Tutorial 5 solutions

1
 Question 1

Three compounds: A, B and C exhibit retention factors on a column having only 500 plates of
kA = 1.40, kB = 1.85, kC = 2.65. Can they be separated with a minimum resolution of 1.05.
Calculate the selectivity factor, α between the peaks
αBA = kB / kA = 1.85 / 1.40 = 1.32
αCB = kC / kB = 2.65 / 1.85 = 1.43
Calculate the resolution between the peaks

𝑁𝑁 αBA − 1 kB
Rs (Between A and B) =
4 αBA 1 + kB

500 1.32 − 1 1.85

=
4 1.32 1 + 1.85
= 0.88
 𝑁𝑁 αCB − 1 kC
Rs Between B and C =
4 αCB 1 + kC

500 1.43 − 1 2.65

=
4 1.43 1 + 2.65

= 1.2

Thus, only peaks B and C can be well separated using N = 500.

 Question 2

A chromatogram reveals a complete resolution between two neighboring peaks, 1 and 2.

Compound 2 is the later eluting species. If k2 = 4 and α = 1.05 and knowing that the
retention time of the second compound is 5 min:

a) Calculate the void time tM of this chromatography.

b) Determine the retention time of solute 1.  N  α − 1 k B 
R s =    
c) Find the plate number using: 
 4  α  1+ k B 

Complete separation Rs = 1.5 (as guideline)

a) Calculate the void time tM of this chromatography.

(t S )2 (t R )2 − t M
k2 = = =4 (t R )2 = 5t M = 5 mins
tM tM

(t R )2 − t M = 4t M t M = 1 min

b) Determine the retention time of solute 1.

(t S )1 (t R )1 − t M
k2 k1 = = = 3.8
α= = 1.05 tM tM
k1

k2 4 (t R )1 − 1
k1 = = = 3.8 = = 3.8
α 1.05 1
(t R )1 = 4.8 min
5
 2 2
2 α   1+ k B 
c) Find the plate number using: N = 16R S    
 α − 1  kB 

Complete separation Rs = 1.5 (as guideline)

2 2
1.05 1+4
N = 16(1.5)2
1.05 − 1 4

= 24806 = 2.5 × 104

6
 Question 3
Consider a chromatogram in which two components with retention factors k1 = 4.0 and
k2 = 5.0 are injected into a column. The retention time for the less retained compound is 10
mins. Calculate void time and retention time of the other compound. Determine the
selectivity factor.
Calculate void time Calculate tR2

t R1 − t M t R2 − t M
k1 = =4 k2 = =5
tM tM

10 − t M tR2 = 6tM = 6×2 = 12 min

=4
tM

t M = 2 mins

7
Determine the selectivity factor.

kB 5
α= = = 1.25
kA 4

8
 Question 4
In a chromatographic analysis of lemon oil a peak for limonene has a retention time of
8.36 min with a baseline width of 0.96 min. g-terpinene elutes at 9.54 min, with a baseline
width of 0.64 min. The column’s void time is 0.31 min. What are the retention factors,
selectivity factor and resolution between the two peaks?

t R1 − t M 8 ⋅ 36 − 0 ⋅ 31
k1 = = = 25 ⋅ 9 2[(t R )B − (t R )A ]
tM 0.31 RS =
WA + WB
t R2 − t M 9 ⋅ 54 − 0 ⋅ 31
k2 = = = 29 ⋅ 8 2[9.54 - 8.36]
tM 0.31 RS = = 1.48
0.96 + 0.64

𝑘𝑘2 29.8
α= = = 1.15
𝑘𝑘1 25.9
Tutorial 6 solutions
GC

10
 Question 1
A standard solution containing 6.3 × 10-8 M iodoacetone and 2.0 × 10-7 M p-dichlorobenzene
(an internal standard) gave peak areas of 395 and 787 respectively in a gas chromatogram.

A 3.00 mL unknown solution of iodoacetone was treated with 0.100 mL of 1.6 × 10-5 M p-
dichlorobenzene and the mixture was diluted to 10.00 mL.
Gas chromatography of the unknown mixture gave peak areas of 633 and 520 for iodoacetone
and p-dichlorobenzene, respectively.

