Ion exchange chromatography separation of different

biomolecules e.g. DNA, Protein, Vitamin and Natural

products
Research code: PHG2306-R9

Course name: Chromatography

Course code: PHG2306

Under supervision

Dr. wael Afifi

NO NAME ID
1 Ahmed Melatam Elzahaby Mohamed 201600585
2 Zeyad Hesham Mohamed 201703271
3 Esraa Abozaid Abdelhamed 201703899
4 Esraa Ramadan Sayed Ahmed 201704237
5 Nada Kazem Ali 201702819

Spring
2020
Page 1 of 17
 List of content

Table of Contents
i. Abstract......................................................................................................page3
ii. Keywords ...................................................................................................page3
iii. Introduction...............................................................................................page4
iv. Ion exchange mechanism...........................................................................page4
v. Basic Principles of Ion-Exchange Chromatography ................................page4
vi. Ion exchange chromatography separation of protein ..........................page5
vii. Ion exchange chromatography separation of natural products...............page8
viii. Ion exchange chromatography separation of vitamin..............................page9
ix. Ion exchange chromatography separation of Nucleotides ..................... page12
x. Conclusion ............................................................................................... page14
xi. References................................................................................................ page15

Figure NO.1……………………………page5
Figure NO.2……………………………page6

Page 2 of 17
Abstract
Ion exchange chromatography separation is a method to
ions. Its used to separate any sample like protein, DNA,
This method needs a specific materials and steps to get
protein, DNA, vitamin and natural products methods to
each.
separate isolate deferentially charged or ionizable
vitamin and natural products to pure components.
the desired results. This research will discuss the
separate and the required materials and steps for

Keywords
Chromatograph, Separation Protein Vitamin Natural Nucleotide

Page 3 of 17
 1- Introduction
Ion exchange chromatography is an essential aspect of ion chromatography Scientific approach to the
isolation and measurement of ionic compounds and chromatography of ion-partition / interaction and
ion-exclusion[1]. Separate ionic chromatography Tion is based on ionic (or electrostatic) interactions
between ionic, ionic and polar analytes Present in eluent and ionic specific groups set to enable
chromatography. Two men Separate processes as follows; interchange of ions through direct ionic
interaction (attraction); and Ion exclusion due to repulsion between analyse ions similarly charged and the
ions fixed on The chromatographic assistance, play a role in the ion chromatography separation. Son: Son
To date, the exchange was a prevalent form of ion chromatography This chromatography is one of the most
powerful methods of adsorption for isolating peptides, proteins , nucleic acids and related biopolymers
charged by molecules of varying molecular size and life[2,3].. The separation is based on ionic forming
Bonds between the charged biomolecular groups and the ion exchange gel / support that bears the opposite
charge[4]. Biomolecules show different degrees of interaction with charged chromatographic media because
of their different charging properties. For many years, exchange chromatography has been used to separate
different ionic compounds; cations and anions, and is still in use. The use of chromatog‐raphy ion exchange
has risen in recent years, since this technique enables the study of a broad variety of molecules in the
pharmaceutical, biotechnology, medical, agricultural and other industries. Ion exchange chromatography
may be used for the isolation and purification from natural or industrial origins of several charged or
ionizable molecules such as proteins, peptides, carbohydrates, nucleotides, DNA, antibiotics, vitamins, etc.
Bioseparation requires the resolution of components in dynamic mixtures contained in biological and
biochemical processes, thereby allowing scientists to evaluate the identification and concentration of each
product, and to separate a specific component from other contaminating molecules for further study or use,
if appropriate. Processes of bioseparation are often dominated by steps in the liquid chromatography ( LC).
LC mixture resolution is based on the idea that individual solutes dissolved in a mobile phase under a
specified range of conditions can associate differently with a chemically changed stationary phase as a result
of variations in individual solvent distribution coefficients (K). In this way, LC takes advantage of intrinsic
variations between biomolecules (e.g., molecular size, hydrophobicity, binding specificity, charge) to
achieve their separation from each other.

