Stepwise elution fractionation

Temperature-risiag elution fractionation (tref) is a technique for obtaining fractions based on short-chain branch content versus molecular weight (96). On account of the more than four days of sample preparation required, stepwise isothermal segregation (97) and solvated thermal analysis fractionation (98) techniques usiag variatioas of differeatial scanning calorimetry (dsc) techniques have been developed. 

The SCB distribution (SCBD) has been extensively studied by fractionation based on compositional difference as well as molecular size. The analysis by cross fractionation, which involves stepwise separation of the molecules on the basis of composition and molecular size, has provided information of inter- and intramolecular SCBD in much detail. The temperature-rising elution fractionation (TREE) method, which separates polymer molecules according to their composition, has been used for HP LDPE it has been found that SCB composition is more or less uniform [24,25]. It can be observed from the appearance of only one melt endotherm peak in the analysis by differential scanning calorimetry (DSC) (Fig. 1) [26]. Wild et al. [27] reported that HP LDPE prepared by tubular reactor exhibits broader SCBD than that prepared by an autoclave reactor. The SCBD can also be varied by changing the polymerization conditions. From the cross fractionation of commercial HP LDPE samples, it has been found that low-MW species generally have more SCBs [13,24]. 

An affinity sorbent based on WPA-PG carrying immobilized human IgG was applied to the isolation of the first component of the complement (Cl) from human serum and for its separation into subcomponents Clr, Cls and Clq by the one-step procedure [126,127]. Cl was quantitatively bound to the sorbent at 0 °C. The activities of subcomponents Clq and Clr2r2 in the unbound part of the serum were found to be 0.8% and 3.3% of the initial activities in serum. This fraction, therefore, could be used as a R1 reagent for determining the hemolytic activity of Cl. Apparently, the neighboring macromolecules of immobilized IgG resemble to some extent an immune complex, whereas Cl formation is facilitated due to the mobility of polymer chains with the attached IgG macromolecules (Cl is usually dissociated in serum by 30%). After activation of bound Cl by heating (30 °C, 40 min) the activated subcomponent Clr is eluted from the sorbent. Stepwise elution with 0.05 mol/1 EDTA at pH 7.4 or with 0.05 mol/1 EDTA + 1 mol/1 NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent Cls and subcomponent Clq. 

Fig. 1. Separation of two exopolygalacturonase groups (Fraction A, Fraction B) on CM-Sephadex C-50. Column size, 20x250 mm. Stepwise elution with 0.05 M acetate buffer, pH 3.8 (starting at arrow marked a), 0.10 M acetate buffer, pH 4.8 (at arrow marked b), 0.15 M acetate, pH 5.6 (at arrow marked c) and the latter buffer plus 1.0 M NaCl (at arrow marked d). Fraction size 6 ml per half hr. Exopolygalacturonase activity determined with sodium pectate, pH 5.0 (o—O) 2nd expressed as A,, .

Figure 19.9. Chromatogram for stepwise elution of bovine serum albumin on a Vistec diethyl aminoethyl cellulose ion-exchanger, using stepwise increases in sodium chloride concentration in the mobile phase to achieve selective desorption. Proteins 1, serum fraction not adsorbed by column (includes y-gobuhn) 2,3,...

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. 

Enzyme fractions obtained by linear gradient elution and stepwise elution were pooled and used later in this work. 

Additional fractionation with Sephadex LH20 using stepwise elution with isopropyl alcohol and dioxane/water was carried out to see whether the majority of the mutagenic activity (neutral fraction) could be separated from the bulk of the organics. The results depicted in... 

The functionality distribution of HTPB 134> can be obtained using the double detection procedure after derivatization of the OH groups with phenyl isocyanate. However, a true fractionation can been obtained by stepwise elution from silica gel n8,120) and, coupled with GPC, it enables establishment of the relationship between functionality and molecular weight distributions. Precipitation fractionation can also be used... 

Fig. 2. Chromatographic diagram of porcine pancreatic juice (13, 14). Abbreviations used for enzyme names are the same as in Fig. 1. The anionic proteins are fractionated on a DEAE-cellulose column equilibrated with 0.005 M phosphate, pH 8.0 and eluted at the same pH with a concentration gradient. The cationic proteins are fractionated on a CM-celluIose column by stepwise elution with buffers of increasing pH s. Ordinates, optical density of the fractions at 280 m/j. Abscissas (on the right diagram), volume of eluate expressed in number of interstitial volumes of the column.

The first peak of enzyme solution was dialyzed against 10 mM sodium succinate buffer, pH 6.0, without EDTA and with 2-mercaptoethanol, then applied to a hydroxyapatite column equilibrated with the same buffer. Stepwise elution with ammonium sulfate in this buffer yielded a single peak in 0.1 M ammonium sulfate fraction. The pooled enzyme was concentrated with 0.8 saturated ammonium sulfate to give 1% protein concentration in imidazole-glycylglycine buffer, pH 7.1. Addition of ammonium sulfate to between 0.5 and 0.55 saturation yielded a turbid solution which in turn yielded crystals after 24 hours at 4 C. The second peak was applied to a Sephadex G-200 column equilibrated with 0.01 M... 

