| | Contents | |
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| | Preface | XV |
| | The Structure of the Book | 1 |
| | Part 1 (general section) | 1 |
| | Part 2 (specific questions) | 1 |
| | In Lieu of an Introduction | 3 |
| | Chromatography Crossword | 4 |
| | Across | 4 |
| | Down | 4 |
| | An HPLC-Quiz | 6 |
| | An HPLC Tale | 9 |
| | The Tale of Peaky and Chromy | 9 |
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| 1 | HPLC Tips | 11 |
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| 1.1 | Stationary Phases and Columns | 11 |
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| Tip No. | | |
| 01 | It improves with age is a rule that applies to port and sometimes to red wine, but how about your C18 column? | 11 |
| 02 | Optimization via column parameters -- what works best? | 14 |
| 03 | Can selectivity always be put down to chemical interactions with the stationary phase? | 17 |
| 04 | A matter of perspective . . . Or: Selectivity and peak symmetry of basic compounds using reversed-phase packing materials | 19 |
| 05 | Separation of isomers | 21 |
| 06 | When should I use a polar C18 phase? | 23 |
| 07 | Are polar RP- C18 phases more suitable for the separation of polar analytes than non-polar phases? | 24 |
| 08 | What about non-endcapped phases -- are they a thing of the past? | 25 |
| 09 | How can I separate acids using RP C18? | 27 |
| 10 | The nitrile phase -- some like it polar | 29 |
| 11 | The selectivity of RP columns | 31 |
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| 1.2 | Buffers, pH Value | 33 |
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| 12 | Does it always have to be potassium phosphate? | 33 |
| 13 | UV cut-off of buffer solutions | 34 |
| 14 | Sources of errors when using buffers | 35 |
| 15 | The drawbacks of using buffers | 37 |
| 16 | Why is the pH value so important, and what does it do? | 40 |
| 17 | Why does the pH value shift even though I am using the correct buffer and the buffer capacity is sufficient? | 42 |
| 18 | Changes to the pH value in the eluent: the extent of the shift and the reasons behind it | 43 |
| 19 | An unintentional pH shift and its consequences | 46 |
| 20 | RP separations in the alkaline medium | 49 |
| 21 | Separation of basic and acidic compounds contained in the same sample | 51 |
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| 1.3 | Optimization, Peak Homogeneity | 53 |
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| 22 | The peaks appear too soon -- what can be done? | 53 |
| 23 | What can I do if the peaks elute late? | 55 |
| 24 | Quick optimization of an existing gradient method | 60 |
| 25 | Increasing efficiency -- often the fast track to success | 63 |
| 26 | Additives to the eluent | 66 |
| 27 | Separating the unknown -- where shall I begin? | 69 |
| 28 | Separation of an unknown sample using a reversed-phase C18 column -- how do I go about it? | 72 |
| 29 | Developing an RP separation -- the two-day-method
Part 1: Choice of column and eluent | 74 |
| 30 | Developing an RP separation -- the two-day method
Part 2: Fine-tuning of the separation | 78 |
| 31 | Quick check on peak homogeneity -- Part 1 | 80 |
| 32 | Quick check on peak homogeneity -- Part 2 | 82 |
| 33 | Tied to a standard operating procedure -- how can a bad separation be improved further? | 84 |
| 34 | More elaborate measures to check peak homogeneity | 86 |
| 35 | First easily digestible tip | 91 |
| 36 | Second easily digestible tip | 94 |
| 37 | Third easily digestible tip | 96 |
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| 1.4 | Troubleshooting | 99 |
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| 38 | How to approach problems in a systematic manner | 99 |
| 39 | Spikes in the chromatogram | 101 |
| 40 | Additional peaks in trace analysis separations | 103 |
| 41 | What causes a ghost peak? | 105 |
| 42 | Ghost peaks in a blank gradient | 107 |
| 43 | Strange behaviour of a peak. What could be the cause? | 108 |
| 44 | When could one expect a change in the elution order of the peaks? | 110 |
| 45 | Tailing in RP HPLC -- Part 1: Fast troubleshooting | 114 |
| 46 | Tailing in RP HPLC -- Part 2: Further causes and time-served cures | 116 |
| 47 | Peak deformation and a shift in retention time due to an unsuitable sample solvent | 119 |
| 48 | Is flushing with water or acetonitrile sufficient? | 123 |
| 49 | Flushing and washing fluids for HPLC apparatus | 125 |
| 50 | When does the peak area change? | 127 |
| 51 | Reasons for a change in either peak height or peak area, but not in both | 129 |
| 52 | Excesses and their pitfalls | 131 |
| 53 | Algae, fungi and bacteria in HPLC | 132 |
| 54 | Does 40 C always mean 40C? | 134 |
| 55 | The most common reason for a lack of reproducibility is a lack of methodological robustness | 135 |
| | Have a break . . . | 138 |
| | Dear Reader | 138 |
| | Complete the sentences | 139 |
| | "Matching pairs | 140 |
| | Has Peaky remembered his lessons correctly? | 141 |
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| 1.5 | General HPLC Tips | 142 |
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| 56 | What changes can you expect when switching from one HPLC system to another? | 142 |
| 57 | What changes can be expected in a chromatogram if the dead volume is larger in one isocratic system than in another? | 144 |
| 58 | Contribution of the individual modules of the system to band broadening | 146 |
| 59 | How to keep retention times constant while reducing the diameter of the column | 148 |
