MAP 2023- Chromatograpy basics.pdf

Chromatography basics Govert W. Somsen Jesper Ruiter Sectie BioAnalytische Chemie Vrije Universiteit Amsterdam [email protected] MAP 3 November 2023 chromatography from Greek: Χρώμα (chroma) = color; γραφειν (grafein) = writing dye separation on calcium carbonate (chalk) using petroleum ether/eth...

Chromatography basics Govert W. Somsen Jesper Ruiter Sectie BioAnalytische Chemie Vrije Universiteit Amsterdam [email protected] MAP 3 November 2023 chromatography from Greek: Χρώμα (chroma) = color; γραφειν (grafein) = writing dye separation on calcium carbonate (chalk) using petroleum ether/ethanol as mobile phase chromatography: a separation technique many (quantitative) analyses require separation of analytes from interferents and matrix components chromatography is the predominant separation technique in analytical chemistry chromatography instruments and columns are found in virtually all chemical laboratories separation of plasma amino acids high performance liquid chromatography (HPLC) Chromatogram of amino acids in plasma. 1) Aspartic acid; 2) Glutamic acid; 3) Asparagine; 4) Serine; 5) Glutamine; 6) Histidine; 7) Glycine; 8) Threonine; 9) Citrulline; 10) Arginine; 11) Alanine; 12) Taurine; 13) Tyrosine; 14) Valine; 15) Methionine; 16) Tryptophan; 17) Phenylalanine; 18) Isoleucine; 19) Ornithine; 20) Leucine; 21) Lysine; I.S. (internal standard): Norvaline. separation of nucleotides fast liquid chromatography separation of peptides (protein fragments) high resolution liquid chromatography tryptic digest of proteins from Escherichia coli bacteria proteomics goal identification of all proteins in an organism or cell approach 1. isolate all proteins by sample preparation 2. cut proteins into fragments (peptides) using the enzyme trypsine 3. separate the complex peptide mixture with liquid chromatography 4. identify the peptides one by one using mass spectrometry 5. identify proteins by their typical peptides separation of oil components two-dimensional gas chromatography before chromatography: sample pretreatment preparing a solution from a sample that can be analysed by chromatography Lecture November 17 basic partitioning: liquid-liquid extraction org aq A A Lecture November 17 P is partition coefficient principle of chromatography partitioning of analytes between a stationary and mobile phases stationary phase stands still, does not move A stat mobile phase moves along/through the stationary phase A mob analyte with higher affinity for stationary phase will on average move slower than analyte with less affinity mobile phase analyte type of chromatography gas in gas phase (high temperature) gas chromatography liquid in solution liquid chromatography stationary phase stationary phase: small particles (1-10 µm diameter) partitioning of analytes between the surface of the stationary phase particles and the flowing mobile phase surrounding the particles stationary phase type of chromatography particles on flat plate thin layer chromatography (TLC) particles packed in column HPLC; early GC coating on capillary wall capillary GC; open tubular LC thin layer chromatography (TLC) thin layer of small particles on a plate solvent front separation chamber for mobile phase mobile phase applying samples to the plate mobile phase mobile phase moves up take plate out before by capillary action front reaches top column chromatography: packed small particles (1 - 10 µm) packed in a tube - steel tubes LC - internal diameter: 1 - 6 mm - length: 5 - 25 cm mobile phase is pushed through column by pressure early GC - steel or glass tubes - internal diameter: 2 - 4 mm - length: 1.5 - 10 m column chromatography: wall-coated thin film of stationary phase (0.1 - 5 µm) - standard format in GC on inner column wall - hardly used in LC - fused-silica capillaries - internal diameter: 0.1 - 1 mm - length: 10 - 100 m stationary phase film fused-silica capillary planar and elution chromatography planar chromatography elution chromatography mobile phase solvent front z0 tR: retention time 0 < Rf < 1 zB zA mobile phase • • • analytes detected on chromatographic bed after stopping mobile phase flow all analytes have the same migration time migration distances are measured • • • • continuous flow of mobile phase analytes detected as they emerge from the column all analytes have the same migration distance retention times are measured HPLC high pressure is needed to push mobile phase through column: high pressure liquid chromatography or high performance liquid chromatography (HPLC) tubing injector pressure: 50-1200 bar pump injection volumes: 0.5-10 µL flow rates: 0.1-2 mL/min mobile phase reservoir column UV absorbance detector data system waste HPLC instrument HPLC separation and detection column detector chromatogram sample injection light continuous flow of mobile phase time detector signal chromatogram time (min) retention time A t0 tR,A B tR,B tR: retention time t0: tR of unretained compound (no retention) t’R: corrected retention time = tR – t0 retention factor k: retention factor of a compound retention factor is: - compound property - dependent on stationary and mobile phase properties - independent of flow rate and of column length and diameter dynamic equilibrium: partition coefficient retention results from dynamic partitioning between stationary and mobile phase Amob stationary phase mobile phase at any time