Spectrophotometry

Ultra Violet and Visible

 UV-Vis
• The spectral range of interest can be
subdivided into three ranges: the near UV, the
Visible and portions of the near infrared (185-
400, 400-700 and 700-1100 nm, respectively
• the measurement of the absorbance based
upon electromagnetic radiation in the
wavelength region of ultraviolet and visible
 Energy absorbed in the UV region
• changes in the electronic energy of the
molecule resulting from transitions of valence
electrons in the molecule
• These transitions consist of the excitation of
an electron from an occupied molecular
orbital (usually a non-bonding, n; or bonding,
p orbital) to the next higher energy orbital (an
anti-bonding, p* or s*, orbital).
Absorbing species

• Absorbing species containing p, s, and n

electrons include organic molecules and ions
as well as a number of inorganic anions
• All organic compounds are capable of
absorbing electromagnetic radiation because
all contain valence electrons that can be
excited to higher energy levels
σ → σ* transition

• The great stability of σ bonds within organic

compounds is reflected by the significant energy
difference between bonding and non-bonding orbitals.
• The transition of an electron between the bonding σ
and non-bonding σ* molecular orbital requires a lot of
energy, thus the transition appears in the far UV.
• This is the reason for saturated hydrocarbons like
hexane or cyclohexane that only contain σ bonds being
transparent in the conventional UV range. For example,
hexane: λmax = 135 nm (ε = 10000).
n → σ* transition

• The promotion of an n electron to the σ*

orbital in O, N, S, and X-containing compound
is observed in the near UV with moderate
intensity. This transition is seen around 180
nm for alcohols, 190 nm for ethers or halogen
derivatives and around 220 nm for amines.
For example, methanol: λmax = 183 nm (ε =
50); ether: λmax = 190 nm (ε = 2000);
ethylamine: λmax = 210 nm (ε = 800).
n → π* transition

• This weak transition is due to the promotion of

an electron from the non-bonding molecular
orbital n to an anti-bonding π* orbital.
• This transition is usually observed in molecules
that contain a heteroatom as part of an
unsaturated system. The most common of these
bands corresponds to the carbonyl band at
around 270 to 295 nm, which can be easily
observed. The molar absorption coefficient for
this band is weak.
π → π* transition

• Most applications of absorption spectroscopy

to organic compounds are based transitions
for n or p electrons to p* excited state,
because the energies required for these
processes bring the absorption peaks into an
experimentally convenient spectral region
(200 to 700 nm).
isolated chromophores
• Chromophores (or oxochromes) are small groups of atoms
responsible for characteristic absorptions.
• By extension, the chromophore in a molecule corresponds
to the site responsible for the electronic transition.
• A chromogene is a species formed by a skeleton on which
many chromopores can be found. For a series of molecules
containing the same chromophore, the position and
intensity of the absorption bands are constant.
• When a molecule contains several isolated chromophores
separated by at least two single bonds, the overlapping of
individual effects is observed.
chromophores in conjugated system

• If several chromophores within the same molecule are

separated by a single bond, this forms a conjugated
system. It results in a strong modification in the
spectrum compared to the overlap of effects produced
by isolated chromophores.
• Spatial delocalisation of electrons increases with the
number of carbon atoms involved in the conjugated
system and this causes a decrease in the energy
difference between the ground and excited states. The
absorption spectrum is displaced towards longer
wavelengths (bathochromic effect) with an increase in
intensity (hyperchromic effect).
 Beer’s Law

• Molecular absorption is based on the

measurement of the transmittance T
• the absorbance A of solutions
• contained in transparent cells having a path
length of b cm
• Ordinarily, the concentration c of an absorbing
analyte is linearly related to absorbance
• The proportionality constant e is known as the
molar absorptivity if c is given in molar
concentration and b is in centimetres;
therefore, e has the units mole-1 cm-1.
• If concentration is expressed in grams of
absorbing material per liter and b is in
centimeters, the proportionality constant is
called the absorptivity and is designed as a in
units of g-1 cm-1.
• C = Molar, maka A = e = absorptivitas molar (L
mol-1cm-1)
• C = g/L, maka A = a = absorptivitas (L g-1cm-1)
• C = % (b/v, g/100mL), maka A = A1% 1cm =
absorptivitas jenis
 Soal
A A 1% b C?
1Cm
0,2 2000 1
0,4
0,8
 Soal
1. Berapakah konsentrasi pembanding yang harus dibuat
bila diinginkan absorban 0.5 dan diketahui absorptivity
= 3000 tebal kuvet 1 cm.
2. Amin-amin membentuk garam dengan asam pikrat
(2,4,6-trinitrofenol). λ maks amin-amin pikrat adalah
359 nm,A1% 545,589, ε 1,25 X 104 liter.mol-1.cm-1.
Sebanyak 0,200 g amin murni diubah secara kuantitatif
menjadi amin pikratnya, dilarutkan dalam air untuk
membuat 50,0 mL. Sebanyak 1,0 mL larutan diencerkan
sampai 100,0 mL. Absorbansi larutan akhir diukur pada
λ 359 nm diperoleh 0,875 pada kuvet setebal 1 cm.
Hitung BM molekul amin tsb!
INSTRUMEN SPEKTROSKOPI
Suatu instrumen fisiko kimia yang mempunyai
celah monokromator (slit) pada bidang datar
yang lebarnya tetap.

Suatu instrumen fisiko kimia yang mempunyai

detektor yang tidak bersifat photoelectric atau
tidak bersifat sensing foton.
Suatu instrumen fisiko kimia yang mempunyai
celah monokromator (slit) pada bidang datar
yang lebarnya bisa diatur.

