Amino Acid Analyser

Amino Acid Analyser

Products

Ion exchange chromatography

Application

An example of application

2000.6.17

석사 3 기박주영

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.

Amino Acid Analyser

Biochrom

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

Open Column Chromatography Thin Layer Chromatography Liquid Chromatography

– Ion exchange chromatographyAmino acid analyer

High Performance Liquid Chromatography

Protein analysis

Determination of

Amino Acid Composition of Proteins1) 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 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 chromatographyAffinity chromatography

– High-performance Liquid Chromatography Electrophoretic methods

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

- 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 .

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

Fig. illustrates the principle of retention by exchange of anions in anion-exchange chromatography,

Ion exchange chromatography 의 구성

Instrumentation– pump– injector– detector– data station

Chemistry components– stationary phase– mobile phase

Instrumentation of IC

Ion Chromatograph(IC), Dionex

Instrumentation of IC

AS40 Automated

Sampler

GS50 Pumps

AutoSelect AS50 Autosampler

EO1 Eluent

Organizer EG40 Eluent Generator

Instrumentation of IC

ED50 Electrochemical Detector

CD25 Conductivity Detector

PDA-100 Photodiode Array Detector

AD25 Absorbance Detector

Ion chromatograph - System 구성

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 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 IC

Functional groups Strong anion-exchanger(SAX)

– quaternary amine functional groups Strong cation exchanger(SCX)

– Sulfonic acid

Stationary phases of IC

Matrixes1. 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

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

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 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 IC

Eluents for Cations Inorganic acids such as nitric acid Organic bases

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 spectrometry

such as ICP MS

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 570nm 에서 분광학적으로 정량할 수 있는 보라색 생성물 루헤만 보라 (Rhhemann’s Purple) 를 형성한다 .

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

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 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.