2- Ion exchange mechanism

Ion-exchange chromatography primarily developed to isolate uniquely charged or ionizable substances
comprises of mobile and stationary phases identical to certain types of column-based liquid chromatography
techniques[5,6].Mobile phases comprises of an aqueous buffer network through which the mixture to be
dissolved is created. The stationary step is normally made of chemically proportional inert organic matrix
with ionizable functional groups (fixed ions) holding displaceable, opposite charged ion[6]. Ions remaining
in a state of equilibrium between the mobile phase and the stationary phase give rise to two potential forms,
anion exchange and cation exchange are considered counter ions[1,7]

3- Basic Principles of Ion-Exchange Chromatography

Ion-exchange chromatography (IEC) was originally developed to isolate differentially charged or ionizable
ions, with its history going back to the 1940s[8.9]. This technique has been used routinely by both chemists
and biochemists to purify proteins[10,11] , enzymes[6,11,12],, antibodies[10,11], peptides[13], amino
acids[14] and nucleic acids, as well as simpler carbohydrates and organic compounds. Its large sample
handling capacity, wide applicability (including high-performance and high-throughput application formats),
moderate cost, powerful resolution and ease of scale and automation have made it one of the most versatile
and widely used LC techniques.
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Esraa Ramadan
As many types of column-based LC (e.g. gel-permeation, association, hydrophobic contact, etc.), this
technique involves both mobile and stationary stages, the former generally an aqueous buffer layer into
which the mixture to be resolved is introduced, and the latter normally a chemically derived inert organic
matrix of ionizable functional groups bearing a displaceable opposite charged counterion. These equivalents
remain in a state of equilibrium between the mobile and stationary stages, culminating in two potential IEC
formats, i.e. anion- and cation-exchange.

Figure NO.1 Ion exchange chromatography separation mechanism

Chromatography through the movement of ions. This is the existence of the counterions expelled from
practical matrix classes that defines the structure of the IEC. Thus, with anion-exchange chromatography
(LHS), the stationary phase matrix shows a positively charged functional group with a negative counterion
that can be replaced by an anionic sample, enabling adsorption of matrixes. Chromatography (RHS) of
cation exchange. The stationary phase matrix shows a negative charged functional group with a positive
counterion that can be replaced by a cationic sample, thereby enabling matrix adsorption again. With either
type, you can achieve sample desorption from the matrix by rising the counterion concentration in the
mobile process.
Esraa Ramadan
4- Ion exchange chromatography separation of protein
Ion exchange chromatography (IEX) is a chromatographic separation process centered basically on the net
protein charge, which is typically used to monitor the development of deamidation which succinimides. Two
forms of IEX, cation exchange and chromatography of anion exchange, exist. The protein is charged
negative (anionic) at buffer pH values above this IP; at pH values below that, the protein is charged
positively (cationic).
Both proteins display a net charge depending on the protein's amino acid composition and any modifications
that are covalently attached. A protein 's net charge is determined by the solvent 's pH in which it is
dissolved, as solvents exchange hydrogen ions with proteins

Page 5 of 17 Ahmed Melatam

 Methods

Figure NO.2 protein ion exchange chromatography separation method

Equilibrium
The first step is on stationary phase equilibrium. Both stationary phase charged groups are linked with
exchangeable counterions, such as chloride or sodium, until equilibrium is achieved. To ensure that proteins
of interest bind to the medium, pH and ionic strength of the start buffer are selected.

Sampling and Washing

In the second stage the aim is to bind the target molecule(s) and wash all unbound material out.

Elections
When ionic pressure rises, the proteins with the lowest net charge at the chosen pH would be the first ones
eluted from the board. Likewise, the proteins with the largest charge at a given pH should be held more
firmly and It will be always kept, and finally elucidated.

Renaissance
A final wash with a high ionic resistance buffer regenerates the column and removes any still bound
molecules. The column is then rebalanced in the start buffer until the next run is begun.[15]

