Cuevas, Regine

PROBLEM SET 2, CHM124

GUIDE QUESTIONS to ANSWER while SELF-STUDYING

I. (1) A mixture yielded two spots when subjected to thin layer chromatography on silica
using hexane-ethyl acetate (50:50) as the developing solvent.

Solvent Front, 4.50 cm

A Analyte A, 3.50 cm

B Analyte B, 1.50 cm

Point of Origin, 0.0 cm

For 2 pts each . . .

[a] which is the more retained analyte, A or B? A because it has a larger Rf value

[b] which is the more polar analyte, A or B? B because it has a lower Rf value.

[c] determine the retention factor of each spot.

SOLUTION
The compound with the larger Rf value is less polar because it does not stick to
the stationary phase as long as the polar compound, which would have a lower
Rf value.

Rf =

Rf value of A = = 0.778

Rf value of B = = 0.333
 [d] which solvent system will increase the retention factor of the more retained
compound hexane: ethyl acetate that is (40:60) or (60:40)?

(60:40) hexane: ethyl acetate is less polar so it will increase the retention factor of
the more retained compound.

(2) Between silica and alumina as the stationary phase in TLC, which one is better for the
separation of weakly polar compounds?
Silica is a better stationary phase for the separation of weakly polar compounds compared to
alumina. Alumina is used in the separation of non-polar compounds.

(3) In paper chromatography, what acts as the stationary phase?

The stationery phase in paper chromatography is the price of paper where mobile phase run.
Mobile phase is thee flow on the stationery phase and carry out the component of the mixture.

II. [A] Enumerate the four general types of chromatography based on the equilibriation
process involved. (4 pts)
 Paper chromatography
 Thin layer chromatography
 Planar chromatography
 Column chromatography

[B] Which equilibriation process is exemplified by each of the following? (1 pt each)

(a) by thin-layer chromatography
mobile phase = liquids or solvents like ethers and toluene
stationary phase = silica gel

(b) by paper chromatography

mobile phase = Mobile phase is thee flow on the stationery phase and
carry out the component of the mixture. It is a liquid.
stationary phase = The stationary phase in paper chromatography is the
strip or piece of paper that is placed in the solvent. It is a solid
(cellulose).

(c) by affinity chromatography

mobile phase = liquid
stationary phase = solid (agarose or porous glass beads on to which are
immobilized molecules like enzymes and antibodies).
 (d) by gravity column chromatography
mobile phase = The mobile phase is a liquid which is added to the top
and flows down through the column by either gravity or external
pressure.
stationary phase = The stationary phase is a solid adsorbent which is
placed in a vertical glass column.

(e) by gas chromatography

mobile phase = The mobile phase is usually an inert gas or an unreactive
gas such as helium, argon, nitrogen or hydrogen.
stationary phase = The stationary phase is a microscopic layer of viscous
liquid on a surface of solid particles on an inert solid support inside a
piece of glass or metal tubing called a column.

(f) by reversed phase HPLC

mobile phase = toluene, 𝑛 –propanol, methanol
stationary phase = a non-polar stationary phase, which is a modified
silica substrate with long hydrophobic long chains.

(g) by gel-permeation chromatography

mobile phase = mobile phase is the buffer that flows in between the
matrix beads.
stationary phase = the stationary phase is composed of a porous matrix.

(h) by vacuum chromatography

mobile phase = liquid
stationary phase = solid

[C] Which equilibriation process: (1 pt each)

(a) separates on the basis of molecular size = size exclusion
chromatography
(b) separates on the basis of charge = ion exchange chromatography
(c) is better for the separation of very nonpolar materials = reversed phase
chromatography
(d) is better for the separation of highly polar materials = normal phase
chromatography
III. Between Gel Filtration and Gel Permeation chromatography, which one is more suited for
the separation of water soluble samples?

ANSWER: Gel filtration chromatography is more suited for the separation of water
soluble samples such as small molecules, proteins, protein complexes, polysaccharides,
and nucleic acids when in aqueous solution.

