Separation of Amino Acids

7 Key excerpts on "Separation of Amino Acids"

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  Electrophoresis in Stabilizing Media

  Paper Chromatography and Electrophoresis

  • John Whitaker(Author)
  • 2013(Publication Date)
  • Academic Press

    (Publisher)

  The electrophoretic mobilities of some amines are shown in Tables II-V. The aromatic compounds in Table V are of special interest because of their potential carcinogenic properties. III. Amino Acids W e shall be concerned here mainly with methods for separating the 20 amino acids which are the building units of proteins. However, it will be seen from the results presented that when satisfactory conditions are achieved for separating these 20 amino acids the other amino acids and amine compounds are readily separated by the same conditions. The acidic and basic amino acids are readily separated from each other and from the neutral amino acids. However, the neutral amino acids are so similar in electrophoretic mobilities that more demanding conditions are needed for their separation. Three ways in which this may be ac-complished are (1) electrophoresis of the mixture under three different conditions, (2) two-dimensional electrophoresis, and (3) electrophoresis combined with chromatography. The experimental procedures presented here appear to us to give the best results among the many that have been proposed. While there has been limited success with separations using low voltage (11-14) particularly with specific groups of amino acids, the re-sults are so much superior when high voltage is used that we shall restrict the discussion to the latter. Several of the neutral amino acids differ in electrophoretic mobilities by 1 to 2 % ; diffusion is sufficient to obscure this difference under low voltage conditions where the time of separation must be prolonged. A. P R E P A R A T I O N OF SAMPLE The hydrolysis of proteins to amino acids (or to the peptide stage by limited treatment) may be performed in acid, base, or enzymatically. The most generally useful method is the one which uses hydrochloric acid.

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  Absorption and Utilization of Amino Acids

  Volume III

  • Mendel Friedman(Author)
  • 2018(Publication Date)
  • CRC Press

    (Publisher)

  vide supra) and with the increasing interest in use of highly sensitive HPLC methods, the contamination can cause problems. Central and important steps, both in traditional and new efficient and simple methods of amino acid analysis, are the extraction, isolation, concentration, and group separation prior to liquid chromatography. The methods are adapted to and comprised amino acids present in foods, body fluids, microorganisms, and animal and plant tissues.

  A. Relatively Large Scale Procedures

  Isolation of pure amino acids and separation of structurally related amino acids require homogenization and solvent extractions using large quantities of solvents, reduction of sample volumes, and use of relatively large columns. These techniques based on different types of ion-exchange columns are described in detail elsewhere.10 , 32 , 33 The separations on strongly acidic cation-exchange resins, including the amino acid analyzers, are mainly determined by pKa1 -values (see Tables 1 and 2 ) and adsorption properties of the amino acids.10 , 32 The separations on strongly basic anion-exchange resins are obtained essentially according to pKa2 -values (Tables 1 and 2 ) and adsorption properties of the amino acid.10 , 15 , 33 , 34 Special and gentle methods are used to avoid artifact problems10 , 19 , 34 during extraction and isolation procedures.

  B. Semimicro Isolation, Purification and Group Separation

  The technique developed for sample preparation and group separation of natural products6 separates amino acids into basic, neutral, and acidic groups.

  Following extraction, the amino acids are isolated (Figure 6 ) with the basic amino acids in the eluate from column (A). The neutral and acidic amino acids from column (B) are separated by re-chromatography on column (C) leaving the neutral amino acids in the water effluent and the acidic amino acids in the pyridine eluate.

  This group separation technique is based on ion-exchange columns. The methods are, however, not traditional ion-exchange techniques. For the columns (A) and (C), the retained ions are released and eluted by use of an eluent (volatile) which removes the charge on the column materials. For the (B) column, it is the positive net charge on the retained compounds which are removed by the elulent (volatile), and by use of the (A) before the (B) column the ions on the (B) column can be eluted by gentle conditions.

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  Applications

  • Z. Deyl(Author)
  • 2000(Publication Date)
  • Elsevier Science

    (Publisher)

