Project Report

1

University Science Instrumentation Centre

          Jawaharlal Nehru

University 

New Delhi-110067 India

SUMMER TRAINING REPORT

On

PRINCIPLE AND WORKING

OF

VARRIOUS ANALYTICAL INSTUMENTS

SUBMITTED BY

SAURABH PANDEY

ROLL NO.-3045

B.tech. in biotechnology Amity university noida (U.P.)

UNDER THE SUPERVISION OF:

Dr. Kavita Arora (Asst. prof.AIF-JNU)

2

University Science Instrumentation Centre

          Jawaharlal Nehru

University 

New Delhi-110067 India

CERTIFICATE

This is to certify that Saurabh pandey of B.tech Biotechnology, 2nd YEAR from

“Amity UNIVERSITY” has successfully completed her 4 weeks of summer

training (11th June to 13 July 2009) in ADVANCED INSTRUMENTATION FACILITY

DEPT. JAWAHARLAL NEHRU UNIVERSITY.

3

ACKNOWLEDGEMENT

By the grace of almighty, I express my profound sense of reverence of gratitude to Dr.Rakesh bhatnagar (prof. SBT-JNU) and Prof. Satyendra Sharma (director AIF) and DR. kavita Arora (Asst prof.) and Dr. Tulika Prasad (Asst Prof) AIF, USIC, JNU New Delhi for their valuable guidance, congenial discussion, incessant help, calm endurance, constructive criticism and constant encouragement through out this investigation right from the imitation of the work to the ship shaping of the manuscript.

No words will suffice to explain the immense help, cooperation and fortitude by the Instrument in charge Mr.Sandeep Sarpal (XRF) and other seniors Mr. Gajendra (CD FLOW), Dr. Manoj pratap Singh (XRD), DR.Neetu (confocal), Dr. Ajay (GCMS).

At last but never the least, words are small trophies to express my deep sense of ineptness and affection to my parents and sister who gave me infinite love to go for this achievement.

Date: 13th July 2009Place: JNU

4

INDEX

1. Time Resolved Fluorescence Spectroscopy Introduction Theory Instrumentation and Working Example Studied Analysis of the unknown sample: Inference:

2. Gas chromatography-Mass spectrometry (GCMS) GC-MS schematic

Analysis

Full scan MS

Selected ion monitoring

Types of Ionization

Electron Ionization Chemical Ionization Positive Chemical Ionization Negative Chemical Ionization

Applications

Environmental Monitoring and Cleanup

Criminal Forensics

Law Enforcement

Food, Beverage and Perfume Analysis

Beam Splitter

5

Example studied

X-Ray Powder Diffraction(XRD) Explanation Two-dimensional powder diffraction setup with flat plate detector

Uses

Advantages and disadvantages Example studied

3. CD Spectrometer Introduction Principle and working Example studied

4. Confocal Microscopy Introduction Principle Theory Fluorescence spectra Brightness of the fluorophore Commonly used fluorophore. Green fluorescent dye / fluorescin Red fluorescent dye/ Tetramethylrhodamine (TMR) Alexa fluor dye Green fluorescence protein (GFP) Instrumentation and working. Image formation Advantages of confocal microscopy Limitations of confocal microscopy Applications with Examples studied.

5. Transmission Electron Microscopy Introduction Principle of electron microscopy Instumentation and working of TEM. Applications:-

X-ray fluorescence

6

Physics of X-ray fluorescence, in a schematic representation.

Characteristic radiation

Primary radiation

Dispersion

Detection

X-ray intensity

XRF in chemical analysis

Energy dispersive spectrometry

Processing

7

Time Resolved Fluorescence Spectroscopy

Introduction

Time resolved spectroscopy is the study of dynamic process in materials or chemical

compounds by means of spectroscopic techniques. Time resolved fluorescence

spectroscopy is an extension of fluorescence spectroscopy. Fluorescence of the sample is

monitored as a function of time after excitation by laser. TRF provides the information

about molecular environment of the fluorophore and also the steady state fluorescence

measurements. Thus, time-resolved fluorescence spectroscopy can be used to investigate

these processes and gain insight into the chemical surroundings of the fluorophore. The

lifetime of the molecule is ve4ry sensitive to its molecular environment, measurement of it

lifetime reveals much about the state of fluorophore. Fluorescence lifetime is an average

time for a molecule to remain in the excited state before emitting a photon. Fluorescence

lifetimes are generally on the order of 1-10 nsec, although they can range from hundreds of

nanoseconds to the sub-nanosecond time scale. Each individual molecule emit radiations

randomly and of different wavelengths.

Theory

8

Dark Count- When there is no sample in the sample chamber and source is not

switched on, still some photon count in seen on the monitor. This is because of the

heat produce due to IR radiations. These radiations are generated due to electronic

errors. To minimize this dark count we need to lower the temperature. This is done

by cooling the PMT using peltier cooling technique.

IRF- IRF stands for Instrumentation Response Time. It depends on the pulse width of

the source which can be measured theoretically. It is the extra time produced due to

heat generated by the instrument. IRF is measured by putting ludox solution in place

of the sample in sample chamber. It depends on the TAC range and PMT. IRF is

comparatively smaller in case of MCP-PMT (Micro Channel Plate – Photo Multiplier

Tube).

Instrumentation and Working

SourcePhoto Diode

Delay

Sample

CFDPMT

TAC MAC

Beam Splitter

Amplifier

Start

Stop

9

The above figure is of TCSPC – Time Correlated Single Photon Counting Technique

module.

An excitation beam with a narrow wavelength range is directed at the sample, where it

excites fluorescence. The fluorescence emission is collected at a 90º angle from the

excitation to prevent light from the excitation source from interfering with the detection of

the weaker fluorescence emission. The collected fluorescence emission enters a

spectrograph and a detector registers the emission spectrum.

The key differences for time-resolved spectroscopy are the replacement of the continuous

light source with a pulsed source and the use of gated detection of the fluorescence

emission. Continuous source is not used because if it is used then molecule will always

remain in the excited state and will always remain in the excited state. Due to this reason we

won’t be able to acquire any time information of the molecule.

The two main types of pulsed-light sources used in time-resolved fluorescence spectroscopy

are the flash lamp and the laser. The characteristics and features of pulsed lasers vary

widely and the experiment setup must be tailored to a specific laser type. If the laser is free-

running, then jitter will be low (jitter is the variation in the time between pulses) and a laser

pre-trigger can be used to trigger the timing generator. If the time between the pre-trigger

and the laser output is long enough, the timing generator can be adjusted to catch each laser

pulse. If the time between the pre-trigger and the laser output is not long enough to account

for all the delays between the pre-trigger and the timing generator, the delay can be adjusted

to catch the subsequent laser pulse. If the laser is a significant source of jitter, data

acquisition can be triggered off the laser pulse itself rather than from the laser pre-trigger.

10

Triggering off the laser pulse is done with the help of beam splitter. It is diverts a small

percentage of the laser beam to a high-speed photodiode. This photodiode acts as a

transducer and covert light signals to electrical signals. The laser pulse must then be

delayed long enough for the photodiode trigger to reach the timing generator, for the

generator to synchronize with the incoming trigger, and for the gate pulse to reach the

detector. Delay of the laser pulse can be accomplished by increasing the laser path in air or

by launching the laser pulse into a fiber optic cable of an appropriate length. This pulse is

when reached to the TAC (Time to Amplitude Converter) act as the start pulse.

Another part of the laser is incident on the sample. Then PMT (Photo Multiplier Tube)

convert the light signals to electrical pulses. These electric pulses are then amplified by

using an amplifier. The extra pulses which act as noise are suppressed using CFD (Constant

Fraction Discriminator). The pulse generated is called stop pulse.

This start and stop pulses controls the functioning of TAC. TAC acts as a capacitor which

starts charging when start pulse is encountered and charging stops when stop pulse is

reached. Depending on the voltage of the photon we get its time information. MCA (Multi

Channel Analyzer) contains many channels, divided on the basis of time resolution.

There are three key desirable features for a detector in time-resolved fluorescence

spectroscopy: sensitivity, repetition rate, and response time. A high sensitivity is necessary

to measure the weak signals, commonly only a few photons of fluorescence per pulse.

Repetition rate is key because most pulsed light sources operate in the kHz range. If the

detector cannot gate faster than the laser, pulses will be missed. In the UV, missing pulses

can be a problem, since image intensifiers are sensitive to UV radiation. Even while the

intensifier is off, light can reach the detector, creating unwanted signal. Time resolution is

important because with a long impulse (either laser-pulse width or intensifier-gate width), a

11

detector with a short time resolution can distinguish the difference between the excitation

pulse and the emission decay.

Example Studied

Preparation of DOPC vesicle:

1. Add 6gms of methanol to C153 as it dissolves in methanol. C153 is the fluorescent

dye, which binds on the head group of the lipid i.e. hydrophobic part. Make

concentration up to 120µM.

