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1
International Journal of Chemical and Environmental Engineering
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
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Contemporary Laboratory Methods for Separation and Purification of Organic Macromolecules A review article
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)
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
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Contemporary Laboratory Methods for Separation and Purification of Organic Macromolecules A review article
Volume (%)
dispersion. The principle of function of this apparatus is 15
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
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)
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)
30
Volume (%)
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Contemporary Laboratory Methods for Separation and Purification of Organic Macromolecules A review article
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
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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
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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