Orgo Lecture 4: biochem and lab techniques

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fatty acids

long carbon chains with a carboxylic acid end.
1) hormones
2) phospholipids and glycolipids of cell membranes
3) fuel for the body

lipolysis

triacylglycerols can be hydrozlied to form glycerol and the corresponding fatty acids in a process called _____. reverse of esterification

saponification

triacylglycerols can be cleaved by the addition of NaOH

amphipathic

contain both a hydrophilic and hydrophobic end.

triacylglyceride

triacylglycerols

fatty acids are stored as tryacylglycerols in adipose cells.

amino acids exist in three forms

4 categories of amino acids

1. acidic
2. basic
3. polar
4. nonpolar

isoelectric point, pI

100% of species two in amino acid type picture.

titration of an alanine with a strong base

carbohydrates

carbons and water. Cn(H₂O)n

ketose

a sugar containing one ketone group per molecule.

aldose

a monosaccharide (a simple sugar) containing one aldehyde group per molecule and having a chemical formula of the form Cn(H₂O)n.

aldohexose

a hexose with an aldehyde group on one end.

anomeric carbon

can be identified as the only carbon attached to two oxygens because its alcohol group may point upwards or downwards on the ring structure resulting in either the α or β anomer.

furanose

a five-membered ring

pyranos

a six-membred ring

glucopyranose

glucose ring.

glycosides

sugars that are acetals (not hemiacetals). name of such sugars end in -oside.

Tollens reagent

a basic reagent that detects aldehydes. aldoses have an aldehyde on their open-chain. promote senediol rearrangement of ketoses so that ketoses also reduce ____ _____. recall acetals used as blocking groups. glycosides (which are closed ring acetals) do NOT reduce Tollens reagent, while nonglycosides do.

disaccharides and polysaccharides

are glycosides where the aglycone is another sugar. the anomeric carbon of a sugar can react with any of the hydroxyl groups of another sugar, but there are only three bonding rearrangements that are common: a 1,4' link; a 1,6' link; and a 1,1' link. the numbers refer to the carbon numbers on the sugars. the linkages are called glycosidic linkages. will only react with Tollens reagent if there is an anomeric carbon that is not involved in a glycosidic bond and is free to react.

sucrose

1,1' glycosidic linkage: glucose and fructose (this linkage is alpha with respect to glucose and beta with respect to fructose. it is more accurately called a 1,2' linkage because teh anomeric carbon on fructose is numbered 2, not 1 like glucose.)

maltose

α-1,4' glucosidic linkage: two glucose molecules

lactose

β-1,4' galactosidic linkage: galactose and glucose

cellulose

β-1,4' glucosidic linkage: a chain of glucose molecules

amylose

α-1,4' glucosidic linkage: a branched chain of glucose molecules with α-1,6' glucosidic linkages forming the branches

glycogen

α-1,4' glucosidic linkage: a branched chain of glucose molecules with α-1,6' glucosidic linkages forming the branches.

methyl α-glucopyranoside

names of reducing sugars

end in -ose

names of nonreducing sugars

end in -oside

nuclear magnetic resonance (NMR) spectroscopy

usually done with H⁺, but can also be done with ¹³C

nuclei with an odd atomic number or odd mass number

exhibit nuclear spin that can be observed by an nmr spectrometer

nuclei aligned with the magnetic field

have a lower energy state state than those aligned against the field.

resonance

a nucleus that is subjected to this perfect combination of magnetic field strength and electromagnetic radiation frequency is said to be in _____. flips face against the external field

an nmr spectrometer can detect

the energy absorption of a nucleus in resonance

example of NMR spectrum

reference compound for NMR

tetramehtylsilane

given an nmr spectrum

you should be able to identify which peaks belong to which hydrogens on a given compound, or which of four compounds might create the given spectrum

each peak represents

chemically equivalent hdyrogens. (enantiotropic)

splitting of peaks

created by neighboring hydrogens

chemical shift

enantiotropic hydrogens are represented by the same peak and have the same ____ _____, the difference between the resonance frequency of the chemically shifted hydrogens and the resonance frequency of hydrogens on the reference compound because it contains many hydrogens that are all enantiotropic and are very well shielded

area under the peak in nmr

is proportional to the number of hydrogens represented by that peak. the more chemically equivalent hydrogen the greater the area.

