Chromatography

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CHROMATOGRAPHY  DEFINITION OF CHROMATOGRAPHY  CHROMATOGRAPHY  Science on the study of separation of molecules based on dierences in their structure structure and/ or composition. It originated from the Greek words “chroma” meaning “chroma” meaning “color”, “color”, and “graphein” meaning “to rite”. rite”. It was coined by the Russian botanist “Mi!hail "em#ono$ich T%$et &T%ett'” during his research about chlorophyll where he separated the dierent plant pigments. Used for separation, identication, identication, and determination determination of the chemical components in comple! mi!ture. It in"ol"es passing a mi!ture dissol"ed in a #mobile phase$ through a #stationary phase$ which separates the analyte to be measured from other molecules in the mi!ture and allows being isolated. %US&' (ened as a procedure by which solutes are separated by a dierential migration process in a system consisting of two phases, one of which mo"es continuously in a gi"en direction and in which the indi"idual substances e!hibit dierent mobility by reason of dierences in adsorption, partition, solubility, "apour pressure, molecular si)e or ionic charge density. It may be preparati$e or anal#tical  Preparati$e Chromatograph#  It seeks to separate the components of a mi!ture for further use *and thus is a form of purication+.  Anal#tical Chromatograph#  It normally operates with smaller amounts of materials and seeks to measure the relati"e proportions of  analytes in mi!tures. •













PRINCIP(E" OF CHROMATOGRAPHY  CHROMATOGRAPHY  It in"ol"es mo"ing a preparation preparation of a materials to be separated %test preparation' o"er a stationary support.  he molecules in the test preparations preparations will ha"e dierent reactions with the stationary support.  his will lead to separation separation of #similar$ #similar$ molecules. (ierent types of molecules can be separated from each other, as they mo"e o"er the support material. •







) PHA"E" IN CHROMATOGRAPHY* "tationar# Pha%e  -i!ed phase %may be porous or nely di"ided solid or a liuid that

has been coated in a thin layer on an inert supporting material. Mo+ile Pha%e &ure liuid or gas or mi!ture of solutions that mo"es through or o"er the !ed phase.

TERMINO(OGIE" Anal#te  he substance to be separated separated during chromatography chromatography Chromatogram  he "isual output/ output/ result result in chromatography E,-ent It is the mobile phase lea"ing the column Mo+ile Pha%e It s the phase which mo"es in a denite direction It may be a liuid, a gas, or a supercritical uid Retention Time Is a characteristic time it takes for a particular analyte to pass through the system *from the column inlet to detector+ under set conditions A.%orption Stationary phase is solid 0obile phase is liuid or gas Retention and separation depends on the ability of the atoms on the surface to remo"e analytes from the mobile phase and adsorb them temporarily by means of electrostatic forces Molec-lar E/cl-%ion 1lso known as “%i0e e/cl-%ion”, e/cl-%ion” , “gel permeation”, permeation”, or “gel 1ltration” Stationary phase is a polymeric substance containing numerous pores of molecular dimensions 0obile phase is a liuid or gas Retention and separation depends on the dierential dierential migration of solute molecules based on molecular si)e Partition Stationary phase is liuid 0obile phase is liuid or gas Retention and separation occur due to the relati"e solubility of the analytes in the two uids as determined by their partition coe2cients PRINCIP(E" OF "EPARATION Ion2E/change

