Chromatography PDF

Chromatography Required reading: Biochromatography [electronic resource] : theory and practice / edited by M.A. Vijayalakshmi. London ; New York : Taylor & Francis, 2002. Chapter 2 Gel permeation chromatography Chapter 3 Ion-exchange chromatography Chapter 6 Affinity...

Chromatography Required reading: Biochromatography [electronic resource] : theory and practice / edited by M.A. Vijayalakshmi. London ; New York : Taylor & Francis, 2002. Chapter 2 Gel permeation chromatography Chapter 3 Ion-exchange chromatography Chapter 6 Affinity Chromatography Chapter 10 Immobilized metal affinity chromatography (IMAC) http://library.polyu.edu.hk/search~S6?/i0415269032/i04152690 32/1,2,2,E/frameset&FF=i0415269032print&1,1, http://cdn.intechopen.com/pdfs-wm/44033.pdf 1 General chromatographic theory ample we wanna separate from -S each other (e g. large & small/-re +ve and chromatography: component molecules (solutes) in a proteins ( sample mixture are transported by a mobile phase over gas liquid) a stationary phase Isolvent : (+ ve) ↳ need heat up to ↑ be volatile Can attract-re protein interaction occurs between the solutes and the stationary phase so that the solute is distributed between the stationary and mobile phase attraction attract from the stationary phaso : we protein - different solutes will move at differing rates since each will have a different affinity for stationary phase with respect to the mobile phase 2 General chromatographic theory mobile prase - & gas and liquid chromatography may rely on different interactions Gas need all volatile => high heat chromatography separation process in either case can still be : described using the same general theory Individual species are retarded by the stationary phase based on various interactions such as: – surface adsorption – relative solubility – charge 3 Molecular Physical Separation Characteristic Property Technique Rely on seperaption Polarity Volatility Gas-liquid chromatography Solubility Liquid-liquid chromatography Adsorptivity = Liquid-solid chromatography ↳ kinding the stationary phase Ionic Charge Ion-exchange chromatography technique Electrophoresis - not chromotography Size (mass) Diffusion Gel-permeation chromatography Dialysis Sedimentation Ultracentrifugation Shape Ligand binding Affinity chromatography b Antibody. bind antigen 4 General chromatographic theory distribution ratio K – conc. of solute in stationary phase behide Stay K = ----------------------------------------------- ↳ achieve separate conc. of solute in mobile phase 5  Theories of chromatography surface : perform condensation ↓ Cooling Plate theory ↑ Plate – proposed in 1941 by Martin and Synge – analogous to the distillation extraction # cle conc.. – an equilibrium between stationary and mobile phase is treated as a theoretical plate (diffent boiling Pt alc and. between waters Rate theory > - mobile phase movement velocity – proposed in 1956 by J. J. van Deemter – consider the dynamics of a separation process Both plate and rate theory has their own advantages and limitations 6  Plate theory In distillation – actual plates exist where vapor passes through a liquid phase cooling = ) + condensation – during this mixing, equilibrium between the phases is assumed – the height of a plate can be directly measured (and no of plate) & Cassum appears – but in chromatography column, the plates cannot be observed - called theoretical plates (egulibrium : ↳ cannot solute keep bind and anything in get off binding and release 7 see achieve ) ↳ result in a different ratio to  Plate theory a column is mathematically equivalent to a plate column no. of theretical plate in the column ~ Total length is divided into N segments each representing an equilibrium stage or theoretical of and release binding ↳ rate plate X same releas binding ~ Different distribution in and (therotical plates An equilibrium is established at each stage as the mobile phase passes from one stage to the next Achieve eg. : K dependent properity to physical of solute 8  Determination of N N is related to the number of equilibria that have taken place during separation N => column more efficient & retention distance) retention distance 2 # retention volume N = [ ----------------------------- ] X 5.54 peak width at half peak height (W 1/2) : N & Retention distance > - binding + releasing 9  OD : 280 nm - volume Injection less desireable ~ (no. of compound) retention distance peak and be wide / narrow ~ tR => cannot compare ~ separation of solute W1/2 therratical same time all => and the sharp column peak > time W 2 t Number of theoretical plates (N) 5.54 R # time/volume > N W 1/2 - H and release ↑ binding 