Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
SPECTROPHOTOMETER MODERN
Class : 2B
By :
Afifah Rizannah
12002
Firdanatul Lailia
12030
12041
12049
Riska Kurnia
12067
Siti Syarifah
12076
CHAPTER 1
INTRODUCTION
A. Background
The spectrophotometer has well been called the workhorse of the modern
laboratory.Inparticular,ultravioletandvisiblespectrophotometryisthemethodof
choicein mostlaboratories concernedwith theidentificationandmeasurementof
organicandinorganiccompoundsinawiderangeofproductsandprocessesin
nucleicacidsandproteins,foodstuffs,pharmaceuticalsandfertilisers,inmineraloils
andinpaint.Ineverybranchofmolecularbiology,medicineandthelifesciences,the
spectrophotometerisanessentialaidtobothresearchandroutinecontrol.
Modernspectrophotometersarequick,accurateandreliableandmakeonly
smalldemandsonthetimeandskillsoftheoperator.However,theuserwhowantsto
optimisethefunctionsofhisinstrumentandtobeabletomonitoritsperformancein
criticalareaswillneedtounderstandtheelementaryphysicsoftheabsorptionprocess
aswellasthebasicelementsofspectrophotometerdesign.
B. Purpose
To know identification of spectrophotometer, Identification Instrument of
spectrophotometer,InstrumentComponentofspectrophotometer,andProcedureby
spectrophotometer
CHAPTER II
DISCUSSION
A. Definition
A spectrophotometer is an instrument for measuring the transmittance or
absorbance of a sample as a function of wavelength. the instrument may be manual or
recording.Spectrophotometer can be used determine the concentration of a solution
through the absorption intensity at certain wavelengths. Depending on the range of
wavelength of light source, it can be classified into two different types:
UV-visible spectrophotometer: uses light over the ultraviolet range (185 - 400 nm)
and visible range (400 - 700 nm) of electromagnetic radiation spectrum.
IR spectrophotometer: uses light over the infrared range (700 - 15000 nm) of
electromagnetic radiation spectrum.
In visible spectrophotometry, the absorption or the transmission of a certain
substance can be determined by the observed color. For instance, a solution sample
that absorbs light over all visible ranges (i.e., transmits none of visible wavelengths)
appears black in theory. On the other hand, if all visible wavelengths are transmitted
(i.e., absorbs nothing), the solution sample appears white. If a solution sample absorbs
red light (~700 nm), it appears green because green is the complementary color of red.
Visible spectrophotometers, in practice, use a prism to narrow down a certain range of
wavelength (to filter out other wavelengths) so that the particular beam of light is
passed through a solution sample.
The wavelength used is the maximum wavelength which gives maximum
absorbance. Generally spectrophotometer is classified into two types: single beam
spectrophotometer and double beam spectrophotometer, IR spectrophotometers are
double beam spectrophotometer. Ordinary spectrophotometer covers a range of 200
nm-800 nm. but if the whole path length is evacuated then below than 200 nm
wavelengths region can be studies. So, spectrophotometer is an instrument that
measures the amount of light absorbed by a substance.
The modern ultr-violet-visible spectrometers consist of light source,
monochromator, detector, amplifier and the recording devices. the most suitable light
sources are tungsten filament lamp or hydrogen-deuterium discharge lamp which
covers the whole of the UV-visible region.
B. Identification Instrument
There
are
many
different
models
of
spectrophotometer.
But,
all
monochromator,
photometer,
sample
area,
and
detector
area.
spectrophotometer contains tungsten lamp (white light), a deuterium lamp (UV light)
or both. A tungsten lamp produces white light, in the visible wavelengths. A
spectrophotometer with a tungsten lamp is called a VIS spectrophotometer because
it produces light in the visible spectrum. A deuterium lamp produces light in the
ultraviolet (UV) part of the spectrum. In a VIS spectrophotometer, when the white
light the prism, it is split into the colours of the rainbow. The wavelength control
rotates the prism directing different colour of light towards the sample. The
wavelengths a light produced by the tungsten lamp a range from 350 nm (purple light)
to 700 nm (red light). All the others colours of the rainbow have wavelength in
between these values. By turning the wavelength control knobs, any wavelength of
visible light between 350 nm and 700 nm can interact with the sample. The molecules
in the sample either absorb or transmit the light energy of one wavelength or another.
The detector senses the amount of light being transmitted by the sample and reports.
That value directly (% transmittance) or converts it to the amount of light absorbed in
absorbance unit.
A spectrophotometer, in general, consists of two devices; a spectrometer and a
photometer. A spectrometer is a device that produces, typically disperses and
measures light. A photometer indicates the photoelectric detector that measures the
intensity of light.
Spectrometer: It produces a desired range of wavelength of light. First a
collimator (lens) transmits a straight beam of light (photons) that passes through a
monochromator (prism) to split it into several component wavelengths (spectrum).
Then a wavelength selector (slit) transmits only the desired wavelengths, as shown
in Figure 1.
Photometer: After the desired range of wavelength of light passes through the
solution of a sample in cuvette, the photometer detects the amount of photons that
is absorbed and then sends a signal to a galvanometer or a digital display, as
illustrated in Figure 1.
