Biochemistry and Lab Techniques

Fatty acids are long carbon chains with a carboxylic acid end.

- nearly all fatty acids have an even number of carbon atoms, most between 12 and 20
- maximum number carbons is 24 in humans!

- In most unsaturated fatty acids, the cis isomer dominates!!

- Unsaturated fatty acids have lower melting points than their saturated
counterparts. The greater the degree of unsaturation, the lower the melting
point!!!

They server three basic functions in the human body:

1) they serve as hormones and intracellular messengers

2) they are components of the phospholipids and glycolipids of cell membranes

3) They act as fuel for the body

- As fuel for the body, fatty acids are stored in the form of triacylglycerols

- Triacylglycerols can be hydrolyzed to form glycerol and the

corresponding fatty acids in a process called lipolysis
 - Notice that this process is the reverse of esterification

- In the lab triacylglyerols can be cleaved by the addition of KOH or NaOH, a

process called saponification. Saponification is the production of soap (salts).

For nomenclature purposes, the carbonyl carbon of a fatty acid is assigned the number 1. The
carbon next to the carbonyl is called the a-carbon (alpha carbon) and the carbon at the opposite
end of the chain is called the W-carbon (omega carbon).

The pKa of most fatty acids is around 4.5, so most fatty acids exist in their anion form in
the cellular environment!!!

The carbon chains on fatty acids may be saturated or unsaturated. Fatty acids are amphiphatic.
Meaning they contain both a hydrophobic and hydrophilic end.

Since the hydrophobic carbon chain predominates, fatty acids are nonpolar.

Fatty acids are highly reduced, which allows them to store more than twice the energy of
carbs or proteins and saturated fats have the highest energy potential, therefore have
high heats of combustion!!!

Note: When we go from a reduced state to oxidized we release energy!!

- THE LONGER THE CHAIN IN THE TRIGLYCERIDE THE MORE

ENERGY IT STORES!!!
Fatty acids are stored as triaclyglycerols in adipose cells.

- Lypolysis of triacylglycerols take place inside the adipose cells when blood levels
of epinephrine, norepinephrine, glucagon or ACTH are high!!

- The fatty acids are linked to Coenzyme A and carried into the mitochondrial
matrix by the g-amino acid L-carnitine. They are then are oxidized TWO
carbons at a time in the KREB cycle, yielding an NADH, FADH2, and acetyl
CoA.

- Each acetyl CoA enters the Kreb cycle for further oxidation by condensation
with oxaloacetate.

Recall: Triglycerides can also be catabolized for ATP. Fatty acids are
converted to acyl CoA along the outer membrane of the mitochondrion
and endoplasmic reticulum at the expense of 1 ATP. Then 2 carbons are
cleaved to make acetyl CoA in the matrix. This reaction also produces
FADH2 and NADH for every two carbons taken from the original fatty acid.

Amino Acids
Amino acids are the building blocks of proteins and are at least diprotic.

A single protein consists of one or more chains of amino acids strung end by end by peptide
bonds. Hence the name polypeptide.

A peptide bond creates a the functional group known as an amide (an amine connected to a
carbonyl carbon)
It is formed via condensation (dehydration) reaction of two amino acids. The reverse
reaction is the hydrolysis of a peptide bond.

Amino acids used by the body are a-amino acids. They are called alpha amino acids
because the amine group is attached to the carbon which is alpha to the carbonyl carbon, similar to
α-hydrogens of ketones and aldehydes.

Since nitrogen is comfortable taking on four bonds and oxygen is comfortable with a partial
negative charge, electrons delocalize creating a resonance that gives the peptide bond a
partial double bond character.

- The double bond character prevents the bond from rotating freely and
affects the secondary and to some extent the tertiary structure of the
polypeptide!!

Notice the R group on each amino acid. The R group is called the side chain of the amino acid.
Nearly all organisms use the same 20 a-amino acids to synthesize proteins. Many amino acids
and amino acid derivatives, such as hydroxyproline and cystine, can be created by post-
translational modifications after the poly peptide is synthesized.
 Note: Cystine is the amino acid dimer formed when a pair of cysteine molecules
are joined by a disulfide bond.

Note: Hydroxyproline differs from proline by the presence of a hydroxyl (OH)

group attached to the C (gamma) atom. Hydroxyproline is a major
component of the protein collagen. Hydroxyproline and proline play key
roles for collagen stability. They permit the sharp twisting of the collagen
helix. This modification of the proline residue increases the stability of the
collagen triple helix

Hydroxyproline

Ten amino acids are essential (can’t be synthesized by the body and must be ingested)!!

