This talks about chromatography the principle use and procedures

1. Column Chromatography
Column chromatography with a support of Polyclar AT, an insoluble
form of the polymer, polyvinylpolypyrrolidone (PVPP), has been used
extensively for the purification of plant hormones [51,52].
From: New Comprehensive Biochemistry, 1999
Related terms:
Peptide, High-Performance Liquid Chromatography, Chromatography, Ethyl Acetate,
Acetone, Thin-Layer Chromatography, Enzymes, Proteins, Polysaccharides,
Phosphoproteins
Column Chromatography
Joe M. Regenstein, Carrie E. Regenstein, in Food Protein Chemistry, 1984
Publisher Summary
Column chromatography is used to separate proteins during preparation. It can also
be used to analyze or characterize different materials and/or reactions, and it can be
used to study the kinetics of an enzyme reaction. This chapter discusses various
techniques and types of columns for column chromatography. Partition
chromatography is used to obtain a continuous series of separatory funnels. Thin-
layer chromatography and paper chromatography use the same partition principle to
separate materials but on a continuous basis. Paper chromatography can be done in
either an ascending or descending mode. Ion exchange chromatography uses the
preferential binding of various positive ions to a particular negative ion bound to the
column (or vice versa) to yield the separation of material. Adsorption chromatography
utilizes the force of attraction of protein to the column support material. It may result
from hydrophobic interaction and/or van der Waals interaction. Molecular exclusion
chromatography, also called gel filtration or molecular sieve chromatography is often
referred to by the trade name of the most commonly used support material,
Sephadex. An adaptation of affinity chromatography is the use of immobilized
enzymes.
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Advances in extraction technologies: isolation and
purification of bioactive compounds from biological
materials
Nishi Srivastava, ... Ravindra N. Kharwar, in Natural Bioactive Compounds, 2021
21.4.2 Column chromatography
Column chromatography is simple and the most popular separation and purification
technique. Both solid and liquid samples can be separated and purified by column
chromatography. Column chromatography consists of a stationary solid phase that
adsorbs and separates the compounds passing through it with the help of a liquid
mobile phase. On the basis of their chemical nature, compounds get adsorbed and

2. elution is based on differential adsorption of a substance by the adsorbent. Various
stationary phases, such as silica, alumina, calcium phosphate, calcium carbonate,
starch, and magnesia, and different solvent compositions based on the nature of
compounds to be separated and isolated, are used in column chromatography.
Optimization of the method is an important task in the separation of different groups
of compounds in extracts. In column chromatography, a cylindrical glass tube, which
is plugged at the bottom by a piece of glass wool or porous disc, is filled with slurry
(adsorbent) and a suitable solvent. Samples to be separated are mixed with silica and
introduced at the top of the column and allowed to move with the solvent. With
polarity differences, compounds are adsorbed at different regions and desorbed with
suitable solvent polarity. The compound of higher adsorption ability will be adsorbed
at the top and that with the lower one will be at the bottom. By adding the solvent at
the top, compounds get desorbed and pass through the column and this process is
called elution. A schematic diagram of column chromatography is shown in Fig. 21.8.
Figure 21.8. Systematic diagram of column chromatography.
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POLYCHLORINATED BIPHENYLS
J. de Boer, in Encyclopedia of Analytical Science (Second Edition), 2005
Carbon column chromatography (CCC)
CCC exhibits excellent selectivity for those CBs for which a planar conformation is
assumed. Different forms of carbon are being used to isolate these congeners. The
sorbents used for CCC include activated carbon, activated carbon/polyurethane foam,
activated carbon/glass fiber, activated carbon/silica gel, activated carbon/celite,
activated carbon/Chromosorb W-HP, and porous graphitic carbon. Activated charcoal
is a popular choice, but it has some serious drawbacks such as low efficiency, severe
tailing of elution profiles, irreversible adsorption, and large batch-to-batch variations.
The use of high-performance liquid chromatography (HPLC) with a porous graphitic
carbon column may help to overcome these drawbacks.

