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Herausgeber: John Wiley & Sons
Kategorie: Wissenschaft und neue Technologien
Sprache: Englisch

Beschreibung

Provides students and practitioners with a solid grounding in the theory of chromatography, important considerations in its application, and modern instrumentation.

Highlights the primary variables that practitioners can manipulate, and how those variables influence chromatographic separations
Includes multiple figures that illustrate the application of these methods to actual, complex chemical samples
Problems are embedded throughout the chapters as well as at the end of each chapter so that students can check their understanding before continuing on to new sections
Each section includes numerous headings and subheadings, making it easy for faculty and students to refer to and use the information within each chapter selectively
The focused, concise nature makes it useful for a modular approach to analytical chemistry courses

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Seitenzahl: 526

Veröffentlichungsjahr: 2016

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Leseprobe

Cover

Title Page

PREFACE

CHAPTER 1: FUNDAMENTALS OF CHROMATOGRAPHY

1.1 THEORY

1.2 BAND BROADENING

1.3 GENERAL RESOLUTION EQUATION

1.4 PEAK SYMMETRY

1.5 KEY OPERATING VARIABLES

1.6 INSTRUMENTATION

1.7 PRACTICE OF THE TECHNIQUE

1.8 EMERGING TRENDS AND APPLICATIONS

1.9 SUMMARY

PROBLEMS

REFERENCES

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Guide

Cover

Table of Contents

Preface

Begin Reading

List of Illustrations

CHAPTER 1: FUNDAMENTALS OF CHROMATOGRAPHY

Source:

Alon McCormick and Peter Carr. Reproduced with permission of U of MN.). It is worth taking time to note the different dimensions involved. For the GC columns, they range from microns (10

−6

m) for the thickness of the stationary phase, to millimeters (10

−3

m) for the column diameter, up to tens of meters for the column length. Note also that LC columns are typically much shorter than GC columns (centimeter versus meter).

Figure 1.2 An example of a chromato

gram

– a plot of signal versus time – measured using a chromato

graph

(the instrument). Each peak represents a different solute that emerges from the column at a different time than the others. The peak width and height are related to the amount of each solute present.

Figure 1.3 This Figure depicts the behavior of phenol and toluene (solutes) partitioning between water and octane (bulk solvents). The water and octane serve as models for the mobile and stationary phases, respectively, in liquid chromatography. The left image depicts the system right after solutes are added to the aqueous phase before equilibrium is established. Once equilibrium is established (right), more toluene than phenol partitions into the nonpolar octane phase. Similarly, more phenol resides in the water due to hydrogen bonding and dipole-dipole interactions.

Figure 1.4 Chromatogram of phenol and toluene. The retention times of phenol and toluene are 3.30 and 5.20 min, respectively. The dead time, the time it takes an unretained solute to pass through the column, is labeled as

Figure 1.5 Effect of decreasing the phase ratio (

) by increasing

and decreasing

on retention (assuming constant

tot

). Solute retention times,

, increase as the volume of stationary phase (

) in the column increases due to solutes spending more time in the stationary phase.

Figure 1.6 The effect of peak width on resolution. The dead time and solute retention times are the same in both chromatograms, meaning that the separation factor is the same in both chromatograms. The lack of resolution in (b) compared to (a) is therefore due to the widths of the peaks.

Figure 1.7 Illustration of the two different measurements of width: (a) baseline width (

) and (b) full width at half maximum (FWHM,

1/2

Figure 1.8 Illustration showing that narrower peaks have smaller

and

1/2

than do broader peaks.

Figure 1.9 A mixture of compounds A, B, C, D, E, and F separated with two different distillation columns. The compound volatility follows the order: volatility of A > volatility of B > … > volatility of F. The column on the left has more plates and smaller plate heights (the distance between plates). This leads to a more complete separation of the components of the mixture compared to that achieved with the column on the left with fewer plates and larger plate heights.

Chromatography columns do not have actual, physical plates inside them like the distillation columns pictured here

, but the concept is borrowed as a way to measure and compare the separation ability of different columns. The columns depicted here have around 10–20 actual plates, whereas GC and LC columns have thousands or hundreds of thousands of “theoretical plates.”

Figure 1.10 Illustration of the effects of broad peaks on peak overlap. The peak maxima occur at the same time in both plots, but the bottom plot has broader peaks resulting from a less efficient column with fewer theoretical plates. It is easier to quantify the peaks in the top chromatogram than in the bottom because in the top, all of the peaks are baseline resolved.

Figure 1.11 Depiction of solute diffusion over time in an open tube. (a) An infinitely narrow plug of solute molecules. (b, c) Solutes diffuse toward regions of lower concentration down the long axis of the column (i.e., longitudinally). The more time that is allowed for diffusion, the broader the distribution of solute molecules, as depicted below each column.

Figure 1.12 The effect of time and diffusion coefficient on band spreading. In case A, the time allowed for diffusion is assumed to be equal. In this case, molecules with higher diffusion coefficients spread out more than do ones with smaller diffusion coefficients. In case B, the diffusion coefficients of the two sets of molecules are assumed to be the same. In this case, the longer the time the molecules are allowed to diffuse, the more broadening occurs. So increased time and higher diffusion coefficients both lead to increased band spreading. Conversely, shorter times allowed for broadening and smaller diffusion coefficients reduce the amount of band broadening.

Figure 1.13 Depiction of the parabolic flow profile. (a) Parabolic flow at a lower mobile phase velocity. (b) With a higher mobile phase velocity, the parabolic flow profile becomes more pronounced and solutes are spread out over a greater distance (assuming at this point that there is no mechanism for combating the spread of molecules).

Figure 1.14 Depiction of radial diffusion in response to concentration gradients caused by parabolic flow. X = solute molecules. Note that in (a), on the far right side, the solute concentration is high in the center of the column and zero at the walls. Conversely, on the far left side, the solute concentration is high near the walls and zero in the center of the column. In both cases, a radial concentration gradient exists. In (b), radial diffusion acts to decrease these concentration gradients.

Figure 1.15 Effect of radial diffusion in the presence of a stationary phase to help reduce zone broadening. In (a), the solute is in equilibrium between the stationary and mobile phases. In (b), the solutes in the mobile phase have moved down the column, meaning that those in the stationary phase lag behind. This causes the solute zone to broaden. Radial diffusion (signified by the squiggly arrows) in response to the concentration gradients that get created mitigates this effect by averaging out the rate of travel. This process of solutes diffusing into and out of the stationary phase in response to concentration gradients gets repeated the entire time that the solutes spend in the column. Note that the stationary phase thickness in these images has been grossly exaggerated. This has been done simply to help the reader picture the dynamic processes occurring in the column. In reality, the stationary phase thickness is nearly negligible compared to the column diameter.

Figure 1.16 An illustration of the process of radial diffusion in response to solute retention caused by the presence of a stationary phase. (a) Solutes in the mobile phase initially partition into the stationary phase. (b) Solutes nearest to the stationary phase are those most likely to partition. When they do, a radial solute concentration exists in the mobile phase, with high solute concentration in the center of the column and low concentration near the walls. (c) Solutes in the mobile phase continue down the column and diffuse to reduce the radial concentration gradient. Similarly, solutes in the stationary phase diffuse out in response to the low concentration in the center of the column that arises because the mobile phase has pushed the rest of the molecules down the column. (d) The molecules that have reentered the mobile phase will now be pushed down the column, while molecules out in front are retained as they approach and interact with the stationary phase. It is important to note that these are static pictures, but in reality, all of these processes, in addition to parabolic flow and longitudinal diffusion, are happening in the column continuously and simultaneously. But breaking them up into discrete steps makes it easier to understand the effects of each process on band broadening.

Figure 1.17

versus plot for an open tube with a thin stationary phase. There are three main contributions: the

, and

terms as shown in Equation 1.18. The

and

terms are both linear with and their contribution to

can be combined as shown. The contribution to

of the term approaches zero at high average linear velocities. The solid black line is the sum of the two contributions, or the total

. Note that at high average linear velocities, the (

) term makes the vast majority of the contribution to the overall

. Conversely, at low average linear velocities, the term dominates. The arrow indicates the optimum average linear velocity (), meaning the velocity that produces the lowest plate height and therefore the narrowest peaks. As noted in the text, however, we frequently operate at linear velocities higher than the optimum, accepting the slightly broader peaks that result in order to decrease the total analysis time.

Figure 1.18 Velocities of the mobile phase vary in different regions of a column packed with particles (gray dots). The different thicknesses of the arrows represent the differing velocities. The existence of multiple flow paths, along which solutes are carried at different velocities, is a source of band broadening in packed columns. This is illustrated by the solutes (X) at the front of each arrow. They are spread out in their location within the column because of the velocity variations. This means that they will also elute at slightly different times, resulting in band broadening. Note: The size of the particles is greatly exaggerated relative to the diameter of the column. Recall that the most common particle sizes are 5 µm and smaller.

Figure 1.19 Depiction of the “flow mechanism” of broadening relaxation. A solute on Path 1 initially experiences a slow velocity due to a high packing density in a particular region of the column. As the packing density decreases, it then experiences a faster velocity. Conversely, a solute on Path 2 initially experiences faster velocity, but then slows as it enters a region of higher packing density. The solute on Path 2 reaches Point A first, but the two solutes might still elute close together in time due to the averaging of velocities that they experience as the move through the column. Again, the size of the particles is exaggerated relative to the column dimensions.

Figure 1.20 Depiction of the “diffusion mechanism” of broadening relaxation. Solutes diffuse in the radial direction (depicted by arrows). In doing so, solutes that had been in fast paths move into slower ones, and conversely, solutes that had been in slow paths move into faster ones. In this way, the velocities experienced by all solutes average out, decreasing the effects of band broadening caused by the existence of different velocity paths through a packed column.

Figure 1.21 A depiction of the events involved in solute retention. A quadrant of a cross section of a particle is shown. Shaded areas represent the silica support particle. White spaces represent the pores, which are filled with stagnant mobile phase. In order to be retained, solutes in the flowing mobile phase must (i) be brought to the solid support particle, (ii) diffuse into the particle pores by diffusing through the stagnant mobile phase that fills the pores, and (iii) diffuse into the stationary phase. The solute must diffuse back through the stagnant mobile phase and out of the pores in order to get back into the flowing mobile phase. When they do, they catch up to other molecules of the same kind that have moved down the column with the mobile phase.

Figure 1.22

versus plot for packed columns. There are three main contributions: the

-, and

- terms as shown in Equation 1.33. The

-term accounts for multiple velocity paths, the

-term accounts for longitudinal diffusion, and the

-term includes the contributions of slow mass transfer in the stationary and mobile phases. This plot is similar to that in Figure 1.17 for flow through an open column coated with a stationary phase, except that here, with a packed column, the

-term is present and makes a constant contribution at all velocities. As in Figure 1.17, the

-term dominates at low average linear velocities, and the

-term dominates at high average velocities. The arrow indicates the optimum average linear velocity (), meaning the velocity that produces the lowest plate height and therefore the narrowest peaks. As noted in the text, however, we frequently operate at linear velocities higher than the optimum, accepting the slightly broader peaks that result in order to decrease the total analysis time.

Figure 1.23 (a) Depiction of the effect of decreasing particle size on plate height, in which the curve labeled (c) corresponds to larger particles and (a) corresponds to smaller particles. The vertical lines show that with smaller particles, a given increase in the linear velocity (Δμ) produces a much smaller corresponding increase in plate height (ΔH) compared to larger particles. This means that smaller particles can be used at higher velocities without significantly increasing band broadening. (b) Experimentally determined van Deemter curves for 5, 3.5, and 1.7 µm particles. The vertical lines correspond to the optimum linear velocity for each column (i.e., the velocity that produces the smallest plate height and thus the narrowest peak). Again we see that the

-term in the high linear velocity region is the least steep for the smallest particles, allowing for higher velocities with minimal additional band broadening. Conditions: 2.1 mm inner diameter column; 50/50 v/v (ACN/water); 210 nm detection; 45

C, benzylphenone as analyte; ultra-high performance LC system. (

Source:

Reprinted with permission from

Amer. Pharm. Rev

. 2008, 11, 24–33, from the authors of that article, and from Dr. Michael Dong who modified the original image for publication in

LC/GC North Am.