Each amino acid differs only at the R group, and each R group has different chemical properties.
Only the three basic amino acids have an isoelectric point above a pH of 7; all others
are below 7!!!

These properties are divided into 4 categories:

1) acidic
a) aspartic acid
b) glutamic acid
2) basic
a) histidine
b) arginine
c) lysine
3) polar
4) nonpolar

All acidic and basic R groups are also polar. Generally, if the side chain contains carboxylic
acids, then it is acidic; if it contains amines, then it is basic.

Polar side groups are hydrophilic and will turn to face an aqueous solution such as cytosol.
Nonpolar side groups are hydrophobic and turn away from an aqueous solution.

- These characteristics affect a protein’s tertiary structure.

Note: The body only has L amino acids! The D forms is its enantiomer

Since the amino acid whose carboxylic acid group participated in the formation of the peptide bond
still has an ammonium group which contains a nitrogen atom, it is called the N terminus of the
peptide. The N terminus is conventionally written to the left. Correspondingly, the amino acid
which still has a free carboxylate group is called the C terminus and is written to the right. When
the order of amino acids in a peptide is written out, it is conventional to write it left to right from
the N terminus to the C terminus.

In the cytosol, amino acids exist in one of three forms:

All of the 20 standard amino acids have α-carboxyl pKa values less than 3.0 and α-amino
pKa values less than 11

As the pH increases, the stronger acid, the carboxylic, is first to lose its proton, creating species 2,
its conjugate base. When species 1 and 2 have reached equivalent proportions, we have reached
the first half equivalence point. As we continue the titration, we remove the proton from all of
the carboxylic acids until we have 100% species 2. The pH at this point is called the isoelectric
point, pI.

- The isoelectric point (pI) is the pH at which a particular molecule or

surface carries NO net electrical charge, and the maximum number of
species are zwitterion.

a) the isoelectric point (pI) is dictated by the side group of the

amino acid!!!

b) The more acidic the side group, the lower the pI; the more
basic the side group, the higher the pI!!

- A zwitterion is a chemical compound that is electrically neutral but carries

formal positive and negative charges on different atoms.

Continuing the titration the base begins to remove a proton from the amine. When species 2 and 3
have reached equivalent proportions, we have reached the second half equivalence point. As we
continue the titration, we remove the proton from all of the amines until we have 100% species 3.
So based upon this chart we can see that it’s a diprotic acid.

Note: The solubility of the protein decreases as we move towards pI and increases
away from it

Carbohydrates
Carbohydrates can be thought of as carbon and water. For every carbon there are one oxygen and
two hydrogens.

- They can’t exist as meso compounds because each end of a carbohydrate is

different from the other.

The formula for a carbohydrate is:

Cn(H2O)m
The carbohydrates most likely to show up on the MCAT is fructose and galatose

or or

D-glucose D-fructose
an aldose a ketose
an aldohexose  a ketohexose

 Carbohydrates are really just polyhydroxyaldehydes, the aldoses, or polyhydroxyketones,

the ketoses.
 So, for a carbohydrate to be an aldose it needs an aldehyde group.
 And to be a ketose it needs a ketone group.
 The chiral centers are marked in the diagrams with *
 The systems are further classified depending on how many C atoms there are
o 4 C = tetrose
o 5 C = pentose
o 6 C = hexose
 The names are commonly combined making glucose an aldohexose.

Only the highest-numbered chiral carbon is given a D or L designation. We also call this carbon
the last chiral carbon (or penultimate carbon). In a Fischer projection, this carbon is the chiral
carbon at the bottom of the structure.

If the hydroxyl group points to the right its D; If it points to the left its L
What about designations for all the other stereocenters in a monosaccharide? Simply stating D-
glucose as the common name specifies that we are talking about the six-carbon sugar that, in the
Fischer projection, has the carbon 2 and 4 hydroxyls on the right and the carbon 3 hydroxyl on the
left. Its enantiomer (L-glucose) has the opposite configuration at every chiral carbon,
not just the last chiral carbon!!.

Note: Most naturally occurring monosaccharides are D-sugars!

The chiral carbon furthest away from the carbonyl may act as a nucleophile and attack the
carbonyl. When this happens, nucleophilic addition to an aldehyde or ketone results and the
corresponding hemiacetal or hemiketal is formed respectively, creating a ring structure.
The aldopentose structures drawn above are all diastereomers.

