History of HPLC

                                        
      
  History of HPLC
Prior to the 1970's, few reliable chromatographic methods were commercially available to the laboratory scientist. During the 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and did not achive sufficiently high resolution to distinguish between similar compounds. During this time, pressure liquid chromatography began to be used to decreaseflowthroughtime, thus reducing purification times of compounds being isolated by columnchromatogaphy. However, flow rates were inconsistent, and the question of whether it was better to have constant flow rate or constant pressure was debated. (AnalyticalChem.vol62, no. 19, Oct 1, 1990). High pressure liquid chromatography was developed in the mid-1970's and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. In the late 1970's, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds. By the 1980's HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far above those obtained using previous techniques. Computers and automation added to the convenience ofHPLC. Additional column types giving better reproducibility were introduced and such terms as micro-column, affinity columns, and Fast HPLC began to immerge. The past decade has seen a vast undertaking in the development of micro-columns, and other specialized columns. The dimensions of the typical HPLC column are: XXX mm in length with an internal diameter between 3-5 mm. The usual diameter of micro-columns, or capillary columns, ranges from 3 μm to 200 μm. Fast HPLC utilizes a column that is shorter than the typical column. A Fast HPLC column is about 3 mm long and is packed with smaller particles. Currently, one has the option of selecting from a lotof columns for the separation of compounds, as well as a variety of detectors to interface with the HPLC in order to obtain optimal analysis of the compound. Although HPLC is widely considered to be a technique mainly for biotechnological, biomedical, and biochemical research as well as forthe pharmaceutical industry,in actual fact these fields currently comprise only about 50% of HPLC users(AnalyticalChem.vol62, no.19, Oct 1, 1990). Currently HPLC is used in a variety of fields and industries including the cosmetics, energy, food, and environmental industries
WHAT IS HPLC?
H: High
P:Performance presure
L : Liquid
C : Chromatography
GC : Gas chromatography
TLC: Thin layer chromatography
IC:Ion chromatography
What is HPLC used for ?.
1. Separation of mixed components
 2. Qualitative analysis /
Quantitative analysis
 3. Preparation of interest components
 Separation analysis and/or preparationof interest components

Parameters used in HPLC
 Retention parametersColumn efficiency parametersPeak symmetry parametersCondition for SeparationRetention : When a component in a sample interacts with the stationary phase in the column and a delay in elution occurs.Column efficiency : Goodness of a column

INDUSTRIAL WASTEWATER CHARACTERISTICS

     