integral trace

line drawn above the peaks that rises each time it goes over a peak. the rise of the ___ ____ is in proportion to the number of chemically equivalent hydrogens in the peak beneath it

digital trace

instrument that records numbers which correspond to the rise in the line.

only the ratio of hydrogens

from one peak to another can be determined using integral trace and digital trace

electron shielding

lateral position can be dteremined by ___ ___, thus limited predictions can be made based upon electron-withdrawing and electron-donating groups.

electron withdrawing groups

tend to lower shielding and thus decrease the magnetic field strength at which resonance takes place. means that hydrogens with less shielding tend to have peaks downfield or to the left.

electron donating groups

tend to increase shielding and increase the required field strength for resonance.

splitting (spin-spin splitting)

results from neighboring hydrogens that are not chemically equivalent. the number of peaks due to splitting for a group of chemically equivalent hdyrogens is given by the simple formula, n+1, where n is the number of neighboring hydrogens that are not chemically equivalent.

neighboring hydrogen

one that is on an atom adjacent to the atom to which the hydrogen is connected.

for proton nmr spectroscopy...

1) identify chemically equivalent hydrogens

2) identify and count neighboring hydrogens that are not chemically equivalent. use n+1 to figure the number of peaks created by splitting for the chemically equivalent hydrogens.

3) if necessary, identify electron withdrawing/donating groups near the chemically equivalent hydroogens. withdrawing groups will move their signal to the left.

aldehyde protons

have a very distinctive shift at 9.5 ppm

for carbon nmr

remember it's carbon 13 and ignore splitting.

infrared radiation

when exposed to ____ _____, the polar bonds within a compound stretch and contract in a vibrating motion.

IR spectroscopy

an infrared spectrometer slowly changes the frequency of infrared light shining upon a compound and records the frequencies of absorption in reciprocal centimeters, cm⁻¹ (number of cycles per turn).

if a bond has no dipole moment

then the infrared radiation does not cause it to vibrate and no energy is absorbed.

carboxylic acid IR

aldehyde IR

alcohol IR

nitrile IR

atoms with greater mass

resonate at lower frequencies

stiffer bonds

such as double and triple bonds, resonate at higher frequencies. bond strength and bond stiffness follow the same order: sp>sp²>sp³

fingerprint region

from 600 to 1400 cm⁻¹. many of the complex vibrations that distinguish compounds are found in this region.

wavelength of ultraviolet light

between 200 and 400 nm

UV spectroscopy

detects conjugated double bonds (double bond separated by a single bond) by comparing the intensities of two beams of light from the same monochromatic light source. one beam is shown through a sample cell and the other is shone through a reference cell. the sample cell contains the sample compound to be analyzed dissolved in a solvent. the reference cell contains only the solvent. the sample cell will absorb more energy from the light beam than the reference cell. the difference in the radiant energy is recorded as a UV spectrum of the sample compound.

the UV spectrum provides limited information about...

the length and structure of the conjugated portion of the molecule.

if a conjugated system is present in the sample,

the sample beam intensity Is will be lower than the reference beam intensity Ir.

absorbance also equals...

the product o concentration of the sample (c), the length of the path of light through the cell (l), and the molar absorptivity (epsilon) (or molar extinction coefficient)

molar absorptivity

a measure of how strongly the sample absorbs light at a particular wavelength.

the longer the chain of conjugated double bonds....

the greater the wavelength of absorption

rule of thumb is that...

each additional conjugated double bond increases the wavelength by about 30 to 40 nm.

an additional alkyl group attached to any one of the atoms involved in the conjugated system

increases the spectrum wavelength by about 5 nm.