10  Peak Same +p l & Desirable Thinner Thicker+ wider of retension distance range  Height equivalent to a theoretical plate (HETP) length of column = > Plate location length HETP = ------------------------------ N = ) No. of plate HETP as the efficiency of the column small volume in ↳ pack the theoretical plate column N Desire : length of 11  Resolution Resolution index RS a measure of how completely 2 neighboring peaks are separated from each other twice the distance between the two peaks RS = ----------------------------------------- sum of the base width of the two peaks RS , better resolution between 2 components 12  Injection tB S tA OR in 100 : 20 Ratio physical equilibrium separation only on : chemical properties after = Only reflect the properties reaching - + ratTo => Need enough topredi Thieve theretical probability * Random WA WB Bind/Release > Ratio will after not change reaching equilibrium - ↑ binding. E. g If solute => the , stationary phase Twice the distance between 2 peaks Resolution (R s ) ↳ Sum of the basewideth of 2 peaks seperatin i 2 (t - t ) B A T each peak sharper W W A B The greater the value for Rs, the better the resolution 13 11vs 11 ~ resolution - higher 1num ~ higher -> resolution  Rate theory of chromatography Plate theory neglects the concepts of solute diffusion and flow paths plate) (unrelated to theretical Rate theory accounts for these and can be used to predict the effect on the column performance factors such as – phase properties phase thickness – solute diffusivities support size – partition coefficients phase velocity – support porosity flow rates 14  van deemter equation reduced form of van deemter equation ↓ H - Choose optimal Nu H = A + B/ + C ↳ comprimise between B and C M 7 depent in opposite manner H Height equivalent to a theoretical plate (HETP) A multipath or eddy diffusion B molecular diffusion C resistance to mass transfer gas flow rate (in gas chromatography) /liquid liquid chromatography in ↳ how fastphase mobile moving is A, B, C are constants but the effect of B and C is dependent on the velocity of the mobile phase ( ) 15  van deemter equation No need to calculate H for each column/eluant combination to be able to use this relationship an understanding of the effects of each term will help you design/select appropriate columns and optimum flows 16 Unequal pathway (multipath) Interaction between solute stationary phase Mobile phase column/ packing of of the the varians Solute Stationary size of stationary phase phase => random path different ↳ arrive at Solutes take different (random) pathways time sharp > X = peak depending on interaction between solute and stationary phase This results in broadening of peaks (Term A) 17  A term (Multipath or eddy diffusion) (when pack is X uniform) the range of possible solute paths results in the broadening of the peak 1 ideal : A ( A= dP takes into account the particle size range, packing uniformity and column dimensions and geometry range packing uniformity Ideal + size a dP is the mean diameter of the stationary phase particles pack regularly Ideal # diameter : + easier to 18  A term (Multipath or eddy diffusion) good separations and minimum band broadening will be achieved using – small particles – with a narrow size range – that are packed uniformly into the column Minimum dead volume once column is packed, A term is fixed a perfect GC column: A term = 0 Clowest possible) 19  Molecular diffusion Forward & backward Concentration Diffusion in mobile phase Profile of band as band moves along Mobile phase Solute Molecular diffusion of solute forward and backward within mobile phase results in broadening of peak (Term B) 20  B term (Molecular diffusion) represents band broadening due to random diffusion of solute in the mobile phase inversely proportional to the velocity of the mobile phase – high flow rate => no time to diffuse) extent of diffusion will be hindered by – the particles of column packing: hindrance factor ( ) (now fast the solute can diffuse – the coefficient of diffusion of the solute in mobile phase (Dg) B = I2 DgS 21  B term (Molecular diffusion) You should keep the flow rate ( ) as high as possible within the limits imposed by the instrument and the C term (Gas) (liquid) B term is more important in GC than in LC since of the large diffusion coefficient of solute in the mobile gas phase Reverse diffusion is more significant than forward diffusion: “trailing tail broadening” ↳ shape of peak is not even 22  Mass transfer ↳ solute (C) Mobile phase (Release) (Binding) Movement off Movement onto stationary phase stationary phase Stationary phase Solute attracted onto high of stationary phase occur