36respuestas
Métodos de separación Cromatografía.
Cromatografía:
La separación de determinados componentes de una mezcla la cual sea homogénea, Técnicaqueseusaparapermitirsepararaquelloscomponentesdeunamezcla,paraello sehacepasaratravésdeunabsorbente(queseadhiereaunasuperficie).
Se conoce y utiliza como metodología más simple es la que usa papel como medio absorbente,elpapeleselfiltroenestaCromatografía,yelsolventeelliquidoalcoholo agua.
Estos componentes se separan cuando estos componentes manifiestan sus diferentes afinidadesporelfiltrodepapelobieneldisolventequeacciona.
Podemos ver que la tinta de plumón parece como totalmente homogénea, sin embargo al estarformadapordistintoscomponentessepuedensepararconfacilidad,paraellosolo requerimosdejarcorrerenunmedioqueseaabsorbenteporaccióndeundisolvente. Nombremos algunos ejemplos que se pueden usar para este método, los productos que se usancomomediodeabsorciónpuedenser,arena,papel,tiza,filtro,etc.
Lomásutilizadoeselpapeldefiltro,siempreenloslaboratoriosdeestudio,seaseuna demostraciónconelpapeldefiltroytintadeplumóndeagua,larazónesqueparapoder separarestoscomponentesdelamezcla,senostornasencilloyaqueutilizamoscomo disolventeelagua.
Paralograrunbuenresultadosedebetenerencuenta,queparalograrlaseparaciónel disolventenopuedenidebeestarencontactoconlamezcla,estedebellegaraellapor mediodelaabsorción.
Separación de Mezclas Cromatografía y Centrifugación Metodos de separacion de Mezcla “Inicio”
Estaentradasepublicóenmezclas,Química,Recomendados,Separación de mezclasy estáetiquetadaconabsorbente,agua,arena,Cromatografía,disolvente,filtro,homogenea, mescla,metodo,MetodosdeseparaciónCromatografía,mezcla,papel,separación,
superficie,tizaenseptiembre10,
@ig*2performance li!uid
c*romatograp*y
"rom OiKipedia, t$e free encyclopedia
>ig$Bperformance liquid c$romatograp$y
n >P#C. "rom left to rig$t pumping device generating a gradient of tLo different solventsB a steel
enforced column and a detector for measuring t$e absorbance.
Acronym >P#C
Analytes organic molecules biomolecules ions polymers +t*er tec*ni!ues "elated C$romatograp$y queous normalBp$ase c$romatograp$y >ydrop$ilic Snteraction C$romatograp$y
Son e*c$ange c$romatograp$y Ai&e e*clusion c$romatograp$y 7icellar liquid c$romatograp$y
@yp*enated #iquid c$romatograp$yBmassspectrometry
modern selfBcontained >P#C.
Ac$ematic representation of an >P#C unit. (+) Aolvent reservoirs, (-) Aolvent degasser, () @radient valve, (0) 7i*ing vessel for delivery of t$e mobile p$ase, (1) >ig$B
pressure pump, (N) ALitc$ing valve in ^inject position^, (NQ) ALitc$ing valve in ^load position^, (G) Aample injection loop, (J) PreBcolumn (guard column), (M) nalytical
column, (+6) /etector (i.e. S2, 8D), (++) /ata acquisition, (+-) Oaste or fraction collector.
@ig*2performance li!uid c*romatograp*y (@1LC' formerly referred to as *ig*2 pressure li!uid c*romatograp*y), is a tec$nique in analytic c$emistry used to separate t$e components in a mi*ture, to identify eac$ component, and to quantify eac$
component. St relies on pumps to pass a pressuri&ed liquid solvent containing t$e sample mi*ture t$roug$ a column filled Lit$ a solid adsorbent material. %ac$ component in t$e sample interacts slig$tly differently Lit$ t$e adsorbent material, causing different floL rates for t$e different components and leading to t$e separation of t$e components as t$ey floL out t$e column.
>P#C $as been used for medical (e.g. detecting vitamin / levels in blood serum), legal (e.g. detecting performance en$ancement drugs in urine), researc$ (e.g. separating t$e components of a comple* biological sample, or of similar synt$etic c$emicals from eac$ ot$er), and manufacturing (e.g. during t$e production process of p$armaceutical and biological products) purposes._+`
C$romatograp$y can be described as a mass transfer process involving adsorption. >P#C relies on pumps to pass a pressuri&ed liquid and a sample mi*ture t$roug$ a column filled Lit$ a sorbent, leading to t$e separation of t$e sample components. !$e active component of t$e column, t$e sorbent, is typically a granular material made of solid particles (e.g. silica, polymers, etc.), -V16 micrometers in si&e. !$e components of t$e sample mi*ture are separated from eac$ ot$er due to t$eir different degrees of
interaction Lit$ t$e sorbent particles. !$e pressuri&ed liquid is typically a mi*ture of solvents (e.g. Later, acetonitrile andFor met$anol) and is referred to as a ^mobile p$ase^. Sts composition and temperature play a major role in t$e separation process by
influencing t$e interactions taKing place betLeen sample components and sorbent. !$ese interactions are p$ysical in nature, suc$ as $ydrop$obic (dispersive), dipoleV dipole and ionic, most often a combination t$ereof.
>P#C is distinguis$ed from traditional (^loL pressure^) liquid c$romatograp$y because operational pressures are significantly $ig$er (16V16 bar), L$ile ordinary liquid
c$romatograp$y typically relies on t$e force of gravity to pass t$e mobile p$ase t$roug$ t$e column. /ue to t$e small sample amount separated in analytical >P#C, typical column dimensions are -.+V0.N mm diameter, and 6V-16 mm lengt$. lso >P#C columns are made Lit$ smaller sorbent particles (-V16 micrometer in average particle si&e). !$is gives >P#C superior resolving poLer L$en separating mi*tures, L$ic$ is L$y it is a popular c$romatograp$ic tec$nique.
