic acid/alginates 
Hyaluronic acid 
Carrageenan

0.10 M Sodium nitrate

Anionic

DEAE dextran 
Polyvinylamine 

Polyepiamine 

n-Acetylglucosamine 

0.80 M Sodium nitrate

0.10% TEA

0.10 M TEA/1% Acetic acid

Cationic

Cationic

Cationic

Polyethyleneimine 
Poly(n-methyl-2-vinyl 
pyridinium) I salt 

Lysozyme 
Chitosan 

Polylysine 

Peptides

0.50 M Sodium acetate/0.50 M Acetic acid

0.50 M Acetic acid/0.30 M Sodium sulfate

5% Ammonium biphosphate/3% Acetonitrile (pH = 4.0)

0.10% TFA/40% Acetonitrile 

Cationic, hydrophobic

Cationic, hydrophobic

Cationic, hydrophobic

Cationic, hydrophobic

Polystyrene sulfonate 
Lignin sulfonate 

Anionic, hydrophobic

Collagen/Gelatin 

Amphoteric

Polyvinyl pyrrolidone 

80:20 0.10 M Sodium nitrate/Acetonitrile

Neutral, hydrophobic

[ TIPS AND FREQUENTLY ASKED QUESTIONS ]

25

Polyisobutylene

Polybutylene

Chlorinated rubber

Polybutadiene

Polyisoprene

Polydimethylsiloxane

Chlorinated polyethylene

Polyethylene–Ethylacrylate

Polyethylene–Vinylacetone

Polyethylene–Methacrylic acid

Polyphenyleneoxide

Poly-4-methylpentene(1)

Polyethylene

Ultra-high Molecular Weight 

      Polyethylene

Polypropylene

Polyetheretherketone

Polyetherketone

Polycarbonate

Polyglycolic acid

Acrylonitrile–Methylmethacrylate

Cellulose acetate

Cellulose acetate–Butyrate

Cellulose acetate–Proprionate

Cellulose nitrate 

Cellulose proprionate

Cellulose triacetate

Diallyl phthalate

Ethyl cellulose

Epoxy

Polyester alkyd

Polybutene(1)

Polybutadiene–Styrene

Phenol–Formaldehyde

Phenol–Furfural

Polymethylmethacrylate

Polypropyleneglycol

Polystyrene

Polysulfone

Polyvinylacetate

Polyvinylbutyral

Polyvinylchloride

Polyvinylchloride–Acetate

Polyvinyldienechloride

Polyvinylformal

Polystyrene acrylonitrile

Polystyrene–Alphamethylstyrene

Polyester thermoset

Phenolics

Rosin acids

Polyglycolic acid

Melamine–Formaldehyde

Nylon (All types)

Polybutylene–Terphthalate

Polyethylene–Terphthalate

Poly acrylonitrile

ABS (Acrylonitrile–Butadiene–Styrene)

ASA (Acrylic–Styrene–Acrylonitrile

ABA (Acrylonitrile–Butdiene–Acrylate)

Carboxymethyl cellulose

ABS/Polycarbonate

Polybutadiene–Acrylonitrile

Polyurethane

Polyacetal

Polyoxymethylene

Polyimide

Polyamide–imide

Polyetherimide

Polyethersulfone

Polyvinyldienefluoride

Polyfuran–Formaldehyde

Polymer

Toluene

Toluene/75 °C

TCB/135–160 °C

TCB/135–160 °C

Phenol/TCB 1:1/145 °C

Methyl chloride

gamma-Butyl lactone

THF/40 °C

Hexafluoroisopropanol + 0.075 M

Sodium trifluoroacetate/55 °C

or

m-Cresol + 0.05 m LiBr/100 °C

DMF + 0.05 m LiBr/85 °C

DMF + 0.05 m LiBr/145 °C

N-Methyl pyrrolidone

+ 0.05 m LiBr/100 °C

Dimethylacetamide/60 °C

GPC Solvent

Non-Aqueous GPC Solvent Selection Guide

26

Frequently Asked Questions

What solvent should I buy my columns packed in, and why? 
Non-Aqueous GPC columns are packed in either:  
 

THF

 

Toluene

 

DMF 

Specialty columns packed in methanol specifically for analysis at room temperature with HFIP (hexafluoroisopropanol) are available. If you are  
using a solvent other than these four for your application, there are a couple of rules-of-thumb to think about. If you are doing a “room temperature” 
application in a solvent such as chloroform or methylene chloride, convert over from THF. If you plan on doing high-temperature work in 
TCB, ODCB, for example, convert over from toluene at ~85 - 90 °C. If you are going to use a solvent that is very polar, such as DMAC (Dimethyl-
acetamide) or NMP (n-methylpyrollidone), convert over from DMF. 

I currently have columns in solvent “A”, can I switch to solvent “B”? 

Generally, one can switch directly from one solvent to another at 0.1 - 0.2 ml/min if the two solvents are miscible (refer to the column’s care 
and use manual). If the solvents are not miscible, an intermediate solvent (which both solvents are miscible in) will have to be used. 

What additives are important and when should I use them?
In certain cases, some mobile-phase additive is required. For example, 0.05 M lithium bromide is added to polar solvents such as DMF, DMAC 
and NMP. These polar solvents are used to analyze polar polymers such as polyurethanes or polyimides, and there is a dipole interaction that 
occurs, causing artificial shoulders to appear on the high molecular weight end of the distribution. This interaction is eliminated with the addition of the 
salt. 

