PCSIR Labs. Karachi Pakistan1. Spectrophotometer Arif Karim Scientific Officer PCSIR Labs. Complex Karachi Pakistan

PCSIR Labs. Karachi Pakistan 1

Spectrophotometer

Arif Karim

Scientific Officer

PCSIR Labs. Complex

Karachi Pakistan


Spectroscopy• Spectroscopy is the use of the

absorption, emission, or scattering of electromagnetic radiation by atoms or molecules (or atomic or molecular ions) to qualitatively or quantitatively study the atoms or molecules.

• The interaction of radiation with matter can cause redirection of the radiation and/or transitions between the energy levels of the atoms or molecules.

• A transition from a lower level to a higher level with transfer of energy from the radiation field to the atom or molecule is called absorption.


• A transition from a higher level to a lower level is called emission where energy is transferred to the radiation field, or nonradiative decay if no radiation is emitted

• Redirection of light due to its interaction with matter is called scattering and may or may not occur with transfer of energy, i.e., the scattered radiation has a slightly different or the same wavelength.


ATOMIC ABSORPTION

MOLECULAR

IR UV VISIBLE

FLAME EMISSION

SPARC

ICP

FLUORESCENCE

SPECTROSCOPY

ABSORPTION EMISSION


Absorption• When atoms or molecules absorb light, the incoming energy excites a

quantized structure to a higher energy level.

• The type of excitation depends on the wavelength of the light. Electrons are promoted to higher orbital by ultraviolet or visible light, vibrations are excited by infrared light, and microwaves excite rotations.

• Absorbance is a ratio of the intensity of light that is measured passing through the sample to the light intensity measured if no sample was present.


Emission• When atoms or molecules absorb light, the incoming energy excites

a quantized structure to a higher energy level.

• Atoms or molecules that are excited to high energy levels can decay to lower levels by emitting radiation (emission or luminescence).

• For atoms excited by a high-temperature energy source this light emission is commonly called atomic or optical emission (atomic emission spectroscopy).

• and for atoms excited with light it is called atomic fluorescence or molecular fluorescence

• For molecules it is called fluorescence if the transition is between states of the same spin and phosphorescence if the transition occurs between states of different spin.


Scattering• When electromagnetic radiation passes through matter, most of the radiation

continues in its original direction but a small fraction is scattered in other directions.

• Light that is scattered at the same wavelength as the incoming light is called Rayleigh scattering.

• Light that is scattered in transparent solids due to vibrations (phonons) is called. Brillouin scattering is typically shifted by 0.1 to 1 cm-1 from the incident light.

• Light that is scattered due to vibrations in molecules or optical phonons in solids is called Raman scattering. Raman scattered light is shifted by as much as 4000 cm-1 from the incident light.


Electromagnetic Spectrum

Radiation Frequency Hz Wavelength Transition

gamma-rays 1020-1024 <10-12 m Nuclear

x-rays 1017-1020 1 nm-1 pm inner electron

ultraviolet 1015-1017 400 nm-1 nm outer electron

visible 4-7.5x1014 750 nm-400 nm outer electron

near-infrared 1x1014-4x1014 2.5 um-750 nmouter electron molecular vibrations

infrared 1013-1014 25 um-2.5 um Molecular vibrations

microwaves 3x1011-1013 1 mm-25 umMolecular rotations, electron spin flips*

radio waves <3x1011 >1 mm nuclear spin flips*


Ultraviolet & Visible light Interactions

• UV-VIS spectroscopy is the measurement of the wavelength and intensity of absorption of near-ultraviolet and visible light by a sample.

• Ultraviolet and visible light are energetic enough to promote outer electrons to higher energy levels.

• UV-VIS spectroscopy is usually applied to molecules and inorganic ions or complexes in solution.

• UV-VIS spectra have broad features that are of limited use for sample identification but are very useful for quantitative measurements. The concentration of an analyte in solution can be determined by measuring the absorbance at some wavelength and applying the Beer-Lambert Law


Infrared Interactions

• IR spectroscopy is the measurement of the wavelength and intensity of the absorption of mid-infrared light by a sample.

• Mid-infrared light (2.5 - 50 µm, 4000 - 200 cm-1) is energetic enough to excite molecular vibrations to higher energy levels.

• The wavelength of IR absorption bands are characteristic of specific types of chemical bonds, and IR spectroscopy finds its greatest utility for identification

of organic and organometallic molecules.


“Spectrophotometer analyze the concentration of solute in a solution by measuring the intensity of a particular light beam after it is directed through and emerges from it.”