, 2014, 32, 553–557, which is the version shown here.)

Figure 1.25 Resolution (

) as a function of the number of theoretical plates (

) on a column for three different values of the retention factor, using the general resolution equation (Equation 1.35). The separation factor,

, is set at 1.1. Note that

rapidly increases as

increases at low plate numbers, but

increases more slowly at higher

. The square root dependence of

mitigates the effect on

of increasing

approaches high values.

Figure 1.27 Effect of separation factor (

) on resolution (

), using the general resolution equation with

= 10,000 at varying values of retention factor (

). (a) This plot shows that as separation factors increase, so does resolution. However, the effect begins to level off at high separation factors. The plot also reinforces that there is little gain in resolution once retention factors exceed

= 10, but significant increases in analysis time result. The inert (b) shows an expanded view of the circled region of plot A. It is clear that small improveents in the separation factor can lead to relatively large improvements in resolution when the separation factor is initially low (i.e., between 1.0 and 1.2). Separation factors are often changed by varying the mobile phase composition (common in LC) or the temperature (common in GC). Recall, baseline resolution is generally associated with

= 1.5. These plots show that even small values of the separation factor can achieve baseline resolution of peaks if the solute retention factors are high enough and

is large enough.

Figure 1.25 Effect of retention factor (

) on resolution (

) at three different separation factors (α), calculated using the general resolution equation (Equation 1.35) with

= 10,000. As illustrated by the dashed lines, increasing the retention factor (Δ

) when the retention factor is low increases

(Δ

) considerably. When retention is already high (

= 6–10), increasing retention has little effect on improving resolution, but it makes the analyses take longer. It is also clear from this graph that small increases in the separation factor even from 1.05 to 1.10, improve resolution substantially at every retention factor. When viewing this plot, recall that a resolution of 1.5 is generally associated with baseline resolution for peaks of the same height. Values under 1.5 suggest overlapped peaks.

Figure 1.27 Depiction of symmetric, fronted, and tailed peaks. The asymmetry factor (AF) is measured as the ratio of

, where

and

are determined as shown in the Figure at 10% of the peak height. Asymmetric peaks often indicate that something is not optimal and the source of the asymmetry should be investigated.

Figure 1.28 Example of a calibration curve based on chromatographic peak area of the analyte.

CHAPTER 2: GAS CHROMATOGRAPHY

Figure 2.1 Chromatogram of premium unleaded gasoline.

Figure 2.2 Depiction of solute partitioning. Squares and circles represent solutes. In this figure, squares have a greater affinity for the stationary phase relative to the gas phase than do the circles.

Figure 2.3 Schematic of a wall-coated open tubular gas chromatography column.

Figure 2.4 The effect of temperature on chromatograms. (a) column at low temperature of 45 °C isothermal separation, (b) column at moderately high temperature 120 °C isothermal separation, and (c) temperature programmed separation from 30 to 180 °C with a rate of 4.8 °C. At 45 °C, the late-eluting peaks are severely broadened and all of the compounds have not yet eluted. At 120 °C, the early-eluting peaks overlap, which complicates quantitative analysis. With temperature programming, all of the peaks are eluted and resolved with narrow peak shapes.

Figure 2.5 Plate height (

) as a function of average linear velocity using N

, He, and H

as the carrier gas. Nitrogen provides the lowest overall plate height possible, but at such a low linear velocity that it would result in very long analysis times. Hydrogen provides good plate heights and the least increase in plate height at high velocities (i.e., shorter analysis times). However, working with hydrogen can be dangerous. Therefore, helium, which offers performance that is comparable to that of hydrogen, is most commonly used as the carrier gas.

Figure 2.6 GC stationary phases from Restek Corporation. Other vendors offer similar stationary phase structures and use a similar numbering scheme, but replace the Restek prefixes (e.g., Rtx and Rxi) with letters specific to their companies. For example, J&W Scientific uses the letters “DB” and Supelco uses “SPB.”

Figure 2.7 Effect of film thickness on

and

. The phase ratio,

, depends significantly on

in wall-coated open tubular capillary chromatography because as

changes a lot (e.g., doubles),

does not show as significant a decrease.

Figure 2.8 Schematic of a gas chromatography system.

Figure 2.9 Schematic of split/splitless injection port.

Figure 2.10 Elimination of solvent tailing using inlet purge. Without purge, there is the potential for the solvent peak “tail” to overlap solute peaks, making quantitation more difficult or impossible.

Figure 2.11 Discrimination of

-alkanes up to C

for filled needle and hot needle injection compared to cold on-column injection. Note the discrimination that occurs for the higher boiling

-alkanes with filled needle injection. With cold on-column injection, all of the solutes are delivered to the column and thus no discrimination based on the relative volatilities of the solutes occurs.

Figure 2.12 Solvent focusing arising from the condensation of the solvent on a cool column followed by evaporation as the column temperature is increased.

Figure 2.13 Chromatograms showing the effect of solvent focusing. On the left, no solvent focusing occurs because the solvent boiling point is lower than that of the column temperature. On the right, however, the peaks for undecane (

-C

) and dodecane (

-C

) are sharpened due to solvent focusing induced by using a solvent,

-octane, which has a boiling point (125 °C) that is higher than the temperature of the column (115 °C). Note that on the right, not only are

-C

and

-C

focused, but minor peaks, possibly arising from solvent impurities, are also focused such that they can be observed on the right but disappear into the solvent tail on the left.

Figure 2.14 Solvent focusing achieved using a retention gap coupled with stationary phase focusing effects.

Figure 2.15 Six-port sampling valves. Configuration (a) is used for sample loading. Note that the sample loop is isolated from the carrier flow (i.e., mobile phase) so that sample can be loaded into the loop. The valve is then rotated to configuration (b) for sample injection. This puts the sample loop into the path of the flowing mobile phase and sweeps the sample onto the column.

