Gradient, separation

Density Gradient Separation. Based on specific density, each cell in a test tube finds its own position (see Fig. 1), ie, red cells at the bottom, then granulocytes, monocytes, lymphocytes, platelets, and plasma on top. Table 2 Hsts average mass density of the cellular components of blood. The actual numbers vary slightly from person to person. 

Many other devices are available for laboratory use. These include the Davis tube, Frantz isodynamic separator, laboratory dmm-type separators, low intensity rotating field separator, and superconducting high gradient separator (2). 

Fig. 2. HPLC ion exchange gradient separation of samples (25 ug of uronic acid) of smaller (G7) citrus pectin oligomers (a), larger (G12) citrus pectin oligomers (b), and the B. fruit extract (c). Detection was by PAD. For each sample the peak co-eluting with the standard galacturonate oc-tamer was designated peak 8, and the remaining peaks were numbered consecutively.

Fig. 3. QAE-Sephadex gradient separation of the B fruit extract. An 18 mg (uronic acid equivalents) sample of extract was dissolved in 20 ml of 125 mM imidazole-HCl buffer (pH 7.0) and applied to the column. The column was then eluted with 50 ml 125 mM buffer followed by a 125 mM to 1.5 M buffer gradient (500ml), and, finally, 50 ml of 1.5 M buffer. Fractions of 5 ml were collected and assayed for uronic acids. Groups of fractions (26-41, 45-50, 53-75 and 84-100) were pooled, concentrated by ultrafiltration and analyzed by HPLC.

Retention time in a Unear-gradient separation (tj can be expressed as foUow ... 

Restricted access phases are another approach to exploiting the differences in characteristics of analytes. Large analytes are excluded from an internal surface on which an adsorptive stationary phase is present. A herbicide analysis for Metsulfuron methyl, Bentazone, Bromoxynil, methylchlorophenoxy acid, and Mecoprop in the presence of humic acid was performed on restricted access reversed phase media.52 The cytostatic compound epirubicin and its metabolites were separated from plasma using a Pinkerton GFF II column.53 Gradient separations of polymers on reversed phase and on normal phase represent an alternative to gel permeation chromatography. Polyesters of noncrystalline materials were separated on a variety of such phases.54... 

Figure 3 Gradient separation of anions using suppressed conductivity detection. Column 0.4 x 15 cm AS5A, 5 p latex-coated resin (Dionex). Eluent 750 pM NaOH, 0-5 min., then to 85 mM NaOH in 30 min. Flow 1 ml/min. 1 fluoride, 2 a-hydrox-ybutyrate, 3 acetate, 4 glycolate, 5 butyrate, 6 gluconate, 7 a-hydroxyvalerate, 8 formate, 9 valerate, 10 pyruvate, 11 monochloroacetate, 12 bromate, 13 chloride, 14 galacturonate, 15 nitrite, 16 glucuronate, 17 dichloroacetate, 18 trifluoroacetate, 19 phosphite, 20 selenite, 21 bromide, 22 nitrate, 23 sulfate, 24 oxalate, 25 selenate, 26 a-ketoglutarate, 27 fumarate, 28 phthalate, 29 oxalacetate, 30 phosphate, 31 arsenate, 32 chromate, 33 citrate, 34 isocitrate, 35 ds-aconitate, 36 trans-aconitate. (Reproduced with permission of Elsevier Science from Rocklin, R. D., Pohl, C. A., and Schibler, J. A., /. Chromatogr., 411, 107, 1987.)...

Figure 12 Gradient separation of bases, nucleosides and nucleoside mono- and polyphosphates. Column 0.6 x 45 cm. Aminex A-14 (20 3 p) in the chloride form. Eluent 0.1 M 2-methyl-2-amino-l-propanol delivered in a gradient from pH 9.9-100 mM NaCl to pH 10.0-400 mM NaCl. Flow rate 100 ml/hr. Temperature 55°C. Detection UV at 254 nm. Abbreviations (Cyt) cytosine, (Cyd) cytidine, (Ado) adenosine, (Urd) uridine, (Thyd) thymidine, (Ura) uracil, (CMP) cytidine monophosphate, (Gua) guanine, (Guo) guanosine, (Xan) xanthine, (Hyp) hypoxanthine, (Ino) inosine, (Ade) adenosine, (UMP) uridine monophosphate, (CDP) cytidine diphosphate, (AMP) adenosine monophosphate, (GMP) guanosine monophosphate, (IMP) inosine monophosphate, (CTP) cytidine triphosphate, (ADP) adenosine diphosphate, (UDP) uridine monophosphate, (GDP) guanosine diphosphate, (UTP) uridine triphosphate, (ATP) adenosine triphosphate, (GTP), guanosine triphosphate. (Reproduced with permission of Elsevier Science from Floridi, A., Palmerini, C. A., and Fini, C., /. Chromatogr., 138, 203, 1977.)...

Figure 21 Analytical scale separation of oligomers of a-(l,4)-linked galacturonic acid. Column 25 x 0.46 cm Dynamax NH2. (A) Gradient separation, pH 5.9, 0.1-0.4 M phosphate in 25 min. UV detection at 220 nm. (B) 0.8 M acetate, pH 5. Refractive index detection. (Reproduced with permission from Elsevier Science from Hotchkiss, Jr., A. T., Hicks, K. B., Doner, L. W., and Irwin, R L., Carb. Res., 215, 81, 1991.)...

Temperature-programmed packed capillary liquid chromatography (TP-CLC), coupled off-line to solvent elimination FTIR (LC Transform ) has recently been used for gradient separations of Irganox 1010/1076/3114 dissolved in DMF with LOD of about 40 ng [511]. Low... 

The reversed-phase gradient separation was run at a flow rate near lOOnL/min and pressure around 23,000 psi. The gradient used to elute the proteins from the... 

FIGURE 17.11 Gradient separation of FAE with different end groups, stationary phase Chromolith C18, 10 x 0.46 cm i.d., mobile phase MeOH H20 (reprinted from Pasch et al., 2005, with permission of European Polymer Federation). 

The two major approaches for HPLC purification are fast gradient separation and parallel purification. Yan et al.178 utilized the former (Figure 1.51). The purification lab received a 96-well plate containing synthesized products at 0.1 to 0.2 mmol/well. A Hydra 96-probe liquid handler prepared QC plates for all samples that were analyzed with a MUX-LCT eight-channel parallel LCMS instrument at a throughput of 2000 samples/day. Only samples with purities above 10% were purified on a... 

---