Salts are also used in aqueous GPC as the methacrylate gels used in the columns have an overall anionic charge. Due to this charge, ion exclu-
sion can occur with anionic samples and ion absorption can occur with cationic samples. The use of sodium nitrate salt can minimize these 
effects as well as pH adjustment of the eluent if any ion-exchange interaction is occurring with cationic samples.

In which order should I place the columns, and why?

Generally, it does not matter what order the columns are placed in. The order will not affect the molecular weight distribution calculations of 
the eluting polymer. It is a good idea, however, to always place the 50 Å or 100 Å columns at the end of the set, as the styrene/divinylbenzene 
gel in these columns tend to be softer and less durable. 

What flow rate should I use in my GPC column? 

It is recommended not to exceed 1.0 mL/min for the 7.8 mm ID analytical columns. The “optimum” resolution for these columns is approxi-
mately 0.70 to 0.80 mL/min. The optimum flow rate for the 4.6 mm ID narrow-bore columns is 0.3 to 0.35 mL/min. Refer to the column’s care 
and use manual for more details. 

When starting up columns, should I gradually increase flow and temperature? 

[ TIPS AND FREQUENTLY ASKED QUESTIONS ]

27

It is mandatory to slowly ramp up the flow rate for analytical GPC columns, particularly the Stryagel HR series. Sudden increase in flow 
(and subsequently pressure) will certainly damage the columns. Temperature ramping is not as critical. Generally, we ramp the flow rate from 
0.0 to  
1.0 mL/min over a 60-second interval, and the temperature from ambient to 150 °C (as an example) over several hours.
How do I choose the column’s pore size range?

The range of pore sizes is chosen by determining the approximate molecular weight range of the sample of interest. Choosing columns that target 
the molecular weight range of the polymer will provide the highest resolution.  For example, if the polymer molecular weight range is low then 
a column set of 50, 500 and 1000 Å would be used; a medium molecular weight range requires a larger pore distribution so a 1000, 
10,000, and 100,000 Å column set would be appropriate. For an unknown molecular weight range it is a good idea to use mixed bed (i.e. 
“linear”, or “extended range”) columns that provide a mixture of pore sizes.. 

The following table lists the molecular weight range of separation for individual pore size columns of styrene/divinylbenzene packings, based 
on polystyrene chain length exclusion limits.

 MW Range

Pore Size 

 MW Range

Pore Size 

 100 - 1000

50 Å

 50,000 - 1,000,000 

100,000 Å

 250 - 2500

100 Å

 200,000 - > 5,000,000

1,000,000 Å

 1,000 - 18,000

500 Å

 500,000 - ~20,000,000

10,000,000 Å

 5,000 - 40,000

1000 Å

 ~1,000 - 10,000,000

Mixed Bed - High

 10,000 - 200,000

10,000 Å

 ~100 - 100,000

Mixed Bed - Low

What is resolution? How much do I need?

In GPC analysis, resolution means range of molecular weight separated in an incremental volume of elution. We would like to maximize 
this whenever possible. The easiest way to maximize this is to add more columns (and therefore analysis time, unfortunately). Another way is 
to use a smaller particle size (~ 5 µm), which will increase efficiency. The trade-off here is column durability and lifetime. In separations where 
oligomers, additives, and multi-modal distributions are present, resolution may be important. If the sample is a high density polyethylene 
with a broad distribution, resolution may not be as important. 

Waters manufactures columns in the high resolution range (HR series) which contain 5-µm particles, the HT series which have ~ 10-µm par-
ticles (good for high temperature work and multiple solvent changeovers), and the HMW series that have 20-µm particles. These are good for 
very high molecular weight samples where shearing is a problem and resolution is not as critical.

Tip: The GPC solvent guide provides typical operating temperature ranges. For GPC analysis, columns are heated (even for room temperature 
applications) to increase resolution by improving analyte permeation. 

What is a “narrow” standard? What is a “broad” standard?

 

Narrow standards are those where the polydispersity is less than ~1.10. The polydispersity is defined as the ratio of the weight average 

molecular weight (Mw) to the number average molecular weight (Mn).

 

Broad standards have polydispersities greater than 1.10 and are usually the same polymer as the sample to be analyzed.

If I use narrow standards, can I inject more than one standard at a time? 

In conventional GPC with RI detection, it is certainly acceptable to inject a mixture of standards, as long as there is sufficient resolution among 
the eluted standards. We would suggest a maximum of three. With advanced detection, such as viscometry, where the area under the curve for 
the standard needs to be known accurately, one standard at a time should be injected.

What standard(s) should I use for my polymer?

For most people, a narrow standard relative calibration is fine. In this case, polystyrene standards are the usual choice for organic 
GPC, but PMMA’s, polyisoprenes, polybutadienes and polyTHF narrow standards may be used. For aqueous GPC, narrow polyethyl-
ene oxides, polyethylene glycols and pullulans (polysaccharides) are available. If the user needs the true molecular weight (relative to the 
calibrant not being good enough), the broad standard (or reference) with the same chemical nature as the samples may be used. 

28

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August 2014  720004331EN  KP-FP