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  1. 1. THE COLUMN
  2. 2. Different particle configurations. Totally porous silica particles are the most common because of their greater column capacity and availability in a wider variety of options (stationary phase, particle and pore size, column dimensions, etc.). The most popular particles have diameters in the 1.5- to 5-μm range.
  3. 3. Pore size and surface area are usually related reciprocally. For compounds have >500 Da, the average pore diameter are about 1.2 nm. For compounds with molecular weights <500 Da, the average pore diameter should preferably be about 9 nm or larger. Larger molecules require larger pores; for example, proteins are usually separated with 30-nm-pore particles. Particle surface-area, average pore-diameter, and pore-diameter distribution typically are measured by the adsorption of nitrogen or argon, using the Brunauer–Emmett–Teller (BET) procedure.
  4. 4. Visual appearance of several silica particles for RPC; magnification in (b)is7×greater than in(a).
  5. 5. Column efficiency as a function of particle size and type. Sample, naphthalene. Conditions: 50 × 4.6-mm, C18 columns; mobile phase is 60%acetonitrile-watermobile phase; 23◦C.
  6. 6. Narrower particle-size- distribution for the superficially porous (Halo™)pack- ing of Figure 5.3, compared with that of a commercial totally porous packing. Cross-section of superficiallyporous (Halo™) particles with 9-nm pores (electron micrograph).
  7. 7. Silica surface showing different types of silanols.
  8. 8. Separation of protonated basic compounds on type-A (a) compared with type-B (b) columns. Sample: four tricyclic antidepressants. Conditions: 150 × 4.6-mm C18 columns; mobile phase is 30%acetonitrile-water with pH-2.5 phosphate buffer.
  9. 9. Aggregation of microparticles to form totally porous particles.
  10. 10. Cross section of representative monolith packings (electron micrographs). (a) Silica-based; (b) polymeric.
  11. 11. RPC packings usually are made by covalently reacting (‘‘bonding’’) an organosilane with the silanols on the surface of a silica particle to form the stationary phase or ligand R: Synthesis of various bonded-phase column packings by the reaction of a silane with silica. (a, d),Monomeric packings; (b, c), potentially polymeric packings.
  12. 12. Some alternative bonded phases based on different reaction conditions.
  13. 13. Some alternative bonded phases based on different reaction conditions.
  14. 14. Options for increasing the stability of alkylsilica columns. (a, b), protection of the—Si–O–bond by a steric-protected bonded phase (for low-pH conditions only); (c, d)protection of the bonded phase by end-capping.
  15. 15. Synthesis of organic/inorganic hybrid particle. Courtesy ofWaters Corporation.
  16. 16. RPC columns classified according to the ligand (figures omit the connecting silane group [–Si(CH3)2–]).
  17. 17. Basis of RPC Column Selectivity (a) hydrophobic interaction (b) steric exclusion of larger solute molecules from the stationary phase (here referred to as ‘‘steric interaction’’) (c) hydrogen bonding of an acceptor (basic) solute group by a donor (acidic) group within the stationary phase (usually a silanol –SiOH) (d) hydrogen bonding of a donor (acidic) solute group by an acceptor (basic) group within the stationary phase (represented here by a group ‘‘X’’) (e) cation-exchange or electrostatic interaction between a cationic solute and an ionized silanol (–SiO−) within the stationary phase; also repulsion of an ionized acid (e.g., R–COO−) (f) dipole–dipole interaction between a dipolar solute group (a nitro group in this example) and a dipolar group in the stationary phase (a nitrile group for a cyano column) (g, h) π –π interaction between an aromatic solute and either a phenyl group (phenyl column) (g), or a nitrile group (cyano column) (h) (i) complexation between a chelating solute and metal contaminants on the particle surface
  18. 18. Comparison of retention on two different C18 columns. Data for 90 different organic compounds. Conditions: 15 × 4.6-mm columns; 50% acetonitrile-water, pH-2.8 phosphate buffer; 2.0 mL/min;35◦C
  19. 19. Characterization of Column Selectivity by means of the Hydrophobic-Subtraction Model
  20. 20. Solute-column interactions that determine column selectivity (figures omit the connecting silane group [–Si(CH3)2–]).
