What should be the column height and diameter ratio in protein size exclusion chromatography?
The efficiency of a column depends on the
1) number of theoretical plates - N
and
2) a parameter known as Height Equivalent to Theoretical Plate (HETP).
HETP = L / N; where L is the length of the column.
For a given particle size, pore size (in case of Size exclusion) and mobile phase viscosity, HETP can be calculated using the Van Deemter Equation.
Thus, the column efficiency improves as the number of theoretical plates increases or by minimizing HETP. Since column lengths and diameters are fixed and since cannot use unwieldily lengthy columns, HETP can be minimized by tighter packing of the column bed. This was what Dominique Liger And Michael Weller alluded to above.
The reader is referred to a book
"Analytical Chemistry" - by Prof. Gary D. Christian, University of Washington
ISBN: 9788126511136, 8126511133
for a lucid explanation of the Principles of Chromatography and methods to calculate column efficiency and other important chromatographic parameters.
Siddhartha
yes it effects. size depends on your protein size. If your protein present in crude extract and protein size is more then you may require lengthy column. Dia depends on your resin which you are using to purify your protein of intrest
Hi there,
Sure dimensions are essential for SEC : height of the column defines the resolution capacity (the bigger the better but the diffusion of molecules also increases with it) and diameter defines the loading capacity (volume and amout of material being injected). The bed volume of the colum defines the void and total volumes between which separation is efficient. For preparative scale, the commercial 16/600 and 26/600 (diameter/height in mm) are the most used. For analytical purpose the 10/300 is used.
GE is providing very good information in its handbook available online:
http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-fr/service-and-support/handbooks/
To improve your separation, you also should consider to decrease viscosity of mobile phase (temperature, dilution), flow rate and bead size (smaller).
Very well explained by all.
Apart from this for protein purification you can consider the new technology developed by Clontech Laboratories which allows you to purify the his-tagged proteins in just 5 mins.
Click on the link below for more details:
http://www.clontech.com/IN/Products/Protein_Expression_and_Purification/His-Tagged_Protein_Purification/Miniprep?sitex=10060:22372:US
The efficiency of a column depends on the
1) number of theoretical plates - N
and
2) a parameter known as Height Equivalent to Theoretical Plate (HETP).
HETP = L / N; where L is the length of the column.
For a given particle size, pore size (in case of Size exclusion) and mobile phase viscosity, HETP can be calculated using the Van Deemter Equation.
Thus, the column efficiency improves as the number of theoretical plates increases or by minimizing HETP. Since column lengths and diameters are fixed and since cannot use unwieldily lengthy columns, HETP can be minimized by tighter packing of the column bed. This was what Dominique Liger And Michael Weller alluded to above.
The reader is referred to a book
"Analytical Chemistry" - by Prof. Gary D. Christian, University of Washington
ISBN: 9788126511136, 8126511133
for a lucid explanation of the Principles of Chromatography and methods to calculate column efficiency and other important chromatographic parameters.
Siddhartha
Yes, length and diameter affect the chromatogram. other factors, such as temperature, flow rate and bead size can affect the result of the separation.
For sure length and diameter are important parameters. However Sandeeps question was about the RATIO between these. I would say that the ratio is not important. You want length enough for good resolution and width enough to handle the sample volume which should be 1-2% of the column volume. Go for a longer column if you need to increase resolution and go for a wider column if you need to increase sample volume. No need to keep the ratio constant when scaling up or down. If you scale up by increasing length the increase in resolution may compensate for an increase in sample load above 2%, but processing time will increase.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins