Table of Contents

Cover

Title Page

List of Contributors

Foreword

The Structure of “The HPLC-Expert 2”

Chapter 1: When Should I Use My UHPLC as a UHPLC?
1. 1.1 Introduction
2. 1.2 What Do I Want to Achieve and What Is a UHPLC Capable of?
3. 1.3 What Is Required from an HPLC Method?
4. 1.4 The UHPLC in Routine Use – A Brief Report
5. 1.5 How Can the Potential of UHPLC Effectively Be Fully Exploited? (See Also Chapters 2, 3, and 9)
6. 1.6 Summary and Outlook
7. References

Part I: Hardware and Software, Separation Modes, Materials

Chapter 2: The Modern HPLC/UHPLC Device
1. 2.1 The Modern HPLC/UHPLC System
2. Acknowledgment
3. References
4. 2.2 The Thermostate of Columns – A Minor Matter
5. Literature
Chapter 3: The Issue of External Band Broadening in HPLC/UHPLC Devices
1. 3.1 Introduction
2. 3.2 Theoretical Background
3. 3.3 Extracolumn Dispersion in (U)HPLC Systems
4. 3.4 Impact of External Contributions in Different Application Areas
5. 3.5 Optimization of HPLC/UHPLC Systems
6. 3.6 Conclusions
7. References
Chapter 4: The Gradient; Requirements, Optimal Use, Hints, and Pitfalls
1. 4.1 Instrumental Influences in Gradient Elution – An Overview
2. 4.2 Gradient Elution Technology and How to Systematically Characterize Gradient Instrumentation
3. References
Chapter 5: Requirements of LC-Hardware for the Coupling of DifferentMass Spectrometers
1. 5.1 Introduction
2. 5.2 From Target Analysis to Screening Approaches
3. 5.3 What Should Be Considered for UHPLC/MS Hyphenation?
4. 5.4 Target Analysis Using Triple-Quadrupole Mass Spectrometry
5. 5.5 Screening Approaches Using LC-MS
6. 5.6 Miniaturization – LC-MS Quo Vadis?
7. References
Chapter 6: 2D chromatography – Opportunities and limitations
1. 6.1 Introduction
2. 6.2 Why Two-Dimensional HPLC?
3. 6.3 Peak Capacity of One- and Two-Dimensional Liquid Chromatography
4. 6.4 Modulation
5. 6.5 Practical Problems of Online LC × LC
6. 6.6 Development of a Miniaturized LC × LC System
7. 6.7 Real Applications
8. 6.8 Advantages of the MS/MS Functionality
9. 6.9 General Comments to Specific Aspects of an LC × LC System
10. 6.10 Method Development and Gradient Programming
11. 6.11 Presentations of the Instrument Manufacturers (in Alphabetical Order)
12. 6.12 2D LC – Quo Vadis?
13. References
Chapter 7: Materials in HPLC and UHPLC – What to Use for Which Purpose
1. 7.1 Introduction
2. 7.2 Requirements for Materials in UHPLC
3. 7.3 Flow Paths in UHPLC Systems
4. 7.4 Low-Pressure Flow Path
5. 7.5 High-Pressure Flow Path
6. 7.6 When and Why Can an Inert UHPLC System Be Required?
7. References

Part II: Experience Reports, Trends

Chapter 8: What a Software has to Possess in Order to Use the Hardware Optimally
1. 8.1 Functionality and Handling
2. 8.2 Data Exchange
3. 8.3 From PCs Scalability to Global Installation
Chapter 9: Aspects of the Modern HPLC Device – Experience Report of an Operator
1. 9.1 Introduction
2. 9.2 Determination of the Gradient Delay Volume
3. 9.3 High-Throughput Separations
4. 9.4 Method Transfer between UHPLC Systems of Different Manufacturers
5. 9.5 Application of Elevated Temperatures
6. 9.6 Large-Volume Injection (LVI)
7. 9.7 UHPLC Separation with 1 mm ID Columns
8. Acknowledgment
9. References
Chapter 10: Experiences of an Independent Service Engineer – Hints and Recommendations for an Optimal Operation of Agilent and Waters-Devices
1. 10.1 Introduction
2. 10.2 The Degasser, Principles
3. 10.3 The Pump, Principles
4. 10.4 The Autosampler, Principles
5. 10.5 The UV Detector, Principles
Chapter 11: The Analyte, the Question, and the UHPLC – The Use of UHPLC in Practice
1. 11.1 Introduction
2. 11.2 When Does It Make Sense to Use UHPLC and When Should I Better Use Conventional HPLC?
3. 11.3 Dissolution Tests in Pharmaceutical Industry
4. 11.4 Method Development and Optimization
5. 11.5 Typical “Classical” Liquid Chromatographic Analysis
6. 11.6 Fast (Most Second) Dimension of Multidimensional Chromatography
7. 11.7 Separation of (Bio)polymers
8. 11.8 Process Analysis (PAT)
9. 11.9 Conclusion
10. References
Chapter 12: Report of Device Manufacturers – Article by Agilent, Shimadzu, and ThermoScientific
1. 12.1 Agilent Technologies
2. References
3. 12.2 HPLC Current Status and Future Development
4. 12.3 Thermo Fisher Scientific, Germering

About the Authors

Index

End User License Agreement

List of Illustrations

Chapter 1: When Should I Use My UHPLC as a UHPLC?