• In the Figure below the red part of the spectrum has been almost completely absorbed by CuSO4 and blue light has been transmitted. Thus, CuSO4 absorbs little blue light and therefore appears blue.

• We will get better sensitivity by directing red light through the solution because CuSO4 absorbs strongest at the red end of the visible spectrum. But to do this, we have to isolate the red wavelengths.

Spectrophotometer




• Io is the incident light and represents 100% of the light striking the cuvette.

• I is the transmitted light. This is the light, which has not been absorbed by the solution in the cuvette and will strike the phototube.

• The photons of light, which do strike the phototube, will be converted into electrical energy. This current, which has been produced, is very small and must be amplified before it can be efficiently detected by the galvanometer.

• The deflection of the needle on the galvanometer is proportional to the amount of light, which originally struck the phototube and is thus an accurate measurement of the amount of light which has passed through (been transmitted by) the sample.


BLANK

• In order to effectively use a spectrophotometer we must first zero the machine, we do this using "the blank."

• The blank contains everything except the compound of interest, which absorbs light. Thus, by zeroing the machine using "the blank," any measured absorbance is due to the presence of the solute of interest.

ABSORPTION SPECTRUM

• Different compounds having dissimilar atomic and molecular interactions have characteristic absorption phenomena and absorption spectra, which differ.

• The point (wavelength) at which any given solute exhibits maximum absorption of light (the peaks on the curves on the figure below) is defined as that compounds particular max.


A cuvette is a kind of cell usually a small square tube, sealed at one end, made of Plastic, glass or optical grade quartz and designed to hold samples for spectroscopic experiments. Cuvette should be as clear as possible, without impurities that might affect a spectroscopic reading. Like a test-tube, a cuvette may be open to the atmosphere on top or have a glass or Teflon cap to seal it shut.

Quartz Cells

170-2700 nm wavelength range

Disposable Cuvettes

UV-Cuvettes for the range between 220-900 nm

VIS-Cuvettes for the range 350-900 nm

Cuvettes


Filters separate different parts of the electromagnetic spectrum by absorbing or reflecting certain wavelengths and transmitting other wavelengths.

• Color Filters

Color filters are glass substrates containing absorbing species that absorb certain wavelengths. A typical example is a cut-on color filter, which blocks short wavelength light such as an excitation source, and transmits longer wavelength light such as fluorescence that reaches a detector.

• Interference Filters

Interference filters are made of multiple dielectric thin films on a substrate. They use interference to selectively transmit or reflect a certain range of wavelengths. A typical example is a bandpass interference filter that transmits a narrow range of wavelengths, and can isolate a single emission line from a discharge lamp. Prisms

Filters


Didymium filters

This glass filter is designed for checking the wavelength calibration of spectrophotometers in both the visible and near infrared regions of the spectrum. The usable range is 430nm to 890nm, instruments of SBW of less than 10nm.

Holmium filters

This filter is intended exclusively for checking the wavelength of moderate to high resolution spectrophotometers.They are custom made to order to required size and supplied either un-mounted or in anodised holders.

Calibration Filters


• Light Source• Monochromator assembly• Sample holder assembly• Detector

Design of a Spectrophotometer


Light SourceLamps convert electrical energy into radiation. Different designs and materials are needed to produce light in different parts of the EMS

Blackbody Sources• A hot material, such as an electrically-heated filament in a light bulb,

emits a continuum spectrum of light. The spectrum is approximated by Planck's radiation law for blackbody radiators:

• The most common incandescent lamps and their wavelength ranges are:tungsten filament lamps : 350 nm - 2.5 mmglowbar : 1 - 40 mmNernst glower : 400 nm - 20 mm

• Tungsten lamps are used in visible and Near-infrared (NIR) absorption spectroscopy and the glowbar and Nernst glower are used for infrared spectroscopy.


Discharge lamps, such as neon signs, pass an electric current through a rare gas or metal vapor to produce light. The electrons collide with gas atoms, exciting them to higher energy levels which then decay to lower levels by emitting light. Low-pressure lamps have sharp line emission characteristic of the atoms in the lamp, and high-pressure lamps have broadened lines superimposed on a continuum.

• Common discharge lamps and their wavelength ranges are:hydrogen or deuterium: 160 - 360 nmmercury : 253.7 nm, and weaker lines in the near-UV and visibleNe, Ar, Kr, Xe discharge lamps : many sharp lines throughout the near-uv to near-IRxenon arc : 300 - 1300 nm

• Deuterium lamps are the UV source in UV-VIS absorption spectrophotometers. The sharp lines of the mercury and rare gas discharge lamps are useful for wavelength calibration of optical instrumentation. Mercury and xenon arc lamps are used to excite fluorescence.