Recall: Diastereomers – have the same formula, bond – bond connectivity, but are not
mirror images of each other, and aren’t the same compound

A more selective term, epimer, is used to designate diastereomers that differ in configuration at
only one chiral center.
 - an anomer is a special type of epimer. It is a stereoisomer (diastereomer, more
exactly) of a saccharide (in the cyclic form) that differs only in its configuration
at the hemiacetal (or hemiketal) carbon, also called the anomeric carbon.

The anomeric carbon can be identified as the only carbon which is attached to 2 oxygens because
its alcohol group may point upwards or downwards on the ring structure resulting in either the a or
B anomer.

example: α- and  β-D-glucose

- So beta and alpha D-glucose are anomers of each other because of the
difference in orientation on their anomeric carbon

The cyclic structures are named according to the number of ring members (including
oxygen): a five-membered ring is called furanose; a six-membered ring is called a
pyranose. So the glucose ring becomes glucopyranose.

Ring Opening/Closure

- As we can see here, when ring opening occurs any –OH groups on the ring
that point downwards are on the right side and any that point upwards are to
the left.

Here is another example:

Sugars that are acetals (not hemiacetals) are called glycosides. The names of such sugars end
in –oside. For instance, if the hydroxyl group on the anomeric carbon of glucose were replaced
with an O-methyl group, it would form methyl glucopyranoside.

a hemi-acetal an acetal

The group attached to the anomeric carbon of a glycoside is called an aglycone.

Tollens’ reagent is a basic reagent that detects aldehydes. Aldoses have an aldehyde on their
open-chain form and reduce Tollens reagent (are oxidized by it). Tollens reagent promotes
enediol rearrangement of ketoses so that ketoses also reduce Tollens reagent (are also
oxidized by it).

Recall from Lecture 3 that acetals are used as blocking groups because they do not react with basic
reducing agents. Since Tollens reagent must react with the open-chain form of a sugar, glycosides
(which are closed ring acetals) do NOT reduce Tollens reagent, while nonglycosides do!!

Reducing Sugar
- Sugars oxidized by Tollens' reagent
- Names of reducing sugars end in –ose

Non-reducing Sugar
- Sugars not oxidized by Tollens' or other reagents.
 - Names of non-reducing sugars ending in –oside.

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 arrangements that are common: 1,4’ link; a 1,6’ link; and a 1,1’ link. The
linkages are called glycosidic linkages.

Note: A disaccharide or polysaccharide will only react with a tollens reagent

if there is an anomeric carbon that is not involved in a glycosidic bond
and is free to react.

There are several disaccharides and polysaccharides for which you should know the common
name:
1) Sucrose
a) 1,1’ glycosidic linkage: glucose and fructose (alpha with respect to glucose
and beta with respect to fructose)

2) Lactose
a) b-1,4’ galactosidic linkage: galactose and glucose

3) Cellulose
a) b-1,4’ glycosidic linkage: a chain of glucose molecules

4) Maltose
a) a-1,4’ glycosidic linkage: two glucose molecules

5) Amylose
a) a-1,4’ glycosidic linkage: a chain of glucose molecules

6) Amylopectin
a) a-1,4’ glycosidic linkage: a branched chain of glucose molecules with a-1,6’
glucosidic linkages forming the branches

7) Glycogen
a) a-1,4’ glycosidic linkage: a branched chain of glucose molecules with a-1,6’
glucosidic linkages forming the branches

If the linkage is pointing upwards it is a beta linkage, if it is pointing downwards it is an

alpha linkage!!!

Glycosidic linkages can be broke via hydrolysis!!!

Without an enzyme, they are broken down only slowly with water. Animals do not possess the
enzyme to break the B-1,4’ glucosidic linkage in cellulose. Some adult humans lack the
enzyme to break the B-1,4’ galactosidic linkage in lactose.

NMR
NMR refers to nuclear magnetic resonance spectroscopy. It identifies protons.

Nuclei with an odd atomic number (# of protons) or odd mass number exhibit nuclear
spin that can be observed by an nmr spectrometer!!! The spin creates a magnetic field
around the nucleus similar to the field by a small magnet. When placed in an external magnetic
field, the nucleus aligns its own field with or against the external field. Nuclei aligned with the
magnetic field have a lower energy state than those aligned against the magnetic field!!!

Note: The stronger the field, the greater the difference between these energy
states.

When photons (electromagnetic radiation) of just the right frequency (energy state) are shone on
the nuclei, those nuclei whose magnetic fields are oriented with the external magnetic field can
absorb the energy of a photon and flip to face against the external field. A nucleus that is subjected
to this perfect combination of magnetic field strength and electromagnetic radiation frequency is
said to be in resonance. An nmr spectrometer can detect the energy absorption of a
nucleus in resonance

In nmr, the frequency of the electromagnetic radiation is held constant while the
magnetic field strength changes!!!