Figure 1.1 Retention (bar) and separation factors (line) of tricyclic antidepressants in acidic acetonitrile/phosphate buffer on differing RP phases; for details, see text.
Figure 1.2 High-resolution 1D-UHPLC separation of a tryptic digestion of five proteins. A chain of four 250 mm columns was constructed using dead volume couplings based on Viper fittings (Thermo Scientific). Stationary phase: Acclaim 120 C18 (Thermo Scientific), temperature: 30 °C. Theoretical peak capacity calculated from the peak width of individual well-resolved peaks.
Figure 1.3 Separation of polystyrene; a good peak capacity is obtained starting with 55% B and using a flat gradient.
Figure 1.4 Initial gradient conditions with the focus on improvement in peak form; Column GeminiNX, 50 mm × 4 mm, 3 µm, 65–100% B (acetonitrile/water).
Figure 1.5 Separation of seven components on a Synergi MAX RP 20 mm × 4 mm, 2 µm column in less than 2 min on a high-pressure gradient system from the beginning of the 1990s – the system had considerable dead volume.
Figure 1.6 Tailing factor depending on the direction of the used capillary. For details, see text.
Figure 1.7 Influence of the direction of capillaries on the asymmetry factor; for details, see text.

Chapter 2: The Modern HPLC/UHPLC Device

Figure 2.1.1 Schematic design of (a) High- and (b) Low-Pressure mixing systems with gradient proportioning valve (GPV).
Figure 2.1.2 Design of the microfluidic optimized Jet Weaver mixer.
Figure 2.1.3 Separation of 13 aldehyde- and ketone 2,4-dinitrophenylhydrazine. Chromatographic parameter: mobile phase: (i) water and (ii) acetone; stationary phase: C18 reversed phase (50 mm × 2.1 mm, sub 2 µm); flow rate: 1 ml min⁻¹; gradient: 5–95% acetone in 30 min; and injection volume: 1 µl; UV detection: at 360 nm.
Figure 2.1.4 Representation of the gradient delay time depending on the flow rate for six different gradient delay volumes.
Figure 2.1.5 Flow path of the mobile phase of a fixed-loop autosampler (pushed-loop design) during the filling of the sample loop and injection.
Figure 2.1.6 Schematic design of (a) pulled-loop design and (b) pushed-loop design during the filling of the sample loop. The arrows show the flow path of the sample.
Figure 2.1.7 Simplified representation of the laminar flow profile of a sample plug into a sample loop.
Figure 2.1.8 Flow path of the mobile phase for a flow-through autosampler during aspiration (a) and injection (b) of the sample.
Figure 2.1.9 Separation of 16 PAHs at different temperatures. Chromatographic parameters: mobile phase: (A) water and (B) acetonitrile; stationary phase: YMC-PAH (50 mm × 2.1 mm, 3 µm); flow rate: 1.3 ml min⁻¹; gradient: 0–0.8 min, 58% B, 0.8–1.13 min, 58–100% B, 1.13–2.0 min, 100% B; injection volume: 0.5 µl; temperature: see figure; and UV detection: at 254 nm. Analytes: (1) naphthalene, (2) acenaphthylene, (3) acenaphthene, (4) fluorene, (5) phenanthrene, (6) anthracene, (7) fluoranthene, (8) pyrene, (9) benz[a]anthracene, (10) chrysene, (11) benzo[b]fluoranthene, (12) benzo[k]fluoranthene, (13) benzo[a]pyrene, (14) dibenz[a,h]anthracene, (15) benzo[g,h,i]perylene, and (16) indeno[1,2,3-c,d]pyrene.
Figure 2.1.10 Schematic design of a UV cell based on the liquid-core optical waveguide technology.
Figure 2.1.11 Overview and function of stainless-steel-based fittings. Shown is only a selection, which does not claim to be complete. * used for e.g. injection or column switching valves.
Figure 2.1.12 Comparison of two isocratic separations at a correct () and an incorrect () installation of the fitting at the column head. Chromatographic parameters: mobile phase: 50/50 (v/v) acetone/water; stationary phase: Agilent Zorbax SB C18 (50 mm × 2.1 mm, 1.8 µm); flow rate: 0.5 ml min⁻¹; injection volume: 0.2 µl; temperature: 30 °C; and UV detection: at 360 nm. Analytes: (1) formaldehyde-2,4-dinitrophenylhydrazone, (2) acetaldehyde-2,4-dinitrophenylhydrazone, and (3) acrolein-2,4-dinitrophenylhydrazone.
Figure 2.2.1 The same HPLC separation at different temperatures.
Figure 2.2.2 Classification of column thermostat types in thermal modes.
Figure 2.2.3 The same HPLC separation in a forced-air thermostat at 70 °C with passive preheater and without preheater.
Figure 2.2.4 Temperature profiles in a column, resulting from the mobile-phase temperature and either ideal-isothermal (left) or ideal-adiabatic (right) mode.
Figure 2.2.5 The same UHPLC separation with passive preheater or without preheater in a forced-air thermostat at 40 °C.
Figure 2.2.6 The same isocratic UHPLC separation in a still-air thermostat with active preheater and without preheater.
Figure 2.2.7 The same isocratic UHPLC separation operated in different column thermostats at 1000 bar.
Figure 2.2.8 Temperature profiles, which occur inside a column that is placed into an ideal-isothermal (left) or ideal-adiabatic (right) column thermostat, further depending on the incoming mobile-phase temperature and the emerging frictional heat due to flow resistance.
Figure 2.2.9 The same HPLC separation in a still-air (on top) and forced-air thermostat (bottom).
Figure 2.2.10 Concentration profiles within a still-air (on top) and forced-air thermostat (bottom).

