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July 2010, Volume 1, No.

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International Journal of Chemical and Environmental Engineering

Contemporary Laboratory Methods for


Separation and Purification of Organic
Macromolecules A review article
M.P.Zacharof¹* and R.W. Lovitt²

Multidisciplinary Nanotechnology Center, Swansea University, Swansea, SA2 8PP, UK¹


School of Engineering, Multidisciplinary Nanotechnology Center, Swansea University, Swansea, SA2 8PP,
UK²
* Corresponding author. e-mail : myrtozacharof1981@yahoo.com

Abstract
Nowadays, there are several laboratory methods suitable for separation and purification of a wide range of chemical substances of
commercial interest from mixtures. These assays include filtration, precipitation, high precision dynamic light scattering (DLS) and
high performance liquid chromatography (HPLC). The main advantage of these methods apart of their cost effectiveness is their
suitability for separation, sizing and purification of a wide range of organic substances such as proteins, oligo- and polypeptides ,
enzymes, antibiotics, dyes, lipids, oils and in general non volatile organic compounds produced either by in vitro synthesis or
biotechnologically. In this study, a description of these techniques will follow and several examples of their use will be given.

Keywords: Filtration, Laboratory techniques, Chromatography, Proteins, Separation, Purification, Membranes, Pore size

1. Introduction In numerous cases there is only one separation


mechanism which drives the separation process. It is
Membranes are thin, porous barriers which perform possible though to combine two or more separation
several degrees of separation (microfiltration, mechanisms for membrane separation, although this
ultrafiltration, and nanofiltration) using differences in procedure will be much more complicated and difficult
concentration, electrical potential and charge and but the separation factor will be large. If there is a
pressure between two compartments they perform combination of primary factors for separation properties
separation to. (Callister, 2004) The driving forces used in of the membranes (porosity, surface charge, chemical
membrane separation which are diffusion, electrical structure, diffusivity) and kind of molecules to be
conductivity, hydraulic pressure. separated, many membrane separation processes can
These forces are interacting with the membrane occur.
creating phenomena such as osmosis, reverse osmosis, According to the major factors which influence the
electrodialysis and electroosmosis. Also other physical bioseparation processes, membranes can be applied into
parameters which influence the permeability of the various operational units. For example the pore size
membrane are the transport number, the membrane provides ultrafiltration, nanofiltration, microfiltration and
potential (surface charge), the streaming current and the gel filtration, diffusivity provides reverse osmosis and
streaming potential. As successful bioseparation process dialysis. Ionic exchange can provide electrodialysis, ion
can be considered only the one where the membrane exchange absorbent, volatility results into distillation,
solvents and molecules of interest interact in equilibrium. vacuum distillation, evaporation, solubility of molecules
Phenomena of ultrafiltration, microfiltration and provides solvent extraction, precipitation and flocculation
nanofiltration are forced by differential pressure, dialysis In the case of microfiltration the pore size of the
and membrane extractions are performed through membranes used ranges between 0.01 μm to 0.04 μm,
concentration differences and ionized chemicals are though sizes smaller to 0.02 μm belong to ultrafiltration.
separated due to electrical potential differences (Oh& The fluxes of the materials range between
Kini, 1996).
Contemporary Laboratory Methods for Separation and Purification of Organic Macromolecules A review article