Figure 2.16 Solid-phase microextraction (SPME) device. The coated fused silica fiber is used to concentrate analytes from a gas or liquid sample. The fiber is then retracted into the needle. The needle is used to pierce the septum of a GC injection port. The fiber is then extended into the port. The solutes are thermally desorbed from the coated fiber in the injection port and swept onto the column by the carrier gas flow.

Figure 2.17 Diagram of a flame ionization detector. Solutes flow up from the column through the detector. They burn in the flame and create ions. The ions are collected by the collector electrode and produce a current. A chromatogram is thus a plot of the detector current (or voltage) versus time.

Figure 2.18 Schematic of a thermal conductivity detector. When the valve is in the “up” position, carrier gas bathes the filament and column effluent is diverted away from the filament. In the “down” position, carrier gas sweeps the column effluent across the filament. If solutes are present, the thermal conductivity of the gas phase changes, leading to a change in the current in the filament.

Figure 2.19 Thermal conductivity detector based on a Wheatstone bridge. As solutes emerge from the column and are swept across the sample filament, the resistance changes due to the mismatch of thermal conductivities between solutes and the carrier gas. When the resistance changes, the voltage between

and

changes and in this way a chromatogram can be recorded by measuring the voltage as a function of time.

Figure 2.20 Simulated chromatograms of complex mixtures containing ketones (indicated by asterisks). (a) Chromatogram resulting from integrating

all

IR signals (i.e., nonselective mode). (b) Chromatogram that might result by plotting only the signal observed at 1715 cm

−1

as a function of time.

Figure 2.21 Electron ionization GC–MS interface. Solutes are ionized by high-energy electrons emitted from the tungsten filament. The resulting ions are accelerated and focused by the accelerating plates and subsequently introduced into the mass analyzer.

Figure 2.22 Schematic of a flame photometric detector. Sulfur- and phosphorus-containing molecules burn in the flame to produce the chemiluminescent species SS and HPO. The chemiluminescence is detected by the photomultiplier tube, the electrical output of which is used to produce the chromatogram.

Figure 2.23 Simulated chromatogram to illustrate Kovats retention index calculations.

Figure 2.24 Kovats retention indices of select compounds on five stationary phases at 80 °C. Crossovers indicate changes in the elution order of solutes as a result of the different blends of intermolecular interactions the solutes experience with each stationary phase. Solutes: () acetone, () dimethylsulfoxide (DMSO), () nitropropane, () hexafluoroisopropanol (HFIPA), () benzene, (•) phenol, () 2-pentanone, (*) butylether, (+) ethanol. Stationary phases: DB-1 = nonpolar dimethylpolysiloxane, DB-17 = intermediate polarity (14% cyanopropylphenyl)-methylpolysiloxane, DB-210 = selective for lone-pair electrons (50% trifluoropropyl)-methylpolysiloxane, DB-225 = polar (50% cyanopropylphenyl)-methylpolysiloxane, DB-wax = polar polyethylene glycol (PEG).

Figure 2.25 Depiction of the permanent dipole of acetone inducing a dipole on the aromatic phenyl rings in a methylphenyl polysiloxane stationary phase.

Figure 2.26 Cartoon illustrating the difference between absorption into the stationary phase and adsorption onto the surface.

Figure 2.27 Schematic of a tunable GC system. Notation: capillary columns with different stationary phases (

and

), inlet (I), detector (D), electronic pressure controller (PC), carrier gas (CG) inlets, capillary pneumatic restrictor (

), vent point (

), inlet pressure (

), outlet pressure (

), and tuning pressure (

is the highest pressure and

is the lowest pressure. By varying the tuning pressure (

), the average carrier gas velocity in

and

is altered. Increasing

decreases the pressure drop across

and increases it across

. This decreases the average carrier gas velocity in

and hence increases the residence time of the sample in

. Concomitantly, an increase in

increases the carrier gas velocity in

which decreases the solute residence time in that column. Thus, by varying

, the relative contributions of the two columns to the overall separation can be tuned to optimize the separation.

Figure 2.28 Micro-GC columns fabricated on silicon chips and a sample separation of six components in 25 seconds.

Figure 2.29 Gold particle coated with thiobutane.

Figure 2.30 (a) Chromatogram of distillate fuel oil from Basra light crude oil. (b) Chromatogram of Iranian heavy crude oil. (c) Chromatogram between nonane and undecane for distillate fuel oil from Basra light crude oil. (d) Chromatogram between nonane and undecane for Iranian heavy crude oil. These chromatograms illustrate that light crude oil from Basra is chemically different than heavy crude oil from Iran and that GC can be used to distinguish them.

CHAPTER 3: LIQUID CHROMATOGRAPHY

Figure 3.1 M.S. Tswett in Keil, Germany, in 1905.

Figure 3.2 Structure of silica.

Figure 3.3 Scanning electron micrograph of spherical porous silica particles (Spherisorb octadecylsilane chemically modified silica). Magnification 700× (a) and 7000× (b). Mean particle diameter 5 µm.

Figure 3.4 Depiction of the porous nature of typical HPLC silica particles (cross-sectional view). Gray-shaded regions represent the solid silica material, in this case with a stationary phase (short black curves) bonded to the surface. Open white spaces represent the pores.

Figure 3.5 Depiction of the adsorption of benzene onto a surface.

Figure 3.6 Depiction of the chemistry for modifying a silica stationary phase with an octylsilane (C-8) linkage. Many other surface modification techniques also exist.

Figure 3.7 Depiction of solute molecules (4-ethylanisole) partitioning into a C-18 stationary phase in RPLC.

Figure 3.8 Depiction of mobile phase modification of the C-18 chains in a typical RPLC column using a typical methanol/water mobile phase. While methanol is shown in this picture, all of the organic modifiers used in RPLC such as acetonitrile and tetrahydrofuran modify the stationary phase to some extent. Also notice that water is attracted to the polar silanol groups that may remain on the surface due to incomplete derivatization with C-18 chains.

Figure 3.9 Electron micrographs of the (a) macroporous and (b) mesoporous structures in a monolithic silica rod.