Chapter 3: The Issue of External Band Broadening in HPLC/UHPLC Devices

Figure 3.1 HETP values vs. linear velocity for particles of 1.8, 3.5, and 5 µm in diameter.
Figure 3.2 Retention factors k for isocratic (a) and k_elution for gradient separations (b). While in isocratic separations, each analyte has a different k value and thus a different peak width, and the k_elution values and thus peak width in gradient separations are similar for all analytes (they depend on the gradient slope b).
Figure 3.3 Schematic representation of a fixed-loop (FL) injector (a) and a flow-through needle injector (b).
Figure 3.4 System variances (from 5 sigma width) for different injector configurations: fixed-loop (a), optimized flow-through needle (b), and standard flow-through needle (c). The injectors were connected to a 250 nl flow cell by a 0.050 mm × 340 mm stainless-steel capillary. The injection volume was 0.15 µl.
Figure 3.5 System variances (from 5 sigma width) for different injection volumes at flow rates of 0.1 (○), 0.5 (Δ), and 1 ml min⁻¹ (◊). The dotted line corresponds to the theoretical values calculated from Equation 3.15.
Figure 3.6 System variances (from 5 sigma width) for configurations with stainless-steel capillaries of different diameters of 0.055 mm (a), 0.070 mm (b), 0.080 mm (c), 0.095 mm (d), and 0.110 mm (e) with lengths of 250, 500, and 1000 mm. The dashed lines represent the theoretical variance derived from the Golay equation.
Figure 3.7 Plot of system variance versus pressure drop for capillaries with different nominal diameters and lengths of 250, 500, and 1000 mm at flow rates of 0.1, 0.5, and 1 ml min⁻¹ (viscosity = 1 cp).
Figure 3.8 Example of a void volume created by nonmatching column capillary connections.
Figure 3.9 Impact of improper capillary connection at the injection valve on separations on a 1 mm column. (a) Isocratic separation with bad connection, (b) gradient separation with bad connection, and (c) isocratic separation with proper connection.
Figure 3.10 System variances (from 5 sigma width) for different heat exchangers.
Figure 3.11 System variances (from 5 sigma width) for different detection cells. (a) 80 nl cell, (b) 250 nl cell, (c) 800 nl and (d) 2400 nl cell connected to a nanovalve with internal groove (square) and to a low-dispersion FTN injector (1290 Infinity II Multisampler) (circle).
Figure 3.12 Impact of data acquisition rate and peak filter on peak width and detection noise.
Figure 3.13 Peak widths (50% of height) on 150 mm Zorbax C18, 3.5 µm columns measured on a system with DAD-UV detection (rectangle) and a QQQ-MS with ESI–Jet Stream Interface (triangle). The lines represent the estimated peak widths (Equation 3.13) without external contributions (– – –) and for an external variance of 0.2 µl² (———).
Figure 3.14 Relation between peak widths (at different heights) and standard deviation of a Gaussian peak.
Figure 3.15 Determination of system variance with and without column. (a) Variance determined without column (5 sigma width), (b) variance determined without column (half-height width), (c) variance determined with column (5 sigma width), and (d) variance determined with column (half-height width). Configuration of system 1 and 2 and conditions are described in the text.
Figure 3.16 Estimated ratio of observed versus intrinsic plate number for different system variances in isocratic mode (50 mm columns with different diameters, particle size = 1.8 µm).
Figure 3.17 Plate numbers determined in isocratic mode on a 2.1 mm × 50 mm column based on 5 sigma width (a) and half-height width (b) with different system configurations. Systems 1 and 2 are described in the text.
Figure 3.18 Comparison of an isocratic separation with system configurations 1 and 2 (as described in text). Conditions: Zorbax Eclipse Plus C18 1.8 µm, 2.1 mm × 50 mm, 0.6 ml min⁻¹, 60% ACN/40% H₂O, 30 °C.
Figure 3.19 Estimated peak capacity ratio (PC_observed/PC_column) in gradient mode for columns 50 mm columns of different lengths with respect to different system variances.
Figure 3.20 Peak capacities determined in gradient mode on a 2.1 mm × 50 mm column based on 5 sigma width (a) and half-height width (b) with different system configurations. Systems 1 and 2 are described in the text.
Figure 3.21 Comparison of a gradient separation with system configurations 1 and 2 (as described in text, Zorbax Eclipse Plus C18 1.8 µm, 2.1 mm × 50 mm, 0.6 ml min⁻¹, 10–90% ACN in 120 s, 30 °C).
Figure 3.22 Comparison of a very fast gradient separation with different postcolumn configurations: (a) 250 nl cell + 0.075 mm × 220 mm capillary (σ² = 0.5 µl²), (b) 800 nl cell + 0.075 mm × 220 mm capillary (σ² = 1 µl²), and (c) 2400 nl cell + 0.110 mm × 220 mm capillary (σ² = 4 µl²). Conditions: PoroShell Eclipse Plus 2.7 µm, 2.1 mm × 50 mm, 1.1 ml min⁻¹, 30–60% ACN in 30 s, 60 °C.

