Practical High-Performance Liquid Chromatography

Veronika R. Meyer

• Publisher: Wiley
• Year: 2013
• ISBN:
  9781118681343
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Analytic Chemistry

Jump into the HPLC adventure!

Three decades on from publication of the 1 st German edition of Veronika Meyer's book on HPLC, this classic text remains one of the few titles available on general HPLC aimed at practitioners.

New sections on the following topics have been included in this fifth edition:

• Comparison of HPLC with capillary electrophoresis
• How to obtain peak capacity
• van Deemter curves and other coherences
• Hydrophilic interaction chromatography
• Method transfer
• Comprehensive two-dimensional HPLC
• Fast separations at 1000 bar
• HPLC with superheated water

In addition, two chapters on the instrument test and troubleshooting in the appendix have been updated and expanded by Bruno E. Lendi, and many details have been improved and numerous references added.

A completely new chapter is presented on quality assurance covering:

• Is it worth the effort?
• Verification with a second method
• Method validation
• Standard operating procedures
• Measurement uncertainty
• Qualifications, instrument test, and system suitability test
• The quest for quality

Reviews of earlier editions

"That this text is written by an expert in both the practice and teaching of HPLC is evident from the first paragraph....not only an enjoyable, fascinating and easy read, but a truly excellent text that has and will serve many teachers, students and practitioners very well." — The Analyst

"…provides essential information on HPLC for LC practitioners in academia, industry, government, and research laboratories…a valuable introduction." - American Journal of Therapeutics

1
Introduction

1.1 HPLC: A POWERFUL SEPARATION METHOD

A powerful separation method must be able to resolve mixtures with a large number of similar analytes. Figure 1.1 shows an example. Eight benzodiazepines can be separated within 70 seconds.

Such a chromatogram provides directly both qualitative and quantitative information: each compound in the mixture has its own elution time (the point at which the signal appears on the screen) under a given set of conditions; and both the area and height of each signal are proportional to the amount of the corresponding substance.

This example shows that high-performance liquid chromatography (HPLC) is very efficient, i.e. it yields excellent separations in a short time. The ‘inventors’ of modern chromatography, Martin and Synge,¹ were aware as far back as 1941 that, in theory, the stationary phase requires very small particles and hence a high pressure is essential for forcing the mobile phase through the column. As a result, HPLC was sometimes referred to as high-pressure liquid chromatography.

1.2 A FIRST HPLC EXPERIMENT

Although this beginner’s experiment described here is simple, it is recommended that you ask an experienced chromatographer for assistance.

It is most convenient if a HPLC system with two solvent reservoirs can be used. Use water and acetonitrile; both solvents need to be filtered (filter with < 1 μm pores) and degassed. Flush the system with pure acetonitrile, then connect a so-called reversed-phase column (octadecyl ODS or C₁₈, but an octyl or C₈ column can be used as well) with the correct direction of flow (if indicated) and flush it for ca. 10 min with acetonitrile. The flow rate depends on the column diameter: 1–2 ml min⁻¹ for 4.6 mm columns, 0.5–1 mlmin⁻¹ for 3 mm and 0.3–0.5 mlmin⁻¹ for 2 mm columns. Then switch to water–acetonitrile 8 : 2 and flush again for 10–20 min. The UV detector is set to 272 nm (although 254 nm will work too). Prepare a coffee (a ‘real’ one, not decaffeinated), take a small sample before you add milk, sugar or sweetener and filter it (< 1 μm). Alternatively you can use tea (again, without additives) or a soft drink with caffeine (preferably without sugar); these beverages must be filtered, too. Inject 10 μl of the sample. A chromatogram similar to the one shown in Figure 1.2 will appear. The caffeine signal is usually the last large peak. If it is too high, inject less sample and vice versa; the attenuation of the detector can also be adjusted. It is recommended to choose a sample volume which gives a caffeine peak not higher than one absorption unit as displayed on the detector. If the peak is eluted late, e.g. later than 10 min, the amount of acetonitrile in the mobile phase must be increased (try water–acetonitrile 6 : 4). If it is eluted too early and with poor resolution to the peak cluster at the beginning, decrease the acetonitrile content (e.g. 9 : 1).

Figure 1.1 HPLC separation of benzodiazepines (T. Welsch, G. Mayr and N. Lammers, Chromatography, InCom Sonderband, Düsseldorf, 1997, p. 357). Conditions: samples: 40 ng each; column: 3cm × 4.6mm i.d.; stationary phase: ChromSphere UOP C18, 1.5 μm (nonporous); mobile phase: 3.5 ml min⁻¹ water–acetonitrile (85:15); temperature: 35°C; UV detector 254 nm. Peaks: 1 = bromazepam; 2 = nitrazepam; 3 = clonazepam; 4 = oxazepam; 5 = flunitrazepam; 6 = hydroxydiazepam (temazepam); 7 = desmethyldiazepam (nordazepam); 8 = diazepam (valium).

Figure 1.2 HPLC separation of coffee. Conditions: column, 15cm × 2 mm i.d.; stationary phase, YMC 120 ODS-AQ, 3 μm; mobile phase, 0.3 ml min^×1 water-acetonitrile (8:2); UV detector 272 nm.

The caffeine peak can be integrated, thus a quantitative determination of your beverage is possible. Prepare several calibration solutions of caffeine in mobile phase, e.g. in the range 0.1–1.0 mg ml⁻¹, and inject them. For quantitative analysis, peak areas can be used as well as peak heights. The calibration graph should be linear and run through the origin. The caffeine content of the beverage can vary within a large range and the value of 0.53 mg ml⁻¹, as shown in the figure, only represents the author’s taste.

After you have finished this work, flush the column again with pure acetonitrile.

1.3 LIQUID CHROMATOGRAPHIC SEPARATION MODES

Adsorption Chromatography

The principle of adsorption chromatography (normal-phase chromatography) is known from classical column and thin-layer chromatography. A relatively polar material with a high specific surface area is used as the stationary phase, silica being the most popular, but alumina and magnesium oxide are also often used. The mobile phase is relatively nonpolar (heptane to tetrahydrofuran). The different extents to which the various types of molecules in the mixture are adsorbed on the stationary phase provide the separation effect. A nonpolar solvent su...