Discharge Lamps


A monochromator is a spectrometer capable of measuring a single wavelength which can be scanned through a wide wavelength range. A common form of monochromator is the Czerny-Turner design, consisting of fixed entrance and exit slits, fixed focussing mirrors and a rotatable diffraction grating. As the grating rotates a different wavelength is focused onto the exit slit.

Monochromator


The wavelength range of a monochromator varies with the choice of grating, but commonly they can scan from 160 nm to 500 nm or ever wider ranges. The spectral resolution depends on the widths of the slits, the choice of grating and focal length, but commonly can be less than 10 pm for high resolution OES. A key to the performance of monochromators is the design of the grating movement: the grating is place on a large drive wheel with motor control, allowing fine and precise positioning of the grating


Monochromator (Optical Spectrometers)

Monochromator parameters

• Bandpass

The wavelength range that the monochromator transmits.

• Dispersion

The wavelength dispersing power, usually given as spectral range / slit width (nm/mm). Dispersion depends on the focal length, grating resolving power, and the grating order.

• Resolution

The minimum bandpass of the spectrometer, usually determined by the aberrations of the optical system.

• Acceptance angle (f/#)

A measure of light collecting ability, focal length / mirror diameter

• Blaze wavelength

The wavelength of maximum intensity in first order.


DetectorSilicon PIN Photodiodes Photoconductive sensorsBlue enhanced for a spectral range from 350nm to 1100nm; designed for low-capacitance, high speed, wide bandwidth applications. Active areas vary from .17 mm² to 100 mm². Applications include: chemical and analytical measurement, laser detection, bar code, smoke detector, appliances, industrial controls, instrumentation,

Silicon PIN Photodiodes Photovoltaic V-Series Blue enhanced for spectral range from 350nm to 1100nm; designed for low-noise, D.C. to medium bandwidth applications. Active areas range from .31mm² to 100mm². Applications include: low light level measurements, particle counting, chemical and analytical measurement and detection.

http://www.photonicdetectors.com/cseries.htm

http://www.photonicdetectors.com/vseries.htm


UV Enhanced Silicon PhotodiodesSpectral enhanced from UV (190nm) response out to Near IR (900nm). Processed for high shunt resistances, low noise and medium electrical bandwidth, these silicon diodes are designed for photovoltaic, low-signal applications. Active areas vary from 0.073mm² to 100mm². Package options include T0-46, T0-18, T0-5, T0-8, Jumbo T0-8, and ceramic packages with quartz and UV transmitting windows. Applications include: pollution monitors, UV exposure meters, water purification, fluorescence, and other spectroscopic applications.

Silicon Carbide (SiC) UV Photodiodes

Standard and custom UV sensors are available with spectral ranges from 200nm to 400nm (optically non-sensitive from 400nm to 1200nm). Active areas include 0.09mm² and special orders for 1.0mm² and larger. Package configurations include: isolated hermetic T0-46 with UV windows. Detector-amplifier hybrid configurations are also available. Applications include: combustion, flame and arc detection, solar blind UV sensing, solar radiation, spectroscopy, sterilization, UV curing detection, and phototherapy control.


Detector-Filter Combination PhotodiodesStandard and custom silicon photodiodes, with integrated optical long-pass (IR), short-pass (VIS), ultra-violet (UV) bandpass, narrow "notch" filters, low-cost plastic long-pass (IR) filters, are offered. Standard configurations include: visible light detectors (500nm), Near-IR (>800nm), UV-A (360nm), UV-B (320nm), UV filter detectors (254 & 310nm), CIE (human eye) response detectors, and neutral-density detector-filter combinations. Active area sizes include 1.55mm² to 100mm². Packages include two-leaded ceramic, T0-46, T0-5, T0-8, Jumbo T0-8, and BNC. Applications include analytical instrumentation, photometry/radiometry, medical instrumentation, and other spectra-radiometry applications.

Gallium Nitride (GaN) UV Detectors This family of Gallium Nitride (GaN) UV Detectors are Schottky processed fully passivated U.V. photodiodes. Spectral range from 200 nm to 365 nm and is ideal for UVA or UVB sensing applications and is packaged with a quartz window.

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Charge-Coupled Devices (CCD)

A CCD is an integrated-circuit chip that contains an array of capacitors that store charge when light creates e-hole pairs. The charge accumulates and is read in a fixed time interval. CCDs are used in similar applications to other detectors, although the CCD is much more sensitive for measurement of low light levels.