  21. 21. Different manifestations of steric exclusion. Shape selectivity (a) compared with steric interaction (b). (c) Separation of a mixture of 13 polycyclic aromatic hydrocarbons on a polymeric column. (d) Separation of same sample with same conditions on a monomericcolumn. (c)and(d
  22. 22. Monitoring different batches of column packing for possible changes in selectivity. Sample: dimethylaniline and toluene. Conditions: 150 × 4.6-mmZorbax Rx-C18 columns; 50%acetonitrile-waterplus pH-7 phosphate buffer; 1.6 mL/min; 22◦ C.
  23. 23. Schematic of equipment for packing columns by the slurry procedure.
  24. 24. REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL (naturally or using mobile phase pH) SAMPLES
  25. 25. Retention in RPC as a function of temperature and the polarity of the solute, mobile phase and column. Sample: as indicated in figure. Conditions: (a-c) 150 × 4.0-mm 5-μm) Symmetry C18 column, and (d) 150 × 4.6-mm (5-μm) Zorbax Stable Bond cyano column; 2.0 mL/min; mobile phase is acetonitrile/water, with mobile-phase composition (%B) and temperature indicated in figure (bolded values represent changes from [a]).
  26. 26. Where kw refers to the (extrapolated) value of k for 0% B (water as mobile phase), S is a constant for a given solute when only %B is varied, and φ is the volume-fraction of organic solvent B in the mobile phase (φ ≡ 0.01% B). Variation of log k with%B. Sample is 4-nitrotoluene. Conditions: 250 × 4.6-mm (5-μm) Zorbax C8 column; mobile phase consists of organic/watermixtures; 35◦ C; 2mL/min.
  27. 27. Separation of a mixture of four nitro-substituted benzenes as a function of solvent strength (%B).Sample: 1, nitrobenzene; 2, 4-nitrotoluene; 3, 3-nitrotoluene; 4, 2-nitro-1,3-xylene. Conditions: 100 × 4.6-mm (3-μm) Zorbax C8 column; mobile phase consists of acetonitrile-water mixtures (varying %B); 35◦ C; 2 mL/min.
  28. 28. Different possibilities for the retention of a solute molecule in reversed-phase chromatography. (a) Solvophobic interaction; (b) adsorption ; (c) partition; (d) comparison of RPC retention (k) with octanol-water partition P; sample; eight amino acids; column: C8; mobile phase: aqueous buffer (pH-6.7); 70 ◦C. it was observed that RPC retention (values of k) correlates with partition coefficients P for the distribution of the solute between octanol and water.
  29. 29. • Relation of K (related to tR) and P (related to polarity of sample) suggests that a partition process best describes RPC retention. • However, later studies showed that correlations of log P versus log k, as in last Figure (for amino acids) are less pronounced when the sample consists of molecules with more diverse structures, which makes the latter argument on behalf of partition less compelling.
  30. 30. A surprising observation was made for the RPC retention of various homologousseries (CH3–[CH2]n − 1 –X), where X representsa functional group such as –OH or –CO2CH3. The plot of log k versusn for a homologousseries and a C8 column; a discontinuity in the expected linear plot (dashed line) is observed (arrow) when n equals 8 for the solute (CH3 –[CH2]7 –X). It was concluded from this observation that the contribution to retention for successive –CH2- groupsin the solute becomes slightly smaller when the length of the solute molecule just exceeds the length of the alkyl ligand. Presumably there is a decreased interaction with the column for solute molecules that are too long to penetrate fully into the stationary phase (or attach to a single column ligand), with a correspondingdecrease in the retention of –CH2-groupsthat ‘‘stick out of’’ the stationary phase.
  31. 31. (a) Illustrative plot of log k versus number of –CH2-groups n for a homologous series CH3 – (CH2)n−1 –X;C8 column; (b) illustration of the ‘‘overlapping’’ of alkyl chains in the solute and column; (c–f ) plots of experimental methylene selectivity αCH2 versus carbon number nc for indicated columns of differing ligand length. Average data for several homologous series; 90%methanol-water as mobile phase; 25◦C. This figuresupports the solvophobic-interaction model.
