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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
2000.6.17

석사3기

박주영

Amino Acid Analyser

Amino Acid Analyser

Products

Ion exchange chromatography

Application

An example of application

amino acid analyser2
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.
aaa accq tag
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

chromatography
Chromatography
  • Open Column Chromatography
  • Thin Layer Chromatography
  • Liquid Chromatography
    • Ion exchange chromatography
      • Amino acid analyer
  • High Performance Liquid Chromatography
protein analysis
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
protein analysis7
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
protein analysis8
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

ion exchange chromatography
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.
slide10
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.
ion chromatograph principles of separation
Ion chromatograph -Principles of separation
  • Fig. illustrates the principle of retention by exchange of anions in anion-exchange chromatography,
ion exchange chromatography12
Ion exchange chromatography의 구성
  • Instrumentation
    • pump
    • injector
    • detector
    • data station
  • Chemistry components
    • stationary phase
    • mobile phase
instrumentation of ic
Instrumentation of IC
  • Ion Chromatograph(IC), Dionex
instrumentation of ic14
Instrumentation of IC

AS40 Automated

Sampler

GS50 Pumps

AutoSelect AS50 Autosampler

EO1 Eluent

Organizer EG40 Eluent Generator

instrumentation of ic15
Instrumentation of IC
  • ED50 Electrochemical Detector
  • CD25 Conductivity Detector
  • PDA-100 Photodiode Array Detector
  • AD25 Absorbance Detector
stationary phases of ic
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.
stationary phases of ic18
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+

stationary phases of ic19
Stationary phases of IC

Functional groups

  • Strong anion-exchanger(SAX)
    • quaternary amine functional groups
  • Strong cation exchanger(SCX)
    • Sulfonic acid
stationary phases of ic20
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.
stationary phase for aaa
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
mobile phases of ic
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
mobile phases of ic23
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-
mobile phases of ic24
Mobile phases of IC

Eluents for Cations

  • Inorganic acids such as nitric acid
  • Organic bases
ion chromatograph detection
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
detection using ninhydrin for aaa
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.
amino acid analyser beckman model
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
application of amino acid analyser
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.
application of amino acid analyser29
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.