Explore key reactions and separations topics for the MCAT, complete with practice problems and explanations to maximize preparation and test-day results.

Reactions and Separations for the MCAT banner

(Note: This guide is part of our MCAT Organic Chemistry series.)

Part 1: Introduction to reactions and separations

Organic chemists use their knowledge of functional groups and reactions to synthesize products. How are these products separated from the reactants and purified?

In this section, we'll cover all that you'll need to know about reactions and separation techniques for the MCAT. From identifying key reagents to understanding the fundamentals of high yield experimental techniques, you will be ready for whatever the MCAT has in store for you on reactions and separations on test day.

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Part 2: Organic reactions

a) Nucleophilic attack

Nucleophiles are species that are especially attracted to positively charged or polarized species. These have high electron density due to the presence of pi bonds and/or electron lone pairs. Since nucleophiles will donate their electrons to form covalent bonds, they are considered Lewis bases.

An Sn1 reaction, or unimolecular nucleophilic substitution reaction, is carried out through two steps. First, a carbocation is formed after a leaving group departs. This is an ionic compound that contains a positively charged carbon. Next, the nucleophile attacks the carbocation. This results in a racemic mixture, or mixture of R and S stereoisomers because the attack can occur on either side of the carbocation.

Since a higher electron density can better stabilize a carbocation’s positive charge, Sn1 reactions tend to prefer more substituted carbons. Additionally, Sn1 reactions favorably occur in protic solvents.

Note that the “S” in the name “Sn2” refers to the substitution that occurs. “N” refers to the nucleophilic attack, while “1” refers to the presence of one and only one species during the rate-limiting step.

Sn2 reactions, or bimolecular nucleophilic substitution reactions, are carried out in one step and they tend to proceed in an aprotic solvent. Essentially, the departure of the leaving group and the nucleophilic attack occur simultaneously. As seen in the figure below, this is a back-sided attack that inverts the stereochemistry of the reactants.

Since both steps of this reaction occur simultaneously, the Sn2 mechanism can also be described as concerted. Note that the rate-limiting step of this reaction involves two species, as indicated with the “2” in “Sn2.”

b) Electrophilic attack

Electrophiles are species that love electrons and negatively charged species. They may be positively charged or positively polarized. For example, a carbonyl carbon does not have a positive charge. However, due to its bond with the more electronegative oxygen atom, it is positively polarized. This makes it electrophilic. Additionally, since electrophiles accept electron pairs, they are considered Lewis acids.

Heterolysis refers to the cleavage of a covalent bond that results in one of the atoms referred to as the leaving group taking both bonded electrons. Weak bases, such as the conjugate bases of strong acids, are good leaving groups because they can stabilize the negative charge from the electrons.

For more information on the functions of electrophiles and nucleophiles, be sure to refer to our guide on the fundamentals of organic chemistry.

c) Saponification and esterification

Saponification and esterification are two key reactions you should be familiar with for test day. For more information on the structure and function of fatty acids, be sure to refer to our guide on lipids and membranes.

Figure: The basic structure of a triglyceride. — **Figure 3** The basic structure of a triglyceride.

Triglycerides are used to store energy in the form of fats. Triglycerides have a distinctive structure, consisting of a molecule of glycerol and three long-chain fatty acids. The length and structure of a fatty acid can vary depending on its function.

Esterification refers to a chemical reaction in which an alcohol and acid form an ester. In biochemistry, esterification allows for the storage of fatty acids. It is analogous to glycogenesis, where glucose is stored in the form of glycogen.

In saponification reactions, fatty acids and potassium hydroxide or sodium hydroxide are mixed to form a salt. Due to the polar head and nonpolar tail of the salt, micelles spontaneously form.

**Figure 4** A fatty acid is mixed with potassium hydroxide (KOH) to form a salt

d) Amino acid synthesis

Most biological macromolecules can be easily synthesized in the laboratory setting. For instance, amino acids can be synthesized through Strecker or Gabriel synthesis. While you won’t be tested on the specifics of these reactions, we’ve included examples below.