   INDUSTRIAL WASTEWATER CHARACTERISTICS
The purposes of pollution control endeavours should
be (1) to protect the assimilative capacity of surface
waters; (2) to protect shellfish, finfish and wildlife;
(3) to preserve or restore the aesthetic and recreational
value of surface waters; (4) to protect humans
from adverse water quality conditions.
The selection and design of treatment facilities is
based on a study of
• the physical, chemical and biological characteristics
of the wastewater
• the quality that must be maintained in the
environment to which the wastewater is to be
discharged or for the reuse of the wastewater
• the applicable environmental standards or discharge
requirements that must be met
The main chemical characteristics of wastewater are
divided into two classes, inorganic and organic. Because
of their special importance, priority pollutants
and volatile organic compounds (VOCs) are usually
considered separately.
Physical characteristics
The principal physical characteristics of wastewater
are its solids content, colour, odour and temperature.
The total solids in a wastewater consist of the
insoluble or suspended solids and the soluble compounds
dissolved in water. The suspended solids
content is found by drying and weighing the residue
removed by the filtering of the sample. When
this residue is ignited the volatile solids are burned
off. Volatile solids are presumed to be organic
matter, although some organic matter will not burn
and some inorganic salts break down at high temperatures.
The organic matter consists mainly of
proteins, carbohydrates and fats.
Between 40 and 65 % of the solids in an average
wastewater are suspended. Settleable solids, expressed
as millilitres per litre, are those that can be
removed by sedimentation. Usually about 60 % of
the suspended solids in a municipal wastewater are
settleable. Solids may be classified in another way
as well: those that are volatilised at a high temperature
(600 °C) and those that are not. The former are
known as volatile solids, the latter as fixed solids.
Usually, volatile solids are organic.
Colour is a qualitative characteristic that can be
used to assess the general condition of wastewater.
Wastewater that is light brown in colour is less than
6 h old, while a light-to- medium grey colour is
characteristic of wastewaters that have undergone
some degree of decomposition or that have been
in the collection system for some time. Lastly, if
the colour is dark grey or black, the wastewater is
typically septic, having undergone extensive bacterial
decomposition under anaerobic conditions. The
blackening of wastewater is often due to the formation
of various sulphides, particularly, ferrous sulphide.
This results when hydrogen sulphide produced
under anaerobic conditions combines with
divalent metal, such as iron, which may be present.
Colour is measured by comparison with standards.
The determination of odour has become increasingly
important, as the general public has become
more concerned with the proper operation
of wastewater treatment facilities. The odour of
fresh wastewater is usually not offensive, but a
variety of odorous compounds are released when
wastewater is decomposed biologically under
anaerobic conditions. The principal odorous compound
is hydrogen sulphide (the smell of rotten
eggs). Other compounds, such as indol, skatol,
cadaverin and mercaptan, formed under anaerobic
conditions or present in the effluents of pulp
and paper mills (hydrogen sulphide, mercaptan,
dimethylsulphide etc.), may also cause a rather
offensive odour. Odour is measured by successive
dilutions of the sample with odour-free water until
the odour is no longer detectable.
The temperature of wastewater is commonly
higher than that of the water supply because warm
municipal water has been added. The measurement
of temperature is important because most
wastewater treatment schemes include biological
processes that are temperature dependent. The
temperature of wastewater will vary from season
to season and also with geographic location. In
cold regions the temperature will vary from about 7
to 18 °C, while in warmer regions the temperatures
vary from 13 to 24 °C.
Chemical characteristics
Inorganic chemicals
The principal chemical tests include free ammonia,
organic nitrogen, nitrites, nitrates, organic phosphorus
and inorganic phosphorus. Nitrogen and phosphorus
are important because these two nutrients are
responsible for the growth of aquatic plants.
Other tests, such as chloride, sulphate, pH and
alkalinity, are performed to assess the suitability of
reusing treated wastewater and in controlling the
various treatment processes.
Trace elements, which include some heavy metals,
are not determined routinely, but trace elements
may be a factor in the biological treatment of
wastewater. All living organisms require varying
amounts of some trace elements, such as iron, copper,
zinc and cobalt, for proper growth. Heavy metals can
also produce toxic effects; therefore, determination of
the amounts of heavy metals is especially important
where the further use of treated effluent or sludge is
to be evaluated. Many of the metals are also classified
as priority pollutants (see below).
Measurements of gases, such as hydrogen sulphide,
oxygen, methane and carbon dioxide, are made
to help the system to operate. The presence of hydrogen
sulphide needs to be determined not only
because it is an odorous gas but also because it can
affect the maintenance of long sewers on flat slopes,
since it can cause corrosion. Measurements of dissolved
oxygen are made in order to monitor and control
aerobic biological treatment processes. Methane
and carbon dioxide measurements are used in connection
with the operation of anaerobic digesters
Volatile Organic Carbons (VOC)
Volatile organic compounds (VOC) such as benzene,
toluene, xylenes, dichloromethane, trichloroethane
and trichloroethylene, are common soil pollutants in
industrialised and commercialised areas. One of the
more common sources of these contaminants is leaking
underground storage tanks. Improperly discarded
solvents and landfills, built before the introduction
of current stringent regulations, are also significant
sources of soil VOCs.
In Table 18.1 a list of typical inorganic and organic
substances present in industrial effluents is presented

Gas Chromatography Columns

                             

Gas Chromatography Columns

Columns

Gas Chromatography.  columns are of two designs: packed or capillary. Packed columns are typically a glass or stainless steel coil (typically 1-5 m total length and 5 mm inner diameter) that is filled with the stationary phase, or a packing coated with the stationary phase. Capillary columns are a thin fused-silica (purified silicate glass) capillary (typically 10-100 m in length and 250 µm inner diameter) that has the stationary phase coated on the inner surface. Capillary columns provide much higher separation efficiency than packed columns but are more easily overloaded by too much sample

Stationary Phases

The most common stationary phases in gas-chromatography columns are polysiloxanes, which contain various substituent groups to change the polarity of the phase. The nonpolar end of the spectrum is polydimethyl siloxane, which can be made more polar by increasing the percentage of phenyl groups on the polymer. For very polar analytes, polyethylene glycol (a.k.a. carbowax) is commonly used as the stationary phase. After the polymer coats the column wall or packing material, it is often cross-linked to increase the thermal stability of the stationary phase and prevent it from gradually bleeding out of the column.

