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Amino Acid Analyser

2000.6.17 석사3기 박주영. Amino Acid Analyser. Amino Acid Analyser Products Ion exchange chromatography Application An example of application. Amino Acid Analyser. AMINO ACID ANALYSER (AAA)

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Amino Acid Analyser

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  1. 2000.6.17 석사3기 박주영 Amino Acid Analyser Amino Acid Analyser Products Ion exchange chromatography Application An example of application

  2. Amino Acid Analyser • AMINO ACID ANALYSER (AAA) • Amino Acid Analyser is a specifically configured system optimised for the analysis of free amino acids. • PRINCIPLE • The system utilises ion-exchange chromatography incorporating post column reaction with ninhydrin and subsequent detection in the visible region spectrum.

  3. Amino Acid Analyser • Biochrom

  4. AAA: AccQ Tag • The AccQTag Method is based on a derivatizing reagent developed specifically for amino acid analysis. Waters AccQFluor Reagent (6-aminoquinolyl-N-hydrozysuccinimidyl carbamate, or ACQ) is an N-hydroxysuccinimide-activated hetrocyclic carbamate, a new class of amine-derivatizing compounds. Waters

  5. Chromatography • Open Column Chromatography • Thin Layer Chromatography • Liquid Chromatography • Ion exchange chromatography • Amino acid analyer • High Performance Liquid Chromatography

  6. Protein analysis Determination of Amino Acid Composition of Proteins 1) break down the polypeptide chain into its constituent amino acids , • hydrolysis (boiling in 6 N HCl) 2) separate the resulting free amino acids according to type, 3) measure the quantities of each amino acid. • chromatography

  7. Protein analysis Methods of protein purification • Differential centrifugation • Diferential precipitaion by soulbility difference • Column procedures • Preparative gel-exclusion chromatography • Column chromatography with protein binding • Ion-exchange chromatography • Affinity chromatography • High-performance Liquid Chromatography • Electrophoretic methods

  8. Protein analysis Determination of Amino Acid Sequence of Proteins • Sequenators (Edman degradation) phenylisothiocyanate + tetrapeptide - phenylthiocarbamoyltetrapeptide mild hydrolysis phenylthiohydantion derivative of NH2-terminal amino acid + original peptide minus NH2-terminal residue

  9. Ion exchange chromatography - 아미노산의 전기적성질 • 중성용액(pH7)에서 카르복시기는 -COO-로, 아미노기는 -NH3+ 로 존재한다. • 따라서 아미노산은 한 분자내에 양이온과 음이온을 동시에 가지지만 전체적으로는 중성인 성질을 띠게 된다.

  10. 이온교환크로마토그래피 • 아미노산이나 다른 용질의 분리에는 종종 이온교환 크로마토그래피를 이용하는데, 이때 대상분자는 전하를 띤 고체지지체의 다른 이온과 교환되었다가 다시 떨어져나온다. • 전형적인 과정에서는 액체상에 녹아있는 용질을 다공성 고체상이 채워진 관을 통과시킨다. • 액체상으로는 물이 보통 쓰이며, 고체상은 하전된 작용기를 가진 합성수지 입자로 된 층이다.

  11. Ion chromatograph -Principles of separation • Fig. illustrates the principle of retention by exchange of anions in anion-exchange chromatography,

  12. Ion exchange chromatography의 구성 • Instrumentation • pump • injector • detector • data station • Chemistry components • stationary phase • mobile phase

  13. Instrumentation of IC • Ion Chromatograph(IC), Dionex

  14. Instrumentation of IC AS40 Automated Sampler GS50 Pumps AutoSelect AS50 Autosampler EO1 Eluent Organizer EG40 Eluent Generator

  15. Instrumentation of IC • ED50 Electrochemical Detector • CD25 Conductivity Detector • PDA-100 Photodiode Array Detector • AD25 Absorbance Detector

  16. Ion chromatograph - System 구성

  17. Stationary phases of IC • 양전하를 가진 수지를 음이온교환체(anion exchanger), 음전하를 가진 고체 지지체는 양이온교환제(cation exchanger)라 한다 • Ion-exchangers are charaterized both by the nature of the matrix used as a support and the nature of the ionic functional groups on the surface.