2. Now, evaporate the methanol and add chloroform to this solution. Make

concentration up to 120 µM.

3. Add this to DOPC as it dissolves in the non - polar solvent i.e. chloroform.

4. Now, heat the solution to evaporate the chloroform.

5. We will observe that a lipid bilayer is formed on the surface (bottom) of the test tube.

6. Add 6ml of water to it. The giant vesicle will be formed.

7. Sonicate it to get small size vesicles and then vortex it.

8. As the vesicles start dissolving the solution will become turbid because the vesicles

formed are comparatively smaller but still large enough to make the solution turbid.

Analysis of the unknown sample:

Sample: NBD-C8 in methanol.

12

Plot rough steady state spectra with the help of the photon count obtained by using PMT.

This gives us a vague idea at which wavelengths the peak is maximum. We got three

wavelengths – 500, 530 and 580 at which we should look for the decay curve. The laser

used is of wavelength 405.8nm.

Initially take the IRF with the help of ludox solution. Set the following parameters:

Emission wavelength = 406nm

Excitation wavelength = 406nm

∆λ = 2

Angle = 0°

Time range = 50ns

Peak count = 20,000

Channels = 4096

Time per channel = 0.01221ns

Select the IRF option

IRF obtained = 317-195 = 122ns

Now take the sample in the quartz cuvette. Close the VND (Variable Neutral Density)

wheel. Put the filter of wavelength of 455nm. Detector used here is MCP-PMT. Now,

change the following parameters:

Angle = 55°

Peak count = 5000

Start rate = 10 MHz

Stop rate = around 10,000 MHz

Keeping these conditions obtain the decay spectrum at three different wavelengths. The

result obtained is as follows:

NBDC8MeOH_500_1.FL

13

 Discrete Components Analysis (Reconvolution)

Fitting range :  [100; 3500] channels² :  1.117

Exp Num B B f (%) f (%) (ns) (ns)

1 -5e-4 1.9e-4 0.192 -0.314 0.312 0.620

2 0.001 2.2e-4 2.522 0.673 2.188 0.101

3 0.018 2.6e-4 97.29 1.459 4.803 8.7e-4

Shift :  -0.015 ns (± 0.043 ns)

Decay Background :  1.263 (± 0.083 )

IRF background :  0.400

14

 Discrete Components Analysis (Reconvolution)

Fitting range :  [100; 3500] channels

² :  1.076

Exp Num B B f (%) f (%) (ns) (ns)

1 -0.004 6.5e-4 0.421 -7.075 0.097 1.648

2 0.001 3.3e-4 2.786 1.010 2.452 0.087

3 0.018 3.6e-4 96.79 1.982 4.820 0.001

Shift :  -0.008 ns (± 0.093 ns)

15

Decay Background :  1.425 (± 0.087 )

IRF background :  0.400

Discrete Components Analysis (Reconvolution)

Fitting range :  [100; 3500] channels

² :  1.091

Exp Num B B f (%) f (%) (ns) (ns)

1 -0.021 0.007 0.856 -33.20 0.037 1.446

2 0.002 7.8e-4 5.282 2.793 3.174 0.049

3 0.017 7.9e-4 93.86 4.304 4.893 0.002

16

Shift :  -0.020 ns (± 0.839 ns)

Decay Background :  1.362 (± 0.088 )

IRF background :  0.400

Inference:Blue end is showing rise and red end is showing decay or fall in shape. This is due to

sample’s own photo physical property. We are getting almost same spectrum in all three

cases. This shows that the solvent is relaxing to produce fluorescence but that is not due to

solvent relaxation.

17

Gas chromatography-Mass spectrometry (GCMS)

Gas chromatography-mass spectrometry (GC-MS) is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation, and identification of unknown samples. GC/MS can also be used in airport security to detect substances in luggage or on human beings. Additionally, it can identify trace elements in materials that were previously thought to have disintegrated beyond identification.

The GC-MS has been widely heralded as a "gold standard" for forensic substance identification because it is used to perform a specific test. A specific test positively identifies the actual presence of a particular substance in a given sample. A non-specific test merely indicates that a substance falls into a category of substances. Although a non-specific test could statistically suggest the identity of the substance, this could lead to false positive identification.

The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer. The gas chromatograph utilizes a capillary column which depends on the column's dimensions (length, diameter, film thickness) as well as the phase properties

18

(e.g. 5% phenyl polysiloxane). The difference in the chemical properties between different molecules in a mixture will separate the molecules as the sample travels the length of the column. The molecules take different amounts of time (called the retention time) to come out of (elute from) the gas chromatograph, and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detecting these fragments using their mass to charge ratio.

GC-MS schematic

These two components, used together, allow a much finer degree of substance identification than either unit used separately. It is not possible to make an accurate identification of a particular molecule by gas chromatography or mass spectrometry alone. The mass spectrometry process normally requires a very pure sample while gas chromatography using a traditional detector (e.g. Flame Ionization Detector) detects multiple molecules that happen to take the same amount of time to travel through the column (i.e. have the same retention time) which results in two or more molecules to co-elute. Sometimes two different molecules can also have a similar pattern of ionized fragments in a mass spectrometer (mass spectrum). Combining the two processes makes it extremely unlikely that two different molecules will behave in the same way in both a gas chromatograph and a mass spectrometer. Therefore when an identifying mass spectrum appears at a characteristic retention time in a GC-MS analysis, it typically lends to increased certainty that the analyte of interest is in the sample.

19

Analysis

A mass spectrometer is typically utilized in one of two ways: Full Scan or Selective Ion Monitoring (SIM). The typical GC/MS instrument is capable of performing both functions either individually or concomitantly, depending on the setup of the particular instrument.

Full scan MS

When collecting data in the full scan mode, a target range of mass fragments is determined and put into the instrument's method. An example of a typical broad range of mass fragments to monitor would be m/z 50 to m/z 400. The determination of what range to use is largely dictated by what one anticipates in being in the sample while being cognizant of the solvent and other possible interferences. A MS should not be set to look for mass fragments too low or else one may detect air (found as m/z 28 due to nitrogen), carbon dioxide (m/z 44) or other possible interferences. Additionally if one is to use a large scan range then sensitivity of the instrument is decreased due to performing fewer scans per second since each scan will have to detect a wide range of mass fragments.

Full scan is useful in determining unknown compounds in a sample. It provides more information than SIM when it comes to confirming or resolving compounds in a sample. During instrument method development it may be common to first analyze test solutions in full scan mode to determine the retention time and the mass fragment fingerprint before moving to a SIM instrument method.

Selected ion monitoring

In selected ion monitoring (SIM) certain ion fragments are entered into the instrument method and only those mass fragments are detected by the mass spectrometer. The advantages of SIM are that the detection limit is lower since the instrument is only looking at a small number of fragments (e.g. three fragments) during each scan. More scans can take place each second. Since only a few mass fragments of interest are being monitored, matrix interferences are typically lower. To additionally confirm the likelihood of a potentially positive result, it is relatively important to be sure that the ion ratios of the various mass fragments are comparable to a known reference standard.

20

Types of Ionization

After the molecules travel the length of the column, pass through the transfer line and enter into the mass spectrometer they are ionized by various methods with typically only one method being used at any given time. Once the sample is fragmented it will then be detected, usually by an electron multiplier diode, which essentially turns the ionized mass fragment into an electrical signal that is then detected. The ionization technique chosen is independent of using Full Scan or SIM.

Electron Ionization

By far the most common and perhaps standard form of ionization is electron ionization (EI). The molecules enter into the MS (the source is a quadrupole or the ion trap itself in an ion trap MS) where they are bombarded with free electrons emitted from a filament, not much unlike the filament one would find in a standard light bulb. The electrons bombard the molecules causing a hard ionization that fragments the molecule, and the way in which a molecule fragment is usually typical for all EI techniques.

Chemical Ionization

In chemical ionization a reagent gas, typically methane or ammonia is introduced into the mass spectrometer. Depending on the technique (positive CI or negative CI) chosen, this reagent gas will interact with the electrons and analyte and cause a 'soft' ionization of the molecule of interest. A softer ionization fragments the molecule to a lower degree than the hard ionization of EI. One of the main benefits of using chemical ionization is that a mass fragment closely corresponding to the molecular weight of the analyte of interest is produced.

Positive Chemical Ionization

In Positive Chemical Ionization (PCI) the reagent gas interacts with the target molecule, most often with a proton exchange. This produces the species in relatively high amounts.

21

Negative Chemical Ionization

In Negative Chemical Ionization (NCI) the reagent gas decreases the impact of the free electrons on the target analyte. This decreased energy typically leaves the fragment in great supply.