3 −1 −1 3 −1 −1 measured. The stirred cell unit was operated in a batch


0.05 m m d to 10 m m d being highly
dependent on the systems configuration and on the dead-end mode. (Morao et al., 2006)
membrane material The calculated Rm value from the inverse of slope of
The majority of membranes are made of polymers water flux hydraulic membrane permeability, (Callister,
but also other materials such as ceramics and stainless 2004)
steel have been used. Polymeric membranes can be easily Lp = J/ΔP = 1/μ* Rm based on viscosity of water
produced but are structurally weak and need solid support 8.937*10-4
in order to function properly. Polymeric membranes can The membrane resistance (Rm) was determined from
be also made very thin and can be applied in a variety for the relationship between pure water flux and pressure.
processes like gas permeation and electrodialysis. According to Darcy’s law, the permeate flux of water is:
J=Δp/μ*Rm J is the permeate flux (L/m2.h), rP is the
Although new materials have been used for the transmembrane pressure (Pa), m is viscosity of water (Pa.
manufacture of membranes, the membranes are not s), Rm is membrane resistance (m-1). (Callister, 2004)
strong enough to support a membrane device alone nor Transmembrane pressure (TMP) was calculated from
can they obtain enough area for mass transfer. So, they following equation TMP = (Pinlet + Poutlet) /2 –Ppermeate
are packaged in several forms like flat, spiral, tubular and
hollow fibber types. The major advantage of this
procedure is that high pressure can be applied. (Morao et
al.2006).
2. Application of Filtration in Laboratory Scale
for Protein Separation
2.1 Design of the stirred cell membrane reactor
The reactor system is composed of an ultrafiltration
stirred cell unit of 200 ml maximum process volume, a
magnetic stirrer and an effective area of 28.7 cm². The
stirred cell unit was Amicon 8200 (Millipore Co., UK)
and able to withstand operating conditions of maximum
pressure 75 psi (approximately 5.17 bar) , maximum
temperature of 85ºC and a pH range 2-10
The stirrer speed was set at 350 rpm through the
series of experiments concerning the nutrient media
filtration, so to avoid foaming due to protein
precipitation. The molecular weight cut-off (MWCO) of
the microfiltration and ultrafiltration polysulphone
membranes in use was 0.02μm, 30 kDa, and 4 kDa and1 Figure1.1.: Photograph of the equipment for the
kDa. The 0.02μm filters were provided from Whatman filtration: Cell unit reactor, pump, scale, Alkali
Limited, Maidstone, England, the 30 kDa were provided solution feed ,Waterbath for temperature control,
from Millipore Co., UK, the 4 kDa were provided computer, electronic meter for pressure
Microdyn-Nadir Co., Germany and the 1 kDa from
General Electric- Osmonics Co.UK. All the membranes
were left for 24h soaking in distilled water to ameliorate Figure 1. : Schematic diagram of the stirred cell
diffusivity of the molecules before each experimental (Amicon cell 8200) (1) cap, (2) pressure relief valve,
procedure. (Hwang et al., 2003, Hwang &Lin, 2005) (3) pressure tube fitting assembly, (4) top o-ring,
The pressure in the system was controlled by an provides seal to maintain pressure in the unit, (5)
electronic pressure adjustment value fitted with a magnetic impeller , provides cross flow conditions ,
pressure gauge. The cell unit was pressurizes by constant (6) main body of the stirred cell , (7) bottom o-ring
compressed nitrogen at 200 kPa. The operating provides seal to maintain pressure in the unit and
temperature was controlled at 25ºC constantly by prevent loss of sample, (8) base with permeate
connecting via rubber tubes the cell unit water jacket with outlet, (9) screw in bottom to secure base in the
a water bath (Grant Water bath, UK).The filtrated main body, (10) permeate line and (11) retaining
solutions were collected into a plastic vessel on an stand prevents displacement of cap when pressure is
precision electronic scale (Santorius, PLS303, UK). The used in the unit
scale was connected with a personal computer in Visual
Basic programming language so the flow rate could be

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Contemporary Laboratory Methods for Separation and Purification of Organic Macromolecules A review article

Each membrane was characterised under different


pressure conditions varying between 0 to 400 kPa with the
following solutions, sterilised distilled water, 10mM
phosphate ( KH 2 PO4 ) buffer (Sigma-Aldrich, UK) and
sterilised basal medium. For each experimental run 50ml
of the selected solution was inserted in the reactor.
2.2. Characterization of the 30 kDa MWCO Cellulose
Acetate Membrane
In order to determine the membrane resistance and
the influence of pressure during operation of the
equipment, membrane characterization studies were
carried out. The permeability of distilled water optimized
nutrient medium and phosphate buffer (10mM) solution
through the membranes of different MWCO was
measured in order to analyze the behavior of the reactor
system.
Primarily, 50 ml of the selected testing solution were
inserted in the unit and all the experiments were
conducted in a 25 °C temperature. During the operation
the flux was measured automatically, through a software Figure1.2.: The effect of pressure on flux during
program installed on a personal computer. The average ultrafiltration of water (♦) Phosphate buffer (■) and
flow rate was used for flux calculation. nutrient medium (▲) in the unit at constant
temperature 25°C
14

y = 0.0339x - 0.2729
12
R2 = 0.9889 2.3 Characterization of 4 kDa & 1kDa MWCO
Polysulfone Membrane Effect of Pressure on Permeate
10 y = 0.0201x + 0.6343 Flux
R2 = 0.988
Using the same technique as used for the 30kDa
Permeate Flux (ml/cm2.h)

