You are on page 1of 38

Dasar dasar Spektrometri

Absorpsi Molekular UV/Vis

An Introduction to Ultraviolet/Visible
Molecular Absorption Spectrometry

Anfisko I/3
Originally, the study of the dispersion of visible light into its
component colors (visible spectrum). Now it means the study of
electromagnetic (EM) and other spectra including mass, electron
and acoustic spectroscopy’s.

Awalnya, studi tentang dispersi cahaya tampak menjadi
komponen komponen warna (spektrum tampak). Sekarang, merup
studi tentang elektromagnetik dan spektrum lain meliputi massa,
elektron, dan spektroskopi akustik (yang berkaitan dg suara).
General properties of EM radiation

Light (EM radiation) has a dual nature

(Cahaya memiliki sifat ganda) :
Light properties of waves and particles
(called photons or quanta).
A. Wave Parameters
+ Wavelength (l)

Electric Field Amplitude (A)

- Time or Distance

Period (p) – the time required for one cycle to pass a fixed point in space.
(wkt yang dibutuhkan satu siklus u/ melewati sebuah titik dalam ruang)
Frequency (n) – the number of cycles which pass a fixed point in space per second.
(jumlah siklus yang melewati suatu titik tertentu per detik)
= 1/p ( s-1 = Hz ) (putaran perdetik)
Wavelength (l) – The distance between two identical adjacent points in a wave
(usually maxima or minima). (jarak antara 2 titik yang
berdekatan yang identik dalam satu gelombang)
Amplitude (A) – The maximum length of the electric vector in the wave (Maximum
height of a wave).
Wavenumber ( n ) - The number of waves per cm in units of cm-1.
• For EM radiation in vacuum and l is in cm, n = 1/l

• For EM radiation in a medium other than vacuum, n = kn, where k is equal

to the reciprocal of the velocity (in cm/s) through the medium.

• Commonly used for infrared spectroscopy (IR) because it is directly

proportional to frequency, and therefore the energy of the radiation.

Power ( P ) - The amount of energy reaching a given area per second.

Intensity ( I ) - The power per unit solid angle.

• Both power and intensity are related to the square of the amplitude (A2) of
an EM wave.
Hubungan antara Energi, frekuensi
dan panjang gelombang
E = h. ν
ν = c/l
E= h.c/l

E= energi radiasi
h = tetapan planck=6,626 x 10-34 Joule
c = kecepatan cahaya = 2,998 x 10-10 cms-1
l= panjang gelombang
 Hitunglah kuantitas-kuantitas berikut untuk suatu radiasi
pada panjang gelombang 380 nm :
a. Panjang gelombang dlm angstrom dan dlm cm
1 nm = 10-7 cm =10oA
380 nm = 3800 0A = 3,8x10-5 cm
b. Bilangan gelombang
1/l = 1/3,8 x 10-5
c. Frekuensi
ν = c/l
d. Energi
E = hc/l
• Types spectroscopic methods based on EM radiation.
Hubungan antara warna dengan panjang gelombang
Panjang Warna yang diserap Warna yang
gelombang diamati/warna
400-435 Ungu (lembayung) Hijau kekuningan
450-480 Biru Kuning
480-490 Biru kehijauan Orange
490-500 Hijau kebiruan Merah
500-560 Hijau Merah anggur
560-580 Hijau kekuningan Ungu
580-595 Kuning Biru
595-610 Orange Biru kekuningan
610-750 Merah Hijau kebiruan

What happens when light is absorbed by

D. Absorption of Radiation
Absorption - The process in which electromagnetic radiation is transferred to the
atoms, ions, or molecules in the sample causing the promotion of these
particles from the ground state to an excited state.

• The atoms, ions, and molecules have only a

limited number of discrete energy (quantum)
levels, so for absorption to occur the energy of
the exciting photon must exactly match the
energy difference between the ground state and
the excited state.
D. Absorption of Radiation
2. Molecular absorption - Absorption spectra are much more complex than for
atomic absorption due to a large number of possible
energy states.

