Table of Contents

Title Page

List of Contributors

The structure of “The HPLC-Expert”

Preface

Chapter 1: LC/MS Coupling

1.1 State of the Art in LC/MS
1.2 Technical Aspects and Pitfalls of LC/MS Hyphenation
1.3 LC Coupled to MS – A User Report
References

Chapter 2: Optimization Strategies in RP-HPLC

2.1 Introduction
2.2 LC Fundamentals
2.3 Methodology of Optimization
2.4 Outlook
References

Chapter 3: The Gradient in RP-Chromatography*

3.1 Aspects of Gradient Optimization
3.2 Prediction of Gradients
References

Chapter 4: Comparison and Selection of Modern HPLC Columns

4.1 Supports
4.2 Stationary Phases for the HPLC: The Historical Development
4.3 pH Stability and Restrictions in the Use of Silica
4.4 The Key Properties of Reversed Phases
4.5 Characterization and Classification of Reversed Phases
4.6 Procedure for Practical Method Development
4.7 Column Screening
4.8 Column Databases
References

Chapter 5: Introduction to Biochromatography

5.1 Introduction
5.2 Overview of the Stationary Phases
5.3 Reversed-Phase Chromatography of Peptides and Proteins
5.4 IEC Chromatography of Peptides and Proteins
5.5 Size-Exclusion Chromatography of Peptides and Proteins
5.6 Further Types of Chromatography – Brief Descriptions
5.7 Summary

Chapter 6: Comparison of Modern Chromatographic Data Systems

6.1 Introduction
6.2 The Forerunners for CDS
6.3 CDS Today
6.4 Advantages and Disadvantages of File-Based CDS
6.5 Advantages and Disadvantages of Database-Supported CDS
6.6 CDS in a Network Environment
6.7 Instrument Control
6.8 Documentation and Compliance
6.9 Brief Overview of Current Systems
6.10 The CDS of Tomorrow
6.11 Special Extensions
6.12 Open Interfaces
6.13 The CDS in 20 Years
Acknowledgment

Chapter 7: Possibilities of Integration Today

7.1 Peak Overlay - Effect on the Chromatogram
7.2 Separation Techniques for Higher-Level Peaks
7.3 Application of Separation Methods
7.4 Chromatogrammsimulation
7.5 Deconvolution
7.6 Evaluation of Separation Methods
7.7 Practical Application of Deconvolution
References

Chapter 8: Smart Documentation Strategies

8.1 Introduction
8.2 Objectives of Documentation
8.3 The Life Cycle Model for Regulated Documents in Practice
8.4 Dealing with Hybrid Systems Comprising Paper and Electronic Records
8.5 Preview
References

Chapter 9: Tips for a Successful FDA Inspection

9.1 Introduction
9.2 Preparation with the Inspection Model
9.3 Typical Course of an FDA Inspection
9.4 During the Inspection
9.5 Post-Processing of the Inspection
Further Readings

Chapter 10: HPLC – Link List

10.1 Chemical Data
10.2 Applications/Methods
10.3 Troubleshooting
10.4 Background Information and Theory
10.5 Literature
10.6 Databases with Costs
10.7 Apps
10.8 Social Media
10.9 Twitter Pages (Examples)
10.10 Facebook Pages (Examples)