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Common techniques and methods for screening of
natural products for developing of anticancer drugs
Monika Singh, ... Rajesh Kumar Singh, in Evolutionary Diversity as a Source for
Anticancer Molecules, 2021
15.6.2 Column chromatography
Column chromatography is the most effective technique used in the separation of
plant extracts into its components in pure form. This is a preparative
chromatographic method and the stationary phase with silica gel is packed in a
column and the mobile phase (called eluent) is passed through the column after
loading the sample (extract) on the top of the stationary phase (i.e., along with silica
gel). The mobile phase carries the compounds present in the mixture at a different
rate based on their affinities to the stationary and mobile phases. Finally, the
components of compounds can be collected along with the mobile phase. Column
chromatography is of two types: Normal column chromatography and flash column
chromatography.
Flash chromatography is also called a medium pressure column chromatography
(MPCC). Here solvent is with a high rate flow with high pressure over the stationary
phase. This is the best method for separating the components of plant material as
plant materials are the complex mixture of multiple unknown components.
There are two types of solvent systems are used in column chromatography: (1)
isocratic and (2) gradient. In an isocratic system, only one solvent is passed through
the stationary phase but in the gradient system. Two solvents with predefined ratios
have been passed through the stationary phase to separate the components. As plant
extracts contain a mixture of complex compounds and they differ in polarities. So
natural product chemists generally use gradient system for isolation of compounds.
A series of steps need to follow for the performance of column chromatography. The
important thing needs to pay attention here is the loading of the column. The loading
may be wet loading and dry loading. In Wet loading, the liquid sample directly placed
onto the top of the column using a pipet, and allow it to percolate through the top of
the sorbent bed. If the sample/plant extract is a solid material it needs to be dissolved
in a less polar solvent (If it is not soluble in low polar solvent better to use a small
portion of high polar solvent). The procedure for dry loading is; the plant extract is
dissolved in a small amount of suitable solvent (polar solvent) which can be easily
removed. A portion of silica gel is also added. The proportion used here is 1:1 or 1:3.
The resultant mixture is placed in a fume hood to evaporate the solvent. Then with
the spatula, the resultant mixture is loaded to the stationary phase. Generally, the
researcher is using the dry loading process for natural product extraction (Euerby and
Petersson, 2003).
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LIQUID CHROMATOGRAPHY | Overview
M.D. Palamareva, in Encyclopedia of Analytical Science (Second Edition), 2005
Classification by the Bed of the Stationary Phase
CC, three-dimensional bed. Conventional CC is performed at atmospheric pressure.
The stationary phase particles are large compared with HPLC: the particle diameter
(d ) is usually in the range 60–200 μm. Thus, atmospheric pressure is sufficient to
overcome the flow resistance of the packed column. This ensures a normal flow rate
(1–3 ml min ) of the mobile phase. HPLC runs at the higher pressure necessary to
overcome the resistance of the smaller particles (usually with d 5 μm) of the
p
−1
p

4. stationary phase. A normal flow rate of the mobile phase is obtained at a pressure of
10–20 MPa. The high efficiency in HPLC is due to the small and uniform size of
particles. Figures 2 and 3 illustrate the equipment necessary for performing
conventional CC and HPLC, respectively. In the first case, a unique instrument
(chromatograph) is not used. The analyte is applied to the top of a glass column
containing the stationary phase. The mobile phase, called the eluent, passes through
the column, and this leads to separation of the solute into its components. The
outcoming solvent (eluate) from the column is collected in separate fractions, and the
compositions are followed using another method. In the case of HPLC, a
chromatograph is used. It is composed of six parts: (1) a mobile-phase delivery
vessel(s), (2) a pump for producing a high pressure, (3) an injector for application of
the analyte, (4) a column containing the stationary phase, (5) a detector (usually UV)
giving signals for the composition of the mobile phase exiting from the column, and
(6) a data station and data processing unit: the record of the separation is called a
chromatogram. The latter is composed of peak(s): any peak corresponds to a specific
compound if a complete separation is achieved (Figure 4).
Figure 2. Schematic representation of conventional CC.

