1. High Performance Liquid Chromatography

2. What is HPLC ? H: High P : Performance (Pressure) L : Liquid C : Chromatography

3. High Performance Liquid Chromatography (HPLC) is one mode of chromatography, the most widely used analytical technique. Chromatographic processes can be defined as separation techniques involving mass-transfer between stationary and mobile phases.

4. HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under high pressure. In the column, the mixture is resolved into its components.

5. In HPLC, several instrument and column chemistry parameters need to be optimized in order to generate a satisfactory separation. Each of the following parameters need to be optimized in order to generate a chromatogram that is suitable for qualitative or quantitative purposes.

6. Mobile phase composition Bonded phase chemistry Column and packing dimensions Injection volume Sample pre-treatment and concentration Mobile phase flow rate Column temperature Detector parameters

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8. 1: The mobile phase composition (usually water and an organic solvent plus other additives) needs to be optimized in order to gain good separation. 2: Degassers are often used to remove air from the mobile phase, leading to better chromatographic baselines. 3: The detector conditions are chosen to give the best response to the analytes of interest and to achieve good sensitivity.

9. 4: The column dimensions and stationary phase chemistry are chosen and optimized to give separations of the quality required. 5: The autosampler introduces a plug of sample into the mobile phase flow which is then swept onto the column. 6: Dual reciprocating pumps are used to deliver the mobile phase at back pressures of up to 400 bar. A steady stream of liquid delivered at a constant flow rate is important.

10. What is HPLC used for ? 1.Separation of mixed components 2.Qualitative analysis / Quantitative analysis 3. Preparation of interest components

12. The ability of HPLC to separate such a wide diversity of species leads to its popularity in a wide range of industries. It is important to note that HPLC is capable of separating analytes in the following categories: High molecular weight (>2000) Low molecular weight (<2000) Polar Non-polar Ionizable Cationic Anionic

13. Selection guide for all of the different chromatographic techniques with liquid mobile phases

14. Normal Phase Chromatography

15. In NP chromatography (also called adsorption or liquid– solid chromatography), the stationary phase is more polar than the mobile phase. The retention increases as the polarity of the mobile phase decreases, and polar analytes are more strongly retained than non-polar ones.

16. 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.

18. Stationary Phases Normal-phase (NP) chromatography generally uses the same types of mobile phase as for the adsorption mode. The difference, however, is the nature of the stationary phase. In NP chromatography, the packing is silica gel that has been bonded with a polar phase. The usual polar phases widely available from many manufacturers include cyano, amino, nitro, and diol phases.

21. Mobile Phases A variety of solvent can be used for adsorption chromatography, ranging non-polar to polar. Adsorption chromatography functions very well for the separation of non-polar and moderately polar compounds using moderately polar eluents. It does not function well with high polarity eluents.

22. In normal phase chromatography the mobile phase is more nonpolar than the stationary phase and therefore made of organic solvents. The intention of the mobile phase is to: Keep the analytes in solution; Transport the analytes through the bed of stationary phase; Contribute to the separation; Compete with the analytes for the adsorption sites on the stationary phase.

23. The strength of the mobile phase is decided by the polarity of the solvent used, and solvents can be ranked according to their solvent strength, Ԑ° (see Table). Solvent strength increases with increasing polarity. Increased solvent strength of mobile phase decreases the retention of the substances.

26. Control of sample retention in adsorption chromatography is achieved almost by modifying the composition of the mobile phase. Minor change in the eluent strength have dramatic effect on k and α values. The elution power of a solvent is measured by its solvent strength parameter ϵ 0. Often a single solvent is unable to effect resolution of sample components and binary and ternary mixtures must be employed.

27. A measure of solvent strength of these mixtures may be determined from the solvent polarity indices The polarity index, P’, is a numerical measure of the relative polarity of various solvents as determined from their solubility in some specific solvents. The polarity index P AB for a mixture can then be determined from the polarity indices of the pure components and their respective volume fractions (ф A,ф B )

28. The following equation can be used: where P’ A and P’ B are the polarity indexes of the two solvents and ɸ A and ɸ B are the volume fractions of solvents A and B.

29. Any desired polarity index can be obtained by mixing the appropriate amounts of solvents. An increase in the polarity of the solvent mixture means a stronger eluent and hence smaller k values. This expressed in the following relation:

30. Thus, a two unit change in polarity index results in a 10-fold change in k.

31. example In a normal-phase partition column, a solute was found to have a retention time of 29.1 min, and an unretained sample had a retention time of 1.05 min when the mobile phase was 50% by volume chloroform and 50% n-hexane. Calculate (i) k for the solute and (ii) a solvent composition that would bring k down to a value of about 10.