Photomultiplier Tubes Photomultiplier Tubes (PMTS) are light detectors that are useful in low intensity applications and due to high internal gain, PMTs are very sensitive detectors.

Design

PMTs are similar to phototubes. They consist of a photocathode and a series of dynodes in an evacuated glass enclosure. Photons that strikes the photoemissive cathode emits electrons due to the photoelectric effect. Instead of collecting these few electrons (there should not be a lot, since the primarily use for PMT is for verly low signal) at an anode like in the phototubes, the electrons are accelerated towards a series of additional electrodes called dynodes.


These electrodes are each maintained at a more positive potential. Additional electrons are generated at each dynode. This cascading effect creates 105 to 107 electrons for each photon hitting the first cathode depending on the number of dynodes and the accelerating voltage. This amplified signal is finally collected at the anode where it can be measured.

Typical specifications

• Wavelength range: 110-1100 nm(wavelength sensitivity dependent on wavelength, uv-sensitive PMTs must have uv-transmitting windows, see optical materials)

• Quantum efficiency (Q.E., number of electrons ejected by the photocathode / number of incident photons): 1-10%

• Response time: 1-15 ns


UV/Visible Spectrophotometer

• It is the measurement of the wavelength and intensity of absorption of near-ultraviolet and visible light by a sample.

• Ultraviolet and visible light are energetic enough to promote outer electrons to higher energy levels.

• UV-VIS spectroscopy is usually applied to molecules and inorganic ions or complexes in solution.

• The UV-VIS spectra have broad features that are of limited use for sample identification but are very useful for quantitative measurements.

• The concentration of an analyte in solution can be determined by measuring the absorbance at some wavelength and applying the Beer-Lambert Law.


• The light source is usually a hydrogen or deuterium lamp for UV measurements and a tungsten lamp for visible measurements.

• The wavelengths of these continuous light sources are selected with a wavelength separator such as a prism or grating monochromator.

• Spectra are obtained by scanning the wavelength separator and quantitative measurements can be made from a spectrum or at a single wavelength.


Optics of UV/Visible Spectrophotometer

The UV-Visible spectrophotometer uses two light sources, a deuterium (D2) lamp

for ultraviolet light and a tungsten (W) lamp for visible light. After bouncing off a mirror (mirror 1), the light beam passes through a slit and hits a diffraction grating. The grating can be rotated allowing for a specific wavelength to be selected. At any specific orientation of the grating, only monochromatic (single wavelength) successfully passes through a slit.


A filter is used to remove unwanted higher orders of diffraction.The light beam hits a second mirror before it gets split by a half mirror (half of the light is reflected, the other half passes through). One of the beams is allowed to pass through a reference cuvette (which contains the solvent only), the other passes through the sample cuvette. The intensities of the light beams are then measured at the end.


In single-beam UV-VIS Absorption Spectroscopy, obtaining a spectrum requires manually measuring the transmittance of the sample and solvent at each wavelength. The double-beam design greatly simplifies this process by measuring the transmittance of the sample and solvent simultaneously. The detection electronics can then manipulate the measurements to give the absorbance.

Dual-Beam UV-VIS Spectrophotometer


Mirrors

• X-rays Ultraviolet aluminium

• Visible aluminium

• Near infrared gold• infrared copper, gold

Lenses

•X-rays Ultraviolet fused silica

(synthetic quartz), sapphire

•Visible glass, sapphire

•Near infrared glass, sapphire

•Infrared CaF2, ZnSe

Optical Materials


AberrationsLenses (and curved mirrors) do not focus light perfectly. Chromatic and spherical aberations occur on-axis and coma and astigmitism occur off-axis.

• Chromatic aberration

Chromatic aberration occurs due to the variation of refractive index with wavelength for a lens material (there is no chromatic aberation in curved mirrors). This wavelength dependence results in slightly different focal lengths for different wavelengths of light. Compound lenses, called achromats, can reduce or eliminate chromatic aberation because the components are chosen such that the variation in refractive index as a function of wavelength cancels out.

• Spherical aberration

Spherical aberation results because the actual focal point of a light ray depends on its distance from the optic axis.


• Coma

Coma is caused by the distortion of a wave front as it encounters an optic asymmetrically. The result for collimated incoming light is a circle instead of a point image. The light rays farther from the optic axis have more severe aberation and the resulting image looks like a comet-shaped series of circles.

