CHEM202 Practice

**IR** SP3;C-H (alkane)

2850-3000 cm^-1

(

**IR** SP2;C-H (alkene)

3000-3100 cm^-1

(>3000 cm^-1)

**IR** SP;C-H (alkyne)

3300 cm^-1

**IR** -OH

rounded, >2700 cm^-1

**IR** C-O, C-N

1000-1200 cm^-1

**IR** -NH

3250-3400 cm^-1

**IR** C=C

1600-1680 cm^-1

**IR** C=O

\~1700 cm^-1

**IR** C-O

1000-1200 cm^-1

**13C NMR** SP3;C-H (alkane)

5-35 ppm

**13C NMR** SP;C-H (alkyne)

60-85 ppm

**13C NMR** C-O, C-halogen

60-80 ppm

**13C NMR** SP2;C-H (alkene)

120-160 ppm

**13C NMR** Aromatic ring

120-160 ppm

**13C NMR** C=O (ketone, aldehyde)

190-220 ppm

**13C NMR** X-C=O (acids, esters, amides)

170-180 ppm

**13C NMR** Carboxylic acid

\~180 ppm

**1H NMR** H-SP3C

0-4.5 ppm

**1H NMR** H-SP2C

4\.5-12 ppm

**1H NMR** H-C SP3 (alkane)

0\.5-1.5 ppm

**1H NMR** H-SPC

1\.7-3.1 ppm

**1H NMR** alkyl-CH3

0\.7-1.3 ppm

**1H NMR** alkyl-CH2

1\.2-1.6 ppm

**1H NMR** H-C-O

2\.3-4.0 ppm

**1H NMR** H-C-C=O (next to carbonyl)

2\.0-2.3 ppm

**1H NMR** CH-C=C (next to alkene)

1\.8-2.2 ppm

**1H NMR** CH-X (X = Cl, Br, I)

2\.5-4.0 ppm

**1H NMR** H-Aromatic ring

6-8 ppm

**1H NMR** H-C=O (aldehyde)

8\.5-10.0 ppm

**1H NMR** -OH

2\.0-5.0 ppm

**1H NMR** -NH

2\.0-5.0 ppm

**1H NMR** HN-C=O (amide)

6\.0-8.0 ppm

**1H NMR** HO-C=O (carboxylic acid)

11\.0-14.0 ppm

Silica gel separates by polarity. Is silica gel (see picture) polar or non-polar?

Polar

Relative to silica gel, are the solvents (see picture) polar or non-polar?

Non-polar

Would a polar compound prefer (choose one):

* To move with the solvent.
* To stick to the silica gel.

To stick to the silica gel.

The **more non-polar** compounds elute **first** in silica gel chromatography. In the pair of compounds (see picture), which would be the **first** to **elute** from a silica gel column?

Compound A

The **more non-polar** compounds elute **first** in silica gel chromatography. In the pair of compounds (see picture), which would be the **first** to **elute** from a silica gel column?

Compound B

The **more non-polar** compounds elute **first** in silica gel chromatography. In the pair of compounds (see picture), which would be the **first** to **elute** from a silica gel column?

Compound A

After performing silica gel chromatography, the student observed the following TLC plate (see picture). What should the student do to get the more polar compound off the column?

Use a more polar solvent system.

In reversed phase chromatography, the **more hydrophobic** the molecule, the **more time it will spend** on the solid support and the higher the concentration of organic solvent that is required to promote de-sorption.

Is C18 gel (see picture) polar or non-polar?

Non-polar

In reversed phase chromatography, solvents such as water (H2O), methanol (CH3OH), or acetonitrile (CH3CN) are used as the mobile phase. Relative to the C18 gel, are the solvents polar or non-polar?

Polar

In reversed phase chromatography, would a **polar** compound prefer (choose one):

* To stick to the C18 gel.
* To move with the solvent.

To move with the solvent.

In reversed phase chromatography, would a **non-polar** compound prefer (choose one):

* To stick to the C18 gel.
* To move with the solvent.

To stick to the C18 gel.

Which of these compounds (see picture) would be the **first** to elute from a C18 reverse-phase column?

Compound B

Which of these compounds (see picture) would be the **first** to elute from a C18 reverse-phase column?