"tationar# pha%e is pha%e  is a polymeric matri! bonded with ionic functional groups *usually %t#rene.i$in#l+en0ene pol#mer+ pol#mer + Mo+ile pha%e is pha%e is always a liuid Retention and separation is mainly due to the electrostatic electrostatic bonds with the functional groups PRINCIPA( PRINCIPA( O34ECTI5E" OF CHROMATOGRAPHY  Resolution of mi!tures into constituent parts (etermination (etermination of homogeneity 3omparison of substances suspected of being identical &urication 3oncentration of substances from dilute solutions Identication and control of technical products 4uantitati"e separation from comple! mi!tures Indication of molecular structure DIFFERENT TECHNI67E" OF CHROMATOGRAPHY  PRINCIP(E" OF "EPARATION  echniues  echniues by 3hromatographic 3hromatographic 5ed Shape 3olumn 3hromatography 3hromatography &lanar 3hromatography &aper 3hromatography  hin 6ayer 3hromatogra 3hromatography phy  echniues  echniues by &hysical State State of 0obile &hase Gas 3hromatography 6iuid chromatography chromatography 7&63 "7PPORT" IN CHROMATOGRAPHIC CHROMATOGRAPHIC PREPARATION" Immo+ili0e. "ilica on Gla%% Plate% 5olatile Ga%e% Paper (i:-i.%; Containing H#.rophilic; In%ol-+le Molec-le%

Thin (a#er Chromatograph# 8T(C9 Ga% Chromatograph# 8GC9 Paper Chromatograph# (i:-i. Chromatograph#

THEORY OF CHROMATOGRAPHY  8!ploits the dierences in partitioning +eha$ior between9  0obile &hase  Stationary &hase •

PHA"E" OF CHROMATOGRAPHY  CHROMATOGRAPHY  Mo+ile Pha%e 1 gas or liuid that passes through the column "tationar# Pha%e 1 solid or liuid that does not mo"e. It refers to the chromatographic chromatographic support. C(A""IFICATION C(A""IFICATION OF CHROMATOGRAPHIC CHROMATOGRAPHIC METHOD"  1dsorption   63 7&63   3olumn 3hromatography 3hromatography  Ion:8!change  7&63  12nity  (;1 12nity 3hromatography  8lectrophoresis  Si)e 8!clusion Gel &ermeation   &artition  7&63  &aper 3hromatography 3A"I" OF INTERACTION" OF AN ANA(YTE ANA(YTE TO THE "TATIONARY PHA"E 3harge Relati"e Solubility Surface 1dsorption THEORIE" OF CHROMATOGRAPHY  CHROMATOGRAPHY  Plate Theor#  he chromatographic chromatographic system system is a series of discrete layers of theoretical plates. %0artin and Synge' Rate Theor#  he dynamics of the solute particle passes through the "oid spaces between the stationary phase particles in a system as well as its kinetics. %Giddings' HO< RATE THEORY AND P(ATE THEORY 9. 8R>9. Retention >actor is the run length of the compound di"ided by the run length of the eluent front. FORM7(A OF RETENTION

 Distancemoved  Distance moved by the compound compound  Rf =  Distancemoved  Distance moved by the solv ent   D 1  Rf =  D 2 "IGNIFICANCE ? IMPORTANCE IMPORTANCE OF RETENTION 1 uantitati"e indication of how far a particular compound tra"els in a particular sol"ent. 1 good indicator of whether an unknown < a known compound are identical or similar. APP(ICATION" APP(ICATION" OF CHROMATOGRAPHY  CHROMATOGRAPHY  (i:-i. Chromatograph#   est  est water samples for pollution Ga% Chromatograph#  5omb detection in airports  Identify/ 4uantify (rugs < 1lcohol  In -orensics, to compare bers found on a "ictim Thin (a#er Chromatograph# (etection of &esticides &esticides < Insecticides  in food  In -orensics, to analy)e dye composition of bers Paper Chromatograph# Separation of 1mino 1cids and 1nions   R;1 -ingerprinting  Separation and testing of histamines < antibiotics •







AD"ORPTION CHROMATOGRAPHY  CHROMATOGRAPHY  "tationar# Pha%e Solid on which sample components are adsorbed Mo+ile Pha%e 0ay be a liuid %liuid:solid' or a gas %gas:solid'

e.g. Col-mn Chromatograph# 8CC9 < Thin (a#er Chromatograph# 8T(C9 PARTITION CHROMATOGRAPHY  "tationar# Pha%e 6iuid supported on inert solid Mo+ile Pha%e Is a liuid %liuid:liuid %liuid:liuid partition' or a gas %gas:liuid' e.g. Paper Chromatograph#  ype  ype of partition partition chromatography chromatography in which the stationary phase is a layer of  water adsorbed on a sheet of paper (i:-i.2(i:-i. Partition Normal2Pha%e Chromatograph# &olar Stationary &hase %e.g. t Gel% De/tran 8"epha.e/9; Agaro%e 8"epharo%e9; or Pol#acrilami.e 83io2Gel9 B G86 -IR1 -I R1I=; I=; "emi2Rigi. or Rigi. Gel%

