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 http://staff.iiu.edu.my/akausar Physical Pharmacy 2 KBA

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

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

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

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

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

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 1.414 and hence the sieve size varies in geometric progression. fixed to a rectangular or circular frame Physical Pharmacy 2 KBA

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

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

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

Microscopy http://www.mos.org/sln/SEM/index.html Physical Pharmacy 2 4/1/2017 Microscopy http://www.mos.org/sln/SEM/index.html 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

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

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

Comparison of microscopes Physical Pharmacy 2 4/1/2017 Comparison of microscopes Feature Light TEM SEM Use morphology Small particles, 40-150 nm Source of illumination Visible light 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 Physical Pharmacy 2 KBA

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

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

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

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

Physical Pharmacy 2 4/1/2017 References SL Flegler, JW Heckman, KL Klomparens, Scanning and transmission electron microscopy, Oxford, New York (1993) http://micro.magnet.fsu.edu/primer/lightandcolor/polarizedlighthome.html Physical Pharmacy 2 KBA