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Estimation of Neurotransmitters in the Brain

Estimation of Neurotransmitters in the Brain

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03/18/2014

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134
Chromatography refers to a group of techniquesused to separate complex mixtures on the basis ofdifferent physical interactions between theindividual compounds and the stationary phaseof the system. The basic components in anychromatography technique are the mobile phase(gas or liquid), which carries the complex mixture(sample); the stationary phase (solid or liquid),through which the mobile phase flows; the columnholding the stationary phase; and the separatedcomponents (eluate).
Modes of separationa. Adsorption chromatography (liquid-solidchromatography)
It is based on the competition between thesample and the mobile phase for adsorptive siteson the solid stationary phase. There is equilibriumof solute molecules being adsorbed to the solidsurface and desorbed and dissolved in the mobilephase. Those molecules, which are most soluble inthe mobile phase, move fastest, whereas those,which are least soluble, move slowest. Thestationary phase can be either acidic polar (eg, silicagel), basic polar (eg, alumina) or nonpolar (eg,charcoal). Commonly, the non-polar organiceluents for example hexane, dicholoromethane,and ethyl actetate are used. It can be used toseparate compounds, which are highly soluble inorganic solvents (eg., Fat-soluble vitamins).
b. Partition chromatography
Partition chromatography is also referred to asliquid-liquid chromatography. Separation of soluteis based on the relative solubility in an organic(nonpolar) solvent and an aqueous (polar) solvent.Modern partition chromatography uses pseudo-liquid stationary phases that are chemically bondedto the support or high-molecular-weight polymersthat are insoluble in the mobile phase. Partitionsystems are called
normal phase
 
when the mobilephase is less polar than the stationary solvent andare termed
reverse phase
when the mobile solventis more polar. When the elution strength of themobile phase is constant throughout theseparation, it is called
isocratic
elution and whenvaried is called as
gradient
elution.In liquid chromatography, the resolution isproportional to the column length (i.e., the numberof theoretical end plates/unit length). Increasingthe surface area leads to an increase in the numberof theoretical plates. An increase in the surface areacan be achieved by decreasing the particle size butcomes with the disadvantage of increasedresistance to flow, which impairs resolution becauseof backpressure. In the recent past, new stationaryphases with small particle size that can withstandhigh pressures and offer better resolution have beendeveloped. This has lead to the development of highperformance liquid chromatography (HPLC).
High Performance Liquid Chromatography(HPLC)
The term High Performance LiquidChromatography (HPLC) was coined to describethe separation of molecules under high pressurein a stainless steel column filled with a matrix andis used for the separation and determination oforganic and inorganic solutes (mol.wt.< 1000)(Figure 1).
Fundamentals of HPLC
1.Selectivity: It is the ability of a column toseparate two components depending on itsaffinity and retention.2.Capacity factor: It is the ability of a column toretain a particular compound.3.Resolution factor: It is the resolving power of acolumn to separate two structurally closelyrelated compounds.
ESTIMATION OF NEUROTRANSMITTERS IN THE BRAIN BYCHROMATOGRAPHIC METHODS
Deepti Nair, Ramkumar K, Srikumar BN, Raju TR and Shankaranarayana Rao BS
Reprinted from: 
 
Brain and Behavior
. Raju TR, Kutty BM, Sathyaprabha TN and Shanakranarayana Rao BS (eds.),National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:134-141.
 
135
Figure 1.
Basic components of the
 
High Performance Liquid Chromatography (Bender, 1972).
Reverse phase High Performance LiquidChromatography
Reverse phase High Performance LiquidChromatography (rpHPLC) is a form of partitionchromatography in which the chemically bondedphase is hydrophobic (nonpolar) and the startingmobile phase is more polar than the stationaryphase.The HPLC consists of these basic components:pump, column, sample injector, detector andrecorder.
Pump
: A pump forces the mobile phase throughthe column at a much greater velocity. The pumpcan be pneumatic syringe type, reciprocating orhydraulic amplifier. The most widely used pumpis the mechanical reciprocating pump, which isnow used as a multihead pump with two or morereciprocating pistons. During pumping, the pistonsoperate put of phase (180º for two heads, 120º forthree) to provide constant flow.
Column
: The stationary phase is packed intolong stainless steel columns. Usually, HPLC is runat ambient temperature, although columns can beplaced in an oven and heated to enhance the rateof partition. Fine, uniform column packing resultsin much less band broadening but requirespressure to force the mobile phase through. Thepacking can also can be pellicular (an inert corewith a porous layer), inert and small particles ormacroporous particles. The most common materialused for column packing is silica gel. It is very stableand can be used as solid packing in liquid - solidchromatography or coated with a solvent, whichserves as the stationary phase (liquid-liquid).Reversed phase HPLC is now very popular. Thestationary phase is made up of nonpolar molecules(eg, octadecyl C-18 hydrocarbon) bonded to silicagel particles. For this type of column packing, themobile phase commonly used is acetonitrile,methanol, water or any combination of solvents.Reverse phase columns are stable in the pH rangeof 2-7 and at elevated temperatures.Reversed phase column can be used to separateionic, non-ionic and ionizable samples. A buffer isused to produce the desired ionic characteristicsand pH for the separation of the analyte. Columnpackings vary in size (3-20mm), with the smallerparticles used mostly for analytical separations andthe larger ones for preparative separations.
Sample injector
: A small syringe can be usedto introduce the sample into the path of the mobilephase that carries it into the column. The best andmost widely used method is the loop injector. Thesample is introduced into a fixed-volume loop.
 