The primary goal of instrument analysis is to quantify an amount of substance. This is done by comparing the relative concentrations among the atomic masses in the generated spectrum. Two kinds of analysis are possible, comparative and original. Comparative analysis essentially compares the given spectrum to a spectrum library to see if its characteristics are present for some sample in the library. This is best performed by a computer because there are a myriad of visual distortions that can take place due to variations in scale. Computers can also simultaneously correlate more data (such as the retention times identified by GC), to more accurately relate certain data.

Another method of analysis measures the peaks in relation to one another. In this method, the tallest peak is assigned 100% of the value, and the other peaks being assigned proportionate values. All values above 3% are assigned. The total mass of the unknown compound is normally indicated by the parent peak. The value of this parent peak can be used to fit with a chemical formula containing the various elements which are believed to be in the compound. The isotope pattern in the spectrum, which is unique for elements that have many isotopes, can also be used to identify the various elements present. Once a chemical formula has been matched to the spectrum, the molecular structure and bonding can be identified, and must be consistent with the characteristics recorded by GC/MS. Typically, this identification done automatically by programs which come with the instrument, given a list of the elements which could be present in the sample.

A “full spectrum” analysis considers all the “peaks” within a spectrum. Conversely, selective ion monitoring (SIM) only monitors selected peaks associated with a specific substance. This is done on the assumption that at a given retention time, a set of ions is characteristic of a certain compound. This is a fast and efficient analysis, especially if the analyst has previous information about a sample or is only looking for a few specific substances. When the amount of information collected about the ions in a given gas chromatographic peak decreases, the sensitivity of the analysis increases. So, SIM analysis allows for a smaller quantity of a compound to be detected and measured, but the degree of certainty about the identity of that compound is reduced.

22

Applications

Environmental Monitoring and Cleanup

GC-MS is becoming the tool of choice for tracking organic pollutants in the environment. The cost of GC-MS equipment has decreased significantly, and the reliability has increased at the same time, which has contributed to its increased adoption in environmental studies. There are some compounds for which GC-MS is not sufficiently sensitive, including certain pesticides and herbicides, but for most organic analysis of environmental samples, including many major classes of pesticides, it is very sensitive and effective.

Criminal Forensics

GC-MS can analyze the particles from a human body in order to help link a criminal to a crime. The analysis of fire debris using GC-MS is well established, and there is even an established American Society for Testing Materials (ASTM) standard for fire debris analysis. GCMS/MS is especially useful here as samples often contain very complex matrices and results, used in court, need to be highly accurate.

Law Enforcement

GC-MS is increasingly used for detection of illegal narcotics, and may eventually supplant drug-sniffing dogs.[1] It is also commonly used in forensic toxicology to find drugs and/or poisons in biological specimens of suspects, victims, or the deceased.

Food, Beverage and Perfume Analysis

Foods and beverages contain numerous aromatic compounds, some naturally present in the raw materials and some forming during processing. GC-MS is extensively used for the analysis of these compounds which include esters, fatty acids, alcohols, aldehydes, terpenes etc. It is also used to detect and measure contaminants from spoilage or adulteration which may be harmful and which is often controlled by governmental agencies, for example pesticides.

23

Example studied

We have got the data for finding various components in Ajowan oil with the help of GC and MS. We compare the two data on the basis of retention time. We take area percentage from the data obtained by GC alone. Because of no contribution of mass it is assumed that GC gives accurate results for area percentage. The names given by the GC-MS analysis are taken into consideration. Thus, we are able to find what amount the different components are present in the compound.

24

X-Ray Powder Diffraction(XRD)

Powder diffraction is a scientific technique using X-ray, neutron, or electron diffraction on powder or microcrystalline samples for structural characterization of materials

Explanation

Ideally, every possible crystalline orientation is represented equally in a powdered sample. The resulting orientation averaging causes the three dimensional reciprocal space that is studied in single crystal diffraction to be projected onto a single dimension. The three dimensional space can be described with (reciprocal) axes x*, y* and z* or alternatively in spherical coordinates q, φ*, χ*. In powder diffraction intensity is homogeneous over φ* and χ* and only q remains as an important measurable quantity. In practice, it is sometimes necessary to rotate the sample orientation to eliminate the effects of texturing and achieve true randomness.

25

Two-dimensional powder diffraction setup with flat plate detector

When the scattered radiation is collected on a flat plate detector the rotational averaging leads to smooth diffraction rings around the beam axis rather than the discrete Laue spots as observed for single crystal diffraction. The angle between the beam axis and the ring is called the scattering angle and in X-ray crystallography always denoted as 2θ. (In scattering of visible light the convention is usually to call it θ). In accordance with Bragg's law, each ring corresponds to a particular reciprocal lattice vector G in the sample crystal. This leads to the definition of the scattering vector as:

Powder diffraction data are usually presented as a diffract gram in which the diffracted intensity I is shown as function either of the scattering angle 2θ or as a function of the scattering vector q. The latter variable has the advantage that the diffract gram no longer depends on the value of the wavelength λ. The advent of synchrotron sources has widened the choice of wavelength considerably. To facilitate comparability of data obtained with different wavelengths the use of q is therefore recommended and gaining acceptability. An instrument dedicated to perform powder measurements is called a powder diffract meter.

Uses

Relative to other methods of analysis, powder diffraction allows for rapid, non-destructive analysis of multi-component mixtures without the need for extensive sample preparation This gives laboratories around the world the ability to quickly analyse unknown materials and perform materials characterization in such fields as metallurgy, mineralogy, forensic

26

science, archeology, condensed matter physics, and the biological and pharmaceutical sciences. Identification is performed by comparison of the diffraction pattern to a known standard or to a database such as the International Centre for Diffraction Data's Powder Diffraction File (PDF) or the Cambridge Structural Database (CSD). Advances in hardware and software, particularly improved optics and fast detectors, have dramatically improved the analytical capability of the technique, especially relative to the speed of the analysis. The fundamental physics upon which the technique is based provides high precision and accuracy in the measurement of interlunar spacing’s, sometimes to fractions of an Angstrom, resulting in authoritative identification frequently used in patents, criminal cases and other areas of law enforcement. The ability to analyze multiphase materials also allows analysis of how materials interact in a particular matrix such as a pharmaceutical tablet, a circuit board, a mechanical weld, a geologic core sampling, cement and concrete, or a pigment found in an historic painting. The method has been historically used for the identification and classification of minerals, but it can be used for any materials, even amorphous ones, so long as a suitable reference pattern is known or can be constructed.

Advantages and disadvantages

Although it possible to solve crystal structures from powder X-ray data alone, its single crystal analogue is a far more powerful technique for structure determination. This is directly related to the fact that much information is lost by the collapse of the 3D space onto a 1D axis. Nevertheless powder X-ray diffraction is a powerful and useful technique in its own right. It is mostly used to characterize and identify phases rather than solving structures. The great advantages of the technique are:

simplicity of sample preparation rapidity of measurement the ability to analyse mixed phases, e.g. soil samples

By contrast growth and mounting of large single crystals is notoriously difficult. In fact there are many materials for which despite many attempts it has not proven possible to obtain single crystals. Many materials are readily available with sufficient microcrystallines for powder diffraction, or samples may be easily ground from larger crystals. In the field of solid-state chemistry that often aims at synthesizing new materials, single crystals thereof are typically not immediately available. Powder diffraction is therefore one of the most powerful methods to identify and characterize new materials in this field.

27

Particularly for neutron diffraction, which requires larger samples than X-Ray Diffraction due to a relatively weak scattering cross section, the ability to use large samples can be critical, although new more brilliant neutron sources are being built that may change this picture.

Since all possible crystal orientations are measured simultaneously, collection times can be quite short even for small and weakly scattering samples. This is not merely convenient, but can be essential for samples which are unstable either inherently or under X-ray or neutron bombardment, or for time-resolved studies. For the latter it is desirable to have a strong radiation source. The advent of synchrotron radiation and modern neutron sources has therefore done much to revitalize the powder diffraction field because it is now possible to study temperature dependent changes, reaction kinetics and so forth by means of time dependent powder diffraction.

Example studied

Potassium choride

When incident x-rays striking the sample satisfies the Bragg eqution, constructive interference take place and peak in the sample is formed. The detector records the x ray signal and converts it to count rate which is output to a device as diffract gram. It helps in determing the PHD (PULSE HEIGHT DISTRIBUTION).

The d-spacing of each peak is then obtained by solution of the Bragg equation for the appropriate value of λ. Once all d-spacings have been determined, automated search/match routines compare the ds of the unknown to those of known materials. Because each mineral has a unique set of d-spacings, matching these d-spacing provides an identification of the unknown sample. A systematic procedure is used by ordering the d-spacing’s in terms of their intensity beginning with the most intense peak. Files of d-spacings for hundreds of thousands of inorganic compounds are available from the International Centre for Diffraction Data as the Powder Diffraction. And by studying the graph obtained, we are able to obtain various components in the sample and also crystal structure of the sample.