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Ahmed Melatam
 Material

stationery phase
Stationary phases consisting of two structural elements; the groups charged involved in the exchange
process and the matrix on which the groups charged are fixed[16]
If they have negatively charged functional groups and possess exchangeable cations, ion exchangers are
called cation exchangeers. Anion exchangers carry anions because their fixed group positively charges[17].
The charged groups determine the specificity and strength of protein binding by its polarity and density
while the matrix determines the stationary phase's physical and chemical stability and flow characteristics,
and may be responsible for unspecific binding effects[16].
General structure (fibrous or beaded form), particle size and variation, pores and dimensions, surface
chemistry (hydrophilic or hydrophobic), matrix swelling characteristics are important factors affecting
chromatographic resolution[18,16].
Porosity of beads sharing ions can be classified as non-porous, microporous, and macroporous.
Porosity of beads sharing ions can be classified as non-porous, microporous, and macroporous.
Matrices obtained by polymerization of polystyrene with different amounts of divinylbenzene are known as
the original matrices for chromatography of the ion exchange. Although these matrices have a much more
hydrophobic surface and due to strong binding, proteins are irreversibly damaged. Ion exchangers focused
on cellulose with hydrophilic backbones are ideally adapted for protein separation matrices. Other matrices
for the ion exchange with hydrophilic properties are based on agarose or dextran[16].
Cellulose; Hydrophilic surface, improved cross-linking stability, cheap
Dextran; important swelling as a function of ionic environment, improved materials through cross-linking)
Agarose; Swelling is almost independently of ionic pressure and pH, strong binding ability produced by
highly porous particle development
Polyacrylamide; Swelling dextran-like behavior
Acrylate copolymer; stability and high with pH
Polystyrene-divinylbenzene; Hydrophobic base, poor protein binding ability
Polystyrene-divinilybenzene coatings; hydrophilic surface
Silica; unstable, rigid particles at pH > 8
Coated Silica; Ground Hydrophilic[16].

Mobile phase
In ion exchange chromatography typically eluents composed of an aqueous solution of an suitable salt or salt
mixtures with the a limited proportion of an organic solvent have been used in which much of the ionic
compounds are stronger dissolved than in those in. Hence it would be much simpler to add various
samples[19,20].
EDTA; tetraacetic acid with ethylenediamine
Polyols; cholesterol, glycerol, and saccharose

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Ahmed Melatam
 Offenders Sparrows Reactifs of
Chloride of urea and guanidinium sulfhydryl Ligae
Hypocrisy Inhibitors of protectiona[16]
Bio-solvents

detector : UV

5- Ion exchange chromatography separation of natural products

Natural medicines, such as traditional Chinese medicine ( TCM) and Ayurveda, have been created and
evolved over thousands of years in the everyday life of ancient people and in the course of fighting diseases,
and have had a significant influence on the development of human society. Today, natural medicines not
only meet the primary health care requirements for the majority of the developing world's people, but have
gained more and more interest in industrialized countries owing to increasing health care expenses and
compulsory financial austerity. In the US, some 49 per cent of the population have sought natural therapies
for disease prevention and treatment[21]. Known Chemicals The "good components" or "working values" in
natural remedies are believed to have therapeutic benefits. Natural ingredients provided the key outlets for
the production of modern medicines. Nearly half of the FDA 's licensed chemical medications for the
treatment of human diseases were produced from or influenced by natural products from the 1940s until the
end of 2014[22, 23]. Natural products offer molecules from combinatorial chemistry with more drug-like
features in terms of functional groups, chirality, and structural complexity[24, 25].
In natural treatments the amounts of chemical compounds are always extremely low. The
laboratory-intensive and time-consuming process of extraction and isolation has been the bottle neck of
natural ingredients being applied to drug development. Efficient and selective methods for the extraction and
insulation of bioactive natural products are desperately required. The study seeks to provide a detailed
description of a range of approaches utilized in natural substance extraction and isolation.

Methods
The extract components according to the above methods are complex and contain a wide range of natural
products that require further separation and purification in order to produce the active fraction or the pure
natural products. The separation depends on the physical or chemical variation of the natural product in
question. The main method used for obtaining pure natural products from a complex mixture is
chromatography, especially column chromatography.
Ion-exchange chromatography (IEC) separates molecules based on their net surface charging differences.
Any natural products, such as alkaloids and organic acids with a functional group able to ionize, can be
isolated by IEC. By changing the ionic strength of the mobile phase ( e.g. changing pH or salt concentration)
the charged molecules could've been caught and released by ion-exchange resin. Cation ion exchange resins
have been used to separate alkaloids, while the anion ion exchange resins have been used to separate natural
organic acids and phenols.
Taking [Kiwifruit] as an example The positively charged anthocyanins in the treated XAD-7 were separated
from the neutral polyphenolic compounds Actinidia melanandra fruit (kiwifruit) extract using the
ion-exchange resin Dowex 50WX8 cation[26]. To isolate (−)epigallocatechin-gallate, Feng and Zhao used
semi-preparatory chromatography and (−)epicatechin-gallate in crude tea extract with a slightly acidic
polysaccharide-based gel CM-Sephadex C-25[27]. A special alkaloid, fumonisin B6 (, together with a
Page 8 of 17

Zeyad Hesham
recognized alkaloid, fumonisin B2 , was separated by IEC over Strata X-C mixed-mode RP-cation-exchange
resin backed by reverse-phase chromatography of the fungus Aspergillus niger NRRL 326 crop
extract[28,29].

