Hello researchers
I have an HPLC-size exclusion column , but i don't know its fractionation range . is there any simple procedure to determine the fractionation range of this column?
thank a lot .
Depends on your purpose.
If you do polymers, I think there are polyethylene samples with known size distributions (probably you find some on the webpage from polymer labs I believe). The same holds I believe for peptides.
If you use it for clean up, as I usually do, you might want to inject stuff you want to separate e.g. fat (butter or rapeseed) and in a separate run your compounds you want to analyse (e.g. pesticides).
enjoy
Kai
It will be different for different types of molecules (i.e. folded vs chains).
The column manufacturer will state the pore size of the stationary phase in the column's instruction and use sheet. This is a good starting place. If you have a specific molecule in mind, then find standards appropriate for use with that conformation or type and run a calibration curve based on on size to define the limits of the specific column.
As already mentiones above, the column manufacturers usually have calibration curves with certein reference materials for their columns of different pore size.
Typically a single pore size column will be able to separate two oders in molar mass.
If it is a conventional SDV based column, you can obtaina first estimate for the Separation range if you know the specified porosity. For a SDV based column the column will usually provide a useful separation +-1 decade from the specified pores ize.
i.e. If you have a comumn with a specified pore size 10^b, you will probably be able to get a separation in the molar mass range 10^(b-1) to 10(b+1)
Note that these limits correspond to narrowly dispersed samples. The separation range required will depend on the dispersity of your sample. In order to separate a sample having a dispersity of D=2, your separation range should span approximately from M(Peak)/10 to M(Peak)*10
thank you all for these answers, unfortunately all of you thought that i have full info of the column , indeed i dont have any info , except that i know it is a size exclusion column . so i need to determine its fractionation range by a suitable and practical method . otherwise i can read the column leaflet if i have it .
Does the column have a label or part number? If so, you should be able to look it up on the web or contact the manufacturer. If not, just run a calibration curve based on size to define the limits of the specific column. You can start with a range of PS stds or other appropriate standards, as applicable.
Then you can only calibrate with a suitable set of standards. The applicable Standards depend on the selection of your mobile phase. For THF, toluene, CHCl3 PS is a suitable calibrant. In DMF, DMSO, DMAC PMMA might be suited. In aqueous solvents PEOs, Pullulans, Polyacrylic acid might work. The column material willmonly tolerate certain solvents. Application of the wrong solvent will ruin the column. E.g. using water on typical SDV columns, will kill your column immediatyl. Thus, you need to have an idea of what column you have. Is there somethinh written on column itself that gives information on coulmn material? Do you have an idea of which solvent is in the column?
Wolfgang Radke ,Bill Letter : thank a lot , the recommended mobile phase is 0.05 M phosphate buffer ,it is from water company but all other info was deleted , i already did several run with this column , i used BSA(66Kd) and lysozyme (44Kd) as standard to check its performance , the method as flowing
0.05 M phosphate buffer PH7
flow rate 1 ml /min
column length 30 cm
both protein gave a good shape peak with a retention time 5 min for BSA and 6 min for lysozyme
but i still need to determine its lower and uper fractionation limit .
For a typical 300x8mm column, you will have an exclusion limit around 5 mL, thus, with your BSA you approach this value already. The Separation limit is expeced around 12 mL. Based on my simple estimate of 2 decades separation range you might be able to separte from a gew hundret molar mass up to you 60k.
Maybe you find a low molar mass PEG Standard in your lab, then you have an indication of the separation cabability of the column at low molar mass end.
The cabability of a GPC column for Separation depneds on the Separation range (that is whether there is sufficient shift in elution volume with molar mass, and on instrumental/column band broadeing. If your coulumn produces broad Peaks, even for a monodisperse substance, you will hav no good Resolution.
Check if (where) ethylene glycol elutes. EG should elute as anarrow Peak allowing determining the plate cout of your column. This gives you a hint, of whether your coliumn is still usefull. Typical GPC colimns yiles 10000 (alraedy low separation cabability) - 50000 (very good, allowing for oligmer separations) plates/meter. An alternative should be Glucose.
thank you Wolfgang Radke for this great clarification . i m gong to try glucose . dose glucose produce the same plate/m range that you mentioned to EG.?
There are minor differences for different analytes, due to different diffusion coefficients etc. Still if you have plate Counts below 1000/m, you waste your time.
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