28

Crystallographic parameters (KCl)

Crystal system: Cubic

Space group: Fm3m

a (Å): 3.1380

b (Å): 3.1380

c (Å): 3.1380

Alpha (°): 90.0000

Beta (°): 90.0000

Gamma (°): 90.0000

29

CD SPECTROMETER

INTRODUCTION

Circular dichroism is the difference in the absorption of left-handed circularly polarized light (L-CPL) and right-handed circularly polarized light (R-CPL) and occurs when a molecule contains one or more chiral chromophores (light-absorbing groups).

Circular dichroism = ΔA (λ) = A(λ)LCPL - A(λ)RCPL, where λ is the wavelength.

Circular dichroism (CD) spectroscopy is used extensively to study chiral molecules of all types and sizes, but it is in the study of large biological molecules where it finds its most important applications. A primary use is in analyzing the secondary structure or conformation of macromolecules, particularly proteins, and because secondary structure is sensitive to its environment, e.g. temperature or pH, circular dichroism can be used to observe how secondary structure changes with environmental conditions or on interaction with other molecules. Structural, kinetic and thermodynamic information about macromolecules can be derived from CD spectroscopy. CD spectra in the far-UV (below 260nm) can be used to predict the percentages of each secondary structural element in the structure of proteins.

30

PRINCIPLE AND WORKING

There is a source of monochromatic linearly polarised light which can be turned into either left- or right-circularly polarised light by passing it through a quarter-wave plate whose unique axis is at 45 degrees to the linear polarisation plane.

150W xenon lamp is used as the source of light. The entire system is purged with nitrogen so as to allow the far UV transmission and prevent the formation of ozone.

Instead of a static quarter-wave plate, a circular dichroism spectrophotometer has a specialized optical element called a photo-elastic modulator (PEM). This is a piezoelectric element cemented to a block of fused silica. At rest, when the piezoelectric element is not oscillating, the silica block is not birefringent. When driven, the piezoelectric element oscillates at its resonance frequency (typically around 50 kHz), and induces stress in the silica in such a way that it becomes birefringent. The alternating stress turns the fused silica element into a dynamic quarter-wave plate, retarding first vertical with respect to horizontal components of the incident linearly polarised light by a quarter-wave and then vice versa, producing left- and then right- circularly polarised light at the drive frequency. The amplitude of the oscillation is tuned so that the retardation is appropriate for the wavelength of light passing through the silica block.

31

On the other side of the sample position there is a light detector. When there is no circularly dichroic sample in the light path, the light hitting the detector is constant. If there is a circularly dichroic sample in the light path, the recorded light intensity will be different for right- and left-CPL. Using a lock-in amplifier tuned to the frequency of the PEM, it is possible to measure the difference in intensity between the two circular polarisations (vAC). The average total light intensity across many PEM oscillations (vDC) can be used to scale the size of the lock-in amplifier signal to take into account variations in total light level. Both signals can be recorded and from them the circular dichroism signal can be calculated easily by dividing the vAC component by the vDC signal.

G is a calibration-scaling factor to provide either ellipticitys or differential absorbance.

APPLICATIONS

Circular dichroism is widely used in the Pharmaceutical industry particularly in biotherapeutic drug development to optimize formulations and understand protein structure and stability.

Additionally the technique has also been used in pharmacokinetics to study in-vitro the potential interactions of small molecule drugs.

Another powerful application for Chirascan CD spectroscopy is to compare different macromolecules, or the same molecule under different conditions, and determine if they have a similar structure. This can be used to ascertain if a newly purified protein is correctly folded, to determine if a mutant protein has folded correctly in comparison to the wild-type.

32

EXAMPLE STUDIED

The interaction of the membrane protein GPI (glycosyl phosphatidyl inositol) with different concentration of manganese metal was studied and the corresponding change in shape was examined. This protein has alpha-helix configuration. CD spectra was taken at the wavelength of 190-260nm. We observed that PBS was the best suited buffer. We avoid using Tris at higher concentration because it increases the voltage at high wavelengths and thus can damage our sample. Quartz cuvette (250μl) was used for this protein. W-shaped curve is obtained confirming the alpha helix configuration of the protein of interest. We take the graph over the given range of wavelength in triplicates and then take the their average.. To eliminate the interaction of the metal with buffer used, we subtract the graph of the buffer with that of the protein with the help of the software. After titrating the protein with manganese metal, an upward shift in the curve was seen due to increase in the CD value. On increasing the manganese concentration, the graph moved upwards each time and hence intensity was reduced. Sometimes, a scattered graph can be seen. This indicates that the protein is getting precipitated after addition of the metal. If there is no change in the shape of the curve on adding the metal, this means that the protein is already saturated.

33

CONFOCAL MICROSCOPY.

INTRODUCTION

Confocal microscopy is a major advance upon normal light microscopy by allowing one to visualize not only deep into cells and tissues, but to also create a three dimensional image of live cells. Confocal microscopes have been in existence for over 20 years. In the late 1980s the first commercially available confocal microscopes became available from Biorad, Leica and Zeiss. It is one of the best techniques to examine the structural details of the cells along with the cellular dynamics. There are numerous applications of confocal microscopy in biology. There are great number of advantages of using confocal microscopy compared to conventional epi-fluorescence microscopy like ability to remove the out of focus noise and increased sensitivity of the machine.

34

PRINCIPLE

The basic principle behind confocal microscopy can be traced back to fluorescence which is the property of a molecule to emit light at specific range of wavelength when hit by the incident light. When a population of fluorophore molecules is excited by an appropriate wavelength of light, some of the molecules will absorb a photon of sufficient energy to boost an electron from ground- energy state to an excited state energy level. The drop back down to the ground state-energy level results in the emission of a photon (fluorescence). The energy of an emitted photon is equal to the difference in the energy levels of the ground and the excited state. This determines the wavelength of the emitted light. One of the distinguishable feature of confocal microscope is the presence of the pinhole. The out of focus light is removed form the image with the help of suitably positioned pinhole. This not only creates the image of excellent resolution .but also allows one to collect optical slices of the object, and to use these slices to create a three dimensional representation of the sample. The focal plane of the pinhole, objective and the detector has the same focal plane. This justifies the name of the instrument.

THEORY

FLUORESCENCE SPECTRA

Fluorescence molecules have a typical excitation or absorption and emission spectra. The amount of light absorbed by the molecule is called as the extinction coefficient. The efficiency with which the molecule converts absorbed light into emitted fluorescent light is called as the quantum yield which is determined by the molecular characteristics ,local environment and wavelength of light used for excitation. The shift in the wavelength between the absorption and the emission spectra is called the “stokes shift”. A larger stokes shift makes it easier to collect the emitted light. The most efficient light for exciting a fluorophore is to use the wavelength where maximum absorption occurs. By changing the excitation wavelength does not alter the emission wavelength.

BRIGHTNESS OF THE FLUOROPHORE

Different fluorophores emit of light of different intensity. The level of the fluorescent light emitted is proportional to both the extinction coefficient and the quantum yield of the fluorophore. Light can be highly destructive to biological samples so it is important to minimize both the amount of light and time of exposure to limit the biological

35

damage.Fluorophore that emits a low level of fluorescence will require higher level of irradiation.

COMMONLY USED FLUOROPHORES.

GREEN FLUORESCENT DYE / FLUORESCEINIt is commonly called as FITC. Its various derivatives are used as cellular markers, or when conjugated to antibodies it is widely for immunolabelling. The FITC absorption maxima are close to the main emission line (488nm blue light) of argon ion and krypton argon lasers. This dye has great quantum yield but the level of fluorescence is influenced by the pH changes in cellular compartments.

RED FLUORESCENT DYE/ TETRAMETHYLRHODAMINE (TMR)It is often used as the second dye in dual labeling experiments with FITC. It has absorption maxima in the yellow region of the spectrum and emits fluorescent light in the red region. It also has sufficient absorption at 488nm (blue) light of the argon ion laser, thus permitting dual labeling when only one laser line is available.

ALEXA FLUOR DYESThese suffocated rhodamine derivatives have improved characteristics compared to FITC and TRITC. These have been designed to be maximally excited by the commonly available lasers in confocal microscopy. For example, alexa488 is designed for excitation by the blue 488nm line of argon ion laser or krypton argon laser and alexa568 is designed to be excited by the yellow 568nm line of the krypton argon laser. The alexa fluor dyes have higher quantum yield than FITC. They are almost unaffected by photobleaching when used with normal laser intensity. They show least fading when applied in a simple buffer solution, making them great for live cells studies as well as fixed specimen analysis. Theses are not sensitive to pH changes and are commercially available as conjugates to a variety of secondary antibodies for immunofluorescence experiments.