Index

End User License Agreement

List of Illustrations

Chapter 1: LC/MS Coupling

Figure 1.1 Analysis of saffron using DIP-APCI with high-resolution QTOF-MS.
Figure 1.2 Polarity range of analytes for ionization with various API techniques. Note: the extended mass range of APLI against APPI and APCI results from the ionization of nonpolar aromatic analytes in an electrospray.
Figure 1.3 Reduction of the droplet size.
Figure 1.4 Reaction mechanism in APCI.
Figure 1.5 Ion suppression in APCI-MS of PAH in urine.
Figure 1.7 Top-down (a) and bottom-up (b) flow path for minimizing the connection tubing length between LC column outlet and MS inlet (ion source).
Figure 1.6 Reduction of re-equilibration time and throughput enhancement by using a second separation column and alternating sample injection (tandem LC); (a) flow scheme, (b) injection interlacing.
Figure 1.8 LC/MS chromatogram of two isomers, m/z = 240.10; (a) PEEK bulk capillary behind the column (0.13 mm I.D.), PEEK fingertight fittings; (b) SST capillary with virtually zero-dead volume connection behind the column (Viper™ fingertight fitting technology, 0.13 mm I.D.).
Figure 1.9 Distinction of isobaric compounds by RP chromatography and UV detection for a reaction control analysis (N-arylation of an E/Z acrylic ester mixture).
Figure 1.10 Separation of a cytochrome C digest by adding 0.05% TFA (a) or 0.1% FA (b) under identical chromatographic conditions.
Figure 1.11 Influence of the dry gas temperature on MS signal quality using FIA of Astemizol in 10 mM aqueous ammonium acetate/methanol 20/80 (v/v) at 50 µl/min on a Triple-Quad instrument; (a) extracted ion chromatogram of Astemizol ([M + H]⁺), m/z 459.3; (b) reconstructed total ion chromatogram.
Figure 1.12 (a) Schematic representation of the quadrupoles of a triple quadrupole instrument. Only two quadrupole rods of the four quadrupole rods are shown for each quadrupole. The first quadrupole (Q1) filters for the precursor ion, the precursor ion is fragmented in Q2 by collision-induced dissociation and Q3 filters for a specific daughter ion. (b) Isomers of hydroxy-eicosatetraenoic acid (HETE). (c) Schematic representation of ion traces by measuring just the precursor ions or daughter ions of HETE isomers.