Find the concentration of iodoacetone in the 3.00 mL of original unknown

6.3 × 10−8 395

−7
= 𝑘𝑘
2.0 × 10 787

k = 0.628
 3.00×10−3 × 𝐴𝐴 �
10.00×10−3 633
0.100×10−3 ×1.6×10−5�
=k
520
10.00×10−3

3.00×10−3 × 𝐴𝐴 �
10.00×10−3 633
0.100×10−3 ×1.6×10−5�
= 0.628 ×
520
10.00×10−3

[A] = 4.07 × 10−7 M

 Question 2
Consider the chromatography of n-C12H26 on a 25 m × 0.53 mm open tubular column
of 5% phenyl-95% methyl poly siloxane with a stationary phase thickness of 3.0 μm
and He carrier gas at 125˚ C. The observed retention factor for n-C12H26 is 8.0.
Measurements were made on plate height H at various values of linear velocity ux
m/s. A least squares curve through the data points is given by
H(m) = (6.0 × 10-5 m2/s)/ux + (2.09 × 10-3 s)ux
From the coefficients of the Van Deemter equation, find the diffusion coefficient of
n-C12H26 in the mobile and stationary phase. Why is one of these diffusion
coefficients so much greater than the other
 B = 2 γ DM
L = 25 m 2k d2
CS =
dcolumn = 0.53 × 10-3 m 3(k + 1) 2 D S
2
Stationary phase thickness = d = 3.0 × 10-6 m 1 + 6k + 11k 2
d
CM =
96(k + 1) 2 DM
k = 8.0

B = 6.0 × 10-5 m2/s

CS + CM = 2.09 × 10-3 s
B = 2 γ DM ≈ 2DM = 6.0 × 10-5 m2/s

DM = 3.0 × 10-5 m2/s

CS +CM = 2.09 × 10-3 s

2k d𝑓𝑓2 2
2×8.0 × 3.0×10−6 5.92×10−13
=
3× 8.0+1 2 𝐷𝐷𝑆𝑆 =
CS = 2
3(k + 1) DS 𝐷𝐷𝑆𝑆

(1 + 6k + 11k2 ) d2𝑐𝑐 (1+6×8.0+11×8.02 ) 0.53×10−3

2
CM = = = 9.07 × 10−4
96(k + 1)2 DM 96 8.0+1 2 × 3.0×10−5

5.92×10−13
+ 9.07 × 10−4 = 2.09 × 10−3
𝐷𝐷𝑆𝑆

DS = 5.01 × 10−10 m2/s

The diffusion coefficient in the mobile phase is

(3.0 × 10-5 m2/s) /5.01 × 10-10 m2/s) = 6.0 × 104

times greater than the diffusion coefficient in the stationary phase.

This makes sense because it is easier for solute to diffuse through He gas
than through a viscous liquid phase.
 Question 3
van Deemter equation for open tubular column,

contains terms A, B and C describing three band broadening mechanisms.

a) Which term is 0 for an open tubular column? Why?
b) Express the value of B in terms of measurable physical properties.
c) Express the value of C in terms of measurable physical quantities.
d) The linear flow rate that produce the minimum plate height is found by setting
the derivative dH/dux = 0. Find an expression of the minimum plate height in
terms of a measurable physical quantities used to answer (b) and (c)

a. The term A, describing multiple flow paths is 0 for an open tubular column.
Multiple paths arise in a packed column when liquid takes different paths
through the column
b. Express the value of B in terms of measurable physical properties.

B = 2 γ DM Where DM is the diffusion coefficient of solute in the mobile phase.

C. Express the value of C in terms of measurable physical properties.

C = CS + CM

2k d2 DS = diffusion coefficient of solute in the stationary phase

CS = k = retention factor
3(k + 1) 2 D S
d = thickness of the stationary phase

1 + 6k + 11k 2
d 2 DM = diffusion coefficient of solute in the mobile phase
CM = k = retention factor
96(k + 1) 2 DM
d = diameter of the column
d. The linear flow rate that produce the minimum plate height is found by setting the
derivative dH/dux = 0. Find an expression of the minimum plate height in terms of a
measurable physical quantities used to answer (b) and (c)

H = B/ux + Cux ux is the linear velocity

Plate height is minimum at the optimum velocity

𝐵𝐵 𝐵𝐵
dH/dux = − 2 + C = 0; ux =
𝑢𝑢𝑥𝑥 𝐶𝐶

The minimum plate height is found by plugging this value of ux back into the
van Deemter equation
𝐵𝐵 𝐵𝐵 2k d2𝑓𝑓 1+6k+11k 2 d2𝑐𝑐
Hmin = B/ux + Cux = + 𝐶𝐶 = 2 𝐵𝐵𝐵𝐵 = 2 2 γ DM × +
𝐵𝐵 𝐶𝐶 3(k+1)2 DS 96(k+1)2 DM
𝐶𝐶
Tutorial 7 solutions
HPLC