GREEN FLOURESCENCE PROTEIN (GFP)

It is used as a marker for sub cellular protein location and trafficking. It is naturally fluorescent protein of 238 amino acids cloned from the jelly fish Aquaria Victoria. This has been modified over past few years to produce a series of proteins of different wavelengths of emission. The fluorescent moiety is an oxidized derivative of tripe tide ser-tyr-gly

36

(residues 65-67of the native protein). The oxidation of GFP needs molecular oxygen. This means that cloned GFP, expressed in a diverse range of organisms, displays the strong fluorescence of the native protein. The residues that become the chromophore on exposure to oxygen are buried deep within the alpha-helix, thus it is protected from photo bleaching. Apart from many advantages, there are some limitations due to the nature of the protein. There is no amplification process in the detection of the fluorescence (i.e. there is only one fluorophore per GFP protein molecule.)

INSTRUMENTATION AND WORKING.

The setup consists of an inverted microscope, confocal scan head which includes scanning mirrors, dichoric mirrors, pinhole, barrier filters and photomultiplier.

1. Scanning mirrors: - there are two scanning mirrors that scan the focused laser light in the x-x direction and x-y direction respectively. the laser must be aligned central to the axis of movement of these mirrors

.

2. Dichotic mirrors :- these are used to direct the laser light towards the objective and to separate the returning fluorescent light into its color components.

3. Barrier filters : - are simply the pieces of colored glass that absorb particular wavelength of light and allow transmission of other wavelengths. These can be broad specificity that allow either short wavelengths to pass through (short pass filters).or they can allow longer wavelengths to pass through (long pass filters).others may allow only narrow range of wavelengths to pass through (narrow band pass filters).

4. Pinhole: - is placed in front of the PMT. The variable size pinhole allows one to adjust the size for confocal scanning.

5. PMT: - are used as very sensitive light detectors. Each imaging channel in the confocal microscope has its own PMT. Fro example: - when dual labeling a dichotic mirror is used to split the light longer wavelength from the shorter wavelength, each of which is directed towards separate PMT tubes.

37

6. Lasers: - three different kinds of lasers are used which are as follows:-

Krypton argon laser: - provides great versatility due to the ready availability of three laser lines (488,568,647nm) these lines are used for single, double, or triple labeling. Each of the laser lines can be selected individually or in combination using suitable filter wheels.

Argon ion laser: - it is suitable for dual labeling and simultaneous transmission imaging. there may be some emission signal bleed through into the other detector due to relatively close wavelengths of the two laser lines(488 and 514nm)

Helium neon laser: - is supplied as a single wavelength laser of either green or red. Used in combination with argon ion laser it is possible to perform triple labeling.

The selected laser lines are focused on the sample using a high quality objective lens. The laser light is scattered in all directions as it strikes the sample. Some of the reflected light can be used to form an image (back scatter image), particularly of the surface topology of the sample. A large portion of laser light passes directly from the sample and is picked up by the PMT.This transmitted light can be used to create the image depicted as gray scale. A small portion of laser light will result in excitation of the fluorophore used to label the sample. The fluorescent light from the labeled sample is emitted in all directions. A small portion of this light is directed towards the objective, back into the scan head where it is spilt into different colors using dichotic mirrors and detected by using highly sensitive PMTS.

IMAGE FORMATION

FLUORESCNECE IMAGING:- in this imaging mode, the selected laser light is scanned on the sample, and the fluorescent light generated from the molecular probe is directed back ,separated into its components and detected by the photomultiplier tubes.

38

TRANSMISSION IMAGING: - gray scale transmission images obtained when laser light is transmitted through the sample. This image will appear out of focus "blur” long before the fluorescence is brought into focus. This image is used effectively to locate the position of the fluorescence label.

BACK SCATTER IMAGING: - image formed by the reflected laser rays after hitting the sample. They show better resolution than transmission images. It allows one to create a series of optical slices. Unstained living cells can be imaged using reflectance mode.

ADVANTAGES OF CONFOCAL MICROSCOPY

The advantages include better resolution, greatly increased sensitivity, obtaining three dimensional information of the cell, quantization of the label.

LIMITATIONS OF CONFOCAL MICROSCOPY

The major limitation is that the resolution is limited by the wavelength of the light, it does not resolve better than 0.1 micrometers. Many sub cellular structures in biology are at or beyond this limit. Objects smaller than this resolution can be viewed is suitable dye is used, but if they are not resolved then two closely associated structures will appear as one.

Another limitation is that a reporter molecule is needed for detection. This molecule has a bulky fluorescence group in addition to the binding site for the molecule of interest. The reporter molecule may alter the location and concentration of the ligand of interest by binding to it.

39

APPLICATIONS WITH EXAMPLES STUDIED.

1.MULTIPLE LABELING:- In this technique, different sub cellular compartments or proteins can be labeled and then imaged simultaneously. The images obtained from multiple labeling, are obtained as separate gray scale images that can be readily merged to create multi color composites images when needed.

2.IMMUNOLABELLING :- We know that antibodies provide specificity for identifying molecules or proteins of interest. There are many ways of visualizing these high specificity antibodies by microscopy by attaching fluorescent probe to the antibody. The probe can be attached to the secondary antibody which has high specificity for primary antibody or it can be attached directly to the primary antibody. These immunofluorescence methods are commonly called as immunofluorescence antibody assays. Immunolabelling can be sometimes combined in dual or triple labeling experiments. for example:- with propidium iodide for staining DNA , or with MitoTracker red for staining the mitochondria.

40

3.PROTEIN COLOCALISATION:- This technique is done by labeling different proteins with different probes and then merging the two colors. If colors are merged well, it indicates that the respective proteins are present at the same location or they are colocalised.

41

TRANSMISSION ELECTRON MICROSCOPY

INTRODUCTION

The unaided human eye can distinguish two points of 0.2mm apart. This distance is called as resolution or resolving power of the eye. By using lens or assembly of lenses in a microscope, it is possible to enlarge this distance to differentiate the points even closer than 0.2mm. Since the resolution of a light microscope cannot be less than half of the wavelength, hence it is around 200nm i.e. 1000 times as compared to the eye.

42

PRINCIPLE FOR ELECTRON MICROSCOPY

It was discovered in 1920s that the accelerated electrons behave like light in the vacuum. They travel in straight lines and have wavelength which is about 100,000 times smaller than that of light. The electric and the magnetic field have the same effect on electrons as glass lenses and mirrors have on visible light. The complex interaction between the accelerated electrons and the specimen results in various physical products like secondary electrons, X-rays, back scattered electrons and auger electrons. Moving electrons are source of illumination is evident from the hypothesis advanced by De-Broglie that moving particles have a definite wave motion.

λ=h/mv where λ= wavelength of particles

h=Planck’s constant

m=mass of electrons

v=velocity of electrons.

INSTRUMENTATION AND WORKING OF TEM.

The basic unit has microscope column, vacuum system and control panel.

Power supply unit:- which generates power supply to various parts of the basic unit including voltage to the electron gun.

Electromagnetic lenses and heaters generate heat. To cool these down, chilled water is circulated. This is done with the help of water chiller. Since the valves at various points in the column are to be opened and closed it is done with the help of the air compressor The microscope column consists of following parts:-

Illumination system:- this system contains two units, the electron gun which is the source of electrons, and the condenser which regulates the intensity of the beam and directs it on the specimen. The electron gun has two components the filament and the anode. The filament (cathode) is V-shaped piece of pure tungsten wire. When voltage is applied, it gets heated up and the electron emission starts. The emitted electrons forms a cloud around the tip of

43

the filament, which is propelled down the column, due to high voltage potential generated(40-120kv) between the cathode and the anode. The electron beam emerging from the gun is focused onto the specimen for illumination by the condenser lens. The intensity of illumination can be adjusted by changing the focal length of the condenser lens. it also depends on the aperture size, more electrons will pass through the condenser and higher will be the intensity of illumination.

Imaging system:- the objective lens forms the initial enlarged image of the illuminated portion of the specimen in a plane that is suitable for further enlargement by the projector lens. by varying the current in the objective lens, the image of the specimen is focused. The projector lens projects the final magnified image on the screen or the photographic emulsion. By varying the current in the projector lens, the size of the image can be varied.

Image translating system: - the information which the electron microscope provides is contained in the final image in the form of variations of electron intensity over its area. To see the information contained by the transmitted electrons they must be converted into visible light. This is done by the fluorescence screen. This screen when bombarded with electrons emits photons of visible light which can be seen by eyes. The image thus seen can be recorded on photographic film which is coated with photosensitive silver halide. The electron beam liberates free silver from silver halides grains, which after developing produces negatives. These negatives can be further processed to get positives on the photographic paper. This image recorded on the photographic paper is known as the electron micrograph.

APPLICATIONS:-

Various biological samples can be studied with the help of electron microscopy. They are viewed at less than 200kv as they may get ruptured at higher voltage. Specific organelles and other sub cellular structures can be examined.

Techniques like immunolabelling are performed with the help of electron microscopy.

