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