Chapter 2: Optimization Strategies in RP-HPLC

Figure 2.1 Schematic representation of ideally symmetrical (a) and real world asymmetrical (b) peak pairs and how the resolution is calculated.
Figure 2.2 Curves to demonstrate the individual influence of the three parameters selectivity α, plate number N, and retention factor k on peak resolution. The constant value of the respective two remaining parameters can be read from the intersection point.
Figure 2.3 Influence of a change in k, N, and α on the chromatogram for a critical peak pair.
Figure 2.4 The three terms of the van Deemter equation expressed in individual curves and the fluidic explanation in related sketches.
Figure 2.5 Band broadening effect of radial temperature inhomogeneity as a result of insufficient mobile phase preheating. The spheres represent the analyte zone.
Figure 2.6 The temperature dependent triangle of interaction in liquid chromatography and some options to vary stationary and mobile phase in RP chromatography.
Figure 2.7 RP Retention of acids and bases as a function of the mobile phase pH. Reproduced from Snyder et al. [3] with permission of John Wiley & Sons.
Figure 2.8 Similar influence of pH and temperature change on the selectivity of a separation of acids and bases. Column: silica based C18, mobile phase: phosphate buffer/ACN 50/50 v/v, peaks: (1) 2-phenylpyridine, (2) ketoprofen, (3) 4-n-pentylaniline, (4) diflunisal, (5) 4-n-butylbenzoic acid, (6) 4-n-hexylanilin, (7) diclophenac acid, (8) 4-n-pentylbenzoic acid, (9) 4-n-heptylaniline, (10) mefenamic acid, (11) 4-n-hexylbenzoic acid. From Dolan [7].
Figure 2.9 Van't Hoff Plot to show the temperature dependence (21–40 °C) for the probe compounds in the Waters column test, chromatographic conditions as in Figure 2.10.
Figure 2.10 Temperature dependence of the Waters column test (according to Uwe Neue). Column: ProntoSIL C18 ACE EPS, 125 mm × 4 mm (Bischoff); Eluent: phosphate buffer 20 mM pH = 7/MeOH 35/65 v/v; peaks: (1) uracil, (2) propranolol, (3) butylparaben, (4) dipropylphthalate, (5) naphthalene, (6) acenaphthene, (7) amitriptyline.
Figure 2.11 Separation of alkylphenones on a C18-modified polyvinyl alcohol (PVA) phase over a wide range of temperature. Column: Shodex® ET-RP1 4D 5 µm, 250 mm × 3 mm; eluent: H₂O/MeOH/25/75 v/v; flow: 0.5 ml/min; peaks: (1) acetophenone, (2) propiophenone, (3) butyrophenone, (4) pentanophenone, (5) hexanophenone, (6) heptanophenone, (7) octanophenone.
Figure 2.12 Van't Hoff Plot to show the temperature dependence of the retention of seven alkylphenones from 30 to 100 °C, chromatographic conditions as in Figure 2.11.
Figure 2.13 Temperature dependence of the plate number of heptanophenone (marked with dot in Figure 2.11) on C18 modified PVA phase (chromatographic conditions as in Figure 2.11).
Figure 2.14 Dependence of the (dynamic) viscosity η on temperature for three representative HPLC solvents and one acetonitrile–water mixture. Curves show the progression as relative values of η related to the viscosity at 25 °C.
Figure 2.15 Van Deemter curves recorded at three different temperatures, column: ProntoSIL KromaPlus C18 5 µm, 250 mm × 4.6 mm, eluent: EtOH/H₂O 80 : 20 (v/v), analyte: pentylbenzene.
Figure 2.16 Dynamic viscosity of mixtures of water with acetonitrile and different alcohols at 20 °C. Please note that the x-axis is in mass-% rather than the more common volume-%.
Figure 2.17 Van Deemter curves recorded at three different isoeluotropic eluent compositions that differ markedly in viscosity, column: ProntoSIL KromaPlus C18 5 µm, 250 mm × 4.6 mm, temperature: 25 °C, analyte: pentylbenzene.
Figure 2.18 (a) Simulated van Deemter curves calculated with Eq. 2.13 for different particle diameters and (b) simulated column pressure curves for a viscosity of 1 cP and a column length of 150 mm.
Figure 2.19 Example for a systematic method speed-up by optimization of particle diameter dp and column length L. Sample: uracil und phenylalkanes C₁–C₅; stationary phase: Prontosil 120-C18-AQ (Bischoff), mobile phase: 80/20 (v/v) ACN/H₂O, temperature: 20°C; L/dp = 25. All other conditions as shown in the figure.
Figure 2.20 Example for a systematic speed-up of a gradient separation. Sample: parabens C₁–C₄; stationary phase: Acclaim 120-C18 (Thermo Scientific, Sunnyvale, USA), gradient: from 60/40 to 20/80 (v/v) H₂O/ACN with the respective t_G; temperature: 40°C; L/dp = 50. All other conditions as shown in the figure.
Figure 2.21 Further speed increase of the separation shown in Figure 2.20. A reduced resolution was tolerated here, as L/dp was reduced from 50 to 25.
Figure 2.22 Speed-up of a gradient separation with transfer from the fully porous stationary phase Hypersil Gold (Thermo Scientific, Runcorn, UK) to the solid core material Accucore RP-MS (Thermo Scientific, Runcorn, UK). Gradient from 65/35 to 40/60 (v/v) H₂O/ACN in the respective t_G; temperature: 30 °C; peaks in order of elution: tebuthiuron, metoxuron, monuron, chlorotoluron, diuron, linuron. All other conditions as shown in the figure.
Figure 2.23 Simulated chromatograms of an original poorly resolving method (R_S = 0.94) showing three different approaches to increase the plate number by factor 2.5 and thus the resolution by factor 1.67. Note that the analysis time and column pressure differs significantly. The virtual mode of the Chromeleon (Thermo Fisher Scientific, Germering, Germany) UCI-50 A/D interface control was used for the chromatogram simulation.
Figure 2.24 Automated off-line 2D-LC separation of an Escherichia coli tryptic digest. Ten fractions of 1 min were collected from first dimension. First dimension run on 300 µm id, 150 mm length capillary columns at 6 µl/min flow, second dimension run on 75 µm id, 150 mm length nano columns at 300 µl/min flow. SCX column was a PolySULFOETHYL Aspartamide 5 µm (PolyLC Inc.) run in a phosphate buffer at pH = 3 with salt gradient up to 0.6 M NaCl in 15 min. All RP columns were Acclaim PA2 5 µm (Thermo Scientific). First dimension RP was run at pH = 9.6 with triethylamine and gradient to 50% acetonitrile in 15 min. Second dimension RP (both methods) was run at pH = 1.9 with TFA and gradient to 50% acetonitrile in 30 min.
Figure 2.25 Venn diagram to compare both 2D-LC methods presented in Figure 2.24 with respect to identified proteins. The percentages are calculated relative to the total number of identified proteins by combination of both methods, but not relative to the number of proteins in E. coli.
Figure 2.26 High resolution 1D-UHPLC separation of a tryptic digest from a mixture of five proteins. A daisy chain of four 250 mm columns was coupled with zero dead volume Viper couplers (Thermo Scientific, Germering, Germany). Stationary phase: Acclaim 120 C18, 3 µm (Thermo Scientific, Sunnyvale, USA), temperature: 30 °C, t_G = 300 min, n_C calculated from peak width of well resolved peptides.
Figure 2.27 Comparison of two generic exploratory methods for traditional Chinese medicine (TCM) run a Vanquish UHPLC system (Thermo Scientific, Germering, Germany) with two different column lengths as specified. Column id: 2.1 mm, L = 400 mm setup: coupling 150 and 250 mm columns with Viper coupling device (Thermo Scientific, Germering, Germany), gradient : from 0% to 100% acetonitrile, flow rate: 670 µl/min, column temperature: 45 °C.