It is commonly used in material sciences for determining the shape of the nanoparticle, crystal lattice, inter atomic distances etc.

44

X-ray fluorescence

X-ray fluorescence (XRF) is the emission of characteristic "secondary" (or fluorescent) X-rays from a material that has been excited by bombarding with high-energy X-rays or gamma rays. The phenomenon is widely used for elemental analysis and chemical analysis, particularly in the investigation of metals, glass, ceramics and building materials, and for research in geochemistry, forensic science and archaeology.

The physics of XRF

45

Physics of X-ray fluorescence, in a schematic representation.

When materials are exposed to short-wavelength x-rays or to gamma rays, ionization of their component atoms may take place. Ionization consists of the ejection of one or more electrons from the atom, and may take place if the atom is exposed to radiation with energy greater than its ionization potential. X-rays and gamma rays can be energetic enough to expel tightly-held electrons from the inner orbitals of the atom. The removal of an electron in this way renders the electronic structure of the atom unstable, and electrons in higher orbitals "fall" into the lower orbital to fill the hole left behind. In falling, energy is released in the form of a photon, the energy of which is equal to the energy difference of the two orbital’s involved. Thus, the material emits radiation, which has energy characteristic of the atoms present. The term fluorescence is applied to phenomena in which the absorption of higher-energy radiation results in the re-emission of lower-energy radiation.

Figure 1: Electronic transitions in a calcium atom. Remember, when electrons are jumping down, one of the electrons in the lower orbital is missing!

Characteristic radiation

Each element has electronic orbital’s of characteristic energy. Following removal of an inner electron by an energetic photon provided by a primary radiation source, an electron from an outer shell drops into its place. There are a limited number of ways in which this can happen, as shown in figure 1. The main transitions are given names: an L→K transition

46

is traditionally called Kα, an M→K transition is called Kβ, an M→L transition is called Lα, and so on. Each of these transitions yields a fluorescent photon with a characteristic energy equal to the difference in energy of the initial and final orbital. The wavelength of this fluorescent radiation can be calculated from Planck's Law:

The fluorescent radiation can be analyzed either by sorting the energies of the photons (energy-dispersive analysis) or by separating the wavelengths of the radiation (wavelength-dispersive analysis). Once sorted, the intensity of each characteristic radiation is directly related to the amount of each element in the material. This is the basis of a powerful technique in analytical chemistry. Figure 2 shows the typical form of the sharp fluorescent spectral lines obtained in the wavelength-dispersive method (see Moseley's law).

Primary radiation

In order to excite the atoms, a source of radiation is required, with sufficient energy to expel tightly-held inner electrons. Conventional x-ray tubes are most commonly used, because their output can readily be "tuned" for the application, and because very high power can be deployed. However, gamma ray sources can be used without the need for an elaborate power supply, allowing use in small portable instruments. When the energy source is a synchrotron, or the X-ray is focused by an optic, like a polycarpellary, the X-ray beam can be very small and very intense, and atomic information on the sub-micrometer scale can be obtained. X-ray tubes are operated at a high voltage (typically in the range 20-60 kV) in order to obtain a more or less continuous spectrum, which allows excitation of a broad range of atoms. The continuous spectrum consists of "bremsstrahlung" radiation: radiation produced when high energy electrons passing through the tube are progressively decelerated by the material of the tube anode (the "target").

Dispersion

In energy dispersive analysis, the fluorescent x-rays emitted by the material sample are directed into a solid-state detector which produces a "continuous" distribution of pulses, the voltages of which are proportional to the incoming photon energies. This signal is processed by a multichannel analyzer (MCA) which produces an accumulating digital spectrum that can be processed to obtain analytical data. In wavelength dispersive analysis, the

47

fluorescent x-rays emitted by the material sample are directed into a diffraction grating monochromatic. The diffraction grating used is usually a single crystal. By varying the angle of incidence and take-off on the crystal, a single x-ray wavelength can be selected. The wavelength obtained is given by the Bragg Equation:

where d is the spacing of atomic layers parallel to the crystal surface.

Detection

In energy dispersive analysis, dispersion and detection are a single operation, as already mentioned above. Proportional counters or various types of solid state detectors (PIN-diode, Si(Li), Ge(Li), Silicon Drift Detector SDD) are used. They all share the same detection principle: An incoming x-ray photon ionizes a large number of detector atoms with the amount of charge produced being proportional to the energy of the incoming photon. The charge is then collected and the process repeats itself for the next photon. Detector speed is obviously critical; as all charge carriers measured have to come from the same photon to measure the photon energy correctly (peak length discrimination is used to eliminate events that seem to have been produced by two x-ray photons arriving almost simultaneously). The spectrum is then built up by dividing the energy spectrum into discreet bins and counting the number of pulses registered within each energy bin. EDXRF detector types vary in resolution, speed and the means of cooling (a low number of free charge carriers is critical in the solid state detectors): proportional counters with resolutions of several hundred eV cover the low end of the performance spectrum, followed by PIN-diode detectors, while the Si (Li), Ge(Li) and Silicon Drift Detectors (SDD) occupy the high end of the performance scale.

In wavelength dispersive analysis, the single-wavelength radiation produced by the monochromatic is passed into a photomultiplier, a detector similar to a Geiger counter, which counts individual photons as they pass through. The counter is a chamber containing a gas that is ionized by x-ray photons. A central electrode is charged at (typically) +1700 V with respect to the conducting chamber walls, and each photon triggers a pulse-like cascade of current across this field. The signal is amplified and transformed into an accumulating digital count. These counts are then processed to obtain analytical data.

48

X-ray intensity

The fluorescence process is inefficient, and the secondary radiation is much weaker than the primary beam. Furthermore, the secondary radiation from lighter elements is of low energy (long wavelength) and has low penetrating power, and is severely attenuated if the beam passes through air for any distance. Because of this, for high-performance analysis, the path from tube to sample to detector is maintained under high vacuum (around 10 Pa residual pressures). This means in practice that most of the working parts of the instrument have to be located in a large vacuum chamber. The problems of maintaining moving parts in vacuo, and of rapidly introducing and withdrawing the sample without losing vacuum, pose major challenges for the design of the instrument. For less demanding applications, or when the sample is damaged by a vacuum (e.g. a volatile sample), a helium-swept x-ray chamber can be substituted, with some loss of low-Z intensities.

XRF in chemical analysis

The use of a primary X-ray beam to excite fluorescent radiation from the sample was first proposed by Glocker and Schreiber in 1928[1]. Today, the method is used as a non-destructive analytical technique, and as a process control tool in many extractive and processing industries. In principle, the lightest element that can be analysed is beryllium (Z = 4), but due to instrumental limitations and low x-ray yields for the light elements, it is often difficult to quantify elements lighter than sodium (Z = 11), unless background corrections and very comprehensive interelement corrections are made.

Figure 4: Schematic arrangement of EDX spectrometer

49

Energy dispersive spectrometry

In energy dispersive spectrometers (EDX or EDS), the detector allows the determination of the energy of the photon when it is detected. Detectors historically have been based on silicon semiconductors, in the form of lithium-drifted silicon crystals, or high-purity silicon wafers.

Processing

Considerable computer power is dedicated to correcting for pulse-pile up and for extraction of data from poorly-resolved spectra. These elaborate correction processes tend to be based on empirical relationships that may change with time, so that continuous vigilance is required in order to obtain chemical data of adequate precision.

Usage

EDX spectrometers are superior to WDX spectrometers in that they are smaller, simpler in design and have fewer engineered parts. They can also use miniature X-ray tubes or gamma sources. This makes them cheaper and allows miniaturization and portability. This type of instrument is commonly used for portable quality control screening applications, such as testing toys for Lead (Pb) content, sorting scrap metals, and measuring the lead content of residential paint. On the other hand, the low resolution and problems with low count rate and long dead-time makes them inferior for high-precision analysis. They are, however, very effective for high-speed, multi-elemental analysis. Field Portable XRF analyzers currently on the market weigh less than 2 kg, and have limits of detection on the order of 2 parts per million of Lead (Pb) in pure sand.

Figure 6: Schematic arrangement of wavelength dispersive spectrometer

50

Wavelength dispersive spectrometry

In wavelength dispersive spectrometers (WDX or WDS), the photons are separated by diffraction on a single crystal before being detected. Although wavelength dispersive spectrometers are occasionally used to scan a wide range of wavelengths, producing a spectrum plot as in EDS, they are usually set up to make measurements only at the wavelength of the emission lines of the elements of interest. This is achieved in two different ways:

"Simultaneous" spectrometers have a number of "channels" dedicated to analysis of a single element, each consisting of a fixed-geometry crystal monochromatic, a detector, and processing electronics. This allows a number of elements to be measured simultaneously, and in the case of high-powered instruments, complete high-precision analyses can be obtained in under 30 s. Another advantage of this arrangement is that the fixed-geometry monochromators have no continuously-moving parts, and so are very reliable. Reliability is important in production environments where instruments are expected to work without interruption for months at a time. Disadvantages of simultaneous spectrometers include relatively high cost for complex analyses, since each channel used is expensive. The number of elements that can be measured is limited to 15-20, because of space limitations on the number of monochromators that can be crowded around the fluorescing sample. The need to accommodate multiple monochromators means that a rather open arrangement around the sample is required, leading to relatively long tube-sample-crystal distances, which leads to lower detected intensities and more scattering. The instrument is inflexible, because if a new element is to be measured, a new measurement channel has to be bought and installed.