during the separation: Astat As Am , nA,stat: number of molecules A in stat phase , nA,mob: number of molecules A in mob phase Vstat: volume stationary phase in column Vmob: volume mobile phase in column stationary phase material most common: microporous silica (SiO2) particles  diameter: 1-10 µm  porous: high specific surface  pressure resistant  compatible with many solvents  stable up to pH 8 crystal structure surface of silica particles is highly polar due to Si-OH (silanol) groups normal phase LC (NPLC) stationary phase: polar mobile phase: apolar e.g. silica e.g. hexane retention en separation governed by polar interactions between analytes and stationary phase - molecule without polar groups has little or no retention (short retention time) - molecule with many polar groups has stronger retention (long retention time) HO retention time order: < OH < OH elution strength of mobile phase in NPLC elution strength is the ability of a solvent to elute compounds (reduce retention times) decrease retention by making the mobile phase more polar (less apolair) increase polarity of mobile phase by adding relatively more polar solvent (modifier) example hexane–ethyl acetate (50:50, v/v) hexane–acetone (80:20, v/v) in NPLC a more polar mobile phase has a higher elution strength effect of elution strength in NPLC column: 150 x 4.6 mm packed with silica particles (5 µm) mobile phase: cyclohexane - ethyl acetate; 2.0 ml/min 25% ethyl acetate 1. 2-aminonaphtalene 2. 2,6-dimethylquinoline 3. 2,4-dimethylquinoline 4. nitrophenol 5. quinoline 40% ethyl acetate 6. isoquinoline quinoline isoquinoline chemical modification of silica surface C18 (or C8, C4, C2) , surface of particles becomes strongly apolar due to alkyl chains reversed phase LC (RPLC) stationary phase: apolar mobile phase: polar e.g. C18-silica water retention en separation governed by hydrophobic interactions between analytes and stationary phase - very polar molecule has little or no retention (short retention time) - molecule with more hydrophobic parts has stronger retention (long retention time) retention time order: < < RPLC is the most widely applied mode of HPLC elution strength of mobile phase in RPLC elution strength is the ability of a solvent to elute compounds (reduce retention times) decrease retention by making the mobile phase less polar decrease polarity of mobile phase by adding less polar solvent (modifier) example water–methanol (50:50, v/v) water–acetonitrile (80:20, v/v) in RPLC a less polar mobile phase has a higher elution strength relative elution strength of solvents in RPLC increasing polarity water H2O methanol (MeOH) CH3-OH acetonitrile (ACN) tetrahydrofuran (THF) CH3-CN increasing elution strength effect of elution strength in RPLC column: 150 x 4.6 mm packed with C18-silica particles (5 µm) mobile phase: water-acetonitrile; 1.0 ml/min 1. 30% acetonitrile 2. 3. 4. 60% acetonitrile 4 5 5. detector signal separation in chromatography A tR,A time selectivity factor  t 'R,B t 'R, A B  t R , B  t0 t R , A  t0 tR,B α α>1 detector sigaal resolution in chromatography (1) detector signal time time tR,A tR,B α is equal, however, separation (resolution) is not the same resolution (RS) depends on: - difference in retention times - width of the peaks , , peak width in chromatography (1) ideal chromatographic peak has   a bell shape according a Gaussian function width of a Gaussian peak is expressed by its standard deviation σ peak width in chromatography (2) h: peak height w0.5: peak width at half height w0.5 h/2 h w0.5 = 2.354σ wb: peak width at base width at base between the tangents of the inflection points wb wb = 4σ resolution in chromatography (2) detector signal ΔtR time , wb,A wb,B tR,A tR,B , , , , , , , resolution in chromatography (3) band broadening in chromatography (1) band broadens along the column x  l2  Hx σl: standard deviation of peak in length units x: distance peak travelled through the column H: proportionality constant band broadening in chromatography (2)   Hx 2 l efficient column: narrow peak, small H less efficient column: wide peak, large H plate height H when peak has passed the entire column x = L, so:   HL 2 l H  l2 L H: plate height σl: standard deviation of peak in length units L: length of column plate height “the capacity of a column to minimize band broadening” abstract term ‘plate’ originates from destillation theory (there are no real plates in chromatography) narrow peak: small H wide peak: large H plate number N (1) ‘number of plates with height H’ per column in chromatographic practice, we measure retention times and peak widths (σ) in time units; transformation of equation to time units (via velocity of mobile phase (in m/s)) yields: N: plate number σt: standard deviation of peak in time units tR: retention time of peak narrow peak: large N wide peak: small N plate number N (2) in chromatographic practice, peak width is measured in a chromatogram as w0.5 or wb w0.5 = 2.354σ wb = 4σ σ = w0.5 / 2.354 σ = wb / 4 resolution in HPLC (3) detector signal ΔtR time , wb,A wb,B tR,A tR,B , if for two closely spaced peaks A and B we assume that wb,A = wb,B it follows that: RS: resolution NA: plate number of A α: selectivity factor kA: retention factor of A resolution in HPLC (4) optimizing Rs by: - increasing N: more narrow peaks - increasing α: larger difference between retention times - increasing k: more retention (up to k~10)