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8/10/2019 Lab2 Chromatography
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Chromatography
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Today
o Extraction
o Chromatography
o Protein concentrationo Spectrophotometry
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Definition:
Chromatography separates the components of a
mixture by their distinctive attraction to the mobile phase
and the stationary phase.
Details:
Compound is placed on the stationary phase
Mobile phase passes through the stationary phase
Mobile phase solubilizes the components
Mobile phase carries the individual components a
certain distance through the stationary phase,
depending on their attraction to both of the phases
Chromatography
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How Does Chromatography Work?
Separation is achieved by interaction betweenthe solute and stationary phase and solute andmobile phase.
Solutes that interact with stationary phasestrongly are not carried too far, those thatinteract with solvent travel farther.
How far the solutes travel and thus how farapart different components are depends on theratio of solute interactions between thestationary phase and the mobile phase.
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Types of Liquid Chromatography
Ion Exchange chromatographyseparation based on
charge1. DEAE
2. CM
Gel Filtration chromatographyseparation based on size
1. Matrix is agarose gel (e.g. Sephadex G-150 beads) allow
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a. Charge
b. Size
c. affinity
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Size Affinity
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Thin Layer Chromatography
Stationary phase-- silica gel (SiO2
)x
Mobile phaseMixture of polar and nonpolar solvents
(e.g.,acetone, toluene, ether)
Non-polar compounds will
-Weakly attract to the plate
-Spend more time in the mobile phase
-Faster and far on the plate.
Polar compounds will
-Strongly adhere to the plate
-Spend less time in the moving phase
-Appear lower on the plate.
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Thin Layer Chromatography
mobile phase
stationary
phase
solvent
front
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Thin-Layer Chromatography: A Two-
Component Mixture
solventfrontoriginmixture
solventfront
componentB
componentA
origin
solventfront
componentB
componentA
origin
Increasing Development Time
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Determination of RfValues (measure ofseparation)
solvent front
component B
component A
origin
dSdB
dA
Rfof component A = dA/ dSRfof component B = dB/ dS
The Rfvalue is a decimal fraction,
generally only reported to two
decimal places
B
A
Rfvalues
should be the same regardless of the
extent of travel of the solvent
in theory are independent of a single
experimental run
depend on the solvent used, and the
type of TLC plate
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Photosynthetic pigment of Spinach
1. ChlorophyllsPrimary pigments1. Chl a
2. Chl b
2. CarotenoidsAccessory pigments1. Carotenes
1. -Carotene
2. Xanthophyllsderivatives of carotenes (contain O2)
1. Lutein2. Violaxanthin
3. Neoxanthin
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Primary spinach pigments
- Aldehyde
- Methyl
Attracted by the mobile phase or the
stationary phase?
Will spend more/less time traveling?
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OH
OH
OH
OH
-caroteneisoprenoid chains
lutein
zeaxanthin
Xanthophylls
Accessory spinach pigments
Polar or non-polar?
Soluble in mobile phase or stationary?
Will spend more/less time traveling?
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Appearance of Pigments on TLC
1. Neoxanthin - Yellow
2. Violaxanthin - Yellow
3. Lutein - Yellow
4. Chlorophyll bOlive green
5. Chlorophyll aBlue green
6. -caroteneYellow Orange
Fi 10 5 Th l t ti t
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Figure 10.5 The electromagnetic spectrum
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Spectrophotometry
-Used to measure the amount of light that a solution
absorbs.
- One of the most common applications of
spectrophotometry is to determine the concentration of
an analyte in a solution.
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Light source Cell Detector
Components of a Spectrophotometer
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Spectrophotometer
Beers law: A=lc1. Molar extinction coefficientVaries by molecules.
- Chl a, 663= 80.17 cm2/mg
- Chl b, 645= 50.93 cm2/mg
2. Path length (1cm)
3. Concentration
Absorbance is directly proportional to concentration .
Reference cuvette and sample cuvette1. Blank is identical to the sample except it lacks the solute that
absorbs light.
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Today
Extract photosynthetic pigments
Separate pigment components using TLC
Determine Rfvalue for each pigment
Determine the absorption spectra of Chla,
Chlb, Xanthophylls and -Carotene.
Determine the maxfor each of the above
Determine concentration of Chla and Chlb
Determine ratio of Chla / Chlb in spinach