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    Red Siling Labuyo Chromatography

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    Lesley Bernadette Gomez
    Column and thin-layer chromatography were used to separate and identify the components of red siling labuyo. Column chromatography separated the extract into yellow, orange, and red eluates. Thin-layer chromatography identified carotenoids like α- and β-carotene, zeaxanthin, lutein, and β-cryptoxanthin, as well as anthocyanin pigments in the extract. Capsanthin, which gives red siling labuyo its red color, was identified as the primary pigment responsible for the red color.

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    Red Siling Labuyo Chromatography

    Uploaded by

    Lesley Bernadette Gomez
    3K views5 pages
    Column and thin-layer chromatography were used to separate and identify the components of red siling labuyo. Column chromatography separated the extract into yellow, orange, and red eluates. Thin-layer chromatography identified carotenoids like α- and β-carotene, zeaxanthin, lutein, and β-cryptoxanthin, as well as anthocyanin pigments in the extract. Capsanthin, which gives red siling labuyo its red color, was identified as the primary pigment responsible for the red color.

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    Formal report on the column and thin-layer chromatography of siling labuyo

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    Column and thin-layer chromatography were used to separate and identify the components of red siling labuyo. Column chromatography separated the extract into yellow, orange, and red eluates. Thin-layer chromatography identified carotenoids like α- and β-carotene, zeaxanthin, lutein, and β-cryptoxanthin, as well as anthocyanin pigments in the extract. Capsanthin, which gives red siling labuyo its red color, was identified as the primary pigment responsible for the red color.

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Download as pdf
    3K views5 pages

    Red Siling Labuyo Chromatography

    Uploaded by

    Lesley Bernadette Gomez
    Column and thin-layer chromatography were used to separate and identify the components of red siling labuyo. Column chromatography separated the extract into yellow, orange, and red eluates. Thin-layer chromatography identified carotenoids like α- and β-carotene, zeaxanthin, lutein, and β-cryptoxanthin, as well as anthocyanin pigments in the extract. Capsanthin, which gives red siling labuyo its red color, was identified as the primary pigment responsible for the red color.

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    DETERMINING THE COMPONENTS OF RED SILING LABUYO THROUGH COLUMN AND

    THIN LAYER CHROMATOGRAPHY


    Christine Joy de Gucena, Nicole Trishia Deparine, Marion Dimayuga, Angelica Gabrielle Gabinete, Jean
    Ira Garcia, Lesley Bernadette Gomez
    2A, Faculty of Pharmacy, University of Santo Tomas

    ABSTRACT
    Chromatography is the most useful and modern means of separating mixtures for purification and
    identification of components. All types of chromatography consist of a stationary phase, fixed medium,
    and mobile phase, the moving medium. Column and thin-layer chromatography were performed in this
    experiment. The bases of this chromatography are solubility, adsorptivity, and polarity of compounds.
    Based on the results obtained, DCM-hexane (1:1) is the least polar among the eluents used and
    DCM:methanol (1:1) is the most polar with the greatest distance traveled. Carotenoids, such as - and carotene, zeaxanthin, lutein and -cryptoxanthin, and anthocyanin are present in red siling labuyo.
    Capsanthin is responsible for its red color.

    I.

    INTRODUCTION

    Column chromatography is the process


    where adsorption in a column process is
    directly related to the affinity of the
    solute for either the stationary adsorbent
    or the flowing solvent. In thin-layer
    chromatography, the mobile phase
    ascends a thin layer of adsorbent.

    II. Chromatography is used to


    establish the amount of individual
    components in a mixture by comparison
    with suitable standards. In all
    chromatographic
    processes,
    the
    stationary phase is the medium fixed in
    the system and the mobile phase is the
    medium that flows through the fixed
    medium. The nature of the interaction
    between the components to be separated
    and the two phases forms the basis for
    classification. Knowledge of the
    mechanism enables predictions about
    the expected behavior of a system and in
    choosing a stationary phase and mobile
    phase combination.
    III. Different
    types
    of
    chromatography
    are
    performed
    according on which two phases are used.
    Examples are column and thin-layer
    chromatography for solid-liquid phases,
    paper and high performance liquid
    chromatography
    for
    liquid-liquid
    phases,
    and
    vapor-phase
    chromatography for gas-liquid phases.

