Stepwise elution

A sample of aequorin (purity > 80%) is first luminesced by adding a sufficient amount of Ca2+. To the spent luminescence solution, ammonium sulfate is dissolved to a concentration of 1M, and then the solution is added onto a column of Butyl Sepharose 4. The apoaequorin adsorbed on the column is eluted stepwise with buffer solutions containing decreasing concentrations of (NH4)2S04 starting from 1M. Apoaequorin is eluted at a (NH4)2SC>4 concentration lower than 0.1 M. The apoaequorin eluted is regenerated with coelenter-azine in the presence of 5 mM EDTA and 2 mM 2-mercaptoethanol... 

Fractionation by Stepwise Elution. Information obtained from the analytical separation was applied for a preparative purification. Lignin peroxidase concentrate was bound to a Q-Sepharose colunm (0= 5 cm, V = 1000 ml) after ultrafiltration and eluted stepwise with 0.08 M, 0.18 M and 0.28 M sodium acetate, pH 6.0. The fraction which was eluted with 0.28 M buffer (V= 3.91, 4400 U/1) was purified further. It was bound to Q-Sepharose and eluted with 0.18 M and 0.3 M sodium acetate. En rnie in the latter fraction was precipitated and dissolved in glycerol as previously described. The volume was 15 ml. 

Equilibrated with 20 mM Tris-HCI, pH 7.5 at flow rate 20 ml min and then eluted stepwise with the buffer containing NaCI. 

II. Purity test of 14C-galactose-l-phosphate dry 10 pi of 14C-galactose-l-phosphate with nitrogen, dissolve in 200 pi of H20. Put 100 pi in a counter vial with 15 ml scintillation liquid (= 100%) and apply 100 pi on a DEAE column. Elute stepwise as described in a) and b) above. Contamination, eluted with 100 mM HC1, should not exceed 0.1-0.2% of the total radioactivity eluted. 

Biological Elution stepwise on silica gel AAS materials column... 

Elution is carried out with a series of successively strohi efe sdly feritl,j-e- stepwise elution. Stepwise elution can provide a complete sofMipn fo the genera elution problem and is quite convenient to carry ioUt,. sirice no special equipment is required. An optimum solvent sequence for the stepwise elution of a particular sample depends upon the /riyaliies of the sample components and the variation of K with solvent coMposition. Section 8-5 provides some general rules for the selection of an optimum solvent series for stepwise elution. 

Figure 5.4-5. Online MDLC using salt-plugs. This HPLC separation mode uses salt-plugs with increasing salt concentrations that are injected by the autosampler onto the lEX-column to elute stepwise stronger bound peptides.

The column was sequentially eluted with CHCI3 (<20 MU), CHCls-MeOH 99 1 (<20 MU), 9 1 (>20 MU), and 1 1 (<20 MU) (each 100 ml). The CHCls-MeOH (9 1) fraction was concentrated by evaporation and applied to a reversed phase column, Develosil LOP ODS (1 x 35 cm, Nomura Chemicals, Seto, Japan) with aqueous solution containing 80% MeCN. The column was eluted stepwise with aqueous 80%, 90%, and 100% MeCN. The fraction eluted in the first 40-60 ml portion of aqueous 80% MeCN was toxic to mice (30-60 MU). A half of this fraction was concentrated by evaporation and applied to another reversed phase column, Cosmosil 5C18-AR-11 (1 X 25 cm, Nakarai Tesque, Kyoto, Japan). The column was eluted by gradient increasing MeCN concentration in water from 70% to 100%. The toxic fraction (10-20 MU) was eluted with aqueous 70-80% MeCN. Aliquots of this fraction were applied to liquid chromatography-diode array detection (LC-DAD), LC/electrospray ionization-mass spectrometry (LC/ESl-MS), and LC/ESl-MS/MS for analysis of polycavemosides. 

Fig. 2. Separation of cathepsin B, H, and L on CM-cellulose chromatography. The mitochondrial lysosomal fraction from rat liver was subjected to seven cycles of freezing and thawing and centrifuged at 105,000 g for 30 minutes. The supernatant solution was concentrated and applied to a column of CM-cellulose chromatography. The column was equilibrated with 0.02 M acetate buffer, pH 5.0, containing 0.05 M sodium chloride and 5 mM 2-mercaptoethanol and eluted stepwise with increasing concentrations of 0.05 to 0.5 M sodium chloride. ( ) /3Ala-Tyr-Met- 3NA, (A) Z-Arg-Arg-jSNA, and (O). BZ-Arg-/3NA were used as substrates for assays of cathepsin B, H, and L.

In principle, the modes and steps in preparative chromatography do not differ from those in analytical chromatography. Four modes are used (Fig. 4) isocratic elution, linear gradient elution, stepwise elution, and displacement development. Frontal analysis is not used for purification of biomolecules, but it is used for... 

Figure 6.7. Separation of the methyl ester derivatives of fatty acids from the phospholipids of mouse brain by reversed-phase HPLC with spectrophotometric detection at 192 nm [67]. In essence, the column of Zorbax ODS phase, maintained at 35 C, was eluted stepwise with acetonitrile-water (7 3, v/v) then with acetonitrile alone. Methyl elaldate was added as an internal standard. (Reproduced by kind permission of the authors and of the Journal of Lipid Research, and redrawn from the original paper).

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