Lectures 7-10

Solvent Extraction

Separation based on differences in solubility and polarity ("like dissolves like")

Liquid-Liquid Extraction (LLE)

Separation using two immiscible liquids (don’t mix) where solutes partition between phases

• Batch extractions - to increase extraction efficiency (recovery)

Solid-Liquid Extraction

Extraction of compounds from a solid using a solvent (e.g. tea/water, beets/water, caffeine/methanol)

Immiscible Solvents

Liquids that do not mix and form two layers (e.g.

Organic Layer

Layer containing organic solvent (often less dense - on top)

Aqueous Layer

Water-based layer in extraction system

Partition Coefficient (Kᴅ)

Ratio of solute concentration in organic phase to aqueous phase at equilibrium

Kᴅ > 1

Compound prefers organic phase (hydrophobic)

Kᴅ < 1

Compound prefers aqueous phase (hydrophilic)

Equilibrium in Extraction

Solute distributes between phases based on relative solubility

Separatory Funnel

Apparatus used to mix and separate immiscible liquids

Green Solvents

Safer

Selective Extraction

Using solvent polarity to isolate specific compounds

Distillation

Separation by vaporization followed by condensation

• General distillation - involves heating a liquid mixture to form vapor and condensing the vapor to collect separated components

  • Components with higher vapor pressure (lower boiling point) vaporize more readily

Volatility

Tendency of a substance to enter the gas phase

Vapor Pressure (Vp)

Pressure exerted by vapor in equilibrium with liquid

Boiling point (BP)

Temperature where vapor pressure equals atmospheric pressure

• lower pressure = higher BP = longer cooking

• higher pressure = lower BP = faster cooking

High vapor pressure

More volatile, lower BP

Low vapor pressure

Less volatile, higher boiling point

Intermolecular Forces (IMFs)

Forces between molecules affecting volatility

Stronger IMFs

Lower volatility, higher BP

London Dispersion Forces

Weak forces from temporary dipoles (present in all molecules) - stickyness

• Shapes of molecules affect the magnitude of the LD forces

• Size: molecules with more surface area (longer hydrocarbon chains) tend to have more LD forces and higher BPs

Dipole-dipole interactions

Attractions between polar molecules

• Are additive

• Partial negative of one molecule interacts with the partial positive of another molecule

Hydrogen bonding

Strong IMF when H is bonded to N, O, or F

• More H bonds = more difficult to get to enter the vapor phase

Ion-dipole forces

Interactions between ions and polar molecules (cation and anion)

Simple distillation

Used when boiling points are very different

Fractional distillation

Used when boiling points are close; uses multiple vaporization steps

• Repeated distillations

  • Temperature gradient as you move up the column (hottest at the bottom)

  • Each time the vapor condenses and vaporizes, the composition of the more volatile liquid in the vapor increases

• Fractioning column is placed between the boiling flask and the condenser

• The fractioning column has added surfaces area for “refluxing”

  • The process in which a liquid is boiled, its vapors are condensed, and the condensate returns to the reaction vessel, allowing prolonged heating at the solvent spoiling point without loss of material

Theoretical plate

Measure of separation efficiency in distillation

Reflux

Continuous vaporization-condensation cycle to improve separation

Color in foods

Result of light interacting with matter and being reflected

White light

Contains all visible wavelengths

Color perception

Brain’s interpretation of reflected light

• reflected wavelengths are perceived as color

Chlorophyll

Pigment responsible for green color

Carotenoids

Pigments responsible for yellow/orange color

Anthocyanins

Pigments responsible for red/blue color (pH dependent)

• pH < 2: flavylium cations (RED) → MORE STABLE

  • More -OCH₃ = redder color

• pH 2.5-6: quinoidal base (BLUE)

  • More -OH = deeper blue color

• pH > 6: chalcone (NO COLOR)

• Stability

  • Increase with more methoxyl groups on the B-ring

  • Decreases with increasing hydroxylation of the B ring

Colorimetry

Science of measuring color objectively

Hue

Type of color (red, blue, green)

Value (lightness)

Brightness or darkness of a color

Chroma

Intensity or saturation of color

Subjective color

Perceived differently by individuals

Objective color

Measured numerically using instruments

Munsell system

Color system based on hue (R, Y, G, B, P), value (lightness), chroma (color intensity)

CIE system

Standardized color measurement system

Tri-stimulus CIE X(red)Y(green)Z(blue) color space

• Dominant wavelength - white light through the object coordinates to the edge

• Distance from white light to the object’s coordinates is % purity

Lab Color system

Uses L* (lightness), a* (red-green), b* (yellow-blue)

Hunter lab values

Factors affecting color

Light source, observer, background, and sample size

Phenolic compounds

Molecules with aromatic ring + hydroxyl group(s)

• Especially abundant in plants

• Many are pigments

• Important for quality, stability, and as antioxidants

  • Involved for browning reactions (converted to quinones)

  • Aroma

    • Quinone + free amino acids = aromas (aldehydes)

Polyphenols vs phenolics

“Phenolic compounds” is more accurate term

Antioxidant function

Donate electrons to neutralize free radicals

Phenolics in food

Affect color, flavor, stability, and astringency

Enzymatic browning

Phenolics oxidized to quinones by PPO

• Tissue damage (cutting, bruising) brings PPO into contact with phenolics

• PPO catalyzes oxidation of phenolics to quinones

• Quinones condense and polymerize to form brown pigments (melanins)