Material
As used in the Ion exchange chromatography separation of protein.

6- Ion exchange chromatography separation of vitamins:

Vitamins are substances that are required by the human body for enhanced growth and development.
Altogether thirteen types of vitamins are essential for the normal functioning of the body.
The vitamins are broadly classified as water-soluble and fat-soluble vitamins which are further distinguished
as vitamin A, B, C, E and K.
The water-soluble and fat-soluble vitamins are separated and purified using various techniques such as
capillary electrophoresis, HPLC and chromatography. Ion-exchange chromatography is being the most
popular. It plays a major role in various environmental applications such as ecological remediation, water
softening, catalysis, hydrometallurgy, wastewater treatment and bimolecular partitions. Various synthetic
resins and several other resins have been used for the separation and purification of the vitamins. An
overview of the different methodologies involved in the process of ion-exchange chromatography used in
the separation and purification of vitamins B1, B2, B6, C and K1 is presented in this chapter.[30]

Experimental:[31]

Apparatus

Model 830 and 840 Liquid Chromatographs (E. I. Du Pont de Nemours & Co., Instrument Products Di-
vision, Wilmington, DE) were used for this study. Bath chromatographs have UV detectors at 254 nm
wavelength and the 830 allo was equipped with a gradient elution device. Important features of these
instruments are described in detail elsewhere in the literature, Samples were introduced onto the column by
syringe injection. A computing integrator (Autolab System IV) was used to determine peak areas and
retention times.

Standards and reagent

The vitamin and phosphate ester standards were obtained from Nutritional Biochemicals (Cleveland, OH),
Cal Biochem (San Diego, CA) and P-L Bio- chemicals, Inc. (Milwaukee, WI) . The salts and buffer
solutions (Fisher Scientific) were all of reagent grade qualitynts

Chromatography of the Water-Soluble Vitamins[32,33,34,35,36,37]

Because of their ionizable nature, the water solu- ble vitamins are well-suited for analysis by high- speed
ion-exchange chromatography.
In general, those vitamins with amine groups chromatograph well by cation exchange while those with
phosphate or car- boxylic groups are best separated by anion-exchange. Isocratic mobile phases are usually
adequate for as- saying single vitamins.

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Esraa Abozaid
However, for complex mixtures, it may be necessary to use gradient elution to elute all of the components in
a reasonable length of time.
Specific mobile phase and column conditions are given for each vitamin in the following sections.
How- ever, the combination pH and ionic strength of the mobile phase can often be varied considerably
from these values while still retaining qadequate separation of a particular vitamin. In many cases it may be
necessary to manipulate the combination of pH and ionic modifier strength to obtain the best resolution of
complex mixtures

Nicotinic Acid (Niacin)

This compound is a dipolar ion and in acid pH below 4 (where both the nitrogen and carboxylic acid group
are protonated) it can be chromatographed on a column of Zipax® SCX . At neutral and basic pH the
vitamin is anionic and can be chro- matographed on an anion exchange column such as Permaphase® AAX
or Zipax® SAX.
In both cases only dilute buffer (less than 0.05M) is required in order to elute nicotinic acid from the col-
umn. Nicotinamide has cationic character in either neutral or acid pH and can be chromatographed on
Zipax® SCX with moderate amounts of a sodium salt modifier such as NaNO3, NaC1O4, or NaH 2PO4

Riboflavin (Vitamin B2)

This vitamin is weakly retained in neutral or slightly acidic pH on a strong cation exchange column. This
vitamin becomes more strongly retained only at pH of 2 or less. Riboflavin has also been retained by anion
exchange chromatog-Raphy

Thiamine (Vitamin B1)

In acid or neutral pH, this vitamin is a quaternary amine and is very strongly retained on a cation ex- change
column. Only in basic pH and high ionic strength is the retention reduced to the point where it can be eluted .
Precautions must be taken not to allow the mobile phase to exceed pH 9 or the column packings may
degrade. At pH greater than 11, thiamine may oxidize to other products

Vitamin B6

The term Vitamin B6 refers to three distinct mo- lecular species; pyridoxine, pyridoxal and pyridox- amine.
All three have vitamin B s biological activity and are found in nature, although pyridoxine is the form
commonly used in vitamin tablets and supple- ments. All three are cationic in nature at pH 4 and separate on
Zipax® SCX at pH 4 in 0.1M KH 2PO4

