Continuous density gradient

Prepare continuous density gradients from Soln. D and Soln. D", cover with the suspension of the crude membrane suspension and centrifuge with 60 000 x and 83 000 x respectively, for 90 min (e.g., Beckman Coulter SW 28 rotor gradient from 16.5 ml Soln. D and 16.5 ml Soln. D" per tube 24 000 rpm). 

Figure 10.10 Rate-zonal centrifugation. (A) Sample layered over a continuous density gradient. (B) Position of zones due to different size of particles at the termination of centrifugation. Note that separation is due to differences in size and shape.

Figure 10.11 Isopymic centrpugation. (A) Sample layered over a continuous density gradient. (B) Position at equilibrium.

Two types of gradients may be prepared discontinuous or step density gradient, or continuous density gradient. 

In order to avoid many of the technical difficulties of sedimentation in homogeneous media (convection, associations, etc.), a continuous density gradient is often used. After a sedimentation run in a buffered gradient, established before the run in the centrifuge tube, a hole is punched in the bottom of the tube, and the solution collected drop by drop. Monitoring is by absorption, determination of radioactivity and enzyme activity, etc., and, frequently, a marker of known molecular weight is added to the solution. 

Continuous density gradient columns can be built and are commercially available. When a sample is dropped into the column, it will fall until it reaches the region of the column containing the same density. From the equilibrium position of the material and its relation to reference floats, the density can be accurately determined (ASTM-D1505-03 2003 150-1183-1 2004 2004). Organic solvents or salt solutions can be used to form the gradient column, but the liquids should be chosen so that they will easily wet the sample but not swell or dissolve the sample (Kholodovych and Welsh 2007). 

Radial density gradients in FCC and other large-diameter pneumatic transfer risers reflect gas—soHd maldistributions and reduce product yields. Cold-flow units are used to measure the transverse catalyst profiles as functions of gas velocity, catalyst flux, and inlet design. Impacts of measured flow distributions have been evaluated using a simple four lump kinetic model and assuming dispersed catalyst clusters where all the reactions are assumed to occur coupled with a continuous gas phase. A 3 wt % conversion advantage is determined for injection feed around the riser circumference as compared with an axial injection design (28). 

Before use, thaw the required number of gradient tubes overnight at 4°, to establish continuous sucrose density gradients. 

Method Density gradient, Rate-zonal Gradient 10-40% continuous sucrose Rotor/run conditions SW 55 Tl rotor at 55000 rpm for approximately 6 hours Potential tube materials Polyallomer, Ultra-Clear... 

Density gradients maybe divided into three types preformed discontinuous gradients (e.g.. Protocol 5.3.1), preformed continuous gradients (e.g.. Protocol 5.3.2) or self-generating gradients (e.g.. Protocol 5.3.3). Materials used for density gradients are classified as ionic and non-ionic media (Table 5.1). 

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