Chapter 4: The Gradient; Requirements, Optimal Use, Hints, and Pitfalls

Figure 4.1 Schematic working principle of a high-pressure (often called binary) gradient or HPG pump and a low-pressure (often called quaternary) gradient or LPG pump. The respective function of the mixer and the consequence of incomplete mixing are also shown.
Figure 4.2 Curves of volume contraction due to excess volumes when mixing water with acetonitrile and water with methanol over the entire range. The resulting mixing volume is shown as percentage of the sum of the partial volumes as a function of the volume ratio of the mixture.
Figure 4.3 Diagram (a) shows the programmed pump parameters of a simple linear gradient running from 100% water to 100% methanol at 1 ml/min flow rate. Diagram (b) shows the real behavior of an HPG relative to an LPG pump with respect to flow rate and eluent composition with this gradient program. Please note that the deviation from composition for the LPG is only schematic and very drastic for better visibility, but its extent does not represent the typical behavior of LPG pumps in the market.
Figure 4.4 Chromatographic effects that occur at the column inlet due to differences in the elution strength of sample solvent and gradient start conditions. (a) These two chromatograms illustrate the focusing effect (right) of a weaker eluting sample solvent to compensate volume overloading (left). (b) This shows the severe peak distortion effect as a consequence of a significantly stronger eluting sample solvent. Both left chromatograms are recorded with the sample dissolved under the start conditions of the gradient.
Figure 4.5 Compressibility curves for water, acetonitrile, and methanol at ambient temperature (up to 2000 bar pressure).
Figure 4.6 Flow rate profile along a UHPLC instrument fluidic path for a method where pure methanol is delivered at 1 ml/min against a column head pressure of 2000 bar (pressure drop in connection tubing is neglected).
Figure 4.7 Dependence of the melting point of water and several organic solvents as a function of pressure up to a level of 2000 bar.
Figure 4.8 Technical scheme of a cam-driven serial 1½-cylinder pump. This pump technology can be applied for both HPG pumps (by combining with a second pump and aligned flow control of both) as well as LPG pumps (by integrating a time-controlled proportioning valve in front of the pump).
Figure 4.9 Schematics of the two common HPLC pump drive principles, cam (a) and lead screw (b), as well as the two common solvent delivery concepts, serial (c) and parallel (d). It is not mandatory that cam-driven pumps have only one motor resulting in coupled movement, but most cam pumps on the market follow this cost-effective principle.
Figure 4.10 Influence of the sample-loop precompression on the recorded pressure trace during the run and more important on the retention time precision on a selected peak (benzophenone). Method: phenone separation in a gradient in 3.6 min from 40 to 100% acetonitrile and a Thermo Scientific Hypersil GOLD 1.9 µm 2.1 × 30 mm column. Flow rate: 0.26 ml/min, temperature: 25 °C, injection volume: 1 µl, detection: UV at 254 nm.
Figure 4.11 The technical and physical steps of solvent aspiration, solvent precompression, and solvent delivery in an HPLC pump operating at elevated pressure level. The scheme illustrates that related thermal effects in the graph on the top. The gray dotted line shows the scenario where the thermal equilibrium is not achieved at the end of the ATEC phase. The ATEC algorithm will then extrapolate the curve and consider the effect for the piston movement in the subsequent delivery phase.
Figure 4.12 Damping of the residual pulsation resulting from compression heat effects to demonstrate the benefit of the automated thermal effect control (ATEC). Pump: Vanquish H Pump (Thermo Scientific), mobile phase: MeCN/H₂O 95/5 v/v, and flow rate: 2.0 ml/min delivered against a restriction capillary at 600 bar.
Figure 4.13 Relationship between the resulting waviness of a linear gradient formed on an LPG and the resulting gradient volume at a given flow rate as a function of the gradient slope. Conditions: F = 600 µl/min, proportioning cycle synchronized to 50 µl stroke, V_G/t_G of (a) 3200 µl/5.3 min, (b) 1600 µl/2.67 min, (c) 800 µl/1.33 min, and (d) 400 µl/0.67 min.
Figure 4.14 Example of a chromatogram with elution of the majority of analytes prior to arrival of the gradient at the column, due to inappropriate GDV. Column: MICRA NPS 1.5 µm ODS 1; 33 × 4.6 mm, flow rate: 4.0 ml/min, gradient: H2O/MeCN 5–100% linear in 0.3 min, GDV: 1200 µl, and peaks: thiourea (1), methylparaben (2), ethylparaben (3), propylparaben (4), and butylparaben (5).
Figure 4.15 Application of the marker pulse method for GDV determination by direct detection of the pure component B (methanol). Instrument: UltiMate 3000 Quaternary SD System (Thermo Scientific), pulse between 0 and 100% from 0.2 and 0.5 min (water to methanol), flow rate: 0.5 ml/min, and detection: UV at 210 nm.
Figure 4.16 Example for a typical Dolan test result. The round edges in the part of the re-equilibration back to initial conditions are also indicative of the gradient mixer characteristics. Deviations from perfect linearity can also be detected but may sometimes be a bias from the determination method (degasser effects).
Figure 4.17 Schematic representation of the technical concept of the SpinFlow™ mixer (Thermo Scientific) [19]. It is a two-stage mixer that combines a radial mixing capillary with a helix structure inside (first stage) with cylindrical or disk-like frit mixer for subsequent longitudinal mixing (second stage).
Figure 4.18 Characterization of the mixing accuracy and the fluidic behavior (sharpness of edges) of a gradient pump. Instrument: Vanquish H pump (Thermo Scientific), solvents: water and water spiked with acetone, flow rate: 2 ml/min, and pressure (generated against restrictor): 1200 bar.
Figure 4.19 Programmed sinusoidal composition pattern generated with an HPG at time period of 1.2 s over the full composition range from 0 to 100% (corresponds to a volume period of 20 µl at the given flow rate of 1 ml/min). The sinusoidal composition change from water to water with marker is traced with a UV detector. The relative change in the amplitude provides a quantitative measure for longitudinal mixing efficiency at the given volume period.
Figure 4.20 Programmed sinusoidal composition pattern generated with an HPG pump with 13 different volume periods between 2000 and 20 µl. The patterns are detected without mixer with the same setup as shown by the gray line in Figure 4.19.
Figure 4.21 Residual pulsation amplitude as a function of mixer dwell volume measured at two different volume periods of pulsation (20 and 200 µl). Zoomed range compares mixers of different vendors at 20 µl period.
Figure 4.22 Amplitude comparison of baseline ripples with TFA to demonstrate the retention effect of the column, here under isocratic conditions of 5% eluent B, but mixed with an HPG pump at 35 µl mixer. Conditions: water/acetonitrile 99:1 v/v with 0.1% TFA (eluent A), acetonitrile with 0.1% TFA (eluent B), flow rate: 1 ml/min, temperature: 35 °C, and column: Acclaim C18, 3 µm, 250 × 3.0 mm.
Figure 4.23 Comparison of detector baseline with two different mixer volumes (same mixer type) in a typical TFA gradient.
Figure 4.24 Residual baseline ripple amplitude in a TFS gradient with and without column in dependence on the mixer dwell volume.
Figure 4.25 Comparison of the absolute baseline ripple from isocratic mixing of 15% acetonitrile with 0.1% TFA to aqueous 0.1% TFA solution for two different HPG pumps, but equipped with identical mixers. The ripple amplitudes with nominal 35 and 200 µl mixers have been experimentally determined, the amplitudes at the other nominal mixer volumes are extrapolated according to the curves determined in Figure 4.24.
Figure 4.26 Chopped protein peak as a consequence of longitudinal discontinuity of mobile-phase composition and demonstration of influence of the gradient mixer volume on this effect. Sample: lysozyme, instrument: Thermo Scientific UltiMate 3000 binary RS system, small mixer: 35 µl SpinFlow, large mixer: 400 µl SpinFlow, flow rate: 1 ml/min, temperature: 30 °C, gradient water with 0.12% TFA to acetonitrile with 0.12% TFA, and “column”: Phenomenex SecurityGuard C18, 4.0 × 3.0 mm.

