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Chromatography: Assignment Analytical Lab Automation
Chromatography: Assignment Analytical Lab Automation
Chromatography: Assignment Analytical Lab Automation
CHROMATOGRAPHY
Arranged by:
KSA,2020
HISTORY1
Chromatography may have had beginnings in 1855 when Runge published his work on
dye separation on paper. Perhaps the real start of chromatographic analysis should be credited to
Coppelsroeder, who presented some of his work in 1861 and Schonbein, who described
separation of substances adsorbed onto filter paper. Fischer and Schmidner were aware of these
works according to their reports which came out in 1892. However, the concept of separations on
columns might be attributed to the work of Reed, who described some his early experiments
using columns in 1893. Day, in 1897, demonstrated the use of columns for separation of
petroleum fractions. A short time later, Engler and Albrecht followed with descriptions of
fractionation by coltecting eluants from a column. There have been other workers during this
time who were practicing “chromatography” before Tswet published his work in the separation
of fragments. The following review presents further development of chromatography which had
a renaissance in the 1930’s. The “rediscovery” of chromatography as it is now known dates from
the reports of separations on columns by Kahn and Lederer. Lederer separated carotenoids from
egg yolk following perusal of Tswett‘s descriptions of chromatography.
1861 1892
1855
Coppelsroeder Fischer and
Runge published dye started Schmidner described
separation paper chromatographic separation of
analysis substance
1930 1897
Tswet published his 1893
Engler and Albrecht
work in the collecting eluants Reed used columns
separation of from a column
fragments
DEFINITION
Separation
Technique A Mixture Components
Important Term
1. Polarity
Shows the separation of positive and negative charge poles of a molecule as a
result of the formation of certain configurations of the constituent atoms
These molecules can be attracted by other molecules that have polarity
The degree of separation of these molecules determines their polarity and
attractiveness
a. Solvents
b. Adsorbents
c. Solutes
2. Partition
The partitioning process depends on the solubility of the solute in two types of
liquid
Sensitive to differences in BM solut
Substances consisting of a series of homologous series are best separated by
partition chromatography.
For example: the separation of various types of amino acids, fatty acids, sugar
TYPES OF CHROMATOGRAPHY2
Chromatography
This division can then be further divided as seen in the following scheme:
1. Gas Chromatography
a. GLC : Gas Liquid Chromatography
b. GSC : Gas Solid Chromatography
2. Liquid Chromatography
a. HPLC : High Performance Liguid Chromatography
b. LLC-PC : Liquid Liquid Chromatography-Paper Chromatography
c. LSC-TLC : Liquid Solid Chromatography-Thin Layer Chromatography
d. Ion Excange
e. Exclusion
GP : Gel Permeation
GF : Gel Filtration
Paper Chromatography
Paper chromatography is chromatography that uses pure cellulose paper which has a
great affinity for water or other polar solvents. Paper chromatography is used to separate the
mixture from its substance into its components.
1. The sample is dissolved in a small amount of solvent, poured through the top of the
column and allowed to flow into the adsorbent (absorbent material).
2. Components in the sample are adsorbed quantitatively by the absorbent material in the
form of a narrow band on the top surface of the column.
3. With the addition of solvents continuously, each component will move down through the
column and a ribbon will be formed which each zone contains one kind of component.
4. Each zone that exits the column can be accommodated perfectly before the other zones
exit the column.
Thin-Layer Chromatography3
Thin-layer chromatography (TLC), first described in 1938, has largely replaced paper
chromatography because it is faster, more sensitive, and more reproducible. The resolution in
TLC is greater than in paper chromatography because the particles on the plate are smaller and
more regular than paper fibers. Experimental conditions can be easily varied to achieve
separation and can be scaled up for use in column chromatography, although thin-layer and
column procedures are not necessarily interchangeable, due to differences such as the use of
binders with TLC plates, vapor-phase equilibria in a TLC tank, etc. There are several distinct
advantages to TLC: high sample throughput, low cost, the possibility to analyze several samples
and standards simultaneously, minimal sample preparation, and that a plate may be stored for
later identification and quantification. TLC is applied in many fields, including environmental,
clinical, forensic, pharmaceutical, food, flavors, and cosmetics. Within the food industry, TLC
may be used for quality control. For example, corn and peanuts are tested for
aflatoxins/mycotoxins prior to their processing into corn meal and peanut butter, respectively.
