Haifa Almubarak and Amani Alghamdi

Amino Acids Analyzer

Is an instrument used to determine the amino acid content in protein or peptide sample, it is also used for the determination of amino acid content in physiological fluid and food stuff.

Amino acid analyzer applications :

Screening tests for amino acid (analysis of amino acids in physiological fluids such as plasma and urine) 2. To measure protein content of food stuffs by quantifying each amino acid and summing the values 3. 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 might be present in a protein or peptide
1.

Objective of amino acid analysis

To analyze amino acid content in a protein or peptide sample. To analyze the free amino acid content in physiological fluids or food stuff samples

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

Sample Preparation
b. Physiological fluid sample As the amino acids already exist in their free state, a purification step is required to remove all protein and peptide (deproteinization) which would otherwise irreversibly bind to the ion exchange resins. Deproteinization methods include: 1. Acid precipitation (5-sulphosalicyilic acid, TCA) 2. Ultra filtration 3. Ultracentrifugation

Amino Acid Analysis. automated method to determine the amino acid content
Ion exchange chromatography

Separation of individual amino acids

derivatize w/ ninhydrin

chromophores

Detected w/ UV-vis

Different amino acids have different chromatographic mobilities (retention times)

7

How the analyzer work

The sample will go through 4 steps inside the amino acid analyzer

Autosampler separation column reaction coil reaction photometer

Autosampler and Calibration procedure

Autosampler and Calibration procedure

Amino acid standards are commercially available for amino acid analysis and typically consist of an aqueous mixture of amino acids Calibration of amino acid analysis instrumentation typically involves analyzing the amino acid standard, which consists of a mixture of amino acids at a number of concentrations. The concentration of each amino acid in the standard is known

Autosampler and Calibration procedure

The analyst dilutes the amino acid standard to several different levels within the expected linear range of the amino acid analysis technique. Peak areas obtained for each amino acid are plotted versus the known concentration for each of the amino acids in the standard dilution

AA are ampholyte
RCHCO2H
NaOH

NH2

HCL

R
NH2 CH COO

H OH

RCHCO2 N H3
+

+ -

R + NH3 CH COOH
Cation, when at low pH

OH

Anion, when at high pH

zwitterion, when at isoelectric point (pI)

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

Isolectric Point (pI) of AAs

anode + + + + + + + + + cathode

RCHCO2H NH2
HOH+ RCHCO2H N+H3

RCHCO2 NH2

RCHCO2 N+H3

Anion in basic soln.

pH > pI

Zwitterion in pI solution. No move to either of Electrode. And with Lowest solubility

Cation in acidic soln.

pH < pI

Separation column reaction

The bead size, and degree of cross-linkage play a role in the separation power of the resin A mixture of aa is loaded onto the column Buffers of varying pH and varying ionic strength are pumped sequentially through the column to effect separation of the aa

Separation column reaction

Na Cl

+ -

Ion-exchange chromatography (IEC) with postcolumn ninhydrin detection is one of the most common methods employed for quantitative amino acid analysis Makes use of a mobile phase (fluid - usually buffer) & a stationary phase (usually small beads). The separation of a.a is done on a cation exchange column. The resin on which the IEC take place consists of small spherical beads of a polystyrene-divinylbenzene to achieve the required degree of cross- linkage

Separation column reaction

Na Cl

+ -

Amino acids are zwitterions; at low pH, they are positively-charged and are bound to the resin by their attraction to the negativelycharged ion-exchange sites. The amino acids are then selectively eluted by increasing the pH and salt concentration with different buffers. With few exceptions, the order of elution follows the isoelectric point of the amino acids, i.e. acidic amino acids first, then neutral and basic.

pH 2
+ +

SO3

Na

H 3N

COOH Ion-exch ange R esin

SO3

H 3N Na
+

COO

pH 4.5

Separation column reaction

A temperature gradient is often employed to enhance separation The column temperature can be varied during the analysis in order to effect the desired separation

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. the quantity of the colored complex produced is directly proportional to the concentration of the particular aa present in the 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

O OH OH O

Ninhydrin Reaction
O O + RCHCOOH NH2 O N 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.

Ninhydrin Reaction
Ninhydrin degrades amino acids into aldehydes, ammonia, and CO2 through a series of reactions; the net result is ninhydrin in a partially reduced form hydrindantin:

Ninhydrin then condenses with ammonia and hydrindantin to produce an intensely blue or purple pigment, sometimes called Ruhemann's purple:

Photometer
After the ninhydrin, the product (the reaction complex) pass through a two channel photometer where the absorption of the colored complex is measured at two wavelength length 570 and 440 nm

Recorder
Photometer is linked to a two channel recorder where a series of peaks representing the a.a are recorded The positions of the peaks on the chart represents the total retention times for the amino acid which gives them a positive identification Measuring the area under each peak gives quantitative information on the amount of each a.a.

Column regeneration
Once the final amino acid is eluted the column should be regenerated. The regeneration solution is passed through the cation-exchange column to regenerate the column and remove any amino acid left in column

Calculation
For quantitative analysis a standard run with a.a of known concentration must be done first 2. The sample run. 3. The area of the peak of each a.a in the standard and the sample is calculated by area = H x W Where H is the height of the peak W is the width at the height
1.
H/2 H W

Calculation
4. The color constant K of each a.a in the standard is = Area/ a.a concentration K= Area/ a.a concentration Then the concentration for the unknown concentration sample of the same a.a = Concentration of a.a = Area/ K of the standard

limitations of the method

1- limitations of the acid hydrolysis step
a) Glutamine and Asparagine are completely converted to their

corresponding acids during acid hydrolysis. b) Tryptophan Is destroyed during the standard acid hydrolysis
is stable in alkali, so samples which require tryptophan determination are hydrolysed in barium hydroxide at 110C for 24 hours under vacuum

(Tryptophan

c) Cysteine/Cystine

are often partially destroyed during standard acid hydrolysis. They must be converted to a more stable form, usually cysteic acid, before standard acid hydrolysis. (Cysteine is usually converted to the acid stable cysteic acid

before standard acid hydrolysis)

limitations of the method

2- The D and L isomers of the amino acids cannot be separated by this method.

Points to be considered when supplying samples for analysis

1- 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 obvious effect on the accuracy of the measurement of glycine. 2- Ammonium salts must be avoided in ..the last stage of purification as large amounts of ammonia will cause the analysis reagent to precipitate out in the reaction coil and cause an instrument failure

Points to be considered when supplying samples for analysis

3- Glycerol and mannitol interfere with the acid hydrolysis process and severe losses of some amino acids are observed. They must be dialysed away before acid hydrolysis can take place

4- Samples with a high salt content can ..adversely affect the ion exchange column and can cause an instrument failure.

General precautions
1- Place the instrument in a low traffic area in the laboratory 2- High purity reagent are necessary (low purity HCl can contribute to glycine contamination) 3- Analytical reagent are changed every few weeks 4-potential microbial contamination and foreign material that might be present in the solvent are reduced by filtering the solvents. 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, and Alanine) 5-The amino acid analyzer instrument and reagent should be kept away from direct sun light (sun light causes enhanced bacterial growth)