• Astigmatism

The projection of an optic off-axis looks squashed in one direction. The squashed direction focuses light to a greater extent than the normal dimension. The result is two line images.

Minimizing aberrations• work on or near the optic axis

• use compound lenses (achromats, doublets, triplets) which can be designed to reduce chromatic aberation, spherical aberation, and coma

• use computer optimized aspheric lenses


Trouble Shooting


• Check the lamp usage hours if it is expired then change it with the new one.(be careful about the warm-up time).

• Check the sample holder and windows, they should be dust free.

• The Cuvettes should be used on the proper side and there should be no scratches on the light exposer side.

• Check the light chopper, the direct exposer also reduces the sensitivity.

• Check the grounding and shielding caps and of detector

• Check the photo cell and its preamplifier circuitry.

Sensitivity problems


Advance Spectrophotometers have their own calibration routines and the user have to just run it, the controller then selects the desired filter and optimized the hardware. In case if there is no auto calibration function one have to use reference sample and calibration filters for adjusting the wavelength scale and intensity readings.

Displacement of peaks (shift in the wavelength)

It is caused due to the miss alignment and over traveling of monochromator arm, knobs, scale or the reference mark, can be removed by calibrating the hardware assembly with the help of

calibration filters (didymium, holmium) or reference samples.

Calibration


• In advanced spectrophotometers the Monochromators are derived through stepper motors. To identify the initial position (home position) a reference hole, mark or notch is usually given. A position sensor (encoder, micro switch, transmitter receiver pair) is installed in the assembly so as to monitor the movement and selection of wavelength. On startup the controller rotates the stepper motor so that to reach the home position or reference mark.

• The common problems are the failure of position sensor, malfunctioning of the stepper motor, and improper homing on startup.

• For proper homing the reference mark and position sensor should be properly aligned and free of dust.

• The working of position sensor can be easily checked by pressing (micro switch), inserting a paper (Optical sensor) and using oscilloscope(encoder).

• The stepper motors used are normally from 5 to 12volt DC(1~5A) and can be checked by applying DC pulses on its winding.

Monochromator Assembly problems


• The common problems in detectors are the damaging and fading of the active areas.

• It is difficult to directly check the response of the detectors, but after some working on the preamplifier circuit one can easily identify the first amplifier IC and by using an oscilloscope the pulsating response can be observed.

• The pulsating response on the output of the amplifier IC is due to the chopping of light from the lamp, and this frequency varies from 50 to 1000Hz.

• Normally one or two variable resistors are given in the Preamplifier section, they are for the adjustment of gain, and offset of the preamplifier.

Detectors and Preamplifier section


• To minimize the effect of stray light a Chopping Technique is normally used in which a fan choppes the light coming from the lamp with frequency corresponding to the number of wings and RPM. This chopping frequency is used as synchronizing pulls for the chopping stabilized opamps.

• For simplicity and minimum component requirement, usually AC fans are used, which gives chopping frequency of 50Hz. Some manufacturer use DC fans with stabilized supply voltages and PWM speed control technique.

• The common problems are the faulty fans, displacement of fan and lamp, or the fan not properly choppes the light beam. This chopping can be observed on the output of first amplifier or synchronizing input. The failure of chopper circuitry misguides the chopper stabilized opamps, which in turn produce the erratic behavior.

Light Chopper assembly


• To stabilized the intensity of lamp through out the analysis the voltage and current regulation is made which uses Regulator IC’s, MOSFET’s or Transistors.

• The common problems are the burnouts of the lamps, shortening of the Transistors, or Regulator IC’s.

• Tungsten lamps can be checked by using its filament resistance(1~200ohms), where as the discharge lamps such as deuterium lamps shows open on its terminal and can only be checked by its rated power supply.

Lamp power supply


• The Acquisition system uses 12bit, 16bit or 18bit Analog to Digital Converters depending on the resolution and accuracy requirements.

• The common problems are the variation or noise in the reference voltage of ADC, malfunctioning of the ADC, RAM or Processor.

• The main program in the EPROM / EEPROM may sometimes be deleted or corrupted due to the frequent power failure or variation.

• To get a rough idea of working and functioning of processor and peripherals, start with the checking of pulses on ALE, Address and Data bus, they should have relation with each other.

• The reset circuitry of the Processor are sometimes triggered by the Power-on, watch-dog and lamp-supply monitoring circuitries, malfunction of these sections lead the processor to hang.

Data Acquisition System


Detector

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PCSIR Labs. Karachi Pakistan1. Spectrophotometer Arif Karim Scientific Officer PCSIR Labs. Complex Karachi Pakistan