Analytical Separation

Question 1

(6) Basis of Separation

Answer 1

1. Size
2. Mass or Density
3. Complex Formation
4. Change in physical state
5. Change in chemical state
6. Partitioning between phases

Question 2

(4) Separation techniques based on size

Answer 2

1. Filtration
2. Dialysis
3. Size-exclusion chromatography
4. Electrophoresis

Question 3

Explain principle of filtration along with the definition of filtrate and retentate

Answer 3

Filtration - used to separate interferent from soluble analytes using a pore size that will retain the interferent

Filtrate - fluid that passes through the filter

Retentate - material that is retained by the filter

Question 4

Explain and differentiate gravity filtration from suction filtration

Answer 4

Gravity filtration - uses the force of gravity to pull liquid through a filter

Suction (or vacuum) filtration - uses a vacuum to speed up the filtration process by drawing the liquid through the filter more quickly.

While gravity filtration is slower and better suited for delicate materials, suction filtration is faster and ideal for removing fine particles or handling larger volumes of liquid.

Question 5

What type of filter is used for particulates too small to be retained by a normal filter paper?

Answer 5

Membrane filter

Question 6

(3) Types of membrane filters

Answer 6

Centrifugal filter - placed in a centrifuge, retains macromolecules with a limit of 10^6 g/mol

Syringe filter - addon on tip of syringe, has a pore size of 0.45µm

Disposable filter system - utilizes vacuum filtration, has a pore size of 0.22µm, filter usually made out of cellulose acetate

Question 7

Explain the principle behind dialysis

Answer 7

Dialysis is based on diffusion through a semi-permeable membrane. Small molecules and ions diffuse across the membrane from an area of higher concentration (inside the solution) to an area of lower concentration (outside the dialysate)

Question 8

Explain the principle behind size-exclusion chromatography

Answer 8

Size-exclusion chromatography separates molecules based on their size and shape.

A column is packed with porous beads, and smaller molecules enter the pores and take a longer path, while larger molecules bypass the pores and elute faster. This method is commonly used to purify proteins, polymers, and other large molecules by exploiting differences in molecular size.

Question 9

Explain the principle of electrophoresis

Answer 9

Electrophoresis separates molecules based on their charge and size by applying an electric field. Molecules such as proteins or DNA fragments migrate through a gel matrix toward the positive or negative electrode depending on their charge. Smaller or more highly charged molecules move faster through the gel, allowing for separation based on size and charge.

Question 10

(1) Separation technique based on Mass or Density

Answer 10

Centrifugation

Question 11

Explain the principle of centrifugation

Answer 11

Centrifugation relies on the principle of centrifugal force to separate components of a mixture based on their density. When a sample is spun at high speed, denser particles move outward toward the bottom of the tube, while lighter particles remain closer to the top. This technique is often used to separate cells, subcellular components, or proteins in a mixture.

Question 12

Principles used in separation technique using complex formation

Answer 12

Masking and Demasking

Question 13

Explain the principle behind masking and demasking

Answer 13

Masking - binding the interferent in a strong soluble complex that prevents it from interfering in the analyte’s determination

Demasking - process of unbinding the masking agent to the interferent

Question 14

(3) Separation techniques based on change in physical state

Answer 14

1. Distillation
2. Sublimation
3. Crystallization

Question 15

Concept behind distillation, sublimation, and crystallization

Answer 15

Distillation separates components of a mixture based on their boiling points. When the mixture is heated, the component with the lower boiling point vaporizes first and is then condensed back into a liquid in a separate container. This method is commonly used to purify liquids or separate volatile substances from non-volatile ones, as in the distillation of alcohol or water purification.

Sublimation separates substances based on their ability to transition directly from a solid to a gas without passing through a liquid phase. When heated, a solid that can sublime (e.g., iodine or dry ice) will vaporize, while non-sublimable impurities remain in the solid phase. The vapor can then be condensed back into a pure solid, making sublimation ideal for purifying substances that have this specific phase behavior.

Crystallization exploits the principle of differential solubility to separate and purify solids. A saturated solution of a solute is allowed to cool or evaporate, causing the solute to form crystals, while impurities remain dissolved or precipitate separately. This method is often used to purify salts, minerals, and other solid compounds by selectively forming well-ordered crystal structures.