32. Reversed-phase chromatography Neutral Compounds

33. RPC is usually a first choice for the separation of both neutral and ionic samples, using a column packing that contains a less polar bonded phase such as C 8 or C 18. The mobile phase in RP chromatography is normally more polar than the stationary phase.

34. Stationary Phases Historically, silica gel was the most common material used in the early development of column liquid chromatography (LC).However, silica is a polar material that contains hydroxyl groups (silanols) that are both acidic and strongly hydrogen-bonding in character.

35. These properties make it unsuitable as a stationary phase for many typical organic molecules that are predominantly hydrophobic compounds. In addition, the silanols interact strongly with basic compounds leading to poor chromatographic results (peak tailing).

36. In order to overcome these undesirable effects of silica and to have a medium more suitable for the separation of a large variety of organic compounds, modification of the surface is necessary to provide a more non-polar (hydrophobic) material.

37. Therefore, chemical modification is the only practical approach to modifying the silica surface in order to create a stationary phase that is compatible with the types of solutes to be separated. The most common method for modifying silica in order to produce a hydrophobic surface is organosilanization.

38. To minimize unwanted interactions with residual silanol groups column packings for RPC are usually endcapped, by a further reaction of the bonded phase with a small silane such as trimethylchlorosilane or dimethyldichlorosilane. 38Imad Abu Reid

39. This procedure decreases the concentration of unreacted silanols, as well as their interaction with retained solute molecules—but does not totally eliminate silanol-solute interaction (end- capping increases the percentage of reacted silanols by only 20–30%,corresponding to a somewhat smaller decrease of unreacted silanols).

40. Reversed-phase stationary phases are more or less hydrophobic, and the degree of this property is characterized by their hydrophobicity H. As a general rule, retention times are longer the more Carbon atoms the bonded stationary phase contains.

41. Within this context, the predominant factors in determining the hydrophobicity are the length of the alkyl chain or the total number of carbon atoms as well as the bonding density.

43. Effect of chain length on retention

45. Mobile Phase The mobile phase generally consist of mixtures of water or aqueous buffer solutions with various water-miscible solvents. The strength of binary mixtures is not a well defined function of %B but depends on analyte and stationary phase properties. Nevertheless, it is possible to give numerical data which allow to obtain a good approximation of mobile phase composition when it is necessary to try more than one solvent in order to find the best selectivity.

47. The following equation can be used: where P’ A and P’ B are the polarity indexes of the two solvents and ɸ A and ɸ B are the volume fractions of solvents A and B.

49. Example In a reversed-phase column, a solute was found to have a retention time of 31.3 min, and an unretained species required 0.48 min for elution when the mobile phase was 30% (by volume) methanol and 70% water. Cal- culate (a) k and (b) a water-methanol composition that should bring k to a value of about 5.

50. Solution

52. Alternatively, a nomogram can be used. It is based on numerous experimental data determined with small organic molecules

53. Solvent strength of binary mixtures for reversed-phase chromatography

54. Problem If a separation with 70% of methanol gives adequate retention but poor selectivity, which other solvent mixtures could be tried?

55. IONIC SAMPLES: REVERSED-PHASE & ION-PAIR CHROMATOGRAPHY 55Imad Abu Reid

56. Ionizable Compounds analysis by RP

57. ACID–BASE EQUILIBRIA AND REVERSED-PHASE RETENTION The RPC retention of neutral samples decreases for less hydrophobic (more polar)molecules. When an acid (HA) or base (B) undergoes ionization (i.e., is converted from an uncharged to a charged species), the compound becomes much more polar or hydrophilic. As a result its retention factor k in RPC can be reduced 10-fold or more: 57Imad Abu Reid

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59. Knowing the pka a substance, its percent ionization at certain pH can be calculated from the equation: 59Imad Abu Reid

61. Effect of pH of the retention of basic analyte 61Imad Abu Reid

62. The retention dependence of basic components on the pH of mobile phase could be subdivided into three regions: A.Fully protonated analyte (cationic form), which shows the lowest retention. The analyte is in the most hydrophilic form. Its interactions with the hydrophobic stationary phase are suppressed. 62Imad Abu Reid

63. B. Partial protonation region. Coexistence of two analyte forms (protonated and deprotonated) in the mobile phase in equilibrium may cause poor peak shape and unstable retention. C. Analyte in its neutral form (the most hydrophobic), which shows the longest retention.