136
When the loop is switched, the sample is placedinto the path of the flowing mobile phase and isflushed into the column. Loop injectors have highreproducibility and are used at high pressures.
Detectors
: Modern HPLC detectors monitor theeluate as it leaves the column and ideally, producean electronic signal proportional to theconcentration of each separated component.Spectrophometers that detect absorbances of visibleor ultraviolet light are commonly used.Fluorescence detectors are also used, because manybiologic substances fluoresce strongly. Anothercommon HPLC detector is the amperometric orelectrochemical detector. These devices measurecurrent produced when the analyte of interest iseither oxidized or reduced at some fixed potentialset between a pair of electrodes.
Recorder
: It is used to record detector signal versusthe time taken for the mobile phase to pass throughthe instrument starting sample injection. The graph iscalled a
chromatogram.
The retention time is used toidentify compounds when compared with standardretention times run under identical conditions. Peakarea is proportional to the concentration of thecompounds that produced the peaks.
ESTIMATION OF AMINO ACIDS IN THEBRAIN
There are several methods that were employedin the past to estimate the levels of differentaminoacids in the brain. The commonly used twomethods are described below.
1. Estimation of glutamate and GABA levels bymultiple development paper chromatographyPrinciple
This method follows the principle of differentpartition coefficients that can be obtained from astationary cellulose phase with a mobile solventphase for different aminoacids, which aid in theirseparation. Extraction and quantification are doneby reacting the aminoacids with ninhydrin(triketohydrindene hydrate). Ninhydrin reacts withan aminoacid to produce CO
2
, NH
2
and its loweraldehyde and ultimately yields a chromophoreknown as Ruhemann’s purple with an absorbancemaximum around 515nm.
Preparation of reagents1.Solvent
: butanol: acetic acid: water (12:3:5):‚To 60 ml of butanol, 15 ml of acetic acid and 25ml distilled water are added.
2.0.25% Ninhydrin
: 200 mg of Ninhydrin isdissolved in 99 ml of acetone. To this solution1ml of pyridine is added.
3.0.005% CuSO4 solution
: 5 mg of cupricsulphate is dissolved in 10 ml 75% alcohol.
Standards
a.2
µ
M glutamate: 2.942 mg of glutamate isdissolved in 10 ml of distilled water.b.2
µ
M GABA: 2.062 mg of GABA is dissolved in10 ml of distilled water.
Assay procedure
For the estimation of amino acids, the originalmethod developed by Sadasivudu and Murthy(1978) is adapted with some modifications in ourlaboratory as described below (Nagaraja andDesiraju, 1993, Shailesh Kumar and Desiraju T,1990, 1992; Shankaranarayana Rao et al. 1998;Sunanda et al 2000).After the dissection of different brain regions,each region is homogenized in 80% double distilledethanol (for every 100mg of the brain tissue, 2mlof 80% alcohol is used). Homogenates aretransferred to polypropylene tubes and centrifugedat 1200rpm for 10 min. 1ml of the supernatant isthen transferred into small test tubes andevaporated to dryness at 70
o
C in an oven. Theresidue is reconstituted in 100 ml distilled waterand 10 ml is used for spotting on Whatman No.1Chromatography paper. Standard solutions ofglutamate and GABA at a concentration of 2 mMare also spotted using an Eppendorf micropipette;the spots are dried with a hair drier. Thechromatograms are then stitched at the sides andplaced in a chromatography chamber containingbutanol: acetic acid: water (65: 15: 25, V/V) assolvent. When the solvent front reached the top ofthe papers, the papers are removed and dried. Asecond run is performed similarly, after which thepapers are dried, sprayed with ninhydrin (0.25%

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