Comprehensive Chromatography in Combination with Mass Spectrometry

Luigi Mondello

• Publisher: Wiley
• Year: 2011
• Print ISBN:
  9780470434079
• Edition: 1
• eBook ISBN:
  9781118003459
• Topic: Technology & Engineering
• Subtopic: Chemical & Biochemical Engineering
• Index: Technology & Engineering

This book provides a detailed description of various multidimensional chromatographic separation techniques. The editor first provides an introduction to the area and then dives right into the various complex separation techniques. While still not used routinely comprehensive chromatography techniques will help acquaint the readers with the fundamentals and possible benefits of multi-dimensional separations coupled with mass spectrometry.

The topics include a wide range of material that will appease all interested in either entering the field of multidimensional chromatography and those looking to gain a better understanding of the topic.

Chapter 1

Introduction

Luigi Mondello and Peter Q. Tranchida

University of Messina, Messina, Italy

Keith D. Bartle

University of Leeds, Leeds, UK

The world surrounding us is characterized by an enormous number of heterogeneous samples, in terms of both complexity and chemical composition. Some mixtures, such as natural fats and oils (e.g., butter, olive oil) are composed of a relatively small number of constituents, whereas others, such as roasted coffee aroma, motor fuels, or protein hydrosylates, are highly complex. Chromatography is, without doubt, the technique of choice for the separation of a real-world sample either into a series of low-complexity subsamples or (ideally) into its individual constituents. To count the number of real-world samples that can be analyzed by using a chromatography technique is akin to counting the number of particles of sand on a beach.

The first use of a chromatographic method, as well as the employment of the word chromatography, were reported over a century ago (Tswett, 1906a,b). Prior to the invention of chromatography, target analyte separation from the rest of the matrix was achieved mainly through distillation, liquid extraction, and crystallization. Mikhail Semenovich Tswett, the inventor of chromatography, described the separation of plant pigments as follows: “Like light rays in the spectrum, the different components of a pigment mixture, obeying a law, are resolved on the calcium carbonate column and then can be qualitatively and quantitatively determined. I call such a preparation a chromatogram and the corresponding method the chromatographic method.” A variety of aspects related to liquid–solid chromatography were discussed in the two initial fundamental papers, suggesting the possibility of achieving two-dimensional chromatography by developing the column with another eluent after the primary separation (Ettre, 2003; Tswett, 1906a,b). As for many revolutionary inventions, the scientific community showed scepticism toward a technique that would have changed the world of analytical chemistry. It was not until the 1930 s that the potential of chromatography was to be fully appreciated, unfortunately long after the death of its first promoter.

1.1 Two-dimensional chromatography–mass spectrometry: a 50-year-old combination

Up until about half a century after the invention of chromatography, the structural elucidation of unknown analytes eluting from a chromatography column was a rather tedious task. Peak assignment was commonly achieved through two approaches: (1) by comparing the retention times of unknown analytes with those of known compounds, using two or more chromatographic columns with different selectivities; or (2) by collecting each chromatographic band eluting from the column and subjecting each fraction to an instrumental identification procedure (e.g., infrared spectroscopy, mass spectrometry). The unreliability of the first option is evident, due mainly to the limited peak capacity of the chromatography systems used in that historical period. Consider the difficulty, or better the impossibility, of qualitatively analyzing a 100-plus component sample on a packed column using only retention times! The second approach, and certainly more preferable, was commonly achieved by using a cold trap, but was characterized by the disadvantages related to excessive sample handling.

It was not until the end of the 1950s that the first online chromatography–mass spectrometry (MS) experiments were reported. In particular, Gohlke (1959) described a two-dimensional gas chromatography (GC)–time-of-flight (TOF) mass spectrometry (MS) system characterized by four parallel packed columns plus a thermal conductivity detector. The TOF MS employed enabled mass unit resolution up to a mass of 200 and generated 2000 spectra per second. A GC–TOF MS chromatogram for a five-compound mixture separated on a 10-ft-long packed column is illustrated in Figure 1.1. The outstanding results reported are certainly not diminished by the fact that probably not more than 50 compounds could have been identified reliably using the two-dimensional method. In fact, a rough visualization of the chromatogram suggests that not more than 15 peaks can be stacked side by side in a 20-min one-dimensional separation space. Furthermore, Gohlke affirmed that “single chromatographic peaks containing two or three components can usually be successfully resolved by a careful examination of several mass spectra obtained at various times during the development of the chromatographic peak.” This was a very interesting statement; clearly, Gohlke fully comprehended the complementary nature of the two analytical dimensions.

Figure 1.1 GC–TOF MS chromatogram of acetone (6), benzene (7), toluene (8), ethylbenzene (9), and styrene (10).

[From Gohlke (1959), with permission. Copyright © 1959 by The American Chemical Society.]

In the field of separation science today, analysts still tend to fall within one of two well-defined groups:

1. Chromatography experts, who tend to have great faith in their capability to optimize the chromatographic process and are inclined to treat the mass spectrometer as little more than a detector. Such an outlook is appropriate as long as the ion source receives analytes resolved entirely (then identified, for example, by using MS libraries). However, problems can arise when extensive peak overlap occurs and a thorough exploitation of the MS dimension becomes necessary.