  55 Chapter 7 AMINO ACIDS AND THEIR DERIVATIVES 2. DEYL GENERAL ASPECTS Separation methods f o r amino acids are well established; the Dreferred proce- dures today are chromatographic. However, electrophoretic separations s t i l l pos- sess considerable importance as they can be used e f f e c t i v e l y f o r screening purposes. Equally, electrophoretic methods can help i n the search f o r the r a r e r amino acids t h a t a r i s e during post-translational reactions i n proteins o r t h a t occur as the r e s u l t o f diverse metabolic processes and disorders. Finally, electrophoresis can be used f o r the separation o f amino acid derivatives t h a t are used i n protein sequence analysis. The approach t o the separation o f underivatized amino acids semains olmast.the same, apart from small variations, whether one i s dealing w i t h the classical twenty o r w i t h r a r e l y occurring amino acids. AMINO ACIDS NATURALLY OCCURRING I N PROTEINS Three independent separations i n d i f f e r e n t b u f f e r systems are necessary i n order t o obtain a complete separation o f the n a t u r a l l y occurring amino acids found i n p r o t e i n hydrolysates. This procedure i s sometimes r e f e r r e d t o as unidi- mensional multi-electrophoretic separation . The f i r s t electrophoresis i s car- r i e d o u t a t pH 5.2 in, e.g., pyridine acetate (paper, running time about 200 min a t 75 V/cm). Under these conditions basic amino acids (lysine, h i s t i d i n e , arginine, hydroxylysine and ornithine) move as cations, and a c i d i c amino acids (cysteic acid, aspartic and glutamic acids) move as anions. Neutral amino acids form an unresolved zone t h a t i s displaced about 1.5-2 cm towards the cathode owing t o the endo-osmotic flow. The second electrophoretic run i s c a r r i e d o u t a t pH 1.85 i n acetate-formate b u f f e r (100 V/cm, 270.min). I n t h i s run most o f the neutral amino acids are separated.

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  Biochemistry

  An Integrative Approach with Expanded Topics

  • John T. Tansey(Author)
  • 2022(Publication Date)
  • Wiley

    (Publisher)

  This chapter begins by discussing amino acids, the building blocks of proteins, then moves on to the basics of protein structure and a brief description of how these macromolecules fold into their specific conformations. Several different examples of different proteins are examined next. The chapter then discusses the basic scheme for conducting a purification, isolating a single protein of interest from a crude mixture. The next section discusses size-exclusion chromatography, a common means of achieving that separation, followed by discussion on use of affinity and ion exchange chromatography techniques to achieve separation. These same techniques can also be used to determine many of the properties of proteins and sometimes their functions.

  Chapter Outline

  3.1 Amino Acid Chemistry

  3.2 Proteins Are Polymers of Amino Acids

  3.3 Proteins Are Molecules of Defined Shape and Structure

  3.4 Examples of Protein Structures and Functions

  3.5 Protein Purification Basics

  3.6 Size-Exclusion Chromatography

  3.7 Affinity Chromatography

  Common Themes

  Evolution’s outcomes are conserved.

  •  Mutations to DNA sequences may result in alterations to a protein’s amino acid sequence. The new protein may function normally, have a new and slightly altered function, or be completely nonfunctional. •  The amino acid sequence of proteins can help establish the evolutionary relationship between organisms. •  Protein structures and the amino acids they are made from have undergone billions of years of natural selection and random change (genetic drift). •  Proteins with conserved amino acid sequences or folds may have similar topology or ligand binding; these attributes can guide the development of purification schemes.

  Structure determines function.

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  Thin Layer Chromatography in Chiral Separations and Analysis

  • Teresa Kowalska, Joseph Sherma(Authors)
  • 2007(Publication Date)
  • CRC Press

    (Publisher)

  12   Chiral Separation of Amino Acid Enantiomers Władysław Gołkiewicz and Beata Polak CONTENTS

  • 12.1   Introduction
  • 12.2   Separation of Amino Acid Enantiomers on the Commercial Chiral Plates
  • 12.3   Separation of Amino Acid Enantiomers on the Impregnated Plates with Chiral Reagent
  • 12.4   Separation of Amino Acid Enantiomers on the Commercial Plates with Chiral Reagent in Mobile Phase
  • 12.5   Most Popular Chiral Reagents Used for Derivatization of Amino Acid Enantiomers
  • 12.6   Conclusions
  • References

  12.1   Introduction

  About 20 L -amino acids are the building blocks of proteins, so there is no need to use the chiral chromatographic techniques to resolve such mixtures; there are many different chromatographic techniques that allow to separate mixture of L -amino acids (for a review see [1 ]).

  On the other hand, the optically active amino acids can be converted into a racemic mixture by racemization reaction [2 ]. In reality, racemization reaction, especially when occurring in fossil or teeth [3 ], takes a lot of time, but it has also become established that racemization takes place with higher reaction rates, at natural pH as well as in dilute acid or base [2 ].

  Racemization in the metabolically stable proteins has also been detected [2 ,4 –6 ].

  Supplementation of livestock feeds with certain amino acid enantiomers has generated interest in analyses of D - and L -amino acids. Nutritional studies on utilization of amino acid enantiomers have shown that e.g. D -phenylalanine has better growth-promoting activity for chicks and rats than D -histidine, whereas D -arginine has no growth-promoting activity in either chicks or rats.