    IV. The objective of this experiment


    to separate the colored components of
    red siling labuyo (jalapenos) leaves
    using column chromatography. It also
    aims to determine the purity of the
    components
    using
    thin-layer
    chromatography. At the end of the
    experiment, Rf values of the colored
    components in TLC are expected to be
    measured.
    V.
    VI.

    MATERIALS AND METHODS


    VII.
    Before extracting the
    pigments of the red siling labuyo, the
    seeds of the red siling labuyo were
    removed in order to lessen its spiciness.
    Four fresh red siling labuyo were

    crushed using the mortar and pestle.


    Enough
    dichloromethane
    (DCM)hexane with a ratio of 1:1 was used as
    the extracting solvent. (See Fig. 1) A
    few drops of the extract were set aside
    for TLC. For the set-up of column
    chromatography, cotton was plugged
    into the column (Pasteur pipette). Silica
    gel was packed up to the indented part
    of the pipette. 0.5 mL of the extract was
    placed on top of the column.
    VIII.
    When
    the
    extract
    reached the silica gel, 3.0 mL each of
    DCM-hexane
    (1:1),
    DCM,
    DCM:methanol (1:1) were introduced
    using a Pasteur pipette. Each eluent was
    incorporated in such a way that the
    column did not run dry. (See Fig. 2)
    Three colored eluates were collected and
    put into separate test tubes labeled with
    the noted number of drops of each
    eluate. The colorless eluate was
    discarded.
    IX.

    Two lines were made in the 5 cm x


    8 cm TLC plate for the 1 cm
    measurement on the top and bottom
    of the plate. The three eluates were
    applied on the bottom line having
    four previously dotted equidistant
    dots. The eluates were spotted 10
    times and the crude extract was
    spotted five times aided by the
    colorless end of the capillary tube.
    (See Fig 3) One capillary tube was
    used per sample during the spotting.
    Prior to the application of eluates,
    the developing chamber was
    prepared. The solvent system,
    DCM-hexane (1:1), was placed into
    a 250-mL beaker. The chamber was
    lined with filter paper and covered
    with a watch glass.
    X. After allowing the filter paper to
    equilibrate, the TLC plate was placed in
    the developing chamber. (See Fig. 4)
    When the solvent system rose up to the
    previously lined upper line, the plate
    was removed. The solvent front was

    marked and the plate was air-dried. UV


    lamp was used to visualize the
    components. To compute for the Rf
    value of the components, this formula
    was used;

    Rf =

    XI.

    XII.
    traveled.

    danalyte
    dsolvent
    wherein, d is the distance

    XIII.
    XIV.

    RESULTS AND DISCUSSION

    XV.In the separation of the colored


    components of red siling labuyo using
    column chromatography, silica gel was
    used as an adsorbent. Adsorbent is a
    porous material that can suck up liquids
    and solutions. Since the adsorbent used
    is packed in a vertical column, there is
    no need for any binders. The particle
    size and size distribution directly affects
    the flow of the solvent. The graph below
    shows the number of drops obtained
    from each colored eluate.
    XVI.

