8L, Extraction & TLC - WPMU DEV
Transcript of 8L, Extraction & TLC - WPMU DEV
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
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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
8L, Extraction & TLC
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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
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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
N
HN
NH
NX
O
O
H3CO
O
O
C20H39
N
N
N
NX
O
O
H3CO
O
O
C20H39Mg2+
Chlorophyll a, X = CH3Chlorophyll b, X = CHO
Pheophytin a, X = CH3Pheophytin b, X = CHO
β-Carotene
HO
OH
Lutein
HO
OH
O
O
Violaxanthin
OHO
•
OHHO
Neoxanthin
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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
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3. TLC (cont’d) (b) Optimal mobile phase for separation on different plates
This week in lab: 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