Exercise 3.

1
3.1) The chromatogram below shows two peaks eluted with the same mobile phase
flow rate on two columns with equal length. tM was 1.3 min; tR for compound A was
8.0 min; and tR for compound B was 10.0 min.

Exercises 3
Efficiency, selectivity and resolution
Acquired from D.C. Harris (Quantitative Chemical analysis, 8th ed. W.H. Freeman)
2
a) Which column gives the highest plate number? tR
b) Which column has the largest plate height? N = 16 Eq (8)
c) Which column provides the best resolution?
wb
d) Which column gives the largest relative retention?
e) Which compound gives has highest retention factor? Column 1 has more
f) What is the value of the separation factor, α? narrow peaks, tR is the
g) What is the value of the unadjusted separation factor, γ? same

Exercise 3.1 Exercise 3.1

3.1) The chromatogram below shows two peaks eluted with the same mobile phase 3.1) The chromatogram below shows two peaks eluted with the same mobile phase
flow rate on two columns with equal length. tM was 1.3 min; tR for compound A was flow rate on two columns with equal length. tM was 1.3 min; tR for compound A was
8.0 min; and tR for compound B was 10.0 min. 8.0 min; and tR for compound B was 10.0 min.

Acquired from D.C. Harris (Quantitative Chemical analysis, 8th ed. W.H. Freeman) Acquired from D.C. Harris (Quantitative Chemical analysis, 8th ed. W.H. Freeman)

a) Which column gives the highest plate number? a) Which column gives the highest plate number?
L 2 (tR(B) – tR(A)) ∆t
b) Which column has the largest plate height? H= Eq (9) b) Which column has the largest plate height? Rs = Rs = (Eq 5)
c) Which column provides the best resolution? N c) Which column provides the best resolution?
wb(A) + wb(B) wb
d) Which column gives the largest relative retention? d) Which column gives the largest relative retention?
e) Which compound gives has highest retention factor? Column 2 has the lowest e) Which compound gives has highest retention factor? Column 1 has more
f) What is the value of the separation factor, α? plate number and f) What is the value of the separation factor, α? narrow peaks, tR is the
g) What is the value of the unadjusted separation factor, γ? therefore has largest H, g) What is the value of the unadjusted separation factor, γ?
same

Column 1 has higher N, α

√N α–1 k(B)
Rs = Eq (11) and k is the same
4 α 1+k(B)

Exercise 3.1 Exercise 3.1

3.1) The chromatogram below shows two peaks eluted with the same mobile phase 3.1) The chromatogram below shows two peaks eluted with the same mobile phase
flow rate on two columns with equal length. tM was 1.3 min; tR for compound A was flow rate on two columns with equal length. tM was 1.3 min; tR for compound A was
8.0 min; and tR for compound B was 10.0 min. 8.0 min; and tR for compound B was 10.0 min.

Acquired from D.C. Harris (Quantitative Chemical analysis, 8th ed. W.H. Freeman) Acquired from D.C. Harris (Quantitative Chemical analysis, 8th ed. W.H. Freeman)

a) Which column gives the highest plate number? a) Which column gives the highest plate number?
b) Which column has the largest plate height? b) Which column has the largest plate height?
c) Which column provides the best resolution? c) Which column provides the best resolution?
d) Which column gives the largest relative retention? The retention is equal d) Which column gives the largest relative retention?
e) Which compound gives has highest retention factor? e) Which compound gives has highest retention factor?
f) What is the value of the separation factor, α? f) What is the value of the separation factor, α?
g) What is the value of the unadjusted separation factor, γ? g) What is the value of the unadjusted separation factor, γ?

tR′ nAnalyte in the stationary phase

k= = Eq (2) Compound B has highest tR
tM nAnalyte in the mobile phase

1
 Exercise 3.1 Exercise 3.1
3.1) The chromatogram below shows two peaks eluted with the same mobile phase 3.1) The chromatogram below shows two peaks eluted with the same mobile phase
flow rate on two columns with equal length. tM was 1.3 min; tR for compound A was flow rate on two columns with equal length. tM was 1.3 min; tR for compound A was
8.0 min; and tR for compound B was 10.0 min. 8.0 min; and tR for compound B was 10.0 min.

Acquired from D.C. Harris (Quantitative Chemical analysis, 8th ed. W.H. Freeman) Acquired from D.C. Harris (Quantitative Chemical analysis, 8th ed. W.H. Freeman)

a) Which column gives the highest plate number? a) Which column gives the highest plate number?
b) Which column has the largest plate height? b) Which column has the largest plate height?
c) Which column provides the best resolution? c) Which column provides the best resolution?
d) Which column gives the largest relative retention? d) Which column gives the largest relative retention?
e) Which compound gives has highest retention factor? e) Which compound gives has highest retention factor?
f) What is the value of the separation factor, α? f) What is the value of the separation factor, α?
g) What is the value of the unadjusted separation factor, γ? g) What is the value of the unadjusted separation factor, γ?