  The two enantiomers of a given amino acid have identical chemical and physical properties in a symmetrical environment. To resolve such a pair of amino acid by chromatography, diastereomers must be formed. Diastereomers can be formed if a chiral reagent (selector) is introduced to either the mobile or the stationary phase. In case of thin-layer chromatography (TLC), latter manner has largely been used for resolution of amino acids, their PTH-, dansyl, and other derivatives [2 ,4 –6

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  Introduction to Proteomics

  • Sudheer Awasthi(Author)
  • 2023(Publication Date)
  • Arcler Press

    (Publisher)

  Figure 3.5. Ion-exchange chromatography diagrammatic representation. Source: Wołowicz A, Wawrzkiewicz M. Screening of Ion Exchange Resins for Hazardous Ni(II) Removal from Aqueous Solutions: Kinetic and Equilibrium Batch Adsorption Method. Processes. 2021; 9(2):285. https://doi.org/10.3390/ pr9020285 Along with a variation in the concentration of ions or pH of the elution buffer, the unattached proteins are eliminated first, followed by the bound proteins. 3.3 DETERMINATION OF THE PRIMARY STRUCTURE OF PROTEINS The main frame of a protein is constituted by the chain of amino acids. This governs how a protein folds and acquires the three-dimensional (3D) shape Methodology for Separation and Identification of Proteins and their ... 95 which regulates a protein’s function. Many additional conclusions might be drawn from the amino acid pattern of a protein; for instance, the protein’s (pI) might be derived from its amino acid pattern. In addition, the existence of numerous hydrophobic amino acids in the sequencing suggests as it is either a membrane protein or a receptor protein (Link, 2002; Bergström et al., 2006). Additionally, the existence of particular amino acids may signal that protein will develop a beta-sheet structure. Thus, a protein’s amino acid pattern and fundamental structure are essential. There are three approaches to identifying a protein’s amino acid sequence. Deciphering the nucleotide pattern of a DNA molecule, Edman degradation, and mass spectrometry are examples of such techniques (Issaq et al., 2005; Capriotti et al., 2011). 3.3.1 Proteomics without Spectrometry 3.3.1.1 Determination of Amino acid Sequence from DNA sequence The amino acid structure of a protein expressed by a gene might be deciphered thanks to our knowledge of gene sequences and our capacity to pattern nucleotides in a DNA part. As long as DNA pattern data was accessible in the GenBank database, it formed common practice to infer the amino acid patterns of various proteins.

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  Pharmaceutical and Biomedical Applications of Capillary Electrophoresis

  • S.M. Lunte, D.M. Radzik(Authors)
  • 1996(Publication Date)
  • Pergamon

    (Publisher)

  J. Chromatogr., 538, 393-402. Stegehuis, D. S., U. R. Tjaden and J. van der Greef (1992). J. Chromatogr. 591, 341-349. Swartz, M. E. and M. Merion (1993). J. Chromatogr., 632, 209-213. Thormann, W., A. Minger, S. Molteni, J. Casiavska and P. Gebauer (1992). J. Chromatogr., 593, 275-288. Towns, J. K. and F. E. Regnier (1992). AreaZ. Chem., 64, 2473-2478. Vinther, A. and H. Soeberg (1991). J. Chromatogr., 559, 27-42. Wang, T. and R. A. Hartwick (1992). J. Chromatogr., 594, 325-334. Weinberger, R., E. Sapp and S. Moring (1990). J. Chromatogr., 516, 271-286. Wemly, P. and W. Thormann (1991). Anal. Chem., 63, 2878-2882. CHAPTER 8 Capillary Electrophoresis in the Study of Amino Acids PAUL L. WEBER Department of Chemistry, Briar Cliff College Sioux City, lA 51104 U.SA. 1 . Introduction 1.1, Perspectives on Amino Acid Analysis The large quantity of literature devoted to the development of analytical methods for the separation and quantitation of amino acids, as well as the variety of commercial analyzers and sequencers, attests to the importance of this field. Regardless of the type of instrumentation employed, these methods have been developed to analyze two basic different types of solubilized samples: a) endogenous solutions of amino acid mixtures, often of bio-logical origin, or b) peptide or protein products such as hydrolysates or amino acid derivatives obtained from sequencing reactions like that used in the Edman degradation. Peptide or protein products contain primarily only the 20 common amino acids or their derivatives and thus represent a narrower range of amino acid structures than are encountered in endogenous samples. Note that over 300 additional amino acids not found in proteins have been observed in cells, where they posses a variety of functions. For example, ornithine and citruUine are important meta-bolic intermediates, while y-aminobutyric acid (GABA) is an intensely studied neurotransmitter.

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