Strecker synthesis requires a precursor with an aldehyde functional group, along with ammonium chloride (NH₃) and cyanide (HCN) as reagents. The overall reaction is shown below:

**Figure 5** An example of Strecker synthesis

Gabriel synthesis requires a precursor that is an alkyl halide—that is, a hydrocarbon alkane bonded to a halogen. The presence of phthalimide as a reagent is required.

**Figure 6** An example of Gabriel synthesis

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Part 3: Key reagents in organic chemistry

a) Oxidizing and reducing agents

Species that gain electrons from other species in a redox reaction are considered oxidizing agents. The permanganate ion (MnO₄^-) and chromium species (Cr₂O₇ ^2-, CrO₃) are strong oxidizing agents. Permanganate will oxidize aldehydes and primary alcohols into carboxylic acids. CrO₃ will oxidize a primary alcohol into an aldehyde and a secondary alcohol into a ketone. The Jones oxidation is an example of such a reaction using CrO₃. However, the reaction conditions consist of CrO₃ dissolved in aqueous H₂SO₄ and then mixed with acetone. This results in primary alcohols being oxidized to carboxylic acids and secondary alcohols being oxidized to ketones.

Species that donate electrons to other species in a redox reaction are considered reducing agents. Lithium aluminum hydride (LiAlH₄) is a very strong reducing agent. It reduces aldehydes, carboxylic acids, and esters into primary alcohols by donating hydride ions (H^-). It also reduces ketones into secondary alcohols and amides into primary amines. Sodium borohydride (NaBH₄) is a weak reducing agent. It can reduce aldehydes into primary alcohols and ketones into secondary alcohols.

Note that oxidizing agents are themselves reduced while reducing agents are themselves oxidized. For more information on this topic, be sure to refer to our guide on oxidation and reduction reactions.

b) Tollens’ reagent and Benedict’s reagent

Tollen's reagent, or [Ag(NH₃)₂]⁺, is used to detect the presence of aldehydes by oxidizing them to carboxylic acids. In carbohydrate biochemistry, Tollen's reagent is used to detect the presence of reducing sugars. These sugars will reduce the reagent and form a silvery mirror.

Benedict’s reagent can also be used to detect the presence of reducing sugars by oxidizing aldehydes into carboxylic acids. It is made up of a mixture that contains Cu(OH)₂ which is reduced to Cu₂O when interacting with aldehydes. This produces a red precipitate.

For more information on this topic, be sure to refer to our guide on carbohydrates.

c) Heat and light

Catalysts speed up chemical reactions without being consumed in a reaction. Enzymes are a key example of biologically based catalysts since they speed up important biochemical reactions by lowering the activation energy.

Heat and light are two non-biological catalysts. Heat works as a catalyst by increasing the kinetic energy of molecules by increasing the temperature. This increased energy will result in more frequent collisions that can allow the occurrence of a reaction. In reaction schemes, heat is symbolized using a “∆” symbol over the reaction arrow.

Light also works as a catalyst by exciting electrons and covalent bonds. In reaction schemes, the presence of light is symbolized using the symbols “hν” over the reaction arrow.

For more information on the role of light, be sure to refer to our guide on atomic structure and periodic trends.

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Part 4: Separation techniques

a) Polyacrylamide gel electrophoresis (PAGE)

Polyacrylamide gel electrophoresis (PAGE) is a separation technique that uses an electrolytic cell to separate compounds based on their size and/or charge. Since this is an electrolytic cell, the cathode is always negatively charged, and the anode is always positively charged. There are three key types of gel electrophoresis you should be familiar with: native PAGE, SDS-PAGE, and isoelectric focusing.