Chapter 3: The Gradient in RP-Chromatography*

Figure 3.6 Regarding the starting conditions with a small number of peaks: Gemini NX, 50 × 4 mm, 3 µm, 65–100%B.
Figure 3.1 Influence of the flow rate, XBridge Shield 150 × 4.6 mm, 5 µm. (a) 0–100%B, 1 ml/min, t_G = 30 min, (b) 0–100%B, 2 ml/min, t_G = 30 min.
Figure 3.2 Influence of the flow rate, (a) 0.6 ml/min, 10 °C, (b) 1 ml/min, 10 °C.
Figure 3.3 Influence of the flow rate, XBridge Shield, 150 × 4.6 mm, 5 µm, (a) 50–90%B, 0.5 ml/min, t_G = 30 min, (b) 50–90%B, 1 ml/min, t_G = 15 min.
Figure 3.4 The gradient duration required, Zorbax SB C8, 150 × 4.6 mm, 5 µm, 40–90%B, t_G = 5 min, 2 ml/min.
Figure 3.5 Effect of initial %B, XBridge Shield, 150 × 4.6 mm, 5 µm, (a) 0–100%B, 0.5 ml/min, t_G = 30 min, (b) 40–100%B, 0.5 ml/min, t_G = 30 min.
Figure 3.7 Effect of initial %B and slope, XBridge Shield, 150 × 4.6 mm, 5 µm, (a) 40–100%B, 2 ml/min, t_G = 15 min, (b) 50–90%B, 2 ml/min, t_G = 15 min.
Figure 3.8 Influence of initial %B and slope, Symmetry C18 150 × 4.6, 5 µm initial %B: (c) 45%, (b) 60%, (c) 70%; final %B in all cases 100%.
Figure 3.9 Effect of initial %B and gradient duration on elution order and resolution, AscentisExpress C18, 50 × 3 mm, 2.7 µm, (c) 20–70%B, t_G = 15 min, (b) 20–90%B, t_G = 10 min, (a) 10–90%B, t_G = 5 min.
Figure 3.10 Influence of column length, (b) Synergi Fusion RP, 150 × 4.6 mm, 4 µm, (a) Synergi Fusion RP, 20 × 4.6 mm, 2 µm.
Figure 3.11 Influence of gradient duration, Synergi MAX RP 20 × 4 mm, 2 µm, (a) gradient duration 2 min, (b) 5 min, (c) 10 min.
Figure 3.12 Parabens on a 150 × 4.6 mm SunFire 2 ml/min 55% MeOH/45% water at room temperature. The peak widths w_50% are 0.078; 0.115; 0.191; 0.365.
Figure 3.13 Parabens on a 150 × 4.6 mm SunFire 2 ml/min 70% MeOH/30% water at room temperature. The peak widths w_50% are 0.052; 0.058; 0.074; 0.098.
Figure 3.14 Detail from “2SIS SunFire MeOH.xls.” The dark grey shaded cells contain the input data. The bar in row 28 is a scroll bar that changes the value of %B in cell B13 and shows dynamically the chromatogram.
Figure 3.15 Parabens on a 150 × 4.6 mm SunFire 2 ml/min 55% MeOH for 5 min, then 70% MeOH at room temperature. The peak widths w_50% are 0.079; 0.116; 0.071; 0.093.
Figure 3.16 Gradient from 5% to 100%B in 20 min in the method file from Labsolutions (Shimadzu).
Figure 3.17 ln(k) versus %B graph on a 150 × 4.6 SunFire 5μ at 30 °C. The four dashed lines are the four parabens in Figure 3.1–3.153.4. The solid lines from bottom to top are phenol, benzaldehyde and benzene, followed by the two closely adjacent straight lines corresponding to toluene and chlorobenzene.
Figure 3.18 Shows the calculation and the predicted chromatogram with a satisfactory separation using the linear data of Figure 3.17 (listed in the lower part of Table 3.2).
Figure 3.19 At the end of the column, the analytes exit delayed by the dwell time t_d and the mobile time t₀.
Figure 3.20 Is identical to Figure 3.17; however, the fitted lines are curvilinear.
Figure 3.21 Cutout from the file “SunFire MeOH 30Grad.” The peaks are shown individually colored in the file for easier identification of overlaps. The four parabens are the four small peaks. The order of the larger peaks is phenol, benzaldehyde, benzene, chlorobenzene, and toluene.
Figure 3.22 Measurement of nine substances on a SunFire column and with a gradient of 45–80% methanol in 25 min with a flow of 1 ml/min at 30°C.
Figure 3.23 After entering the retention time in Figure 3.1 and 3.2, with DryLab the Resolution Map and the predicted isocratic chromatogram at 65.95% MeOH can be seen. The predicted retention times agree very well with those of Figure 3.3.
Figure 3.24 ChromSword prediction for a gradient from 45 to 80% methanol in 25 min based on the linear model. With one click the quadratic function is selected and the last two peaks come closer together, as in the real measurement shown in Figure 3.22. The input data are those from Table 3.4.