5. Figure 3. Schematic representation of the components of HPLC.
Figure 4. HPLC chromatogram of a compound.
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Detection of gluten
Herbert Wieser, in Gluten-Free Cereal Products and Beverages, 2008
Column chromatography
Column chromatography has been used for a long time to characterize, separate, and
quantitate cereal protein fractions. In particular, gel permeation (GP)
chromatography, separating according to different molecular weights, and reversed-
phase (RP) chromatography, separating according to different hydrophobicities, have
been widely used. Applications on the basis of HPLC (Kruger and Bietz, 1994) have
considerably reduced the time of analysis (often less than 30 minutes). The detection
and quantitation of proteins eluted from the column is carried out by UV absorbance
in the range of 200–220 nm. At these wavelengths, the absorbance units are highly
correlated with protein quantity (Wieser et al., 1998). The detection limit is around 1–
2 μg protein. A disadvantage is that the detection technique cannot differentiate
between gluten and non-gluten proteins and is, therefore, not applicable for the
analysis of complex foods. Nevertheless, column chromatography is a valuable aid, for

6. example, to determine composition and quantity of a reference protein (van Eckert et
al., 2006) or to judge the results of other methods (Wieser et al., 1994).
In special cases, however, column chromatography can be applied for gluten
determination. GP-HPLC on Superdex 200 HR was used to quantify both gliadins
and total gluten in a series of wheat starches by means of the following steps (Wieser
and Antes, 2002): extraction of 1 g with 10 mL of 60% ethanol (gliadins) or 50% 2-
propanol plus reducing agent (total gluten), centrifugation, drying of 4 mL
supernatant in a vacuum centrifuge, dissolving in 500 μL elution solvent, injection of
100–200 μL, UV absorbance at 210 or 205 nm. The analyzed 23 starch samples had
gliadin contents between 15 and 574 mg/kg (Wieser and Seilmeier, 2003). The
average coefficient of variation resulting from two determinations was ±2.6%.
According to the ratio of gliadins to glutenins, strong differences were found (0.2–
4.9), demonstrating that the calculation gliadin ×2 = gluten proposed by the Draft
Revised Codex Standard is not justified. In addition to wheat starch, other raw
materials used for the production of gluten-free food were tested. Gluten
determination was, in principle, possible for apple fiber, buckwheat groats, spice
mixture, chestnut, millet, and rice flour. Skim milk powder and maize flour, however,
contained components that prevent correct analysis by means of GP-HPLC. In
conclusion, column chromatography can serve as an alternative method for gluten
analysis in special cases and can help to control other methods.
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The Polygalacturonases and Lyases
J.K. Burns, in The Chemistry and Technology of Pectin, 1991
d. Molecular Weight and pI
Column chromatography followed by isoelectric focusing has revealed multiple forms
or isozymes of endo-PL, and this has been the case for enzyme isolated from Bacillus
polymyxa, Pseudomonas fluorescens, Erwinia carotovora, and E. chrysanthemi (Keen and
Tamaki, 1986; Lei et al., 1988; Liao, 1989; Nagel and Wilson, 1970; Thurn et al., 1987;
Willis et al., 1987). The isoelectric points of most of the isozymes range from 8.8 to
10.0 (Keen and Tamaki, 1986; Liao, 1989; Thurn et al., 1987). One form of pectate
lyase in E. chysanthemi and the enzyme from Aspergillus nidulans had an acidic pi of 4.2.
In Cytophaga johnsonae, a pi of 6.7, and in E. carotovora, a pi of 6.6 was reported (Dean
and Timberlake, 1989b; Liao, 1989; Thurn et al., 1987; Willis et al., 1987). Molecular
weights of the enzyme and associated isozymes are 40,000 to 47,500 (Dean and
Timberlake, 1989b; Keen and Tamaki, 1986; Lei et al., 1987, 1988; Thurn et al., 1987).
The molecular weight of C. johnsonae endo-PL was reported as 35,000 (Liao, 1989).
Slightly lower molecular weights have been deduced in processed protein arising
from DNA sequencing (Lei et al., 1987, 1988).
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PHOSPHOLIPIDS | Determination
B.F. Szuhaj, in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003
Quantitative Analysis of Phospholipids
Column Chromatography
Column chromatography precedes TLC in the separation of phospholipids. The
techniques are slow and require good skill with column preparation, flow rates, and
solvent removal. Commercial lecithins can be separated by dissolving the crude
mixture in petroleum ether and passing it through a deactivated silica gel column
with petroleum ether. The phospholipids are adsorbed and do not pass through the

