Gravity Chromatography

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Overview

Gravity chromatography is appropriate for laboratory-scale separations and involves minimal instrumentation. Gravity chromatography relies primarily on hand-packed columns and is particularly well suited for single-step purifications like desalting and affinity applications. This section provides an overview of gravity chromatography and selection guides to help you build your own gravity chromatography setup.

Related Topics: Low Pressure Chromatography, Medium Pressure Chromatography

Gravity Chromatography Technique


Typical gravity column.

As pictured to the right, a typical gravity column involves a buffer reservoir at the top of the column with flow controlled using a stopcock or a tubing pincher at the column outlet. There are a number of refinements available to assist with the general design:

  • Graduated reservoirs for column packing and buffer delivery
  • Flow adaptors to minimize head space above the gel bed and minimize disruption of the gel bed during sample loading
  • Column bed supports/frits to retain media
  • Integral water jacket for temperature control
  • Stopcocks to control flow rate
  • Luer fittings for tubing and serial column connections
  • Fraction collectors

 

Gravity Chromatography Considerations

  • Simplicity — a gravity column is easy to design, set up, and operate
  • Low cost — costs are limited to the researchers' needs. No injectors, pumps, detectors, or fraction collectors are required
  • Convenience — many disposable prepacked and empty polypropylene columns are available
  • Gravity chromatography is ideal for single-step purifications like desalting and affinity

Advantages of Gravity Chromatography

Gravity chromatography is:

  • A manual process requiring constant monitoring
  • Not a high-resolution method
  • Time consuming. No automated injectors, pumps, or detectors are used to control sample delivery, flow rate, or fraction analysis

Reproducibility is an issue if care is not taken to normalize methods. Manual sample loading can be a challenge due to the disruption of the column bed both before and after sample loading. Use a flow adaptor to minimize these effects.

Adjusting flow rate

  • With a gravity flow system, the buffer reservoir must be placed above the column to ensure flow
  • The flow rate can be controlled manually using a stopcock or a simple tubing pincher at the column outlet
  • It is highly recommended to measure the volume and flow rate by counting drops/unit time:
    1 drop = 50 µl. Normalized methods will lead to more reproducible separation methods
  • Fraction collectors are available that can count drops and dispense fractions defined by drop number or volume

Sample loading

  • Manual sample loading is not recommended in size exclusion/gel filtration chromatography. It is critical that the column bed remains undisturbed or the resolution will be compromised
  • Bind and elute techniques work well, however, additional column washes become necessary due to the inherent sample dilution impact with this technique

Elution and fraction collection

  • It is highly recommended that the user employ a timer during elution and fraction collection to avoid the possibility of the column running dry

Gravity Chromatography Column Selection

Bio-Rad offers a complete line of prepacked and empty columns for gravity chromatography. Use the selection tables below to help find the best gravity chromatography setup for your application.

Empty Column Selection Guide

Column Application Requirement Pressure Range Volume Range Best Used With
Bio-ScaleMT High-resolution column 0–1,000 psi 1–22 ml High-performance system
Econo-Column® General purpose glass column 0–15 psi 1–1,374 ml Low pressure or gravity
Econo-Pac® General purpose plastic column 0–15 psi 1–20 ml Low pressure or gravity
Poly-Prep® Disposable plastic column 0–15 psi 2 ml Gravity
Bio-Spin® Disposable plastic column 1.2 ml Swinging bucket centrifuge
Micro Bio-Spin Disposable plastic column 0.8 ml Standard micro-centrifuge
EasyPack Process scale 3 bar (glass)
8.7 bar (stainless steel and acrylic)
0.320–3,800 L Process chromatography system

Prepacked and empty columns.

Prepacked and empty columns.

Glass Econo-Column Columns Selection Guide
Choose the correct Econo-Column chromatography column by length and inner diameter The corresponding flow adapter is shown with the columns. A flow adapter is not needed for ID's of 0.5 or 0.7 cm. A dash ( – ) indicates that the column is not available in that configuration (L, ID, or package type).

  Inner Diameter (ID)
    0.5 cm 0.7 cm 1.0 cm 1.5 cm 2.5 cm 5.0 cm
Length Type            
5 cm Pkg of 4 737-4506 737-4706 737-4006 737-4150 737-4250
Pkg of 2 737-0507 737-0707 737-1007 737-1507 737-2507
  Flow adapter 738-0014 738-0016 738-0017
10 cm Pkg of 4 737-4511 737-4711 737-4011 737-4151 737-4251
Pkg of 2 737-0512 737-0712 737-1012 737-1512 737-2512
Pkg of 1 737-5011
Flow adapter 738-0014 738-0016 738-0017 738-0018
15 cm Pkg of 4 737-4516 737-4716 737-4156
Pkg of 2 737-0517 737-0717 737-1517
Flow adapter 738-0016
20 cm Pkg of 4 737-4521 737-4721 737-4021 737-4152 737-4252
Pkg of 2 737-0522 737-0722 737-1022 737-1522 737-2522
Pkg of 1 737-5021
Flow adapter 738-0015 738-0016 738-0017 738-0018
30 cm Pkg of 4 737-4731 737-4031 737-4153 737-4253
Pkg of 2 737-0732 737-1032 737-1532 737-2532
Pkg of 1 737-5031
Flow adapter 738-0015 738-0016 738-0017 738-0018
  Inner Diameter (ID)
    0.5 cm 0.7 cm 1.0 cm 1.5 cm 2.5 cm 5.0 cm
Length Type            
50 cm Pkg of 4 737-4751 737-4051 737-4155
Pkg of 2 737-0752 737-1052 737-1552 737-2551
Pkg of 1 737-5051
Flow adapter 738-0015 738-0016 738-0017 738-0018
70 cm Pkg of 1 737-5071
Flow adapter 738-0018
75 cm Pkg of 2 737-1576 737-2576
Flow adapter 738-0016 738-0017
100 cm Pkg of 2 737-1091 737-1591 737-2591
Flow adapter 738-0015 738-0016 738-0017
120 cm Pkg of 2 737-1093 737-1593 737-2593
Flow adapter 738-0015 738-0016 738-0017
170 cm Pkg of 2 737-1598
Flow adapter 738-0016

Jacketed Econo-Column Chromatography Columns Selection Guide
Choose the correct jacketed glass Econo-Column chromatography column by length and inner diameter. The corresponding flow adapter is shown with the columns. A dash ( – ) indicates that the column is not available in that configuration (L, ID, or package type).

  Inner Diameter (ID)
    0.7 cm 1.0 cm 1.5 cm
Length Type      
15 cm Pkg of 1 737-6108 737-6116
Flow adapter 738-0015
30 cm Pkg of 1 737-6131
Flow adapter 738-0015
50 cm Pkg of 1 737-6151
Flow adapter 738-0016

Related Content

Connecting a Flow Adaptor
This tutorial shows you how to attach a flow adpator to an Econo-Column chromatography column.
 
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