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![[Figure 4]](ju5081fig4mag.jpg)
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Figure 4
Illustration of the effects of user-modifiable optimization parameters on the separation quality of the benchmarking protein BSA (as monomer, dimer, and higher oligomers). The protein is detected by monitoring UV absorption at 280 nm; a 10 kDa membrane is used for all runs. (a) Effect of the rate of the Cross Flow, for 20 µL of BSA 4.6 mg ml−1 in PBS buffer, employing a 350 µm-thick channel spacer, and a detector flow of 0.5 ml min−1 with a 35 min elution time. Satisfactory separation is reached in this example at 3.0 ml min−1 Cross Flow rate. Inset: zoom on the peak region. (b) Separation function F for the monomer and dimer peaks [after Giddings (1960  )], as a function of the flow rate. (c) Effect of the channel height set by the spacer thickness, for 20 µL of BSA 5.0 mg ml−1, in PBS buffer, employing for a 25 min elution a Cross Flow of 3.0 ml min−1, Detector Flow 0.5 ml min−1. (d) Separation function F as a function of the spacer thickness. (e) Effect of the injection volume at constant 5.0 mg ml−1 BSA concentration, run in PBS buffer with 350 µm spacer, Cross Flow of 3.0 ml min−1, Detector Flow 0.5 ml min−1. (f) Lack of a significant effect on the separation function F within this range of injection volumes. (g) Effect of the elution time for a 20 µL injection of 5.0 mg ml−1 BSA in PBS, employing a 350 µm spacer, Cross Flow of 3.0 ml min−1, Detector Flow 0.5 ml min−1. No significant changes in the elution times of the peaks of interest are observed; however, particles large enough not to elute until the Cross Flow is switched off are better separated for longer elution times. (h) Lack of significant effect on the separation function F for the elution times tested. In all plots showing the values of F, the uncertainties are propagated from the fit errors. |
![[Figure 4]](ju5081fig4.jpg)
 | JOURNAL OF SYNCHROTRON RADIATION |
ISSN: 1600-5775
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