Method and material :[32]

Chemicl

RP‐WAX‐modified silica material was prepared in our laboratory using an optimized proprietary
manufacturing process
Mobile phases for chromatography were prepared from HPLC grade acetonitrile, disodium hydrogen
phosphate and sodium dihydrogen phosphate (Merck). Vitamins were purchased from Sigma‐Aldrich
(Steinheim, Germany).
Log D values were calculated for pH 4.4 with ACD/Labs 7.00 software from Advanced Chemistry
Development (Toronto, ON, Canada).

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Esraa Abozaid
 Instrumentation

chromatographic experiments were carried out on a VWR (Darmstadt, Germany) Hitachi LaChrom Elite
HPLC system (three channels pump L‐2130 with degasser, autosampler L‐2200, oven L‐2350, diode array
detection (DAD) system L‐2455).
The gradient optimization was performed on a VWR Hitachi LaChrom Ultra HPLC system (two single‐
channel pumps L‐2160U, autosampler L‐2200U, oven L‐2300, diode array detection system L‐2455U).

Sample preparation

Stock solutions of single vitamins were prepared in methanol at concentrations up to 3 mg/L or up to the
limit of solubility. The working solutions with single vitamins were prepared at the desired concentrations
by dilution of the stock solutions. The vitamin mixtures were prepared at the desired concentration directly
from pure compounds. All solutions were stored at −20°C after sonicating and filtered on a 0.2‐μm PTFE
syringe filter
before being placed in the autosampler. The solutions were freshly prepared every 2 days except for the
sample stability study.
The vitamin tablets (see Table 2 for ingredients) were dissolved in 20 mL water. After sonication and
filtration on a 0.2‐μm PTFE syringe filter, the mixture was directly injected onto the column.

Chromatographic conditions

A column filled with the RP‐WAX material described previously (125×4 mm, 5 μm particle size, 100 Å)
was used for the analysis. The injection volume was 10 μL and the flow rate was set to 1.0 mL/min.
Four different wavelengths (260, 270, 300, and 310 nm) were evaluated. The best results were obtained at
270 nm for almost every vitamin.
Vitamin A‐acetate exhibits very low absorbance at this wavelength and the validation was therefore
performed at 310 nm for this molecule.
The simultaneous determination was performed at both wavelengths.
Mobile phase composition, pH, temperature, and gradient were optimized . Best results were obtained with a
mobile phase containing water and acetonitrile in the presence of phosphate buffer . Dead volume t 0 was
determined by injecting pure methanol (t 0 = 1.70).
Selectivity: Standard vitamin solutions were first injected onto the column and the evaluation of the
obtained chromatogram allowed us to ensure the purity of the stock solutions.
The selectivity of the method was then demonstrated by comparison of the chromatograms of the vitamin
mixture and of the real samples with chromatograms monitored for each vitamin standard solution.

Robustness

The robustness of the vitamins quantification must be assessed through small deliberate variations of the
method parameters 19.
We investigated the effect of small variations of the temperature, the pH, the buffer concentration, and the
slope of the gradient, which we identified as critical parameters.
The relative standard deviation of the retention time was then calculated for each vitamin and the limit of
deviation was set up to 5%

Range

The range is given in mg/L as interval of concentrations for which precision is under 2%, accuracy is in the
95–105% range, recovery is stable, and between the LLOQ and the ULOQ 26.

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Zeyad Hesham
 Storage stability

To assess storage stability, the evolution of the peak area was compared for each vitamin within the mixture
during 15 h at room temperature and during 5 days in four different conditions. For the 15‐h measurements,
the mixture remained in the autosampler at RT.
For the 5‐day measurements, the mixture was prepared at day 0 and it was aliquoted in autosampler vials.
One set of vial was stored at −28°C in the absence of light and another one was stored in the dark at +4°C.
The two last sets of vials were stored at room temperature, with and without light, respectively. For each
measurement, the vial was put in the autosampler
shortly before injection.