Chapter 5: Requirements of LC-Hardware for the Coupling of DifferentMass Spectrometers

Figure 5.1 Matrix effect chromatogram of a house dust extract. Selected mass transitions of different mycotoxins are shown.
Figure 5.2 Typical system setup of LC-MS hyphenation.
Figure 5.3 Optimized system design for the hyphenation of a flexible UHPLC system with a mass spectrometer.
Figure 5.4 LC-MS/MS chromatogram of 182 MRM transitions on a 50 mm × 2.0 mm Chromolith FastGradient RP18 column. Chromatographic parameters: temperature: 40 °C; injection volume: 20 µl; mobile phase: A = water + 0.1% formic acid, B = methanol + 0.1% formic acid; gradient: 5–95% B in 20 min; flow rate: 400 µl min⁻¹; mass spectrometric parameters: pause time: 5 ms; and dwell time: 100 ms.
Figure 5.5 Extracted LC-MS/MS chromatogram of 182 MRM transitions on a 50 mm × 2.0 mm Chromolith FastGradient RP18 column. Chromatographic parameters: temperature: 40 °C; injection volume: 20 µl; mobile phase: A = water + 0.1% formic acid, B = methanol + 0.1% formic acid; gradient: 5–95% B in 20 min; flow rate: 400 µl min⁻¹; mass spectrometric parameters: pause time: 5 ms; and dwell time: 10 ms.
Figure 5.6 Extracted LC-MS/MS chromatogram of 182 MRM transitions on a 50 mm × 2.0 mm Chromolith FastGradient RP18 column. Chromatographic parameters: temperature: 40 °C; injection volume: 20 µl; mobile phase: A = water + 0.1% formic acid, B = methanol + 0.1% formic acid; gradient: 5–95% B in 20 min; flow rate: 400 µl min⁻¹; mass spectrometric parameters: pause time: 5 ms; MRM detection window: 60 ms; and target scan time: 2 s.
Figure 5.7 Comparative presentation of the multiple reaction monitoring (MRM) principles. (a) Retention-time-independent MRM mode; (b) retention-time-independent MRM mode including a classification of the chromatogram into periods; and (c) retention-time-dependent MRM mode with variable detection windows.
Figure 5.8 General workflow of an LC-MS- and MS/MS-screening analysis.
Figure 5.9 Comparison of (a) the obtained peak width using a conventional (▪) and highly efficient LC separation (•). In addition, the corresponding cycle times of combined MS full-scan and MS/MS acquisition data are shown for four (b) and eight (c) information-dependent MS/MS experiments.
Figure 5.10 Comparison of commercially available emitter tips. (a) Conventional emitter tip with an inner diameter of 100 µm, compatible with 1/16″ fittings and (b) miniaturized emitter tip with an inner diameter of 25 µm, compatible with 1/32″ fittings.
Figure 5.11 System design for the separation of selected pharmaceuticals on a monolithic nano-HPLC column (150 mm × 0.1 mm).
Figure 5.12 LC-MS/MS chromatogram of a separation for 50 pharmaceuticals on (a) 50 mm × 2.0 mm Chromolith FastGradient RP18 column. Chromatographic parameters: temperature: 40 °C; injection volume: 10 µl; mobile phase: A = water + 0.1% formic acid, B = methanol + 0.1% formic acid; flow rate: 500 µl min⁻¹; mass spectrometric parameters: pause time: 5 ms; dwell time: 20 ms and (b) 150 mm × 0.1 mm Chromolith CapROD C18 column. Chromatographic parameters: temperature: room temperature; injection volume: 100 nl; mobile phase: A = water + 0.1% formic acid, B = acetonitrile + 0.1% formic acid; flow rate: 5 µl min⁻¹; mass spectrometric parameters: pause time: 5 ms; and dwell time: 20 ms.

Chapter 6: 2D chromatography – Opportunities and limitations

Figure 6.1 Matrix effect chromatogram of a tea extract. For further details, see text.
Figure 6.2 Chromatogram of a pesticide standard. The deconvoluted extracted ion chromatograms are marked by vertical gray lines. The bold black line represents the total ion current chromatogram.
Figure 6.3 Schematic illustration of heart-cut 2D LC. For further details, see text.
Figure 6.4 Schematic illustration of comprehensive 2D LC. For further details, see text.
Figure 6.5 Schematic illustration of the correlation of a hypothetical scattering of compounds across a two-dimensional separation space. (a) Low correlation of both retention mechanisms (orthogonal system) and (b) high correlation (no orthogonal system) between first and second separation. For further details, see text.
Figure 6.6 Schematic illustration of the principal setup of a comprehensive online LC × LC system. For further details, see text.
Figure 6.7 Total ion current chromatogram of the two-dimensional separation of the reference standard. For further details, see text.
Figure 6.8 Analyte map on the basis of the extracted ion current chromatograms of all compounds detected in the reference standard. For further details, see text.
Figure 6.9 Single chromatogram of the micro-LC separation in the second dimension. Different extracted masses originating from the 29^th fraction are shown. For further information, see text.
Figure 6.10 Typical extracted ion current chromatogram across five fractions. What can be seen is the nonmodulated signal as registered continuously by the detector. For further details, see text.
Figure 6.11 Total ion current chromatogram of the two-dimensional separation of the wastewater sample. For further information, see text.
Figure 6.12 Analyte map on the basis of the extracted ions for all compounds that have been identified according to a suspected target screening. For further details, see text.
Figure 6.13 (a) Envelope of the extracted ion current chromatogram with m/z 261.0323 across six fractions. What can be seen is the nonmodulated signal as registered continuously by the detector. For further details, see text. (b,c) Product ion spectra for the precursor ion with m/z 261.0. For further details, see text.