In native PAGE, proteins are placed in the polyacrylamide gel without any modifications. Thus, they retain their native structure and are separated based on size and charge. In SDS PAGE, a detergent, sodium dodecyl sulfate (SDS), is used to denature the proteins. This gives them a uniform charge and separation is based on size alone. Finally, isoelectric focusing uses a different gel from normal PAGE. It uses one with a pH gradient where the anode is acidic (positively charged) and the cathode is basic (negatively charged). When a protein is at a point in the gel gradient where the pH is below its isoelectric point (pI), it will have a positive charge causing it to be attracted to the cathode. As it migrates towards the cathode, however, it will stop once its charge has been neutralized. The point at which it stops in the gel corresponds with its pI.

The function and setup of PAGE are extensively discussed in our comprehensive guide on biochemistry lab techniques.

b) Extraction

Extractions allow us to separate compounds. Using immiscible solvents, or solvents that are unable to mix together, polar and nonpolar compounds can be separated. The polar layer is referred to as the aqueous phase and it will dissolve any polar compounds. Water is a common example of an aqueous phase in extractions. The nonpolar layer is referred to as the organic phase and will dissolve any nonpolar compounds. Ethers are a common example of organic phases in extractions.

The two immiscible solvents, along with the mixture to be separated, are poured into the top of a separatory funnel and allowed to separate. Opening the spigot at the bottom of the funnel allows the bottom-most layer of the separated mixture to be collected.

If the compound of interest is polar, then it will be found in the aqueous phase. If the compound of interest is nonpolar, then it will be found in the organic phase.

c) Distillation

Distillation is a separation technique used to separate substances in the liquid phase based on their boiling points. The mixture to be separated is placed in a distilling flask and heated using a heat source. As the sample heats, substances in the mixture reach their boiling point and vaporize. The vapors are collected in a condenser, which collects and cools the vapors.

The liquid that has the lowest boiling point will be vaporized and collected first. This sample is fairly pure and is referred to as the distillate. There are three different types of distillation that can be used.

The first is simple distillation. This technique is used when the boiling points for the liquids are 25°C apart from one another and both are under 150°C.

If the boiling points for the liquids are less than 25°C apart, then fractional distillation can be used. Thus, if two mixed liquids have boiling points of 100°C and 110°C, it is preferable to use fractional distillation to separate them. If two mixed liquids with boiling points of 100°C and 135°C, simple distillation may be used to separate them.

Finally, vacuum or column distillation is used when the boiling points of the liquids are above 150°C. This technique slowly decreases the pressure of a system, thereby lowering the boiling points of the liquid. This prevents degradation of the products.

d) Chromatography

There are many different types of chromatography that you should be familiar with for the MCAT. Fortunately, they all share a few fundamental principles. There is a stationary phase and a mobile phase. Typically, the stationary phase will be solid.

The mobile phase, on the other hand, will typically be a liquid or gas that runs through the stationary phase and contains the dissolved sample. Compounds in the sample that share their features with the stationary phase will bind to it. Alternatively, compounds in the sample that share their features with the polar phase will bind to it.

Normal-phase paper chromatography uses a polar cellulose paper as the stationary phase medium. A nonpolar solvent is used as the mobile phase. It runs through the paper via capillary action. The figure below illustrates the process of normal-phase paper chromatography.

**Figure 9** Normal-phase paper chromatography

Samples are placed at the black “O” marks. As the nonpolar mobile phase moves through the medium, it carries nonpolar samples with it. The further up a sample goes, the more nonpolar it is. Thus, those that do not move very far are more polar. Compounds can be compared to each other using the retention factor (R_f). This can be calculated using the equation:

R_f = (Distance sample moved) ÷ (Distance mobile phase moved)
A compound will always have the same R_f value. Therefore, it can be used to narrow down the identity of an unknown compound. Note that the R_f value cannot be used to definitively confirm the identity of a molecule, since multiple compounds may share the same R_f value.

Thin-layer chromatography (TLC) is very similar to paper chromatography, where the stationary phase is polar, and the mobile phase is nonpolar. The major difference is that it uses a silica gel or alumina as the polar stationary phase.