Figure 3.10 Separations representative of five ternary mobile phases. Peaks: (1) benzyl alcohol, (2) phenol, (3) 3-phenylpropanol, (4) 2,4-dimethylphenol, (5) benzene, and (6) diethyl

-phthalate.

Figure 3.11 Depiction of common RPLC stationary phases. (a) From top to bottom: C-1, C-4, C-8, C-18, aminopropyl, cyanopropyl, phenylhexyl, and a polar embedded phase. (b) Horizontally polymerized trifunctional silanes.

Figure 3.12 Depiction of the retention of K

via cation exchange on a sulfonic acid exchange resin (strong acid type). The competing ion in this case is H

, which is in the mobile phase. In (a), the eluent ion (H

) is interacting with the phase, whereas in (b), the solute ion (K

) is being retained. This equilibrium continues throughout the timeframe of the separation, with the solute moving down the column when it is displaced from the stationary phase and into the mobile phase by the eluent ions. Separation of solute ions (e.g., between Ag

and K

) occurs because of their different affinities for the charged stationary phase and thus their different abilities to displace the mobile phase counterion from the stationary phase. Note: Other ions such as Na

or Li

are frequently used in place of H

as the eluent ion in the mobile phase.

Figure 3.13 Functional groups of ion exchange resins.

Figure 3.14 Effect of mobile phase salt concentration on the retention of various food additives in ion-exchange chromatography. Column: 100 × 0.21 cm Zipax coated with 1% quaternary ammonium liquid-anion-exchanger; mobile phase: 0.01 M sodium borate (pH 9.2) with added NaNO

in water, 24 °C; 0.85 mL/min.

Figure 3.15 (a) Depiction of the elution of a solute band containing the analyte (nitrate ions in this case). (b) Ion exchange inside the suppressor column reduces the conductivity of the mobile phase but not that of the analyte (nitrate) zone, leading to a chromatogram of conductivity versus time.

Figure 3.16 Expanded view of the micromembrane suppressor for ion exchange chromatography.

Figure 3.17 Depiction of the water-rich layer on the surface of the pores in bare silica particles in HILIC. This image also depicts two of the different retention modes; partitioning of the solute from the organic-rich phase into the water-rich layer, and ion exchange (specifically cation exchange in this instance).

Figure 3.18 Depiction of size exclusion chromatography. The shaded area represents a porous particle. Small circles represent small solutes that can enter all pores, medium circles represent solutes that can enter only some of the pores, and large circles represent solutes that are too large to enter any of the pores and thus are not retained. Mobile phase flow carries the solutes around the porous particles. If the particles are of appropriate size, they can diffuse into pores. If they are too large, they continue past the particles and elute at the dead time.

Figure 3.19 Retention as a function of solute size in SEC.

Figure 3.20 Depiction of affinity chromatography. Only those solutes that are complementary to the molecules immobilized on the stationary phase are strongly retained. In this depiction the solid black “solutes” are complementary to the immobilized arrows. All other solutes ideally pass through the column unretained.

Figure 3.21 Schematic of a liquid chromatography system.

Figure 3.22 Operation of reciprocating pumps. (a) The pump fills with mobile phase during the back stroke as solvent is drawn in from the solvent reservoirs and through the proportioning and mixing valves. (b) The mobile phase is pushed out to create flow through the column during the forward stroke of the piston.

Figure 3.23 HPLC manual sample injection loop. Left: Load position in which the syringe is used to fill the sample loop. Note that the mobile phase from the pump goes directly into the column, bypassing the sample loop in this position. Right: Injection position in which the mobile phase flow from the pump flows through the sample loop, pushing the sample onto the column.

Figure 3.24 A variety of depictions of superficially porous particles. (b) is a tunneling electron micrograph of a cross-section through a 2.6 mm Kinetex core–shell particle from Phenomenex.

Figure 3.25 van Deemter curves comparing fully porous particles to superficially porous particles (SPPs). Note that the SPPs have essentially the same performance characteristics as the sub-2 µm particles. This is particularly important in the high flow rate region (i.e., high linear velocities) as it enables high-speed LC with the larger SPPs without sacrificing resolution and without the high back pressures encountered with smaller particles.

Figure 3.26 Flow cell for UV–Visible detection in HPLC.

Figure 3.27 Absorbance data as a function of wavelength and time acquired using a diode array detector.

Figure 3.28 A deflection-type refractometer.

Figure 3.29 Schematic of an electrospray ionization (ESI) interface. Droplets in the solvent spray acquire a charge from an electrode. Eventually the charge is transferred to solutes through a process of solvent evaporation and droplet explosion. Charged solutes are drawn through the capillary sampling orifice and into the mass analyzer.

Figure 3.30 Schematic of an atmospheric pressure chemical ionization (APCI) interface. Droplets are ionized by the corona discharge needle. Charge is transferred from the solvent to solute molecules which are eventually drawn into the sampling capillary and passed into the mass analyzer.

Figure 3.31 (a) Depiction of an Orbitrap mass analyzer and the oscillatory nature of an ion with it. (b) Alexander Makarov holding his invention.

Figure 3.32 Example of the advantage of high-resolution mass spectrometry to discriminate a target molecule from a matrix component of equivalent nominal mass. With lower resolution (10,000), the presence of norfloxacin in the sample could not be confirmed, but high resolution (100,000) makes this possible.

Figure 3.33 Schematic of an evaporative light scattering detector (ELSD). (a) Column effluent is nebulized after eluting from the column. (b) The aerosol droplets pass through a heated drift tube where solvent evaporation occurs. (c) Sample particle enter an optical cell where they pass through a laser beam. Scattering from the sample particles is detected and generates the electrical signal that generates the chromatogram.

Figure 3.34 Comparison of an evaporative light scattering detector (ELSD) to a UV detector at 254 nm used to analyze the same sample. Note that the saponins are not detected by the UV detector because they do not absorb 254 nm radiation, but they are detected by the ELSD, which is based simply on light scattering rather than on absorption.

