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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Corporation. Ultrahydrogel, HSPgel, Envirogel, Ultrastyragel, TruView, LectraBond, BioSuite, Protein-
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©2014 Waters Corporation. Printed in the U.S.A.
August 2014 720004331EN KP-FP
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