  32. 32. • Increasing amounts of the B-solvent (e.g., acetonitrile) are taken up by the stationary phase as %B increases. Likewise some solutes may interact with underivatized silanols present on the particle surface. • Thus there is not a clear distinction between partition and adsorption in RPC
  33. 33. Role Stationary Phase The use of mobile phases that are predominantly aqueous (φ ≈ 0) can lead to greatly reduced sample retention—the opposite of that predicted. When first observed, this reduced retention was attributed to ‘‘phase collapse,’’ whereby alkyl ligands clump together and tend toward a horizontal rather than vertical alignment with the particle surface.
  34. 34. Selectivity The most effective way to improve the resolution (or speed) of a chromatographic separation is to initiate a change in relative retention (selectivity).
  35. 35. Separation of a moderately irregular sample (mixture of eight nitro-aromatic compounds) as a function of solvent strength (%B).Sample: 1, nitrobenzene; 2, 2,6-dinitrobenzene; 3, benzene (shaded peak); 4, 2-nitrotoluene; 5, 3-nitrotoluene; 6, toluene; 7, 2-nitro-1,3- xylene; 8, 1,3-xylene. Conditions: 100 × 4.6-mm (3-μm) Zorbax C8 column; mobile phase consists of acetonitrile/water mixtures; 35◦ C; 2 mL/min. Regular samples are often composed of structurally similar molecules; for example, in the last separation Figure that the sample is a mixture of mono-nitro alkylbenzenes. Here we have irregular separation because there are different compounds.
  36. 36. Separation of a mixture of substituted benzenes as a function of solvent strength (%B). Sample: 1, p-cresol; 2, benzonitrile; 3, 2-chloroaniline; 4, 2-ethylaniline; 5,3,4-dichloroaniline; 6, 2-nitrotoluene; 7, 3-nitrotoluene; 8, toluene; 9,3- nitro-o-xylene; 10,4-nitro-m-xylene.. Solvent-type selectivity: Separation of a mixture of substituted benzenes with methanol or mixtures of methanol- acetonitrile as mobile phase. Solvent-Type Selectivity
  37. 37. Solvent-typeselectivity: fine-tuning the B-solvent.Same sample and conditions as in Figures upper (peaks 1–4 only), plus added figure (d); (b) is 30%MeOH + 23%ACN, and (d) is 35%MeOH + 20%ACN.
  38. 38. Solvent-strength nomograph for reversed-phase HPLC (adapted from [28]).Two mobile phases of equal strength (46%ACN and 57%MeOH) marked by •,asanexample
  39. 39. Temperature Retention of polycyclic aromatic hydrocarbonsas a function of separation temperature. (a) Sample (fused-ringaromatic hydrocarbons): 1, anthracene; 2, fluoranthene; 3,triphenylene; 4, chrysene; 5, 3,4-benzofluoranthene; 6, 1,2,5,6-dibenzoanthracene. (b) (b) Sample same as (a), plus added poly-aryls: A,1,1 ,-dinaphthyl; B, 1,3,5- triphenylbenzene; C, 9,10- diphenylanthracene.
  40. 40. Separation of a mixture of 10 organic compoundsof diversestructure on four differentcolumns. Sample: 1, 4-nitrophenol; 2, 5,5- diphenylhydantoin; 3, acetophenone; 4, benzonitrile; 5, 5- phenylpentanol; 6,anisole; 7, toluene; 8, cis-chalcone; 9, ethylbenzene; 10, trans- chalcone. Column Selectivity
  41. 41. Separation of isomers with a cyclodextrin-bonded column. Conditions: 250 × 4.6-mm (5-μm) Cyclobond I column; 30% acetonitrile–pH-4.5 buffer; 35◦C; 2.0 mL/min. While it is possible to achieve the baseline separation of some isomers by RPC with alkylsilica columns, the use of a cyclodextrin column may be a better choice Silver-ion complexation of olefins has been found to be a useful means for enhancing the RPC separation of cis-from trans-olefin isomers.
  42. 42. Column selectivity changes: (1) a change in column source (i.e., part number (2) a change in separation conditions (‘‘method adjustment’’).
