Saurav Datta

Department of Biotechnology, IITR

F-8, Old Biotechnology Building
Email: sdattfbt@iitr.ac.in
 Bioseparations: Downstream Processing for Biotechnology – Belter, Cussler and Hu
 Principles of Bioseparations Engineering – Raja Ghosh
 Transport Processes and Separation Process Principles – Geankoplis
 Feed Raffinate
Extracting
Extract
Solvent

 Liquid-liquid (L-L) extraction is a separation process, where solute

from a liquid solution (feed) selectively transfers to a second liquid
(solvent) phase by intimate contact between the two phases
 The two liquid phases are either completely immiscible or slightly
miscible
 Driving force for extraction is chemical potential gradient that is
expressed in terms of concentration gradient
 Separation is achieved by the distribution of the solute between the
two liquid phases, i.e. by partitioning
 Equilibrium is attained – equilibrium governed operation
 After extraction, the solute-rich phase is known as Extract and the
treated residual feed solution is called Raffinate
 Separation of the raffinate and extract phases are achieved by density
difference
 Purification of Antibiotics – Penicillin is purified from
fermentation broth using methyl isobutyl ketone (MIBK) or
butyl acetate or amyl acetate
 Purification of Alkaloids – Purification of morphine,
caffeine, nicotine by acid/base extraction
 Purification of Proteins – Using aqueous two-phase
systems
 Purification of Lipids
 Purification of DNA
 Biofuels recovery
 Selectivity – Selectivity of the solute for the added solvent over the
career (feed solvent). Expressed in terms of partition coefficient
(distribution coefficient)
 Immiscibility – If the mutual solubility of the solvents is low, they can
be easily separated
 Density – Higher the density difference between the solvents, better
is the separation in the “Settler” as the driving force for phase
separation is density gradient
 Others - Low viscosity, less expensive, non-toxic/non-corrosive,
low volatility/flammability

 Aqueous/non-aqueous solvent extraction – Low molecular weight

compounds, such as antibiotics, alkaloids, small peptides
 Aqueous two-phase extraction – Macromolecules, such as proteins
and nucleic acids
 Supercritical fluid extraction – Special cases where normal solvent
systems do not function efficiently
 F
Feed eed
Case 1 - Batch Extraction of steroid
Methylene dichloride solvent Mixer
Mixer-settler arrangement (Feed + Solvent)
High mixing - one dispersed phase
and another continuous phase –
FeExtract
enhanced mass transfer
Equilibrium attained Raffinate
Separation of phases based on Fe Settler
density difference Mixer-Settler
Separatory
Funnel
Mixer
Provides higher interfacial surface area per unit volume for solute transfer –
enhanced mass transfer, and hence solute flux
Provides improved hydrodynamic conditions – enhanced mass transfer
coefficient, and hence solute flux
Settler
Enables coalescence of the droplets of the dispersed phase
Allows phase separation
Case 2 - Sequential Reversed Phase Extraction
(i) Purification of penicillin
(ii) First extraction using organic solvent, such as amyl acetate, (pH 2.0)
(iii)Second extraction in aqueous phase (pH 7.0)

Fresh solvent Solvent Spent org. broth

recycle (Raffinate 2)

Extractor-1 Extractor-2 To Crystallizer

Pure Penicillin
Penicillin in
Penicillin in aqueous solution
fermentation
solvent (Extract 2)
broth (pH=2)
(Extract 1)
Spent aq. broth
Buffer (pH=7)
(Raffinate 1)

Sequential Reversed Batch Extraction of Penicillin

Chemical potential of the solute in two solvents are equal at equilibrium

Greater the difference in chemical potential, better is the extraction

Solute transfer to the extract phase is favorable at lower temperature

Phase Rule: F=C-P+2

Degrees of freedom (F) for one solute system is 3

Temperature, pressure and concentration of the solute in any one phase
Partition Coefficient (or Distribution Coefficient)
For dilute solutions, in general