• The energy of a band in a molecular

absorption spectrum is the sum of three
different energy components.
E = Eelectronic + Evibrational + Erotational

• The sharpness of molecular absorption

spectra also depends on the state of the
sample. In condensed states the spectra
broaden due to molecular collisions.
 Sinar UV dan vis memberikan energi yang cukup
u/ terjadinya transisi elektronik
 Jika suatu molekul sederhana dikenakan REM
maka molekul tsb akan menyerap REM yang
energinya sesuai
 Interaksi antara molekul dg REM akan
meningkatkan energi potensial pada keadaan
 Jika hanya terjadi transisi elektronik dari satu
gugus yang terdapat dlm molekul  terjadi satu
absorpsi  disebut garis spektrum
 Spektrum uv/vis disebut pita spektrum
terjadinya eksitasi elektron lebih dari satu
macam gugus molekul yang kompleks,
sehingga memiliki lebih dari satu panjang
Aspek Kualitatif Spektrofotometer
 Data spektra UV – vis tersendiri tidak dpt
digabung u/ identifikasi
 Data yang diperoleh dari spektrofotometer
uv-vis sebagai parameter analisis kualitatif :
adalah panjang gelombang maksimum
Aspek Kuantitatif

• These methods require two measurements:

P0 – the power of the radiation beam before it passes through the sample

P – the power of the radiation beam after it passes through the sample medium
containing the analyte.

• There are two terms commonly used with absorption methods to describe the
relationship of P0 and P.

1. Transmittance

2. Absorbance
B. Absorption Methods
1. Transmittance - The fraction of incident radiation transmitted
through the sample medium.

Commonly expressed as a percentage:

%T = T x 100
• Major limitation – transmittance has a nonlinear relationship with concentration
of analyte in the sample.
I. Measurement of Transmittance and Absorbance
A. Transmittance (T)
T and %T  T  100
Where: P0 = Power of incident beam
P = Power of emergent beam
Power – Energy (in ergs) of radiation
impinging on a 1 cm2 area of
the detector per second.

• In reality, we do not measure the true P0. We measure PSolvent, the power of
the beam passing through a cell containing solvent only.
B. Absorption Methods
2. Absorbance - A measurement of the amount of radiant power
absorbed by the sample defined as the negative log
of transmittance.

A = -log10T

• Absorbance has a linear relationship with sample concentration defined by

Beer’s Law.
A = ebc
e = molar absorptivity
b = sample path length
c = concentration
B. Absorbance (A)
PSolution PSolvent 1
A   log T   log  log  log
PSolvent PSolution T

C. Beer’s Law
• The amount of radiation absorbed is proportional to the thickness of the
absorbing layer (b), the molecular concentration (c) of the analyte, and the
molecular absorbing coefficient (a) of the species, which is characteristic of
the species at a given wavelength.

A  abc
• When concentration (c) is expressed in molarity (mol/L) and thickness or
pathlength (b) is expressed in centimeters, the molecular absorbing coefficient
(a) is called molar extinction coefficient (e) and has units of liters per moles
per centimeter [ L/(mol•cm) ].

A  ebc
• For mixtures Beer’s Law is additive.
ATotal  A1  A2  A3......  An
ATotal  e1b1c1  e 2b2c2  e 3b3c3......  e nbncn

• Limitations of Beer’s Law

• According to Beer’s Law absorbance (A) is linear with respect to pathlength
(b) and concentration (c), yet there are some real limitations to this
1. A has no limitation with respect to b.
• Use short pathlength cells for samples of high concentration (absorbance).
• Use long pathlength cells for samples of low concentration (absorbance).

Example: If A = 0.410 in a 1.0 cm cuvette (b = 1.0 cm)

Then if: b = 2.0 cm, A = 0.820

b = 0.1 cm, A = 0.041
• Limitations of Beer’s Law
2. Chemical Deviations
• A is linear with respect to concentration (c) except under conditions of
high concentration or when chemical reactions are occurring.

a. A usually becomes nonlinear with respect to concentration when

c > 0.10 M

• Above concentrations of 0.10 M the distance between analyte

molecules decreases to the extent that they change each others charge
distribution effectively changing the way they absorb radiation
(i.e. e changes).

• The same effect is observed if the salt concentration in the solvent (or
buffer) is too high.
• Limitations of Beer’s Law
2. Chemical Deviations
• A is linear with respect to concentration (c) except under conditions of
high concentration or when chemical reactions are occurring.

b. A becomes nonlinear when chemical reactions occur.

• If the analyte associates, dissociates, or reacts with the solvent or other
components in the solution, deviations from Beer’s Law will occur.


HIn  H  In -
Color 1 Color 2
3. Instrument Deviations
a. Effect of Polychromatic Radiation
• Ideally, a monochromator will pass radiation of a single wavelength, but in
reality the monochromator passes a band of radiation. The bandwidth of the
spectrometer will affect the linearity of Beer’s Law.

• Measuring absorption off the wavelength of maximum absorption (lmax)

can affect the linearity of the Beer’s Law because of the change in e wrt

• e will vary across the bandwidth, so a plot of A vs. c can deviate from
• Beer’s Law will be linear when the bandwidth is < 1/10 the width of the
absorption peak at half height.
3. Instrument Deviations
b. Scattered Light
• Scattered light that reaches the detector will increase power reaching the

• Long pathlength cells cause more scattered light, which causes deviations
from linearity in the relationship between A and b.

