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    Experiment 1 PH Measurement and Buffer Preparation

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    The document summarizes an experiment on preparing buffer solutions and determining pH electrometrically and colorimetrically. Key points: 1. A primary phosphate buffer with a desired pH of 3.0 was prepared using phosphoric acid, sodium hydroxide, and distilled water. Its pH was measured using a pH meter and adjusted as needed. 2. The pH of the buffer and distilled water samples was also determined colorimetrically using acid-base indicators that change color at specific pH ranges. 3. Both electrometric and colorimetric methods were used to measure pH in order to compare the results from each approach.

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    Experiment 1 PH Measurement and Buffer Preparation

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    Anonymouscat
    1K views5 pages
    The document summarizes an experiment on preparing buffer solutions and determining pH electrometrically and colorimetrically. Key points: 1. A primary phosphate buffer with a desired pH of 3.0 was prepared using phosphoric acid, sodium hydroxide, and distilled water. Its pH was measured using a pH meter and adjusted as needed. 2. The pH of the buffer and distilled water samples was also determined colorimetrically using acid-base indicators that change color at specific pH ranges. 3. Both electrometric and colorimetric methods were used to measure pH in order to compare the results from each approach.

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    Original Title

    Experiment 1 Ph Measurement and Buffer Preparation

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    The document summarizes an experiment on preparing buffer solutions and determining pH electrometrically and colorimetrically. Key points: 1. A primary phosphate buffer with a desired pH of 3.0 was prepared using phosphoric acid, sodium hydroxide, and distilled water. Its pH was measured using a pH meter and adjusted as needed. 2. The pH of the buffer and distilled water samples was also determined colorimetrically using acid-base indicators that change color at specific pH ranges. 3. Both electrometric and colorimetric methods were used to measure pH in order to compare the results from each approach.

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    1K views5 pages

    Experiment 1 PH Measurement and Buffer Preparation

    Uploaded by

    Anonymouscat
    The document summarizes an experiment on preparing buffer solutions and determining pH electrometrically and colorimetrically. Key points: 1. A primary phosphate buffer with a desired pH of 3.0 was prepared using phosphoric acid, sodium hydroxide, and distilled water. Its pH was measured using a pH meter and adjusted as needed. 2. The pH of the buffer and distilled water samples was also determined colorimetrically using acid-base indicators that change color at specific pH ranges. 3. Both electrometric and colorimetric methods were used to measure pH in order to compare the results from each approach.

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    of 5

    PREPARATION OF BUFFER AND ELECTROMETRIC AND COLORIMETRIC

    DETERMINATION OF PH
    Eunice Aurelle T. Basco, Ian Lindley C. Cabral, Aira Mina A. Cayago,
    Jardine Mariel L. Ching, Leomariss M. Chua and Filjosh R. Cucueco
    Group 2
    2A-Medical Technology
    Biochemistry Laboratory
    ABSTRACT
    Preparation of buffer solution, determination of pH of the buffer and samples electrometrically and colorimetrically
    were done in the experiment. The standard reagent was prepared by diluting 60g of NaOH pellets. The primary
    phosphate buffer was prepared by using 1.69 mL of 14.8 M H3PO4 and 0.88g NaOH pellets. To obtain the desired pH of
    3.0, the buffer was measured electrometrically using the pH meter and was manipulated by using either 6.0 M HCl or
    6.0 M NaOH. The pH of the prepared buffer solution was measured colorimetrically using acid-base indicators such as
    thymol blue, bromophenol blue, bromocresol green, bromocresol purple, phenol red, methyl red, methyl orange, and
    phenolphthalein. The sample, distilled water with a pH of 5.7, was subjected to colorometric determination giving the
    result colors of yellow for thymol blue, violet for bromophenol blue, blue for bromocresol green, yellow for
    bromocresol purple, yellow for phenol red, yellow for methyl red, orange for methyl orange, and colorless for
    phenolphthalein.

