IC Troubleshooting

Principles and Troubleshooting Techniques in ION CHROMATOGRAPHY
2002 Dionex Corporation
Document No. 034461 January 2002
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January 2002
Contents
Table of Contents
1 Introduction
What is Chromatography? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2
2 The Process of Ion Chromatography

2.1 Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 2.2 6 Functions of the Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 9
The Chromatographic Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 2.2.2 2.2.3
Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Thermodynamic Factors of Chromatography . . . . . . . . . . . . . 12 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3 The Chromatography System

3.1 Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.1.1 3.1.2 3.1.3 3.2 3.3 Function of the Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Preparation of Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Troubleshooting Eluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Injection Valve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Principles of Ion Chromatography 1/2002
iii
Contents
3.4
Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3.4.1 3.4.2 3.4.3 3.4.4 Headspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Column Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.5
Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3.5.1 3.5.2 3.5.3 3.5.4 3.5.5 Conductivity Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Amperometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Absorbance Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Fluorescence Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Other Detection Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4 Method Development
4.1 4.2 Define Goals of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Selecting the Appropriate Separation Mode . . . . . . . . . . . . . . . . . . . . 55 4.2.1 4.2.2 4.2.3 4.3 Ion Exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Reverse Phase Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 57 Column Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
iv
1 Introduction
Course Objectives
Outline the basic concepts involved in chromatography and develop them with respect to Ion Chromatography Define chomatography Overview the basic chromatographic process
Discuss the factors affecting chromatographic separation Discuss the components of a chromatography system and their roles in separation and detection.
1.1
What is Chromatography?
Chromatography is the separation of a mixture of compounds into its individual components based on their relative interactions with an inert matrix. A mobile phase, usually a liquid or gas, is used to transport the analytes through the stationary phase. The matrix , or stationary phase, is generally an inert solid or gel and may be associated with various moieties, which interact with the analyte(s) of interest. Separation results from the differential migration of the compounds contained in a mobile phase through a column uniformly packed with the stationary matrix. Interactions between the analytes and stationary phase are non-covalent and can be either ionic or non-ionic in nature depending on the type of chromatography being used. Components exhibiting fewer interactions with the stationary phase pass through the column more quickly than those that interact to a greater degree. Various forms of chromatography can be used to separate a wide variety of compounds, from single elements to large molecular complexes. By altering the qualities of the stationary phase and/or the mobile phase it is possible to separate compounds based various physiochemical characteristics. Among these characteristics are size, polarity, ionic strength, and affinity to other compounds.
Principles and Troubleshooting Techniques of Ion Chromatography
Figure 1. Separation resulting from differential migration of compounds Chromatography is the separation of the components of a mixture by differential partitioning between a mobile and stationary phase
1.2
History
The development of chromatography as an analytical tool began in 1903 when Michael Tswett (1872-1919), a Russian botanist, discovered that he could separate colored leaf pigments by passing a solution through a column packed with adsorbent particles. Since the pigments separated into distinctly colored bands, Tswett named the new method chromatography (chroma color, graphy writing). Several developments were made over the next few decades but it wasnt until the early 1970s that ion chromatography began to be seen as a viable process for ion separation and analysis, due mainly to the difficulties involved with the detection of ionic species in an ionic mobile phase. Throughout the development of chromatography, technological advances have been limited to a great extent by the ability to detect and measure the analytes of interest. Tswetts initial experiments involved direct visual detection and did not require a means of quantitation. Other detection methods were developed that exploited a compounds radioactivity, fluorescence, or its ability to absorb light in the UV spectrum. Compounds not inherently possessing any of these characteristics could sometimes be subjected to post-separation reactions that rendered directly
1 Introduction
measurable products. These characteristics were easily discernable from the general levels of background noise contributed by the mobile phase, allowing a higher degree of sensitivity. The separation of ions, however, relies on the use of an ionic mobile phase that bears the same characteristic (the capacity to act as a conductor) as the analytes of interest. Although adequate separation of these species was attainable, the significant background signal generated by the mobile phase caused their detection and quantitation to be either impossible or, at best, impractical. The early 1970s saw the introduction of a process that could allow direct conductivity of ions. This technology utilized a second ion exchange column after the separator column that reduced the overall conductivity of the mobile phase without adversely affecting that of the analyte. Eluent suppression , as it came to be known, allowed low levels of common inorganic ions to be separated and detected using a standard ionic mobile phase.