a no (wanna time > - dist the physical chemical property) Lachieve equilibrium ( Mass transfer of solute onto and off stationary phase affects the overall movement of solute in mobile phase (Term C) 23 C term (Resistance to mass transfer) 8 k ×df 2 C ( I× 2 (1 k ) ×D l –k capacity factor (retention factor) 4) how likely solute go into stationary phase df liquid phase effective film thickness 4) thickness of stationary phase Dl diffusion coefficient of solute in liquid phase) (stationary phase for GC) I Stationary 24  C terms it takes time for a solute to reach an equilibrium between the mobile and stationary phases thick or viscous stationary phases have larger C terms minimize the effect of C term by: – using “thin” coatings of the stationary phase on a solid support – use less viscous phases – keep the flow rate ( ) as low as possible - limited by the effect of the B term 26  Optimum flow rate ( opt) opt is the flow rate at the Hmin best flow rate is a function of the van deemter equation and practical conditions. You need to have a useable analysis: too low the flow rate => too long the analysis time 27  Optimum flow rate ( opt) A – small and uniformly packed particle support (minimize dead volume) B – particle support and mobile phase (minimize diffusion of solute) – maximize flow rate C – thin coating of stationary phase – less viscous stationary/mobile phase – minimize flow rate 28  Optimum velocity The best velocity is a function of the van deemter equation and practical conditions. You need to have a useable analysis Also, since the effects of B are greater than C, it is best to set the flow rate a little on the high side in case it changes slightly during the analysis opt * - Less H * than ↑ th e H B C A 29  Types of chromatography Adsorption chromatography – Thin Layer Chromatography (TLC) => separation by polarity ) use ↳ cellulose/paper Exclusion (permeation) chromatography – gel filtration = Separation by size Ion exchange chromatography (IEC) vel-ve > - 2 types : + => Separate by charge Affinity chromatography antibody > antigen/metal ion - protein - > - use Partition chromatography – Liquid-liquid chromatography (LC) – Gas-liquid chromatography (GLC S 30  Adsorption chromatography thin layer chromatography (TLC) or column mobile: solvent (liquid) – low polarity (e.g. hexane) mix in – intermediate polarity (e.g.dichloromethane) ratio – high polarity (e.g. butanol) stationary: adsorbent (solid) – silica gel, aluminum oxide, calcium carbonate, cellulose – hydroxyapatite (crystalline calcium phosphate) Separating dsDNA from ssDNA 7 (single Strand) (Double strand Also used in purifying protein Salt conc. ssDNA dsDNA – Binds preferentially to basic proteins low (Binding/Equilibration ( Mainly qualitative, ‘low tech’ medium (washing) X high (Elution] X X ) remove ↳ solute off the 31 column  => solvent front ↳ further solvent go Stationary phase = S powder Spotting a TLC plate with sample: Running the TLC plate in solvent 32 http://www.chem.vt.edu/chem-ed/sep/tlc/tlc.html  Outline of a TLC procedure sample application via glass capillary tube drying of application spot plate is placed vertically in a tank with solvent (mobile base) at bottom solvent rises by capillary action post-chromatographic detection (coloured – visual, fluorescence, radiation, chemical development 33  Applications of TLC Monitor reaction progress not getge Do Fee ? Figure 2.8: a) Reaction scheme of the acetylation of ferrocene, b) TLC plate (visualized with UV light) with 3 lanes: F for pure ferrocene, Co for the co-spot, and AF for the acetylferrocene product. https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Book%3A_Organic_Chemistry_Lab_Techniques_(Nichols)/02%3A_C hromatography/2.02%3A_Thin_Layer_Chromatography_(TLC)/2.2.0B%3A_2.2B%3A_Uses_of_TLC 34  Applications of TLC Detection of drugs in serum and urine – TLC, GC-MS, LC-MS identification separation If I coupled equipment TLC GC-MS (Gas chromatography - Mass HPLC-MS Mass Spectrometry ( (Liquid chromatography - spectrometry Qualitative Qualitative + Quantitative Qualitative + Quantitative (what molecules (no, of molecule Separation by polarity of Gold standard for volatile For both volatile and non- drugs relative to solid (non-polar) drugs (high neatr) volatile drugs (e.g opiates) phase (silica) Urine (preferred), serum Body fluids, hair Body fluids, hair Skilled experiences Highly sensitive and Highly sensitive and needed to interpret result specific specific; Less sample preparation and faster GC : compound separate differentretention time by https://www.pathologyoutlines.com/topic/chemistrytoxicology.html 35 MS : Mass for detection (highly accurate)  Applications of TLC Detection of amino acids; needs post- chromatographic staining with ninhydrin 36  Surface adsorption processes van der Waals forces dipole diploe interactions intermolecular hydrogen bonding 37  Advantages of TLC minimum sample preparation, fast, cheap simultaneous analysis of standards and samples high sensitivity (samples retained on plate) ↳ loss/no nothing trap greater post-chromatographic possibilities G when (solvent removed) solute need not possess a chromatographic group (post-chromatographic reactions used for location)need detection afterwards. 