CE = Equilibrium solute concentration in extract, kg/m3

(1) CR = Equilibrium solute concentration in raffinate, kg/m3

Batch
FeedExtraction – Completely Immiscible Solvents
F
eed

Feed + Solvent CR0 = Solute concentration in initial feed, kg/m3

R = Volume of feed or raffinate, m3
E = Volume of added solvent or extract, m3

Fe Extract

Raffinate
Mixer-Settler
Fe
Assumptions
Extract and raffinate are in equilibrium
Negligible entrainment of the other phase
K is independent of concentration
ECE0 + (2) [CE0 = 0, for most of the cases]

For equation (1) type of equilibrium relationship,

(3)

(4)

(5)

(6)
LLE Problem 1: Determine the fraction extracted if a 10 L of fermentation broth
of a hormone (conc. 0.1 g/L) is contacted with 1 L of an organic solvent and the
concentration of the hormone in the extract at equilibrium is 0.7 g/L.

LLE Problem 2: In a food processing industry, 100 L of aqueous citric acid

solution (concentration of solute = 1 g/L) is contacted with 10 L of an organic
solvent. The equilibrium relationship is given by CE=100 CR2, where CE and CR
are citric acid concentrations (in g/L) in the extract and raffnate, respectively.
Calculate: (a) CE and CR; (b) The fraction of citric acid extracted.
If the extract thus obtained is then contacted with another
batch of the aqueous citric acid solution (same volume and concentration),
calculate: (c) CE and CR for the second stage of extraction.
Non-linear equilibrium relationship – Graphical solution for
Batch Extraction

(7)
(from Equation 2)
Continuous Single Stage Extraction – Completely Immiscible Solvents

QECE0 + (8)

(9)
[for linear equilibrium relationship]
Assumptions
Extract and raffinate are
in equilibrium (10)
Negligible entrainment of
the other phase
K is independent of
(11)
concentration
For non-linear equilibrium relationship,

Operating line is represented by (12)

LLE Problem 3: Fermentation broth enters a continuous mixer-settler unit at a
flow rate of 100 L/min. The broth contains 20 g/L antibiotic and its pH has
been adjusted to 3. Butyl acetate, which is used as the extracting solvent, enters
the extractor at a flow rate of 10 L/min. At pH 3, the equilibrium relationship is
given by CE=40CR, where CE and CR are antibiotic concentrations (in g/L) in the
extract and raffnate, respectively. Calculate: (a) CE and CR; (b) The fraction of
antibiotic extracted.
The organic extract from the first extraction step enters a
second mixer-settler unit. An aqueous solvent of pH 7.0 is fed at a flow rate of
5 L/min. At pH 7.0, the equilibrium relationship is given by CE=37CR. Calculate:
(c) CE and CR for the second stage of extraction; (d) The overall fraction of
antibiotic extracted.
Counter-current Continuous Multi-Stage Extraction – Immiscible Solvents

Assumptions
Extract and raffinate
streams from each stage
are in equilibrium
Negligible entrainment
CRn+1 of the other phase
K is independent of
solute concentration
 to (13)

(14)

(15) [From Equation (13 and (14)]

(16)

(17)

(Power Series)

(overall extraction)

(18)
For non-linear equilibrium relationship, graphical method is used
to determine the number of stages required for a given
extraction
to

(19)
LLE Problem 4: An antibiotic is to be extracted from an aqueous solution using
pure amyl acetate. The equilibrium relationship is given by CE=32CR, where CE
and CR are expressed in g/L. The antibiotic concentration in the fed is 0.4 g/L,
the feed flow rate is 500 L/h and the solvent flow rate is 30 L/h.
(a) How many ideal counter-current stages are required for 97 % extraction of
the antibiotic?
(b) If 3 counter-current stages are used, what will be the fraction extracted?

