Derivatization

What is derivatization?

The chemical modification of an existing compound to produce a new compound having properties that are suitable for a specific analytical procedure.

What are the reasons for derivatizing substances?

1. To impart volatility
2. To increase adsorption
3. To improve resolution
4. To increase stability
5. To improve detectability.

Define Resolution

A measure of the separation of two peaks of different retention time in a chromatogram

Define co-elution

when two (or more) compounds do not chromatographically separate due to both species having retention times that differ by less than the resolution of the method

What are the issues that may arise in regard to peak shape?

Peak broadening, peak fronting, and peak tailing.

What causes peak broadening?

1. Dead volume
2. Column issues

Dead Volume

All the volume in a chromatographic system from the injector to the detector other than the column.

What are some causes of peak tailing?

1. Poor column cut at inlet
2. Improperly placed column
3. Polar analytes interacting with active sites within inlet

What are some causes of peak fronting?

1. Column overload (injection of a high concentration)

T/F: Derivatization procedures involve the substitution of active hydrogens on the compound with a variety of functional groups.

TRUE

What are the derivatization techniques?

1. Silylation
2. Acylation
3. Alkylation

Advantages of silylation

1. Easily prepared
2. Excellent thermal stability
3. Variety of reagents available

Disadvantages of silylation

1. Silylation reagents are moisture sensitive
2. TMR derivatives are easily hydrolyzed
3. Derivatives cannot be made in aqueous solution

List the reactivity of functional groups with silylation reagents.

Alcohols > Phenols > Carboxylic Acids > Amines > Amides

Commonly Used Silylation Reagents

BSTFA and MSTFA

Advantages of acylation

1. Stability
2. Increased sensitive with added MW
3. Increased detectability using fluorines

Disadvantages of acylation

1. More difficult to prepare
2. Reagents are moisture sensitive
3. Reagents are hazardous and odorous

Commonly Used Acylation Reagents

1. TFAA
2. PFAA
3. HFAA

Solvents for Derivatization

1. Acetonitrile
2. Pyridine
3. Ethyl acetate
4. Tetrahydrofluran
5. Dimethylformamide
6. Dimethylsulfoxide

Advantages of alkylation

1. Wide range of reagnets available
2. Reaction conditions can vary widely (acidic to basic)
3. Derivatives are stable

Disadvantages of alkylation

1. Limited to acidic hydroxyls
2. Reagents often toxic
3. Optimization for particular compounds often necessary