  43. 43. A change in column source Example of the use of values of Fs to select columns of similar selectivity for possible replacement in a routine HPLC assay. Gradient separations where only the column is changed for the separations of a–d. Asterisks mark peaks of interest, values of Fs calculated from Equation (5.5) (ionic [not neutral] sample).
  44. 44. Example of method adjustment for a seven-component mixture of neutral compounds. Sample: 1, oxazepam; 2, flunitrazepam; 3, nitrobenzene; 4, 4 nitrotoluene; 5,benzophenone; 6, cis-4- nitrochalcone; 7, naphthalene. Conditions: 150 × 4.6-mm C18 column (B differs from A only in a 10% lower ligand coverage); 2.0 mL/min; acetonitrile-water mobile phases; other conditions shown in figure. Method adjustment
  45. 45. Comparison of separation by an original versus ‘‘orthogonal’’ method. Gradient separations where the column and organic solvent are changed (mobile-phase pH = 6.5 for both a and b). Asterisks mark gradient artifacts (not solute peaks). Orthogonal Separation
  46. 46. General method-developmentapproach for use in this and following chapters.
  47. 47. Multiple-Variable Optimization Multiple-variable optimization in each case relies on an experimental design: a plan for the required experiments, as illustrated for certain combinations of conditions that affect selectivity for neutral samples.
  48. 48. Experimental designs for the simultaneous optimization of various separation conditions for optimum selectivity. (a) Solvent strength (%B) and temperature (T); (b) solvent strength and solvent type (MeOH and ACN); (c) solvent type (MeOH, ACN, and THF).
  49. 49. Preferred solvents for maximum change in solvent-typeselectivity. Tetrahydrofuran (THF) is used less often because of its higher UV cutoff,susceptibility to oxidation, slower column equilibration when changing the mobile phase (e.g., from THF/water to ACN/water), and incompatibility with PEEK tubing . Mixtures of Different Organic Solvents
  50. 50. Use of seven solvent-type-selectivity experiments for the separation of a mixture of nine substituted naphthalenes. Sample substituents are: 1,1-NHCOCH3; 2,2-SO2CH3; 3,2-OH; 4,1- COCH3; 5,1-NO2;6,2-OCH3;7, -H (naphthalene); 8,1-SCH3; 9, 1-Cl. Mobile phases (circled): 1, ACN; 2,MeOH; 2 exchange: 1, ACN; 2,MeOH; 3, 39%tetrahydrofuran/water; 4,1:1mixture of 1 and 2; 5, 1:1 mixture of 2 and 3; 6, 1:1 mixture of 1 and 3; 7, 1:1:1 mixture of 1, 2, and 3.
  51. 51. Separation of six steroids by changes in solvent strength (%B) and type. Sample: 1, prednisone; 2, hydrocortisone; 3, cortisone; 4, dexamethasone; 5, corticosterone;6, cortexolone.
  52. 52. Separation of a mixture of 6 organic compounds of diverse structure by changes in solvent strength (%B) and temperature. Sample: 1, methylbenzoate; 2, benzophenone; 3, toluene; 4, naphthalene; 5, phenothiazine; 6, 1,4-dichlorobenzene. Conditions: 125 × 3.0-mm C18 column; mobile phase acetonitrile/watermixtures; 1.0 mL/min.
  53. 53. Optimized separation of a mixture of 10 organic compounds of diverse structure on four different columns by varying solvent strength (%B) and temperature. Sample and conditions as in Figure 6.14, except as indicated in figure.
  54. 54. Illustrations of a change in column conditions to either improve resolution or decrease run time. Sample components (non-ionized for these conditions; pH-2.6): 1,phthalic acid; 2, 2- nitrobenzoic acid; 3, 2-fluorobenzoic acid; 4, 3-nitrobenzoic acid; 5;2-chlorobenzic acid; 6, 4- chloroaniline; 7, 3-fluorobenzic acid; 8, 2,6-dimethylbenzoic acid;9, 2-chloroaniline; 10, 3,4- dichloroaniline. Conditions: 4.6-mm C18 columns (5-μm) withindicated lengths L; mobile phase is 30%ACN-buffer for (a)and(b); 40%ACN-buffer for (c)and (d); 40◦Cin(a)and(b),30◦ Cin(c)and(d); flow rates indicated in figure
  55. 55. NONAQUEOUS REVERSED-PHASE CHROMATOGRAPHY (NARP) The mobile phase for NARP separations will thereforeconsist of a mixture of more polar (A-solvent) and less polar (B-solvent) organic solvents.Often the A-solvent will be ACN or MeOH, while the B-solvent can be THF, methylene chloride, chloroform,methyl-t-butyl ether (MTBE), or other less polar organic solvents.