Pol#%t#rene; Gla%% 3ea.%; or Al!#late. De/tran > G86 &8R081I=; Mo+ile Pha%e 6iuid or Gas Used for the separation of solutes with dierent molecular si)e Used e!tensi"ely for the separation of macromolecules macromolecules or biological origin and for purication of synthetic:organic synthetic:organic polymers AFFINITY CHROMATOGRAPHY  CHROMATOGRAPHY  Utili)es high specic interactions between one kind of solute molecule and a second molecule co"alently attached %immobili)ed' to the stationary phase Immobili)ed molecule can be an antibody to a particula p articularr protein TECHNI67E" IN I N PARTITION PARTITION CHROMATOGRAPHY  COMMON TYPE" OF PARTITION PARTITION CHROMATOGRAPHY  &aper 3hromatography  hin 6ayer 3hromatogra 3hromatography phy Gas:6iuid 3hromatography Gel 3hromatography "PECIA( TYPE" OF PARTITION PARTITION CHROMATOGRAPHY  6iuid 3hromatography %63' 7igh &erformance 6iuid 3hromatography %7&63' Si)e 8!clusion 3hromatography %S83' 3olumn 3hromatography %33' TECHNI67E" IN I N PARTITION PARTITION CHROMATOGRAPHY  =peration of a column %a tube lled with adsorbent and sol"ent' 1 solution containing the solute is layered on top of the sorbent and is allowed to enter the sorbent  he sol"ent is is allowed to to pass continually continually through the column PARTITION COEFFICIENT 8=9 If two phases are in contact with one another, and if one or both phases contain a solute, the solute will distribute itself between two phases It is the ratio of the concentrations of the solute in the two phases Concentrationof solute ∈the

 K =

stationary phase Concentrationof solute ∈the mobile phase

MATERIA(" 7"ED IN PARTITION CHROMATOGRAPHY  3olumns containing a matri! that adsorb solutes (iatomaceous 8arth %3elite' Silica Gel 3ellulose &owder 3ross:6inked (e!trans %Sephade! 67 [email protected]' Stationary phase created by suspending the support or washing the column with a proper sorbent 7ydrophobic Sol"ent %5en)ene' 7ydrophilic 7ydrophilic Sol"ent %1lcohol' 0obile &hase 1lcohols < 1mides > for non:polar materials &uried Aater > for polar materials P(ANAR CHROMATOGRAPHY  CHROMATOGRAPHY  1 separation techniue in which the stationary phase is present as or on a plane  he plane can can be a paper, paper, ser"ing ser"ing as such or impregnated by a substance as the stationary bed or a layer of solid particles spread on support such as a glass plate •



TECHNI67E" IN PAPER CHROMATOGRAPHY  PAPER CHROMATOGRAPHYDE"CRIPTION CHROMATOGRAPHYDE"CRIPTION  his method in"ol"es in"ol"es spotting the the sample solution onto a strip of chromatographic paper  he paper is is placed into into a Bar, containing containing a shallow layer of sol"ent and then sealed 1s the sol"ent rises through the paper, it meets the sample mi!ture, which starts to tra"el up the paper with the sol"ent (ierent compounds in the sample mi!ture tra"el dierent distances, according to how strongly it interacts with the paper  his allows the calculation calculation of the Rf "alues, and compare with a standard 1 method in"ented by the 5ritish biochemists, Archer 4ohn Porter Martin and Richar. (a-rence Millington "#nge 1n analytical techniue for separating and identifying mi!tures mi!tures that or can be colored, especially pigments T Chromatogram  he components which which ha"e been been separated dier in their retention factor or Rf "alues, which are then computed using9