38  Limitations of TLC Cannot separate enantiomers (chrial) Solutedistancetravelea (Ratio : To identify specific compounds, Rf values of compounds of interest needs to be distance determined beforehand. solute (X solvent front => Not accurate) Short stationary phase; length of (small no. of N separation is limited compared to other chromatography. https://microbenotes.com/thin-layer-chromatography/ 39  Liquid chromatography (liquid as mobile phase) 1. Pre-packed column e.g. HiTrap (5 ml) Desalting column from GE adding buffert continue y & Take out sample can be same of different ↑ Ton exchange : 2. Packing a Chromatography Column salt high conc. 40 http://www.chem.vt.edu/chem-ed/sep/lc/lc.html  Exclusion (permeation) chromatography separation based on molecular size & shape + column desalting – gel particles or porous glass granules acting as “molecular sieve” large molecules stationary phase in – completely excluded from the pores and will pass through the column quickly smaller molecules – distributed between the solvent inside and outside the molecular sieve and will elute slowly (pores) 41  Exclusion (permeation) chromatography distribution coefficient, Kd is a function of the solute’s molecular size spend (time outside inside and the port elution volume (buffer) – volume of solvent required to elute the analyte larger molecules have smaller elution volume Stay => outside of pores 42  Large Small elution times Clongest (travel inside polymers ~cannot insid travel =>> fast will almost Salt never come out from the (Remove salt from protein column > - Desalting 43 Large Small Materials used in exclusion chromatography uniform more > - porous, easy to pack (spherical in shape), no adsorption, no charged group, cause no denaturation to analytes “pore” size larger pore - size limit to enter smaller - – e.g. Sephadex 200, Sephadex 25 - dextrin : polymer of (kcl) carbohydrate – determines size exclusion limit “particle” size – e.g. superfine, fine, medium and coarse packed Isuperfine ↳ better more in separation – determines how well the column can be packed ↳ small deal volume ↳ difficult to pass through => resolution ↳ need pump 44  Materials used in exclusion chromatography dextrans (e.g. Sephadex) – cross-linked polymer of polysaccharide – varying degree of crosslinking results in differences in pore sizes – pore size: Sephadex 200 > 100 > 50 > 25 > 10 agarose (e.g. Sepharose, Bio-Gel A) – linear polysaccharrides containing alternating residues of D-galactose and 3,6-anhydro L-galactose polyacrylamide (e.g. Bio-Gel P) polystyrene (Bio-Bead S) Control pore size > - what protein suitable 45 for separation  -3 Polymer T rade name Fractionation range (Mr x 10 ) * fractional ↑ size Dextran Sephadex range G-10 last ↳ of ion exchange chromatographelution used high come salt water -> unfavourable + desaltion (permeable step chromotography) 47 Ion exchange chromatography (IEC) separation based on charge differences of different analytes a process wherein a solution of an electrolyte is brought into contact with an ion exchange resin ↳ stationary phase and active ions on the resin are replaced by ↳eg.. Nat , C normally attact to stationary phase before chromatograph ions of similar charge from the analyte solution ↳ protein 48 Ion-exchange chromatography cone (nigh elutionion (Replace protein slow salt keep resin volume attached) washing > I - ↳ activit a I L lessness) (+ve/neutral) & & ! ↓ ↳ Need Desalt 49  Types of IEC cation exchanger (bind cation > - -ve) – -SO3Na, -OH, -CH2CO2H (Carboxy Methyl; CM) anion exchanger (bind arion + tre) – CH2CH2NH(CH2CH3)2+ (Diethylaminoethyl; DEAE) choice of cation or anion exchanger – charge of analytes changes with pH – e.g. if analytes are more stable above its isoelectric point, giving it negative charge => use anion exchanger – analytes stable over wide range of pH => use either one – strong or weak ion exchanger 50  +1 -1 Strong ion exchangers are charged in pH 1 to 13; Can be used in all pH ranges. 