Reverse-phase paper chromatography uses a nonpolar stationary phase and polar mobile phase. Thus, it is essentially the reverse of normal-phase paper chromatography. As a result, the further up a sample travels, the more polar it is.

Gas chromatography, also known as gas-liquid chromatography, uses a column to separate volatile compounds that are vaporizable. The stationary phase is a liquid or solid that is adhered to a column. The mobile phase is an inert gas. Compounds with a lower boiling point will be eluted first. Gas chromatography can be used to separate racemic mixtures, which contain both enantiomers of a particular compound in equal concentrations. A stationary phase can be used that only binds to one enantiomer, such as the R confirmation, allowing the other enantiomer, such as the S confirmation, to be isolated and eluted.

HPLC, or high-pressure liquid chromatography, is a more modern and sophisticated technique. It is the optimal method for small sample sizes and compounds that are very similar. The procedure can be very complex. The primary difference between this and gas chromatography is that the mobile phase is a liquid instead of a gas. In normal-phase HPLC, the stationary phase is polar, and the mobile phase is nonpolar. In reverse-phase HPLC, the stationary phase is nonpolar, and the mobile phase is polar.

**Figure 10** High-pressure liquid chromatography

One final type of chromatography, column chromatography, separates compounds based on charge, affinity, and size using a column lined with silica or alumina beads as a stationary phase. In this separation technique, the mixture is pumped through a column using a nonpolar mobile phase. The column is filled with gel beads with specific physical properties. The mixture then separates based on its interactions with the column. It then flows out the bottom of the column and is referred to as eluent.

In ion-exchange chromatography, the beads are coated with a charged substance that attracts compounds with the opposite charge. Thus, if the beads are coated with a positively charged substance, then a more negatively charged compound will adhere to the stationary phase, and more positively charged compounds will remain in the mobile phase.

In affinity chromatography, the beads are coated with a substance that the compound of interest has a high affinity for. Compounds in a sample that do not have an affinity for the substance that coats the beads will be eluted with the mobile phase.

Finally, in size-exclusion chromatography, the beads contain tiny pores. These pores form a complex maze that traps smaller compounds and slows their migration through the column. In contrast, larger compounds can avoid passing through the pores and instead make their way in between the beads. Thus, the largest compounds elute first, while the smallest compounds elute last.

Table 1 Different types of column chromotography

	Separation based on	Description
Ion-exchange chromatography	Charge	Beads coated with a charge substance make up the stationary phase and bind with compounds of opposite charge.
Affinity chromatography	Affinity	Beads coated with a substance that a compound of interest has a high affinity for.
Size-exclusion chromatography	Size	Contains porous beads that slow down the flow of smaller compounds and allows larger compounds to elute first

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Part 5: High-yield terms

Nucleophile: a species that is strongly attracted to positively charged or positively polarized species

Sn1 (unimolecular nucleophilic substitution) reaction: a two-step nucleophilic substitution reaction that results in the formation of an intermediate carbocation

Sn2 (bimolecular nucleophilic substitution) reaction: one-step nucleophilic substitution reaction that results in a racemic mixture of products

Heterolysis: cleavage of covalent bonds in which one of the atoms takes both bonded electrons

Esterification: a chemical reaction in which an alcohol and an acid form an ester

Saponification: a chemical reaction in which a fatty acid is reacted with potassium hydroxide or sodium hydroxide to form a salt

Oxidizing agent: species that gains electrons from other species in a redox reaction

Reducing agents: species that donates electrons to other species in a redox reaction

Tollen’s reagent: detects the presence of aldehydes by oxidizing them to carboxylic acids; forms a silvery mirror when the test is positive

Native PAGE: separates proteins based on size and charge; maintains the native structure of the protein

SDS-PAGE: uses sodium dodecyl sulfate (SDS) to denature and neutralize proteins and separate them based on size alone

Isoelectric focusing: separates proteins based on their isoelectric point

Extractions: separate compounds using immiscible solvents; the compound of interest may be found in a polar aqueous layer or a nonpolar organic layer