8 MWCO membrane, to characterize the ultrafiltration


membranes of 4kDa and 1 kDa MWCO the results are
6 demonstrated in the graphs below and the numerical
values are as well tabulated.
4 y = 0.0055x + 0.4277
R2 = 0.9787
1.4
y = 0.0028x + 0.07
2 2
R = 0.9975
1.2

0
0 50 100 150 200 250 300 350 400 450 1
Flow rate (ml/cm2.min)

Pressure (kPa)

0.8
y = 0.0024x - 0.0369
R2 = 0.9774

0.6
The effect of pressure on the flux for water, 10 mM
KH2PO4 and optimized medium is shown on the Figure 0.4
y = 0.0012x + 0.0236
.Flux values from both linearly increased with increasing R 2 = 0.9821

pressure. For pure water the flux increased from 7.90 to 0.2

28.00 ml/cm².min with an increase in pressure from 50 to


0
400 kPa. For phosphate buffer solution the flux increased 0 100 200 300 400 500
Pressure (kPa)
from 1.95 to 9.05 ml/cm².min with an increase in pressure
from 50 to 400 kPa. While operating with optimized
nutrient medium the flux is lower from 0.79 to 2.80 Figure 1.4 The effect of pressure on flux during
ml/cm².min with an increase in pressure from 50 to 400 ultrafiltration of water (♦) Phosphate buffer (■)
kPa respectively. and nutrient medium (▲) in the unit at constant
temperature 25°C

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Contemporary Laboratory Methods for Separation and Purification of Organic Macromolecules A review article

2.4 Application of Dynamic Light Scattering (DLS) in Statistics Graph (8 measurements)


Laboratory Scale for Protein Sizing
25
The HPPS system is an instrument for measuring the
20
size of molecules in solution or the size of particles in

Volume (%)
dispersion. The principle of function of this apparatus is 15

based on Dynamic Light Scattering (DLS) (also known as 10


PCS - Photon Correlation Spectroscopy, or QELS - 5
Quasi-Elastic Light Scattering) which measures Brownian
0
motion of the particles in a solution and relates this to the 1 10 100 1000 10000
size of the particles. This is done by illuminating the Size (d.nm)
particles with a laser beam and analysing the intensity
fluctuations of the scattered light. An important feature of Mean w ith Max-Min error bar
Brownian motion for DLS is that small particles move
quickly and large particles move more slowly. (Callister,
2004) The relationship between the size of a particle and
its speed due to Brownian motions defined in the Stokes- Statistics Graph (10 measurements)
Einstein equation
50

kT 40
Dr =

Volume (%)
3πηd 30

20
Where k (1.3807*10¯²³ J/K) is the Boltzmann constant, T 10
is the absolute temperature in Kelvin (K), and η is the 0
viscosity (8.94*10‾ ⁹ kg/ms) of the medium in which the 1 10 100 1000 10000

particles of diameter d (meters, m) are suspended. Size (d.nm)

The HPPS system measures the rate of the intensity Mean w ith Max-Min error bar
fluctuation and then uses this to calculate the size of the
particles. The size of the particles is graphical represented Figure1.6 Size distribution of protein sources
into curves where the highest peak represents the majority
of molecules in the specific size given buy the peak.
(Moachon et al.2001)
Statistics Graph (10 measurements)

In order to measure the size of the molecules 4 ml of 50


samples, in these figures bacteriocins are placed in plastic
40
cuvettes and put in the apparatus. The apparatus was
Volume (%)

30
connected with a personal computer equipped with the
20
special software programme (Malvern Instruments Ltd.
DTS 4.20 2002) and all the measurements were done 10

automatically. 0
1 10 100 1000 10000
Size (d.nm)
Statistics Graph (9 measurements)

40 Mean w ith Max-Min error bar

30
Volume (%)

The sizing of the proteins is also checked by the


20 correlation function. The correlation function is
10
demonstrating the rate of decay of the Brownian
movement of the particles in a solution. It is related to the
0
1 10 100 1000 10000
size of the particles as the rate of decay is much faster for
Size (d.nm)
small particles than it is for large particles. As it can be
seen by the above graphical representation, the filtration
Mean w ith Max-Min error bar of the media is successful.