51 http://www.proteinchemist.com/tutorial/iec.html Changes of the net charge of proteins in different pHs Isoelectric ~ same charge (p1 point) plt : value neutral in charge Point ↳ differen from every protein Binds to Anion + Net Charge on Protein Exchanger separate since only charge can - different in will bind to ~ protein I (cation exchanger anion exchanger pH 2 4 6 8 10 12 - Binds to Cation Exchanger 52  Matrix of IEC charged groups are linked to a solid matrix which should be inert, neutral and gives good packing properties polystyrene – e.g. Dowex50: sulphonated polystyrene cellulose – e.g. CM-cellullose, DEAE-cellulose 53 Type Polymer Functional group Examples of commercial products Weakly acidic Polyacrylic acid -COO- Amberlite IRC 50, Bio-Rex 70, Zeocarb 226 (cation exchanger) Cellulose or dextran -CH2COO- CM-Sephadex, Cellex CM Agarose -CH2COO- CM-Sepharose Strongly acidic Polystyrene -SO3- Amberlite IR 120, Bio-Rad AG 50 (cation exchanger) Dowex 50, Zeocarb 225 Cellulose or dextran -CH2CH2CH2SO3- SP-Sephadex Weakly basic Polystyrene -CH2NHR2 Amberlite IR 45, Bio-Rad AG 3, (anion exchanger) + Dowex WGR Cellulose or dextran + -CH2CH2NH(CH 2CH3)2 DEAE-Sephadex Cellex D Agarose + -CH2CH2NH(CH2CH3)2 DEAE-Sepharose Strongly basic Polystyrene -CH2N(CH3)3 Amberlite IRA 401, Bio-Rad AG 1 (anion exchanger) + Dowex 1 + Amberlite IRA 410, Bio-Rad AG 2 CH2N(CH3)2 Dowex 2 CH2CH2OH Cellulose or dextran + QAE-Sephadex CH2CH2N(CH2CH3)2 CH2CH(CH)CH3 + Cellex T CH2CH2N(CH2CH3)3 Table showing examples of ion-exchangers of biochemical importance CM-cellulose (weakly acidic) papers, as well as cellulose phosphate (strongly acidic), cellulose citrate (weakly 54 acidic) and aminoethylcellulose (weakly basic) papers. Resin-impregnated papers are also commercially available, for example Amberlite SA-2 (strongly acidic) and Amberlite SB-2 (strongly basic). IEC used in protein purification e.g. for a protein X with pKa = 5 – use a DEAE-cellulose column (anion exchange) – use a low ionic strength buffer at pH = 9 – protein X will be negative in charge and bind to DEAE-cellulose – other “junk” proteins will be either neutral or positive in charge => not binding to DEAE-cellulose – Elution use either high ionic strength buffer or low pH buffer (protein X will be neutral or positive in charge) 55  (andifferent rate) (elution) Conductivity salt I how many pass through Buffer A = 20 mM Tris, pH =8.0 Buffer B = 20 mM Tris, 1 M NaCl, pH=8.0 Equilibrate column in buffer A. Bind protein dialyzed against buffer A. Elute with a linear gradient of A to 60% B in a flow rate of 1mL/min (total 20 mL) not binding as G strong as and Advantage of gradient elution: remove non-specific bands GRADIENT GRADIENT 56  STEP ISOCRATIC 57  ↳ cocluted needed ↳ 2 pumps - merged Tatter separated http://www.intechopen.com/books/protein-engineering-technology-and-application/chromatography-method 58  Affinity chromatography exploits the unique property of extremely specific biological interactions antibodya leg antigen/ to achieve separation. relies on a detailed knowledge of the structure and biological specificity of the analyte 59  Affinity chromatography Istationary phase e.g. immobilize ligand (substrate, inhibitor, ↳ small moleculesA coenzyme) on the matrix can be used to purify an enzyme that can bind the ligand specifically. elution of enzyme can be done by excess ↳ high conc. ) compeating ligand or changes in pH/ionic strength to ↑ disrupt ligand-enzyme interaction 60  flexible long/short linert chemical gp , , attract to enzyme > - binding ligand > - stick out purpose -> * accessible close to martis In binding ligand >. hidden - If X , excess free ligand (e g.. salt conc ). from come out column ↓ shape changed I due to pl > X recongise ligand = ↳ come off used membrane with pore ↳ only free ligand pass enzyme trapped => > - collect 15 mmo/ salt) (most pH7 , 61 Group specific ligands commonly used in affinity chromatography Ligand Affinity + 1. 5’ AMP Adenosine monophosphate, cofactors NAD -dependent dehydrogenases and certain kinase 2. 2’, 5’ ADP NADP+ -dependent dehydrogenases. Adenosine diphosphate & OH 3. B Compounds with coplanar cis-diol groups, e.g. sugars, OH nucleosides, nucleotides, catecholamines 4. Proteins containing SH-groups HgCl 5. Poly(U) mRNA which contain a poly(A) tail => mamalian feature 6. Poly(A) Oligo(dT) base pair purification mRNA of E Ribonucleic acids which contain a poly(U) sequence; RNA-specific proteins such as nucleic acid polymerases 7. Lysine rRNA; plasminogen 8. Concanavalin A Glycoproteins and glycopeptides; membrane fragments carbohydrates containing -D-mannopyranosyl and ↳ kind to purified dicocilated protein Ye - -D-glucopyranosyl residues 9. Calmodulin Proteins regulated by calmodulin 10. Heparin A wide range of