2. MS experts, who tend to have great faith in their capability to untangle a multicomponent band that enters the ion source, because the mass analyzer can then resolve a group of ions on a mass basis. It is true that mass spectrometry can be very useful for the unraveling of overlapping analytes. However, the reliability of peak identification is inversely proportional to the extent of compound coelution. In truth, chromatography and MS processes are equally important and complementary, and should be pushed to their full potential.

1.2 Shortcomings of one-dimensional chromatography

At present, one-dimensional chromatography is the method most commonly employed for the separation of real-world samples. However, in the past three decades it has become increasingly clear that the baseline separation of all the constituents of a sample or of specific target analytes from the rest of the matrix is often an unreasonable challenge when using a single chromatography column. The two fundamental aspects that govern all chromatography processes are peak capacity (nc) and stationary-phase selectivity. The former parameter is related to the column characteristics (i.e., length, internal diameter, particle diameter, stationary-phase thickness, intensity of analyte–stationary phase interactions, etc.) and to the experimental conditions (i.e., mobile-phase flow and type, temperature, outlet pressure, etc.). The other feature is related to the chemical composition of the stationary phase, and hence to the specific type of analyte–stationary phase interactions (i.e., dispersion, dipole–dipole, hydrogen bonding, electrostatic, size exclusion, etc.). Selectivity is also dependent on analyte solubility in the mobile phase, whenever this type of interaction occurs. Ideally, a chromatographic analysis will be achieved in the minimum time for a given sample if the column is characterized by the most appropriate separation phase and generates the minimum peak capacity required. Nothing more than this goal is sought by all chromatographers.

An experienced chromatographer with a knowledge of basic theory will easily get the best out of a column or, in other words, will maximize the number of peaks that can be stacked side by side (with a specific resolution value) in a one-dimensional space. However, it has been emphasized that such an analytical capability will fall far short of the peak capacity requirements for many applications. In inspirational work, Giddings demonstrated from a theoretical viewpoint that “no more than 37% of the peak capacity can be used to generate peak resolution” and that “many of the peaks observed under these circumstances represent the grouping of two or more close-lying components,” concluding that “s (the number of single component peaks) can never exceed 18% of nc (Giddings, 1990). Although such a value does not take stationary-phase selectivity into account, it provides an excellent indication of the separation power of a one-dimensional chromatography system.

The well-known master equation for the calculation of resolution between two compounds with retention factors equal to k1 and k2, is

1.1

The different degrees of influence of N, α, and k on Rs can be observed in the excellent example shown in Figure 1.2, where the separation of two analytes (k1 = 4.8;k2 = 5.0; α = 1.05) on a GC column (N = 20,000) under fixed conditions is considered. If we direct our attention to the three variables contained in Eq. (1.1) and to the effects of their variation on resolution (visualized in Figure 1.2), we can draw the following conclusions

Figure 1.2 Resolution: influence of N, α, and k.

[From Sandra and David (1990), with permission. Copyright © 1990 by Taylor & Francis Group LLC.]

:

k	If the column phase ratio is reduced (or a lower temperature is used), leading to an increase in the retention factors, the benefits gained are very limited in terms of resolution. An increase in k has a substantial effect on Rs only for analytes with low k values ( ≤ 3).

α

If a more selective stationary phase is employed, thus increasing the separation factor, resolution will benefit greatly. From Eq. (1.1) it can be concluded that at lower values, an increase in α will lead to a considerable improvement in resolution, up to an α value of about 3. At higher separation factor values, the function tends to level off. Of the three variables, selectivity has the greatest effect on resolution, and thus it is fundamental to select the most suitable stationary phase for a given separation. However, it must also be noted that Eq. (1.1) is valid only for a single pair of analytes and not for a complex mixture of compounds; in the latter case, a stationary-phase change will often lead to an improvement in resolution for some analytes and a poorer result for others. The choice of the most selective stationary phase has the best effects only when a low-complexity sample is subjected to separation.

N

If the column length is extended fourfold, leading to an increase in N by the same factor, resolution of the two analytes is only doubled. It follows that an evident improvement in peak resolution can only be achieved by extending the column length considerably. Such a modification is usually not desirable and certainly is not a practical solution in view of the greatly increased analysis time. However, enhancing the plate number is without doubt the best choice whenever a highly complex mixture is subjected to chromatography. In fact, an increase in N will lead to the same percentage increase in resolution for all the constituents of...

Table of contents

• Cover
• Series Page
• Title Page
• Copyright
• Contributors
• Preface
• Chapter 1: Introduction
• Chapter 2: Multidimensional Gas Chromatography: Theoretical Considerations
• Chapter 3: Multidimensional Liquid Chromatography: theoretical considerations
• Chapter 4: History, Evolution, and Optimization Aspects of Comprehensive Two-Dimensional Gas Chromatography
• Chapter 5: Flow-Modulated Comprehensive Two-Dimensional Gas Chromatography
• Chapter 6: Comprehensive two-dimensional gas chromatography combined with mass spectrometry
• Chapter 7: Detector Technologies and Applications in Comprehensive Two-dimensional Gas Chromatography
• Chapter 8: History, Evolution, and Optimization Aspects of Comprehensive Two-Dimensional Liquid Chromatography
• Chapter 9: Comprehensive Two-Dimensional Liquid Chromatography Combined with Mass Spectrometry
• Chapter 10: Comprehensive two-dimensional liquid chromatography applications
• Chapter 11: Other Comprehensive Chromatography Methods
• Chapter 12: Comprehensive Chromatography Data Interpretation Technologies
• Wiley Series
• Index