Small gaseous species can be separated by gas-solid chromatography. Gas-solid chromatography uses packed columns containing high-surface-area inorganic or polymer packing. The gaseous species are separated by their size, and retention due to adsorption on the packing material.

GC Columns

Stationary Phases

The most common stationary phases in gas-chromatography columns are polysiloxanes, which contain various substituent groups to change the polarity of the phase. The nonpolar end of the spectrum is polydimethyl siloxane, which can be made more polar by increasing the percentage of phenyl groups on the polymer. For very polar analytes, polyethylene glycol (a.k.a. carbowax) is commonly used as the stationary phase. After the polymer coats the column wall or packing material, it is often cross-linked to increase the thermal stability of the stationary phase and prevent it from gradually bleeding out of the column.

Small gaseous species can be separated by gas-solid chromatography. Gas-solid chromatography uses packed columns containing high-surface-area inorganic or polymer packing. The gaseous species are separated by their size, and retention due to adsorption on the packing material.

GC Columnsadsorbent, are mostly used for gas analysis. As a result of the simpler injection procedure and the more precise sampling method, the packed column tends to give greater quantitative accuracy and precision. However, despite its problems with sample injection, the open tubular column is seen as the 'state of the art' column and is by far the most popular column system in general use. The length of open tubular columns range from about 10 m to 100 m and can have internal diameters from 100 mm to 500 mm. The stationary phase is coated on the internal wall of the column as a film 0.2 mm to 1 mm thick.

The Packed GC Column

Packed columns are usually constructed from stainless steel or Pyrex glass. Pyrex glass is favored when thermally labile materials are being separated such as essential oils and flavor components. However, glass has pressure limitations and for long packed columns, stainless steel columns are used as they can easily tolerate the necessary elevated pressures. The sample must, of course, be amenable to contact with hot metal surfaces. Short columns can be straight, and installed vertically in the chromatograph. Longer columns can be U-shaped but columns more than a meter long are usually coiled. Such columns can be constructed of any practical length and relatively easily installed. Pyrex glass columns are formed to the desired shape by coiling at about 700˚C and metal columns by bending at room temperature. Glass columns are sometimes treated with an appropriate silanizing reagent to eliminate the surface hydroxyl groups which can be catalytically active or produce asymmetric peaks. Stainless steel columns are usually washed with dilute hydrochloric acid, then extensively with water followed by methanol, acetone, methylene dichloride and n-hexane. This washing procedure removes any corrosion products and traces of lubricating agents used in the tube drawing process. The columns are then ready for packing.

walls and then initiating polymerization either by heat or an appropriate catalyst. This locks the stationary phase to the column wall and is thus completely immobilized. Polymer coatings can be formed in the same way using dynamic coating. The techniques used for immobilizing the stationary phases are also highly proprietary and little is known of the methods used by companies that manufacture the columns. In any event, most chromatographers do not want the trouble of coating their own columns and prefer to purchase proprietary columns.

Very difficult separations can be achieved using the capillary column, and in a relatively short time. An example of the separation of a complex mixture on a capillary column is shown in figure 17. The column used was designated as a VOCOL column and was 60 m long, 0.75 mm I.D. and carried a film of stationary phase 1.5 micron thick. The column was held a 10^˚C for 6 minutes and then programmed to 170˚C at 6˚C per minute. The carrier gas was helium at a flow rate 10 ml/min. The detector employed was the FID. This chromatogram demonstrates the clear advantages of capillary columns over packed column. Not only does the column produce exceeding high efficiencies but they are also achieved with reasonable separation times.

Open Tubular Column Types

Open Tubular columns are broadly split into two classes, the wall coated open tubular columns or WCOT Columns (which have already been described and are by far the mot popular,) and the porous layer open tubes or PLOT Columns. The two types of column are shown diagramatically in figure 18. The PLOT columns are largely used for gas analysis and the separation of low molecular weight

Liquid Ammonia as a Solvent

                                

Liquid Ammonia as a Solvent

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Ammonia has a reasonable liquid range (-77 to –33 °C), and as such it can be readily liquefied with dry ice (solid CO₂, T_sub = -78.5 °C), and handled in a thermos flask. Ammonia’s high boiling point relative to its heavier congeners is indicative of the formation of strong hydrogen bonding, which also results in a high heat of vaporization (23.35 kJ/mol). As a consequence ammonia can be conveniently used as a liquid at room temperature despite its low boiling point.