proteins including lipoproteins, lipase, Protein Gl coagulation proteins and steroid receptors 11. Protein A phase limmobilize > - stationary ↳ bind antibodies ) lgG and molecules which contain the Fc region of lgG (a protein isolated from cell walls protein A/G purify protein > column - of Staphylococcus aures) 12. Cibacron Blue F3GA Nucleotide-requiring enzymes; blood coagulation factors; albumin 13. Lectin Cells and macromolecules containing N-acetyl- -glucosamine62 or N-acetyl- -galactosamine residues  Polyhistidine tag for protein purification ↳ for protein similar to size and ion charge. Ni-NTA column I /Cu-NTA (to bind to polyhistidine- tagged protein) Poly Histadline > 6112 Histadine. ↓> - M 1 tag : let protein bind to column specifically NTA Stationary ~ phase blue ) cannot Everything assure = 3 2 (InducedBacteriaFlowthroug , Bounded/Eluted Show any a Clone the polyhistidine tag (e.g. HHHHHH or H12) at the N- or C-terminus of protein > - confirm by tag Anti- Protein purification overview Histidine tag https://www.youtube.com/w antibody 63 atch?v=PVvpEKeOzEM ↳ protein dye (transfer protein > - membrane)  Gas Chromatography (GC) 1941 Martin and Synge proposed that rapid separation can be achieved using gas instead of liquid as mobile phase Istationary phase) (mobile phase) partition of compounds between liquid and gas phase high sensitivity, reproducible, speed ( can very high flow rate) most suitable for separation of compounds of low polarity e.g. lipids optimum carrier gas flow rate for max. column efficiency (ie min. HETP) http://www.shsu.edu/~chm_tgc/sounds/chrom.mov http://www.edusolns.com/gc/ http://www.uga.edu/srel/AACES/GCtutorial/page1.html http://www.youtube.com/watch?v=08YWhLTjlfo&feature =channel_page 65  Gas Chromatography analyte must be volatile (some analytes may need ↳ may derivatization) not gas orginally ↳ to add under a temp change by gp- voatile , compounds characterization based on retention time Retention Index (RI) of carbon no -. – retention time relative to n-alkanes ) know ↳ RI under fixed condition 4) as standard 66  (liquid I gas Sample ladjust flow rate - sample vaporize sample) > = * temp (vaporize (in gas cylinder -> in high (N2/He = iner + >Pressure (separation) (further separate) elution time =>> record * Retention time 67 https://slideplayer.in.th/slide/2124045/  In Out wires 3m ( insulating layer 68  Components of GC carrier gas (mobile phase) - inert gas : N2, He, Ar detectors in GC – flame ionization detector (FID) ionization (burning) of solutes results in changes in current – thermal conductivity detector (TCD) presence of solutes in carrier gas => thermal conductivity of carrier gas => loss of cooling efficiency => temperature of a heated wire – electron capture detector (ECD) radioactive source ionizes N2, releasing e- e- are “captured” by solute => current 69  Components of GC Modern capillary GC columns made of 2 parts Polyimide coating ( fragite( A fused silica glass (1) tube with external Fused silica tubing polyimide coating Stationary phase (2) A stationary phase made of a thin film of high molecular weight, thermally stable polymer coated on the inner wall 70  71 1 Chapter 24 GC Gas Chromatography. 2 GC Mechanism of separation is primarily volatility. Difference in boiling point, vapor pressure etc. What controls. - ppt download (slideplayer.com) Common stationary phases in GC Intermediate polarity I /Strate Strongly polar Strongly polar 72 The fused silica tubing typically has an internal diameter of 0.1 – 0.53 millimeters and can reach lengths between 10 – 150 meters. The external polyimide coating dramatically increases the flexibility of the glass column and allows it to be tightly coiled and housed in the GC oven for temperature control. As the sample plug flows through the column, the different components separate from one another based on the strength of their interaction with the stationary phase. Many different types of stationary phases exist for GC and are usually described by their degree of polarity. While the intermolecular forces governing solubility equilibrium is a complex topic, a high-level description is polar molecules are more soluble in polar systems while non-polar molecules are more soluble in non-polar systems (“like dissolves like”). When a GC employs a polar stationary phase, the more polar molecules in a sample flowing through the column will partition more into the stationary phase compared to the non-polar molecules and will take longer to flow through the column. The degree of partitioning is also temperature-dependent, and the temperature of the oven that houses the column can be leveraged to facilitate improved separation between specific compounds with similar polarities. A linear temperature ramp of the column oven is common in many GC applications. 