Chapter 7: Materials in HPLC and UHPLC – What to Use for Which Purpose

Figure 7.1 Low-pressure and high-pressure flow path in a UHPLC system with fixed-loop (pulled-loop) autosampler. The low-pressure flow path is shown in light gray; the high pressure flow path is shown in dark gray. Reproduced with permission of Thermo Fisher Scientific.
Figure 7.2 Comparison of the mobile-phase flow path (a, dark-gray) and the sample flow path (b, light-gray) in a UHPLC system with fixed-loop (pulled-loop) autosampler.
Figure 7.3 LC-MS detection of diisononyl phthalate and erucamide in FEP solvent tubing extract. The FEP solvent tubing had been filled with H₂O/ACN 20/80 (v/v) and incubated at 40 °C for 24 h. Erucamide: m/z = 338.34 [M+H]⁺ and 675.67 [2M+H]⁺ and diisononyl phthalate: m/z = 441.30 [M+Na]⁺.
Figure 7.4 LC-MS detection of erucamide in PEEK-frit extract. The PEEK frit was incubated in H₂O/ACN 20/80 (v/v) at 40 °C for 24 h. Erucamide: m/z = 338.34 [M+H]⁺. Besides erucamide, other unidentifiable substances had been detected.
Figure 7.5 Components of a low-pressure gradient pump. Shown are degasser unit, gradient proportioning valve, pump head with working and equilibration cylinder, purge valve, and mixer.
Figure 7.6 End face mechanical seal with seal and spring.
Figure 7.7 Two-position-six-port rotary shear valve including rotor and stator.
Figure 7.8 Schematics of a rotor with two types of leakages: radial leakage (shown in dark-gray) and cross-port leakage (shown in light-gray).
Figure 7.9 Image of a new, unused rotor (a) and a used rotor (b) with defects, which cause cross-port leakage.
Figure 7.10 Results of a flow injection analysis of caffeine (concentration 100 mg l⁻¹ in water, flow 0.5 ml min⁻¹) with two different 75 mm × 0.18 mm stainless-steel capillaries with a smooth and a rough interior surface. The peak volume increases by about 13% when the capillary tubing with rough interior surface is used.
Figure 7.11 End faces of different stainless-steel tubings: (a) file-cut tubing, (b) tubing cut by a commercially available tube cutter, and (c) factory precut tubing.
Figure 7.12 Schematic representation of a three-piece fitting system (A) consisting of a nut (a), a collet (b), and a ferrule (c) and a cross section of a tubing, which is fixed by the three-piece fitting system VHP-200 from IDEX (B).
Figure 7.13 Influence of a noninert LC system on the baseline and noise of an electrochemical detector.
Figure 7.14 Chromatography of litronesib and detection of nitroso derivates. Chromatographic conditions: mobile phase: (a) 0.05% ammonium hydroxide in water, (b) acetonitrile; stationary phase: waters X-bridge C18, 4.6 mm × 75 mm, 2.5 µm; flow rate: 1 ml min⁻¹; gradient: 0–1 min 75% A, 1–23 min 75–20% A, 23–25 min 20% A, 25–28 min 75% A; injection volume: 10 µl; temperature: 30 °C; and UV-detection: 290 nm. (a) LC-UV chromatogram (290 nm) and (b) UV/VIS spectra of litronesib und nitroso derivates.

