Reference Books X-ray Methods

① Instrumental Methods of Analysis (5th Edition, Chapter 10)

By Bobart H. Willard, Lynne L. Merrit, John A. Dean
② Principles of Instrumental Analysis (6th Edition, Chapter 12)
by Skoog, Holler and Crouch
③ Modern Instrumental Analysis (Volume 47, Chapter 7)
by S. Ahuja and N. Jespersen
Find other reference book from the library, related to this topic.

1
Content X-ray Methods
• Fundamental principles • Type of scattering of x-rays:
•Ways to excite atoms Elastic & Inelastic

•Production of x-ray • Diffraction of x-ray (Bragg’s Law):

Constructive &
• Characteristics of wavelengths Destructive Interference
•X-ray beam on crystal structure •Instrumentation (X-ray spectroscopy)
• Methods of x-ray analysis: •X-ray diffraction:
(1) X-ray emission spectroscopy (XES) Sample preparation
(2) Auger emission spectroscopy (AES) Analysis procedure
(3) X-ray fluorescence spectroscopy • X-ray crystallography:
(XRF)
Factors inhibit the crystallization
(4) Electron spectroscopy for chemical
analysis (ESCA) •X-ray fluorescence (XRF)
(5) X-ray absorption Principles, advantages, example
(6) X-ray diffraction
2
Fundamental principles X-ray Methods
• X-ray spectroscopy is based on measurement of emission,
absorption, scattering, fluorescence, and diffraction of
electromagnetic radiation.

• When an atom is excited by the removal of an electron from an

inner energy level, it may return to its normal state by:
(1) Transferring an electron from The energy transition appears as:
some outer level to the vacant X-ray emission spectroscopy
inner level (XES).
(2) Ejecting a second electron The energy transition appears as:
Auger emission spectroscopy
from the outer shell (AES).

• X-ray wavelengths are characteristic of certain element

QUALITATIVE

• X-ray intensities are proportional to the number of excited atoms

QUANTITATIVE 3
Production of x-ray (Schematic #1) X-ray Methods

2 Accelerated electrons
Hit metal target (anode)

1 This circuit are used

to heat the filament & X-rays
to accelerate the electrons
to the target (anode) 3 X-rays are produced
(also known as Bremsstrahlung)

Schematic of x-ray tube 4

(Coolidge tube) Clearer figure on next page…
Production of x-ray (Schematic #2) X-ray Methods
• X-ray tube can be in a variety of shapes and forms.
• Carried out in vacuum. Coolant

Ground

Anode (+) Window

Glancing
θ angle
Electron beam X-ray beam
Slit
Hot tungsten
cathode (-) Shield

Glass or Evacuated volume

ceramic insulator
Schematic of x-ray tube (Coolidge tube) 5
Characteristics of wavelengths X-ray Methods

• X-rays have wavelength in the range of 0.01 to 10 nanometers.

• Corresponding to frequencies in the range 30 petahertz to

30 exahertz (3 x 1016 Hz to 3 x 1019 Hz).

• Energies in the range 120 eV to 120 keV.

6
X-ray beam on crystal structure X-ray Methods
• Every atom in a crystal structure scatters an x-ray beam incident
upon it in all directions.

① ②
Incident x-ray beam Reflected x-ray beam

θ θ A
Crystal plane

d θ θ
Lattice C D
B Crystal plane
spacing
d
dsinθ
Crystal plane
Diffraction of x-rays from a set of crystal planes (Bragg’s Law) 7
Methods of x-ray analysis X-ray Methods
(1) X-ray emission spectroscopy (XES)
•Primary electron beams eject e- from inner energy level.
•Secondary x-rays are emitted as outer-level e- fall into
vacant inner levels.
(2) Auger emission spectroscopy (AES)
•Primary x-rays eject e- from inner energy level.
•When e- fall into vacant inner levels by nonradioactive process,
excess energy ejects electrons from outer levels.
Primary
electron Primary X-ray

Outer
level

Inner
level
Primary Secondary
process process
8
XES AES 8
Methods of x-ray analysis X-ray Methods
(3) X-ray fluorescence spectroscopy (XRF)
•Primary x-rays eject e- from inner energy level.
•X-rays are emitted as e- fall from outer levels to vacant inner
level.
(4) Electron spectroscopy for chemical analysis (ESCA)
•Primary x-rays eject e- from inner energy level.
•Energy of the emitted e- is measured.
Primary X-ray Primary X-ray

XRF ESCA 9
Methods of x-ray analysis X-ray Methods
(5) X-ray absorption
•The intensity of x-rays is diminished (lessen) as they pass
through material.
•Discontinuities in absorptivity occur when x-rays have
sufficient energy to eject e- as in XES, AES, XFS, or ESCA.