Compound A

Which of these compounds (see picture) would be the **first** to elute from a C18 reverse-phase column?

Compound A

In the reversed phase chromatography lab, the impurity used was benzophenone.

* Benzophenone is _____ (polar/non-polar).
* In the experiment, the benzophenone eluted _____ (before/after) the polar unknowns.

* non-polar
* after

In the enzymatic resolution lab, the two enantiomeric starting alcohols (choose one):

* Interact differently on silica gel.
* Have the same physical properties and cannot be separated.
* Have different melting points.
* Have different boiling points.

Have the same physical properties and cannot be separated.

Which of the following represents a racemic mixture (see picture)?

**Racemic mixture**: a mixture that has equal amounts of left- and right-handed enantiomers of a chiral molecule.

Mixture C

Determine the stereo-chemical designation (R or S) of the following compounds (see picture).

Compound A: R

Compound B: S

Determine the stereo-chemical designation (R or S) of the following compounds (see picture).

Compound A: R

Compound B: S

In the enzymatic resolution lab, the enzyme should convert one of the isomeric alcohols to an ester but the other alcohol should remain unreacted. However, if you leave it long enough, the enzyme will slowly react with the isomer also.

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A student was monitoring their enzymatic resolution laboratory using TLC (see picture).

At what time should the student stop the reaction?

1\.5 hours

The following is a TLC of a student’s enzymatic resolution reaction visualized under UV light (see picture).

The **top** spot of the TLC is (choose one):

* The alcohol starting material.
* The ester product.
* The acyl donor starting material.

The ester product.

The following is a TLC of a student’s enzymatic resolution reaction visualized under UV light (see picture).

The **bottom** spot of the TLC is (choose one):

* The alcohol starting material.
* The ester product.
* The acyl donor starting material.

The alcohol starting material.

The following compound was isolated (see picture).

Its literature optical rotation value is -43.9 degrees.

The calculated optical rotation value is -20.9 degrees.

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What is the optical purity of this sample?

47\.6%

For the following enzymatic reaction (see picture), identify what the following shapes represent in the reaction (acyl donor, ester product, alcohol with incorrect stereochemistry, enzyme, and alcohol with correct stereochemistry).


1. Black square and circle
2. White triangle
3. Large, rounded shape
4. White circle
5. Black circle

1. **Black square and circle**: Ester product
2. **White triangle**: Acyl donor
3. **Large, rounded shape**: Enzyme
4. **White circle**: Alcohol with incorrect stereochemistry
5. **Black circle**: Alcohol with correct stereochemistry

Size exclusion chromatography is based on the molecular size of the components. Separation is achieved by the differential exclusion from the pores of the packing material, of the sample molecules as they pass through a bed of porous particles. It is generally used to separate biological molecules and to determine molecular weights and molecular weight distributions of polymers.

Molecules that are **smaller** than the pore size can enter the particles and therefore have a **longer path** and take a **longer time** to elute than larger molecules that cannot enter the particles (see picture).

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In the size exclusion lab, you purified a smaller peptide from a mixture of a protein and a peptide using a size exclusion gel column. Which compound will elute first, the peptide or the protein?

Protein

In a mixture of the five proteins listed below, which should elute second size exclusion (gel filtration) chromatography?

* Cytochrome C: MW = 13,000
* Immunoglobin G: MW = 145,000
* Ribonuclease A: MW = 13,700
* RNA Polymerase: MW = 450,000
* Serum albumin: MW = 68,500

Immunoglobin G

The best way to increase the resolution of your chromatography column in size exclusion would be to (choose one):

* Use gel beads with larger holes so that all the molecules in the sample can enter some of the holes.
* Use a longer column.
* Run the buffer through the column at a faster rate.
* Load more sample into the column.

Use a longer column.

In the size exclusion lab, your peptide was not colored. How can you determine which fractions eluted from the column contain the purified peptide (choose one)?

* Keep them all and get NMR of each sample.
* Keep them all and get MS of each sample.
* Taste them.
* UV-Vis to detect any molecules that absorb in the UV-Vis region.

UV-Vis to detect any molecules that absorb in the UV-Vis region.