"Sequential" spectrometers have a single variable-geometry monochromator (but usually with an arrangement for selecting from a choice of crystals), a single detector assembly (but usually with more than one detector arranged in tandem), and a single electronic pack. The instrument is programmed to move through a sequence of wavelengths, in each case selecting the appropriate X-ray tube power, the appropriate crystal, and the appropriate detector arrangement. The length of the measurement program is essentially unlimited, so this arrangement is very flexible. Because there is only one monochromator, the tube-sample-crystal distances can be kept very short,

51

resulting in minimal loss of detected intensity. The obvious disadvantage is relatively long analysis time, particularly when many elements are being analyzed, not only because the elements are measured in sequence, but also because a certain amount of time is taken in readjusting the monochromatic geometry between measurements. Furthermore, the frenzied activity of the monochromator during an analysis program is a challenge for mechanical reliability. However, modern sequential instruments can achieve reliability almost as good as that of simultaneous instruments, even in continuous-usage applications.

Sample presentation

In order to keep the geometry of the tube-sample-detector assembly constant, the sample is normally prepared as a flat disc, typically of diameter 20-50 mm. This is located at a standardized, small distance from the tube window. Because the X-ray intensity follows an inverse-square law, the tolerances for this placement and for the flatness of the surface must be very tight in order to maintain a repeatable X-ray flux. Ways of obtaining sample discs vary: metals may be machined to shape, minerals may be finely ground and pressed into a tablet, and glasses may be cast to the required shape. A further reason for obtaining a flat and representative sample surface is that the secondary X-rays from lighter elements often only emit from the top few micrometers of the sample. In order to further reduce the effect of surface irregularities, the sample is usually spun at 5-20 rpm. It is necessary to ensure that the sample is sufficiently thick to absorb the entire primary beam. For higher-Z materials, a few millimeters thickness is adequate, but for a light-element matrix such as coal, a thickness of 30-40 mm is needed.

Figure 7: Bragg diffraction condition

52

Example studied

Following is the result obtained by EDXRF of the soil near Yamuna River. It shows that iron and manganese are present in large amount in the sample. Copper, nickel, zirconium, etc are present in the trace amounts. So by the application of XRF, we can get the various component of the sample. The obtained concentration can be compared with standard concentration of each component. The increase in either of component can lead to pollution. And we emanate spate step in order to nullify the reasons leading to pollution.

Scanning electron microscope

53

The scanning electron microscope (SEM) is a type of electron microscope that images the sample surface by scanning it with a high-energy beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition and other properties such as electrical conductivity.

The types of signals produced by an SEM include secondary electrons, back scattered electrons (BSE), characteristic x-rays, light (cathodoluminescence), specimen current and transmitted electrons. These types of signal all require specialized detectors that are not usually all present on a single machine. The signals result from interactions of the electron beam with atoms at or near the surface of the sample. In the most common or standard detection mode, secondary electron imaging or SEI, the SEM can produce very high-resolution images of a sample surface, revealing details about 1 to 5 nm in size. Due to the way these images are created, SEM micrographs have a very large depth of field yielding a characteristic three-dimensional appearance useful for understanding the surface structure of a sample. This is exemplified by the micrograph of pollen shown to the right. A wide range of magnifications is possible, from about x 25 (about equivalent to that of a powerful hand-lens) to about x 250,000, about 250 times the magnification limit of the best light microscopes. Back-scattered electrons (BSE) are beam electrons that are reflected from the sample by elastic scattering. BSE are often used in analytical SEM along with the spectra made from the characteristic x-rays. Because the intensity of the BSE signal is strongly

54

related to the atomic number (Z) of the specimen, BSE images can provide information about the distribution of different elements in the sample. For the same reason, BSE imaging can image colloidal gold immune-labels of 5 or 10 nm diameter which would otherwise be difficult or impossible to detect in secondary electron images in biological specimens. Characteristic X-rays are emitted when the electron beam removes an inner shell electron from the sample, causing a higher energy electron to fill the shell and release energy. These characteristic x-rays are used to identify the composition and measure the abundance of elements in the sample.

Scanning process and image formation

In a typical SEM, an electron beam is thermionic ally emitted from an electron gun fitted with a tungsten filament cathode. Tungsten is normally used in thermionic electron guns because it has the highest melting point and lowest vapor pressure of all metals, thereby allowing it to be heated for electron emission, and because of its low cost. Other types of electron emitters include lanthanum hex boride (LaB6) cathodes, which can be used in a standard tungsten filament SEM if the vacuum system is upgraded and field emission guns (FEG), which may be of the cold-cathode type using tungsten single crystal emitters or the thermally-assisted Scotty type, using emitters of zirconium oxide.

The electron beam, which typically has an energy ranging from a few hundred eV to 40 keV, is focused by one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. The beam passes through pairs of scanning coils or pairs of deflector plates in the electron column, typically in the final lens, which deflect the beam in the x and y axes so that it scans in a raster fashion over a rectangular area of the sample surface.

When the primary electron beam interacts with the sample, the electrons lose energy by repeated random scattering and absorption within a teardrop-shaped volume of the specimen known as the interaction volume, which extends from less than 100 nm to around 5 µm into the surface. The size of the interaction volume depends on the electron's landing energy, the atomic number of the specimen and the specimen's density. The energy exchange between the electron beam and the sample results in the reflection of high-energy electrons by elastic scattering, emission of secondary electrons by inelastic scattering and the emission of electromagnetic radiation, each of which can be detected by specialized detectors. The beam current absorbed by the specimen can also be detected and used to create images of the distribution of specimen current. Electronic amplifiers of various types

55

are used to amplify the signals which are displayed as variations in brightness on a cathode ray tube. The raster scanning of the CRT display is synchronised with that of the beam on the specimen in the microscope, and the resulting image is therefore a distribution map of the intensity of the signal being emitted from the scanned area of the specimen. The image may be captured by photography from a high resolution cathode ray tube, but in modern machines is digitally captured and displayed on a computer monitor and saved to a computer's hard disc.

Magnification

Magnification in a SEM can be controlled over a range of up to 6 orders of magnitude from about x25 to x 250,000 and exceptionally to 2 million times in the Hitachi S-5500 in-lens Field Emission SEM, imaging a specimen area about 60nm wide with resolution up to 0.4 nm. Unlike optical and transmission electron microscopes, image magnification in the SEM is not a function of the power of the objective lens. SEMs may have condenser and objective lenses, but their function is to focus the beam to a spot, and not to image the specimen. Provided the electron gun can generate a beam with sufficiently small diameter, a SEM could in principle work entirely without condenser or objective lenses, although it might not be very versatile or achieve very high resolution. In a SEM, as in scanning probe microscopy, magnification results from the ratio of the dimensions of the raster on the specimen and the raster on the display device. Assuming that the display screen has a fixed size, higher magnification results from reducing the size of the raster on the specimen, and vice versa. Magnification is therefore controlled by the current supplied to the x,y scanning coils, and not by objective lens power.

Sample preparation

56

An insect coated in gold, having been prepared for viewing with a scanning electron microscope.

An instrument for carbon coating

All samples must also be of an appropriate size to fit in the specimen chamber and are generally mounted rigidly on a specimen holder called a specimen stub. Several models of SEM can examine any part of a 6-inch (15 cm) semiconductor wafer, and some can tilt an object of that size to 45 degrees.

For conventional imaging in the SEM, specimens must be electrically conductive, at least at the surface, and electrically grounded to prevent the accumulation of electrostatic charge at the surface. Metal objects require little special preparation for SEM except for cleaning and mounting on a specimen stub. Nonconductive specimens tend to charge when scanned by the electron beam, and especially in secondary electron imaging mode, this causes scanning faults and other image artifacts. They are therefore usually coated with an ultrathin coating of electrically-conducting material, commonly gold, deposited on the sample either by low vacuum sputter coating or by high vacuum evaporation. Conductive materials in current use for specimen coating include gold, gold/palladium alloy, platinum, osmium,[5] iridium, tungsten, chromium and graphite. Coating prevents the accumulation of static electric charge on the specimen during electron irradiation.

