amino acids analysis

8/6/2019 Amino Acids Analysis

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a a mu ara an man g am


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Amino Acids Analyzer

Is an instrument used to determine the amino acidcontent in protein or peptide sample, it is also used forthe determination of amino acid content in

.


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Amino acid analyzer applications :

1. Screening tests for amino acid (analysis of amino acidsin physiological fluids such as plasma and urine)

.

each amino acid and summing the values3. To quantify the amount of each amino acid in food

stuffs (useful for measuring essential amino acid)

4. Specific tests to detect atypical amino acids that mightbe present in a protein or peptide


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Objective of amino acid analysis

To analyze amino acid content in a protein or peptidesample.

o ana yze t e ree am no ac content nphysiological fluids or food stuff samples


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Sample Preparation

a. Protein and peptide sample Step one (hydrolysis step)

It is necessary to hydrolyze the protein/peptide to its

individual amino acid constituents before amino acidanalysis.

Acid hydrolysis is the most common method forhydrolyzing a protein sample before amino acid analysis(6N HCl at 110 C for 24 h under vacuum.


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Sample Preparation

b. Physiological fluid sample As the amino acids already exist in their free state, apurification step is required to remove all protein and

e tide de roteinization which would otherwise

irreversibly bind to the ion exchange resins. Deproteinization methods include:

1. Acid precipitation (5-sulphosalicyilic

acid, TCA)2. Ultra filtration3. Ultracentrifugation


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Amino Acid Analysis. automated method to

determine the amino acid content

derivatize w/ninhydrinSeparation ofindividualamino acids

Ion exchangechromatography chromophores

7

Detected w/UV-vis

Different aminoacids have differentchromatographicmobilities (retentiontimes)


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How the analyzer work

The sample will go through 4 steps inside the aminoacid analyzer

Autosamplerseparation column reaction

coil reactionphotometer


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Autosampler and Calibration procedure


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Amino acid standards are commercially available foramino acid analysis and typically consist of an aqueous

m x ure o am no ac s Calibration of amino acid analysis instrumentation

typically involves analyzing the amino acid standard,which consists of a mixture of amino acids at a number ofconcentrations.

The concentration of each amino acid in the standard isknown


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The analyst dilutes the amino acid standard to severaldifferent levels within the expected linear range of theamino acid analysis technique.

Peak areas obtained for each amino acid are plotted versusthe known concentration for each of the amino acids inthe standard dilution


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AA are ampholyte

R R

RCHCO2H

NH2NaOHHCL

Anion, when athigh pH zwitterion, when at

isoelectric point (pI)

Cation, when atlow pH

NH2 CH COO-OH

- NH3+ CH COO-H

+

OH-RCHCO2

-

N+H3

H

NOTE: peptide or protein also have both acid and base properties. Theyshare the same property of being positively charged at low pH andnegatively charged at high pH.


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Isolectric Point (pI) of AAs

RCHCO2H

NH2

+

+

+

--

--

anode cathode

Anion in

basic soln.

RCHCO2-

N+H3

H+

RCHCO2H

N+H3

HO-

RCHCO2-

NH2

+

+

+

+

+

-

-

-

-

-

-

Zwitterion in

pI solution. Nomove to either

of Electrode.

And with Lowest

solubility

Cation in

acidic soln.

pH < pIpH > pI


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Separation column reaction

The bead size, and degree of cross-linkage play arole in the separation power of the resin

m xture o aa s oa e onto t e co umn

Buffers of varying pH and varying ionic strength are

pumped sequentially through the column to effectseparation of the aa


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Separation columnreaction Na

+Cl

-

Amino acids are zwitterions; at low pH, theyare positively-charged and are bound to theresin by their attraction to the negatively-charged ion-exchange sites.

The amino acids are then selectively eluted byincreasing the pH and salt concentration withdifferent buffers.

With few exceptions, the order of elutionfollows the isoelectric point of the aminoacids, i.e. acidic amino acids first, then neutral

and basic.