Overview The basic process of ion chromatography involves introducing the sample into a moving stream of mobile phase. This mixture passes into a column that is uniformly packed with particles coupled to an active site with an opposite charge than that of the analyte. Thus, for cation analysis a column is used that has negatively charged active sites. The mobile phase, or eluent, is made up of an aqueous solution of ion salts and serves several functions in the separatory process. Following the column, the mixture proceeds through a suppressor (suppressed ion chromatography) and to the detector (typically conductivity detection for ion chromatography). All ion chromatography systems consist of the same basic components:
Eluent Pump Injection Valve Columns Suppressor Detector Data Collection System
Eluent
Pump
Injection Valve
Column
Suppressor
Detector
Data Collection
Figure 2. Components of an Ion Chromatograph
2.1
Eluent
2.1.1 Functions of the Eluent Stabilize sample ions in a solution
Provide kinetic flow of sample ions through a system Provide counter-ions to compete with analytes for active site on a stationary phase
Different analytes in the sample mixture will pass through the column at different rates depending on their relative interactions with either the mobile (eluent) or stationary phases. The rates of analyte migration can be affected by altering eluent composition and/or using different formulations of stationary phase (Figures 3 and 4).
Comparison of Anion Analysis With Varying Eluent Concentrations
Figure 3. Effect of Eluent Concentration on an AS14A separation
Comparison of Anion Analysis with Varying Stationary Phase

Eluent: 1.8 mM Na2CO 3 /1.7 mM NaH CO3 Flow Rate: 2.0 ml/min
(a) AS4A-SC
(b) AS4A
Figure 4. Effect of varying column (stationary phase) on anion separation
2.2
The Chromatographic Separation

This process of separation can result in three possible outcomes:
The solutes will be completely resolved (Figure 6a) The solutes will be partially resolved or (Figure 6b) No resolution will take place (Figure 6c)
2.2.1
Resolution
Resolution is the measure of separation of any two given solutes and can be defined by the equation:
R = (2)(flowrate)(T2 - T1 ) (W1 + W 2)
where: V = the elution volume of the peak W = the width of the peak at the baseline
where: T1 = retention time of peak 1 T2 = retention time of peak 2 W 1 = peak Width at baselie of peak 1 W 2 = peak width at baseline of peak 2 Figure 5. Resolution
Two peaks are considered to be completely resolved when a distinct baseline can be observed between the peaks, indicated by an R value near 1.5 (Figure 6a).
Figure 6a. Complete Resolution
10
Figure 6b. Partial Resolution
Total Eluent 12.56mM Carbonate Ratio: 90.4%, Resolution 0.090

Bulanesulfonate
Propanesulfonate Pentanesulfonate
Figure 6c. Poor Resolution
11
The resolution of any two solutes is dependent on their respective retention profiles and peak shapes, which are, in turn, affected in a composite manner by the kinetic and thermodynamic factors inherent in the chromatographic system. These factors, known as capacity (retention characteristics), selectivity, and efficiency will be unique for every combination of mobile/stationary phase and will vary based on the physical conditions of separation (i.e. flow rate, temperature, etc.).
Figure 7. Thermodynamic and Kinetic Factors determining resolution
2.2.2
Thermodynamic Factors of Chromatography