Immiscible solvents: solvents that are unable to mix together

Simple distillation: used when the boiling points of both compounds are 25°C apart and under 150°C

Fractional distillation: used when the boiling points for both compounds are less than 25°C apart

Vacuum distillation: used when the boiling points for both compounds are above 150°C

Distillate: compound with the lowest boiling point and is collected first during a distillation

Normal-phase paper chromatography: separates compounds based on their affinity for a polar stationary phase and nonpolar mobile phase; uses cellulose paper as the medium for the stationary phase

Reverse-phase paper chromatography: separates compounds based on their affinity for a nonpolar stationary phase and polar mobile phase

Gas chromatography: uses a column to separate volatile compounds that are vaporizable

High-pressure liquid chromatography: computerized separation technique that is optimal for small sample sizes and similar compounds

Ion-exchange column chromatography: uses beads coated with a charged substance to attract oppositely charged compounds

Affinity column chromatography: uses beads coated with a substance that a compound of interest has a high affinity for

Size-exclusion column chromatography: uses porous beads that retard the flow of smaller compounds

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Part 6: Passage-based questions and answers

The mTOR signaling pathway plays an important role in skeletal muscle hypertrophy. This pathway is stimulated by key amino acids, including leucine. Current research indicates that other amino acids such as lysine and arginine may trigger muscle hypertrophy via the mTOR signaling pathway.

A student investigating the mTOR signaling pathway begins with a mixture of free lysine and leucine. To separate the mixture, she utilizes a column chromatography setup in which the stationary phase contains beads coated with a negatively charged substance. The mobile phase of the chromatography uses an uncharged liquid.

Next, the student identifies the isoelectric points of these two amino acids using isoelectric focusing gel electrophoresis. The results of this experiment are shown in Figure 1.

**Figure 1** Isoelectric focusing of leucine and lysine

Passage created for educational purposes only

Question 1: Which of the following is the structure of arginine at physiological pH?

A)

B)

C)

D)

Question 2: Which of the following amino acids would be found in the same phase as leucine during column chromatography?

A) Alanine

B) Lysine

C) Histidine

D) Arginine

Question 3: What is the isoelectric point of leucine

A) 2.0

B) 3.0

C) 6.0

D) 10.0

Question 4: Which of the following accurately characterizes the type of chromatography the student used and the expected results?

A) The student used ion-exchange chromatography, in which leucine would be found in the stationary phase and lysine in the mobile phase.

B) The student used ion-exchange chromatography, in which lysine would be found in the stationary phase and leucine in the mobile phase.

C) The student used affinity chromatography, in which leucine would be found in the stationary phase and lysine in the mobile phase.

D) The student used affinity chromatography, in which lysine would be found in the stationary phase and leucine in the mobile phase.

Question 5: If the student were interested in separating the amino acids based on size alone, which of the following methods could she use?

A) SDS-PAGE

B) Native PAGE

C) Simple distillation

D) Reverse-phase chromatography

Answer key for passage-based questions

Answer choice A is correct. It is critical to memorize the structures of all naturally occurring amino acids. Arginine contains an sp²-hybridized carbon atom bonded to three nitrogen atoms (choice A is correct). Lysine contains a single NH₃ group at the end of an alkyl chain (choice B is incorrect). Leucine is characterized by the presence of an isobutyl side chain (choice C is incorrect). Asparagine contains a carboxamide functional group (choice D is incorrect).
Answer choice A is correct. Since leucine is nonpolar and uncharged, an amino acid sharing those properties is expected to be found in the same phase. Alanine is a nonpolar and uncharged amino acid (choice A is correct). Lysine is a positively charged amino acid and thus should not be found in the same phase (choice B is incorrect). Histidine and arginine are also positively charged amino acids (choices C and D are incorrect).
Answer choice C is correct. Refer to Figure 1 for the isoelectric point of leucine and lysine. Since leucine is a nonpolar and neutral compound, it is expected to have a lower isoelectric point than lysine. Thus, the dot that has been focused on a pH of 6.00 should represent leucine. The dot that has been focused on a pH of 9 should be lysine (choice C is correct).
Answer choice B is correct. The passage states that the student used charged beads in the column. Thus, she must have used ion-exchange chromatography (choices C and D are incorrect). Since the stationary phase consists of charged beads, leucine would be found in the mobile phase. Lysine would be found in the neutrally charged stationary phase (choice B is correct, choice A is incorrect).
Answer choice A is correct. SDS-PAGE neutralizes charges and separates proteins based on size alone (choice A is correct). Native PAGE maintains the structure of proteins and separates them based on both size and charge (choice B is incorrect). Simple distillation separates compounds based on their boiling points (choice C is incorrect). Reverse-phase chromatography separates compounds based on polarity (choice D is incorrect).

