Spectrophotometry: An Analytical Tool

Proses penyerapan sinar oleh larutan

concentration 2 concentration 1 blank where Io = I
with sample I < Io

light source

detector

Io
b
Cell with Pathlength, b, containing solution

I
As concentration increased, less light was transmitted (more light absorbed).

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Hukum Lambert-Beer

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Beers Law

A = ambc
where am molar absorptivity, b pathlength, and c molar concentration

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Some terminology
I intensity where Io is initial intensity T transmission or %T = 100 x T
(absorption: Abs = 1 T or %Abs = 100 - %T)

T = I/ Io

A absorbance A = - log T = -log I/ Io

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Analyze at what wavelength?

Scan visible wavelengths from 400 650 nm (detector range) to produce an absorption spectrum (A vs. l)
Crystal Violet Absorption Spectrum
1.4 1.2

Absorbance

0.8 0.6 0.4 0.2 0 200 250 300 350 400

lmax
phototube detector range
wavelength, nm
450 500 550 600 650 700 750

lmax - wavelength where maximum absorbance occurs

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BLANKO
Mengandung semua zat kecuali analit Digunakan untuk mengatur serapan = nol: Ablank = 0 Hal ini menghilangkan semua serapan sinar oleh sel dan substansi selain analit Semua serapan yang terukur disebabkan oleh analyte.
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The components of a Spec-20D

Light source - white light of constant intensity slits

filter

occluder Grating

Phototube detects light & measures intensity

slits Sample

Separates white light into various colors Rotating the grating changes the wavelength going through the sample

When blank is the sample Io is determined PGCC CHM 103 otherwise I is measured Sinex

IR

What does the absorbed light (electromagnetic radiation) do to the molecule?

700 nm visible Energy increasing

UV 400 nm

high energy UV ionizes electrons low energy UV and visible promotes electrons to higher energy orbitals
(absorption of visible light leads to a colored solution)

IR causes molecules to vibrate (more later)

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UV/visible light absorption

Valence electrons
In organic molecules, electronic transitions to higher energy molecular orbitals double bonds: p p* In transition metals, hydrated ions as Cu++ have splitting of d orbital energies and electronic transitions weak absorption In complexed transition metals, charge transfer of electrons from metal to ligand as Cu(NH3)4++ strong absorption
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Aplikasi spectrofotometri sinar tampak

Analysis of unknowns using Beers Law calibration curve (Been there, done that!) Absorbance vs. time graphs for kinetics Single-point calibration for an equilibrium constant determination Spectrophotometric titrations a way to follow a reaction if at least one substance is colored sudden or sharp change in absorbance at equivalence point, a piece-wise function
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Kinetics of Crystal Violet Reaction

purple

CV+ + OH- CV-OH

colorless

colorless

Follow concentration of crystal violet over time as it reacts by measuring its absorbance. How will absorbance change with time? For a absorbance vs. time plot, how will you determine the rate of the reaction?
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Chime structures

CV+ + OH CV-OH purple colorless colorless This is tracking reaction progress over time. absorbance Since the absorbance is related to concentration, rate or DA/Dtime is the slope of a regression line.

Short run times to get initial rates.

time
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STELLA model

Single-point calibration
Standard with measured absorbance Astd = abcstd Unknown with measured absorbance Aunk = abcunk Ratio the two equations Aunk/ Astd = abcunk /abcstd Aunk/ Astd = cunk /cstd Solve for cunk
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When calibration curves go bad!

The linear Beers Law relationship starts to show curvature at high concentrations
1

Absorbance

0.8 0.6 0.4 0.2 0 0 0.2

Calibration Curve

Non-linear
linear curved Linear (linear)

concentration

0.4

0.6

0.8

Single-point calibration assumes a linear calibration curve

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Practice Examples
1. Tentukan koefisien ekstinsi molar, E, pada 351 nm for aquocobalamin dlm 0.1 M bufer fosfat. pH = 7.0 dari data berikut which were obtained in 1 Cm cell. Solution A B C x 105 M 2.23 1.90 Io 100 100 I 27 32

2. Koefisien ekstinsi molar, E, senyawa riboflavin 3 x 103 Liter/cm x Mole. Bila absorbansi terbaca (A) pada 350 nm 0.9 menggunakan sel 1 cm, Tentukan konsentrasi senyawa riboflavin dlm sampel?

3. Konsentrasi senyawa Y 2 x 10-4 moles/liter dan absorbansi larutan pada 300 nm menggunakan 1 cm quartz cell 0.4. What is the molar extinction coefficient of compound Y? 4. Calculate the molar extinction coefficient E at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH =7.0 from the following data which were obtained in 1 Cm cell. Solution A C x 105 M 2.0 I0 100 I 30

Soal latihan: 1. Larutan yang mengandung 2 g/L zat yang dapat menyerap sinar dalam 1 cm kuvet pada panjang gelombang tertentu mentransmisikan sinar sebanyak 75%. Tentukan banyak sinar yang ditransmisikan oleh larutan yang mengandung zat yang dapat menyerap sinar dengan konsentrasi: a) 4 g/L b) 1 g/L c) 6 g/L d) 5,4 g/L e) bila masa molekul relatif senyawa tersebut 250, tentukan absorptivitas molarnya 2. Suspensi bakteri mengandung 400 mg berat kering per L mempunyai serapan 1 (1 cm kuvet, 450 nm). Tentukan densitas suspensi sel yang dapat mentransimisikan sinar sebanyak 30% dalam 3 cm kuvet
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3. Larutan mengandung NAD+ dan NADH mempunyai optical density dalam 1 cm kuvet 0,311 pada 340 nm dan 1,2 pada 260 nm. Tentukan konsentrasi koenzim bentuk teroksidasi dan reduksi dalam larutan. NAD+ dan NADH menyerap pada 260 nm dan hanya NADH menyerap pada 340, dengan koefisien ekstingsi sbb:

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4. 10 g lemak telah disaponifikasi, fraksi yang tidak tersaponifikasi di ekstrak dengan 25 mL kloroform. Absorbansi larutan kloroform dalam 1 cm kuvet adalah 0,53 pada 328 nm dan 0,48 pada 458 nm. Tentukan konsentrasi vitamin A dalam lemak tersebut. Koefisien ekstingsi karoten dan vit A pada panjang gelombang tsb adalah:

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Menghitung pH dalam Titrasi Asam Lemah-Basa Kuat

Soal: Menghitung pH dalam titrasi 40,00 mL 0,100 M asam asetat (Ka = 1,8 x 10-5) setelah penambahan 0,100 M NaOH dengan volume: (a) 0,00 mL (b) 20,00 mL (c) 40,00 mL
CH3COOH (aq) + NaOH (aq) CH3COONa (aq) + H2O (l) CH3COOH (aq) + OH- (aq) CH3COO- (aq) + H2O (l)

CH3COO- (aq) + H2O (l)

OH- (aq) + CH3COOH (aq)

2. Seorang alhi tanah membutuhkan larutan buffer karbonat pH 10 untuk mempelajari pengaruh hujan asam terhadap tanah yang kaya akan batuan gamping. Berapa g Na2CO3 harus ditambahkan ke dalam 1,5 L larutan segar NaHCO3 0,2 M Untuk membuat larutan buffer karbonat? Diketahui Ka HCO3- = 4,7 x 10-11

3. Tentukan apakah larutan zinc formate, Zn(HCOO)2 bersifat asam, basa atau netral. Diketahui Ka Zn(H2O)6 1x10-9 dan Ka HCOOH 1,8x10-4

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