Hydrogen atoms within different compounds experience unique surrounding-electron densities and
are also uniquely affected by the magnetic fields of other nearby protons. The electrons shield
the protons from the magnetic field. As a result, the external field must be strengthened for a
shielded proton to achieve resonance. Thus protons within a compound absorb
electromagnetic energy of the same frequency at different magnetic field strengths.

The nmr spectrum is a graph of the magnetic field strengths absorbed by the hydrogens of specific
compounds at a single frequency. The field strength is measured in parts per million, ppm, and
despite decreasing numbers, the field strength increases from left to right.

EWG H’s are more downfield and EDG H’s are more upfield

Unless otherwise specified by the MCAT, nmr is concerned with hydrogens.

Determining Equivalent Hydrogens in 1H NMR Spectroscopy

The hydrogens in the structure of a molecule can be grouped together based on their
individual molecular environments (i.e., where each hydrogen is located in the molecule’s
 structure). Hydrogens that are in identical molecular environments in a molecule are chemically
equivalent. Chemically equivalent hydrogens have the same chemical shift in a 1H NMR
9spectrum, so they show up as a single signal.

For example, all six hydrogens in ethane are chemically equivalent; they are all in the
same molecular environment, so only one signal is seen in ethane’s 1H NMR spectrum.

Chemically equivalent hydrogens are called homotopic hydrogens. Two hydrogens must be
in identical molecular environments for them to be homotopic.

Hydrogens in a molecule that are in different molecular environments are chemically

nonequivalent. Chemically nonequivalent hydrogens have different chemical shifts in the 1H
NMR spectrum of the compound and show separate signals. For example, the eight hydrogens
in propane are not chemically equivalent. The six methyl hydrogens are chemically equivalent,
as are the two methylene hydrogens, but the two methylene hydrogens are in a different
molecular environment than the six methyl hydrogens.

The methyl hydrogens and the methylene hydrogens in propane are chemically nonequivalent.
These two groups of nonequivalent hydrogens have different chemical shifts and will show up
as two separate signals in a 1H NMR spectrum. Chemically nonequivalent hydrogens are
called heterotopic hydrogens. In order for two hydrogens to be heterotopic, they must be in
different molecular environments. (In theory, every group of nonequivalent hydrogens gives
rise to a separate signal in the 1H NMR spectrum, but in some cases the chemical shift
differences between nonequivalent hydrogens is so small that the separate signals for these
nonequivalent hydrogens are not detectable.)

You can often tell from simple inspection of a molecule's structure whether or not certain
hydrogens are chemically equivalent to each other. A substitution test is an easy
way to determine if two hydrogens in a molecule are in the same or different molecular
environments. The hydrogens in question in the structure are replaced, one at a time, by a
test group like F, and then the resulting structures are compared. If the resulting
structures are identical, then the hydrogens in question are chemically equivalent; if the
resulting structures are not identical, then the hydrogens in question are chemically
nonequivalent.

To illustrate this test, let's look at the hydrogens that are labeled a, b, and c in the
structure of propane below.

Substituting F first for the hydrogen labeled a and then for the hydrogen labeled c gives the
following two structures:

Both of these structures represent the same compound, 1-fluoropropane. Therefore the
hydrogens labeled a and c are chemically equivalent and they give the same signal in propane’s
1
H NMR spectrum.

Substituting F first for the hydrogen labeled a and then for the hydrogen labeled b in
propane gives the following two structures:

In the first case, the substitution gives 1-fluoropropane, while in the second case the substitution
gives 2-fluoropropane. These structures represent constitutional (structure) isomers of each
other, so the hydrogens labeled a and b are chemically nonequivalent. These hydrogens give
different signals in the 1H NMR spectrum.
 Now let’s add in something more: stereochemistry.

A hydrogen’s molecular environment depends not only on its location in the molecule (i.e., the
constitution of the molecule) but also on the hydrogen’s orientation in the molecule (i.e., the
configuration of the molecule).

Constitutionally equivalent hydrogens oriented with different configurations are not in

identical molecular environments. Therefore, they are not chemically equivalent.

In propene, for example, it is easy to see that the CH3, the CH, and the CH2 hydrogens are
in nonequivalent environments, and each group will show a different signal in the 1H NMR
spectrum. This difference is easily shown by using the substitution test which gives 3-
fluoropropene, 2-fluoropropene and 1-fluoropropene as the resulting structures.