73 Gas Chromatography Fundamentals | Agilent  Flame Ionization Detector ↳ burn sample The schematic diagram below shows a flame ionization detector for gas chromatography. The eluent from the column mixes with H2 and is burned in the presence of excess air. Combustion of organic analytes produces a flame containing electrons and organic cations, presumably CHO+. Applying a potential of approximately 300 volts between the flame tip and the collector, generates a small current of roughly 10–9 to 10–12 amps. that, when amplified, provides a useful analytical signal proportional to the concentration of cations—and thus the concentration of analyte— Burning > - emission of Ton in the flame. - ↳ ↑ currey to measure (out) 74 Flame Ionization Detector | Image and Video Exchange ForumImage and Video Exchange Forum (asdlib.org) Electron Capture Detector Sample capture electron) The effluent from GC is exposed to slow electrons generated by the ionization of the carrier gas (Ar or N2) by a constant flux of beta rays from a radio in current - drop 75 chemicals 76  Derivatization of samples many samples cannot be analyzed by GC directly because of non-volatility, or too strongly attracted to stationary phase => “tailing” derivatization: modify samples before GC – increase volatility of sample – reduce thermal instability of sample => reduce thermal degradation – increase detector response by incorporating into the derivative functional groups e.g. CF3 groups for ECD 77  Derivatization of samples silylation => less polar, more volatile – ROH + Cl-SiMe3 => R-O-SiMe3 + HCl acylation => detectable in ECD – R-OH + O-(COCF3)2 => R-O-COCF3 78  GC applications drugs lipid urine detect pollutants , , , Quantifying pollutants in drinking and waste water Drugs and their metabolites in blood and urine (pharmacological and forensic applications) Identification of unknown organic compounds in waste forecinicpharamatical dumps Air quality control Mainly for volatile, non-polar samples that do not need derivatization, e.g. pesticides – ?maximum residue levels (MRL) not be exceeded 79  Rely on time > - Same condition > - identify peak no ↓ accurcy may > - e r ro r 1. Cyprodinil => mass spect 2. Myclobutanil 3. Quinoxyfen 4. Tebuconazole 2 4 1 3 80  Gas chromatography – mass spectrometry (GC-MS) GC-MS, LC-MS – Use a mass spectrometer to detect the solute – Identification based on mass/charge (m/z) – Higher sensitivity (due to MS) – Accurate (m/z is unique to the solute) – Multiple components can be identified simultaneously (by monitoring multiple m/z) https://www.youtube.com/watch?v=bVKASwadjQY 81  HPLC L High Performance (Pressure) Liquid Chromatography ↳ protein FPLC (Fast Protein Liquid Chromatography) X – HPLC with lower pressure, mainly for protein < -2 pumps) > - Medium pressure resolving power of a chromatographic column by protein & separation N – column length & no. of theoretical plate / unit length – achieved by surface area of the stationary phase i.e. smaller solid support Problem effector B – smaller solid support => resistance to eluant flow – Solution flow rate by pump pressure – increasing flow rate also reduces diffusion of analytes 82 ↳ too slowly https://www.youtube.com/watch?v=eCj0cRtJvJg move  HPLC Problem – high pressure => solid support collapses – solution: use better matrix for solid support Problem – high pressure => traditional “open column” not suitable – solution: column under sealed/closed steel ( (material stainless environment : * open leakage column avoid 83  HPLC HPLC achieve high resolution by: – small solid support made with better matrix – high pressure in a “closed column” – Versus “open column” in conventional LC HPLC is an instrumentation setup, not a separation method – HPLC can be applied to adsorption, partition, ion-exchange, exclusion & affinity chromatography http://www.youtube.com/watch?v=kz_egMtdnL4&feature=relmfu 84  HPLC versus GC HPLC: liquid; GC: gas as mobile phase HPLC has wider applicability (adsorption, partition, ion-exchange, exclusion, affinity) than GC (adsorption and partition) HPLC does not require sample volatility, therefore widening the range of sample that can be analyzed. In general, no sample preparation is needed for HPLC, whereas, derivatization is needed for many samples in GC 85  HPLC versus GC > collect same back HPLC’s detector is non-destructive, measure - therefore facilitating sample collection HPLC is in general more expensive than GC, but price of the former is dropping due to its ever-increasing popularity HPLC’s efficiency