Fig. 4 Stepwise elution profile of HDLs and LDLs by hydroxyapatite chromatography. Column Bio-Gel HTP DNA-grade hydroxyapatite (5.0 x 2.5 cm I.D.) eluents 75 and 290 mM potassium phosphate buffers at pH 7.4 flow rate 1.0 mL/ min sample 1.4 mL concentrated HDL-LDL CCC fraction.

Analogous to HPLC, CCC fractionation permits the use of either stepwise elution or gradient elution, provided that some precautions are taken. Eor example, in the isolation of extracts of Ginkgo biloba, one starts with water as a stationary phase, eluting with ethyl acetate with... 

There are three principal elution procedures isocratic (from the Greek isochros, meaning equal strength) stepwise (or fractional) and gradient. 

A part of the cell wall fraction was extracted with 0.25M sodium hydroxide at 4°C for 24hr and the extract was then subjected to pronase digestion to remove protein. The digested extract was chromatographed on a DEAE cellulose column by stepwise elution with water, sodium bicarbonate and sodium hydroxide. The yield of the major fraction, which was eluted with 0.5M sodium bicarbonate, SE, was 20.3% of the cell wall by weight. Another part of ACW was extracted successively with 45% phenol-water(PE), then with hot water in an autoclave(HE), and finally with 1% potassium carbonate at room temperature for 24hr(PCE). The yields of these fractions, PE, HE and PCE were 0.47%, 0.27% and 4.4%, respectively. 

Figure L Elution profiles of the bulk mannans of three C. albicans strains (Batch I) by DEAE—Sephadex chromatography (A-50, acetate, 4 X 25 cm) using a stepwise elution system consisting of water and NaCl solutions, Ten-fxL aliquots of fractions were assayed for carbohydrate content with phenol-sulfuric acid reagent (13) (A) C. albicans NIH A-207 (A-strain) (B) C. albicans NIH B-792 (B-strain) (C) C. albicans J-1012 (J-strain) (11),...

Elute peptides from the SPE column by stepwise elution as described in Section 3.1.1., step 6. Add to each fraction 5 pL of 2-MTE, cap, shake well, and store at 4 C (see Note 1). 

The countercurrent chromatography is used in the separation and purification of the natural products of notopterol and isoimperatorin from notopterygium forbessi Boiss, a Chinese medicinal herb used as an antifebrile and anodyne. The stepwise elution of two solvent systems, 5 5 4.8 5 and 5 5 5 4 of light petroleum-EtOAc-MeOH-water, respectively, was employed. The separation process took several hours and gave pure fractions of notopterol and isoimperatorin with the purity of 8%. 

Figure 6. Chromatography of BSA on DEAE-cellulose using stepwise elution. The starting buffer was 0.005 M sodium phosphate buffer pH 6.2 followed by 2, 0.0175 M sodium phosphate buffer pH 6.2 3, 0.075 M NaCl in 0.0175 M phosphate buffer pH 6.2 4, 0.2 M NaCl in 0.0175 M sodium phosphate pH 6.2. A Cohn fraction V lot No. 4394 from ICN. B Crystalline BSA, lot No. 46C-8090 from Sigma. C Fatty-acid-free BSA lot No. 46C-7450 from Sigma. From Habeeb (unpublished work).

Figure 19. A Gel filtration pattern of a 40X1 tryptic hydrolysate (1 hr) of bovine serum albumin. The hydrolysate (0.3 g) was applied onto a column (2.7 x 80 cm) of Sephadex G-100 which was eluted with 0.01 M NH HCOs. Fractions (7.5 ml each) were analyzed continuously by an ultraviolet monkor. B Chromatogi hic pattern on DEAE-cellulose of peak 3 from A. The material (100 mg) was applied on a column (1.5 x 20 cm) which was subjected to stepwise elution. Initial elution was with 0.005 M sodium phosphate buffer at pH 6.2. At positioa 2 the column was eluted with 0.0175 M phosphate buffer pH 6.2, and at portion 3 the ehient was changed to 0.0175 M phosphate pH 6.2 plus 0.077 M NaCl. Fractions (3 ml) were read continuously by an ultraviolet monitor. From Habeeb and Atassi (19766).

Serum albumins from goat, pig, horse, and human showed heterogeneity reflected in charge differences by chromatography on DEAE-cellulose. Each albumin was separated into three fractions using the stepwise elution system as that for BSA (Habeeb, 1978 ). All the albumins revealed the same elution pattern, showing three main components of which the first and third components eluted in a symmetrical peak while the second component trailed (Fig. 25). 

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