Chapter 12. Separation of Mixtures
Recommended Article : 【Chemistry】 Chemistry Table of Contents
1. Filtration
2. Dialysis
4. Salting Out
5. Distillation
1. Filtration: Heterogeneous Mixture
⑴ Definition : Separation of heterogeneous solid and liquid mixtures
⑵ Method : Passing the mixture through a filter paper with tiny holes (filtering paper)
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2. Dialysis: Colloid
⑴ Definition : Diffusion of substances that can pass through a semipermeable membrane
⑵ Dialysate Concentration ＞ Solution Concentration : Movement of substances from dialysate to solution
⑶ Dialysate Concentration = Solution Concentration : No movement of substances
⑷ Dialysate Concentration ＜ Solution Concentration : Movement of substances from solution to dialysate
⑸ Renal Hemodialysis is a representative method.
3. Electrophoresis: Colloid
4. Salting Out : Colloid
Figure. 1. Concentration of salt and solubility
⑴ Low Concentration of Salt
① Solubility increases as salt is added
② Reason : Salt-induced changes in substances aid water penetration
⑵ High Concentration of Salt
① Solubility decreases as salt is added
② Reason : Salts surround the entire substance, decreasing interaction with water
⑶ The higher the amount of target substance, the larger the peak value of solubility
⑷ Initial means of purification, frequently using ammonium sulfate
⑸ Application : Adding magnesium chloride (MgCl2) to tofu production
5. Distillation: Solution
⑴ Definition : Method of separating liquids based on their boiling point differences
⑵ When heating a solution, the substance with the lower boiling point vaporizes first
6. Chromatography: Solution
⑴ Classification based on polarity and non-polarity
① Terms
○ Stationary phase : Fixed substance
○ Mobile phase : Moving substance
○ Partition coefficient : Affinity difference between stationary and mobile phases for various substances
○ Chromatography : Method of separation based on time differences due to partition coefficients
② Normal-phase Chromatography: When the mobile phase is non-polar and the stationary phase is polar
○ Substances with greater distance traveled have stronger hydrophobicity
○ Toluene is frequently used as a mobile phase
○ Example : Ion-exchange chromatography
③ Reversed-phase Chromatography: When the mobile phase is polar and the stationary phase is non-polar
○ Substances with greater distance traveled have stronger hydrophilicity
○ Example : Partition chromatography
⑵ Classification based on form
① Thin-layer Chromatography (TLC): Used to monitor the progress of reactions
○ Principle : Interaction difference between sample and silica gel
○ Purpose : Determine reaction progress and termination
○ Normal phase silica gel: Strongly polar, affinity with the sample increases with sample polarity
○ Stationary phase is silica gel (strongest polarity), mobile phase is eluent (non-polar)
○ Definition of Rf : Distance traveled by the sample ÷ Distance traveled by the solvent
○ Comparing Rf for different samples
○ Acids, bases, metal salts < Carboxylic acids, amines, amides < Alcohols < Aldehydes, ketones < Alkyl halides < Esters < Alkenes ＜ Alkynes
○ Trend 1. The higher the sample’s polarity, the stronger the interaction with silica gel, leading to a lower Rf value
○ Trend 2. Amines and hydroxyl groups have less polarity compared to nitro groups, but due to hydrogen bonding, they have lower Rf values
○ Trend 3. trans alkenes have lower Rf values than cis alkenes (because they interact more)
○ Example : In a paper chromatography and chlorophyll separation experiment, Rf values are in the order: carotinoid > xanthophyll > chlorophyll a > chlorophyll b (using toluene as the solvent)
○ Comparing Rf values with organic solvents
○ Water ＜ Acetic acid ＜ Alcohols ＜ Ethyl acetate ＜ CH2Cl2 ＜ Toluene ＜ CCl4 ＜ n-Hexane
○ Trend 1. The higher the polarity of the eluent, the more the sample moves, leading to an increased Rf value
○ Ethyl acetate: Representative polar eluent
○ n-Hexane: Representative non-polar eluent
② Column Chromatography: Divided into ion-exchange, affinity, and size exclusion chromatography
③ Ion-exchange Chromatography
