Chromatography, Centrifugation, Spectrophotometry PDF
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Mahmoud A. Alfaqih
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Summary
This document provides an overview of chromatography, centrifugation, and spectrophotometry, including their principles, applications, and related concepts. It covers topics such as types of chromatography, retardation factor, and the separation of components in a mixture.
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CHROMATOGRAPHY, CENTRIFUGATION, SPECTROPHOTOMETRY Mahmoud A. Alfaqih BDS PhD Department of Medical Biochemistry Chromatography ■ Chroma= color, graph=writing (Greek). ■ Separation technique (molecules/components) ■ Molecules pass through stationary phase ■ Molecules are carried by mobile phase ■...
CHROMATOGRAPHY, CENTRIFUGATION, SPECTROPHOTOMETRY Mahmoud A. Alfaqih BDS PhD Department of Medical Biochemistry Chromatography ■ Chroma= color, graph=writing (Greek). ■ Separation technique (molecules/components) ■ Molecules pass through stationary phase ■ Molecules are carried by mobile phase ■ Uses of Chromatography: diagnostic tool (may separate body fluids, proteins) ■ Separation of components of mixture: – Affinity to solvent (mobile phase) → water, alcohol..etc – Adsorption to stationary phase → filter paper, gel..etc Types of chromatography ■ Column chromatography. ■ Ion-exchange chromatography. ■ Gel-permeation (molecular sieve) chromatography. ■ Affinity chromatography. ■ Paper chromatography. ■ Thin-layer chromatography. ■ Gas chromatography. ■ Dye-ligand chromatography. Paper chromatography Mobile phase→ water Stationary phase→ paper Chromatography: Retardation Factor Rf = Distance traveled by a component from origin (mm) Distance traveled by mobile phase from origin (mm) ■ If Rf is great → Solubility is great, and Adsorption is small ■ If Rf is small → Solubility is small, Adsorption is great Centrifugation ■ Separation based on density ■ The molecules are forced to move by centrifugal force ■ Dense molecules displace light molecules reaching the bottom of tube. ■ The light molecules at the top of tube. ■ Heamatocrit: Red cells + Buffy coat ≈ 45% of total volume of centrifuged blood Spectrophotometry ■ Measures light Absorption or Transmission in specific wavelength band by using a photometer ■ A photometer is device that measures light intensity. ■ Helps to find sample properties (hemoglobin concentration) by measuring the specific wavelength light absorption or transmission of a sample. ■ Hb Conc.: M: 16+ 2 g/dL F: 14+ 2 g/dL ■ Anemia: Low Hemoglobin. ? Low Iron Intake Spectrophotometer