 Distancetraveled by solute solute  Rf value=  Distancetraveled by solvent  solvent  TECHNI67E" IN THIN (AYER CHROMATOGRAPHY  THEORIE" Aidely used laboratory techniue, similar to paper chromatography chromatography Stationary phase used is a thin layer of adsorbent on an inert, at substrate %glass plate'9 Silica Gel 1lumina 3ellulose AD5ANTAGE" OF THIN (AYER CHROMATOGRAPHY  Rapid Sensiti"e 8!cellent Resolution Simplicity 3heap THIN (AYER CHROMATOGRAPHY  "tationar# Pha%e &T(C Plate%' It consists of a thin layer of adsorbent  material immobili)ed onto at, inert carrier sheet.   63 &lates •







 63 &lates are are made by mi!ing the adsorbent, such as %ilica gel with gel with a small amount of inert binder like calci-m %-l>ate &g#p%-m' and water.   he mi!ture mi!ture is spread spread as thick thick slurry on an unreacti"e carrier sheet, usually gla%%, gla%%, thic! al-min-m >oil or pla%tic or pla%tic,, and the resultant plate is then dried and acti"ated by heating in an o"en for [email protected] mins. at [email protected]3.   he thickness of the adsorbent adsorbent layer is typically around 2) mm for mm for anal#tical p-po%e% and p-po%e%  and around 2) mm for mm for preparati$e T(C. T(C.  o ensure the stationary stationary adheres adheres rmly rmly   o on the backing plate and does not ake o during de"elopment, +in.er% are added to the adsorbent.  3alcium Sulfate %gypsum'  Starch  3arbomethylcellulose Mo+ile Pha%e   he common sol"ents sol"ents are the the following9  7eptane  7e!ane  Isooctane  3yclohe!ane  0ethanol  1cetic 1cid  Aater  8thyl 8ther  3hloroform  1cetone  33lJ  oluene   oluene  5en)ene  1cetonitrile  &yridine  8thylene 3hloride  8thanol  I:&ropanol  (io!ane  8thyl 1cetate  -or mi!tures of unknown composition, +en0ene or +en0ene or chloro>orm ith  ethanol is ethanol is the best sol"ent for e!plorotatory runs. "ample Preparation an. Application  (ierent compounds in the sample mi!ture tra"el at dierent rates due to the dierences in their attraction to the stationary phase and because of dierences dierences in solubility in the sol"ent.  Separation of compounds is based on the competition of the solute and the



mobile phase for binding places on the stationary phase. "pecial Detecting Metho.%  Charring  In"ol"es the spraying of concentrated sulfuric acid and heating the plate   he result result is seen by the charring charring of the spots  7%e o> Io.ine 5apor  In this method, the chromatogram is placed in a closed container holding a few iodine crystals spots react react with the   he sample spots iodine "apor and form brown spots   he reaction reaction is re"ersible re"ersible  E/amination -n.er 75 Ra.iation  Useful for compounds that uoresce   wo  wo UK light sources sources are are useful and and commercially a"ailable9 75 %hort2a$e &)nm' 75 long2a$e &Jnm'

Preparation o> Deri$ati$e% F7NCTIONA ( GRO7P" Aci.% Al.eh#.e% an. =etone% Amine% an. Amino Aci.% Al!aloi.% 3ar+it-rate % Car+oh#.ra te% (ipi.% "teroi.%

REAGENT" 3romcre%ol Green );2 .initrophen#lh#. ra0ine Ninh#.rin

Merc-ric Ni Nitrate Diphen#lcar+a0o ne Aniline Phthalate 3romth#mol 3l-e Antimon# Trichlori.e

CO(OR PROD7CED  Yello  Yello  Yello  Yello

Fl-ore%cen t Yello to to 3ron P-rple Gra#23lac!  (ight Green 5ario-%

DETECTION OF "POT" ON THIN (AYER CHROMATOGRAPHY  5y its natural color

5y uorescence 5y spraying with "isuali)ation reagents "PECIFIC 5I"7A(IING AGENT" Ninh#.rin Rho.amine 3 Anti Antimo mon# n# Ch Chlori lori.e .e "-l>-ric aci. K Heating Pota%%i-m Permanganate in "-l>-ric Aci. Ani%al.eh#.e in "-l>-ric Aci. 3romine 5apor