′
tR(B) k(B) 10.0 – 1.3 tR(B)
α= = Eq (6) α=
8 – 1.3
= 1.3 γ= Eq (7) γ = 10.0
8.0
= 1.25
′
tR(A) k(A) tR(A)

Exercise 3.2 Exercise 3.2

x) Substances A and B have retention times of 16.40 (A) and 17.63 (B) minutes, on a x) Substances A and B have retention times of 16.40 (A) and 17.63 (B) minutes, on a
30-cm column. An unretained compound passes through the column in 1.30 min. 30-cm column. An unretained compound passes through the column in 1.30 min.
The peak widths at baseline, wh, are 1.11 min for A and 1.21 min. for B. The peak widths at baseline, wh, are 1.11 min for A and 1.21 min. for B.
a) Calculate the resolution between A and B a) Calculate the resolution between A and B
b) Calculate the average number of plates in the column b) Calculate the average number of plates in the column
c) Calculate the plate height c) Calculate the plate height
d) Calculate the length of the column required to give a resolution of 1.5 d) Calculate the length of the column required to give a resolution of 1.5
e) Calculate the time required to elute substance B on the column that gives Rs of 1.5 e) Calculate the time required to elute substance B on the column that gives Rs of 1.5
f) Calculate the plate height required for a resolution of 1.5 on the original 30-cm f) Calculate the plate height required for a resolution of 1.5 on the original 30-cm
column and with the original time column and with the original time

A A
B B
tM tM

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 min 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 min

2 2 2
tR 16.40 17.63
2 (tR(B) – tR(A)) ∆t 2 (17.63 – 16.40) N = 16 NA = 16 = 3493 NA = 16 = 3397
Rs = = wb 1.11 1.21
Rs = = 1.06
w b(A) + w b(B) wb 1.11 + 1.21
Navg = (3493+3397)·0.5 = 3445

Exercise 3.2 Exercise 3.2

x) Substances A and B have retention times of 16.40 (A) and 17.63 (B) minutes, on a x) Substances A and B have retention times of 16.40 (A) and 17.63 (B) minutes, on a
30-cm column. An unretained compound passes through the column in 1.30 min. 30-cm column. An unretained compound passes through the column in 1.30 min.
The peak widths at baseline, wh, are 1.11 min for A and 1.21 min. for B. The peak widths at baseline, wh, are 1.11 min for A and 1.21 min. for B.
a) Calculate the resolution between A and B a) Calculate the resolution between A and B
b) Calculate the average number of plates in the column b) Calculate the average number of plates in the column
c) Calculate the plate height c) Calculate the plate height
d) Calculate the length of the column required to give a resolution of 1.5 d) Calculate the length of the column required to give a resolution of 1.5
e) Calculate the time required to elute substance B on the column that gives Rs of 1.5 e) Calculate the time required to elute substance B on the column that gives Rs of 1.5
f) Calculate the plate height required for a resolution of 1.5 on the original 30-cm f) Calculate the plate height required for a resolution of 1.5 on the original 30-cm
column and with the original time column and with the original time

A A
B B
tM tM

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 min 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 min

√N α–1 k(B)
Rs =
L 30 cm 4 α 1+k(B)
H= = = 0.087 mm
N 3445 k and α will be the same, so increased resolution must come from increased N

2
 Exercise 3.2 Exercise 3.2
x) Substances A and B have retention times of 16.40 (A) and 17.63 (B) minutes, on a x) Substances A and B have retention times of 16.40 (A) and 17.63 (B) minutes, on a
30-cm column. An unretained compound passes through the column in 1.30 min. 30-cm column. An unretained compound passes through the column in 1.30 min.
The peak widths at baseline, wh, are 1.11 min for A and 1.21 min. for B. The peak widths at baseline, wh, are 1.11 min for A and 1.21 min. for B.
a) Calculate the resolution between A and B a) Calculate the resolution between A and B
b) Calculate the average number of plates in the column b) Calculate the average number of plates in the column
c) Calculate the plate height c) Calculate the plate height
d) Calculate the length of the column required to give a resolution of 1.5 d) Calculate the length of the column required to give a resolution of 1.5
e) Calculate the time required to elute substance B on the column that gives Rs of 1.5 e) Calculate the time required to elute substance B on the column that gives Rs of 1.5
f) Calculate the plate height required for a resolution of 1.5 on the original 30-cm f) Calculate the plate height required for a resolution of 1.5 on the original 30-cm
column and with the original time column and with the original time

A A
B B
tM tM

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 min 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 min

√N α–1 k(B) tR′ = tR – tM ⇒ tR = tR′ + tM

Rs =
4 α 1+k(B)
Both t′R and tM will double if the column length is doubled (under isocratic conditions)
The original resolution (1.06) must be increased 1.42 times
⇒ tR(new) = 17.63 min · 2 = 35.26 min
Rs ∝ √N ⇒ N must be increased 1.422 times = 2.00 times
Since plate height is the same, this will mean that the column length must be
doubled to double the N ⇒ new length is 60 cm

