Amino Acid Analyser
2000.6.17
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Amino Acid Analyser
Products
Ion exchange chromatography
Application
An example of application
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 (6aminoquinolyl-N-hydrozysuccinimidyl
carbamate, or ACQ) is an Nhydroxysuccinimide-activated
hetrocyclic carbamate, a new class of
amine-derivatizing compounds.
Waters
Chromatography



Open Column Chromatography
Thin Layer Chromatography
Liquid Chromatography
– Ion exchange chromatography
 Amino

acid analyer
High Performance Liquid Chromatography
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 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 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 존
재한다.
 따라서 아미노산은 한 분자내에 양
이온과 음이온을 동시에 가지지만
전체적으로는 중성인 성질을 띠게
된다.

이온교환크로마토그래피

아미노산이나 다른 용질의 분리에는 종종 이온교환 크로마
토그래피를 이용하는데, 이때 대상분자는 전하를 띤 고체
지지체의 다른 이온과 교환되었다가 다시 떨어져나온다.

전형적인 과정에서는 액체상에 녹아있는 용질을 다공성 고
체상이 채워진 관을 통과시킨다.

액체상으로는 물이 보통 쓰이며, 고체상은 하전된 작용기
를 가진 합성수지 입자로 된 층이다.
Ion chromatograph - Principles of separation

Fig. illustrates the principle of retention by exchange of anions in anionexchange chromatography,
Ion exchange chromatography의 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), 음
전하를 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
Sulfonic acid -SO3- H+
Carboxylic acid -COO- H+
Phosphonic acid PO3- H+
Phosphinic acid HPO2- H+
Phenolic -O- H+
Arsonic -HAsO3- H+
Selenonic -SeO3- H+
Anion exchanger
Quaternary amine -N(CH3)3+ OHQuaternary amine - N(CH3)2(EtOH)+OHTertiary amine -NH(CH3)2+ OHSecondary amine -NH2(CH3)2+ OHPrimary amine -NH3+ OH-
Stationary phases of IC
Functional groups

Strong anion-exchanger(SAX)
– quaternary amine functional groups
 Strong cation exchanger(SCX)
– Sulfonic acid
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

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 있
다.

닌히드린(혹는 트리케토히드린덴 수화물)은 매우 강한 산화제로서, 알
파 아미노 작용기의 산화적 탈아민화를 일으킨다.

이 반응의 생성물은 알데히드, 암모니아, 이산화탄소, 그리고 닌히드
린의 환원된 유도체인 히드린단틴(hydrindantin)이다.

이때 생성되는 암모니아는 히드린단틴과 또다른 닌히드린 분자와 반
응하여 570nm에서 분광학적으로 정량할 수 있는 보라색 생성물 루헤
만 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.