64. Effect of pH of the retention of acidic analyte 64Imad Abu Reid

65. Similar retention curves can be obtained for acidic components, but obviously their retention dependence will be the mirror image of that for basic analytes 65Imad Abu Reid

66. Knowledge of the pKa of the analytes in the mixture is very important. Significant changes in retention and even reversals in elution order can be observed. Take, for example, two analytes: one basic and one acidic, both with pKa values of approximately 4.

67. If a mixture of these two analytes were analyzed at pH 2.3 and pH 6.0, the base would show lower retention at pH 2.3 and higher retention at pH 6.0, and the acidic component would show higher retention at pH 2.3 and lower retention at pH 6.0

69. Choice of Buffers Whenever acids or bases are separated, it is necessary to buffer the mobile phase in order to maintain a constant pH and reproducible retention during the separation. In selecting a buffer for RPC separation, several buffer properties may prove relevant: 69Imad Abu Reid

70. Factors to be considered pKa and buffer capacity solubility UV absorbance (when UV detection is used) volatility (when mass-spectrometric or evaporative light-scattering detection is used) ion-pairing properties stability and compatibility with the equipment Imad Abu Reid70

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74. ION-PAIR CHROMATOGRAPHY (IPC) 74Imad Abu Reid

75. Ion-pair chromatography (IPC) can be regarded as a modification of RPC for the separation of ionic samples. The only difference in conditions for IPC is the addition of an ion-pairing reagent R + or R − to the mobile phase, which can then interact with ionized acids A − or bases BH + in an equilibrium process: 75Imad Abu Reid

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77. The use of IPC can thus create similar changes in sample retention as by a change in mobile-phase pH,but with greater control over the retention of either acidic or basic solutes, and without the need for extreme values of mobile-phase pH (e.g., pH 8). Typical ion-pairing reagents include alkylsulfonates R–SO - 3 ( −R − ) and tetraalkylammonium salts R 4 N + (R + ), as well as strong (normally ionized) carboxylic acids (trifluoroacetic acid, TFA; heptafluorobutyric acid, HFBA [R − ]),and so-called chaotropes (BF 4 −, ClO 4 −, PF 6 − ). 77Imad Abu Reid

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79. Basis of Retention Two possible retention processes or ‘‘mechanisms’’ exist for separation by IPC.As an example, we will use the ion-pairing of an ionized acidic solute A − by a tetraalkylammonium IPC reagent R +. The ion-pairing of a protonated basic Solute B + by an alkylsulfonate IPC reagent R − can be described similarly. 79Imad Abu Reid

80. The analytes form hydrophobic ion-pairs in the eluent, and the paired ions are retained on a hydrophobic stationary phase surface by their hydrophobicity. Usually less than 0.1 wt% of the counter-ion and buffer concentration is employed. The selection of a stationary phase material is simple and a variety of hydrophobic stationary phase materials for reversed-phase liquid chromatography can be used. 80Imad Abu Reid

81. The organic modifier concentration depends on the hydrophobicity of stationary phase materials used and the ion pair reagent. Increasing the size of the counter-ion increases the retention time. The maximum retention time is reached when the counter-ion concentration reaches the micelle condition. 81Imad Abu Reid

82. Relationship between analytes and counter- ions in ion-pair liquid chromatography 82Imad Abu Reid

83. Example: ion-pair liquid chromatography of amino acids. Amino acids are zwitterions. The amino group can form an ion-pair with an alkanesulfonate ion (such as octanesulfonate), and the carboxyl group can form an ion-pair with a tetrabutylammonium ion, depending on the pH of the solution. 83Imad Abu Reid

84. In reversed-phase liquid chromatography, the ionization of the solute decreases the retention. The addition of counter-ion under these conditions forces the formation of an ion-pair between the ionized solute and counter-ion, and then the retention of the analyte increases as the paired-ion is retained. 84Imad Abu Reid

85. Two mechanisms for retention in reversed-phase ion-pair liquid chromatography have been considered. One is the adsorption of the hydrophobic paired ion on the hydrophobic surface of stationary phase material. In the second mechanism, the hydrophobic counter-ion is held on the surface of the hydrophobic stationary phase, and the analyte ion is retained by ion-ion interactions. 85Imad Abu Reid

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87. The separation conditions available for the control of selectivity in IPC include: pH IPC reagent type (sulfonate, quaternary ammonium salt, chaotrope) IPC reagent concentration solvent strength (%B) solvent type (ACN, MeOH, etc.) temperature column type buffer type and concentration 87Imad Abu Reid