20
 Question 1
A chromatography column with a length of 10.3 cm and diameter of 4.61 mm is packed
with a stationary phase that occupies 61.0% of the volume. If the volume flow is 1.13
cm3/min:

a) Find the linear flow velocity in cm/min.

b) How long does it take for solvent (which is the same as unretained solute) to pass
through the column?
c) Find the retention time for a solute with a retention factor, k of 10.0

a) Linear flow velocity

F = uoAε = uo × πr2 × ε

1.13 = uo × [3.142 × (0.461/2)2 ] × (1 − 0.610)

uo = 17.36 cm/min
 b) Time taken for solvent to pass through column
L
uo =
tM
10.3
17.36 =
tM

tM= 0.593 min

c) Retention time of solute

tR − tM
k=
tM

tR − 0.593
10.0 =
0.593

𝑡𝑡𝑅𝑅 = 6.52 min

22
 Question 2
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:
Calculate
a. The number of plates from each peak.
Retention Width of peak base
b. The mean and the standard deviation for N.
time, min (W), min
Non- 3.1 - c. The plate height for the column.
retained
d. The retention factor for A, B, C and D.
A 5.4 0.41
B 13.3 1.07 e. The resolution of B and C.
C 14.1 1.16
f. The selectivity factor of B and C.
D 21.6 1.72
g. The length of the column necessary to separate
the two species B and C, with a resolution of 1.5.
a. The number of plates from each peak
tR W N N2
A 5.4 0.41 2.78 ×103 7.701 × 106 2
tR
B 13.3 1.07 2.472×103 6.111 × 106 N = 16 2
W
C 14.1 1.16 2.363×103 5.584 × 106
D 21.6 1.72 2.523 ×103 6.366 × 106 2
 N

1.013×104 25.762 ×106
N
 ∑ xi 
 i =1 
b. The mean and the standard deviation for N ∑ x 2
i −
N
s = i =1
𝑁𝑁 = 1.013 × 104 /4 = 2.53 × 103 N −1

25.762 × 106 − (1.013 × 104 )2 /4

𝑠𝑠 = = 189.5
4 − 1

= 0.2 × 103 𝑁𝑁 = 2.5 (±0.2) × 103

c) The plate height for the column.
H = 24.7 cm/2534 plates = 9.747 × 10−3 cm = 9.75 × 10−3 cm
d) The retention factor for A, B, C and D

e) The resolution of B and C

RS = 2(14.1 − 13.3)/(1.07 + 1.16) = 0.717 = 0.72

f) The selectivity factor of B and C.

g) The length of the column necessary to separate the two species B and C, with a
resolution of 1.5

( RS )1 N1 0.717 2534
= = =
( RS ) 2 N2 1.5 N2
2534 × (1.5) 2 /(0.717) 2 =
N2 = 1.11 × 104
11090 =

H = 9.75 × 10−3 cm/plate

L = 11090 × 9.75 × 10−3 = 108 cm

 Question 3
Two components in an HPLC separation have retention times that differ by 22 s. The first
peak elutes in 10.5 min and the peak widths are approximately equal. Given the void
time as 1.05 min, use a spread sheet to find the minimum number of theoretical plates
needed to achieve the following resolution Rs values 0.50, 0.75, 0.90, 1.0, 1.10, 1.25,
1.50, 1.75, 2.0 and 2.5. Explain the relationship between the resolution and number of
theoretical plates.

tR1 = 10.5 min

tR2 = 10.5 + (22/60) =10.87 min  N  α − 1 k B 
tM = 1.05 min R s =  
  
 4  α  1 + k B 
α = (10.87 − 1.05)/(10.5−1.05) = 1.039
kB= (10.87 − 1.05)/1.05 = 9.35
 Rs N
0.50 3478.7
tR1 = 10.5 min 0.75 7827.1
tR2 = 10.5 + (22/60) =10.87 min
tM = 1.05 min 0.90 11271.0
α = (10.87 − 1.05)/(10.5−1.05) = 1.039 1.00 13914.9
kB= (10.87 − 1.05)/1.05 = 9.35 1.10 16837.0
1.25 21742.0
1.50 31308.5
 N  α − 1 k B  1.75 42614.3
R s =  
  
 4  α  1 + k B  2.00 55659.5
2.50 86967.9

Resolution is directly proportional to the number of plates