The two vinylic hydrogens on C-1 need further inspection. Even though the two vinylic
hydrogens on C-1 are constitutionally equivalent, they are not chemically equivalent. If we
draw out the trigonal planar configuration for the carbon-carbon double bond in propene, and
then use the substitution test to check the equivalence of the two hydrogens on C-1, we get the
following result:

Substituting F for Ha gives cis-1-fluoropropene, while substituting F for Hb gives trans-1-

fluoropropene. These two structures represent stereoisomers of each other. Therefore, the
hydrogens labeled a and b are chemically nonequivalent, and they will show separate signals in
the 1H NMR spectrum. The two structures that result from the substitution test are diastereomers,
so the hydrogens labeled a and b above in propene are diastereotopic hydrogens. Diastereotopic
hydrogens are in nonequivalent environments, they are heterotopic hydrogens and they give
separate signals in the 1H NMR spectrum. Diastereotopic hydrogens often arise in compounds
that have restricted rotation because of the presence of carbon-carbon double bonds or
rings.
The presence of a stereogenic center in a molecule can also give rise to diastereotopic
hydrogens. In R-2-butanol, the methylene hydrogens on C-3 are constitutionally equivalent,
but they are not chemically equivalent. Substituting F in the structure for each of these two
hydrogens in turn gives structures that are diastereomers: the (R,R) and the (R,S) stereoisomers.

The two methylene hydrogens in 2-butanol are in chemically different environments and they
will give rise to separate signals in the 1H NMR spectrum.

Differences in configuration can result in hydrogens that are in chemically different environments
even when the compound lacks a stereogenic center, a double bond, or a ring. Constitutionally,
the six methyl hydrogens in butane are in a different molecular environment than the four
methylene hydrogens. Using the substitution test to check the chemical equivalence of a pair of
methylene hydrogens gives the following two structures:

The resulting structures both have the constitution 2-fluorobutane, but they do not represent the
same compound. These two structures represent a pair of enantiomers – they are
nonsuperimposable mirror images of each other. Since substituting each of these methylene
hydrogens with an F gives rise to structures that represent a pair of enantiomers, these
hydrogens are called enantiotopic hydrogens. Enantiotopic hydrogens are not chemically
equivalent. Enantiotopic hydrogens differ from each other only by being in mirror image
molecular environments.

Under typical achiral conditions, the 1H NMR spectrophotometer cannot distinguish between
mirror image molecular environments in a molecule, so enantiomeric hydrogens have the
same chemical shift and show the same signal in the1H NMR spectrum!!!

Note: Chemical shift is the difference between the resonance frequency (energy state)
of the chemically shifted hydrogens and the resonance frequency (energy state)
of hydrogens on a reference compound. It indicates the electronic
environment of the molecule

However, if an optically active solvent is used, then these enantiotopic hydrogens will be
seen as nonequivalent by the 1H NMR spectrometer and will show different signals in the
compound’s spectrum!!!

Cyclopropanol is another example of a compound that shows the different ways that
the three-dimensional structure of a molecule can influence the molecular environments of the
hydrogen atoms in the molecule. The methine hydrogen at C-1 is obviously in a chemically
different environment than the pairs of methylene hydrogens at C-2 and C-3, but what are the
chemical relationships between the four methylene hydrogens in cyclopropanol?

The ring structure here prevents free rotation so we treat it as a double bond sort of.
Hydrogens a, b, c, and d are all in different chemical environments. The pair of hydrogens
labeled a and b, and the pair of hydrogens labeled c and d, are both pairs of diastereotopic
hydrogens since using the substitution test gives two different structures, either cis-2-
fluorocyclopropanol or trans-2-fluorocyclopropanol.

The pair of hydrogens labeled a and c, and the pair of hydrogens labeled b and d, are also pairs
of diastereotopic hydrogens for the same reason.

The pair of hydrogens labeled a and d, and the pair of hydrogens labeled b and c, are
constitutionally equivalent but they differ in configuration. Using the substitution test for either
the hydrogens labeled a and d, or for the pair of hydrogens labeled b and c, gives a pair of
enantiomers.
 Even though these hydrogens are on different carbon atoms in the structure, they are
enantiotopic hydrogens and will show the same signal in the 1H NMR spectrum.

Therefore, cyclopropanol will show three sp3-CH hydrogen signals in its 1H NMR
spectrum: one for the methine hydrogen, one for the enantiotopic pair of
hydrogens labeled a and d, and one for the enantiotopic pair of hydrogens labeled
b and c.