IV. In Gel Permeation chromatography, which type of solutes is eluted first: larger or smaller?

ANSWER: large molecules are excluded from all pores and hence elute first.

V. In Affinity chromatography, which solute is eluted first: bound or unbound?

ANSWER: bound solute is eluted first.

VI. In Reverse Phase chromatography, which solute is eluted first: less polar or the more polar?

ANSWER: the more polar solute is eluted first.

VII. The following data are for a liquid chromatographic column:

Length of packing 24.7 cm
Flow rate 0.313 mL/min
VM 1.37 mL
VS 0.164 mL

A chromatogram of a mixture of species A, B, C, and D provided the following data:

Retention Time, min Width of Peak Base (W), min
Non retained 3.1 ----
A 5.4 0.41
B 13.3 1.07
C 14.1 1.16
D 21.6 1.72
Calculate:
a) The number of plates from each peak.

N, the number of theoretical plates, is one index used to determine the

effectiveness of columns and its performance. It is calculated using the equation
shown below:

SOLUTION FOR A:

N = 16 (retention time in minutes / width of peak)^2

N = 16 (5.4/0.41)^2
N = 2775.49

SOLUTION FOR B:

N = 16 (retention time in minutes / width of peak)^2

N = 16 (13.3/1.07)^2
N = 2472.04

SOLUTION FOR C:

N = 16 (retention time in minutes / width of peak)^2

N = 16 (14.1/1.16)^2
N = 2363.97

SOLUTION FOR D:

N = 16 (retention time in minutes / width of peak)^2

N = 16 (21.6/1.72)^2
N = 2523.31

b) The mean and the standard deviation for N.

SOLUTION:

Mean = sum of the terms/number of terms

Mean of N = (2775.49 + 2472.04 + 2363.97 + 2523.31)/ 4
Mean of N = 2533.70

Standard Deviation Formula

Standard Deviation of N =
( ) ( ) ( ) ( )
√ = 150.98
c) The plate height for the column.

SOLUTION:

Plate height of the column, H = where L is the length of column and

N is the number of theoretical plates.

= 0.00889 cm/plate

= 0.0102 cm/plate

= 0.0104 cm/plate

d) The retention factor

SOLUTION:
𝑛 𝑛
𝑛 𝑛 𝑛 𝑛

Retention factor =

Retention factor of A = = 1.348

Retention factor of B = = 4.783

Retention factor of C = = 5.130

Retention factor of A = = 8.391

 e) The distribution constant

SOLUTION:

Ka = retention factor
= retention time
= non retained time/void time
= volume in mobile phase
𝑛 𝑛

Ka = and Ka = (Ka)( )

( )( )
Ka = = 0.742 and Ka =

( )( )
Kb = = 3.29 and Kb = = 27.48

( )( )
Kc = = 3.55 and Kc = = 29.65

( )( )
Kd = = 5.97 and Kd = = 49.87

Calculate for species B and C:

f) The resolution

SOLUTION:

Resolution, R = = =
( ) ( )

g) The selectivity factor

SOLUTION:

𝑛 𝑛
𝑛 𝑛 4.783

Selectivity factor = = 1.072

h) The length of column necessary to separate the two species with a resolution of
1.5.

SOLUTION:

R1 = , the resolution we have calculated for B and C

R2 = 1.5, given
L1 = 24.7, length of column
L2 = ?

( )

( )

( ) ( )

(L2) 0.2304 = 24.7

L2 = 107.20 cm

i) The time required to separate the two species on the column in part (h).

SOLUTION:
Calculate for species C and D:
j) The resolution.

SOLUTION:

Resolution, R = = =
( ) ( )

k) The length of column necessary to separate the two species with a resolution of
1.5.

SOLUTION:

R1 = , the resolution we have calculated for C and D

R2 = 1.5, given
L1 = 24.7, length of column
L2 = ?

( )

( )

( ) ( )

(L2) 12.05 = 24.7

L2 = 2.05 cm