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Chromatography Part II

The document discusses the principles of chromatographic separation, emphasizing the importance of minimizing band broadening to achieve sharp, symmetrical peaks for optimal separation. It introduces the concept of theoretical plates and their relationship with column efficiency, resolution, and peak width. Additionally, it provides calculations for various chromatography scenarios, including retention factors, selectivity factors, and average number of plates.

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sanjaysr292617
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2 views

Chromatography Part II

The document discusses the principles of chromatographic separation, emphasizing the importance of minimizing band broadening to achieve sharp, symmetrical peaks for optimal separation. It introduces the concept of theoretical plates and their relationship with column efficiency, resolution, and peak width. Additionally, it provides calculations for various chromatography scenarios, including retention factors, selectivity factors, and average number of plates.

Uploaded by

sanjaysr292617
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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❖ To obtain optimal separations, sharp, symmetrical


chromatographic peaks must be obtained. This means that
band broadening must be limited.
❖ View column as divided into a number (N) of adjacent
imaginary segments called theoretical plates.

❖ Within each theoretical plates complete equilibration of


analyte occurs between stationary and mobile phase.
❖ Greater separation occurs with:
▪ Greater number of theoretical plates (N)
▪ As plate Height or the Height Equivalent to a Theoretical
Plate (H or HETP) becomes small

L=N×H (or) H = L/N

Where,
L - Length of the column
N - Number of plates
H - Plate height or Height Equivalent of Theoretical Plates
❖ Solute moving through a column spreads into a
Gaussian shape with standard deviation σ, and the
variance is σ2.
❖ The variance per unit length of column is the
measurement of column efficiency.

 2
H =
L
▪ A number of independent factors such as sample-
injector and detector characteristics, temperature and
column retention processes, contribute to the dispersion
of molecules in a band and band broadening.

▪ The cumulative effect of small variations is these factors


is described in statistical terms as the variance, σ2, in the
process.
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Band spreading - the width of bands increases as their retention
time increases

N = L/H number of pates


2
t 
N = 16  R 
W1/2 W 
2
 tR 
N = 5.54  
 W1/ 2 

Common measures of breadth are:


❖ The width w½ measured at half-height.
❖ The width w at the baseline between tangents drawn to the
steepest parts of the peak (inflection points).
------------------------------------------------------------------------------------------
 = Standard deviation in distance
 = standard deviation in time

=L
tR

 = tR
L

The smaller HETP, the narrower the eluted peak


W = 4
WL
 =
4 tR
2W2 L
H = =
L 16 t R2
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❖ Successfully accounts for the peak shapes and rate of
movement.

❖ Does not account for the “mechanism” causing peak


broadening.

❖ No indication of other parameters’ effects.

❖ No indication for adjusting experimental parameters.


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1. Substances A and B have retention times of 13.01 and 14.31
min, respectively, on a 25 cm column. The peak widths at base
for A and B are 1.45 and 1.61 min, respectively.
Calculate (a) The column resolution
(b) The average number of plates in the column
(c) The plate height
Ans: Given, (tR)A = 13.01 min; (tR)B = 14.31 min
L = 25 cm
WA = 1.45 min; WB = 1.61 min
(a) Resolution; RS = 2 [(tR)B ⎯ (tR)A] / (WA + WB) = 0.85
(b) Number of Plates NA = 16 [(tR)A / W]2 = 1288
NB = 16 [(tR)B / W]2 = 1264
Average number of plates; NAverage = [NA + NB] / 2 = 1276
(c) L = H x N
H = L / Naverage = 25 / 1276 = 2.35 x 10-2 cm
2. Substances A and B have retention times of 16.40 and 17.63 min,
respectively, on a 30 cm column. An unretained species passes through
the column in 1.30 min. The peak widths at base for A and B are 1.11
and 1.21 min, respectively.
Calculate (a) The column resolution
(b) The average number of theoratical plates in the column
(c) The plate height
Ans: Given, (tR)A = 16.40 min; (tR)B = 17.63 min; tM = 1.30 min
L = 30 cm
WA = 1.11 min; WB = 1.21 min
(a) Resolution; RS = 2 [(tR)B ⎯ (tR)A] / (WA + WB) = 1.06
(b) Number of Plates NA = 16 [(tR)A / W]2 = 3493
NB = 16 [(tR)B / W]2 = 3397
Average number of plates; NAverage = [NA + NB] / 2 = 3445
(c) L = H x N
H = L / Naverage = 30 / 3445 = 8.7 x 10-3 cm
3. Two components A and B are separated by elution with hexane from a
column packed with silica-gel (water adsorbed on silica surface).
Distribution coefficients (K = Caq/Corg) of compounds A and B in
water/hexane system are 5.99 and 6.16 respectively. The ratio VS/VM for
the packing is 0.425.
Calculate (a) Retention factor for A and B
(b) Selectivity factor
(c) Length of the column if the number of theoretical plates
and plate height of packing are 9.03 x 104 and 1.53 x 10-3 cm
respectively.
Ans: Given, KA = 5.99; KB = 6.16; VS/VM = 0.425
(a) Retention factor; kA = KA x (VS/VM) = 2.546
kB = KB x (VS/VM) = 2.618
(b) Selectivity factor; a = kB / kA = 1.03
(c) L = HN = 9.03 x 104 x 1.53 x 10-3 = 138.159 cm
4.

Ans:
▪ NA = 16 [(tR)A / W]2
▪ L=HxN
▪ H = σ2/L
5. From the above data (question no. 4); Calculate
(a) Retention factor
(b) The distribution constant.
Ans. (a) Retention factor; kA = [(tR)A – tM]/tM = tS/tM
Similarly, kB, kC, kD can be calculated
(b) Distribution constant; KA = kA (VM/VSa
) =
KB
KA
Similarly, KB, KC, KD can be calculated
kB
a =
6. From the above data (question no. 4); Calculate
(a) The resolution
kA
(b) The selectivity factor (t R ) A - t M
kA = and
Ans. (a) Resolution; tM
(b) Selectivity factor;
(t R ) B - t M
a =
(t R ) A - t M
7.

Ans.
▪ NA = 16 [(tR)A / W]2
▪ L=HxN
▪ H = σ2/L
8. From the above data (Question no. 7); Calculate the resolution for
(a) Methylcyclohexene and methylcyclohexane
(b) Methylcyclohexene and toluene
(c) Methylcyclohexane and toluene

Ans. Resolution;
9. If VS and VM for the column in the problem 7 are 19.6 and 62.6
mL, respectively, and a non retained air peak appears after 1.9
1
min, Calculate v = µ ´
1+ k A
(a) The retention factor for each compound
(b) The distribution constant for each compound
L L 1
a = K´B
(c) The selectivity factor for methylcyclohexane
= and
methylcyclohexene t R t MK 1 + k A
A
t Rk - t M tS
Ans. (a) Retention factor, kaA == B =
k tAM tM
- /V
(tKR )xA (V tM ) (t R ) B -
k A A= A
(b) Distribution constant, k = S M and k B =
tM tM
(t R ) B - t M
(c) Selectivity factor, a =
(t R ) A - t M

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