Hydroxybenzoic Acids (C6–C1)

Phenol + carboxylic acid structure

Hydroxycinnamic Acids (C6–C3)

Aromatic ring + propionic acid side chain

• Predominantly present as esters

Flavonoids (C6–C3–C6)

Major class of plant phenolics with 3-ring structure

• Color properties are related to the conjugated systems

  • Double bonds are conjugated when they are separated by one single bond (C=C-C=C)

  • Color is only present when the C ring is conjugated with both the A and B rings

• Classes:

  • Flavonols

  • Isoflavones

  • Flavan-3-ols

  • Flavonones

  • Anthocyanidins

Flavan Nucleus

Two aromatic rings + heterocyclic ring

Anthocyanidins

Flavonoids responsible for red/blue colors

Tannins

Phenolics that bind proteins and cause astringency

• Phenolic compounds in plant extracts that can convert animal skin into leather (tanning)

Hydrolysable Tannins

Breakable by hydrolysis (ester bonds)

• Bind protein

Condensed Tannins

Polymers of flavan-3-ols (non-hydrolyzable)

• Proanthocyanidins

• Bind protein

Astringency

Dry, puckering sensation from tannins binding proteins

Polyphenol Oxidase (PPO)

Enzyme that catalyzes browning reactions

% Recovery & Yield

Drying agents

Water removal from solvents

• Wash the organic layer with anhydrous salt

  • Add a drying agent (salt) to the organic solvent

  • Salt pulls the water from the organic layer

Techniques for extracting volatiles - Purge and Trap

• Used to isolate volatile organic compounds (VOCs) from liquids or solids, especially for flavor and aroma analysis

• How purge and trap works

  • An inert gas (usually helium or nitrogen) is bubbled through the sample

  • Volatile compounds are stripped (purged) from the sample into the gas phase phase

  • The gas carrying the volatiles passes through a sorbent trap

  • Volatile compounds are captured and concentrated on the trap

• Distortion and analysis

  • Trap is rapidly heated

  • Trapped compounds are released (desorbed) and transferred to an instrument, typically GC or GC-MS

Head space analysis

Technique used to measure volatile compounds present in the gas phase (headspace) above a sample

• Sample is sealed in a closed vial

• Volatile compounds partition from the sample into the headspace

• After equilibrium is reached, a portion of the headspace gas is sampled

Fractional Distillation Columns

• Open tubular column

  • Low surface area

  • Low efficiency (plates)

• Vigreux column

  • Moderate surface area

  • Moderate efficiency

• Packed column

  • Increased surface area

  • High efficiency

Cones

Color

• Blue: 420-440 nm

• Green: 530-540 nm

• Red: 560-580 nm

Rods

Sensitive to brightness

Monochromatic

Light

EMR (electromagnetic receptors) which stimulate the retina of the eye

What influences color?

• Light source differences

• Observer differences

• Size difference

• Background differences

CIE Standardized Geometrics for Reflectance Instruments: 45/0 and 0/45

• 45 illumination/0 measure

  • Use to minimize specular (gloss) reflection (apples, tomatoes)

  • When surface texture or shine could distort color readings

• 0 illumination/45 measure

  • When illumination needs to be uniform and normal to the surface

  • When sample shape or orientation varies (grains, flat matte foods)

Examples of Polyhydroxylated Phenols

Phenolic compounds in tea

Lignin

• Part of the plant cell wall, non-digestible material

• Defined as dietary fiber

Glycosylated Phenolic Compounds

• Glycosylated forms

  • Phenolics are bound to one or more sugars (mono-, and oligosaccharides)

  • Aglycone form - not bound to a sugar

Antioxidant properties of phenolic compounds

• Many exhibit antioxidant properties

• Phenolic compounds act as reducing agents (donate H+ and e-)

• Phenolics donate the H+/e- from the -OH groups on the aromatic ring

• Phenolic becomes a free radical that is stabilized through resonance

Controlling enzymatic oxidation reactions

1. Eliminate oxygen (PPO requires oxygen)

2. Addition of metal complexing compounds (PPO is a metal low enzyme therefore, substances that complex metal ions will inactivate the enzyme)

3. Reduce the pH (most PPO enzymes have a pH optimum between pH five through seven, reducing pH, add adding acid will limit the rate of the enzyme

4. Heat-denature the enzyme (denatures above 40°C)

5. Add ascorbic acid (reduces the Keynote back to the o-diphenol state)

6. Add sulfite (binds with amino acids in the active site of PPO and deactivates it)

Final compounds and taste

• Some phenolic compounds can be bitter (citrus fruit)

• Glycoylated flavones/flavanones

• Glycosyl bond is responsible for the bitterness

Total phenolic compounds assay (Folin-Ciocalteu)

• Measuring the reduction of reagent and alkaline conditions by phenolic compound → blue color

• Not specific to phenolics:

  • Also detect other reducing agents

  • Report reported as total phenolics

2,2-Diphenyl-1-picryhydrazyl radical assat (DPPH)

• Radical scavenging acids

• DPPH is a stable free radical with an unpaired electrons (blue/purple)

• When an antioxidant is added to the assay, the antioxidant donates a hydrogen atom and an electron to the DPPH

• This neutralizes (quenches) the DPPH radical

• Absorbance decrease is measured at 517 nm

• Measures radical scavenging (antioxidant) activity

• More specific to antioxidants than FC