1. Kulliyyah of Pharmacy, IIUM
Physical Pharmacy 2
4/1/2017
Particle Size Sizing Technique 2: hydrodynamic chromatography sieving microscopy
Kausar Ahmad
Kulliyyah of Pharmacy, IIUM
Physical Pharmacy 2
KBA

2. Hydrodynamic Chromatography
Physical Pharmacy 2
4/1/2017
Hydrodynamic Chromatography
a 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
Results are independent of particle density because in hydrodynamic chromatography, flow of particles depends on the rate of diffusion. Diffusion is independent of particle density.
Physical Pharmacy 2
KBA

3. Physical Pharmacy 2
4/1/2017
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.
Physical Pharmacy 2
KBA

4. Results generated by HDC
Physical Pharmacy 2
4/1/2017
Results generated by HDC
Physical Pharmacy 2
KBA

5. Liposome PL-PSDA (Polymer Lab: HDC mechanism)
Physical Pharmacy 2
4/1/2017
Liposome PL-PSDA (Polymer Lab: HDC mechanism)
What is the volume average diameter?
What is the polydispersity index?
Physical Pharmacy 2
KBA

6. Physical Pharmacy 2
4/1/2017
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.
Physical Pharmacy 2
KBA

7. Physical Pharmacy 2
4/1/2017
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 and hence the sieve size varies in geometric progression.
fixed to a rectangular or circular frame
Physical Pharmacy 2
KBA

8. Sieving procedure Sieves stacked pan Maximum size Minimum size
Physical Pharmacy 2
4/1/2017
Sieving procedure
Sieves stacked
Maximum size g f
Wire mesh e d c b
The sieves are arranged one above the other in such a way that the lowest size sieve opening is at the bottom and the highest at the top.
The bottom pan is stacked below the finest sieve.
Known amount of powder sample is loaded on the top sieve and then closed with the lid.
This arranged sieves set is placed in a mechanical sieve shaker and it is operated for a definite time (ca. 5 min).
Then the amount of the material present in each sieve is weighed separately and the values are tabulated along with their corresponding sieve opening. a
pan
Minimum size
Physical Pharmacy 2
KBA

9. Table of Sieve Results Sieve no./size (um) Weight of sieve (g)
Physical Pharmacy 2
4/1/2017
Table of Sieve Results
Sieve no./size (um)
Weight of sieve (g)
Wt of sieve & sample (g)
Wt of sample (g)
% Wt of sample
% cumulative weight
(<25) na a
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
Physical Pharmacy 2
KBA

10. Interpretation of Sieve Results
Physical Pharmacy 2
4/1/2017
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.
Cumulative weight (%)
Weight (%)
Physical Pharmacy 2
KBA

11. Microscopy http://www.mos.org/sln/SEM/index.html
Physical Pharmacy 2
4/1/2017
Microscopy
The deer tick, is about the size of a freckle.
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 shells of radiolarians, single-celled animals.
Physical Pharmacy 2
KBA

12. Physical Pharmacy 2
4/1/2017
Principles of Microscopy
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 .
Physical Pharmacy 2
KBA

13. Using light microscopy equipped with graticule to determine size
Physical Pharmacy 2
4/1/2017
Using light microscopy equipped with graticule to determine size
Physical Pharmacy 2
KBA

14. Comparison of microscopes
Physical Pharmacy 2
4/1/2017
Comparison of microscopes
Feature
Light
TEM
SEM
Use
morphology
Small particles, nm
Source of illumination
Visible light
High speed electrons
Best resolution
200 nm
0.2 nm
3-6 nm
Magnification X
,000X
20-150,000X
Depth of field nm 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
Physical Pharmacy 2
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15. Scanning Electron Microscope
Physical Pharmacy 2
4/1/2017
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.
Physical Pharmacy 2
KBA

16. Physical Pharmacy 2
4/1/2017
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.
Physical Pharmacy 2
KBA

17. Physical Pharmacy 2
4/1/2017
Inside the SEM
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.
Physical Pharmacy 2
KBA

18. Information given by SEM
Physical Pharmacy 2
4/1/2017
Information given by SEM
pore diameter
particle size
shape
surface condition.
Physical Pharmacy 2
KBA

19. Physical Pharmacy 2
4/1/2017
References
SL Flegler, JW Heckman, KL Klomparens, Scanning and transmission electron microscopy, Oxford, New York (1993)
Physical Pharmacy 2
KBA