Chapter 9: Aspects of the Modern HPLC Device – Experience Report of an Operator

Figure 9.1 Determination of the gradient delay volume of a UHPLC system. Chromatographic parameters: stationary phase: none, replaced by a zero dead volume union (ZDV); mobile phase: (A) deionized water and (B) deionized water with 50 mg l⁻¹ caffeine; flow rate: 0.2 ml min⁻¹; gradient: 0–5 min, 0% B; 5–15 min, from 0% to 100% B; injection volume: no injection, injection needle, and sample loop are switched into the flow path; temperature: 30 °C; and detection: UV at 270 nm.
Figure 9.2 Separation of six antineoplastic drugs. Chromatographic parameters: stationary phase: Shimadzu Shim-pack XR-ODS, (a,b) 50 mm × 3.0 mm, 2.2 µm and (c) 50 mm × 2.0 mm, 2.2 µm; mobile phase: (A) deionized water + 0.1% formic acid and (B) acetonitrile with 0.1% formic acid; flow rate: 0.3 ml min⁻¹; gradient: see figure; injected absolute mass per analyte: (a) 12.5 ng, (b) 5 ng, and (c) 0.002 ng; temperature: (a) 35.0 °C and (b,c) 31.3 °C; detection: MS, MRM. Maximum pressure during the gradient: (a) 150 bar, (b) 95 bar, and (c) 172 bar; and analytes: (1) gemcitabine, (2) ifosfamide, (3) cyclophosphamide, (4) etoposide, (5) paclitaxel, and (6) docetaxel. Note to (c): differences in the peak height ratio of the analytes are caused by adjustments of the MS source parameter.
Figure 9.3 Chromatographic separation of 13 aldehyde- and ketone-2,4-dinitrophenylhydrazones on (a) UHPLC system from manufacturer A and (b) UHPLC system from manufacturer B. Chromatographic parameters: stationary phase: Agilent Zorbax SB C₁₈(50 mm × 2.1 mm, 1.8 µm); mobile phase: (A) deionized water and (B) acetone; flow rate: 1.2 ml min⁻¹; gradient: see figure; injection volume: 1 µl; temperature: (a) 33.4 °C and (b) 33.0 °C; detection: UV at 360 nm. Maximum pressure of the method: (a) 1100 bar and (b) 1080 bar. Analytes: (1) formaldehyde-2,4-DNPH, (2) acetaldehyde-2,4-DNPH, (3) acrolein-2,4-DNPH, (4) acetone-2,4-DNPH, (5) propionaldehyde-2,4-DNPH, (6) crotonaldehyde-2,4-DNPH, (7) methacrolein-2,4-DNPH, (8) 2-butanone-2,4-DNPH, (9) butyraldehyde-2,4-DNPH, (10) benzaldehyde-2,4-DNPH, (11) valeraldehyde-2,4-DNPH, (12) m-tolualdehyde-2,4-DNPH, and (13) hexaldehyde-2,4-DNPH. Ψ: impurities of the standard which are not considered during method development.
Figure 9.4 Chromatographic separation of 13 aldehyde- and ketone-2,4-dinitrophenylhydrazones. Chromatographic parameters: stationary phase: Agilent Zorbax SB C₁₈ (50 mm × 2.1 mm, 1.8 µm); mobile phase: (A) water and (B) acetone; flow rate: 0.5 ml min⁻¹; gradient: 5–95% B in 5 min; injection volume: 1 µl; temperature: see figure; and detection: UV at 360 nm. Maximum pressure during the gradient: (a) 567 bar and (b) 320 bar.
Figure 9.5 Chromatographic separation of 13 aldehyde- and ketone-2,4-dinitrophenylhydrazones. Chromatographic parameters: stationary phase: Agilent Zorbax SB C₁₈ (50 mm × 2.1 mm, 1.8 µm); mobile phase: (A) water and (B) acetone; flow rate: 0.5 ml min⁻¹; gradient: 5–95% B in 10 min; injection volume: 1 µl; temperature: (a,b) 60 °C and (c,d) 90 °C; and detection: UV at 360 nm. (a,c) No preheating and (b,d) passive preheating in a volume of 1.6 µl.
Figure 9.6 Separation of four antineoplastic drugs (a) without and (b) with online enrichment [17]. Chromatographic parameters: stationary phase: (a) Waters XBridge C₁₈ (50 mm × 2.1 mm, 5 µm) and (b) ThermoHyperCarb (10.2.1 mm, 5 µm) coupled to a Waters XBrdige C₁₈ (50 mm × 2.1 mm, 3.5 µm); mobile phase: (A) deionized water + 0.1% trifluoroacetic acid and (B) acetonitrile + 0.1% trifluoroacetic acid; flow rate: 0.5 ml min⁻¹; gradient: 0–99% B in 10 min, 10–20 min, 100% B; injection volume: 1000 µl; temperature: 35 °C; detection: MS. Analytes: (1) gemcitabine, (2) ifosfamide, (3) cyclophosphamide, and (4) fenofibrate.
Figure 9.7 Separation of four antineoplastic drugs [17]. Chromatographic parameters: stationary phase: ThermoHyperCarb (10 2.1 mm, 5 µm) coupled to a Waters XBrdige C₁₈ (50 mm × 2.1 mm, 3.5 µm); mobile phase: (A) deionized water + 0.1% trifluoroacetic acid and (B) acetonitrile + 0.1% trifluoroacetic acid; flow rate: 0.5 ml min⁻¹; gradient: 0–90% B in 10 min, 10–20 min, 100% B; injection volume: 1000 µl; temperature: see figure; and detection: MS. Injected absolute amount of substance: 25 ng. Analytes: (1) gemcitabine, (2) ifosfamide, (3) cyclophosphamide, and (4) fenofibrate.
Figure 9.8 Chromatographic separation of 13 aldehyde- and ketone-2,4-dinitrophenylhydrazones. Chromatographic parameters: stationary phase: (a) Agilent Zorbax SB C₁₈ (50 mm × 2.1 mm, 1.8 µm) and (b,c) Agilent Zorbax SB C₁₈ (50 mm × 1.0 mm, 1.8 µm); mobile phase: (A) deionized water and (B) acetone; flow rate: (a) 1.2 ml min⁻¹ and (b,c) 0.272 ml min⁻¹; gradient: see figure; injection volume: 1 µl; temperature: 33.0 °C; and detection: UV at 360 nm. Maximum pressure of the method: (a) 1080 bar and (b,c) 825 bar. Volume of the detector cell (a,b) 9 µl and (c) 1 µl. Analytes: (1) formaldehyde-2,4-DNPH, (2) acetaldehyde-2,4-DNPH, (3) acrolein-2,4-DNPH, (4) acetone-2,4-DNPH, (5) propionaldehyde-2,4-DNPH, (6) crotonaldehyde-2,4-DNPH, (7) methacrolein-2,4-DNPH, (8) 2-butanone-2,4-DNPH, (9) butyraldehyde-2,4-DNPH, (10) benzaldehyde-2,4-DNPH, (11) valeraldehyde-2,4-DNPH, (12) m-tolualdehyde-2,4-DNPH, and (13) hexaldehyde-2,4-DNPH. Ψ: impurities of the standard.

Chapter 11: The Analyte, the Question, and the UHPLC – The Use of UHPLC in Practice

Figure 11.1 Flowchart of a typical HPLC analysis.

Chapter 12: Report of Device Manufacturers – Article by Agilent, Shimadzu, and ThermoScientific

Figure 12.1.1 Schematic representation of the extended rinsing procedure: (a) rinse the needle by means of gradients, (b) rinse the outer needle surface in the wash port, and (c) backflushing of the needle seat and the needle seat.
Figure 12.1.2 Agilent Infinity II Multicolumn Thermostat with eight built-in columns, low dispersion heat exchanger, A-Line Quick-Connect Fittings und column-switching valve.
Figure 12.1.3 Agilent Multiple Heart-Cutting Solution Valve Setup. The two sample loops are replaced by two complete six-column selection valves with six sample loops each.
Figure 12.1.4 Heart-Cutting 2D-LC without “peak parking.” The red marked peaks cannot be analyzed in the second dimension, as the actual 2D run is not finished.
Figure 12.1.5 Heart-Cutting 2D-LC with “peak parking.” All peaks can be analyzed in the second dimension, peaks are worked off subsequently.
Figure 12.1.6 A-Line Quick-Connect Fitting.
Figure 12.2.1 Nexera MP system with LCMS-8050 triple-quadrupole mass spectrometer.
Figure 12.2.2 Nexera method scouting system.
Figure 12.2.3 Shimadzu i-Series/Controller screen and i-PAD screen.
Figure 12.3.1 Setup of the Thermo Scientific Vanquish Horizon UHPLC system following the concept of integrated modularity. The fluidic path is marked with a white line for better traceability.
Figure 12.3.2 Design of the universal and tool-free Thermo Scientific Viper UHPLC fittings. The photo shows the first version of Viper for a clearer view on the different parts. In the current version, the PEEK ring, the force adapter, and the cylinder with the screw are connected for functional reasons.