| 60 | Has 3 m material been developed sufficiently to be used in routine separations? | 150 |
| 61 | Miniaturization may be all well and good -- but when does it really work and does it make sense in routine separations? | 152 |
| 62 | Why is it that peaks appear later with a new column? | 154 |
| 63 | Column length, flow and retention times in gradient separations | 155 |
| 64 | Column dimensions and gradient separations | 159 |
| 65 | What is the difference between dead time and dead volume on the one hand and selectivity and resolution on the other? | 161 |
| 66 | Troublesome small peaks | 163 |
| 67 | Lowering the detection limit by optimizing the injection | 164 |
| 68 | Setting the parameters of an HPLC instrument | 167 |
| 69 | The right wavelength -- old hat to some, a revelation to others | 171 |
| 70 | Characteristics of refraction, fluorescence and conductivity detectors | 175 |
| 71 | Does it always have to be HPLC? | 177 |
| 72 | Methanol versus acetonitrile | 180 |
| 73 | Tale of a foursome pub-crawl -- can peaks elute before the front? | 183 |
| | References to HPLC-Tips | 185 |
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| 2 | LC-MS Coupling, Micro- and Nano-LC, Quantification | 187 |
| | 2.1 by Friedrich Mandel, 2.2 by Jürgen Maier-Rosenkranz, 2.3.2--2.3.4 by Hans-Joachim Kuss | |
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| 2.1 | LC-MS Coupling | 187 |
| | LC-MS -- The one and only universal tool? | 187 |
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| 74 | Choosing the right LC-MS interface | 189 |
| 75 | Which mobile phases are compatible with LC-MS? | 195 |
| 76 | Phosphate buffers -- the exception | 197 |
| 77 | Paired ions | 198 |
| 78 | Using additives to enhance API-electrospray ionization | 200 |
| 79 | How can I enhance sensitivity of detection? | 203 |
| 80 | No linear response and poor dynamic range? | 205 |
| 81 | How much MSn do I need? | 208 |
| | Need more help? | 210 |
| | References to LC-MS | 211 |
| | Internet addresses of interest for LC-MS coupling | 212 |
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| 2.2 | Micro- and Nano-LC | 213 |
| | A short introduction | 213 |
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| 82 | Lower efficiency -- plate number too low
Part 1: Effects of dead volume in the connecting parts.
Which column diameter should be used with which capillary diameter? | 214 |
| 83 | Lower efficiency -- plate number too low
Part 2: Effects of injection amount and injection volume | 215 |
| 84 | Lower efficiency -- plate number too low
Part 3: Impact of flow cell (UV, fluorescence, radio detection) | 216 |
| 85 | No gain in sensitivity: flow cell -- path length -- S/N | 218 |
| 86 | Fused silica and PEEK capillary connections | 219 |
| 87 | Fast sample loading due to column switching | 220 |
| 88 | Injection system: full loop injection, partial loop fill injection, timed programmed injection, direct injection | 222 |
| 89 | Protecting the system: cleaning-up guard column, saturation column | 223 |
| 90 | Retention time shift: gradient delay volume, mixing chamber volume, gradient accuracy | 224 |
| 91 | Transferability -- downscaling: correct gradients | 226 |
| | References to Micro- and Nano-LC | 228 |
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| 2.3 | Quantification | 229 |
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| 2.3.1 | Practical aspects of quantification in HPLC | 229 |
| 2.3.1.1 | Peak area or peak height? | 229 |
| 2.3.1.2 | What factors have an impact on the peak area? | 230 |
| 2.3.1.3 | Formulae and short statements or comments with respect to the quantification methods | 231 |
| 2.3.1.4 | Examples with actual figures | 233 |
| 2.3.2 | Quantification in Chromatography | 236 |
| 2.3.2.1 | Optimum separation -- correct peak acquisition | 236 |
| 2.3.2.2 | Understanding the “mind” of the integration system | 240 |
| 2.3.2.3 | Setting parameters and their effect on peak area and peak height | 241 |
| 2.3.2.4 | Where can mistakes be made? | 243 |
| 2.3.3 | Methods of Quantification | 244 |
| 2.3.3.1 | What is the 100% method? | 244 |
| 2.3.3.2 | What is the external standard method? | 244 |
| 2.3.3.3 | Why use an internal standard? | 245 |
| 2.3.3.4 | In which cases should the additions method be used? | 247 |
| 2.3.4 | Weighted Regression | 249 |
| 2.3.4.1 | What about the F-test? What are the other possibilities? | 249 |
| 2.3.4.2 | How do we weight the individual values? | 250 |
| 2.3.4.3 | How to use Excel for weighted regression | 250 |
| 2.3.5 | Solutions to quantification problems | 253 |
| | References | 253 |
| 3 | Appendix | 261 |
| 3.1 | Solutions to the Problems | 261 |
| 3.1.1 | Crossword -- the solution | 261 |
| 3.1.2 | An HPLC quiz -- the solution | 261 |
| 3.1.3 | An HPLC tale with Peaky and Chromy -- the solution | 264 |
| 3.1.4 | Complete the sentences | 265 |
| 3.1.5 | “Matching pairs” | 266 |
| 3.1.6 | Did Peaky remember his lessons correctly? | 267 |
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| 3.2 | From Theory to Practice -- Empirical Formulae, Rules of Thumb and Simple Correlations in Everyday HPLC | 269 |
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| 3.3 | Information Resources for Analysis/HPLC | 277 |
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| 3.4 | Analytical Chemistry Today | 281 |
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| 3.5 | Trends in HPLC | 285 |
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| 3.6 | Thoughts About a Dead Horse | 290 |
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| | Index | 291 |