Chapter 4: Comparison and Selection of Modern HPLC Columns

Figure 4.1 Test of polar selectivity: (a,b) neutral (black), acidic (dark gray), and basic (light gray) analytes.
Figure 4.3 Neue test: comparison between reversed phase with bedded polar group and fluoro phase.
Figure 4.4 Charakterization of different RP stationary phases. Reproduced from Kromidas [6], with permission of Wiley.
Figure 4.5 Hydrophobicity and Methylen group selectivity. Reproduced from Kromidas [6], with permission of John Wiley & Sons.
Figure 4.6 On the bandwith of retention and separation factors by the separation of metabolites of tricyclic anti-depressants in acetonitrile/acidic phosphate buffer. Reproduced from Kromidas [6] with permission of John Wiley & Sons.
Figure 4.7 On the suitability of k- and α-values for a differentiation of stationary phases; for comments, see text. Reproduced from Kromidas [6] with permission of John Wiley & Sons.
Figure 4.8 Plot of the separation factors of analyte pairs that differ as much as possible, on two columns; for explanation see text.
Figure 4.9 Plot of the separation factors of two characteristic analyte pairs on several columns; for explanation see text.
Figure 4.10 Two simple tests for the characterization of RP-phases.
Figure 4.11 POPLC scheme.
Figure 4.12 HILIC scheme.
Figure 4.13 Procedure with classical reversed-phase chromatography.
Figure 4.16 Procedure with ion-pair chromatography or by the use of mixed-mode columns.
Figure 4.17 Screenshot of the PQRI-database.

Chapter 5: Introduction to Biochromatography

Figure 5.1 Levels of protein structure Source: Wikipedia 2012.
Figure 5.2 Particle size distribution meassured by laser diffraction.
Figure 5.3 Protein loading capacity of RP-HPLC materials of different particle sizes. Source: Protein and Peptide Analysis and Purification, Vydac Reversed Phase Handbook, 5th edition, W.R. Grace, 2013.
Figure 5.4 Inverse size-exclusion chromatography from two different polystyrene phases. Source: J. Maier-Rosenkranz, PhD thesis, Tübingen, 1992.
Figure 5.5 (a) Separation of paracelsin on a silica-based C8 phase. (b) Separation of paracelsin on a PS/DVB phase. Source: J. Maier-Rosenkranz, PhD thesis, Tübingen, 1992.
Figure 5.6 Influence of change %B on k.
Figure 5.7 Influence of the gradient slope on resolution. Vydac 218TP C18, 4.6 × 150 mm, 10 µm; 25–50% ACN in 0.1% TFA ml/min. Sample: Subunits of cytochrome c oxidase. Source: Protein and Peptide Analysis and Purification, Vydac Reversed Phase Handbook, 5th edition, W.R. Grace, 2013.
Figure 5.8 Effect of the combination of ion-pair reagents. LC–MS TIC of peptides. Column: Vydac® Everest® 238 EV, 300 Å, C18, 5 µm 150 × 1 mm. Mobile phase: A: 95:5% water: ACN with ion-pair reagents. B: 20:80% water:ACN with ion pair reagents. Flow: 50 µl/min, gradient: 12.5–50% B in 30 min. Source: Protein and Peptide Analysis and Purification, Vydac Reversed Phase Handbook, 5th edition, W.R. Grace, 2013.
Figure 5.9 Separation of a peptide mixture at different pH values. Column Vydac® 218TP, 300 Å, C18, 4.6 × 250 mm, 5 µm. Mobile phase/gradient: 15–30% ACN in 20 mM phosphate, (a) pH 2.0, (b) pH 4.4, (c) pH 6.5. (1 ml/min). Sample: 1. bradykinin, 2. oxytocin, 3. angiotensin II, 4. neurotensin, 5. angiotensin I. Source: Protein and Peptide Analysis and Purification, Vydac Reversed Phase Handbook, 5th edition, W.R. Grace, 2013.
Figure 5.10 Example of ion-exchange chromatography. Source: Grom Analytik + HPLC GmbH, Application 09105; 2004.
Figure 5.11 IEC of nucleotides using salt-, pH- and organic solvent-gradient Source: Grom Analytik + HPLC GmbH, Application 07127; 2004.
Figure 5.12 Principle of size-exclusion chromatography.
Figure 5.13 SEC separation. Source: Grom Analytik + HPLC GmbH, Application 09046b; 2004.
Figure 5.14 SEC curve. Source: Application Note No 005, Grom Analytik + HPLC GmbH, 2003.
Figure 5.15 Example of an HIC protein separation. Source: Grom Analytik + HPLC GmbH, Application 09106; 2004.
Figure 5.16 Chromatogram of bradykinin in the HILIC mode. Column: Alltima HP HILIC, 5 µ, 150 × 4.6 mm. Mobile phase: (70:30) acetonitrile: 10 mM ammonium acetate w/0.2% acetic acid; flow rate: 0.75 ml/min. Detector: ELSD. Source: Alltech Application Data Base, W.R. Grace; 2007.