    INTRODUCTION
    pH is defined as a way of expressing low
    concentration of hydrogen ions. [1] The term was
    first described by Danish biochemist Sren Peter
    Lauritz Srensen in 1909. [2] pH is an
    abbreviation for "power of hydrogen" where "p" is
    short for the German word for power (potenz)
    and H is the element symbol for hydrogen. The H
    is capitalized because it is standard to
    capitalize element symbols. [2]
    A buffer system/solution is a mixture of a weak
    acid and its conjugate base, or a weak base and
    its conjugate acid. [1] Since its components
    neutralize the excess hydrogen (H+) and
    hydroxyl ion (OH-), it allows solutions to resist
    large changes in pH. Therefore, a buffer helps
    maintain a near constant pH upon the addition of
    small amounts of H+ or OH- to a solution. [3]
    For buffers, The Henderson-Hasselbach equation
    shows the relationship of the pH of a solution to
    the pK of an acid and the ratio of the
    concentrations of the acid and its conjugate base.
    [1] The equation provides a convenient way to
    think about buffers and pH:

    pH= pKa+ log

    The Henderson-Hasselbalch equation is used to


    determine if an aqueous solution of a conjugate
    acid/base pair is functioning as a buffer. [3]
    With these in mind, we prepared different buffer
    solutions, determined the pH of the buffers and
    samples colorimetrically (by using liquid
    indicators) and electrometrically (by using the pH
    meter), and calculated the buffer capacity of the
    prepared buffer solutions in this experiment.

    EXPERIMENTAL
    A. Compounds, solvents, and solutions
    utilized and consumed
    For the preparation of reagents, the samples
    used, solvents or compounds tested in the
    experiment were distilled water (H2O), Sodium
    hydroxide pellets (NaOH) and 12.2 M
    concentrated Hydrochloric Acid (HCl).
    For the preparation of primary phosphate
    buffer, the samples used, solvents or compounds
    tested in the experiment were distilled water
    (H2O), Sodium hydroxide pellets (NaOH) and
    14.8 M concentrated Phosphoric Acid (H3PO4).
    For the electrometric determination of pH, the
    samples used, solvents or compounds tested in
    the experiment were distilled water (H2O), Coca
    Cola soft drink (assigned sample), prepared 250
    mL Primary phosphate buffer, prepared 250 mL
    6.0 M Hydrochloric acid reagent, and prepared
    250 mL 6.0 M Sodium hydroxide reagent.
    For the colorimetric determination of pH, the
    samples used, solvents or compounds tested in
    the experiment were the prepared 250 mL
    Primary phosphate buffer and the different acidbase indicators: Thymol blue (C27H30O5S),
    Bromophenol blue (C19H10Br4O5S), Bromocresol
    green (C21H14Br4O5S), Bromocresol purple
    (C21H16Br2O5S), Phenol red (C19H14O5S), Methyl
    red (C15H15N3O2), Methyl orange (C14H14N3NaO3S),
    and Phenolphthalein (C20H14O4).

    B.iProcedure
    1. Preparation of Reagents
    The group was assigned to prepare a 250 mL
    6.0 M NaOH aqueous solution. First, the amounts
    needed to prepare the reagents were computed

    using the dilution factor as well as the formula for


    getting the molar concentration.
    For the preparation of 250 mL 6.0M NaOH
    solution, 60 g of Sodium hydroxide (NaOH)
    pellets were measured using a triple beam
    balance and was then dissolved with distilled
    water (H2O) in a 250 mL beaker. The aqueous
    solution was transferred to a 250 mL volumetric
    flask were additional distilled water (H2O) was
    added to fill up to the 250 mL mark. Afterwards,
    the 250 mL 6.0M NaOH reagent was transferred
    to a reagent bottle and then labeled properly.
    Given:
    NaOH pellets (40 MM)
    250 mL 6.0 M NaOH aqueous solution

    moles Buffer Solution=( L solution)( M solution )


    moles Buffer Solution=(0.250 L)(0.1 M )
    moles Buffer Solution=0.0250 moles
    [conjugate base]
    pH= pKa+ log
    [weak acid]