    Volume of eluate (drops)


    160
    140
    120
    100
    80
    60
    40
    20
    0

    Volume of eluate (drops)

    XVII.
    Eluate is the solution
    obtained by eluation while eluent is the
    portion of the mobile phase that carries
    the components with it. Errors such as
    less extract placed than the desired
    amount, allowing the column to run dry,

    and incorrect counting of drops collect


    may have caused the inconsistent
    volume of eluates. The addition of
    DCM-hexane produced a yellow eluate
    while DCM produced an orange eluate
    and DCM:methanol (1:1) produced a
    dark orange to red eluate. The eluates
    represent the colored pigments found in
    red siling labuyo which are yellow,
    orange, and red.
    XVIII.
    Chromatography
    is
    frequently used to confirm either the
    presence of absence of a compound in a
    sample. The number of spots in thinlayer chromatography indicated the
    minimum number of components. The
    purity of a compound can be checked
    and the presence of a single spot is taken
    as an indication of purity. In this
    experiment, minimum of one spot per
    eluate manifested. Both in the first and
    second eluate, there is only one visible
    color but in the third eluate there are
    separate yellow and orange spots. On
    the other hand, the crude extract showed
    several pigments with different distance
    traveled from the origin. The position of
    an analysis on the TLC plate is
    characterized by the retention factor Rf.
    The Rf value varies between 0 (analyte
    remains on the start) and 1 (analyses
    move with the front of the mobile phase.
    (See Fig. 5)
    XIX.
    The
    first
    pigment
    produced by the first eluate traveled 6.9
    cm while the longest distance of the
    pigment from the crude extract travele
    7.0 cm. The proximity of these two
    accounts to the eluent used since DCMhexane (1:1) was the first to be
    introduced into the column and the
    extracting solvent used on the first
    procedure is DCM-hexane (1:1) as well.
    These two favored the mobile phase
    which consists of the same solvent.
    Also, the distance traveled of the
    pigment of the light orange eluate is
    close to the distance traveled by the
    orange eluate. The polarity of DCM and

    DCM:methanol (1:1) may be the reason


    behind this.
    XX.
    The eluents used have
    increasing polarity caused by the
    functional groups present. DCM-hexane
    is the least polar among the three
    because the non-polarity property of
    hexane affects the polarity of DCM and
    DCM:methanol is the most polar
    because methanol is an alcohol which
    has high polarity. Red siling labuyo or
    ripened jalapenos is a cultivar of the
    species Capsicum annuum. Chili pepper
    fruits undergo metabolic transformations
    in terms of pigment composition.
    Anthocyanins produce purple pigments,
    and the yellow-orange colors are
    provided by - and -carotene,
    zeaxanthin, lutein and -cryptoxanthin.
    Capsanthin is a red color pigment that is
    lipophilic and is mostly synthesized and
    accumulated in red Capsicum spp. Refer
    to Figure 6 and Figure 7 for the
    molecular structure of the pigments
    present in red siling labuyo.
    XXI.
    8
    7
    6
    5
    4
    3
    2
    1
    0

    XXII.
    XXIII.
    XXIV.
    XXV.
    XXVI.
    XXVII.

    Distance of
    component
    from origin
    (cm)
    Rf value

    XXVIII.
    XXX.
    XXXI. REFERENCES
    XXXII. Colombini, M., & Modugno, F.
    (2009). Organic Mass Spectrometry
    in Art and Archaeology. West
    Sussex, United Kingdom: John
    Wiley & Sons, Ltd
    XXXIII.
    XXXIV.
    Cserhati, T. (2007). Liquid
    Chromatography
    of
    Natural
    Pigments and Synthetic Dyes.
    Amsterdam,
    The
    Netherlands:
    Elsevier
    XXXV. Robards, K., Haddad, & P., Jackson
    P. (1994). Principles and Practice of

    XXIX.
    Modern Chromatographic Methods.
    London,
    United
    Kingdom:
    Academic Press Limited
    XXXVI. Shugar, G., & Ballinger, J.
    (1990).
    Chemical
    technicians
    Ready Reference Handbook (3rd
    Ed.). USA: McGraw-Hill, Inc.
    XXXVII. Solvent Miscibility and Polarity
    Chart. Retrieved September 22,
    2014
    from
    http://www.perfinity.com/downloads
    /Solvent%20Miscibility%20and
    %20Polarity%20Chart

    XXXVIII.
    XXXIX.

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