Gradient volume

Vq and to are the gradient volume and the gradient time, respectively, during which the concentration 9 changes from A to the concentration 9g=A-H A9 at the end of the gradient A9 is the gradient range... 

The sample should contain about 8 x 10 cells in 1-ml culture medium per 12 ml total gradient volume and 0.5 ml sample (2-10 mg protein per milliliter) per 10 ml gradient volume. If selfgenerating gradients are used, separation is done with a g t-value of 1.35 X 10 g min (30 000 rpm, 45 min), in case of pre-formed gradients run with 800 gav for 15 min. Allow the rotor to decelerate without brake to avoid swirling of the bands. 

The gradient volume VG is the volume of eluent flushed through the column during the gradient run (60) ... 

P-Casein (13 mg) containing 0.036 fid M-P-C was subjected to plasmin hydrolysis for 45 min and the reaction mixture was dissolved in 7 mL column buffer (5mM Tris—3mM NaCl—urea, pH 8.55) together with 100 mg whole casein that had been alkylated with iodoacetamide. The sample solution was applied to a column (1.6 X 50 cm) of DEAE-cellulose equilibrated with column buffer. Elution was with a NaCl gradient (3.0-155mM) in column buffer (gradient volume, 1.0 L) 5.0 mL fractions were collected. Under the conditions used as as-caseins remained adsorbed to the column K-caseins were eluted in Fractions 35-56 Am measurement (----------) radioactivity (--) (28). 

Linear gradient experiments can also be employed to determine K and v.52 Equation (3) relates a solute retention volume (Vg) to initial and final carrier salt concentrations (x xf), the gradient volume (Vg), the column... 

FIGURE 12 Purification of a murine monoclonal antibody against IgE form hybridoma culture supernatant on a phenyl Sepharose HP. The feedstock has been added with ammonium sulfate to a final concentration of 0.5 M. The sample volume was 130 mL. The column was operated at 100 cm / hr. As equilibrium buffer a 20 mM potassium phosphate buffer, pH 7.0, supplemented with 0.5 M ammonium sulfate was used. Elution was performed by a linear gradient with a 20 mM potassium phosphate buffer, pH 7.0. The gradient volume was equal to 10 column volumes. 

Decrease Vm by decreasing column length, L, with a proportional decrease in tg. If column diameter is also changed, the ratio of gradient volume to column volume (tg F/Vm) must remain constant. Thus, F must be adjusted with change in column diameter. 

Although the use of zonal rotors is somewhat more involved than conventional swinging bucket rotors it is important to emphasize that the end result is the same. An example of this is shown in Figure 9-32, which depicts the elution profile of a stepped sucrose gradient that was used to separate mitochondria, proplastids, and glyoxysomes. This profile is very similar to that shown in Figure 9-16A, but in that experiment the gradient volume was 54 ml. Here it is 1760 ml and accommodates the crude particulate fraction yielded from 1 lb of fresh tissue. 

In addition to the gradient volume and to the column plate number gradient shape can be adjusted [85,94]. 

Fig. 1.30. (A) The resolution window diagram for the gradieni-elulion separalion of a mixture of eight phenyluiea herbicides on a Separon SGX. 1.5 tm. silica gel column (150 x 3.3 mm i.d.) in dependence on the initial concentration of 2-propanol in n-hcpiane at ihe slart of the gradicni. A. with optimum gradient volume Vc, - 10 ml. Column plate number N = 5000. compounds as in Fig. 1.23. (B. C) The separation of the eight phenylurea herbicides with optimised gradient-elution conditions (maximum resolution in (A)) with gradients from 12 to 38.6 2 2-propanol in n-hcptanc in 7 min (B) and from 25 to 37.5 2 2-propanol in fi-heptane in 5 min (C). Flow rale I ml/min.

Fig. 1.35. Top the re.solution window diagram for the elution. strength ternary gradieni-elulion. separation of a mixture of twelve phenylurea herbicides in dependence on the initial sum of concentrations of methanol and acetonitrile in water at the start of the gradient. Ay. with the concentration ratio of aceionitrile, X = Vaccionimte/tVaccioniiriie + Vmethanoi) = 0.4 optimised for isocratic ternary mobile phases (Fig. I..34) and optimum gradient volume V(-, = 31 ml. Column and sample compounds as in Fig. 1.33. Bottom the separation of the twelve phenylurea herbicides with optimised ternary gradient from 18.6% methanol + 12.4% acetonitrile in water to 60% methanol + 40% acetonitrile in water in 73 min. Flow rate I ml/min.

Rotor Gradient volume ml Gradient length cm Speed rev min X 10 Time of run hr... 

Resolution is also affected by the total gradient volume (gradient volume X flow rate). Although the optimum value must be determined empirically, a good rule of thumb is to begin with a gradient volume that is approximately 10-20 times the column volume. The slope can then be increased or decreased in order to optimize the resolution. 

Fig. 2 RP gradient elution separation of 1,2-naphthoylenebenzimidazole alkylsulphonamides. Column Lichrosorb RP-18, 10 pm (300 X 4 mm id). Linear gradients of metbanol in water with a constant gradient range but different gradient volumes (A-C), and with a constant gradient steepness (1.67% metbanol/min) but different initial concentrations of metbanol (D-F). Flow rate= 1 mL/min. Tbe number of peaks agrees with tbe number of carbon atoms in alkyls.

Fig. 3 Top The resolution window diagram for RP gradient elution separation of phenylurea herbicides on a Separon SGX Cl8 7.5-pm column (150 x 3.3 mm ID) in dependence on the initial concentration of methanol in water at the start of the gradient A with optimum gradient volume Vq = 13 mL. Column plate number A = 5000 sample compounds hydroxymetoxuron (1), desphenuron (2), phenuron (3), metoxuron (4), monuron (5), monolinuron (6), chlorotoluron (7), metobromuron (8), diuron (9), linuron (10), chlorobromuron (11), and neburon (12). Bottom The separation with optimized binary gradient from 24% to 100% methanol in water in 73 min. Flow rate = 1 mL/ min T=40°C.

I-pH Unit Gradients Volumes of Immobiline for 15 ml Each of Starting Solution (2 gels)... 

For gradient runs steeper slope and/or reduction of gradient volume (see Tip No. 45)... 

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