-------------------------------------------------------------------------------------------------

In summary, the substitution test allows you to easily determine between the
identical, enantiotopic, and diastereotopic hydrogens in a molecule’s structure and
thereby predict the number of signals that you would see in the compound’s 1H NMR
spectrum.

• If the two hydrogens are not constitutionally equivalent, then they are
chemically nonequivalent and will give different signals in the 1H NMR
spectrum.

• If the two hydrogens are constitutionally equivalent, you now have to check
their stereochemical relationship.

• If the hydrogens are in identical configurations, then they will show

the same signal in the 1H NMR spectrum.

• If the hydrogens are enantiotopic, then they will show the same signal
in the 1H NMR spectrum (unless a chiral solvent is used; in this case
they will show up as two separate signals in the spectrum).

• If the hydrogens are diastereotopic, then they will show different

signals in the 1H NMR spectrum regardless of the conditions.

Things to understand:

- each peak represents one or a group of chemically equivalent

hydrogens
a) such hydrogens are said to be enantiotropic

b) they have the same chemical shift (peak on the graph)

- Chemical shift is the difference between the

resonance frequency of the chemically shifted
hydrogens and the resonance frequency
 of hydrogens on a reference compound
such as tetramethysilane

c) A hydrogen atom is "different" to another hydrogen atom if it

is not in an identical environment to the other hydrogen. This
could mean it's attached to a different type of atom (e.g. CH
vs OH, or sp3 CH vs sp2 CH) or due to the number of
adjacent H (e.g. CH3- vs -CH2- ) or cis hydrogens vs. trans
hydrogens or just at a different point in a chain (e.g. compare
the H in the methylene (CH2) groups in CH3CH2CH2OH

d) Remember to consider the three dimensional nature of the

molecule

- Splitting of peaks is created by “neighboring hydrogens”

a) Also called spin-spin splitting results from

neighboring hydrogens that are NOT chemically
equivalent!!!!

b) The number of peaks due to splitting for a group of

chemically equivalent hydrogens is given by the formula,
n+1, where n is the number of neighboring hydrogens that
are NOT chemically equivalent.

c) The deal with hydrogens that are attached to O or N is

that they normally do not influence splitting. (That is,
they don't split other H’s and themselves are not split).

Note: spin-spin coupling is the same thing except tha it also includes
hydrogens that are chemically equivalent, but the MCAT
wont test the distinction.

The area under a peak is proportional to the number of hydrogens

represented by that peak. The more chemically equivalent hydrogens, the greater
the area!! The tallest peak doesn’t necessarily correspond to the greatest area. The
integral trace line drawn above the peaks that rises each time it goes over a peak. The
rise of the integral trace is proportional to the number of chemically equivalent hydrogens
A newer instrument, called a digital trace, records the numbers which correspond to the
rise in the line.

For example, if the heights were 0.7 cm, 1.4 cm and 2.1 cm, the ratio of the peak
areas would be 1:2:3.

That in turn shows that the ratio of the hydrogen atoms in the three different
environments is 1:2:3.
Electron-withdrawing groups tend to lower shielding and thus decrease the
magnetic field strength at which resonance takes place, thus more downfield **

Electron-donating groups tend to increase shielding and thus increase the magnetic
field strength at which resonance takes place, thus more upfield

For proton nmr spectroscopy, follow these steps:

1) identify chemically equivalent hydrogens

2) identify and count neighboring hydrogens that are not chemically
equivalent. Use n+1 to find the number of peaks for that group
3) identify electron withdrawing/donating groups near the chemically
equivalent hydrogens and their effect will be more downfield/upfield
respectively.
Aldehyde protons have a very distinct shift at 9.5 ppm. Watch for it!!!

In a rare situation, carbon nmr may also appear on the MCAT. Remember, the nucleus
must have an odd atomic number or odd mass number to register on nmr, so carbon-13 is
the only carbon isotope to register. Treat carbon nmr the same way as hydrogen nmr
except with NO splitting.

Recall:

Mass number = protons + neutrons

Atomic number = # of protons

IR spectroscopy
When exposed to infrared radiation, the polar bonds within a compound stretch and
contract in a vibrating motion. Different bonds vibrate at different frequencies.

When the resonance frequency of the oscillating bond is matched by the frequency
of the infrared radiation, the IR energy is absorbed.

Note: This is useful to identify functional groups.

In 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.

If a bond has no dipole moment, then the infrared radiation doesn’t

cause it to vibrate, and thus no energy is absorbed!! However, energy can
also be absorbed due to other types of stretching and scissoring motions of the molecules
in a compound.
The most predictable section of the IR spectrum is the 1600 to 3500 cm^-1 region.