Separation and Purification of Nucleotides

Nucleotides

Nucleotides are a unit charged ionic atoms comprising of a chemical element base, a 5-carbon
monosaccharide sugar, and oxygen acid. Four of the bases (A, T, C, and G), substituted with
2-deoxy-α-d-ribose, polymerize to provide polymer (DNA), and 3 of those additionally to U (A, U, C, and
G), substituted with α-d-ribose, be a part of to border ribonucleic acid. The progressive nucleotides of each
ribonucleic acid and DNA area unit covalently connected to every alternative once a compound (RNA or
DNA polymerase) catalyzes arrangement of a phosphor di ester bond between the 3-hydroxyl cluster of 1
monosaccharide and therefore the 5-hydroxyl of the subsequent monosaccharide. The reaction of phosphate
bonds within the precursors—that is, the ester triphosphates—drives the DNA and ribonucleic acid enzyme
responses. Nucleotides to boot assume a focal half in metabolism at the cell level. They convey various
energy within the form of nucleoside triphosphates ATP,GTP,CTP, and UTP all through the cell to the
various cell capacities that need energy, that incorporate integration amino acids, proteins and cell films and
parts; moving the cell and cell organelles, each inside and intercellular ;separating the cell , and soon .There
are many commercially used activity techniques for the separation and purification of nucleotides like action,
particle exclusion, HPLC, and reversed-phase HPLC []. Among these, action is that the most ordinarily used
thanks to its Brobdingnagian applications[44].[45]

Separation of Nucleotides

For analysis of cyclic nucleotides and connected compound exercises, ion-exchange chromatography is that
the most typically used separation procedure. Cation exchange and additionally anion-exchange resins are
often used for cyclic ester detachments. Dowex-50 column created with acidic solvents is especially
valuable if the solutions contain a great deal of electrolytes since such examples will specifically be
connected. Anion-exchange resins have in addition been used for cyclic ester partitions. These materials are
used notably in examining adenylate and guanylate cyclase and of cyclic ester phosphor di esterase reactions.
Their application to the filtration of cyclic nucleotides from tissue concentrates is tough as a result of these
excess electrolytes should be expelled out before sample application within the column. For this reason,
surface assimilation of the nucleotides to charcoal under acidic conditions and extraction into a mix
containing ammonia and alcohol has been applied. A charcoal advance are often dodged with definitely
essential anion-exchange materials, as an example, QAE-Sephadex, if tissue homogenization and ester
extraction don't seem to be performed in acid, however rather in an exceedingly Zn ethanoic acid
derivation liquor arrangement. The water used for recovery of the resins need to be deionized. For the last
stages of the organic compound recovery and for the event of the elution liquids, in any case, the water of
glass-redistilled quality needs to be used. A QAE-Sephadex column is often used for a fast and flourishing
detachment of the marked glycoside from the remaining cyclic ester [42]

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Nada Kazem
 Cation-Exchange Resin

He Associate in a nature of nucleotides at an acidic pH is owing to variations within the cationic charge of
the bases and therefore the anionic charge of the phosphate cluster connected to the carbohydrate moiety;
but, variations within the web negative charges of nucleotides can be traditional. The charge variations
among nucleotides, in conjunction with their variable segments between mobile eluent and stationary
. organic compound matrix, ar attributes for natural process variation [44]

Anion-Exchange Materials

The main divisions of nucleotides were acquired by anion-exchange activity. The technique has been in
wide application and adjusted to suit divisions of bases and nucleosides during a similar examination.
Hence, the anion-exchange activity with smaller and uniform beads was reinvestigated to form frameworks
which will be utilized to isolate the important nucleotides in single eluent and to isolate most nucleosides,
nucleotides, and completely different chemical reaction things during a single analysis[39]

Method
1) A technique for DNA purification during which DNA in answer is sublimate by initial contacting the
DNA answer with a rosin suspension comprising a porous ion exchange resin and a porous ion exchange
resin, then removing the resins.
2) A technique for DNA purification during which DNA in answer is sublimate by initial contacting the
DNA answer with a resin suspension comprising a porous ion exchange resin, then contacting the answer
with a resin suspension comprising porous ion exchange resin, then removing the resins.
3) The method of claim one during which the resin suspension to boot includes linear polyacrylamide.
4) The method of claim two during which either or each resin suspensions more comprise linear
polyacrylamide.[40]

Anticipated Results
He expected yield for a preparation of genomic DNA from class cell culture is ∼30 to 40 µg per 107 cells.
Low-copy-number plasmid and cosmid preparations from cells grown long in avoirdupois unit medium
yield 0.2 to 1 µg DNA per millilitre of long culture, while high-copy-number plasmids ready under these
conditions yield 3 to 5 µg/ml. When grown in super-rich medium, the yield of cosmids and
low-copy-number plasmids is redoubled to 2.5 to 5 µg DNA/ml, which of high-copy-number plasmids to 5
to 25 µg/ml.[42]