  18. Stationary phases of IC Functional groups Cation Exchanger Anion exchanger Sulfonic acid -SO3- H+ Quaternary amine -N(CH3)3+ OH- Carboxylic acid -COO- H+ Quaternary amine - N(CH3)2(EtOH)+OH- Phosphonic acid PO3- H+ Tertiary amine -NH(CH3)2+ OH- Phosphinic acid HPO2- H+ Secondary amine -NH2(CH3)2+ OH- Phenolic -O- H+ Primary amine -NH3+ OH- Arsonic -HAsO3- H+ Selenonic -SeO3- H+

  19. Stationary phases of IC Functional groups • Strong anion-exchanger(SAX) • quaternary amine functional groups • Strong cation exchanger(SCX) • Sulfonic acid

  20. Stationary phases of IC Matrixes 1. Silica-based • better chromatographic efficiency, stability and durability in high pressure • limited pH range : 2< pH <7 2. Polymer-based • chemically derivatization of synthetic organic polymers • most widely used types of ion-exchaners • subject to pressure limitations • tolerance towards eluents and samples with extreme pH, between 0-14.

  21. Stationary phase for AAA • 양이온 교환매질 • 강산성, 폴리스티렌 수지(도웩스-50) • 약산성, 카르복시메틸렐룰로오스(CMC) • 약산성, 킬레이트화, 폴리스티렌수지(켈렉스-100) • 음이온 교환매질 • 강염기성, 폴리스티렌 수지(도웩스-1) • 약염기성, 디에틸아미노에틸(DEAE) 셀룰로오스

  22. Mobile phases of IC Properties of Mobile phases • compatibility with the detection mode • nature of the competing ion • concentration of the competing ion • mobiles phase’s pH • buffering capacity of the monile phase • ability to complex the ionic sample components • organic modifiers

  23. Mobile phases of IC Eluents for Anions • Aromatic carboxylic acids and their salts • mostly widely employed eluent • low conductances • ex) lithium hydroxide • Aliphatic carboxylic acid • Aromatic and aliphatic sulfonic acids • Potassium hydroxide • Polyol-borate complexes • Ethylenediaminetetraacetic acid -EDTA • Inorganic salts such as Cl-, SO42- or PO43-

  24. Mobile phases of IC Eluents for Cations • Inorganic acids such as nitric acid • Organic bases

  25. Ion chromatograph -Detection • Conductivity detection • Difference in the ionic conductance • Electrochemical detection • Electric oxidation-reduction potential • Spectroscopic detection • UV-VIS Absorption • Fluoescence • Refractive Index(RI) • combication with various forms of atomic spectrometrysuch as ICP MS

  26. Detection using ninhydrin for AAA • 아미노산은 닌히드린과의 반응을 통해서 쉽게 확인되고 정량될 수 있다. • 닌히드린(혹는 트리케토히드린덴 수화물)은 매우 강한 산화제로서, 알파 아미노 작용기의 산화적 탈아민화를 일으킨다. • 이 반응의 생성물은 알데히드, 암모니아, 이산화탄소, 그리고 닌히드린의 환원된 유도체인 히드린단틴(hydrindantin)이다. • 이때 생성되는 암모니아는 히드린단틴과 또다른 닌히드린 분자와 반응하여 570nm에서 분광학적으로 정량할 수 있는 보라색 생성물 루헤만 보라(Rhhemann’s Purple)를 형성한다.

  27. Amino Acid Analyser (Beckman model) • Detector • Wavelength range: 190-700 nm • Max. Sensitivity : 0.001 Absorbance units full scale(AUFS) • Autosamper : up to 80 samples • Possible range of flow rates : 0.01-10 ml/min • Sample • nature: liquid • size: 1-2 ml

  28. Application of Amino Acid Analyser • Physiological sample (blood, urine, CSF...) • Duran M., Dorland L., De Bree P.K., and Berger R. Selective screening for amino acid disorders. European Journal of Pediatrics. 1994; 153(1): S33-S37. • Food • M. Ghojale and M.Sayhoon. Comparative assessment of irradiated proteins in potato tuber with untreated control by High Performance Liquid Chromatography (HPLC) and gel electrophoresis. Radiation Physics and Chemistry. 1995 October-December; 46: 4-6.

  29. Application of Amino Acid Analyser • Molecular study • Wu T, Wu Y, Yuan Y, He H. and Zhang G. Study on amino aicd compositon of HSP70 and the level of plasma free amino acids of workers with long-term exposure to harmful factors. J Tongji Med Univ 1998; 18(4): 204-7. • 기타 • J. Csapo and Zs. Csapo-Kiss. Use of amino acids and their racemisation for age determination in archaeometry. Trends in Analytical Chemistry 1998; 17(3): 140-148.

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