Applications of TLC to the analysis of a variety of compounds, including lipids, carbohydrates,
vitamins, amino acids, and natural pigments, are discussed in reference.
Gas chromatography (GC), including analytical tools. Analysis can be divided into:
Gas chromatography is aligned as a method of analysis that can be used to analyze organic
compounds. Based on the stationary phase, there are two types of gas chromatography known
that is :
In both cases the mobile phase is gas (so they are called gas chromatography) but the
stationary phase is different. Nevertheless both methods have many similarities in how they
work. In GSC we have absorption (absorption) and in GLC we have partition (solution).
GSC operation requires a higher level of expertise than GLC, so it is relatively rarely used.
the cause is GSC based on solid stationary phase, so that the analyte mooring force is based on
physical adsorption. This mooring force is semi-permanent to polar or active molecules so that
the adsorption process is non-linear, consequently the elution pic becomes very tailed. GLC is
relatively easy to operate so it is widely used in all fields of science, and the name GLC is often
abbreviated as Gas Chromatography (GC).
1.1 Gas Solid Cromatography, GSC
Conditions 1 and 2 are very difficult to standardize. These are the things that cause
the low "Reproducibility" of the GSC. Frequently encountered are:
a. The caudate peaks are due to the inhomogeneous active surface of the absorber.
b. Retention time is relatively long.
c. Retention time is very dependent on the number of footage.
d. It is possible that the absorbent can act as an active catalyst
That is why the use of the GSC is very limited, both for compounds that have low
or high boiling points. However there are advantages to GSC when compared to
GLC. The stationary phase cannot be evaporated, because the vapor pressure from the
solid is very low. Until it can use sensitive detectors, because here does not happen
"column bleeding". In recent years the GSC has become more important, after
finding better absorbents. Examples of absorbents are:
1. Highly polar compounds such as H20, NH3, R-NH2, R-OH and glycols,
and low fatty acids.
2. Also for like CO2, N2O, O2 as well as others.
The oldest gas chromatography used was the GSC. In 1800 the gases were
purified by absorbing the stationary phase in the form of: alumina (AI203), or
silica gel (Si O, purified sand). Sometimes these absorbents become inactive
when exposed to water.
Sensitivity:
GLC is very sensitive. The simplest tool can detect concentrations in 0.01% size
(= 100 ppm). More complicated GLC tools can detect compounds whose
concentrations are only a few specific tools that can now be made with the ability to
detect "parts per billion", up to a picogram range of 10 g. Due to the high sensitivity
of the GLC it only requires a small amount of footage, usually in microliter size.
Liquid Liquid Chromatography (LLC)
LLC is division chromatography in which the partition occurs between the mobile phase
and the stationary phase of both the liquid. In this case the stationary phase must not dissolve in
the mobile phase.
Generally as a stationary phase water is used and as a mobile phase is an organic solvent.
For example in paper chromatography, as the stationary phase is water that is absorbed in the
cellulose fibers from the paper.
In terms of equipment the HPLC system includes column chromatography because the
stationary phase is loaded or packed in the column. And when viewed from the separation
process, HPLC is classified in the adsorption or partition chromatography. HPLC is a very useful
tool in analysis. This section explains how the implementation and use and principles of HPLC
are the same as thin layer chromatography and column chromatography.
HPLC is also the most widely used chromatography technique, with advantages:
4. Size-exclusion chromatography or gel chromatography (for solutes with Mr > 1000) which
can be separated again into gel filtration and gel permiation.
Ion-exchange chromatography
This technique uses zeolitas, organic or inorganic resins as ion exchangers. Compounds
which have ions with different affinity for the resin used can be separated. Analysis of amino
acids is common in this way. Other examples are nucleic acids and analysis of inorganic salts.
Exclusion chromatography
In this technique, many porous nonionic gels of the same size are used to separate
mixtures based on differences in molecular size. Small molecules will enter the pores of the gel
while large molecules will pass through the sidelines of the gel more quickly when compared to
molecules that pass through the pores. So the initial elution sequence is the larger molecule, the
medium molecule, and finally the smallest molecule. If as a filter hydrophilic gel (Sephadex) is
used, this technique is called gel filtration chromatography and when using a hydrophobic gel
(polystyrene-divinylbenzene) is called gel permeation chromatography.