(6) X-ray diffraction

•X-rays are diffracted by the planes of a crystal.
Primary X-ray

Material

Primary X-ray

Planes of a
crystal

X-ray absorption X-ray diffraction 10

Type of scattering of x-rays X-ray Methods
Two type of scattering of x-rays:
(1) Elastic:
•The scattered x-rays have the same energy and thus same
wavelength, as the incident x-rays.
•Example: X-ray crystallography.

(2) Inelastic:
•The scattered x-rays have neither the same energy nor the
same wavelength, as the incident x-rays.
• Useful for probing such excitations matter, but not in
determining the distribution of scatterers within the matter,
which is the goal of X-ray crystallography.

When light hits the pattern it gets diffracted.

11
Diffraction of x-ray (Bragg’s Law) X-ray Methods
Crystal structure
•X-rays are diffracted by the planes of a crystals.
• Sir William Henry Bragg & William Lawrence Bragg - The 1915 Nobel Prize in
Physics:"for their services in the analysis of crystal structure by means of
X-rays."
Incident X-ray beam Reflected X-ray beam

θ θ A
Sir William Henry Bragg
Crystal
plane
Lattice
d θ θ
spacing D
C
Crystal
B
plane
d
dsinθ
Crystal
William Lawrence Bragg
plane
Diffraction of x-rays from a set of crystal planes (Bragg’s Law) 12
Diffraction of x-ray (Bragg’s Law) X-ray Methods
Constructive & Destructive Interference
• Atoms of a crystal are regularly arranged in space, as lying in parallel sheets
separated by a definite and defined distance.
• The effect of the constructive or destructive interference intensifies because
of the cumulative effect of reflection in successive crystallographic planes of
the crystalline lattice.
•The path difference between two waves undergoing interference is given by
2dsinθ, where θ = scattering angle.
•Bragg’s Law describes the condition on θ for the constructive interference
to be at its strongest: 2dsinθ = nλ Where : n = a positive integer
λ = wavelength of incident wave.
+
+
+
+ -

Constructive interferences Destructive interferences

According to the 2θ deviation, the phase shift causes constructive (left figure) or
destructive (right figure) interferences. 13
Diffraction of x-ray (Bragg’s Law) X-ray Methods

Conditions for diffraction

• d should be equal to λ

Contribution for diffraction

(1) Atoms located exactly on the crystal planes contribute
maximally to the intensity of the diffracted beam.

(2) Atoms located exactly halfway between the planes exert

maximum destructive interference.

(3) Those at some intermediate location interfere constructively or

destructively depending on their exact location but with less
than their maximum effect.

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Diffraction of x-ray (Bragg’s Law) X-ray Methods
Position of diffraction beam from a crystal depends on:
(1) the size and shape of the repetitive unit of crystal, and
(2)the wavelength of the incident x-rays beam.

Intensities of diffraction beam depends on:

(1) the type of atoms in the crystal, and
(2) the location of the atoms in the fundamental repetative unit.

15
Instrumentation (X-ray spectroscopy) X-ray Methods

Electric Voltage High- X-ray tube

power stabilizer voltage
generator Specimen
Current Slit (crystal
stabilizer Collimator powder)

Amplifier Scaler Printer

Ratemeter Recorder

Instrumentation for X-ray spectroscopy.

16
X-ray diffraction equipment (XRD) X-ray Methods
X-ray beam Detector

Sample stage

Sample holder (tower)

Only certified technicians are allowed to operate this equipment.
X-ray = radiation
nd
School of Physic, USM, 2 Floor.
Model : Bruker D8 Advance XRD.
Watch the video:
https://www.youtube.com/watch?v=u80SBINbTzQ
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Example of X-ray powder diffraction pattern X-ray Methods
Counts per second

Scattering angle (degrees 2θ)

Example of X-ray powder diffraction pattern

18
X-ray diffraction X-ray Methods

• Primary tool for the study of solid-state crystallography

(understanding of the structural aspects).

• To comprehend must Ability of a crystalline solid to

the power of this understand act as a diffraction grating for
technique the electromagnetic radiation
of appropriate wavelength.