    3.00=2.12+ log

    [ primary phosphate]
    [ phosphoric acid]

    [ primary phosphate] 7.5858 M


    =
    [ phosporic a cid ]
    1M

    [Buffer solution ]=[ primary phosphate ] +[ phosphoric acid ]


    [Buffer solution]=7.5858 M+ 1 M
    moles NaOH =(Molarity NaOH )(Volume NaOH )moles NaOH
    =( 6.0solution]=8.5858
    M ) (0.250 L)
    [Buffer
    M
    moles NaOH =1.5 moles NaOH

    Computation:

    Moles of Primary Phosphate (H2PO4-1)

    mass NaOH=(moles NaOH )(molar mass NaOH )


    mass NaOH =(1.5 moles)(40 g /moles)
    mass NaOH=60 g NaOH
    2. Preparation of Buffer Solution
    The group was assigned to prepare a 250 mL
    primary phosphate buffer solution (pK=2.12)
    with a desired pH of 3.00. After identifying the
    weak acid and conjugate base components of the
    buffer solution, the amounts needed to prepare
    the buffer were computed using the HendersonHasselbalch equation, dilution equation, and as
    well as the formula for getting the molar
    concentration.
    For the preparation of 250 mL primary phosphate
    buffer solution, 0.88 g of Sodium hydroxide
    (NaOH) pellets was reacted with 1.69 mL of 14.8
    M concentrated Phosphoric Acid (H3PO4). It was
    then diluted with distilled water (H2O) in a 250
    mL beaker. Then, the aqueous solution was
    transferred to a 250 mL volumetric flask were
    additional distilled water (H2O) was added to fill
    up to the 250 mL mark. Afterwards, the 250 mL
    primary phosphate buffer solution was
    transferred to a reagent bottle and then labeled
    properly.

    [Primary phosphate] moles primary phosphate


    =
    [buffer solution]
    moles buffer solution
    7.5858 M moles primary phosphate
    =
    8.5858 M
    0.0250 moles
    Moles Primary phosphate=0.022 moles
    Moles of Phosphoric Acid

    [Phosphoric acid ] moles phosphoric acid


    =
    [ buffer solution ]
    moles buffer solution
    1M
    moles primary phosphate
    =
    8.5858 M
    0.0250 moles
    Moles Phosphoric acid=0.0029moles
    Volume of Acid Used

    Volume Phosphoric acid=

    0.0250 moles
    14.8 M

    Volume Phosphoric acid=0.00169 L1.69mL


    Mass of Base Needed

    Given:
    14.8 M Concentrated H3PO4 (pK = 2.12)
    NaOH pellets (40 MM)
    250 mL 0.10 M buffer solution
    Desired pH = 3.00
    Weak acid = Phosphoric acid (H3PO4)
    Conjugate base = Primary Phospate (H2PO4-1)

    Mass Sodium hydroxide=(0.022moles)(40 g/moles)


    Mass Sodium hydroxide=0.88 g
    3. Electrometric Determination of pH
    The pH meter was calibrated first at pH 4, 7,
    and 10. First, the pH and temperature of the
    distilled water (H2O) was measured and noted.

    The same procedure was done for the Coca Cola


    soft drink (assigned sample) and prepared
    Primary phosphate buffer. For each of the pH
    measured, the [H+] were calculated using the
    formula 10-pH.
    Using a pH meter, the pH of the prepared
    buffer solution was measured. The buffer solution
    was then manipulated to pH 3.0 (which is the
    desired pH) using the 6.0M NaOH solution (to
    make it more basic, increase pH) and 6.0M HCl
    solution (to make it more acidic, decrease pH).
    4. Colorimetric Determination of pH
    Table 1. Organic Dyes Used for Colorimetric
    Determination of pH
    Indicator

    pH Range
    Acidic: 1.2-2.8

    Thymol Blue
    Alkaline: 8.0-9.6
    Bromophenol
    Blue
    Bromocresol
    Green
    Bromocresol
    Purple