isolated double bonds...

do not increase the absorption

UV spectra lack...

detail

samples must be extremely pure or...

the spectrum is obscured

a compound's absorbance with ______ moves into the visible spectrum

8 or more double bonds

mass spectrometry gives

the molecular weight, and, in the case of high resolution mass spectrometry, the molecular formula.

mass spectrometry mechanism

the molecules of a sample are bombarded with electrons, causing them to break apart and ionize. the largest ion is the size of the original molecule but short one electron.

molecular ion

largest ion that is the size of the original molecule but short one electron. for methane, would be CH₄⁺

mass to charge ratio (m/z)

the radius of curvature of their path depends upon the .....

a computer records the...

amount of ions at different magnetic field strengths as peaks on a chart.

base peak

the largest peak.

parent peak

the peak made by the molecular ions. look for it all the way to the right of the spectrum.

abundances

all peaks are assigned abundances as percentages of the base peak. abundance of 10 means that peak is 10% as high as the base peak

mass spec pick

chromatography

the resolution of a mixture by passing it over or through a matrix t hat adsorbs different compounds with different affinities. ultimately altering the rate at which they lose contact with the resolving matrix. the mixture is usually dissolved into a solution to serve as the mobile phase, while the resolving matrix is often a solid surface

typically, the more polar compounds elute...

more slowly because they have a greater affinity for the stationary phase.

column chromatography

where a solution containing the mixture is dripped down a column containing the solid phase (usually glass beads). the more polar compounds in the mixture travel more slowly down the column, creating separate layers for each compound. each compound can subsequently be collected as it elutes with the solvent and drips out of the bottom of the column

paper chromatography

a small portion of the sample to be separated is spotted onto paper. one end of the paper is then placed into a solvent. the solvent moves up the paper via capillary action and dissolves the sample as it passes over it. as the solvent continues to move up the paper, the more polar components of the sample move more slowly because they are attracted to the polar paper. the less polar components are not attracted to the paper and move more quickly. the result is a series of colored dots representing the different components of the sample with the most polar near the bottom and the least polar near the top

Rf factor

can be determined for each component of the separation by dividing the distance traveled by the component by the distance traveled by the solvent. nonpolar components have an Rf factor close to one; polar components have a lower Rf factor. always between 0 and 1

thin layer chromatography

similar to paper chromatography except that a coated glass or plastic plate is used instead of paper, and the results are visualized via an iodine vapor chamber

gas chromatography

the liquid phase is the stationary phase. the mixture is dissolved into a heated carrier gas (usually helium or nitrogen) and passed over a liquid phase bound to a column. compounds in the mixture equilibrate with the liquid phase at different rates and elute as individual components at an exit port.

Distillation

separation based upon vapor pressure. a solution of two volatile liquids with boiling point differences of approximately 20°C or more may be separated by slow boiling.

teh compound with the lower point (higher vapor pressure)

will boil off and can be captured and condensed in a cool tube.

azeotrope

solution of two volatile liquids exhibits a positive deviation to Rault's law and the solution boils at a lower temperature than either pure compound. cannot be separated by distillation. can also form when the solution has a higher boiling point than either pure substance.

fractional distillation

a more precise method of distillation. the vapor is run through glass beads allowing the compound with the higher boiling point to condense and fall back into the solution.

crystallization

pure substances form crystals. think of an iceberg. very inefficient method of separation. crystallization of most salts is an exothermic process

extraction

based upon solubility due to similar polarities. like dissolves like.

Extraction step 1:

add a acid base and shake. the acid protonates bases like amines in the organic layer, making them polar. the polar amines dissolve in the aqueous layer and are drained off

Extraction step 2:

add a weak base. the base deprotonates only the strong acids like carboxylic acids, making them more polar. the polar carboxylic acids dissolve in the aqueous layer and are drained off.

Extraction step 3:

add a strong base. the strong base reacts with the rest of the acids (hopefully all weak acids like phenol). these acids dissolve in the aqueous layer and are drained off.

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