Figure 3.35 Amperometric detector for LC. Solutes are oxidized or reduced at the working electrode (W), which creates a current that is measured to produce the chromatogram.

Figure 3.36 R & S enantiomers of thalidomide.

Figure 3.37 Depiction of the different intermolecular interactions experienced by enantiomers on a chiral stationary phase.

Figure 3.38 An example of a Pirkle-type chiral stationary phase; l-Naphthylleucine.

Figure 3.39 Examples of separations of enantiomers using Pirkle-type chiral stationary phases.

Figure 3.40 (a) α-Cyclodextrin (β- and γ-cyclodextrin have 7 and 8 glucose units). (b) Depiction of the cup-like 3D shape of cyclodextrins. (c) Depiction of cyclodextrins chemically bonded to the surface of a chromatographic support particle and their ability to selectively bind one enantiomer more strongly than the other.

Figure 3.41 Structure of vancomycin. Note the number of chiral centers and the multiple sites capable of donating and accepting hydrogen bonds. Because of these numerous chiral and hydrogen bonding sites, the molecule is able to bind different enantiomers of the same molecule with different strengths and thus help achieve their separation.

Figure 3.42 Reversed-phase enantiomeric separation of (from left to right) bromacil, devrinol, and coumachlor on a vancomycin chiral stationary phase. Column: 25 cm × 0.44 cm column, Silica particles: 5-µm, mobile phase: 10:90 acetonitrile: 1% pH 7 triethylammonium acetate buffer (v/v). Flow: 1.0 mL/min, temp: ambient, detection: UV at 254 nm.

Figure 3.43 Scale up of a reversed-phase separation of two xanthines. Shown are (a) analytical column separations and (b) the preparative separation resulting from scale-up calculations. (a) Column: 150 mm × 3 mm, 5 μm

Zorbax SB-C18 (Agilent Technologies, Wilmington, Delaware); mobile phase: 90:10 (v/v) water-acetonitrile; flow rate: 0.6 mL/min; detection; UV absorbance at 270 nm; pathlength: 10 mm; ambient temperature. (b) Column: 150 × 21.2 mm, 5 μm

, Zorbax SB-C18; mobile phase: 90:10 (v/v) water-acetonitrile; flow rate: 25 mL/min; detection; UV absorbance at 270 nm; pathlength: 3 mm; ambient temperature.

Figure 3.44 Scale-up of a separation of three antibiotics. (a) The structures of the antibiotics and (b) scale-up separations on an analytical column. (a) Column: 150 mm × 4.6 mm, 5 µm

Zorbax Eclipse XDB-C18; mobile phase: 65:35 (v/v) water–acetonitrile, both with 0.1% trifluoroacetic acid; flow rate: 1 mL/min; detection; UV absorbance at 254 nm; injection volume 30 μL; ambient temperature.

Figure 3.45 Effect of particle size on plate height (i.e., the height equivalent to a theoretical plate, HETP). (a) A 250 mm × 4.6 mm column packed with 10-µm particles, (b) a 125 mm × 4.6 mm column packed with 5 µm particles, and (c) a 100 mm × 4.6 mm column packed with 3 µm particles.

Figure 3.46 Simulation of a three-component separation obtained using (a) conventional and (b) fast LC conditions. Note the very different time scales in each chromatogram (15 min vs 1.5 min). Column (a): 150 mm × 4.6 mm, 5 µm particles, flow rate: 1.0 mL/min; column efficiency: 5400; operating pressure: 1200 psi; minimum resolution: 1.51; extracolumn volume: 70 μL. Column (b): 30 mm × 4.6 mm, 3 µm particles, flow rate: 2.0 mL/min; column efficiency: 2700; operating pressure: 1900 psi; minimum resolution: 1.07; extracolumn volume: 7 μL.

Figure 3.47 Block diagram of a tandem-column LC system. The contribution of the two columns to the overall solute retention is controlled by controlling the temperature of each column. Lower temperatures lead to greater contributions to retention.

Figure 3.48 Chromatograms showing the separation of a mixture of triazine herbicides on (a) C-18 at 30 °C and a 1-mL/min flow rate, (b) carbon-coated zirconia at 60 °C and a 1 mL/min flow rate, and (c) a thermally tuned tandem set with a C-18 column at 30 °C and a carbon-coated zirconia column at 125 °C, and a 3 mL/min flow rate. Mobile phase: 30:70 (v/v) acetonitrile–water; detection wavelength: 254 nm. Peaks: 1 = simazine, 2 = cyanazine, 3 = simetryn, 4 = atrazine, 5 = prometon, 6 = ametryn, 7 = propazine, 8 = turbulazine, 9 = prometry, 10 = terbutryn.

Figure 3.49 Simulation of comprehensive sampling of the effluent from one column (the first dimension) with subsequent analysis of that portion on a second column (the second dimension). In this case, samples are collected every 30 s, as indicated by the dashed lines. The interest in 2D-LC lies in the potential to observe compounds that partially or entirely overlapped under one set of chromatographic conditions by further separating them under a substantially different set of conditions. Here, the subsequent analysis of only two portions of the first dimension is being shown, but all eight portions that clearly contain at least one compound could be analyzed with the second column for comprehensive analysis.

Figure 3.50 Offline HILIC × RP separation of a Chinese medicine extract. Fractions from the first dimension were collected once every minute and subsequently analyzed by RPLC on a C-18 column. Each line running from left to right therefore represents a separate RPLC separation. If the peaks are compressed or condensed onto the left axis labeled “Fraction” by mentally sweeping them from right to left, one would have an idea of what the chromatogram from the first dimension looked like. Clearly, by looking at some fractions (like 7 and 10), many compounds eluted during the minute that the effluent was collected. Thus, it is likely several peaks partially or entirely overlapped, obscuring the true complexity and chemical makeup of the sample. The third axis (mAU) is the detector response at 280 nm.