* Note that all instrument deviations lead to negative absorbance errors.

II. Instrumentation
A. Four Basic Types of UV-Vis Instruments
• Single-beam
• Double-beam in space
• Double-beam in time
• Multi-channel
1. Single-beam Instrument
• These can be either single channel or scanning instruments.

• 0% T is set with shutter in the beam path.

• 100% T is set with a reference in the beam path.
• Measurement is then made with the sample in the beam path.
2. Double-beam in space instrument

• Sample and reference are measured simultaneously and the signal from the
reference is subtracted from the sample signal.

• A major drawback of this type of instrument is the requirement of two

detectors, which makes the instrument more expensive.
3. Double-beam in time instrument

• Most common type of double-beam instrument commercially available.

• Advantages of a double-beam over a single-beam instrument:
a) Compensates for variations in the source intensity.
b) Compensates for drift in the detector and amplifier (DB in time only).
c) Compensates for variation in intensity as a function of wavelength.
4. Multi-channel instrument

• Able to “scan” (collect) an entire spectrum in ~ 0.1 sec.

• Uses signal averaging over a period of 1 sec or more to enhance signal-to
noise ratio.
• Have high throughput of radiant energy due to the minimal optics.
• Typically use a deuterium lamp source for a spectral range of 200 nm 
820 nm and have a spectral bandwidth (resolution) of 2 nm.
B. Components of UV-Vis Spectrometers
1. Source
a. Tungsten Lamp
• Used for the visible region of the spectrum (350 – 800 nm).
• Usually constructed of a quartz or glass bulb containing a tungsten
b. H2 or D2 lamp
• Used for the ultraviolet region of the spectrum (160 – 350 nm).
• The excited D2 will dissociate to give a continuous band of radiation.
• Constructed of a quartz bulb filled with deuterium and a pair of

c. Xe lamp
• Used when a high intensity lamp is required and emits a continuous
band of radiation from 200  1000 nm.
• Not very common because they are expensive and have a short lifetime.
B. Components of UV-Vis Spectrometers
2. Sample containers are usually cuvettes.
• Constructed of either quartz or glass.
- Glass is used for the visible region because it is transparent from 350 
2000 nm.
- Quartz (silica) is used for the UV region because it is transparent below
350 nm.
• Cuvettes are commercially available
in pathlengths of 0.1  10 cm.

3. Detectors
• Use either a photomultiplier tube or a diode array.
B. Some Typical Instruments
• Two major classifications of instruments:
1. Photometers - Simple instruments that use filters to select wavelength.
They can only detect a single wavelength at a time, have a
high throughput energy due to the simple optics (good
S/N), and are inexpensive.

2. Spectrophotometers - Instruments that contain a monochromator or

dispersive element that allow them to scan
various wavelengths. More expensive than
photometers and usually have a lower S/N due to
the more complex optics.
1. Photometers
a. Visible Photometers
1. Photometers
b. UV Photometers
• Common detector used for high performance liquid chromatography.
• Usually contain a Hg lamp as a source and the emission line at 254 nm is
isolated by a filter.
• Good for the detection of organic
molecules, which absorb at 254 nm.
c. Probe Type Photometers
• Contain fiber optics and a mirror to
project the source beam through the
• Commonly found in field instruments
and eliminate the need for a sample cell.
2. Spectrophotometers
a. Visible Spectrophotometers
• Inexpensive ($500 to $3000)
• Range: 380 – 800 nm
• Resolution: 8 –20 nm
2. Spectrophotometers
b. Single-beam and Double-beam UV-Vis Spectrophotometers
• Commonly use exchangeable tungsten and D2 lamps.
• Range: 200 – 900 nm
• Cost: $3000 - $8000 for SB, $4000 - $15,000 for DB
• Resolution: 0.5 to 8 nm for SB, 0.1 to 3 nm for DB
• Accuracy: ±0.005 A for SB, ±0.003 A for DB
2. Spectrophotometers
c. Double-dispersing UV-Vis Spectrophotometers
• Light passes through the monochromator twice resulting in very high
• Expensive: > $10,000
• Bandwidth: 0.07 nm
• Stray light: 0.0008%
2. Spectrophotometers
d. Diode Array UV-Vis Spectrophotometers
• Can be very compact in size.
• Fast scanning (~ 0.1 s)
• Range: 200  820 nm
• Bandwidth: 2 nm
• Cost: $3000  $20,000