88. DETECTION

90. characteristics of an ideal HPLC detector have high sensitivity and predictable response respond to all solutes, or else have predictable specificity be unaffected by changes in temperature and carrier flow respond independently of the mobile phase not contribute to extra-column peak broadening 90Imad Abu Reid

91. be reliable and convenient to use have a response that increases linearly with the amount of solute be nondestructive of the solute provide qualitative information on the detected peak

92. 4.6.2 Detection Techniques There are four general techniques that are used for HPLC detection: bulk property or differential measurement sample specific hyphenated techniques 92Imad Abu Reid

93. 1. Bulk Property Detectors A bulk property detector can be considered a universal detector as it measures a property that is common to all compounds. The detector measures a change in this property as a differential measurement between the mobile phase containing the sample and that without the sample. The most familiar of the bulk property detectors is the refractive index detector.Bulk property detectors have the advantage that they detect all compounds. 93Imad Abu Reid

94. 2. Sample-Specific Detectors Some characteristic of a sample is unique to that sample, or at least is not common to all analytes, and the sample- specific detector responds to that characteristic. The UV detector is the most commonly used sample-specific detector. It responds to compounds that absorb UV light at a specific wavelength. Other common sample-specific detectors rely on the ability of an analyte to fluoresce (fluorescence), conduct electricity (conductivity), or react under specific conditions (electrochemical). 94Imad Abu Reid

95. 3. Hyphenated Techniques Hyphenated techniques refer to the coupling of an independent analytical instrument to the HPLC system to provide detection, and often are abbreviated with a hyphenas LC-(plus the technique). The most common hyphenated technique is LC-MS, where a mass spectrometer is coupled with an HPLC system. Other less widely used techniques are LC-IR or LC-FTIR and LC- NMR. 95Imad Abu Reid

96. UV-Vis detectors are the most commonly used detectors in HPLC. Solutes which absorb UV or visible radiation (typically 190 - 600 nm) can be detected. The degree of absorption is a function of the molar absorptivity of the sample molecule, the path length of the detector flow cell and the solute concentration. The solute concentration is directly proportional to the absorbance allowing quantification. UV- Vis detectors can routinely achieve detection of only a few nanograms. They have a large linear dynamic range and are very robust. I. UV Detector 96Imad Abu Reid

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99. variable wavelength UV detector 99Imad Abu Reid

100. diode-array UV detector 100Imad Abu Reid

101. Diode Array Spectral Capabilities 101Imad Abu Reid

102. General UV-Detector Characteristics 102Imad Abu Reid

103. II. Fluorescence detectors The fluorescence detector is a highly sensitive and specific detector for HPLC. A 1000 fold increase in sensitivity over UV detection is possible. About 20% of compounds can naturally absorb UV radiation becoming excited and subsequently emitting radiation at a lower energy and longer wavelength than the excitation energy. Many others can be made to fluoresce through derivatization. Radiation from a deuterium or xenon source is focused onto the first grating. 103Imad Abu Reid

104. Schematic of fluorescence detector 104Imad Abu Reid

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106. III. ELECTROCHEMICAL (AMPEROMETRIC) DETECTORS Many compounds that can be oxidized or reduced in the presence of an electric potential can be detected at very low concentrations by selective electrochemical (EC) measurements. By this approach the current between polarizable and reference electrodes is measured as a function of applied voltage. Because a constant voltage normally is imposed between the electrodes, and only the current varies as a result of solute reaction, EC detectors are more accurately termed amperometric devices. EC detectors can be made sensitive to a relatively wide variety of compound types. 106Imad Abu Reid

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108. Schematic of electrochemical detector 108Imad Abu Reid

109. IV. CONDUCTIVITY DETECTORS Conductivity detectors are most commonly used for detection of inorganic and organic ions usually after ion exchange chromatography. This detector measures the conductance of the mobile phase. The sensitivity of the detector is largely dependent upon the initial conductance of the mobile phase. 109Imad Abu Reid

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111. V. REFRACTIVE INDEX DETECTORS The refractive index detector is one of the most universal LC detectors. Anything that changes the refractive index of the mobile phase can be detected. It is also one of the least sensitive LC detectors. Refractive index detectors must always be thermostatically controlled as the refractive index will change with temperature. The most common type of refractive index detector is the beam deflection device. The Fresnel prism can be used for microbore work. The laser interferometer is the most sensitive but can be the least reliable. 111Imad Abu Reid

112. Schematic of refractive index detector 112Imad Abu Reid

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