Alcohol:

Carboxylic Acid:

Aldehyde:
Nitriles:
Amines:
 3300  N - H
1700  C = O, sharp dip
3200  O-H, broad dip
2200  CN, sharp dip
2800-3000  saturated C – H

Limited predictions about vibrations can be made based upon the mass of the atoms
involved and the stiffness of the bonds between them.

- Atoms with greater mass resonate at lower frequencies.

- stiffer bonds, such as double and triple bonds resonate at a

higher frequency.

- Bond strength and stiffness follow the same order: sp > sp2 >
sp3 For example, The stretching frequency for a triple bond between
two carbons is in the range of 2100-2260 cm-1 whereas the double
between two carbons is in the range of 1620-1680 cm-1. See Fig 1
above.

Many of the complex vibrations that distinguish one come from another are found in the
600 to 1400 cm^-1 region, called the fingerprint region (unique to every compound).

The region that is useful for identifying functional group is from 1600 to 3500 cm^-1

UV Spectroscopy
The wavelength of ultraviolet light is between 200 and 400 nm, much shorter than
infrared light and at a much higher energy level.

UV spectroscopy detects conjugated double bonds (double bonds separated by one

single bond) by comparing the intensities of two beams of light from the same
monochromatic light source.

One beam is shone 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 solvent. The
reference cell contains only the solvent. The sample cell will absorb more energy from
the light beam than the reference cell.

When a photon collides with an electron a molecule in the sample, the

electron may be bumped to a vacant molecule orbital and the photon
absorbed. These are typically p-electron movements from bonding to
nonbonding orbitals ( to *).
Electrons in sigma-bonds usually require more energy to reach the next
highest orbital, and thus are typically unaffected by wavelengths greater than
200nm.

Conjugated systems with bonds on the other hand, have vacant orbitials at
energy levels close to their highest occupied molecular orbital (HOMO)
energy levels. The vacant orbitals are called LUMO (lowest unoccupied
molecular orbital).

UV photons are able to momentarily displace electrons to the LUMO, and the energy is
absorbed. If a conjugated system is present in the sample, the sample beam intensity
(Is) will be lower than the reference beam intensity (Ir).

The absorbance is given by A = log(Is/Ir) or it also equals A = ecl (where e = molar

absorptivity, l is length of the path of light through the cell, and c = concentration of the
sample)

The molar absorptivity is a measure of how strongly the sample absorbs light at a
particular wavelength.

** UV conjugated absorption starts at around 217 nm with butadiene **

Conjugated systems absorb at even longer wavelengths. The longer the chain of
conjugated double bonds, the greater the wavelength of absorption!

The rule of thumb is that each additional conjugated double bond increases the
wavelength by about 30-40 nm. Additional alkyl groups attached to any one
of the atoms involved in the conjugated system increases the absorbed
wavelength by about 5 nm. **

** Isolated double bonds do NOT increase the absorption wavelength **

C=O systems also absorb light in the UV region. Electrons can be excited from an
unshared pair into a nonbonding orbital (n to *). Acetone has a broad absorption
peak at 280 nm.

If a compound has eight or more double bonds, its absorbance moves into the
visible spectrum!!

B-carotene, a precursor of vitamin A, has 11 conjugated double bonds and has a

maximum absorbance at 497. When white light passes through or is reflected by a
colored substance, a characteristic portion of the mixed wavelengths is absorbed. The
remaining light will then assume the complementary color to the wavelength(s)
absorbed. This relationship is demonstrated by the color wheel shown on the right. Here,
complementary colors are diametrically opposite each other. Thus, absorption of 420-430
nm light renders a substance yellow, and absorption of 500-520 nm light makes it red

Mass Spectrometry
Mass spectrometry gives the molecular weight, and, in the case of high resolution
mass spectrometry, the molecular formula. Mass spectrometry is becoming
increasingly important in biochemistry as well; sequencing of proteins using this
technique alone allows protein structures to be determined on a virtually single-cell scale.

A mass spectrometer is designed to do three things:

1) Convert neutral atoms or molecules into a beam of positive or negative ions

2) Separate the ions on the basis of their mass-to-charge (m/z) ratio!!
3) Measure the relative abundance of each type of ion

In mass spectrometry, the molecules of a sample are bombarded with electrons, causing
them to break apart and to ionize. The largest ion is the size of the original
molecule, but short one electron!!! This cation is called the molecular ion (a
type of radical cation). If the molecular ion is too unstable then it can fragment to give
other smaller ions. 