• Application of x-ray diffraction :

(1) Characterization of single crystals.
(2) Study of powdered crystalline solids.

19
Instrumentation
X-ray diffraction spectroscopy and its components
(1) X-ray production tube
•Large vacuum tube containing heated cathode(-) & anode(+).

(2) Collimator
• Consist of a series of closely spaced paralell metal plates.
• Function to limit the divergence of the rays that reach crystal.

(a) Without a collimator. (b) With a collimator.

Collimators filters a stream of rays.
20
Instrumentation X-ray Methods
(3) Mount and rotator
•Mount made of glass, aluminium, or bronze.
•A modern approach is to scoop the crystal up in a tiny loop,
made of nylon or plastics and attached to a solid rod, that is
the flash-frozen with liquid nitrogen.
•This freezing reduces the radiation damage of the x-rays, as
well as the noise in the Bragg peaks due to thermal motion.
Crystal
Tiny loop

Mount
Rotator
21
Instrumentation X-ray Methods
(4) Detector
•Classical photographic film can be used to measure the
intensity of radiation.

•Record the scattered x-rays into a pattern of spots or reflections.

•Direct linear relationship between blackening of the developed

film and the intensity of radiation.

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 Instrumentation X-ray Methods
(4) Detector

A debye-Scherrer powder camera.

Holes in film for entrance and exit tubes.

The film strip after development. D2, D1, T indicate positions
of the film in the camera. 23
 Sample preparation X-ray Methods
(1) Crystalline sample is ground to a fine homogeneous powder.
(2) When X-ray beam passes through the material, a significant
number number of the particles are oriented in such ways as to
fullfill the Bragg condition for reflection.
(3) Samples are usually placed in a sample holder that uses a
depression or cavity to mount the sample.
(4) Excess powder are eliminated and the surface is flattened.
(5) Alternatively, samples may be mixed with non-crystalline binder
and molded into an appropriate shape.
Glass slide

Sample holder24
Homogenization Eliminate excess powder Flat surface 24
Analysis procedure X-ray Methods
(1) Multiple data sets have to be collected & each set containing
tens of thousands of reflections.
(2) These data are combined computationally with complementary
chemical information to produce & refine a model of the
arrangement of atoms within the crystal.

(3) One image of spots is insufficient to reconstruct the whole

crystal; it represents only a small slice of the full crystal.

(4) To collect all the necessary information, the crystal must be

rotated step-by-step through 180°, with an image recorded
at every step.

(5) However, if the crystal has a higher symmetry, a smaller

angular range such as 90° or 45° may be recorded.

25
X-ray crystallography X-ray Methods
Formation of macromolecular crystallization for x-ray
diffraction technique &
the factors that inhibit the crystallization:

• Numerous factors during crystal growth which affect both size &
quality of the crystal.

• The most important factors:

(1) in terms of organic compounds are purity
(2) the solvent chosen for recrystallization Will be discuss in
(3) the number of nucleation sites detail in the
(4) mechanical agitation to the system, and following pages...
(5) time.

26
X-ray crystallography X-ray Methods
Factors inhibit the crystallization:
(1)Purity
• Be sure that the flask and solvent are dust free.
• Eliminate other possible contaminant.

(2) The solvent chosen for recrystallization

• Typically, it is good to choose a solvent in which the compound is
moderately soluble.
• If the solute (a dissolve substance) is too soluble, crystal size
will tend to be small.
• Avoid solvents in which the compounds form saturated solutions,
because saturated solutions typically yield crystals which are too
small.
• Exception: When a compound is soluble in most liquids, then
slow cooling of saturated solutions can yield good
single crystals. 27
 X-ray crystallography X-ray Methods
Factors inhibit the crystallization:
(3)The number of nucleation sites
• The fewer nucleation sites at which crystals begin to grow results
in fewer crystals of large size. It is highly desirable to produce
large crystal size.

• Conversely, many nucleation sites result in a smaller average

crystal size, and it is not desirable.

• Dust in the laboratory and microscopic paper fillings (from paper

filters) are factors that cause to produce small crystals.

• Minimize dust and extraneous particulate matter in the solution

of the compound and the growing vessel.

28
 X-ray crystallography X-ray Methods
Factors inhibit the crystallization:
(4)Mechanical agitation
• Mechanical disturbance of the crystal growing vessel can result in
ruining all of the effort to produce large crystal size.

• Let the crystals grow with minimum disturbance.

→avoid placing the growing vessel next to the vacuum pump.