Figure 1.5: Size distribution of protein sources

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Contemporary Laboratory Methods for Separation and Purification of Organic Macromolecules A review article

Figure 1.7. Correlation Function.

Figure 1.8: Photograph of HPLC Equipment: Delivery pumps, Injection loop, Column, Waste, Solvent
delivery system
mixture of substances. Having connected the
2.5 Application of High Performance Liquid chromatographic bed with a light detector
Chromatography (HPLC) in Laboratory Scale for Protein (Ultraviolet/Visible lamp) an injection loops (varying in
Purification size between 10 μl to 100 μl) and with two pressure
A popular separation and also purification method pumps the apparatus is ready to use.
for proteins is High Performance Liquid Chromatography According to their size, molecular weight and charge
(HPLC). HPLC is a separation process in which the the substances are entrapped in different parts of the
sample mixture is distributed between two phases in a chromatographic bed. Each substance is then
chromatographic bed. (Column) One phase is stationary demonstrated as a peak which according to the properties
whilst the other (mobile phase) passes through the of the substance varies in size. (Height and volume) In
chromatographic bed, carrying through the sample order to exactly identify the nature and the type of the

60
Contemporary Laboratory Methods for Separation and Purification of Organic Macromolecules A review article

separated substances, several tests are made with a variety during the time length of the process. The flow rate of the
of known substances. The peaks that are found are used as mobile phases well may vary between 1ml to 2 ml per
reference so to exactly identify the substances existing in minute, depending mainly on the pressure the system can
the samples. The establishment of a protocol using the withstand.
previously described method is necessary for the accurate Apart from testing the mobile phase, its absorbance,
and successful use of HPLC method as identification and flow mode and flow rate the time length of the process
separation process, as HPLC is mainly qualitative should be tested as well. Usually most protocols for
analytical method that is only partially automated. peptide identification and separation vary between 10 min
In spite of testing various substances so to fabricate a to 40 min
standard protocol, similar testing has to be done as to The stationary phase is either a solid, porous, a
determine the most suitable mobile phase (solvent) that surface –active material in particle form or a very thin
can successfully carry the substances without dissolving, film or liquid coated on a solid material supporter column
deactivating or destroying them. wall. The mobile phase can be either consisted of gas or
Water is often described as the strongest elution liquid. If a gas is used, the technique is called as Gas
medium for chromatography, though it is mainly applied Chromatography (GC) though if the mobile phase is
for adsorption process. The mobile phase generally liquid is called High Performance Liquid
consists of mixtures of water or aqueous buffer solutions Chromatography. (HPLC) The results, meaning the size of
with various water miscible solvents such as methanol, the peaks are calculated according to the retention
acetonitrile, isopropanol and ethanol. In order to separate time(Er) of a solute in HPLC which is defined as the
proteins reverse phase chromatography is used. In this necessary for maximum elution of the particular solute.
type of chromatography the stationary phase is less polar
than the mobile phase. The effectiveness of mobile phase 2.6 HPLC Unit Operation
is tested prior to its application on the system. The HPLC system was connected with a UV/Vis
Testing the mobile phase is majorly achieved though detector ( Dionex, UK) and fitted with a C18 reverse
testing the absorbance of the mobile in a range of phase column (Vydac 238 TP54, HPLC columnswhich is
Ultraviolet (UV) to Visible (Vis) light wavelength used to detect small polypeptides less than 4,000-5,000
although it is far more common for macromolecules to be MW, enzymatic digest fragments, natural and synthetic
detected in a range of wavelength between 210nm to peptides and complex carbohydrate
320nm.Another parameter that should be examined as The solvent (mobile phase) delivery system was

8
WI:
mVolts c :\ my d o c ument s \nis in new lamp \ s olut io n- 5 - 2 0 0 9 0 1 0 .run File: c :\ my d o c ument s \nis in new lamp \ s olut io n- 5 - 2 0 0 9 0 1 0 .run
Channel: 1 = UV R esult s
Last recalc: NA
1.386