Chapter 7: Possibilities of Integration Today

Figure 7.1 Effect of peak overlay on the determination of the peak height and retention time.
Figure 7.2 For available separation techniques for higher-level peaks and their comparison ((a) vertical separation, (b) tangential separation, (c) valley-to-valley separation, (d) Gauss separation method, (e) exponential separation, (f) comparison, cumulative curve with single peak).
Figure 7.3 Simulation of the report of Westerberg [4] using MS-Excel.
Figure 7.4 The user window of hi|chromet after completion of the Chromatogrammsimulation.
Figure 7.5 Chromatogram with forward simulation in hi|skim.
Figure 7.6 To-be-evaluated Chromatogrammbereich set to 23–27 min.
Figure 7.7 Added a step gradient of linear fits to the baselines development.
Figure 7.8 Complete the separation by means of deconvolution (variation coefficient: 0.9992).
Figure 7.9 Overlay of the chromatograms with different concentrations of 6,7-dehydro-EE.

Chapter 8: Smart Documentation Strategies

Figure 8.1 Hierarchical document structure in regulated organizations.
Figure 8.2 Document life cycle of regulated documents.
Figure 8.3 Data model of instrumental raw data, calculated result data, and report data.

Chapter 9: Tips for a Successful FDA Inspection

Figure 9.1 Inspection model for structuring the topics.
Figure 9.2 Typical course of an FDA inspection.

List of Tables

Chapter 1: LC/MS Coupling

Table 1.1 Suitability and purpose of various mass spectrometer types
Table 1.2 Recommended re-equilibration volume for high-throughput and high-resolution columns under typical MS-compatible conditions
Table 1.3 Volumes and backpressure of a 30″/750 mm capillary with different I.D. in the maximum viscosity of water/acetonitrile and water/methanol mixtures
Table 1.4 Applicable and ideal working ranges for selected ionization processes
Table 1.5 Common gas phase adducts at positive (left) and negative (right) polarity

Chapter 2: Optimization Strategies in RP-HPLC

Table 2.1 Example case study for resolution optimization by changing the individual parameters that control selectivity
Table 2.10 Retention dependencies in RP chromatography
Table 2.2 Example for the attempt to improve resolution through efficiency increase when transferring from HPLC to UHPLC. All numbers are theoretically modeled on the basis of the artificial method #1 (*method #2 is not feasible in practice)
Table 2.3 Comparison between methods (a) and (b) from Figure 2.27 for number of identified peaks and minimum level of resolution between all adjacent peak pairs as quality criteria to assess the value of different peak capacities
Table 2.4 m_LOD for an analyte of 300 Da in conventional HPLC using a 250 mm × 4.6 mm column packed with 5 µm particles on a UV detector with 10 mm light path and 50 μAU baseline noise at different k and significantly different
Table 2.5 m_LOD for an analyte of 300 Da in modern UHPLC using a 100 mm × 2.1 mm column packed with 1.7 µm particles on a UV detector with 10 mm light path and 10 μAU baseline noise at different k and significantly different .
Table 2.6 Peak volume and injection volume levels for representative column formats
Table 2.7 Instrument related volumes and detector settings
Table 2.8 Columns, particle sizes and their operational conditions
Table 2.9 Method speed-up
Table 2.11 Peak resolution and dependencies on fundamental method parameters
Table 2.12 Peak capacity in 1D-LC and 2D-LC

