particle size sizing technique 2: hydrodynamic chromatography sieving microscopy
DESCRIPTION
Particle Size Sizing Technique 2: hydrodynamic chromatography sieving microscopy. Kausar Ahmad Kulliyyah of Pharmacy, IIUM http://staff.iiu.edu.my/akausar. Hydrodynamic Chromatography. HDC Operation. Results generated by HDC. Liposome PL-PSDA (Polymer Lab: HDC mechanism). - PowerPoint PPT PresentationTRANSCRIPT
Physical Pharmacy 2 1
Particle Size Sizing Technique 2:
hydrodynamic chromatography sieving
microscopy
Kausar Ahmad
Kulliyyah of Pharmacy, IIUM
http://staff.iiu.edu.my/akausar
Physical Pharmacy 2 2
Hydrodynamic Chromatographya technique for separating particles based on their size, eluting in the order: largest to smallest. – compare with SEC
Measures complex particle size distributions in the range 5nm to 2µm
Makes no assumptions regarding the shape of the particle size distribution
Results are independent of particle density
Analysis time less than 10 minutes
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HDC Operation
A water-based eluent
is continuously
pumped through the system at
constant flow rate.
sample & a small
molecule marker
solution, are introduced
into the system.
The unit contains a separating 'cartridge'.
dynamic operating
range from e.g. 20nm to
1.2µm.
UV detector response is
used to calculate
concentration of particles of different size
present in sample.
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Results generated by HDC
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LiposomePL-PSDA (Polymer Lab: HDC mechanism)
What is the volume average diameter?
What is the polydispersity index?
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Sieving POWDER can be separated
into various size fractions by vibrating sieve loaded with sample to enable the particles of size less than that of the mesh openings to pass through and the over size to remain in the sieve.
Dry sieving is adopted for free flowing powder samples of size range varying from e.g. 4mm down to 25μm.
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Sieves A standard test sieve is generally made of a
woven wire mesh cloth specified wire thickness with square openings
Standard sieves are made according to recommended norms to maintain opening size interval between successive sieves.
The ratio between successive sieves is kept as a constant such as 1.414 and hence the sieve size varies in geometric progression.
fixed to a rectangular or circular frame
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Sieving procedure
Sieves stacked
pana
b
c
d
e
f
g
Wire mesh
Minimum size
Maximum size
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Table of Sieve ResultsSieve no./size (um)
Weight of sieve (g)
Wt of sieve & sample (g)
Wt of sample (g)
% Wt of sample
% cumulative weight
(<25) na na a ax100/M ax100/M
25 x1 y1 b (a+b)x100/M
41 x2 y2 c (a+b+c)x100/M
60 x3 y3 d
116 x4 y4 e
450 x5 y5 f
2000 x6 y6 g 100
Total M
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Interpretation of Sieve Results
From the % cumulative weight, the mean diameter can be obtained i.e.
d(0.5) – the diameter at which 50% of the samples, based on weight falls below it.
Wei
ght
(%)
Cumulative weight (%)
Microscopyhttp://www.mos.org/sln/SEM/index.html
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To fly, moths must be light. A close-up of the Cecropia Moth scale shows that it is mostly air, adding very little weight to the moth
The deer tick, is about the size of a freckle.
The shells of radiolarians, single-celled animals.
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Principles of Microscopyhttp://micro.magnet.fsu.edu/primer/lightandcolor/polarizedlighthome.html
The microscope is an instrument designed to make fine details visible.
The Concept of Magnification
• The image of an object can be magnified when viewed through a simple lens.
• By combining a number of lenses in the correct manner, a microscope can be produced that will yield very high magnification values.
Lenses and Optics
• The action of a simple lens, is governed by the principles of refraction and reflection .
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Using light microscopy equipped with graticule to determine size
Comparison of microscopesFeature Light TEM SEM
Use morphology Small particles, 40-150 nm
morphology
Source of illumination
Visible light High speed electrons
High speed electrons
Best resolution 200 nm 0.2 nm 3-6 nm
Magnification 10-1000X 500-500,000X 20-150,000X
Depth of field 0.002-0.05 nm 0.004-0.006 0.003-1 mm
Lens type Glass Electromagnetic Electromagnet
Image ray-formation spot
On eye by lenses
On phosphorescent plate by lenses
On cathode tube by scanning device
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Scanning Electron Microscope
Conventional light microscopes use a series of glass lenses to bend light waves & create a magnified image.
The SEM creates the magnified images by using electrons instead of light waves.
The SEM shows detailed 3D images at much higher magnifications than is possible with a light microscope.
The images created without light waves are rendered black and white.
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SEM Technique
Samples have to be prepared
to withstand vacuum inside
SEM.
Specimens are dried to
prevent from shriveling.
Because SEM use electrons, they must be
made to conduct
electricity. Samples are coated with a very thin layer
of gold by sputter coater.
Now the prepared
specimen is ready.
The sample is placed inside
the microscope's
vacuum column
through an air-tight door.
Inside the SEM
Physical Pharmacy 2 17
After air is pumped out of
column, an electron gun emits
a beam of high energy electrons. This beam travels downward through
a series of magnetic lenses
to focus electrons to a fine spot.
Near the bottom, a set of scanning coils moves the focused beam back and forth
across the specimen, row by
row.
As the electron beam hits each
spot on the sample,
secondary electrons are
knocked loose from its surface. A
detector counts these electrons and sends the signals to an
amplifier.
The final image is built up from the
number of electrons emitted from each spot on
the sample.
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Information given by SEM
pore diameter
particle size
shape
surface condition.
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References
SL Flegler, JW Heckman, KL Klomparens, Scanning and
transmission electron microscopy, Oxford, New York
(1993)
http
://micro.magnet.fsu.edu/primer/lightandcolor/polarizedlig
hthome.html