29
X-ray crystallography X-ray Methods
Workflow for solving the structure of a molecule by x-ray crystallography

Diffraction pattern

Crystal

Protein model or Electron density

Atomic model map

X-ray crystallography can be use to study the structure of a protein or

atomic model. 30
X-ray crystallography X-ray Methods
Information obtained from the analysis of single-crystal
x-ray diffraction:
(1) Information on the 3-D conformation of the compound.
The 3-D structure of penicillin, solved
by Dorothy Hodgkin in 1945.

(2)A complete summary of the bond angles (°) & bond length (Å)
of the compound.

Guanine-cytosine base pair.

(3) Detailed information regarding how the molecules assemble

to yield the complete crystal. 31
Flow chart of the x-ray crystallography process X-ray Methods
Workflow for solving the structure of a molecule by x-ray crystallography

Diffraction pattern

Crystal

Protein model or Electron density

Atomic model map

X-ray crystallography can be use to study the structure of a protein or

atomic model. 32
 Flow chart of the x-ray crystallography process X-ray Methods
(1) Selecting a crystal
-Crystal were examine under microscope
-Single crystal with clear shape were selected.
-Organic crystal 0.3 mm3 → mount on the difractometer
(2) Obtaining the unit cell
-Fast scanning: at least 10 data framework are collected and unit cell are determined.

(3) Determining the data collection strategy

-Computer will count the data collection strategy

(4) Data collection

-Reflections are collected from crystal lattice.
-Depends on several factors: symmetric crystal lattice, crystal structure, and type of
difractometer.
-Time required: 45 minutes-several days or weeks.

(5) Data Processing

-Combination, normalization, and background absorption correction are apply to the
collected data.

33
 Flow chart of the x-ray crystallography process X-ray Methods
(6) Obtaining .hkl file
-After data processing, one file .hkl are written, which contain all diffracted information.
(7) Determining the space group
-Based on the .hkl file, instructional file (.ins) are then prepared, which contain unit
cell dimension & contents of the crystal (type of element)
(8) Structure solution
-this process will produce result file (.res), which contain information regarding cell
unit, content of the crystal, & coordinates of atoms.
(9) Refinement
- Repetition process; small changes were made, based on the electron density
map, using .ins & .res files. (Statistics).
- Protein model or atomic model are then analyze for the interaction of hydrogen
bond and π arrangement.
(10) Generating final file
- .cif or .pdb (protein crystal)

(11)Publishing
i) Sending crystal data to the database, Cambridge Structural Database (CSD)

Related videos: Understanding Crystallography - Part 1: From Proteins to Crystals

https://www.youtube.com/watch?v=gLsC4wlrR2A 34
Flow chart of the x-ray crystallography process X-ray Methods
Activities:
(1) Why do we use x-ray radiation instead of light microscope to
analyze the dimensional structure of lysozyme?

35
Flow chart of the x-ray crystallography process X-ray Methods
Activities:
(1) Why do we use x-ray radiation instead of light microscope to
analyze the dimensional structure of lysozyme?
- The size of lysozyme is far to small to be observed under light microscope
- The wavelength of light is larger than the size of the lysozyme molecule.
- The wavelength of x-ray radiation is much smaller than wavelength of light,
thus, it will permit the observation of lysozyme structure.
Visible light wavelength 1.5x10-6 m X-ray wavelength 10-10 m

36
X-ray fluorescence (XRF) X-ray Methods
Principles & Advantages
(1) Non-destructive method
(not causing destruction of material being investigated or treated).
(2) XRF is an analytical method to determine the elemental analysis in all
kinds of materials in the form of solid (biomass fuel, ceramics, pigments
used in paintings), liquid (paint, oil, petrol blood, wine), powdered, filtered,
etc.

(3) XRF is a fast method which requires minimum sample amount and
simple sample preparation.

(4) The elements that can analysed and detected ranged from Beryllium to
Uranium (depends on the type or model of the instrument) and the
concentrations ranged from ppm to 100 %.

(5) High precision, high accuracy, and high reproducibility of results are
possible with good standard samples.

37
X-ray fluorescence (XRF) X-ray Methods
Principles & Advantages
Periodic table of elements
Atomic number

38
X-ray fluorescence (XRF) X-ray Methods
Example of result
X-ray fluorescence analysis

39
X-ray fluorescence (XRF) X-ray Methods
Example of result

SQX analysis result

Before
Content Analysis standardization
name value Unit

Total : 100 40