  56. 56. Non-aqueousreversed-phase(NARP) separations of carotenes.Conditions: 250 × 4.6-mm C18 column; 8%chloroform-ACNmobile phase; 2.0 mL/min; ambient temperature.
  57. 57. SPECIAL PROBLEMS • (1) poor retention for very polar samples (k ≥ 1) SOLVING: a. changing mobile phase pH, b) ion pair reagents, c) using higher surface area (smaller pore diameter), d) Using normal phase chromatography • (2) peak tailing (asymmetry factors As >2) SOLVING: a) Using TFH or TEA for acid and base samples interaction with sp, b) Using another column or guard column (new one)
  59. 59. NPC used for (1) analytical separations by thin-layer chromatography (TLC, Section 1.3.2), (2) the purification of crude samples (preparative chromatography and sample preparation), (3) the separation of very polar samples (4) the resolution of achiral isomers.
  60. 60. Packing NPC • Inorganic: alumina, magnesia, magnesium silicate (Florisil), and diatomaceous earth (Celite, kieselguhr), • Synthetic (unbonded) silica: a more neutral, less active surface, with less likelihood of undesirable sample reactions during separation strong particles of controlled size and porosity that can withstand the high pressures.
  61. 61. Three polar-bonded-phase packings (1) cyano columns, where –(CH2)3–C ≡N groups are bonded to silica particles, (2) diol columns bonded with –(CH2)3 –O–CH2 – CHOH–CH2OH groups, and (3) amino columns with –(CH2)3 –NH2 ligands.
  62. 62. mono-substituted benzenes (substituents indicated for each peak; e.g., –H is benzene, –Cl is chlorobenzene). Conditions: 150 × 4.6-mm silica (5-μm particles); 20%CHCl3-hexane mobile phase; ambient temperature; 2.0 mL/min. (a) Chromatogram is recreated from data of [1]; (b) retention of (a) compared with RPC retention from Figure 2.7c for benzenes substituted by the same functional group (50%acetonitrile-water as RPC mobile phase).
  63. 63. • Because the column in NPC is more polar than the mobile phase, more- polar solutes will be preferentially retained or adsorbed—the opposite of RPC. • But the correlation of Figure 8.1b is moderately strong (r2 = 0.76), there is also significant scatter of the data. That is, NPC separation cannot be regarded as the exact opposite of RPC retention.
  64. 64. Differences NPC vs RPC (1) Different Polarity of mp and sp (2) Different behaviour for the number n of alkyl carbons in the solute molecule (its carbon number Cn), (3) Difference conditions for isomeric solutes separation.
  65. 65. Comparison of NPC separation (a) with RPC separation (b–d) for a mixture of alkyl-substituted anilines. Conditions: 150 × 4.6-mm C8 column (5-μm particles) in (a), 150 × 4.6-mm cyano column (5-μm particles) in (b–d); mobile phase is 60% methanol–pH-7.0 buffer in (a), and 0.2%isopropanol-hexane in (b); ambient temperature and 2.0 mL/min in (a) and (b). Sample (peak numbers): 1–3, 2-, 3- and 4-methylaniline; 4, 2,6-dimethylaniline; 5, 2-ethylaniline; 6, 2,5-dimethylaniline; 7, 2,3-dimethylaniline; 8, 2,4-dimethylaniline; 9, 3-ethylaniline; 10, 4-ethylaniline; 11, 3,4-dimethylanilne; 12, 2,4,6- trimethylaniline; 13,2-i-propylaniline; 14,4-i-propylaniline.