Figure 3.51 Examples of 2D-LC separation. (a) Separation of low-molecular-weight components from a sample of human urine. Discovery HS-F5 (pentafluorophenylpropyl) and ZirChrom-CARB (carbon coated zirconia) phases were used as the two dimensions. (

Source:

From D.R. Stoll,

Anal. Bioanal. Chem

., 2010, 397, 979–986. Reproduced with permission of Springer.) (b) Separation of peptides from a tryptic digest of bovine serum albumin (BSA). The two dimensions were a strong cation exchange (SCX) and a RPLC column. (

Source:

From R.J. Vonk et al.,

Anal. Chem

Figure 3.52 Three different sampling modes for 2D-LC. (a) The comprehensive mode (LC × LC) in which the first dimension is sampled at regular time intervals and analyzed on the second dimension column. (b) The heartcutting mode, in which only a single or a very small number of time segments or peaks from the first dimension are targeted for analysis on the second dimension. (c) The selective comprehensive mode, in which multiple regions of the first chromatogram are sampled regularly and analyzed on the second dimension.

Figure 3.53 Location of 139 sampling sites in U.S. Geological Survey study of organic contaminants in streams.

Figure 3.54 Structure of sulfonamides (SAs), also known as sulfa drugs, and tetracyclines (TCs).

Figure 3.55 Approximate p

values for sulfonamides.

86,89

Figure 3.56 Sulfanilyl fragment ion with

= 156.

List of Tables

CHAPTER 1: FUNDAMENTALS OF CHROMATOGRAPHY

Table 1.1 Common RPLC and GC Characteristics

Table 1.2 Physical Significance of the Terms in the van Deemter Equation

Table 1.3 Factors That Impact Band Broadening

CHAPTER 2: GAS CHROMATOGRAPHY

Table 2.1 Linear Velocities Converted to Flow Rates as a Function of Column Diameter

Table 2.2 Typical Characteristics for Common GC Columns

Table 2.3 Volume of Gas Occupied by 1 μL of Liquid Solvent

Table 2.4 Characteristics of Common GC Injection Modes

Table 2.5 Characteristics of Common GC Detectors

7,16

Table 2.6 Relative Response of ECDs to Solutes

Table 2.7 Kovats Retention Indices Based on Figure 2.23

Table 2.8 Kovats Retention Indices at 80 °C

Table 2.9 A McReynolds Probe Solutes, Parameter Symbols, and Interactions They Represent

Table 2.9 B McReynolds Constants for Common GC Stationary Phases

Table 2.10 Illustration of the Effects of Injection Variation on Peak Area

Table 2.11 Utility of Internal Standards for Quantitative Analysis

CHAPTER 3: LIQUID CHROMATOGRAPHY

Table 3.1 Characteristics of Common Silica Particles for Liquid Chromatography

Table 3.2 Characteristics of Major Modes of Liquid Chromatography

Table 3.3 Common LC Stationary Phases

Table 3.4 SEC Polymeric Particle Pore Sizes and the Molecular Weight Range They Can Separate

Table 3.5 Characteristics of Common LC Detectors

Table 3.6 Some General Classes of Chiral Stationary Phases

CHROMATOGRAPHY

Principles and Instrumentation

Mark F. Vitha

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Names: Vitha, Mark F.

Title: Chromatography : principles and instrumentation / Mark F. Vitha.

Description: Hoboken, New Jersey : John Wiley & Sons, Inc., [2017] | Includes bibliographical references and index.

Identifiers: LCCN 2016011576| ISBN 9781119270881 (cloth : alk. paper) | ISBN 9781119270898 (pdf : alk. paper) | ISBN 9781119270904 (epub : alk. paper)

Subjects: LCSH: Chromatographic analysis. | Gas chromatography. | Liquid chromatography.

Classification: LCC QD79.C4 V58 2017 | DDC 543/.8-dc23 LC record available at http://lccn.loc.gov/2016011576

About the cover: A two-dimensional liquid chromatography (LC × LC) separation of maize seed extract. Ground seed was extracted using methanol mixed with a phosphate buffer (pH 5.7) followed by solid phase extraction prior to analysis. The first dimension separation used a Zorbax SB-C3 column while a carbon-clad core-shell silica column was used for the second dimension. Additional experimental details about the extraction and chromatographic system can be found in Huang, Y.; Gu, H.; Filgueira, M.; Carr, P.W. J. Chromatogr. A, 1218, 2011, 2984–2995 and Filgueira, M.; Huang, Y.; Witt K.; Castells C.; Carr, P.W. Anal Chem. 83, 2011, 9531–9539. The author thanks the creators of this image for their permission to use this image and Marcelo Filgueira for the work he put into it for this book.

PREFACE

Chromatography is the most widely used technique in modern analytical chemistry. In this book, you will learn the fundamental principles underpinning chromatography and be able to connect those principles to the design and use of chromatographic systems.

The focus of this book is current theory and the modern practice of chromatography. The successful practice of chromatography depends upon an understanding of molecular-level processes such as partitioning, band broadening, and the effects of temperature and mobile phase modifiers on solute retention. In every chapter, you will find figures and problems designed to help you visualize these processes and identify the key variables in any given chromatography problem.

Sections such as those on microfabricated gas chromatography (GC) separations, two-dimensional liquid chromatography (2D-LC), superficially porous particles, ultra-high performance liquid chromatography (UHPLC), and Orbitrap mass spectrometry ensure that you are introduced to the most recent developments in chromatography and are thus well prepared to enter the workforce.

Case studies illustrate the range of applications addressed by chromatography. Examples such as the analysis of performance-enhancing drugs in sports and the detection of deliberate contamination of food with melamine and Sudan dyes are discussed in the context of liquid chromatography (LC). Chapter 3 ends with a detailed discussion of the analysis of pharmaceutical compounds found in waterways across the United States as part of a U.S. Geological Survey study. Chapter 2 describes a case study centered on the analysis of Middle Eastern oil samples. This case was part of an international incident that involved the United States, Iraq, Iran, and Russia, and it shows the impact of analytical chemistry in general, and chromatography, specifically, on world affairs. All of the case studies demonstrate how the principles and instrumentation presented earlier in the chapters contribute to solving practical, real-world problems.