LLE Problem 5: A pharmaceutical ingredient is produced in an enzyme

bioreactor as an aqueous solution (concentration = 20 wt%). This ingredient is
to be extracted with an organic solvent using a staged counter-current
extraction with 4 stages. The equilibrium relationship is given by CE=12CR,
where CE and CR are expressed in mass ratio. QR = 100 kg/h and QE = 20 kg/h.
Calculate
(a) Composition of the final extract and the raffinate
(b) Fraction extracted

LLE Practice Problem: An antibiotic is to be extracted from fermentation media

(concentration 1 g/l) to an organic solvent using staged counter-current
extraction. Feed flow rate is 550 l/h, while the solvent flow rate is 80 l/h.
Equilibrium relationship is given by CE=((25CR)/(1-CR)), where CE and CR are in
g/l. Determine the number of theoretical stages required for 90% extraction of
the antibiotic.
 Partially Miscible Solvents – Ternary Phase Diagram
A → Solute, B → Initial solvent
C → Extracting solvent
Single phase
region Points A,B,C → Pure components A
xB
Lines AB, BC, CA → Two component
Plait Point

Two phase
region

Tie Lines Binodal xA

Solubility
curve
B C
L
xC C
Ternary Diagram for partially miscible solvents

The phases are in equilibrium

Concentrations are either in mole fraction or mass fraction
Two phase region – within the Binodal Solubility curve
Single phase region – outside the Binodal Solubility curve
Overall composition of mixture represented by H – Contents of A, B and C are
proportional to HL, HJ and HK, respectively
Mixture represented by a point, such as H contains two phases in equilibrium –
Composition of the two phases are represented by P and Q
P and Q are obtained from the points of intersection of the binodal curve with the
tie line (PQ) that passes through H
Tie Lines are the straight lines that connect the composition of the two phases in
equilibrium
 Batch Extraction – Partially Miscible Solvents

A → Solute
B → Initial solvent
C → Extracting solvent

Ternary Diagram for Batch Extraction with

C partially miscible solvents

The phases are in equilibrium

F → Feed solution composition; C → Extracting solvent composition; M → Overall
composition; FM/CM = E/R
E=no. of moles of extracting solvent; R=no. of moles of the feed
R1 and E1 represent composition of raffinate and extract, respectively
If second extraction stage is needed to extract solute further from R1 using pure
solvent, add R1 and C
New overall composition is represented by N
Tie line passing through N represents composition of R2 and E2 in equilibrium
Highlights
Substances above its critical temperature and pressure – water: Tcp=374
0C and P =217 atm, CO : T =31 0C and P =73 atm
cp 2 cp cp
No distinct phase boundary – Homogeneous phase
Supercritical fluid (SF) possesses beneficial properties of both liquid and
gas
Gaseous property – Ability to penetrate substances easily and high
solute diffusivity
Liquid property – Ability to dissolve substances easily
Solubility increases significantly with pressure
Environment friendly – alternative to toxic organic solvents

Pressure-Temperature Phase Diagram Pressure vs. solubility curve for supercritical fluid
Applications
Separation and purification of pharmaceutical ingredients
Food and beverage processing – e.g. production of decaf-
coffee and alcohol-free beer
Extraction of flavoring compounds
Extraction of essential oils
Concentration of biofuel (ethanol)
Pretreatment of lignocellulosic biomass for enhanced
production of biofuels
Cleaning of oil/grease from devices

(Adiabatic
Compression)

Concentration of ethanol using SF Extraction

 Bioseparations: Downstream Processing for Biotechnology – Belter, Cussler and Hu
 Principles of Bioseparations Engineering – Raja Ghosh
 Ladisch
 It is a solute fractionation technique which depends on the dynamic
distribution of the solute molecules between two phases based on
some specific mechanism of interaction
 Two phases – Stationary (Binding) phase and Mobile (Carrier) phase
 Mobile phase is pumped continuously – Feed is added either in pulse
or step mode
 Relative “affinity” of the solute molecules between the two phases
 Mechanism of separation depends on the type of interaction: Size
Exclusion, Ion Exchange, Reversed Phase, Hydrophobic, Affinity

Ref.: http://separationprocesses.com
 Types of chromatography based on application
◦ Analytical: Analysis of complex mixtures
◦ Preparative: Separation of molecules for manufacturing purpose

 Substances separated using chromatography

◦ Proteins, nucleic acids, lipids, antibiotics, hormones, sugars,
alcohols, organic acids, etc.