Question 16

(4) Types of distillation

Answer 16

1. Simple distillation
2. Fractional distillation
3. Vacuum distillation
4. Steam distillation

Question 17

Define the (4) types of distillation

Answer 17

Simple distillation - applies to an analyte that does not undergo decomposition before its boiling point (bp difference at least 150°C)

Fractional distillation - also called rectification, and utilizes fractionating column, applicable for two compounds that have very similar vapor pressure characteristics
(bp difference <25°C)

Vacuum distillation - applied to substances that boil above 200°C

Steam distillation - passing dry steam through the sample whereby the steam volatile compounds are volatilized

Question 18

(3) Conditions in using simple distillation

Answer 18

1. must be relatively pure (containing no more than 10% liquid contaminants)
2. has a non-volatile component such as solid contaminant
3. is mixed with another liquid with a boiling point that differs by at least 150°C

Question 19

(4) Separation techniques based on change in chemical state

Answer 19

1. Precipitation
2. Electrodeposition
3. Volatilization
4. Separation by chemical reactivity

Question 20

Concept behind precipitation, electrodeposition, volatilization, and separation by chemical reactivity

Answer 20

Precipitation - a target compound is separated from a solution by converting it into an insoluble solid, called a precipitate, through a chemical reaction. The precipitate is then filtered and removed, leaving the other substances dissolved. This method is based on differences in solubility between the target substance and other solution components.

Electrodeposition - involves using an electric current to reduce ions of a target metal from a solution and deposit them onto an electrode. This process separates the metal from the solution, concentrating it in solid form on the electrode surface. The principle relies on the difference in reduction potentials of various metal ions.

Volatilization - separates compounds based on their ability to vaporize when heated. Substances that are easily converted to gases at certain temperatures are volatilized and removed from the mixture, while less volatile components remain. This method is used when the target compound has a lower boiling point compared to other components.

Separation by chemical reactivity - exploits differences in the reactivity of substances with specific reagents. A target compound reacts to form a new product, while other components do not react under the same conditions, allowing selective separation. This technique is often used in cases where the target substance has unique chemical properties.

Question 21

(2) Separation techniques based on partitioning between phases

Answer 21

1. Extraction
2. Chromatography

Question 22

Concept behind extraction, and chromatography

Answer 22

Extraction - transfer of solute from one phase to another

Chromatography - separates compounds based on their differential affinities between a stationary phase (solid or liquid) and a mobile phase (gas or liquid). As the mobile phase moves over the stationary phase, different components travel at different speeds, causing them to separate. The method relies on factors like molecular size, polarity, or charge of the substances.

Question 23

(4) Types of extraction

Answer 23

1. Liquid-solid
2. Solid-liquid
3. Liquid-gas
4. Liquid-liquid

Question 24

Define the (4) types of extraction

Answer 24

refer to as:
(sample - extractant)

Liquid-solid: sample is passed through a cartridge that contains a solid sorbent; also known as solid phase extraction (SPE)

Solid-liquid: continuous extraction of a solid sample is carried out using soxhlet extractor

Liquid-gas: removal of volatile organic compounds (VOCs) using purge and trap method

Liquid-liquid: solute partitions itself between two immiscible phases; one is usually the aqueous solvent, and the other is organic solvent, carried out using separatory funnel

Question 25

(3) steps of solid phase extraction

Answer 25

1. the sample is added to the cartridge containing the solid adsorbent
2. the sample is washed to remove interferents
3. the analytes are eluted

Question 26

(3) main sections of a soxhlet extractor

Answer 26

1. Percolator - (boiler and reflux) which circulates the solvent
2. Thimble - (usually made of thick filter paper or membrane) which retains the solid to be extracted
3. Siphon mechanism - which periodically empties the thimble

Question 27

Formula for partition coefficient

Answer 27

K = C1/C2

the higher the K, the better for extraction

Question 28

Formula for concentration remaining

Answer 28

q=(V1/V1+KV2)^n

Question 29

Formula for distribution of base between two phases

Answer 29

D= (Kd*Ka) / (Ka+[H+])

Question 30

Formula for distribution of acid between two phases

Answer 30

D= (Kd*[H+]) / (Ka+[H+])

Question 31

many _ _ _ _ _ extractions are more effective than a few _ _ _ _ _ extractions

Answer 31

many small extractions are more effective than a few large extractions