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- Posts: 12
- Joined: Mon Dec 29, 2014 3:45 pm
Hello,
I'm using a Shimadzu SPD-20A that's giving good 280nm peaks but negative 214nm peaks for the same region. The buffer is 25mM Tris, 10mM NaCl.
When switching from PBS or water to the Tris buffer, the signal jumps (from the my own perspective and the instrument's read out) over the detection limit resulting in a noisy "baseline" at 214nm but a flat one on the 280nm channel.
When the protein solution (CSF diluted 1:5 in the Tris buffer, roughly 200ug/ml protein content, varies by sample) passes through the detector on its way to the depletion set up, the 214nm peaks are negative while the 280nm peaks remain positive. My first thought was that the plug of injected, dilute CSF is absorbing less than the background Tris buffer, resulting in the negative peaks. Given the 1:5 dilution into the Tris buffer this doesn't seem right, though.
Also, my boss is dubious of this explanation so I'm hoping someone else has encountered this before or can chime in.
Thanks!
I'm using a Shimadzu SPD-20A that's giving good 280nm peaks but negative 214nm peaks for the same region. The buffer is 25mM Tris, 10mM NaCl.
When switching from PBS or water to the Tris buffer, the signal jumps (from the my own perspective and the instrument's read out) over the detection limit resulting in a noisy "baseline" at 214nm but a flat one on the 280nm channel.
When the protein solution (CSF diluted 1:5 in the Tris buffer, roughly 200ug/ml protein content, varies by sample) passes through the detector on its way to the depletion set up, the 214nm peaks are negative while the 280nm peaks remain positive. My first thought was that the plug of injected, dilute CSF is absorbing less than the background Tris buffer, resulting in the negative peaks. Given the 1:5 dilution into the Tris buffer this doesn't seem right, though.
Also, my boss is dubious of this explanation so I'm hoping someone else has encountered this before or can chime in.
Thanks!