The unknowns for the size exclusion lab had chromophores, structures that absorb light. Tyrosine, Phenylalanine, and Tryptophan all absorb in the UV region (see picture).

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Which wavelength(s) should be monitored (choose one)?

* 254, 290
* 200-300
* 260, 280
* 220, 280

260, 280

This is the MS of a protein (see picture).

The largest peak (most abundant form) of this protein has a+18 charge. If the m/z ratio of that peak is 950 Da, what is the mass of the protein (choose one)?

* 17,100 Da
* 932 Da
* 950 kDa
* 52.78 Da
* 0.01895 Da

17,100 Da

Affinity columns are designed to bind only a particular type of protein. This protein will stick to the column while all of the others will wash through.

What are the steps of affinity chromatography?

1. The mobile phase, a mixture of many proteins, is brought into contact with the prepared resin. The target protein binds strongly to the “key” ligands on the resin.
2. The column is washed. Other proteins and contaminants, not being fixed to the protein, are washed away, leaving only the protein of interest behind.
3. Elution, the final step, releases the purified protein from the resin so it can be collected for further purification or other processing.

A single pass of a complex mixture of proteins through an affinity column can achieve greater than 1000-fold purification of a specific protein.

An affinity column made with 3-aminophenylboronic acid will bind tightly to glycoproteins (proteins that have sugar molecules attached to it) (see picture).

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* A student receives a mixture of ovalbumin (a glycoprotein) and myoglobin and places the mixture on an affinity column constructed with 3-aminophenylboronic acid residues. When the student washes the column with the wash buffer, which protein elutes?
* In a second step, an elution buffer is added to disrupt interactions between the bound target biomolecules with the ligand on the resin. When the elution solution is added, which protein elutes?

* Myoglobin
* Ovalbumin

In affinity chromatography, elution buffers dissociate the protein from the ligand-resin by disrupting the binding. Some methods include extremes of pH (low or high), high salt (ionic strength), or detergents that denature one or both of the molecules, removal of a binding factor, or competition with a counter ligand.

Which of these methods is the **LEAST** likely to denature your protein (choose one)?

* High salt concentration
* Detergent
* Low pH
* A competitive ligand
* High pH
* Low salt concentration

A competitive ligand

After a protein is purified using affinity chromatography, the sample is analyzed using SDS-PAGE gels.

**SDS Polyacyrlamide Gel Electrophoresis (PAGE)** is an analytical method in biochemistry for the separation of charged molecules in mixtures by their _____ in an electric field.

molecular masses

**SDS (sodium dodecyl sulphate, also called lauryl sulphate)** is an ionic detergent that coats proteins and imparts a _____ charge to all the proteins in the sample. These charges swamp the inherent charge of the proteins and give every protein a negative charge.

negative

**Mobility** becomes a function of _____. As a **charged** protein moves through the gel under the influence of an electric field, it experiences draw that **slows** it down. The **larger** the protein, the greater the drag, and the **slower** it moves.

We can estimate the **size** of the protein based on how far it moves.

molecular weight

SDS-PAGE can tell you two pieces of information. What are they?

1. The number of compounds present.
2. An approximate molecular weight when compared to known standards of specific molecular weights.

Once treated with SDS, do your protein samples migrate toward the positive or negative electrode?

Positive

In polyacrylamide gel electrophoresis (PAGE), will large MW proteins or small MW proteins migrate faster?

Small MW proteins

A student prepared a PAGE gel of their unknown protein (see picture, right lane). In the left lane, a molecular weight size marker was added.

Estimate the MW of the protein represented by the band in the right lane.

40 Da

For a determination of the molecular weight, the relative migration distances of the individual protein bands are measured in the separating gel.

The relative mobility (called Rf value or Rm value) is the distance of the band of the protein divided by the distance of the buffer front. The distances of the bands and the buffer front are each measured from the beginning of the separation gel. The distance of the buffer front roughly corresponds to the distance of the bromophenol blue contained in the sample buffer. The relative distances of the proteins of the size marker are plotted against their known molecular weights.

By comparison with the linear part of the generated graph or by a regression analysis, the molecular weight of an unknown protein can be determined by its relative mobility (see picture).

What is the MW of the unknown protein that is circled on the graph (choose one)?

