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Methods to determinate a.a.

R. of reactive groups:
a)General: amino- and carboxyl groups
b)Specific: other groups

a)R. of amino groups
-Ninhydrin R.
-Formol titrations
-Deamination of a.a. w. FDNB
-Danzyl reaction

b)Detection of other reactive groups
-Identification of sulphydryl groups
-Million´s R for detection of tyrosine


Ninhydrin R.
-What does ninhydrin do?

-Deamines alpha-a.a. by condensation
-Purple, with proline yellow. Collidine-ninhydrin gives various colours w. diff. a.a.
-Chromotograph, ninhydrin sol., hairdryer, vaporizer


Formol titration
-What does the formaldehyde do?
-How can the a.a. conc. be calculated?

-R. w. amino groups and form methylol compounds
-H+ obtained in the R. can be titrated.
-A.a. sol, formaldehyde, NaOH, Phenolphtalein indicator


Deamination of a.a. w. FDNB
-FDNB stands for?
-What does FDNB do?
-How can we determine the Rf?

-R. w. N-term. of a.a. and form DNP-a.a.
-Alkaline milieu, room temp.
-Yellow colour
-With chromotography
-Resistant to acidic hydrolysis


All labtests?

-Ninhydrin R.
-Formol titration
-Identification of sulphydryl groups


Danzyl reaction
-What does the danzyl-reagent do?
-How can the prod. be visualized?

-R. w. N-term. of a.a. and form danzyl-a.a.
-Alkaline milieu
-With flueroscence
-Resistant to acidic hydrolysis


Identification of sulphydryl groups
-Which a.a.?

-Alkaline milieu
-Two cysteine bound together form cystine, two H+ released reduced Fe3+ to Fe2+.
-Purple (free -SH with Fe3+) -> colourless (Fe2+)
-Cysteine sol., Na2CO3, Fe2(SO4)3


Million´s R for detection of tyrosine

-Comp. cont. p-hydroxyphenyl groups R. with mercuric salts in nitric acid forming red complex
-Diluted blood serum, tyrosine, glycine, Million´s reagent


Hopkins-Cole´s R. for detection of tryptophan

-Tryptophan forms a violet condens. prod. w. aldehydes in alkaline milieu
-Milk(tryptophan), acetic acid, sulphuric acid


Sep. of a.a. from their mixture

1.Thin layer chomotography
2.Ion exchange chomotography


Thin layer chromatography
-Support media?
-Which flow fastest?
-How is the a.a. faciliated in the sep.?
-How is the components visualized?
-How is the retention factor measured?

-Cellulose, silicagel, alumina oxide layer on glass/plastic
-Princ.: Solubility of diff. a.a. depends on solvent
-Fastest: Apolar solvents->furthest away from start line
-Faciliated by the fact they have diff. affinity to the medium. Apolar a.a. binds with lower strength.
-Visualized by staining R.-ninhydrin R.
-Rf=dist. moved by solvent/dist. moved by a.a.(Rf


Ion exchange chromatography
-Support media?
-What does the strength of the bond depend on?
-How can the a.a. be eluted?

-Diff. synthetic resins cont. diff. ionized groups
-A medium w. neg.charged group binds to a.a.-cations. (DEAE-cellulose, w. SO3^-).
-Depends on pH and dissociation properties of a.a.
-Eluted by: gradually increasing pH.