7. column, whilst triglycerides and sterol esters are eluted. The phospholipids are
subsequently quantified by TLC and wet phosphorus analysis.
High-Performance Liquid Chromatography (HPLC)
Newer technologies have found that HPLC can separate and quantify phospholipids
more quickly and accurately. Separation is carried out on several types of columns,
including silica gel and an amino group bonded to the silica surface (μBondapak-
NH ). The columns are eluted with chloroform/methanol gradients,
acetonitrile/methanol/85% phosphoric acid, or acetonitrile/methanol/ water. The
eluent is measured at 205 nm or detected with flame ionization. Figure 3 shows an
HPLC separation of commercial lecithin, using ultraviolet detection. The mass
detector, an evaporative analyzer, has also been successfully used for the HPLC
determination of phospholipids. (See CHROMATOGRAPHY | High-performance
Liquid Chromatography.)
Figure 3. High-performance liquid chromatography of deoiled soya bean lecithin. PC,
phosphatidylcholine; Pl, phosphatidylinositol; PA, phosphatidic acid; PE,
phosphatidylethanolamine. Column, μ Porasil 10 μ 3.98 × 300 mm; mobile phase,
hexane/2-propanol/acetate (8:8:1, v/v/v), buffer pH 4.2; detection, ultraviolet (206 nm);
injection, 10 μl; flow rate, 1 ml min . Retenton time (RT) in minutes. Courtesy of P.
Balazs, Central Soya, Food Research, Fort Wayne, Indiana, USA.
Densitometry
Densitometric scanning has been used as an indirect method for determining
phospholipid content on TLC plates. While the method has some promise, a problem
is the quanitative charring of the phospholipid spots. Each phospholipid has a
different charring density and this depends on fatty acid composition. Only with
proper standards can this method be useful.
Thincography
Thin-rod TLC combines TLC with quantification by flame ionization detection. Rods
are used rather than plates but controversy still exists over the suitability of the
technique for routine lipid analyses.
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2
−1
Methods
S.W. Lewis, C.E. Lenehan, in Encyclopedia of Forensic Sciences (Second Edition), 2013

8. Recommended publications
Industrial Crops and Products
Journal
Fish & Shellfish Immunology
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•
Column (or Liquid–Solid) Chromatography
Column chromatography is commonly used in sample purification and cleanup,
particularly in synthetic organic laboratories. One of the simplest
chromatographic techniques to set up, column chromatography utilizes
commonly available laboratory materials to effect the separation. The column
typically comprises either a laboratory pipette or a burette that is filled with a solid
stationary phase (typically alumina, silica, or celite). The mixture to be separated is
dissolved in an appropriate solvent and added to the top of the column. Mobile
phase is continually added to the top of the column and liquid flow is continued
through the column. Compounds are separated on the basis of their differential
migration through the column. Compounds with a strong affinity for the
stationary phase take a longer time to be eluted. The eluted compounds can be
collected, recovered, and examined.
In the 1960s and 1970s, toxicologists routinely used column chromatography for the
isolation and purification of drugs and pesticides from biological fluids. More
recently, commercially prepared ‘solid-phase extraction’ (SPE) chromatography
columns have become the most commonly used column chromatography system for
sample cleanup in the forensic laboratory. Ready to use SPE columns of varying
stationary phase composition can be purchased from a variety of chromatographic
suppliers. The choice of stationary phase is dependent on the analyte of interest, with
SPE columns suitable for proteins, drugs, and pesticides being readily available.
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STARCH OLIGOSACCHARIDES: LINEAR,
BRANCHED, AND CYCLIC
KEIJI KAINUMA, in Starch: Chemistry and Technology (Second Edition), 1984
a Charcoal Column Chromatography
Since charcoal column chromatography was first introduced to carbohydrate research
by Whistler and Durso (43), the technique has been widely used for the preparation of
oligosaccharides. In the original method, stepwise elution by ethanol was employed,
but later gradient elution with n- and tert-butanol or n-propanol (44, 45) was found
to improve resolution. The basic technique of the charcoal column chromatography is
described elsewhere (46). Charcoal column chromatography is especially useful
because of the high loading capacity of the column. The amount of carbohydrate that
can be loaded on the column is roughly several percent of the weight of the packed
charcoal, but for sharp separations, about 0.5% is recommended.
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9. South African Journal of Botany
Journal
Biochimica et Biophysica Acta (BBA) - Bioenergetics
Journal
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