Chapter 3: The Gradient in RP-Chromatography*

Table 3.2 The input cells are in the upper part in the range of 0% to 100% MeOH
Table 3.1 Contains in the upper part the retention times of the eight gradient chromatograms with an instrumental gradient slope of 5% MeOH/min
Table 3.4 Is part of the original file “GS curve delay temperature.xls” by U.D. Neue, which forms the basis of the prediction of a simple linear gradient shown in this chapter
Table 3.3 Retention times of the prediction according to Neue, according to the linear model and measured values, as well as percentage deviations from the measured values

Chapter 4: Comparison and Selection of Modern HPLC Columns

Table 4.1 Examples (!) of column assortments for special cases and/or conditions
Table 4.2 Examples (!) of column assortments for special cases and/or conditions

Chapter 5: Introduction to Biochromatography

Table 5.1 Biomolecules - monomeres
Table 5.2 List of the 20 proteinogenic amino acids
Table 5.3 Overview different base materials for Stationary phases

Chapter 7: Possibilities of Integration Today

Table 7.1 Comparison of area integration of Vertical skim and the deconvolution
Table 7.2 Comparing the surfaces of the respective separation method with deviation
Table 7.3 Summary of results from hi|chromet to the deviation of soldering methods for the 6,7-dehydro-EE-peak

Chapter 8: Smart Documentation Strategies

Table 8.1 Analytic process in an HPLC laboratory, according to the SIPOC-model
Table 8.2 Communication interfaces, according to the SIPOC model
Table 8.3 Meta-data model of data traceability in an HPLC laboratory
Table 8.4 Comparison of the requirements for documentation under GLP versus GMP regulation
Table 8.5 Required definitions for documents
Table 8.6 Overview of the dependencies of paper and electronic records

Dr. Stavros Kromidas
Consultant, Saarbrücken
Breslauer Str. 3
66440 Blieskastel
Germany

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List of Contributors

Torsten Beyer
Dr. Beyer Internet-Beratung
Weimarer Str. 30
Ober-Ramstadt
Germany

Mike Hillebrand
Sanofi-Aventis Deutschland GmbH
Industriepark Höchst
K703
65926 Frankfurt
Germany

Andreas Hofmann
Novartis Institutes for BioMedical Research
Novartis Campus
Basel
Switzerland

Stavros Kromidas
Breslauer Str. 3
Blieskastel
Germany

Hans-Joachim Kuss
Maximilians-Universität München
Innenstadtklinikum der Ludwig
Nussbaumstr. 7
München
Germany

Stefan Lamotte
BASF SE
Carl-Bosch Str. 38, Competence Center Analytics
GMC/AC-E210
67056 Ludwigshafen
Germany

Jürgen Maier-Rosenkranz
Grace Discovery Sciences
Alltech Grom GmbH
In der Hollerhecke 1
Worms
Germany

Markus M. Martin
Thermo Fisher Scientific
Dornierstraße 4
Germering
Germany

Alban Muller
Novartis Institutes for BioMedical Research
Novartis Campus
Basel
Switzerland

Iris Retzko
create skills
Simpsonweg 4c
Berlin
Germany

Oliver Schmitz
University of Duisburg-Essen
Faculty of Chemistry
S05 T01 B35
Universitätsstraße 5
Essen
Germany

Stefan Schmitz
CMC Pharma GmbH
M5, 11
Mannheim
Germany

Arno Simon
beyontics GmbH
Altonaer Str. 79–81
Berlin
Germany

Frank Steiner
Thermo Fisher Scientific
Dornierstr. 4
Germering
Germany

The structure of “The HPLC-Expert”

This book contains the following chapters:

Chapter 1 (LC/MS coupling) is dedicated to the most important coupling technique of the modern HPLC. In the first part of the chapter, Oliver Schmitz overviews the state of the art of LC/MS coupling and opposes different modes. In the second part, Markus Martin shows Pitfalls of LC/MS coupling and provides precise and specific hints on how LC/MS coupling can successfully be established in a daily routine. LC/MS coupling is often linked to life science and environmental analysis. Alban Muller and Andreas Hofmann show a concrete example of LC/MS coupling in ion chromatography as an unfamiliar application.