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SO3-

N a+

C O O H

H 3 N+

pH 2

Ion-exchange Resin

SO3-

N a+

C O O-

H 3 N+

pH4.5


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Separation column reaction

A temperature gradient is often employed to enhance

The column temperature can be varied during theanalysis in order to effect the desired separation


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Detection The column effluent is mixed with ninhydrin solution, then the

resultant mixture is pumped through a high temperature reaction

coil When the amino acid present in the elute reacts with ninhydrin,

the reactant has characteristic purple or yellow color.

t e quant ty o t e co ore comp ex pro uce s rect y proportional to the concentration of the particular aa present inthe original mixture

Amino acids, except Proline & hydroxyproline, give a purple color,

and show the maximum absorption at 570 nm. proline give a yellow color, and show the maximum absorption at

440 nm


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Ninhydrin ReactionRCHCOOH

NH2

O

O

OH

OH

+

O

O

N

O

O

+ RCHO + CO2 + 3H2O

Note:

1, Ninhydrin solution is made in basic solution of phosphate buffer (pH8.04).

2, The reaction products of all AAs except proline, are purpule-bule(540nm), and for

proline is yellow(440nm)

3, protein and peptide also can occur this reaction due to their containing amino

group.4. This reaction can be used to quantitative and qualitative analysis, with the help of

spectrophotometer, TLC.


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Ninhydrin Reaction Ninhydrin degrades amino acids into aldehydes, ammonia,

and CO2 through a series of reactions; the net result isninhydrin in a partially reduced form hydrindantin:

Ninhydrin then condenses with ammonia and hydrindantin toproduce an intensely blue or purple pigment, sometimes called

Ruhemann's purple:


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Photometer After the ninhydrin, the product (the reaction

complex) pass through a two channel photometer where the absorption of the colored complex is


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Recorder Photometer is linked to a two channel recorder where

a series of peaks representing the a.a are recorded

e pos t ons o t e pea s on t e c art represents t etotal retention times for the amino acid which givesthem a positive identification

Measuring the area under each peak gives quantitativeinformation on the amount of each a.a.


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Column regeneration Once the final amino acid is eluted the column should

be regenerated.

The regeneration solution is passed through the-

remove any amino acid left in column


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Calculation1. For quantitative analysis a standard run with a.a of

known concentration must be done first

2. The sample run.

3.

e area o t e pea o eac a.a n t e stan ar anthe sample is calculated by area = H x W

Where H is the height of the peak

W is the width at the height

H

WH/2


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Calculation4. The color constant K of each a.a in the standard is =

Area/ a.a concentration

= rea a.a concentrat on

Then the concentration for the unknown concentration

sample of the same a.a =

Concentration of a.a = Area/ K of the standard


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limitations of the method1- limitations of the acid hydrolysis step

a) Glutamine and Asparagine are completely converted to theircorresponding acids during acid hydrolysis.

b) Tr to han Is destro ed durin the standard acid h drol sis

(Tryptophan is stable in alkali, so samples which require tryptophandetermination are hydrolysed in barium hydroxide at 110C for 24 hoursunder vacuum

c) Cysteine/Cystine are often partially destroyed during

standard acid hydrolysis. They must be converted to a morestable form, usually cysteic acid, before standard acidhydrolysis. (Cysteine is usually converted to the acid stable cysteic acidbefore standard acid hydrolysis)


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limitations of the method 2- The D and L isomers of the amino acids cannot be

separated by this method.


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Points to be considered whensupplying samples for analysis1- Glycine buffers should never be used during ..the work

up of proteins that have to be amino acid analysed.Glycine is very difficult to remove afterwards, with

glycine.

2- Ammonium salts must be avoided in ..the last stage of

purification as large amounts of ammonia will causethe analysis reagent to precipitate out in the reactioncoil and cause an instrument failure


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Points to be considered whensupplying samples for analysis3- Glycerol and mannitol interfere with the acid

hydrolysis process and severe losses of some aminoacids are observed. They must be dialysed away before

4- Samples with a high salt content can ..adversely affectthe ion exchange column and can cause an instrumentfailure.


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General precautions1- Place the instrument in a low traffic area in the laboratory2- High purity reagent are necessary (low purity HCl can contribute

to glycine contamination)3- Analytical reagent are changed every few weeks

- tential micr bial c ntaminati n and f rei n material that

might be present in the solvent are reduced by filtering thesolvents.5-keep pipette tips in a covered box, the analyst may not handle

pipette tips by hands, the analyst may wear powder free latex

gloves (dust can contribute to elevated Glycine, Serine, andAlanine)5-The amino acid analyzer instrument and reagent should be kept

away from direct sun light (sun light causes enhanced bacterial

growth)

amino acids analysis

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