Exercise 3.2 Exercise 3.3

x) Substances A and B have retention times of 16.40 (A) and 17.63 (B) minutes, on a x) Four peaks are eluted by LC under the following conditions: flow rate, 0.313 mL/min;
30-cm column. An unretained compound passes through the column in 1.30 min. Column length, L=24.7 cm; volume of stationary phase, VS = 0.164 mL, volume of
The peak widths at baseline, wh, are 1.11 min for A and 1.21 min. for B. mobile phase in column, VM = 1.37 mL. An unretained compound elutes at 3.1 min.
a) Calculate the resolution between A and B The following data were acquired for the four compounds:

b) Calculate the average number of plates in the column Compound A, tR=5.4 min, wb = 0.41 min

c) Calculate the plate height Compound B, tR=13.3 min, wb = 1.07 min

d) Calculate the length of the column required to give a resolution of 1.5 Compound C, tR=14.1 min, wb = 1.16 min

e) Calculate the time required to elute substance B on the column that gives Rs of 1.5 Compound D, tR=21.6 min, wb = 1.72 min

f) Calculate the plate height required for a resolution of 1.5 on the original 30-cm a) Calculate the resolution between compound B and C
column and with the original time b) Calculate the selectivity factor, α, between compound B and C
c) The number of plates, N, for each peak
A
B d) The mean and the standard deviation for N C
tM e) The plate height for the column

2 (tR(B) – tR(A)) ∆t
Rs = Rs =
w b(A) + w b(B) wb B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 min

Average plate number was 3445 (from b) 2 (14.1 – 13.3)

Rs = = 0.717
The number of plates must be doubled to achieve R s of 1.5 (from d) ⇒ N = 6890 1.07 + 1.16
The column length is the same (30 cm)
1.16
1.07
L 30 cm 13.3
14.1
H= = = 0.044 mm
N 6890

Exercise 3.3 Exercise 3.3

x) Four peaks are eluted by LC under the following conditions: flow rate, 0.313 mL/min; x) Four peaks are eluted by LC under the following conditions: flow rate, 0.313 mL/min;
Column length, L=24.7 cm; volume of stationary phase, VS = 0.164 mL, volume of Column length, L=24.7 cm; volume of stationary phase, VS = 0.164 mL, volume of
mobile phase in column, VM = 1.37 mL. An unretained compound elutes at 3.1 min. mobile phase in column, VM = 1.37 mL. An unretained compound elutes at 3.1 min.
The following data were acquired for the four compounds: The following data were acquired for the four compounds:
Compound A, tR=5.4 min, wb = 0.41 min Compound A, tR=5.4 min, wb = 0.41 min
Compound B, tR=13.3 min, wb = 1.07 min Compound B, tR=13.3 min, wb = 1.07 min
Compound C, tR=14.1 min, wb = 1.16 min Compound C, tR=14.1 min, wb = 1.16 min
Compound D, tR=21.6 min, wb = 1.72 min Compound D, tR=21.6 min, wb = 1.72 min
a) Calculate the resolution between compound B and C a) Calculate the resolution between compound B and C
b) Calculate the selectivity factor, α, between compound B and C b) Calculate the selectivity factor, α, between compound B and C 2
tR
c) The number of plates, N, for each peak c) The number of plates, N, for each peak N = 16
d) The mean and the standard deviation for N C d) The mean and the standard deviation for N wb
e) The plate height for the column e) The plate height for the column

′
tR(B) 14.1 – 3.1 min ±174 or N = 2534±
N = 2534± ±7%
α= = = 1.08
′
tR(A) 13.3 – 3.1 min B
N = 2775 N = 2364
tR=5.4 min tR=14.1 min
wb=0.41 min wb=1.16 min

N = 2472 N = 2523
tR=13.3 min tR=21.6 min
wb=1.07 min wb=1.72 min

1.16
1.07
14.1
13.3

0 2 4 6 8 10 12 14 16 18 20 22 min

3
 Exercise 3.3
x) Four peaks are eluted by LC under the following conditions: flow rate, 0.313 mL/min;
Column length, L=24.7 cm; volume of stationary phase, VS = 0.164 mL, volume of
mobile phase in column, VM = 1.37 mL. An unretained compound elutes at 3.1 min.
The following data were acquired for the four compounds:
Compound A, tR=5.4 min, wb = 0.41 min
Compound B, tR=13.3 min, wb = 1.07 min
Compound C, tR=14.1 min, wb = 1.16 min
Compound D, tR=21.6 min, wb = 1.72 min
a) Calculate the resolution between compound B and C
b) Calculate the selectivity factor, α, between compound B and C
L
c) The number of plates, N, for each peak H=
d) The mean and the standard deviation for N N
e) The plate height for the column

±174 or N = 2534±
N = 2534± ±7%
H = 24.7 cm / 2534 = 0.097 mm
N = 2775 N = 2364
tR=5.4 min tR=14.1 min
wb=0.41 min wb=1.16 min

N = 2472 N = 2523
tR=13.3 min tR=21.6 min
wb=1.07 min wb=1.72 min

0 2 4 6 8 10 12 14 16 18 20 22 min