8L, Extraction & TLC

L4-1

CHEM 8L, Lecture 4 Experiment 3 – Extraction & TLC Analysis of Spinach Pigments Last time…GC Analysis of Citrus Oils GC Sample Calculations Provided in lab – work on these during distillation

- Higher temperature – poor separation of terpenes New & Improved Conditions for You!

- Lower oven temperature, sample spends longer on the column, better separation

Positive Peak Identification: Sample Spiking – Intentionally contaminate (pre-coat) syringe with standard (ex. Pinene): new peak or enhanced peak?

(c) What if spiked standard is not in the sample? Chromatography Overview

Classification

Nature of Stationary

Phase

Nature of

Mobile Phase

Property for

Separation

GC Partition

TLC Adsorption

8L, Extraction & TLC

L4-2

Spinach Experiment 1. Solid to liquid extraction of pigments from spinach 2. Liquid – liquid extraction of pigments from unwanted water-soluble components 3. TLC analysis / separation of individual pigments

1. Solid to liquid extraction

2. Liquid – Liquid Extraction, Based on Partition Coefficients (KH/W) - Ratio of solubilities of pigments in organic solvent (ex. Hexane) vs. water KH/W = (solubility of pigments in hexane) / (solubility in water) >> 1

Methanol(CH3OH)

Break cell wallsRemoves H2O from cells

1st extract(liquid waste)

Hexanes (Hex)&

CH3OH

Extract pigmentsfrom chloroplasts

*quick* vacuum filtration

spinach waste(cellulose)

add di H2O

screw-captest tube

crude extractpigments

in hex / CH3OH

ORGANIC LAYER

AQUEOUSLAYER

ORG

AQ AQ ORGAQ

firstwash

add di H2O

ORG

AQ AQ

firstwash

ORGonly

anhydrousNa2SO4

dry ORG layer5 min

gravity filter

may need furtherconcentration

rotary evaporator(rota-vap)SPINACH

EXTRACT!

- g- g- d- -3

8L, Extraction & TLC

L4-3

Spinach Pigments

Relative Polarity of Functional Groups

Functional Group Structure Example of Organic Solvent

(Common TLC Mobile Phase) Alkanes R-H LEAST POLAR Hexanes

Alkenes R-CH=CH-R Aromatic

Hydrocarbons Ar-H Toluene

Ethers R-O-R Diethyl Ether Esters R-COOR Ethyl Acetate

Ketones R-CO-R Acetone Aldehydes R-CO-H

Amides R-CO-NH2 Amines R-NH2

Alcohols R-OH Methanol Carboxylic Acids R-COOH

Amino Acids H3N+-CHR-COO- MOST POLAR

8L, Extraction & TLC

L4-4

3. Thin-Layer Chromatography (TLC) Analysis Goal: separate as many pigments as possible Mini-experiment (a) optimal spot application

Spotting the Plate

Running the plate

Retention Factor (Rf) = distance from origin to spot (cm) distance from origin to solvent front (cm)

originalmixture

origin

solvent front

solvent front

= more polar compound

= less polar compound

Before During Final

light pencilmark at ORIGIN

light pencilmark at ORIGIN

spot width: 2-3 mm

6.5 cm

2.5 cm

8L, Extraction & TLC

L4-5

3. TLC (cont’d) (b) Optimal mobile phase for separation on different plates

Next time in lab (10/23-10/25): Exp 2, Day 2 - GC Analysis of Citrus Oils - Day 2 notebook prep & typed responses to pre-lab questions - Cooperate / rotation to share GC instruments: 4-6 per lab - TA will demo proper use of instrument & etiquette - Bring Intro Packet – write the abstract during lab - Refer to the Technical Writing Guidelines, Part C - 4-6 sentences: purpose, methods, results, conclusion - The main results in the abstract: recovery of oil (g and %) and % composition of the oil by GC. Example results sentence: “Orange oil was isolated as a clear liquid (xx mg, xx% recovery). GC analysis revealed the oil contained α-pinene (xx%), β-pinene (xx%), limonene (xx%)…”

HexanesHex / EtOAc(9:1)

Hex / EtOAc(7:3)

Ethyl Acetate(EtOAc)

already run!

Optimal Solvent for Separation

Nonpolar(ex. Hexane)

Polar(ex. EtOAc)

Medium polarity(ex. Hex / EtOAc)

MOBILE PHASE:

= more polar

= less polar