○ Definition : Chromatography using beads with attached target substance (e.g., protein)
○ Anion-exchange or positive ion exchange resin chromatography
○ Utilizes negatively charged beads
○ Aims to separate positively charged molecules (e.g., proteins with high isoelectric points)
○ Molecules with more negative charge are eluted faster
○ Cation-exchange or negative ion exchange resin chromatography
○ Utilizes positively charged beads
○ Aims to separate negatively charged molecules (e.g., proteins with low isoelectric points)
○ Molecules with more positive charge are eluted faster
④ Affinity Chromatography
○ Utilizes differences in affinity with the adsorbent to separate target substances; highly effective separation method
○ Example 1: His-tag Chromatography
○ Structure: 〈Ni2+〉 - 〈His and Protein X〉 - 〈Protein Y〉
○ Ni2+ and His form a strong chelation: large formation constant K
○ Example 2: Glutathione-GST
○ Structure: 〈Glutathione〉 - 〈GST and Protein X〉 - 〈Protein Y〉
○ Glutathione and GST form a strong chelation: large formation constant K
○ Example 3: Biotin-Streptavidin
○ Structure: 〈Biotin〉 - 〈Streptavidin and Protein X〉 - 〈Protein Y〉
○ Biotin and Streptavidin form a strong chelation: large formation constant K
○ Avadin can be used instead of Streptavidin
○ Utilized in DAB immuno-histochemical technique
○ Example 4: Diels-Alder Reaction
○ Example 5: EDC/NHS - Carboxylic Acid Click Reaction
○ Example 6: Thiol - Maleimide Click Reaction
⑤ Size Exclusion Chromatography (SEC)
○ Uses a porous matrix that only allows small molecules to pass through: larger molecules have faster elution
○ Molecular weight range of samples: 1.2 × 102 ~ 1.1 × 106
○ Examples: PD-10 desalting column, gel-filtration chromatography, Sephacryl S300 column chromatography
⑥ High Performance Liquid Chromatography (HPLC)
○ Measures presence and molecular weight of trace substances based on differences in their movement on a stationary phase
○ Molecular weight range of samples: 6 × 101 ~ 104
○ Components: solvent, pump, injector, column, detector, recorder
○ UV detector most widely used in HPLC
○ RI detector is sensitive to environmental changes like pressure and temperature, but not very sensitive to solute concentration
⑦ Gas Chromatography (GC)
○ Definition : Separation of evaporated sample components through distribution between a stationary phase and a moving gas phase in a separation tube
○ Carrier gas is usually helium, hydrogen, nitrogen, or argon: typically helium is used as it is an inert gas
○ Reason 1. Light gas molecules like H2 and He have high diffusion coefficients, leading to faster separation
○ Reason 2. Light gas molecules like H2 and He have high thermal conductivity, so the preheating time is short
○ Reason 3. Gases like H2 and O2 can react with the sample
○ Advantages: High resolution
○ Disadvantages: Requires volatile samples, so molecules should be small
○ Molecular weight range of samples: 100 ~ 103
○ Generally, when molecular weight exceeds 500, gas chromatography becomes difficult
○ Application 1: GLC (gas-liquid chromatography) and GSC (gas-solid chromatography)
○ Application 2: Molecular weight can be measured using gas chromatography: utilizing the relationship between retention time and molecular weight
○ Application 3: GC-MS (gas chromatography mass spectrography)
○ 1st. Substances separated in gas chromatography are ionized or chemically ionized, then sorted based on mass
○ 2nd. Sorted substances form unique mass spectra
○ 3rd. This is compared to accumulated library data for structural information or quantitative analysis
○ 1st ionization is selective, and 2nd ionization is performed to quantify very small amounts of components
⑧ Paper Chromatography
○ Origin of chromatography: derived from “chroma” meaning color; relates to paper chromatography
○ Used in paper chromatography and chlorophyll separation experiment: Rf values are in the order: carotinoid > xanthophyll > chlorophyll a > chlorophyll b (using toluene as the solvent)
○ Proved Law of Segregation
⑨ Supercritical Fluid Chromatography (SFC)
○ Uses supercritical CO2 as the mobile phase
○ Molecular weight range of samples: 5 × 101 ~ 104
Input: 2019.08.16 21:36