  66. 66. That is, NPC can separate solutes of differing functionality, but differences in solute carbon number have much less effect on retention. NPC permits the group-separation of petroleum samples into saturated hydrocarbons, olefins, benzenes, and various polycyclic aromatic hydrocarbons—according to the number of double bonds in the molecule, but with little effect of differences in alkyl substitution or solute molecular weight. Similarly lipid samples can be resolved into mono-, di-, and tri- glycerides (as well as other compound classes).
  67. 67. Theory Retention in NPC is best described by a displacement process, based on the fact that the silica surface is covered by a monolayer of solvent molecules that are adsorbed from the mobile phase. Consequently, for a solute molecule to be retained in NPC, one or more previously adsorbed solvent molecules must be displaced from (leave) the silica surface in order to make room for the adsorbing solute.
  68. 68. Hypothetical examples of solute retention on silica for chlorobenzene (a,b non- localized) and phenol (c,d localized). Mobile phase in (a,b) is a less-polar solvent (CH2Cl2); mobile phase in (c,d) is a more-polar solvent (tetrahydrofuran, THF).
  69. 69. Retention differs for a more-polar mobile-phase solvent such as THF and a more polar solute such as phenol. Here the interaction of solvent and solute molecules with surface silanols will be stronger, as indicated by the arrows that connect the two interacting species—in contrast to the weaker and less specific interactions shown for phenol sample in CH2Cl2 (as mp). As a result there is a ratio 1 :1 interaction of a surface silanol with a polar group in a molecule of either solute or mobile phase—called localized adsorption. Under these conditions adsorbed molecules can assume a vertical rather than flat configuration.
  70. 70. Solvent nomograph for normal-phase chromatography and silica columns.
  71. 71. Solvent-strength selectivity in normal-phase chromatography.Sample: 1,2- aminonaphthalene; 2, 2,6-dimethylquinoline; 3, 2,4-dimethylquinoline; 4, 4- nitrophenol; 5, quinoline; 6, isoquinoline. Conditions: 150 × 4.6-mm silica column (5-μm particles); ethylacetate (B)-cyclohexane (A) mixtures as mobile phase; ambient temperature; 2.0 mL/min. Peaks 1 and 4 are shaded to emphasize their change in relative retention as %B is varied.
  72. 72. Corresponding separations by TLC and column chromatography (involving the same sample, mobile phase, temperature, and especially the same silica as stationary phase) should yield similar values of k for each compound in the sample. The RF value of a solute in TLC is defined as its fractional migration from the original sample spot (point at which the sample is applied) toward the solvent front (end of solvent migration during TLC). Use of TLC Data for Predicting NPC Retention
  73. 73. Solvent-strength selectivity in normal-phase chromatography. Sample: 1,2-aminonaphthalene; 2, 2,6- dimethylquinoline; 3, 2,4-dimethylquinoline; 4, 4-nitrophenol; 5, quinoline; 6, isoquinoline. Conditions: 150 × 4.6- mm silica column (5-μm particles); ethy-lacetate (B)-cyclohexane (A) mixtures as mobile phase; ambient temperature; 2.0 mL/min. Peaks 1 and 4 are shaded to emphasize their change in relative retention as%B is varied.
  74. 74. • Experimental studies have shown that solvent- type selectivity in NPC depends mainly on the strength of the B-solvent (ε0 B). • As ε0 B increases, the B-solvent becomes more strongly attached to a specific silanol, resulting in localized adsorption of the B-solvent.
  75. 75. Exampleof solvent-type selectivity for normal-phasechromatography. Sample: 1, 2-methoxynapthalene; 2, 1-nitronapthalene; 3, 1,2- dimethoxynapthalene; 4,1,5-dinitronapthalene; 5, 1-naphthaldehyde; 6, methyl-1-naphthoate; 7, 2-naphthaldehyde; 8, 1-naphthylnitrile;9, 1- hydroxynaphthalene; 10, 1-acetylnapthalene; 11, 2- acetylnapthalene; 12, 2-hydroxynaphthalene. Conditions: 150 ×4.6-mm silica column (5-μm particles); mobile phases (%v) indicated in figure (50% water-saturated), except that (c) contains 6% added CH2Cl2to achievemiscibilityof ACN (hexane is the A-solvent in each case) 35◦ C; 2 mL/min.(a–c) Separations withindicated mobilephases;(d–f ) correlations of retention data from (a–c).