!$e sc$ematic of an >P#C instrument typically includes a sampler, pumps, and a detector. !$e sampler brings t$e sample mi*ture into t$e mobile p$ase stream L$ic$ carries it into t$e column. !$e pumps deliver t$e desired floL and composition of t$e mobile p$ase t$roug$ t$e column. !$e detector generates a signal proportional to t$e amount of sample component emerging from t$e column, $ence alloLing for
quantitative analysis of t$e sample components. digital microprocessor and user softLare control t$e >P#C instrument and provide data analysis. Aome models of mec$anical pumps in a >P#C instrument can mi* multiple solvents toget$er in ratios c$anging in time, generating a composition gradient in t$e mobile p$ase. Darious detectors are in common use, suc$ as 8DFDis, p$otodiode array (P/) or based on mass spectrometry. 7ost >P#C instruments also $ave a column oven t$at alloLs for adjusting t$e temperature t$e separation is performed at.
Contents
• - !ypes o -.+ Partition c$romatograp$y o -.- =ormalVp$ase c$romatograp$y o -. /isplacement c$romatograp$y o -.0 2eversedBp$ase c$romatograp$y (2PC) o -.1 Ai&eBe*clusion c$romatograp$y o -.N SonBe*c$ange c$romatograp$y o -.G ;ioaffinity c$romatograp$y
o -.J queous normalBp$ase c$romatograp$y
• Ssocratic and gradient elution
• 0 Parameters o 0.+ !$eoretical o 0.- Snternal diameter o 0. Particle si&e o 0.0 Pore si&e o 0.1 Pump pressure • 1 Aee also • N 2eferences • G "urt$er reading • J %*ternal linKs
+peration
!$e sample mi*ture to be separated and analy&ed is introduced, in a discrete small volume (typically microliters), into t$e stream of mobile p$ase percolating t$roug$ t$e column. !$e components of t$e sample move t$roug$ t$e column at different velocities, L$ic$ are function of specific p$ysical interactions Lit$ t$e sorbent (also called
stationary p$ase). !$e velocity of eac$ component depends on its c$emical nature, on t$e nature of t$e stationary p$ase (column) and on t$e composition of t$e mobile p$ase.
!$e time at L$ic$ a specific analyte elutes (emerges from t$e column) is called its
retention time. !$e retention time measured under particular conditions is considered an identifying c$aracteristic of a given analyte.
7any different types of columns are available, filled Lit$ sorbents varying in particle si&e, and in t$e nature of t$eir surface (^surface c$emistry^). !$e use of smaller particle si&e pacKing materials requires t$e use of $ig$er operational pressure (^bacKpressure^) and typically improves c$romatograp$ic resolution (i.e. t$e degree of separation
betLeen consecutive analytes emerging from t$e column). Sn terms of surface c$emistry, sorbent particles may be $ydrop$obic or polar in nature.
Common mobile p$ases used include any miscible combination of Later Lit$ various organic solvents (t$e most common are acetonitrile and met$anol). Aome >P#C
tec$niques use LaterBfree mobile p$ases (see =ormalBp$ase c$romatograp$y beloL). !$e aqueous component of t$e mobile p$ase may contain acids (suc$ as formic, p$osp$oric or trifluoroacetic acid) or salts to assist in t$e separation of t$e sample
components. !$e composition of t$e mobile p$ase may be Kept constant (^isocratic
elution mode^) or varied (^gradient elution mode^) during t$e c$romatograp$ic analysis. Ssocratic elution is typically effective in t$e separation of sample components t$at are not very different in t$eir affinity for t$e stationary p$ase. Sn gradient elution t$e composition of t$e mobile p$ase is varied typically from loL to $ig$ eluting strengt$. !$e eluting strengt$ of t$e mobile p$ase is reflected by analyte retention times Lit$ $ig$ eluting strengt$ producing fast elution (Es$ort retention times). typical gradient profile in reversed p$ase c$romatograp$y mig$t start at 14 acetonitrile (in Later or
aqueous buffer) and progress linearly to M14 acetonitrile over 1V-1 minutes. Periods of constant mobile p$ase composition may be part of any gradient profile. "or e*ample, t$e mobile p$ase composition may be Kept constant at 14 acetonitrile for +V min, folloLed by a linear c$ange up to M14 acetonitrile.
!$e c$osen composition of t$e mobile p$ase (also called eluent) depends on t$e
intensity of interactions betLeen various sample components (^analytes^) and stationary p$ase (e.g. $ydrop$obic interactions in reversedBp$ase >P#C). /epending on t$eir
affinity for t$e stationary and mobile p$ases analytes partition betLeen t$e tLo during t$e separation process taKing place in t$e column. !$is partitioning process is similar to t$at L$ic$ occurs during a liquidVliquid e*traction but is continuous, not stepBLise. Sn t$is e*ample, using a LaterFacetonitrile gradient, more $ydrop$obic components Lill elute (come off t$e column) late, once t$e mobile p$ase gets more concentrated in acetonitrile (i.e. in a mobile p$ase of $ig$er eluting strengt$).
!$e c$oice of mobile p$ase components, additives (suc$ as salts or acids) and gradient conditions depends on t$e nature of t$e column and sample components. ?ften a series of trial runs is performed Lit$ t$e sample in order to find t$e >P#C met$od L$ic$ gives adequate separation.