Buffer exchange columns

Dialyze the sample against 0.0175 M buffer pH 6.3 or conversely, the buffer may be exchanged using a Sepadex PD-10 column or another prepacked buffer exchange column. 

Check the column packing quality Buffer exchange before testing... 

Figure 3. Typical chromatogram of UV-ahsorhing constituents of a 0.15-ml sample of urine. Conditions 150-cm anion exchange column (1215 fi, Aminex A ZI) bujfer, acetate at pH 4.4 gradient, buffer concentration from 0.015M to 6M flow rate, 10.5 ml/hr, coUimn temperature, 25°C for first 5 hr and 60°C thereafter (34).

To the flasks for the crop and soil samples (Section 6.1), add 2mL of 0.01 M Tris-HCl buffer solution (pH 7.7) and 50 and 100 qL of 1M Tris-HCl buffer solution for wheat grain, bariey grain and rice straw, and for soil, respectively. Adjust the pH to about 7.7 (confirm the pH with a pH test paper using the sample of untreated area). Homogenize the residue with ultrasonication and transfer the homogenate to the top of an ion-exchange column. Wash the flask twice with 2mL of 0.01 M Tris-HCl buffer solution and transfer the washings to the column. Elute the column with 40 mL of the same buffer solution. Discard this eluate. 

Hutchens, T. W., Li, C. M., and Paige, P. K., Performance evaluation of a focusing buffer developed for chromatofocusing on high-performance anion-exchange columns, /. Chromatogr., 359, 169, 1986. 

The polyamines putrescine, cadaverine, spermidine, and spermine, which are seen at elevated levels in some victims of cancer, were separated on a Technicon (The Technicon Company Chauncey, NY) TSM Amino Acid Analyzer packed with an 8% divinylbenzene-co-polystyrene sulfonated resin with post-column ninhydrin detection.111 Amines such as ethanolamine, noradrenaline, hexamethylene diamine, methoxytryptamine, spermine, and spermidine were separated from amino acids on a DC-4A cation exchange resin.112 A similar approach, using a Beckman Model 121M amino acid analyzer equipped with an AA-20 column, was also successful.113 A Polyamin-pak strong cation exchange column (JASCO) was eluted with a citrate buffer for the detection of putrescene, spermine, cadaverine, and 1,5-diaminohex-ane from rat thymus.114 A post-column o-phthaldehyde detection system was used. 

In an off-line configuration, a complex peptide mixture from a proteomic sample is loaded onto a SCX column and fractions collected (Fig. 11.1). After the collection of fractions, they are then loaded into an autosampler and analyzed via the traditional RP/ MS/MS approach. Using this system, a variety of buffers and elution conditions may be used (Table 11.1). For example, one may use a volatile salt such as ammonium formate (Adkins et al., 2002 Blonder et al., 2004 Fujii et al., 2004 Yu et al., 2004 Qian et al., 2005a and b) or ammonium acetate (Cutillas et al., 2003 Coldham and Woodward, 2004), collect SCX fractions, lyophilize, resuspend in low acetonitrile and acid, and then directly analyze via RP/MS/MS. In most of the cases, when ammonium acetate or ammonium formate are used, a 20-minute wash period is used to remove the ammonium acetate or ammonium formate prior to the reversed-phase gradient (Table 11.1). However, because fractions are collected and can be buffer exchanged,... 

IEC was applied to determine biogenic polyamines such as putrescine (4a), cadaverine (4b), tyramine (5), histamine (6), spermidine (38), agmatine (39) and tryptamine (40), contained in aqueous trichloroacetic extracts of leafy vegetables, such as cabbage and lettuce. A cation exchange column loaded with potassium ions and a special buffer were used. Spermidine (38) was the major amine detected in this group (7-15 Xg/g fresh weight)144. 

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