Liquid ammonia is a good solvent for organic molecules (e.g., esters, amines, benzene, and alcohols). It is a better solvent for organic compounds than water, but a worse solvent for inorganic compounds. The solubility of inorganic salts is highly dependant on the identity of the counter ion 

Soluble in liquid NH3	Generally insoluble in liquid NH3
SCN-, I-, NH4+, NO3-, NO2-, ClO4-	F-, Cl-, Br-, CO32-, SO42-, O2-, OH-, S2-
Table 1: General solubility of inorganic salts in liquid ammonia as a function of the counter ion.

The difference in solubility of inorganic salts in ammonia as compared to water, as well as the lower temperature of liquid ammonia, can be used to good advantage in the isolation of unstable compounds. For example, the attempted synthesis of ammonium nitrate by the reaction of sodium nitrate and ammonium chloride in water results in the formation of nitrogen and water due to the decomposition of the nitrate, Equation 1. By contrast, if the reaction is carried out in liquid ammonia, the sodium chloride side product is insoluble and the ammonium nitrate may be isolated as a white solid after filtration and evaporation below its decomposition temperature of 0 °C, Equation 2. 

Ammonation

Ammonation is defined as a reaction in which ammonia is added to other molecules or ions by covalent bond formation utilizing the unshared pair of electrons on the nitrogen atom, or through ion-dipole electrostatic interactions. In simple terms the resulting ammine complex is formed when the ammonia is acting as a Lewis base to a Lewis acid, Equation 3 and Equation 4, or as a ligand to a cation, e.g., [Pt(NH₃)₄]²⁺, [Ni(NH₃)₆]²⁺, [Cr(NH₃)₆]³⁺, and [Co(NH₃)₆]³⁺.

(3)

(4)

Ammonolysis

Ammonolysis with ammonia is an analogous reaction to hydrolysis with water, i.e., a dissociation reaction of the ammonia molecule producing H⁺ and an NH₂^- species. Ammonolysis reactions occur with inorganic halides, Equation 5 and Equation 6, and organometallic compounds, Equation 7. In both case the NH₂^- moiety forms a substituent or ligand.

(5)

(6)

(7)

The reaction of esters, Equation 8, and aryl halides, Equation 9, are also examples of ammonolysis reactions.

(8)

(9)

Homoleptic amides

A homoleptic compound is a compound with all the ligands being identical, e.g., M(NH₂)_n. A general route to homoleptic amide compounds is accomplished by the reaction of a salt of the desired metal that is soluble in liquid ammonia (Table 1) with a soluble Group 1 amide. The 

solubility of the Group 1 amides is given in Table 2. Since all amides are insoluble (except those of the Group 1 metals) are insoluble in liquid ammonia, the resulting amide may be readily isolated, e.g., Equation 10 and Equation 11.

(10)

(11)

Amide	Solubility in liquid ammonia
LiNH2	Sparingly soluble
NaNH2	Sparingly soluble
KNH2	Soluble
RbNH2	Soluble
CsNH2	Soluble
Table 2: Solubility of Group amides in liquid ammonia.

Redox reactions

Ammonia is poor as an oxidant since it is relatively easily oxidized, e.g., Equation 12 and Equation 13. Thus, if it is necessary to perform an oxidation reaction ammonia is not a suitable solvent; however, it is a good solvent for reduction reactions.

(12)

(13)

Liquid ammonia will dissolve Group 1 (alkali) metals and other electropositive metals such as calcium, strontium, barium, magnesium, aluminum, europium, and ytterbium. At low concentrations (ca. 0.06 mol/L), deep blue solutions are formed: these contain metal cations and solvated electrons, Equation 14. The solvated electrons are stable in liquid ammonia and form a complex: [e^-(NH₃)₆].

(14)

The solvated electrons provide a suitable and powerful reducing agent for a range of reactions that are not ordinarily accomplished, e.g., Equation 15 and Equation 16