Fraction collection amino acids

However, the use of a HPLC separation step enabled a remarkable acceleration of the deconvolution process. Instead of preparing all of the sublibraries, the c(Arg-Lys-O-Pro-O-P-Ala) library was fractionated on a semipreparative HPLC column and three fractions as shown in Fig. 3-2 were collected and subjected to amino acid analysis. According to the analysis, the least hydrophobic fraction, which eluted first, did not contain peptides that included valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine residues and also did not exhibit any separation ability for the tested racemic amino acid derivatives (Table 3-1). 

A similar ion-exchange resin method was used by Ling in 1955 (LI) for the examination of combined amino acids in urine. According to this procedure urine was desalted and simultaneously freed from amino acids by using Amberlite IR-112, H+-form resin. The effluent collected from the column was then fractionated on Amberlite IRA, OH--form resin, by successive elution with 0.16 N acetic acid, 0.08 N formic acid, 0.25 N formic acid, 0.08 N hydrochloric acid, and finally with 0.16 N formic acid. The solutions of all acids contained 10% of acetone. The collected fractions were hydrolyzed with hydrochloric acid and the liberated amino acids identified by means of paper chromatography. 

The chemical structures of the amino acids found in soil-solids are shown in Fig. 6 [25] while the quantities of amino acids found in HS extracted from various solid phases are represented in Fig. 7 (data were collected from Ghosh and Schnitzer [37] and Schnitzer et al. [38]). High levels of amino acid nitrogen were found in HA, FA, and humin fractions, indicating incorporation of common acidic and some neutral amino acids, particularly glycine, alanine, and valine. 

The separation power of CIEF often generates a high number of peaks even when relatively pure samples are analyzed. As already discussed, one of the advantages of CIEF is its potential micropreparative capabilities. Capillary IEF allows the collection of fractions that can be further analyzed by other methods. Some of the most widely used characterization tools include MS, peptide mapping, and amino acid analysis. 

Separation of a purified hydrolyzate of demineralized bovine dentin by cation exchange chromatography at pH 5.25. Gradient 0.0 - 0.5 M NaCl in 0.05 M HAc/NaAc, 1 mM NaNs, pH 5.25. Column SP Sephadex C25 (34 x2.6cm). Flow rate approx. 30 ml/h. The 4-ml fractions were assayed for amino acids by ninhydrin reaction (—) and for NaCl by electric conductivity measurement after 75-fold dilution (—). Fractions collected for further characterization are denoted by bars and Roman numerals. 

Fig. 2. NAPI facilitates H2A, H2B release from nucleosomes that are on positively coiled DNA (A) but not negatively coiled DNA (B). The positively coiled DNA (6.0 kb) with a superhelical density of + 0.05 and negatively coiled DNA (6.0 kb) with a superhelical density of -0.05 were reconstituted with lysine, arginine-labeled histones H3, H4, H2A, H2B by NaCl dialysis from 2.0 M to 1.2 M to 0.6 M to 0.1 M NaCl over a 14 h period. The samples were incubated with NAPI at 35 °C for 5 min and applied to a 5-20% sucrose/100 mM NaCl/40 mM Tris, pH 7.8 gradient. After sedimentation at 200,000 X g for 5 h, fractions were collected and the distribution of DNA (bottom panel) was determined on agarose gel and the distribution of protein (top panel) on SDS-PAGE followed by fluorography. These data are unpublished observations (V. Levchenko and V. Jackson). The deg-H2A is degraded H2A in which a 15 amino acid peptide of the C terminal has been proteolytically removed. When H2A, H2B is no longer present in a nucleosome, the C terminal region is sensitive to proteolysis [126] from a protease which is a minor contaminate in the NAPI preparation.

The amino acid is recrystallized by dissolving all the crude material in 12.5 1. of water heated to 950 on a steam cone. The hot solution is treated with 20 g. of Norite for thirty minutes and filtered hot. An equal volume of 95 per cent alcohol is added immediately, and the flask is placed in the icechest overnight. The crystalline material is collected on a filter and washed with 200 cc. of 95 per cent alcohol. The yield of pure leucine in this fraction is 290-300 g. An additional crop is obtained by evaporating the mother liquors under reduced pressure until considerable solid separates (liquid volume about 1 1.), adding an equal volume of alcohol, and cooling. This crop is washed with 100 cc. of cold water and then with 200 cc. of alcohol it amounts to 60-65 g- The total yield of pure leucine is 350—365 g. (43-45 per cent of the theoretical amount). It decomposes at 290-292° (uncorr.) in a sealed capillary (Note 4). 

The completion of a chromatographic experiment calls for a means to detect the presence of solutes in the collected fractions. The detection method used will depend on the nature of the solutes. Smaller molecules such as lipids, amino acids, and carbohydrates can be detected by spotting fractions... 

Prior to the chromatographic separation of amino acids on Dowex 50 columns, Carsten (C5) first desalts the urine sample on Amberlite IR 100 or Duolite C 3 and removes most of the nitrogenous bases on Amberlite IRA 400. This preliminary treatment allows for amino acid separations at ordinary temperatures using 2M and 4M HC1 on H+ columns for elution, instead of buffer mixtures a single column of 25 g of Dowex 50 is sufficient for all amino acids and 350-375 one-milliliter fractions are collected. The resolving power of this method does not seem to be as satisfactory as Moore and Stein s procedures, and it is not less time nor labor consuming. 

The feasibility of the approach has been demonstrated with base-line resolved separations of tagged amino acids at a electric field strength of 50 V/cm applied across the separation length (bed width) of 1 cm [81]. However, it is clear that high resolution separations will not be the domain of this technique, for the absolute magnitude of the separation voltage across the separation bed is limited to -100-200 V (see Eq. 8). The flow through the outlet channels was combined into one hole in the cover plate, therefore fraction collection experiments were not possible in this study. 

Determination of the molecular weight difference between the wild- type and the variant TTR obtained by ESI MS limited postulated amino acid substitutions to a relatively small number. Analysis of the tryptic digest of the protein by LC-ESI MS was used to locate more precisely the site of the mutation. Comparison of the tryptic map obtained for the variant with that of a normal sample facilitated detection of the modified tryptic peptide. MS-MS was carried out on the variant tryptic peptide contained in the appropriate chromatographic fractions collected during the LC-ESI MS experiment. 

Inspection of the UV chromatogram from the LC-ESI MS analysis of the tryptic digest of the protein revealed a peak not present in the digestion of wild-type TTR, at retention time 25.95 min. The [M + H]+ of this variant peptide (m/z 1392.6) was consistent with a 27 Da increment to the normal tryptic peptide T4, 22GSPAINVAVHVFR34. The location of the mutation was confirmed by MALDI MS analysis of the chromatographic fraction collected between retention times 25.0-27.5 min. In the MALDI mass spectrum, peaks at m/z 1365.2 (wild-type T4) and m/z 1392.1 (variant T4) are both present (Fig. 8). This observation confirmed that the mass difference between the variant and the wild type peptides is indeed +27 Da. The only possible amino acid substitution that would give rise to a +27 Da shift in that peptide is Ser23 —> Asn. 

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