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Part 7: Standalone questions and answers

Question 1: Which of the following agents can reduce a carboxylic acid into a primary alcohol?

A) Chromium trioxide

B) Lithium aluminum hydride

C) Sodium borohydride

D) Permanganate

Question 2: A researcher is interested in separating two liquid compounds. One of the compounds has a boiling point of 142°C, and the other has a boiling point of 106°C. Which separation technique should be used?

A) Fractional distillation

B) Vacuum distillation

C) Simple distillation

D) Size-exclusion chromatography

Question 3: Which of the following amino acids would elute last during size-exclusion chromatography?

A) Phenylalanine

B) Alanine

C) Glycine

D) Tryptophan

Question 4: Which of the following most accurately describes the function of Tollens’ reagent?

A) It detects the presence of reducing sugars by oxidizing aldehydes into carboxylic acids

B) It detects the presence of reducing sugars by reducing aldehydes into carboxylic acids

C) It detects the presence of oxidizing sugars by oxidizing aldehydes into carboxylic acids

D) It detects the presence of oxidizing sugars by reducing aldehydes into carboxylic acids

Question 5: What is the retention factor of compound A in the figure shown below?

A) 1/4

B) 1/2

C) 5/6

D) 6/7

Answer key for standalone questions

1. Answer choice B is correct. Lithium aluminum hydride is a very strong reducing agent that can reduce aldehydes, carboxylic acids, and esters into primary alcohols (choice B is correct). Chromium trioxide and permanganate are oxidizing agents (choices A and D are incorrect). Sodium borohydride is a weak reducing agent. It can reduce aldehydes into primary alcohols and ketones into secondary alcohols. However, it is not able to reduce carboxylic acids (choice C is incorrect).

2. Answer choice C is correct. Since the two liquids can be separated using their different boiling points, a distillation technique should be used (choice D is incorrect). Specifically, simple distillation should be used because both compounds have boiling points that are under 150°C and at least 25°C apart from one another (choice C is correct). Fractional distillation is used when the boiling points of multiple compounds are less than 25°C apart (choice A is incorrect). Vacuum distillation is used when the boiling points of the compounds are above 150°C (choice B is incorrect).

3. Answer choice C is correct. In size-exclusion chromatography, larger compounds elute first as they are not hindered by the pores in the beads coating the column. Thus, the smallest amino acid, glycine, would elute last (choice C is correct).

4. Answer choice A is correct. Tollen’s reagent is used in the detection of reducing sugars (choices C and D are incorrect). This is done by oxidizing aldehydes into carboxylic acids (choice B is incorrect).

5. Answer choice C is correct. The mobile phase moved a total of 12 units, from 2 units to 14 units. Sample A moved a total of 10 units, from 2 units to 12 units. These values can be entered into the formula for the retention factor:

R_f = [distance sample moved] ÷ [distance mobile phase moved]
R_f = 10 ÷ 12
R_f = ⅚

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Reactions and Separations for the MCAT: Everything You Need to Know

Explore key reactions and separations topics for the MCAT, complete with practice problems and explanations to maximize preparation and test-day results.

Table of Contents

Part 1: Introduction to reactions and separations