I get very small peaks if I load 0.3 microgram (total concentration)
0.3 microgram is not a unit of concentration. In any case, it is a very, very small amount of protein for chromatography. I'm surprised you could detect any peaks at all.
Protein concentration is 3microgram/microliter. I am getting small peaks and from your answer, I understand that I can increase the concentration to get a good peak?
Hi there,
It's not the concentration of the sample you need to increase but the total amount of protein in the injection loop. In other words : what is the total amount of protein you have in hands?
Hi Liger. Thanks for your answer. My loop size is 100 microliter and the total amount of protein is 0.2µg.
I would think the SEC column that you use would influence the outcome. If you are using either of the Sephacryl S-200 16/60 or 26/60 you're looking at 120 & 320 mL bed volumes. If you have to use such small concentrations I would suggest using a much smaller column like an S200 increase 3.2/300 with a bed volume of 2.4 mL and can be loaded in volumes from 5 - 50 µL (if you have the loop) which means that the sample can be concentrated higher. Equivalent Shodex columns are also much better than the GE columns.
Although reading back to the top, 0.3 µg/µL (0.3 mg/mL) is a fair amount of protein. What is the volume of your samples at that concentration?
3mg/milliliter, its not enough to get much higher peak, if u need higher peak u should increase your protein concentration level from 3mg/milliliter to 15mg/milliliter. Here i also attached some other document that may be useful to you.
Sephacryl S-200 HR
Column: BP 113/120 packed with Sephacryl S-200 HR, bed height 100 cm
Sample: Albumin fraction from previous ion exchange step. Sample load
equivalent to 4% of Vt (Vt = column volume).
Sample concentration: 15 mg/ml
Buffer: 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.5
Flow rate: 7.5 cm/h
If your protein sample is at a concentration of 3 mg/ml and your injection loop is 100 ul, then you are injecting at most 300 ug of protein. That's not much. If that's all you have, you should use the smallest volume column you can find to minimize dilution and collect fractions with a suitably small volume, say 200-300 ul (4-6 drops).
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