Sasse: Methods of Resolution, Chemical Methods used in Analysis and Identification, Amino Acid Analysis

3 methods of resolution

physical

chemical

biological

physical

fractional crystallisation

mechanical separation of R/S crystals

chromatographic separation

fractional crystallisation

crystal of pure S enantiomer is added into saturated solution

fractional crystallisation induced

S enantiomers crystallise, R stay in solution

filter

chromatographic separation

create a chiral solid support column

enantiomers form diastereomeric complexes with the chiral support

different elution times for pure enantiomers

what does the chiral solid support column consist of

optically active stationary support in which the the 2 enantiomers interact with differently

chemical methods

formation of diastereomeric salts

describe how an amino acid ionizes itself

carboxylic acid reacts with amino group and protonates it

can't do much with this molecule

what are the main steps in the formation of diastereomeric salts

1. protect amine group by forming amide

2. treatment of optically active amine

3. separation of salts by fractional crystallisation

4. remove brucine

5. hydrolyse/ deprotect amino acid

how do we protect the amine group

reacting it with another acid

what is the result of the protection of the amine group

nitrogen of the amide cannot grab a hydrogen from carbox acid

carbox acid is now available to form a solute with an organic base

biological

acylase cleaves any amino acid that is acylated but only S enantiommer

R remains unreacted

separation is easy due to different chemical properties

5 methods used in analysis and identification

metal complex salt formation

reactions with alpha amino acids with carbonyl compounds

reaction with 2,4-dinitrofluorobenzene

reaction with ninhydrin

peptide bond formation

metal complex formation

amino acids can form complexed due to presence of amino and carbox acid group

Get a ratio of 1 (metal ion) : 2 (amino acids)

Gives a colour

Then do UV spectroscopy

can you titrate amino acid

no due to their zwitterionic nature

carbox acid protonates the amino group

amino group = v weak acid

carbox acid group = v weak base

so in acidic aqueous medium we cannot quantitatively protonate or deprotonate

what should i do if i want to titrate in amino acids

use a solution other than water i.e. ethanol

-> acid gets more acidic and base gets more basic

reaction of a-amino acids with carbonyl compounds

- formol titration

titrate potassium oxide in the presence of formaldehyde

formaldehyde reacts with amino group -> imminium ion

imminium ion is much more acidic

titration now possible

reaction with sanger reagent

make a derivative of amino acid using sanger reagent

nucleophilic aromatic substitution of the fluorine and amino -> yellow product with aromatic ring which can be identified using HPLC

quantitation by UV

when is the reaction with sanger reagent used

sequencing of amino acids in peptides and proteins

reaction with ninhydrin

react amino acid with ninhydrin reagent under heated conditions

reagent grabs N of the amino acid

product has a lamina max of 570 - absorbs yellow light which appears purple to us

-> used as a spraying agent or in a test tube

exception to the purple colour seen in ninhydrin

proline

-> has a cyclic structure where the amino group is secondary and therefore cant give up N

lamina max - 440, yellow colour

identification tests of phenylalanine

optical rotation - check if we have pure enantiomer

IR

TLC with ninhydrin as spraying agent

purity tests for phenylalanine

optical rotation

amino acid analysis

amino acid analysis

methodology used to determine amino acid composition or content of protein, peptide and other pharmaceutical preparations

what can amino acid analysis be used for

quantify proteins and peptides

determine identity of protein or peptide

support protein and peptide structure analysis

evaluate fragmentation strategies

detect atypical amino acids

how can we separate different amino acids without using TLC

ion exchange chromatography

followed by detection with ninhydrin

2 types of cation exchange systems used for amino acids

lithium - complex aa

sodium - simple aa -easy, quick, cheap

how are the amino acids separated in ion exchange

combination of changes in pH and cation strength

temp gradient also used

2 types of derivatisation

pre column

post column

post column

amino acids into separation column - elute at different times

flushed through derivatisation fluid - ninhydrin/ OPA

post column advantages

good reproducibility, good quantification, can use samples that contain small amounts of buffer components because they are filtered out in the beginning

disadvantages

need quite a lot of protein

use a lot of reagent

cannot use HPLC

only 2 reagents

pre column derivatisation

derivatisation occurs before it is put into the column

lots of different reagents can be used

why can ninhydrin not be used in pre column derivatisation

if u react amino acids with ninhydrin u get one product which is the same for all aa

stick into separation column - all elute at the same time

pre column advantages

v sensitive

consumes less reagent

simple instrument configuration

lots of reagents

can use HPLC

Disadvantage

can be influenced by buffers and salts

can be unstable