* 0.00047 kDa
* 4.7 kDa
* 50 kDa
* 4.7^10 kDa

50 kDa

Like most column chromatography methods, ion exchange chromatography relies on Columbic interactions. Column’s Law is based on size of charge and distance between the charges.

In cation exchange chromatography, _____ charged molecules are attracted to a negatively charged solid support. Conversely, in anion exchange chromatography, _____ charged molecules are attracted to positively charged solid support (see picture).

* positively
* negatively

In ion exchange chromatography, based on the charges of coordination complexes, determine which compound would interact more strongly with a negatively charged column (see picture).

(see picture)

Co has a +3 charge.

In ion exchange chromatography, determine which compound would interact more strongly with a negatively charged column (see picture).

(see picture)

Co has a +3 charge.

To determine how a complex will interact with your Dowex 50Wx4 (100-200 mesh) cation exchange resin, you need to know the charges on your complexes.

The charge of the complex is the charge of the structure inside the brackets. What is the charge of the complexes used in the ion-exchange lab (see picture)?

1. -3
2. +3
3. +1
4. +1
5. +1
6. +3
7. -1

Assuming a negatively charged column, what would be the order of elution for the following compounds (see picture)?

A, C, then B

Assuming a negatively charged column, what would be the order of elution for the following compounds?

A, B, then C

In order to remove highly charged compounds from an ion exchange column, the elution solution should be high or low ionic strength?

High ionic strength

In ion exchange chromatography, what would happen if the highest ionic strength elution solution is used first (choose one)?

* All the charged compounds come off the column.
* Nothing.
* Only the most charged compound comes off the column.
* Only the least charged compound comes off the column.

All the charged compounds come off the column.

Using UV-Vis Spectroscopy in ion exchange chromatography, you will quantify the complex ions that you purified earlier in the lab.

The **Beer-Lambert Law** states that the intensity of the light passing through sample cell is also measured for that wavelength - given the symbol, *I*. If this is less than *I*0 (light through a cuvette), then the sample has absorbed some of the light.

This absorbance can be used to determine the concentration of the sample.

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A = Ecl

* A = ?
* E = ?
* c = ?
* l = ?

* A = absorbance
* E = molar absorption constant (a constant that is known for each possible complex ion)
* c = concentration of solution
* l = length of the cuvette

Guanosine has a maximum absorbance of 275 nm. E275 = 8400M^-1 cm^-1 and the path length is 1 cm. Using a spectrophotometer, you find that A275 = 0.70.

What is the concentration of guanosine (choose one)?

* 5.88 x 10^3 mol/L
* 12 x 10^3 mol/L
* 8.33 x 10^-5 mol/L
* Impossible to determine without the molecular weight.
* 5.88 x 10^3 mol/L

8\.33 x 10^-5 mol/L

In chromatography, there are two phases, a stationary phase and a mobile phase. The stationary phase can be solid or liquid supported on a solid. What is the mobile phase (choose one)?

* Solid
* Liquid
* Gas
* Solid or liquid
* Liquid or gas

Liquid or gas

Which of the following types of chromatography involves the separation of substances in a mixture over a 0.2 mm thick layer of adsorbent (choose one)?

* Gas chromatography
* Column chromatography
* Affinity chromatography
* TLC (thin layer chromatography)
* Ion exchange chromatography

TLC (thin layer chromatography)

True or false: Chromatography can be used to purify volatile substances.

True

In gas chromatography, the concentration of a substance can be determined by (choose one)…

* measurement of the height of the peak produced by the substance.
* comparison of the Rf of the substance with that of the standard.
* comparison of the area under the peak produced by the substance with the areas under the peaks produced by standard solutions.
* calculation of the Rt value of the substance.

comparison of the area under the peak produced by the substance with the areas under the peaks produced by standard solutions.

Thin layer chromatography can be used to distinguish between different molecules. If a particular molecule has low solubility in the mobile phase used, then it (choose one)…

* will move at a speed close to that of the solvent
* will have a low Rf value.
* must have a high molecular mass.
* will spend more time dissolved in the mobile phase than attached to the stationary phase.

will have a low Rf value.

Which of the following statements about mass spectrometry is incorrect (choose one)?