 Industrial applications
◦ Pharmaceuticals and other biomolecules production
◦ Foods and nutraceuticals production
◦ Biomedical detection and analysis
◦ Environmental analysis
 Well developed technology
 Higher resolution even for a complex mixture of biomolecules
 Sensitive and lower response time
 Wide choices of chromatographic systems, and stationary and carrier
phases
 Ease of use

 Expensive columns
 Dilution
 Diffusion dominated operation – slow for industrial scale operations
 Difficult to scale up
 High pressure drop (energy intensive)
 Normal Load Inject

 Online (flow-through) analysis:

Online analysis based on physical
property, which could be correlated
to concentration. UV-VIS absorbance,
fluorescence, refractive index,  Chromatogram is a plot of
conductivity and light scattering concentrations in effluent as a function of
time or effluent volume
Time of maximum concentration of a
solute in the effluent stream is known as
Retention Time
 Packed bed column
 Packed Capillary
Column
 Open tubular
(capillary) column
 Membrane
 Monolith

 High pressure: Frequently referred as High Performance Liquid

Chromatography (HPLC). Pressure requirement of greater than 1
MPa, which is maintained by a plunger pump
 Medium pressure: Pressure requirement is comparatively lower (0.1
to 1 MPa), which is maintained by plunger/diaphragm/peristaltic
pump
 Low pressure: Pressure requirement is less than 0.1 MPa, hence
peristaltic pump or gravity flow is used
Separation in chromatography is governed by the
interaction of the solute molecules with the stationary
phase in comparison to the mobile phase
Size Exclusion Chromatography (SEC)
Based on size
Gel Filtration or Gel Permeation Chromatography
Inert porous material as stationary phase
Support material: Cellulose and derivatives
Smaller solute molecules can enter the pores of the
stationary phase, while larger molecules are excluded
Smaller molecules spend longer time within the
column than larger molecules and hence appear later
at the effluent
Size exclusion limit – The molecular weight of solute
that can be resolved by the column
All molecules larger than the size exclusion limit will
travel at the same velocity and appear in the effluent
at the same time
Molecules smaller than the size exclusion limit will
resolute based on their individual molecular weight
Single step fractionation
Ion Exchange (IEX) Chromatography
Based on electrostatic interaction
Stationary phase consisting of charged groups
attached to insoluble support material
Support material: Cellulose and derivatives, agarose,
acrylic resins, cross-linked dextran, etc.
Cation exchange: Negative charged – sulfonate and
carboxylate groups
Anion exchange: Positively charged – quaternary
ammonium and diethylaminoethyl (DEAE) groups
pKa, pH and ionic strength have major influence on
IEX chromatography
pH needs to be adjusted to maintain proper charge
on the solute molecules depending on the nature of
the stationary phase
Ionic strength needs to be lowered for efficient Anion exchange
binding, because of the shielding effect of the charged chromatography
ions
Multi-step process: Case Study:
Binding/Loading Separation of HIgG (pI 7) and HSA
Washing
(pI 4.9)
Separation of lysozyme (pI 11)
Elution: Change in composition
from other egg proteins (pI below 6)
(salt conc. or pH)
Reversed Phase Chromatography
Based on polarity
Used for non-polar molecules
Support material: Silica based
Uses hydrophobic, non-polar stationary phase (C4-
C18 aliphatic hydrocarbons)
Feed consists of polar solvent (water/methanol)
containing non-polar substance (s)
Non-polar solute molecules favorably partition into
non-polar stationary phase
Solute molecules are eluted from the stationary
phase using non-polar solvents
(acetonitrile/isopropanol)
Not used for sensitive macromolecules, such as large
proteins and nucleic acids – used for small proteins,
hormones, antibiotics
For separation of polar solutes, polar stationary
phase is used

Multi-step process: Case Study:

Binding/Loading
Washing
Elution: Change in solvent
Separation of insulin from aqueous
fermentation broth using silica-C18
stationary phase and 80% acetonitrile
+ 20 % isopropanol as eluent
Hydrophobic Chromatography
Based on hydrophobicity (hydrophobic patches on the
solute molecules as well as on the stationary phase)
Support material: Agarose
Stationary phase: butyl, octyl and phenyl groups
containing alkyl and aromatic hydrocarbons
Used for proteins
In aqueous protein solution, hydrophobic amino acid
residues are shielded with structured water layer
Anti-chaotropic salts (>1M) are added to disrupt the
water layer and expose the hydrophobic
residues/domains
For elution, salt concentration is reduced

Multi-step process: Case Study:

Binding/Loading Separation of recombinant
Washing epidermal growth factor (rEGF) from
Elution: Change in anti- aqueous fermentation broth in
chaotropic salt concentration presence of ammonium sulfate
Affinity Chromatography
Based on specific recognition and binding (affinity)
between ligand in the stationary phase and the target solute
molecules
Support material: Cross-linked agarose, dextran and
cellulose
Ligands: Antigen, antibody, dye, protein A/G,
Complimentary nucleotide base pair sequence, etc.
Recognition is based on stereo-specificity, i.e. shape of the
ligand is complimentary to the target solute (or a part of it)
Binding due to a combinatorial force arising from hydrogen
bonding, hydrophobic and van der Waals interactions
Specific vs. general affinity
Elution by harsh conditions of pH or chaotropic salts (urea,
guanidine)
Affinity tags, such as biotin or histidine, are often used to
avoid harsh elution – Expensive process

Multi-step process: Case Study:

Binding/Loading Purification of antigens for vaccines
using corresponding antibodies
Washing
Separation of IgG-antibodies using
Elution: Change in chaotropic
Protein A/G
salt concentration or pH
Summary of Various Chromatographic Separation Mechanisms

Type Basis for Resolution/Speed/ Application

separation Capacity
SEC Size Moderate/good/low Protein, DNA,
Plasmid
IEX Charge High/high/high Protein, peptide,
nucleic acid,
hormone
Hydrophobic Hydrophobicity Good/good/high Polypeptide
Reversed Polarity Excellent/high/high Antibiotics,
Phase hormones, peptides
Affinity Bio-affinity Excellent/high/high Antigen-antibody,
nucleic acids

(Reference: Separation Process Principles: Henley, Seader and Roper)

Isocratic and gradient elution: Constant composition of mobile phase –

isocratic elution, whereas variable composition of mobile phase – gradient
elution
Sometimes combination of different chromatographic is needed to
obtain the required purity of the target solute
Binary Chromatography
 Chromatographic separation using two mobile phases
 Initial mobile phase for promoting interaction between the target solutes
and stationary phase
 Switch to a second mobile phase for sequential elution of the bound solutes

Binary chromatography
with four solutes Loading Elution

A → Non-interacting
B → Weakly-interacting
C → Strongly-interacting
D → Very strongly-interacting

Selection of mobile phases in binary chromatography

IEX → Low ionic strength/High ionic strength; Particular pH/pH on the other side of pI
Reversed → Polar/Non-polar
Hydrophobic → High anti-chaotropic/Low or no anti-chaotropic salt
Affinity → Physiological conditions/aggressive condition or chaotropic salts

Modes of introducing the second mobile phase

Step change
Linear gradient
Non-linear gradient
Solute-stationary phase interaction relative to the solute-mobile phase
interaction, i.e. distribution/partition of solute is the governing factor
for chromatographic separation
Solute-stationary phase interaction for each solute is expressed in
terms of a physically measurable parameter, that is the respective
retention time of the solutes
Chromatogram, containing the retention times for the solutes, is
critically analyzed to obtain the feasibility of a chromatographic
separation
Hydrodynamic conditions and column properties are important

Assumption: Equilibrium is attained – concentration of solute in

stationary phase and mobile phase can be correlated by partition
coefficient
Following analysis is valid for isocratic chromatography
Capacity Factor or Retention factor (kʹ)
Capacity (Retention) Factor, kʹ is the ratio of the quantity of
solute bound to the stationary phase (ns) to the quantity of
solute present in the mobile phase (nM) (a)