C. Instrumental Component
1. Source
The requirements are that the source should be stable during the
measurement period, i.e. that the intensity of emitted radiation should not
fluctuate, and that there should be adequate intensity over as large a wavelength
region as possible.Ultraviolet light is generally derived from a deuterium arc that
provides emission of high intensity and adequate continuity in the 190 - 380 nm
range. A quartz or silica envelope is necessary not only because of the heat
generated but also to transmit the shorter wavelengths of the ultraviolet radiation.
The limiting factor is normally the lower limit ofatmospheric transmission at
about 190 nm.
Visible light is normally supplied by a tungsten lamp or, in modern
systems, by a tungsten-halogen (also
described asquartz-iodine) lamp which
has higher relative output in the crossover region (320 - 380 nm). The long
wavelength limitis usually the cut-off of
the glass or quartz envelope, normally
well beyond the useful visible limit at
900 nm.
In
most
modern
spectrophotometers the
power supply arrangements, including
any necessary
Figure 2. UV/Vis Light Sources
start-up sequences for arc lamps, as well as the cross-over between sources at the
appropriate wavelength, are automatic mechanical sequences. Lamp sare usually
supplied on pre-set focus mounts or incorporate simple adjustment mechanisms
for easy replacement.
2. Monochromator
The function of a monochromator is to produce a beam of monochromatic
(single wavelength) radiation that can be selected from a wide range of
wavelengths. The essential components are entrance slit, collimating device (to
produce parallel light), a wavelength selection or dispersing system, focusing lens
or mirror and an exit slit.
characteristics
related
of
the
convenient
c. Diffraction Grating
Gratings provide an alternative means of producing monochromatic light. A
diffraction grating consists of a series of parallel grooves (lines) on a reflecting
surface that is produced by taking a replica from a master carefully prepared using
a machine or, increasingly, from one which is holographically generated. The
grooves can be considered as separate mirrors from which the reflected light
interacts with light reflected from neighbouring grooves to produce interference,
and so to select preferentially the wavelength that is reflected when the angle of
the grating to the incident beam is changed.
Among the advantages that gratings offer (compared to prisms) are better
resolution, linear dispersion and therefore constant bandwidth and simpler
mechanical design for wavelength.
Figure 4.Diffraction Grating Monochromator
2. Careful selection of the blaze angle (the angle at which the groove is cut)
will peak the energy at the wavelength of the blaze, typically 250 nm for
instruments of the kind under discussion.
3. Both the energy and the resolution of a grating are directly proportional
to the number of lines.
For maximum efficiency the line separation should be as close as
possible to one wavelength and for UV/Vis gratings the line density is
typically1200 per mm.
Gratings have the following advantages over prisms:
(a) Better resolution and energy transfer.
(b) Linear dispersion and therefore constant bandwidth.
(c) Less complicated wavelength drive mechanism is required.
(d) Stray light is limited to imperfections at the grating surface.
d. Optical geometry
As all absorption measurements are ratio dependent (I/Io), it is necessary to
record a reference solution before bringing the sample under test into the light
path. These measurements are done using a cuvette (matched, if possible, to that
containing the test sample) in the light path filled with the appropriate solvent.
The reference intensity (Io) varies with wavelength in a complicated multifunction way (due mainly to source energy, monochromator transmission, slit
width and detector response), so it is essential, when measuring absorption, to remeasure the reference for each discrete wavelength at which measurement is to be
made. All modern instruments are microprocessor based, and have the facility to
store a baseline, that is 100%T or 0 A set at each wavelength in the range,
overcoming this requirement. Thishas allowed single beam spectrophotometers to
compete on performance with the more expensive double beaminstruments.
Additional advantages of microprocessor handling of the detector output are the
ability to introduce component factors (e.g. concentration or molar absorption
data) and to present results in alternative formats without additional
manualcalculation. An important consideration in some laboratories is the ability
to interface with personal computers, so that results can be incorporated into a
laboratory information management system (LIMS) or transferred to diskfor
archiving or data manipulation purposes.
1. Single beam optics
The development of the microprocessor has made it possible to achieve
excellent results using a single beam configuration when compared to a double
beam configuration; this results in greater optical and mechanical simplicity.
The process of comparison between reference and sample cells can be
achieved with single beaminstrumentation by feeding the post detector signal
to a microprocessor which stores the reference data for subtractionfrom the
sample signal prior to printing or displaying the reference corrected result (the
baseline). Signal levels can becompared between different samples at one
wavelength, at a series of predetermined wavelengths or, if wavelength driveis
provided, a complete absorption spectrum can be obtained.
Double beam
1. Determination of the
absorption
time.
time.
2. The price is more expensive.
3. Good for quantitative analysis,
2. Cheaper price
3. Good for qualitative analysis
3. Sampling handling
In practice, by far the greater part of all measurements will be made on
samples in solution. Vapours and solids can be accommodated, but most
instruments are designed with a standard cell
(or cuvette) as the normal sample container.
The design, construction and material of the
cuvette
are
all
important
to
accurate
CHAPTER III
CONCLUSION
ATTACHMENT
Type of Spectrophotometer
DAFTAR PUSTAKA