* Isotropic distribution patterns are observed in mass spectra.
* Mass spectrometry gives information about fragmentation patterns which can be compared to the reference spectra.
* Mass spectrometry provides direct information about complete structure and connectivity.
* Parent ions (the molecular weight) are not always observed in the mass spectra of compounds.

Mass spectrometry provides direct information about complete structure and connectivity.

A mass spectrometer is often linked to a gas chromatogram (GC). This is done so that the (choose one)…

* gas used as the mobile phase in the GC is first purified by the mass spectrometer.
* substances can be separated in the GC and then the mass spectrometer can be used to analyze the mass of each pure compound.
* exact mass of the mixture is determined by mass spectrometry before it is passed into the GC.
* substances can be separated in the mass spectrometer and then passed through the GC for analysis.

substances can be separated in the GC and then the mass spectrometer can be used to analyze the mass of each pure compound.

A student used an ion exchange resin with an SO3^-1 group.

A mixture of three transitional metal complexes (see picture) was eluted through the column.

Which order will these compounds elute from the column?

1. C
2. B
3. A

An affinity column is used to separate a mixture of 4 proteins, A-D. The ligand attached to the bead has the molecular characteristics shown (++, non-polar, OHs) in the geometry depicted (see picture).

The fall through fraction and the proteins that bound to the ligand on the column were then eluted through an SDS-PAGE gel. Lane 1 was the protein mixture, lane 2 was the “fall through” from the column, and lane 3 was the material eluted with the free ligand.

What would be expected for lane 3 (choose one)?

* Proteins A and B
* Proteins A and C
* Proteins B and D

Proteins A and C

A cartoon of a lipase with an acylating agent and a racemic mixture of an alcohol is shown (see picture).

What are the likely products of the reaction?

(see picture)

Four organic compounds (2-butanone, pentane, butanol, and propanoic acid) were separated by silica gel column chromatography with benzene as the eluent.

What is the order of elution from the column?

1. Pentane
2. 2-butanone
3. Butanol
4. Propanoic acid

A sample was loaded on a reversed phase chromatography column (see picture).

Was A, B, or C most polar?

A

You synthesize a liposome sample and analyze it by DLS. You then run the sample through an extruder. What is the most likely scenario, before and after (choose one)?

* Before: diameter = 1200 nm; polydispersity = 0.35; after, diameter = 300 nm; polydispersity = 0.15
* Before: diameter = 1200 nm; polydispersity = 0.15; after, diameter = 300 nm; polydispersity = 0.35
* Before: diameter = 300 nm; polydispersity = 0.15; after, diameter = 1200 nm; polydispersity = 0.35
* Before: diameter = 300 nm; polydispersity = 0.35; after, diameter = 1200 nm; polydispersity = 0.15

**Before**: diameter = 1200 nm; polydispersity 0.35

**After**: diameter = 300 nm; polydispersity = 0.15

Two solutions of the same compound were made up. Solution A was of concentration 0.98 x 10^-4 mol dm^-3, and solution B was was 1.66 x 10^-2 mol dm^-3.

The UV spectrum of solution A contained one absorbtion at λmax = 230 nm, while the spectrum of solution B contained absorbtions at λmax = 230 and 365 nm.

The difference in the spectra was because (choose one)…

* the value of λmax depends on concentration.
* electronic spectroscopic data are not always reproducible.
* Emax for the absorbtion at 365 nm is much smaller than Emax for the band at 230 nm.
* the value of Emax depends on concentration.

Emax for the absorbtion at 365 nm is much smaller than Emax for the band at 230 nm.

Which of the following peaks would be diagnostic for the IR spectrum of diethyl amine (choose one)?

* two sharp peaks at 3300-3400 cm^-1
* one sharp peak at 3300-3400 cm^-1
* two sharp peaks at 2700-2900 cm^-1
* one broad peak at 1600 cm^-1

one sharp peak at 3300-3400 cm^-1

Which of the following compounds can be characterized by UV-Vis spectroscopy (choose one)?

* hexaaquaaluminum complex ion
* trans-1,2-dibenzolyethylene
* Chromium (Cr)
* penta-1,4-diene

trans-1,2-dibenzolyethylene