Experiment
In 2011, Zhong et al. aimed at removal of RNA impurities from pDNA sample without using RNase by
firstly, precipitation of pDNA takes place with isopropanol as a pre-purification step . Secondly, anion
exchange membrane chromatography was implemented to get rid of the RNA impuritie [38]

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Nada Kazem
 Materials
In 2011, Zhong et al. aimed at removal of RNA impurities from pDNA sample without using RNase by
firstly, precipitation of pDNA takes place with isopropanol as a pre-purification step . Secondly, anion
exchange membrane chromatography was implemented to get rid of the RNA impuritie [41]

Materials
The stationary phase is an anion exchange membrane incorporated with 3-
acrylamidopropyl-trimethylammonium chloride packed in stainless steel membrane disk holder. The elution
system is a gradient increase in sodium chloride concentration from 50 mM NaCl in Tris-HCl to 1 M NaCl
at pH 8. The recovery of pDNA is 84%.[43].[42]

Method
it's not convenient for separation of pDNA isoforms . Aiming at purification of huge pDNA molecules (25
kbp), Ongkudon et al. innovated a brand new round shape monolith column used in one step for this purpose,
by reducing the wall channel size of stone column .The whole sample was diluted with buffer A (25 metric
linear unit TriseHCl, 2 mMEDTA, pH 7) so washed by eluting buffer A: buffer B (25 metric linear unit
Trise HCl, 2 m M EDTA, 1.0 M NaCl, pH 7) (8:2) to get rid of the impurities and finally extraction was
done by admixture buffer A with buffer B to wash the pDNA. The gentle conditions of the extraction system
allowed the researchers to separate giant size pDNA (25 kbp) in an exceedingly cheap purity and recovery
as shown in such giant pDNA molecules could not be separated in an exceedingly smart yield in forceful
ways like affinity chromatographic ways. This method is rapid, cheap and efficient. [44].[47]

7- Conclusion
This research has been explained the methods to [Ion exchange chromatography separation] for each of
protein, DNA, vitamin and natural products .Ion-exchange chromatography primarily developed to isolate
uniquely charged or ionizable substances comprises of mobile and stationary phases identical to certain
types of column-based liquid chromatography techniques.Natural products as natural treatments the amounts
of chemical compounds are always extremely low. The laboratory-intensive and time-consuming process of
extraction and isolation has been the bottle neck of natural ingredients being applied to drug development.
Efficient and selective methods for the extraction and insulation of bioactive natural products are desperately
required. The study seeks to provide a detailed description of a range of approaches utilized in natural
substance extraction and isolation.
Ion exchange chromatography (IEX) is a chromatographic separation process centered basically on the net
protein charge, which is typically used to monitor the development of deamidation which succinimides. Two
forms of IEX, cation exchange and chromatography of anion exchange, exist. The protein is charged
negative (anionic) at buffer pH values above this IP; at pH values below that, the protein is charged
positively (cationic). The vitamins are broadly classified as water-soluble and fat-soluble vitamins which are
further distinguished as vitamin A, B, C, E and K.The water-soluble and fat-soluble vitamins are separated
and purified using various techniques such as capillary electrophoresis, HPLC and
chromatography.Ion-exchange chromatography is being the most popular. For analysis of cyclic nucleotides
and connected compound exercises, ion-exchange chromatography is that the most typically used separation
procedure.

Page 14 of 17

Nada Kazem
 8- References

1. Haddad PR, Jackson PE. Ion chromatography. Elsevier; 1990 Jul 16.updated in october 2012
2. Okada T. Nonaqueous ion-exchange chromatography and electrophoresis: Approaches to nonaqueous
solution chemistry and design of novel separation. Journal of Chromatography A. 1998 Apr 24;804(1-
2):17-28. updated in 2010

3. Bruch T, Graalfs H, Jacob L, Frech C. Influence of surface modification on protein retention in ion-
exchange chromatography: Evaluation using different retention models. Journal of Chromatography A.
2009 Feb 6;1216(6):919-26.updated in 2015

4. Stanton P. HPLC of Peptides and Proteins. Methods in Molecular Biology. New Jer‐sey: Humana
Press. 2004.updated in 2011

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