Retardation Factor, Rf, is the ratio of the quantity of solute

in mobile phase to the total quantity in system
(b)
(c)
For planar chromatography, Rf is ratio of the distance travelled by the solute to that by the solvent
Extent to which a solute molecule interacts with the stationary phase is
quantified in terms of kʹ
K=partition coefficient; nS, nm=moles of solute in
stationary and mobile phases, respectively; CS,
(1) CM=Conc. of solute in stationary and mobile
phases, respectively; VS, VM=Volume of stationary
and mobile phases, respectively

If, ε=void fraction of the column=void volume or mobile phase volume/column volume

ε=VM/VC=VM/(VM+VS), so, VS/VM=(1- ε)/ ε (2)

From (1) and (2), (3)
For a non-interacting solute, ns=0, so kʹ=0. Typically, for interacting solutes, kʹ=1-10
Solutes with higher kʹ, have stronger interaction with the stationary phase, have delayed
elution, and often need gradient elution
Retention Time
tM=mobile phase retention time, tRʹ=additional
Retention time, (4) retention time due to solute-stationary phase
interaction

It has also been demonstrated that kʹ= tRʹ/tM, hence, tRʹ = kʹtM (5)

Greater K indicates greater

From (3), (4) and (5), (6) retention time

Mobile phase retention time, tM, is dependent on hydrodynamic condition,

such as flow rate, and the column properties, such as volume and void
fraction
(7)

From (6) and (7), (8)

Using equation (8), retention time, tR, for a solute can be calculated by
knowing the column properties, flow rate of the mobile phase and the
partition coefficient
Selectivity

Selectivity Parameter, (9)

Selectivity is a thermodynamic property

Selectivity depends on the difference in interaction of the two solutes
with the stationary phase relative to the mobile phase
It is independent of the flow rate of the mobile phase and the solute
concentration in the feed
α>1: Solute 2 has stronger interaction with the stationary phase
compare to solute 1, and therefore, solute 1 elutes faster than solute 2

If α is close to unity, changing hydrodynamic conditions or column

properties will not improve the feasibility of separation. Instead,
changing the temperature (used often for GC) or the composition of
the mobile phase (used often in LC) or the properties of the stationary
phase (enhanced interaction) will enable significant improvement
Mathematical models for chromatography

Equilibrium Analysis (Discrete Stage Analysis): Chromatographic

column is not a continuous packed bed. It consists of number of
discrete hypothetical theoretical stages (plates), which are individually
well mixed. In each stage, solute in mobile phase and stationary phase
remains in equilibrium
oKinetic Analysis (Reaction and Diffusion Analysis): Concentration
profile at the effluent of column chromatography is the result of
interaction between solute and stationary phase (chemical reaction)
and mass transfer resistances (diffusion and dispersion)

Solute mass balance is conducted in both of the models and the

following expression for concentration profile is obtained

tR

Plate theory predicts that a chromatographic peak could be

expressed as a Gaussian function, which leads to Normal distribution
(Gaussian distribution)
Gaussian Distribution Function

1. Width at the inflection

points (w = 2σ)
2. Width at half of the peak
height (w = 2.354σ)
3. Width at the intersection of
the tangent drawn through
the inflection points to the
base (w = 4σ)

σ =standard deviation
σ2=variance
Peak Width and Peak Resolution
Peak width (w1 and w2) means
width at the base intercept
(segment of the peak base
intercepted by the tangents drawn
to the inflection points on either
side of the peak)
Peak width depends on
interaction between the solute and
the stationary phase and transport
properties

Spatial separation of the two peaks, i.e. whether they overlap or not, is
measured in terms of the Resolution Parameter, R

(10)

Greater R signifies better spatial resolution of the peaks

R<1: Peaks overlap
R=1: Peaks just resolve
R>1: Peaks resolve properly
Height Equivalent of Theoretical Plates (HETP) and No. of Plates
A chromatographic column is assumed to be consists of a number of
discrete theoretical plates (hypothetical). Greater the number of plates in
a column, better is the separation

Height equivalent of theoretical plates (HETP), (11)

l=length of the column
No. of theoretical plates, N can be determined from the chromatogram,