"PEICIFC COMPO7ND" IDENTIFIED Amino Aci.% (ipi.% "ter "teroi oi.% .%;; Ter Terpi pin noi.% oi.% 7ni$er%al 5i%-ali0ing Agent >or Mo%t Organic "-+%tance% H#.rocar+on%

Car+oh#.rate% Ole1n%

AD5ANTAGE" OF THIN (AYER CHROMATOGRAPHY  Great resol"ing power because spots are smaller Greater speed of separationL 7igher resolution Aider choice of materials as sorbents 8asy detection of spots 8asy isolation of substances from the chromatogram •



• • •

DR7G" ANA(YED IN THIN (AYER CHROMATOGRAPHY  1nalgesics 1ntipyretics 1spirin, &henacetin, 1cetaminophen 1nti:Inammatory 1nti:Inammatory (rugs Uricosuric (rugs 3aeine and 3aeine:3ontaining (rugs • • • • • •

GA"2(I67ID CHROMATOGRAPHY PRINCIP(E" GA" CHROMATOGRAPHY  1lso known as #ga%2li:-i. # ga%2li:-i. chromatograph#$ chromatograph#$ or #ga%2li:-i. #ga%2li:-i. partition chromatograph#$ chromatograph# $ 1 separation techniue in which the mobile phase is gas It is used in the analysis of gaseous and "olatile substances Mo+ile Pha%e  his is the carrier carrier gas 3onsiderations in the choice of carrier carrier gas9 Safety &urity Inertness 1"ailability 3ost

(etector to be used SampleMs matri! 3arrier gases employed in G39 7elium ;itrogen 7ydrogen 1rgon 1ir "tationar# Pha%e It is adhered to the inside of a9 Small diameter glass tube %Capillar# Col-mn' Col-mn' Solid matri! inside a larger metal tube %Pac!e. %Pac!e. Col-mn' Col-mn ' 3haracteristics9 3hemically stable 7igh boiling point 6ow "iscosity Specic sol"ent properties towards the components to be separated 8!amples9 7ydrocarbons Silicone oils and gums &olyesters &olyalcohols *carbowa!+ TYPE" OF GC CO(7MN Pac!e. Col-mn% 6arge core columns with a length of D.F:[email protected] meters and an internal diameter of ?:J mm  he tubing is usually made made of %tainle%% %teel or gla%% and gla%% and contains a packing of nely di"ided, inert, solid support material that is coated with a stationary phase Capillar# Col-mn% -le!ible columns with a "ery small internal diameter diameter of @.?:@.F mm and a length of ?F:[email protected] meters  he tubing is made of >-%e.2%ilica ith a pol#imi.e o-ter coating  ypes9  ypes9 o this slows down their mo"ement through the column 0olecules are eluted from the column in the order of9  (ecreasing si)e  (ecreasing molecular weight %for relati"ely constant shapes > Glo+-lar or Ro.' Ro.'

MATERIA(" MATERIA(" 7"ED IN M" GC Gel% 1 E( network with a "ery random structure. 3onsist of cross:linked polymers that are9 Inert ;on:Reacti"e Uncharged Used for aueous solution De/tran &olysaccharide composed of o glucose residues. &roduced by fermentation fermentation of glucose by Leuconostoc mesenteroides

&repared by "arious degrees of cross:linking to control pore si)e o "epha.e/ : "epha.e/ : tradename Agaro%e o =btained from seaweeds o Its pore si)e is larger than Sephade! Useful for the analysis < o separation of (;1 o "epharo%e 3iogel > 3iogel  > tradenamesL supplies as wet beads Pol#acr#lami.e Its pore si)e is determined by o the degree of cross:linking o &repared by cross:linking 1crylamide with N;N2 Meth#lene23i%2Acer#lami.e 3iogel P : tradename o o

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