does not change with solvent flow rate whereas GC’s efficiency depends heavily on the gas flow rate 86  Components of an isocratic HPLC Sample injection loop Solvent reservoir Detector Column High pressure pump Sample collector Recorder and data system 87  Components of a HPLC solvent reservoir – mobile phase depends on chromatographic technique used high pressure pump – critical part of HPLC – high, sustainable, reproducible pressure – dual pumps for gradient elution sample injection port – to maintain the pressure of the column during sample injection Copen directly pressure > - lost door. no door no. 2 ↓ ↓ sample > - Apressure HPLC 88  components of a HPLC column – stationary phase depends on chromatographic technique used detector (with recorder) – UV-VIS absorbance, fluorescence, refractive index sample collector > - Auto collect 89  pump for maintaining high flow rate that is crucial for the high resolution of HPLC (expensive) Staable * need to output at least 5000 p.s.i. (pound per square inch) need to deliver sustainable and constant pressure 90  column made of stainless steel to withstand the high pressure column packing materials: – microporous supports Porous materials like silica, alumina or sephadex – pellicular (superficially porous) porous particles are coated on an inert solid support (silica, glass, polystyrene) – bonded phases Stationary phase chemically bonded onto insert solid support (e.g. silica) 91  bonded phases stationary phase chemically bonded onto insert solid support (e.g. silica) stationary phase will not be washed off with repeated uses; ideal for liquid-liquid chromatography normal phase – stationary: polar (e.g. alkyl nitrile) – mobile: non-polar (e.g. hexane) reverse phase – stationary: non-polar (e.g. C18 hydrocarbon) ↳ hydrophobic – mobile: polar (e.g. acetonitrile/water) * 92 hydrophilic sample  solvent (mobile phase) choice depends on the chromatographic technique used 2 main types of solvent elution methods – isocratic elution: single composition of solvent used throughout the elution – gradient elution: composition of the developing solvent is continuously changed “degassing” of solvent is needed ↳ desolve leave solvent gas – e.g. by warming, ultrasonic, vacuum 93  detector UV-VIS spectrometer, fluorimeter, refractive index monitor connected in series to anther mass spectrometer (HPLC-MS) for the identification of the chemical nature of eluant as they come off the HPLC electrochemical detection: current changes when analyte passes through detector weight whole protein Ms HPLC-MS- weight : If same I diffent parts weighting ↳ release amino acids. 94  Choice of a chromatographic system Non-volatile Volatile Gas-liquid Biological Affinity chromatography Sample specificity Solubility Differing Differing molecular Hydrophobic Hydrophilic size molecular size Gel permeation Gel permeation Organic Aqueous chromatography chromatography solvent solvent Different Homologous Weakly Ionic Strongly function groups series or polar ionic Adsorption Normal phase Reverse phase Ion-exchange chromatography partition partition chromatography chromatography chromatography 95 Choice of a chromatographic system Non-volatile Volatile Gas-liquid Biological chromatography Sample specificity Affinity Solubility Differing Differing Hydrophobic Hydrophilic molecular molecular size Gel permeation size Gel permeation Organic Aqueous chromatography chromatography solvent solvent Different Homologous Weakly Ionic Strongly function groups series or polar ionic Adsorption Normal phase Reverse phase Ion-exchange chromatography partition partition chromatography chromatography chromatography 96 Choice of a chromatographic system Non-volatile Volatile Gas-liquid Biological chromatography Sample specificity Affinity Solubility Differing Differing Hydrophobic Hydrophilic molecular molecular size Gel permeation size Gel permeation Organic Aqueous chromatography chromatography solvent solvent Different Homologous Weakly Ionic Strongly function groups series or polar ionic Adsorption Normal phase Reverse phase Ion-exchange chromatography partition partition chromatography chromatography chromatography 96 Choice of a chromatographic system Non-volatile Volatile Gas-liquid Biological chromatography Sample specificity Affinity Solubility Differing Differing Hydrophobic Hydrophilic molecular molecular size Gel permeation size Gel permeation Organic Aqueous chromatography chromatography solvent solvent Different Homologous Weakly Ionic Strongly function groups series or polar ionic Adsorption Normal phase Reverse phase Ion-exchange chromatography partition partition chromatography chromatography chromatography 96