In Chapter 2, Frank Steiner, Stefan Lamotte, and Stavros Kromidas go in detail into optimization strategies for RP-HPLC and discuss, on the basis of selected examples, which parameters seem promising in which case.

Chapter 3 is devoted to the gradient elution. Stavros Kromidas, Frank Steiner, and Stefan Lamotte discuss about aspects of gradient optimization in a dense form in the first part and offer simple “to-do” rules. In the second part, Hans-Joachim Kuss shows that predictions of gradients runs with excel can be very unerring and that the often used linear model represents a simplified approximation.

Chapter 4 is about the comparison and choice of modern HPLC columns; Stefan Lamotte, Stavros Kromidas, and Frank Steiner give an overview of different columns and come forward with proposals for pragmatic tests for columns as well as column portfolios, depending on the separation problem.

In Chapter 5, Juergen Maier-Rosenkranz introduces separation techniques in the biochromatography, illustrates their characteristics compared with RP-HPLC, and describes the advantages and disadvantages of the individual modes.

Evaluation programs have several strengths, extents, and opportunities. In Chapter 6, Amo Simon shows as a neutral insider advantages and disadvantages of the most known software on the market: modern HPLC-Software programs – characteristics, comparison, outlook.

During integration of peaks, which are not separated by base line, there might amount enormous and often undetected mistakes. Mike Hillebrand presents in Chapter 7 prospects of the “right” integration nowadays. At the same time, he introduces among other things two software tools, which allow to determine objectively the deviation from desired value as well as the identification of the “true” peak area.

Chapter 8 is a question of HPLC in the regulated field. In the first part, Stefan Schmitz shows opportunities and gives a great many of hints in terms of intelligent documentation. Iris Retzko and Stefan Schmitz also give many hints for a successful FDA inspection in the second part. Especially, psychology and some simple tricks act a crucial part.

To gather information in an intelligent way is not only for secret services of prime importance. Efficient information collecting in the era of web 2.0 at the example of HPLC is the topic of Torsten Beyer in Chapter 9. Some links are presented, which might be useful to find specific information and the quality of these sources is also examined.

MS coupling has difficulties with isobar compounds; furthermore, there are some interesting molecules that are not UV active and finally refraction index detectors cannot be used in case of gradient elution. In Chapter 10, trends of detection techniques, Stefan Lamotte is giving a short overview of aerosol detectors und presents advantages as well as disadvantages.

The reader is not obliged to read the book linear. Every chapter represents a self-contained module, so jumping in between chapters is always possible. In this way, the character of the book gives justice to meet the requirements of a reference work. The reader may benefit thereof. At the end: some of the readers might want to use the EXCEL-Makro of Hans-Joachim Kuss for predicting gradient runs. Also the software tools of Mike Hillebrand to estimate integration errors might have drawn the interest of the reader. After all, Torsten Beyer's collection of useful links might be worth one's weight in gold and save unnecessary search. We want to give you the opportunity to use these tools online. WILEY-VCH makes the following link available: http://www.wiley-vch.de/textbooks/, where you can find the original-makro of Hans-Joachim Kuss for prediction of gradient runs, a demo version of the two integration tools of Mike Hillebrand as well as a list of links from Thorsten Beyer. We hope this offer obtains approval.

Preface

The HPLC-user fortunately can find nowadays many and good textbooks for the HPLC-methodology. Also applied literature, for example, for the pharma-analytics or for techniques such as UHPLC or gradient elution is available.

In this book, we cover different topics in the field of modern HPLC. The purpose is to demonstrate current developments and dwell on techniques which recently found their way to the HPLC-laboratory or will do in near future.

At the same time, we offer knowledge in condensed form. In 10 chapters experts address the skilled user and the laboratory head with practical attitudes, who are searching for profound (background-)knowledge and new insights.

Our purpose is on the one hand to point out for the reader unknown mistakes and on the other hand to offer him latest tips, which are hard to get in this condensed form. I hope this choice of topics meets the audience with approval.

My acknowledgments belong to the colleagues who placed their experience and knowledge at the disposal. Special thanks go to WILEY-VCH and especial Reinhold Weber for the extraordinary good cooperation.

Blieskastel, February 2016
Stavros Kromidas

The HPLC Expert

Possibilities and Limitations of Modern High Performance Liquid Chromatography

List of Contributors

The structure of “The HPLC-Expert”

Preface