    3.0-4.6
    3.8-5.4
    5.2-6.8

    Color Change
    Acidic: Red to
    yellow
    Alkaline: Yellow to
    blue
    Yellow to Blueviolet

    The preparation of the buffer was done by


    altering the amount of weak acid and its
    conjugate base in the solution. The pH of the
    buffer was obtained by the use of a pH meter.
    This is a device utilized to measure the
    expression of the degree of activity of an acid or
    base according to hydrogen ion [H+] activity or
    the acidity or alkalinity of a solution - also known
    as pH [5]. pH is the unit of measure that
    describes the degree of acidity or alkalinity of the
    given solution and is measured on a scale of 0 to
    14 [5].
    In the experiment, if the actual pH was less than
    the desired pH, the buffer would be treated with
    an appropriate amount of the standard Sodium
    hydroxide (NaOH). However, if the actual pH was
    greater than the desired pH, the buffer would be
    treated with a small amount of the standard
    Hydrochloric acid (HCl).

    Yellow to Blue
    Yellow to Purple

    Phenol Red

    6.4-8.2

    Methyl Red
    Methyl Orange
    Phenolphthalei
    n

    4.8-6.0
    3.2-4.4

    Yellow to Redviolet
    Red to yellow
    Red to yellow

    8.2-10.0

    Colorless to pink

    a. Preparation of Color Standards Using the


    Buffer Solutions
    A certain number of vials (8) was prepared and
    properly labeled with their respective acid-base
    indicators. Using a serological pipette and an
    aspirator, each vial was filled with 5mL of the
    prepared primary phosphate buffer solution. A
    certain amount (2 drops) of acid-base indicator
    was dropped in the corresponding labeled vials.
    The vials were shaken and the color was noted
    down.
    b. Determination of the pH of samples
    The sample used in this experiment was
    distilled water (H2O). A certain number of vials
    (8) was prepared and properly labeled with their
    respective acid-base indicators. Using a
    serological pipette and an aspirator, each vial was
    filled with 5mL of distilled water (H2O). A certain
    amount (2 drops) of acid-base indicator was
    dropped in the corresponding labeledvials. The
    vials were shaken and the color was noted down.

    RESULTS AND DISCUSSION


    1. Electrometric Determination of pH

    Figure 1. A pH meter connected


    combination pH electrode.

    to

    2. Colorimetric Determination of pH
    Colorimetry is a technique that is commonly used
    in biochemistry. This process makes use of a
    device called colorimeter that measures the
    absorbance of specific wavelengths of light by a
    specific
    solution
    and
    thus, involves
    the
    quantitative estimation of colors [4] [6]. The
    produced difference in color results in the
    variation in the absorption of light, which is made
    use of here in this technique. Moreover, this
    method is usually applied to determine the
    concentration of a known solute in a given
    solution by the adaptation of the Beer-Lambert
    law
    [4].
    The colorimeter is based on Beer-Lambert's law,
    according to which the absorption of light
    transmitted through the medium is directly
    proportional to the medium concentration. The
    results depicted in Table 1 shows that at pH=8,
    the acid-base indicators, when mixed with the
    solution, yielded their corresponding colors and
    those colors are due to the concentration of the

    colored complex that was formed. The color or


    the amount of light absorbed is also related to
    the concentration of the absorbing compound,
    therefore it is one of the most useful techniques
    in obtaining the pH.
    According to Beers law when monochromatic
    light passes through the colored solution, the
    amount
    of
    light
    transmitted
    decreases
    exponentially with increase in concentration of
    the colored substance [6]. In addition, the
    Lamberts law states that the amount of light
    transmitted decreases exponentially with increase
    in thickness of the colored solution [6].
    Colorimetric determination of pH exhibits the
    varying color changes an acid-base indicator
    undergoes when added to a solution of a certain
    pH. This property is used to distinguish different
    substances by adjusting their pH ranges.