Suatu instrumen fisiko kimia yang mempunyai

detektor yang bersifat photoelectric atau
bersifat sensing foton.
Spektrofometer UV-Vis Spektrofotometer UV-Vis
konvensional Photo Diode Array (PDA)
 Sistem optik Single beam

Sistem optik Double beam

(CELL / KUVET)
VACUUM PHOTOTUBES
PHOTOMULTIPLIER TUBES (PMT)
PHOTO DIODE ARRAY (PDA)
UV / Visible spectrophotometer

• Instruments for measuring the absorption of

ultraviolet and visible are made up of one or
more (1) sources, (2) wavelength selectors, (3)
sample containers, (4) radiation transducers,
and (5) signal processors and readout devices.
 Sources
• Deuterium and Hydrogen Lamps
A continuum spectrum in the ultraviolet region is produced
by electrical excitation of deuterium or hydrogen at low
pressure. Both deuterium and hydrogen lamps produce a
useful continuum spectrum in the region of 160 to 375 nm.
• Tungsten Filament Lamps
The most common source of visible and near-infrared
radiation is the tungsten filament lamp. A tungsten filament
lamp is useful for the wavelength region between 350 and
2500 nm.
• Xenon Arc Lamps
This lamp produces intense radiation by the passage of
current through an atmosphere of xenon. The spectrum is
continuous over the range between about 200 and 1000
nm, with the peak intensity occurring at about 500 nm.
 Wavelength Selectors

• For most spectroscopic analyses, radiation

that consists of a limited, narrow, continuous
group of wavelengths called a band is
required. Ideally, the output from a
wavelength selector would be radiation of a
single wavelength or frequency. Two types of
wavelength selectors, filters and
monochromators, are used to vary the
wavelength of radiation continuously over a
considerable range.
 Sample Containers

• In common with the other optical elements of an

absorption instrument, the cells, or cuvettes that hold the
sample and solvent must be constructed of a material that
passes radiation in the spectral region of interest.
• Quartz or fused silica is required for work in the ultraviolet
region (below 350 nm); both of these substances are
transparent in the visible region.
• Silicate glasses can be employed in the region between 350
and 2000 nm. Plastic containers have also found application
in the visible region. The most common cell length for
studies in the ultraviolet and visible regions is 1 cm.
 Radiation Transducers/Detector

• The ideal transducer would have a high

sensitivity, a high signal-to-ratio, and a
constant response over a considerable range
of wavelength. In addition, it would exhibit a
fast response time and a zero output signal in
the absence of illumination. Finally, the
electrical signal produced by the transducer
would be directly proportional to the radiant
P.
Signal Processors and readouts Devices

• The signal processor is ordinarily an electronic

device that amplifies the electrical signal from
the transducer
 Kalibrasi spektofotometer

1 kalibrasi skala absorbansi yaitu:

senyawa kalium bikromat digunakan
untuk mengkalibrasi skala absorbansi
pada spektofotometer uv.
2 kalibrasi skala panjang gelombang
yaitu: skala panjang gelombang
pada spektofotometer uv-vis diuji
dengan menentukan panjang
gelombang maksimum spesifik dari
suatu larutan halmium perklorats
 Analisis Kualitatif
• Penentuan Panjang Gelombang
• Grafik
 SEBAGAI DATA PENDUKUNG

1. Curve fitting (menghitung harga MF)

2. Perhitungan lmaksimum
Kaidah Woodward
Kaidah Fieser & Kuhn
Tidak tergantung pada struktur molekul akan tetapi sangat
tergantung pada struktur elektronik molekul.
 Analisis kuantitatif
• One point method
• Kurva kalibrasi
INTERAKSI REM DENGAN SAMPEL
SETELAH MELALUI MEDIA
 HUKUM

T = I / Io = e -kbc
A= - log T = -log I / Io = log Io / I = e b c
Tidak ada gangguan Background absorption
Ada gangguan Background absorption

Simple Simultaneous Equation ( SSE )

Least Square ( LSQ )
Maximum Likelihood ( MLH )
1. Penentuan dilakukan pada lmaksimal
2. Nisbah SBW/NBW ≦ 0,1
3. Rentang dinamik (Instrumental Dynamic Range)
atau IDR AU = 0,2 – 1,5
4. Hindari penentuan pada daerah radiasi sesatan
(stray radiation) l = 220 nm dan 340 nm
 Pada l maksimum diperoleh kepekaan yang
maksimal

lnm
Pada l maksimum deviasi terhadap hukum Beer kecil
1. Dengan perbandingan AU pada lmak.
(AU)Smp/ (AU)Std= ( C )Smp/ ( C )Std
Harga perbandingan 0,5 – 2 kalinya
2. Dengan membuat kurva baku standar pada
rentang 25% - 175% terhadap kadar sampel.
3. Dengan membandingkan harga absorbansi
E1%1cm = AU10ppmx 1000 (di pustaka resmi)
dengan sampel.
4. Dengan membandingkan harga absorbansi
molar (e)= E 1%1cmx Mr x 10–1(di pustaka
resmi ) dengan sampel.
1) Sampel dianalisis dengan Sp UV. A sample adalah 0,42.
Hitung konsentrasi sampel jika standar
dibuat dari 50 mg dalam 100 ml , diambil 1 ml dan dilarutkan ke
dalam 10 ml diperoleh A= 0.56
2). 100 mg serbuk tablet ibuprofen dilarutkan dalam 100 ml. kemudian
Diambil 1 ml dan dilarutkan ke dalam 10 ml dan diukur diperoleh pada
panjang gelombang 246 nm diperoleh A=0.6
a)Hitung kadar ibuprofen per tablet. Bila seharusnya kadar/tablet 500
mg dan bobot tablet 750 mg b) Hitung kemurniannya
Konsentrasi (mikrogram/ml) A
10 0.20
20 0.39
30 0.61
40 0.79
50 0.90