This book also complements a modular approach to teaching. Rather than being encyclopedic, this focused book allows instructors to integrate selected sections with other modular books focused on techniques such as spectroscopy or mass spectrometry, or to supplement the information provided in this book with their own materials. It also facilitates its use in courses such as interdisciplinary laboratory courses, biochemistry, and introductory analytical chemistry courses.

The book has three chapters. Chapter 1 provides an in-depth description of the processes governing chromatography. It includes a systematic development of band broadening, drawing extensively from the work of J. Calvin Giddings. Numerous figures help you visualize the processes that contribute to band broadening and link those processes to key equations. Problems embedded within the chapter reinforce the connections between theory, visualization, and the contributions of different factors to the overall breadth of a peak.

Chapter 2 focuses on gas chromatography. It introduces the components of GC instruments in the context of the function they accomplish and discusses the theory behind instrumentation, instrument design, and practical aspects such as temperature programming, injection volumes, and the selection of stationary phases and detectors that practitioners must consider when conducting GC analyses.

The final chapter addresses liquid chromatography. It emphasizes reversed-phase liquid chromatography (RPLC) because of its prominence, but you will also find descriptions of all of the common modes of LC, including hydrophilic interaction chromatography, which has great attributes for the separation of small, polar molecules, and which may prove to be quite valuable as a mode that is orthogonal to RPLC for two-dimensional separations. This chapter also discusses topics such as sub-2-µm particles, superficially porous particles, UHPLC, and 2D-LC as part of the mainstream modern practice of LC, preparing you with the knowledge needed to operate in modern laboratories.

There are many people to thank for the completion of this book. The sacrifices my mother made and her unflagging commitment to my education provided the foundation upon which this book exists. Each page of this book embodies her love, support, and dedication. My wife, Maura Lyons, has been a stalwart supporter throughout the process. Her love, encouragement, and even keel are irreplaceable. Thanks also to Greg Febbraro, who tracked the progress of this book through all its ups and downs. I wish we could celebrate its publication together – your friendship is missed. Frank Settle, whose Instrumental Methods of Analysis textbook provided the genesis for this project, has been unwavering in his support. Some figures and text from that work have been included in this book. I owe much to Peter Carr, both for the specific work he put into reviewing material in this book and for the years he has spent teaching me chromatography. Leah Carr fostered an academic family and I, like many, miss her support and affection. I am grateful to Joseph Brom and Gary Mabbott for the research opportunities they provided that introduced me to instrumentation generally and to separation science specifically. The influence of their teaching made this book possible. John Dorsey, Stephen Weber, Dwight Stoll, Charles Lucy, Paige Diamond, Teresa Golden, Brian Gregory, Brian Lamp, Yinfa Ma, David McCurdy, and James Miller all provided valuable feedback as reviewers. I appreciate the time and effort they spent on my behalf. Much of what is right in this book is thanks to them, and all errors are mine. Susan Boyer provided significant feedback regarding the prose. Dwight Stoll and Paul Boswell shared an Excel chromatogram generator that I used to create many of the chromatograms in this book. Neal Byington at the U.S. Customs and Border Protection was exceptionally helpful in discussions regarding the analysis of oil samples from the Middle East related to the case study in Chapter 2.

Mark F. VithaDes Moines, IowaOctober 4, 2016

CHAPTER 1

Many “real-world” samples are mixtures of dozens, hundreds, or thousands of chemicals. For example, medication, gasoline, blood, cosmetics, and food products are all complex mixtures. Common analyses of such samples include quantifying the levels of drugs – both legal and illegal – in blood, identifying the components of gasoline as part of an arson investigation, and measuring pesticide levels in food.

FUNDAMENTALS OF CHROMATOGRAPHY

Chromatography is a technique that separates the individual components in a complex mixture. Fundamental intermolecular interactions such as dispersion, hydrogen bonding, and dipole–dipole forces govern the separations. Once separated, the solutes can also be identified and quantified. Because of its ability to separate, quantify, and identify components, chromatography is one of the most important instrumental methods of analysis, both in terms of the number of instruments worldwide and the number of analyses conducted every day.

1.1 THEORY

Chromatography separates components in a sample by introducing a small volume of the sample at the start, or head, of a column. A mobile phase, either gas or liquid, is also introduced at the head of the column. When the mobile phase is a gas, the technique is referred to as gas chromatography (GC) and when it is a liquid, the technique is called liquid chromatography (LC). Unlike the sample, which is injected as a discrete volume, the mobile phase flows continuously through the column. It serves to push the molecules in the sample through the column so that they emerge, or “elute” from the other end.

Two particular modes of LC and GC, known as reversed-phase liquid chromatography (RPLC) and capillary gas chromatography, account for approximately 85% of all chromatographic analyses performed each day. Therefore, we focus on these two techniques here and leave discussions of specific variations to the chapters that describe LC and GC in greater detail.

In GC, the mobile phase, which is typically He, N2, or H2 gas, is delivered from a high-pressure gas tank. The gas flows through the column toward the low-pressure end. The column contains a stationary phase. In capillary GC, the stationary phase is typically a polymer film that is 0.25–5 µm thick (see Figure 1.1a). It is coated on the interior walls of a fused silica capillary column with an inner diameter of approximately 0.5 mm or smaller. The column is usually 10–60 m (30–180 ft) long.

Figure 1.1 Representations of typical capillary gas (a) and liquid (b) chromatography columns. Figure (c) is a depiction of a cross section of a porous particle (shaded areas represent the solid support particles, white areas are the pores, and the squiggles on the surface are bonded alkyl chains. Figure (d) is an scanning electron microscope (SEM) image of actual 3 µm liquid chromatography porous particles. Note that the lines across the particle diameters have been added to the image and are not actually part of particles. (Source: Alon McCormick and Peter Carr. Reproduced with permission of U of MN.). It is worth taking time to note the different dimensions involved. For the GC columns, they range from microns (10−6 m) for the thickness of the stationary phase, to millimeters (10−3 m) for the column diameter, up to tens of meters for the column length. Note also that LC columns are typically much shorter than GC columns (centimeter versus meter).

RPLC is the most common mode of liquid chromatography. In RPLC, the mobile phase is a solvent mixture such as water with acetonitrile (CH3

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