  76. 76. Comparison of NPC separation (a) with RPC separation (b–d)for a mixture of alkyl-substituted anilines. Conditions: 150 × 4.6-mm C8 column (5-μm particles) in (a), 150 × 4.6-mm cyano column (5-μm particles) in (b–d); mobile phase is 60% methanol–pH-7.0 buffer in (a), and 0.2%isopropanol-hexane in (b); ambient temperature and2.0mL/min in (a)and(b). Sample (peak numbers): 1–3, 2-, 3- and 4-methylaniline; 4, 2,6-dimethylaniline; 5, 2-ethylaniline; 6, 2,5-dimethylaniline; 7, 2,3-dimethylaniline; 8, 2,4-dimethylaniline; 9, 3-ethylaniline; 10, 4-ethylaniline; 11, 3,4-dimethylanilne; 12,2,4,6- trimethylaniline; 13,2-i-propylaniline; 14,4-i-propylaniline.
  77. 77. • Isomeric solutes of identical alkyl-carbon number (e.g., C1, consisting of o-, m-, and p- methylanliline) are seen to be bunched together, while solutes of differing carbon number (e.g., C1 vs. C2) are well separated.
  78. 78. Column Selectivity Comparison of retention and selectivity among different NPC columns. Sample: 1, chrysene; 2, perylene; 3, 1-nitronaphthalene; 4, 1-cyanonaphthalene; 5,2- acetonaphthalene; 6, naphthalene-2,7- dimethylcarboxylate; 7, benzyl alcohol. Conditions: 150 × 4.6-mm columns (column type indicated in figure); hexane mobile phase; 35◦ C; 2.0 mL/min. Chromatograms (a − c) reconstructed from data of [26]; (d) estimated from data of [1] (note extreme change in retention range for silica column d vs. polar-bonded columns a–c).
  79. 79. Factors that contributeto isomer selectivity for NPC separation on silica columns. (a, b) Steric hindrance; (c, d) electron donation; (e, f ) relative positions of polar groups within the solute molecule; (g) intramolecular hydrogen bondingof two polar groups.
  80. 80. In the case of polar-bonded-phaseNPC columns, steric hindrance effects (Figs. 8.13a,b) will be less important because the silica surface is further removed from the polar cyano, diol, or amino group of the stationary phase—hence contributingless to steric hindrance between the solute and the stationary phase. Similarly the matching of polar groupsin the solute molecule with polar groupsin the stationary phase (Figs. 8.13e,f ) will be easier for a polar- bonded-phasecolumn (with less effect on isomer selectivity) because the cyano, diol, or amino groupsare not rigidly positioned on the surface but are connected to the silica surface by a flexible –CH2 –CH2 –CH2 –linkage. Finally, the attraction of polar groupsin the solute molecule to the polar stationary phase is weaker for polar-bonded-phasecolumnsthan for silica, which in turn reduces the effect of each of the contributions to isomer separation in Figure 8.13.
  82. 82. HYDROPHILIC INTERACTION CHROMATOGRAPHY (HILIC) Hydrophilic interaction chromatography (HILIC) can be regarded as normal-phase chromatography with an aqueous-organic mobile phase; for this reason it is sometimes referred to as ‘‘aqueous normal-phase chromatography.’’
  83. 83. Separation of a mixture of derivatized oligosaccharides by HILIC with mobile phases of varying%-water. Conditions: 200 × 4.6-mm PolyHydroxyethyl A column (5-μm particles); mobile phases are water-acetonitrile as indicated in the figure; 2 mL/min.
  84. 84. Columns Subsequently a variety of different bonded-silica packings have been employed for HILIC, which can be categorized as follows: bare silica, polar neutral (e.g., cyanopropyl), diol-bonded, amide-bonded, polypeptide-bonded, positively charged amine-bonded (anion-exchange), negatively charged (cation- exchange), and zwitterionic phases.
  85. 85. HILIC Problems • Peak shape (both fronting and tailing) • Column bleed • Irreversible sorption • Slow • equilibration of the column