50

40

30
4.657

9.627
9.325
7.818

20

10

X : 1 1 .0 8 5 0 M inut es
Y : 3 .3 8 mV o lt s
-8
5 10 15 20 25 30

Minutes

Figure 1.9. Correlation Function Testing the Vydac C18 Reverse phase column function HPLC Testing
standard peptide mixture by Sigma Aldrich (1:8 dilution) (negative peak indicates water)

well is the flow and the flow rate of the mobile phase in constructed by 2 pumps (pumps A and B) (Varian Co.
the system. There are two main modes of flow, isocratic Canada.) with a pressure operation range between 1500
and gradient. Isocratic flow is described as an equal and 1900 mbar. Temperature control of the solvents was
amount of the mobile phase mixture (solvents A & B) maintained with a hotplate (Millipore Co., UK) at 25°C.
being injected in the system simultaneously though in the An example of mobile phase can be the following.
case of gradient flow a percentage of each solvent (E.g. The mobile phase was represented by two solutions,
30% solvent A & 70% of solvent B) is being injected in solvent A consisted of 99% pure acetonitrile (ACN) 10%
the system. The amount of the percentage may vary v/v in distilled water and 1% v/v of standard buffer

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Contemporary Laboratory Methods for Separation and Purification of Organic Macromolecules A review article

solution and solvent B of 99% pure acetonitrile (ACN) References:


75% in distilled water and 1% v/v of standard buffer
[1] Morao A. C., Alves A.M.B., Manuel, C.C., Cardoso J.P.,
solution. The standard buffer solution was consisted of Nanofiltration of a clarified fermentation broth. Journal of
7.5% trifluroacetic acid (TFA) 5 % v/v (TEA) and 65% of Chemical Engineering Science 2006, 61, 2418-2427.
99% pure acetonitrile (ACN) in distilled water. The [2] Callister W. D. J., Fundamentals of Materials Science and
solutions were delivered to the pumps via rubber tubes Engineering: An Integrated Approach. 2nd ed., John Wiley
and valves. The mobile phase was organised as gradient, and Sons: 2004, p 155-172.
consisting of 65% of solvent A and 35% of solvent B. The [3] Hwang, K. J., Cheng, Y.H., Tung K.L., Modelling of cross-
flow rate of the samples and of the mobile phase was set flow microfiltration of fine particle/macromolecule binary
at 1.5 ml/min for 15 minutes, and the wavelength used suspension. Journal of Chemical Engineering of Japan 2003,
was 220nm. 36, 1488-1497.
The operation of the system was controlled [4] Hwang K. J., Lin L.W., Separation of protein from
automatically using Prostar Workstation Data analysis microbe/protein binary suspension using a cross flow
microfiltration Journal of Chemical Engineering of Japan
software package (Varian Co., Canada). Every cycle of 2005, 11, 894-902.
measurements lasted for 17 min. All the samples were
injected in the system via a sterile HPLC plastic syringe [5] Li S. Z., Li X.Y., Cui Z.F., Wang D.Z., Application of
ultrafiltration to improve the extraction of antibiotics.
(1 ml sterile syringe, Fischerbrand, UK)at a 20 μl Separation and Purification Technology 2004, 34, 115-123.
injection loop connected with the HPLC system All the
diluted samples (1:15 dilution rate) were injected in the [6] Oh J. T., Kim, W.S., Production of poly-β-hydroxybutyrate
(PHB) by fed-batch fermentation using hollow fibber
injection loop in a quantity of 20 μl. Each measurement membrane system. Journal of Chemical Engineering of
lasted for 20 min. After the end of the measurements and Japan 1996, 29, 893-897.
between each sample measurement the equipment was [7] Kwon S., Yoo I.K., Woo G.L., Chang H.N., Chang, Y.K.,
cleaned using the mobile phase solutions. High rate continuous production of lactic acid by
The above figure is typical protocol result that clearly Lactobacillus rhamnosus in a two stage membrane cell
indicates the successful separation achieved from the recycle bioreactor. Biotechnology & Bioengineering Journal
2001, 73, 25-34
chromatographic bed of the various peptides existing in
the standard peptide mixture used. The first negative peak
indicated water that is used as diluents of the sample. The
remaining peaks (1 to 5, left to right) are indicating
glycine, valine, met-enkefalin, leucine and angiotensin.

3.0 Conclusion
In this article a review was made on the main laboratory
methods used for separation and purification of organic
compounds from complicated mixtures. The principle of
function of each method was explained and specific
scientific results were as well given in order to
demonstrate the exact function of each method.

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