List of Tables

Chapter 2: The Modern HPLC/UHPLC Device

Table 2.1.1 Comparison of the individual properties of low- and high-pressure mixing systems, with reference to [3]
Table 2.1.2 Comparison of the advantages and disadvantages of a fixed-loop and flow-through autosamplers with reference to [10, 11]
Table 2.1.3 Overview of the common HPLC-/UHPLC detectors

Chapter 3: The Issue of External Band Broadening in HPLC/UHPLC Devices

Table 3.1 Typical values for peak standard deviation in isocratic and gradient separation separations

Chapter 5: Requirements of LC-Hardware for the Coupling of DifferentMass Spectrometers

Table 5.1 Overview of the different LC-MS strategies and workflows

Chapter 6: 2D chromatography – Opportunities and limitations

Table 6.1 Overview of the identified analytes by 1D-HPLC-MS and 2DnLC × μLC-MS. Detailed list of detected targets is given in [47]

Chapter 7: Materials in HPLC and UHPLC – What to Use for Which Purpose

Table 7.1 Wetted materials (selection) typically used in UHPLC systems
Table 7.2 Wetted materials (selection) typically used in the low-pressure flow path of UHPLC systems
Table 7.3 Wetted materials (selection) typically used in UHPLC pumps
Table 7.4 Chemical composition of stainless steel type 304, 316, 316 L, and 316 Ti [2]
Table 7.5 Chemical composition of titanium alloys [17–19]
Table 7.6 Wetted materials (selection) typically used in UHPLC autosamplers
Table 7.7 Wetted materials (selection) typically used in tubing and fittings
Table 7.8 Chemical composition of MP35N [39]
Table 7.9 Selection of aqueous buffer systems used in biochromatography
Table 7.10 Passivation methods with citric acid, phosphoric acid, and nitric acid for stainless-steel-based tubing or UHPLC systems

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List of Contributors

Siegfried Chroustovsky

Techno Trade and Service

Lichtenklinger Str. 13

Siedelsbrunn, 69483 Wald-Michelbach

Germany

Monika Dittmann

Agilent Technologies

Hewlett-Packard-Straße 8

76337 Waldbronn

Germany

Björn-Thoralf Erxleben

Shimadzu Europa GmbH

Albert-Hahn-Str. 6-10

47269 Duisburg

Germany

Tobias Fehrenbach

Schraudolphstr. 38

80799 Munich

Germany

Michael Heidorn

Thermo Fisher Scientific

Dornierstr. 4

82110 Germering/Munich

Germany

Terence Hetzel

Institut für Energie- und Umwelttechnik e.V., IUTA

Bliersheimer Str. 58-60

47229 Duisburg

Germany

Stavros Kromidas

Consultant

Breslauer Str. 3

66440 Blieskastel

Germany

Stefan Lamotte

BASF SE, Global Comp. Center Analysis

GMC/AC-E210

67056 Ludwigshafen

Germany

Juri Leonhardt

Institut für Energie- und Umwelttechnik e.V., IUTA

Bliersheimer Str. 58-60

47229 Duisburg

Germany

Christoph Portner

Tauw GmbH

Richard-Löchel-Straße 9

47441 Moers

Germany

Arno Simon

Beyontics GmbH

Altonaer Straße 79-81

13581 Berlin

Germany

Frank Steiner

Thermo Fisher Scientific

Dornierstr. 4

82110 Germering/Munich

Germany

Thorsten Teutenberg

Institut für Energie- und Umwelttechnik e.V., IUTA

Bliersheimer Str. 58-60

47229 Duisburg

Germany

Jens Trafkowski

Agilent Technologies

Hewlett-Packard-Straße 8

76337 Waldbronn

Germany

Jochen Tuerk

Institut für Energie- und Umwelttechnik e.V., IUTA

Bliersheimer Str. 58-60

47229 Duisburg

Germany

Steffen Wiese

Institut für Energie- und Umwelttechnik e.V.

IUTA, Bliersheimer Str. 58-60

47229 Duisburg

Germany

Foreword

In “The HPLC-Expert,” we discussed several topics of the modern HPLC and illustrated new developments. In this book, “The HPLC-Expert 2” we focus on the modern HPLC/UHPLC device.

Our objective is to give detailed information about the modern HPLC/UHPLC equipment, so that our HPLC Colleagues can use their device optimal depending on their requirement. On the one hand, we present in 12 chapters how HPLC-Hardware and also particular modules can be run at the maximal resolution and peak capacity and, on the other hand, the procedure, if robustness is the main focus.

Practice is put forward, and theoretical background information is only given to an extent that we considered absolutely necessary. I hope that practice-oriented laboratory supervisors and experienced operators will find inspiration and hints about chances and constraints of the modern HPLC/UHPLC devices.

My special thanks go to Klaus Illig for his critical hints to the manuscript and to my author colleagues who contributed their experience and knowledge. Also, I want to thank WILEY-VCH and in particular Reinhold Weber for the good and close collaboration.

Stavros Kromidas
Blieskastel, February 2016

The HPLC Expert II

Find and Optimize the Benefits of your HPLC/UHPLC

List of Contributors

Foreword