(12) N depends on retention time and peak width

Resolution Parameter, R, can be correlated to the number of plates, N, as

follows

(13)
Theoretical Plate Height Based on van Deemter Equation
Theoretical plate height, H can also be determined from van Deemter
equation,
A=Eddy diffusion component (m), B=Axial diffusion
constant (m2/s), L=Transfer constant (s), u=mobile
(14) phase velocity (m/s)

“A” depends on the variability of the path length taken by the mobile phase –
depends on particle diameter, size distribution and packing density
“B” depends on diffusion of the solute molecules on the direction of flow of the
mobile phase and is significant at lower mobile phase velocity
“L” depends on the solute mass transfer between the mobile and stationary phases

At lower u, increasing the velocity

decreases the plate height – efficiency of
separation increases with increase in velocity
– second term, B/u, dominates
Plate height reaches to a minimum value of
Hmin at uopt
Further increase in u results in increase in
H, because in this range the third term, Lu,
dominates
Numerical problems

1. Egg white proteins are being separated by isocratic chromatography using a

10 cm long column having 250 theoretical plates. The distribution
coefficients for the proteins are: Kovalbumin = 0, Kconalbumin = 1, Klysozyme = 5. If
the voidage fraction of the column is 0.45 and the mobile phase retention
time is 10 min, predict the retention time of the three proteins. Comment on
the selectivity and the resolution of separation.
2. A plasmid has a retention time of 10 min in a chromatographic column of
volume 0.01 m3 and voidage fraction of 0.3. The distribution coefficient of
the plasmid is 2. Calculate the mobile phase retention time and the flow rate
at which the above separation is carried out. If the same column-mobile
phase system is used to separate plasmid from RNA (with capacity factor
4.66), what will be the feasibility of separation?
3. Albumin is separated from IgG by isocratic chromatography in a 50 cm long
column with a voidage of 0.25 and diameter of 10 mm. The distribution
coefficients of IgG and albumin are 1 and 0.1, respectively. (a) Mobile phase
flow rate is 10 ml/min. If the albumin peak has a characteristic peak width
of 0.52 min, predict the selectivity and resolution. (b) When the mobile
phase flow rate is increased to 20 ml/min, the HETP increases by 80 %.
Predict the selectivity and resolution at the higher flow rate.
Yield and Purity of Solutes

The eluted solutes are collected as separate fractions

Fractions are designated in terms of (i) time and (ii) number of bed
volumes
Information from chromatogram is utilized to determine the purity and
yield of the solutes

Plate theory predicts that a chromatographic peak could be expressed

as a Gaussian Function, which leads to Normal Distribution

tR
(15)

C0=Max. solute conc. in the peak,

tR=Retention time or the time for max.
conc., σ=Std. dev. of the peak,
σ2=variance, w=4σ
Total amount of a solute eluted in a peak, (16)

Q=mobile phase flow rate, C=concentration of the solute=C(t)

Yield of a solute in effluent collected over a time interval of t1 and t2 is,

(17)

Purity of solute A in the binary separation of solutes A and B in the

effluent collected over a time interval of t1 and t2 is,

(18)
Numerical problem
4. Chromatograms (given below) for two different proteins, A and B, were
obtained by injecting pure individual samples into a 30 cm long column
(void fraction 0.3) at the same mobile phase flow rate (residence time 2
min). If A and B have to be separated from a mixture under the same
conditions as stated above, calculate (a) the theoretical plate height of the
column, (b) selectivity, and (c) resolution parameter. If the column effluent is
collected from the start to 7 min onto the run, calculate (d) the purity of A in
the sample, (e) the percent yield of A in the sample. (f) Assuming 50 % of the
solutes are eluted at the respective retention times, recalculate the yield and
purity mentioned above.
Error Function Table
Numerical problem
5. We plan a large scale production of urease using packed column of
polyacrylamide beads. Following data are obtained. The bed volume is 20 L.
Find VR, σV and the yield at 174 and 200 L assuming 50 % of urease elute at VR.

Volume eluted (L) Concentration (mol/L)

174 0.0063

190 0.0152 (maximum)