Two important reasons for coating, even when there is more than enough specimen conductivity to prevent charging, are to maximize signal and improve spatial resolution, especially with samples of low atomic number (Z). Broadly, signal increases with atomic number, especially for backscattered electron imaging. The improvement in resolution arises because in low-Z materials such as carbon, the electron beam can penetrate several

57

micrometers below the surface, generating signals from an interaction volume much larger than the beam diameter and reducing spatial resolution. Coating with a high-Z material such as gold maximizes secondary electron yield from within a surface layer a few nm thick, and suppresses secondary electrons generated at greater depths, so that the signal is predominantly derived from locations closer to the beam and closer to the specimen surface than would be the case in an uncoated, low-Z material. These effects are particularly, but not exclusively, relevant to biological samples.

An alternative to coating for some biological samples is to increase the bulk conductivity of the material by impregnation with osmium using variants of the OTO process. No conducting specimens may be imaged uncoated using specialized SEM instrumentation such as the "Environmental SEM" (ESEM) or field emission gun (FEG) SEMs operated at low voltage. Environmental SEM instruments place the specimen in a relatively high pressure chamber where the working distance is short and the electron optical column is differentially pumped to keep vacuum adequately low at the electron gun. The high pressure region around the sample in the ESEM neutralizes charge and provides an amplification of the secondary electron signal. Low voltage (LV) SEM of non-conducting specimens can be operationally difficult to accomplish in a conventional SEM and is typically a research application for specimens that are sensitive to the process of applying conductive coatings. LV-SEM is typically conducted in an FEG-SEM because the FEG is capable of producing high primary electron brightness even at low accelerating potentials. Operating conditions must be adjusted such that the local space charge is at or near neutral with adequate low voltage secondary electrons being available to neutralize any positively charged surface sites. This requires that the primary electron beam's potential and current be tuned to the characteristics of the sample specimen.

Embedding in a resin with further polishing to a mirror-like finish can be used for both biological and materials specimens when imaging in backscattered electrons or when doing quantitative X-ray microanalysis.

Biological samples

For SEM, a specimen is normally required to be completely dry, since the specimen chamber is at high vacuum. Hard, dry materials such as wood, bone, feathers, dried insects or shells can be examined with little further treatment, but living cells and tissues and whole, soft-bodied organisms usually require chemical fixation to preserve and stabilize

58

their structure. Fixation is usually performed by incubation in a solution of a buffered chemical fixative, such as glutaraldehyde, sometimes in combination with formaldehyde and other fixatives, and optionally followed by post fixation with osmium peroxide. The fixed tissue is then dehydrated. Because air-drying causes collapse and shrinkage, this is commonly achieved by critical point drying, which involves replacement of water in the cells with organic solvents such as ethanol or acetone, and replacement of these solvents in turn with a transitional fluid such as liquid carbon dioxide at high pressure. The carbon dioxide is finally removed while in a supercritical state, so that no gas-liquid interface is present within the sample during drying. The dry specimen is usually mounted on a specimen stub using an adhesive such as epoxy resin or electrically-conductive double-sided adhesive tape, and sputter coated with gold or gold/palladium alloy before examination in the microscope.

If the SEM is equipped with a cold stage for cryo-microscopy, cry fixation may be used and low-temperature scanning electron microscopy performed on the cryogenically fixed specimens. Cryo-fixed specimens may be cryo-fractured under vacuum in a special apparatus to reveal internal structure, sputter coated and transferred onto the SEM cryo-stage while still frozen. Low-temperature scanning electron microscopy is also applicable to the imaging of temperature-sensitive materials such as ice (see e.g. illustration at right) and fats.

Freeze-fracturing, freeze-etch or freeze break is a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. The preparation method reveals the proteins embedded in the lipid bilayer.

Gold has a high atomic number and sputter coating with gold produces high topographic contrast and resolution. However, the coating has a thickness of a few micrometres, and can obscure the underlying fine detail of the specimen at very high magnification. Low-vacuum SEMs with differential pumping apertures allow samples to be imaged without such coating and without the loss of natural contrast caused by the coating, but are unable to achieve the

resolution attainable by conventional SEMs with coated specimens.

Materials

Back scattered electron imaging, quantitative x-ray analysis, and x-ray mapping of geological specimens and metals requires that the surfaces be ground and polished to an

59

ultra smooth surface. Geological specimens that undergo WDS or EDS analysis are often carbon coated. Metals are not generally coated prior to imaging in the SEM because they are conductive and provide their own pathway to ground.

Fractography is the study of fractured surfaces that can be done on a light microscope or commonly, on an SEM. The fractured surface is cut to a suitable size, cleaned of any organic residues, and mounted on a specimen holder for viewing in the SEM.

Integrated circuits may be cut with a FIB or other ion beam milling instrument for viewing in the SEM. The SEM in the first case may be incorporated into the FIB.

Metals, geological specimens, and integrated circuits all may also be chemically polished for viewing in the SEM.

Special high resolution coating techniques are required for high magnification imaging of inorganic thin films.

ESEM

The accumulation of electric charge on the surfaces of non-metallic specimens can be avoided by using environmental SEM in which the specimen is placed in an internal chamber at higher pressure than the vacuum in the electron optical column. Positively charged ions generated by beam interactions with the gas help to neutralize the negative charge on the specimen surface. The pressure of gas in the chamber can be controlled, and the type of gas used can be varied according to need. Coating is thus unnecessary, and X-ray analysis unhindered.

Detection of secondary electrons

The most common imaging mode collects low-energy (<50 eV) secondary electrons that are ejected from the k-orbital’s of the specimen atoms by inelastic scattering interactions with beam electrons. Due to their low energy, these electrons originate within a few nanometers from the sample surface. The electrons are detected by an Everhart-Thornley detector which is a type of scintillator-photomultiplier system. The secondary electrons are first collected by attracting them towards an electrically-biased grid at about +400V, and then further accelerated towards a phosphor or scintillator positively biased to about +2000V. The

60

accelerated secondary electrons are now sufficiently energetic to cause the scintillator to emit flashes of light (cathodoluminescence) which are conducted to a photomultiplier outside the SEM column via a light pipe and a window in the wall of the specimen chamber. The amplified electrical signal output by the photomultiplier is displayed as a two-dimensional intensity distribution that can be viewed and photographed on an analogue video display, or subjected to analog-to-digital conversion and displayed and saved as a digital image. This process relies on a raster-scanned primary beam. The brightness of the signal depends on the number of secondary electrons reaching the detector. If the beam enters the sample perpendicular to the surface, then the activated region is uniform about the axis of the beam and a certain number of electrons "escape" from within the sample. As the angle of incidence increases, the "escape" distance of one side of the beam will decrease, and more secondary electrons will be emitted. Thus steep surfaces and edges tend to be brighter than flat surfaces, which results in images with a well-defined, three-dimensional appearance. Using this technique, image resolution less than 1 nm is possible.

Detection of backscattered electrons

Comparison of SEM techniques:* Top: backscattered electron analysis - composition* Bottom: secondary electron analysis - topography

Backscattered electrons (BSE) consist of high-energy electrons originating in the electron beam, that are reflected or back-scattered out of the specimen interaction volume by elastic scattering interactions with specimen atoms. Since heavy elements (high atomic number) backscatter electrons more strongly than light elements (low atomic number), and thus appear brighter in the image, BSE are used to detect contrast between areas with different chemical compositions.[16] The Everhart-Thornley detector, which is normally positioned to one side of the specimen, is inefficient for the detection of backscattered electrons because few such electrons are emitted in the solid angle subtended by the detector, and because the

61

positively biased detection grid has little ability to attract the higher energy BSE electrons. Dedicated backscattered electron detectors are positioned above the sample in a "doughnut" type arrangement, concentric with the electron beam, maximizing the solid angle of collection. BSE detectors are usually either of scintillator or semiconductor types. When all parts of the detector are used to collect electrons symmetrically about the beam, atomic number contrast is produced. However, strong topographic contrast is produced by collecting back-scattered electrons from one side above the specimen using an asymmetrical, directional BSE detector; the resulting contrast appears as illumination of the topography from that side. Semiconductor detectors can be made in radial segments that can be switched in or out to control the type of contrast produced and its directionality.

Backscattered electrons can also be used to form an electron backscatter diffraction (EBSD) image that can be used to determine the crystallographic structure of the specimen.

Example studied

The oily samples were filtered and the solids subjected to analysis by SEM/EDS.The new unused oil contained large amounts of agglomerated carbon richComponents. While the used samples contained large amounts of carbon, sulfurAnd phosphorus rich particles. The below given image shows the SEM of particle in used oil. So basically the comparison can be done between the component of used oil and fresh oil. It is concluded that, fresh oil contains; carbon containing component whereas used oil contains carbon, sulfur and phosphorus.

62

SEM of Large Carbon, Sulfur and Phosphorus Rich VarnishParticle. The background shows 0.2 micron holes in silver filter pad.

SEM of Large Carbon Rich Polymer (fiber like)Substance in Fresh Oil