DETERMINACIÓN DE ESTRUCTURAS ORGÁNICAS(ORGANIC SPECTROSCOPY)

LIQUID CHROMATOGRAPHY

Hermenegildo García GómezHermenegildo García GómezDepartamento de QuímicaDepartamento de Química

Instituto de Tecnología QuímicaInstituto de Tecnología QuímicaUniversidad Politécnica de ValenciaUniversidad Politécnica de Valencia

46022 Valencia46022 Valencia

EE--mail: mail: hgarcia@qim.upv.eshgarcia@qim.upv.esTelephone: +34 96 387 7807 or ext. 78572/73441Telephone: +34 96 387 7807 or ext. 78572/73441

Fax: + 34 96 387 Fax: + 34 96 387 78097809

Types of Chromatography

1. Gas Liquid Chromatography

2. Liquid Column Chromatography

3. Others

LIQUID COLUMN CHROMATOGRAPHY

A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.

With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.

Diagram of Simple Liquid Column ChromatographyDiagram of Simple Liquid Column Chromatography

The 4 basic liquid chromatography modes are named according to the mechanism

of separation involved:

1. Liquid/Solid Chromatography (adsorption chromatography)

2. Liquid/Liquid Chromatography (partition chromatography)

3. Ion Exchange Chromatography

4. Gel Permeation Chromatography (exclusion chromatography)

FOUR BASIC LIQUID CHROMATOGRAPHY

ADSORPTION CHROMATOGRAPHY

The surface contains silenol groups that are able to form hydrogen bonds.

LIQUID SOLID CHROMATOGRAPHY

Si - OH

HEXANE

OH

C-CH3

CH3

CH3- CCH3

CH3

OH

OH

CH3

CH3

Hydrogen Bond

LessPolar

SOLVENTS

Polar Solvents

Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile

Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane

REVERSE PHASE CHROMATOGRAPHY

Silanization renders the silice particle “hychophobic”Polar molecules do not interact with reverse phase silicaApolar molecules interact strongly with the silanized particles

HO Si

OH

OH O

O

OO

OOH

OH

OH

Si

HO

HO

(Silanization)

Polar

NORMAL VERSUS REVERSE PHASE CHROMATOGRAPHY

Ketones Aromatics Alkanes

Evolution order

Carboxylicacids

Carboxylicacids

KetonesAromaticsAlkanes

WATER-SOLUBLE VITAMINS

1. Niacinamide 2. Pyridoxine

N

CONH2

N

CH2OH

CH2OH

HO

H3C

3. Riboflavin

N

NNH

NCH2

HOCHHOCHHOCH

CH2OH

O

OH3C

H3C

ClN

S

N

NH3C

CH2

NH2

CH3

CH2CH2OH

4. Thiamin

WATER-SOLUBLE VITAMINS

PARTITION CHROMATOGRAPHY ODPN(oxydipropionylnitrile)

Normal Phase LLC Reverse Phase LLC

NCCH3 CH2 OCH2 CH2 CN(Normal)CH3 (CH2 )16 CH3 (Reverse)

The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase.

Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.

ION-EXCHANGE CHROMATOGRAPHY

Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).

Ionic bond

MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS

SO3-

SO3-

Na+

COO-

H3N+

Na+

COOHH3N

+

pH2

pH4.5

Ion-exchange Resin

H 3 N +

SO 3-

SO 3-

SO 3-

SO 3-

SO 3-

SO 3-

H 3 N +

COOH

OH

COOH

COOHH 3 N +

H 3 N +OH

COO -Na +

H 3 N +

COO -

Na +

Na +

H + OH - = H 2 O

H + OH - = H 2 O

Na +

Na +

pH3.5

Mobile PhaseStationary Phase

Exchange Resin

pH4.5

Chromatography of Amino AcidsChromatography of Amino Acids

Porous ParticlesPorous Particles

GEL-PERMEATION CHROMATOGRAPHY

Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules.

Schematic Diagram of Liquid Chromatography

1. Ultraviolet DetectorDiode Array

200-400nm 254 nm

2. Refractive Index Detector

Universal Detector

DetectorsDetectors

flow400

220

Led detector

High Performance Liquid Chromatography

• Waters• Varian• Agilant

Chromatogram of Orange Juice Compounds

Retention Time

Time required for the sample to travel from the injection port through the column to the detector.

Response

Retention Time

5 10 15 20 25

A

B

C

D

SELECTIVITY (α)

Ratio of Net Retention Time of 2 components.

X 2 - X 0

X 1 X 0-α =

R e s p o n s e

R e ten tio n T im e

X

X

X

1 3 6

2

1

0

SelectivitySelectivity

Solvant

RESOLUTION

HEIGHT EQUIVALENT TO A THEORETICAL PLATE

Length of a column necessary for the attainment of compound distribution equilibrium (measure the efficiency of the column).

Theoretical plates (N) = 16 ( )XY

2

X

Y