The radius of curvature of their path depends on the mass to charge ratio of the ion
(m/z). Most of the ions have charge of +1.
The magnetic field is altered to allow the passage of different size ions through the
flight tube!!!

The largest peak is the base peak. The peak made by the molecular ions is called the
parent peak. Look for the parent peak all the way to the right of the spectrum. Only
heavier isotopes will be further right.

For M+2 peaks if it is a 3:1 ratio to the parent peak then there is a chlorine atom if
its 1:1 then it’s a bromine atom

All peaks are assigned abundances as percentages of the base peak. In the diagram on
the previous page, the parent peak has an abundance of 10 because it is 10% as high as
the base peak.

Chromatography
Chromatography is the resolution (separation) of a mixture by passing it over or
through a matrix (usually solid) that 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.

The resolving matrix adsorbs compounds from the mixture, establishing the stationary
phase. The compounds in the mixture that have a greater affinity for the surface move
more slowly. Typically, the more polar compounds elute more slowly b/c they have a
greater affinity for the stationary phase.

The result of chromatography is the establishment of separate and distinct layers, one
pertaining to each component of the mixture.

Different types of chromatography include:

1) column chromatography (solid to liquid)

a) Where the solution is dripped down a column containing the solid
phase (usually glass beads).
b) The more polar compounds in the mixture travel more slowly down
the column, creating a separation of layers for each compound.
c) Each substance can be collected as it elutes

2) paper chromatography (solid to liquid)

a) One end of the paper is placed in the solvent and the solvent moves up
the paper via capillary action and dissolves the same as it passes over
it.
b) As the solvent moves 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.

c) The result is a series of colored dots representing different components

of the solvent with the most polar down at the bottom and the least
polar near the top.

d) An Rf factor can be determined for each component of the separation

by dividing the distance traveled of the component by the distance
traveled of the solvent. The Rf factor is always between 0 and 1
 - nonpolar components have a Rf value close to one
- polar components have a lower Rf factor

3) thin layer chromatography (solid to liquid)

a) Is similar to paper chromatography except that a coated glass or plastic
plate is used instead of paper (i.e. silica gel membrane which is
polar), and the results are visualized via an iodine vapor chamber.

4) gas chromatography (Gas to liquid)

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

Distillation

Distillation is separation based upon vapor pressure. A solution of two volatile fluids
with boiling point differences of approximately 20 degrees celcius or more may be
separated by slow boiling. The compound with the lower boiling point (higher vapor
pressure) will boil off and can be captured and condensed in a cool tube.

If a solution of two volatile liquids exhibits a positive deviation to Raults law, the
solution will boil at a lower temperature than either pure compound. The result will
be a solution with an exact ratio of the two liquids called an azeotrope.

Note: An azeotrope is a mixture of two or more liquids (chemicals) in such a

ratio that its composition cannot be changed by simple distillation.
This occurs because, when an azeotrope is boiled, the resulting
vapor has the same ratio of constituents as the original mixture!!!

An azeotrope can also form when the solution has a higher boiling point than either
pure substance. 5% ethanol and 95% water form an azeotrope

Fractional distillation is a more precise method of distillation . In fractional

distillation, the vapor is run through glass beads allowing the compound with the higher
boiling point to condense and fall back into the solution.

Can never separate it 100%

Crystallization
Crystallization is based upon the principle that pure substances form crystals more
easily than impure substances, so they have higher melting points.
Crystallization is an inefficient method for separation; it is very difficult to arrive at a
pure substance via crystallization. You can also never retrieve 100% of the substance
you want because there will always be a little left over in the solvent due to the
solubility.

** Crystallization/precipitation of most salts is an exothermic process **

Extraction
Extraction is based upon solubility due to similar polarities. We start with an organic
mixture on top of an aqueous layer. They have different polarities and don’t mix.

There are three steps:

1) add a strong acid and shake (ex. HCl)

a) the acid protonates bases, like amines, in the organic layer, making
them polar
b) the polar amines dissolve into the aqueous layer and are drained off

2) add a weak base (ex. NaHCO3)

a) the base protonates only the strong acids like carboxylic acids, making
them more polar.
b) the polar carboxylic acids dissolve into the aqueous layer and are
drained off

3) add a strong base (ex. NaOH)

a) the strong base protonates the rest of the acids, hopefully all weak
acids (like phenol), making them more polar.
b) These acids dissolve into the aqueous layer and are drained off

Note: A strong base will deprotonate all acids. And to separate something
into the aqueous phase we need to induce a charge on the molecule.