    Figure 1.
    Indicators

    Distilled water with Acid-Base

    Figure 2. Buffer solution with AcidBase Indicators


    Table 2. Results of Colorometric

    Colorimetric Analysis use the acquired various


    Determination of pH (R = Red, O=
    colors as a means of determining the pH since
    Orange, G= Green, Y = Yellow, b = Blue,
    the intensity of the color of a solution changes
    P=Pink, V= Violet, C= Colorless, YO=
    with its concentration or pH. The color may be
    Yellow Orange, OY= Orange Yellow,
    due to inherent property of a substance in the
    solution or due to the formation of a product as a
    GB= Green Blue, BV= Blue Violet, VB=
    result of the addition of a suitable reagent or
    Violet Blue) (Tb = Thymol Blue, Bb=
    acid-base indicator. The
    Acid
    pH Color Standard
    Bromophenol
    pH of a solution can be
    Base
    2
    3
    5
    7
    7.5
    8
    12
    D.
    blue, Bg=
    determined
    by Indic. 2.2
    3
    4.6 6.9 7.4 7.5 7.9 12.1 H2O Bromocresol
    comparing
    the
    color
    green, Bp =
    Tb
    O
    YO
    Y
    Y
    Y
    Y
    Y
    B
    Y
    intensities
    of
    the
    Bromocresol
    Bb
    G
    Y
    B
    VB
    V
    V
    V
    V
    V
    solution with unknown
    purple, Pr =
    Bg
    Y
    Y
    GB
    B
    B
    B
    B
    B
    B
    pH with the intensities
    Phenol Red, Mr
    Bp
    Y
    Y
    Y
    BV
    V
    V
    V
    V
    Y
    of the solutions with
    Pr
    Y
    Y
    Y
    YO
    R
    R
    P
    P
    Y
    known pH.
    = Methyl Red,
    Mr
    P
    P
    R
    Y
    Y
    Y
    Y
    Y
    Y
    Mo= Methyl
    Table 1. List of AcidMo
    R
    R
    OY YO
    O
    O
    O
    O
    O
    Orange, Pp =
    Base Indicators with
    Pp
    C
    C
    C
    C
    C
    C
    C
    P
    C
    their
    corresponding
    Phenolphthalein)
    colors
    Acid - Base Indicator

    pH 8.0

    Thymol blue

    Yellow

    Bromophenol blue

    Purple

    Bromocresol green

    Blue

    Bromocresol purple

    Purple

    Phenol red

    Red

    Methyl red

    Yellow

    Methyl orange

    Orange

    Phenolphthalein

    Colorless

    REFERENCES
    [1] Crisostomo, A. D., Daya, M. L., de Guia, R.
    M., Farrow, F. L., Gabona, M. G., Ysrael, M.C.
    (2009). Laboratory Manual in General
    Biochemistry. Ph Measurement and buffer
    preparation, 1, 1.
    [2] Anne Marie Helmenstine, Ph. D. (2015,
    November 13). What Does pH stand for?
    Retrieved from
    http://chemistry.about.com/od/ph/f/What-DoesPh-Stand-For.htm
    [3] Chemistry Department-Xavier University of
    St. Louisiana (2014, December 3). Preparation
    of buffers at a desired pH. Retrieved from

    www.xula.edu_Chemistry_documents_biolab_Car
    oll...(14-12-03pdf).pdf
    [4] Chhabra, N. (September, 2015). ColorimetrySimplified. Retrieved from
    http://www.namrata.co/colorimetry-simplified/
    [5] No author (2003). pH Meter. Retrieved from
    http://www.omega.co.uk/prodinfo/ph-meter.html
    [6] No author (June, 2011). Principles of
    Colorimetry. Retrieved from

    https://ecoplants.wordpress.com/2011/06/23/pri
    nciples-of-colorimetry/

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