2.2.2.1 Distribution Coefficient (K D)
The flow rate of the eluent and the distribution of the solute between the mobile and stationary phases determine a solutes retention time. In a system without flow, a solute will achieve equilibrium between the two phases. This equilibrium can be described as the distribution coefficient K D and is defined by the equation:
12
K D = CS/CM where : C S = the concentration of solute in the stationary phase C M = the concentration in the mobile phase. The distribution is influenced by the ionic attraction to the active sites on the column packing. A solute with a high K D is more likely to be found associated with the stationary phase at any given moment. A a low K D indicates a solute that favors the mobile phase. An analytes retention time is determined by the eluent flow rate and by the distribution of solute between the mobile and stationary phases. Given a particular combination of mobile and stationary phases, any two analytes will generally have distinct distribution coefficients. This difference in K Ds is the basis for the differential migration of various components.
An analyte with a relatively low K D favors distribution in the mobile phase of the system where it is subject to the influence of eluent flow. This analyte will be pushed through the column more quickly than one with a higher KD An analyte with a higher K D favors distribution towards the stationary phase. This analyte elutes at a slower rate. The KD describes the ratio of sample in either phase at equilibrium under a given set of conditions. Thus, although a solute favors the stationary phase, it is still present to an extent in the mobile phase and can flow through the column.
Under ideal conditions the K D of a molecule within a system composed of a stationary and mobile phase at a constant temperature will be constant. We see, in fact, that this only true for a small minority of molecules. It is observed that in most systems a molecules K D will vary over a range of solute concentrations. The relationship between the K D of a molecule and its concentration can be described by a function called an isotherm.
13
Figure 8. Isotherms Figure 8 depicts the three types of isotherms with KD represented by the slopes of the lines.
Isotherm A represents an ideal state where K D remains constant

throughout the concentration range.
Isotherm B is a more accurate representation of most molecules in ion

chromatography. Here we see that as the concentration of component in the sample increases its K D will decrease, resulting in an increased distribution of the solute into the mobile phase.
Isotherm C is a less common situation in which lower concentrations of

solute actually favor the stationary phase over the eluent. The importance of isotherms will be established in later sections when we discuss the kinetic factors influencing peak shape.
14
2.2.2.2 Capacity Factor
Another way to describe the retention characteristic of an individual component is by its capacity factor, K, which is a comparison of the elution time of the solute with the void volume of the column. The capacity factor gives us a measure of the time the analytes spends in the stationary phase versus the mobile phase.
K = (Te - T0)/T 0
Figure 9. The capacity factor is a comparison of the elution time of the solute with the void volume of the column. The equation for K is
K = (Ve-Vm )/Vm
where: Ve = the elution volume of the solute V m = the void volume of the column. Given a constant flow rate we can substitute the times into this equation to yield
k = (Te-T0)/T0
where: T0 = the time needed to flush one column volume (this is the duration of time from the injection to the water dip). Te = the resolution time of the solute Analytes with higher capacity factors will elute farther from the void volume. This may improve separation, but it will also lengthen analysis time and lead to increased peak broadening.
15
2.2.2.3 Selectivity
Selectivity is described as the ratio of two analytes capacity factors. The selectivity factor, , is defined by the equation:
= (T2-T0)/ (T 1-T0)
If = 1, is equal to one there is no resolution between the analytes. Increasing values of indicate analytes that would be more thoroughly resolved.
Figure 10. Selectivity determines analyte elution order
The elution order of a mixture of analytes is determined by the selectivity of a stationary phase to each analyte in that mixture under a given set of conditions (mobile phase composition, etc.). Early theorists postulated that the size of the hydrated analyte determined its relative attraction to the stationary phase, with the smaller hydrated ions maintaining more stable associations with the stationary phase and, thus, eluting later than the larger ones. This theory, however, did not explain the tendency for ions to change elution order when structural changes were made to the stationary phase (no alteration to the active site).
16
Elution order most likely results from a combination of analyte hydration energy and electrostatic attraction Further research suggested that selectivity is influenced by the relative hydration energy of the ions as well as by electrostatic attraction between the analyte and the active site on the column packing. There is some thought that the higher hydration energy exhibited by small ions enables them to enter the highly structured water matrix of the mobile phase. Larger ions, with lower energies, are not as able to reorient water molecules within the eluent in a manner that permits stability and are displaced toward the stationary phase. As the larger ions approach the stationary phase they are more subject to the electrostatic attraction with the active sites, thus enhancing the retentive effects on the ions travel through the system. It stands to reason that an increase in the ionic strength of the mobile phase would cause more of its water to be tied up in the hydration of eluent salts, thus allowing the later eluting components more freedom to enter the mobile phase. Conversely, lowering the concentration of the eluent would cause the ions to become less stable in the mobile phase, resulting in an increased retention time.
Factors Controlling Selectivity:

Counterion composition/concentration Nonionic modifiers in mobile phase (isopropanol, etc.) Temperature of mobile phase Structure of stationary phase/active site Chemical composition of active site
17
2.2.2.4 Efficiency
In an ideal system each component would travel through the column in discrete band with a constant concentration and it would be possible to completely resolve compounds with very little differences in their K Ds (Figure 11).
Figure 11. Separation in an ideal system 1. In actuality it is observed that the concentration of an analyte varies throughout its region of occupation in the column.
For solutes with a type A isotherm (KD is constant throughout the

concentration range) the concentration distribution varies such that the eluted peak is Gaussian in nature. This phenomenon is known as band broadening and can lead to the loss of resolution between closely eluting peaks. (Figure 12)
18
Figure 12. Band broadening can lead to loss of resolution 0. Band Broadening
Under a given set of conditions peak width is found to be directly

proportional to both the length of the column and the particle size of the stationary phase.
Peak width will tend to vary directly with changes in the eluent
flow rate. 0. Efficiency
Efficiency is the ability of a column to separate a component

without spreading it out. Efficiency is measured by calculating the number of theoretical plates in the column. A theoretical plate is an abstract term describing a complete step of equilibrium exchange of a solute between the mobile and stationary phases. The Craig Distribution model illustrates this process. (Figure 13)
19
Figure 13. Craig Distribution Model of Theoretical Plates Consider a series of squares each representing a site of exchange between two phases. Solute is introduced into the mobile phase of the first compartment and achieves an equilibrium between the two phases, with the amount in each phase determined by the K D. Solute remaining in the mobile compartment is transferred to the next stage by eluent flow where it undergoes the same equilibrium process. Likewise, solute remaining in the first stationary compartment is free to establish an equilibrium with fresh eluent entering its associated mobile compartment and the process is repeated. Because the quantity of solute transferred to any successive stage is dependent on the amount remaining in the mobile compartment under equilibrium conditions, over many stages the concentration will assume a binomial, or Gaussian, distribution.
As the number of theoretical plates increases, we can expect more broadening to occur. The most common method of increasing the number of plates is to increase the length of the column. While we do see a broadening of peaks, increasing the plate number is often beneficial in that it can allow better separation between components with closely related distribution coefficients.
20
The term Height Equivalent Theoretical Plate (HETP) is used to describe the efficiency of different columns and is calculated by dividing the columns length by the number of theoretical plates.
HETP = L/N
Lower values of HETP indicate more efficient separation. The amount of band broadening is found to be proportional to the square root of the column length. Maximizing the number of theoretical plates (better separation) in the shortest length possible will maximize the efficiency of a column. This can be done by optimizing the composition of mobile and/or stationary phases for a particular application.
Plates per Column

Solute Fluoride Chloride Nitrate Phosphate Sulfate 3.5 mM Carbonate 1480 2220 1900 2930 3960 3.5 mM Bicarbonate 2520 3060 3120 2660 3140 Standard AS4A Eluent 1050 2850 3130 2130 4050
Increasing the number of theoretical plates without increasing the length of the column will allow a more efficient separation to the stationary phase active site.
21
2.2.2.5 Flow
It is important to stress the effect of eluent flow rate on loss of efficiency. Given a situation with no flow, an analyte will assume an equilibrium distribution between the mobile and stationary phases determined by its distribution coefficient. When we introduce directional flow of the eluent, the portion of solute in the mobile phase will be advanced ahead of the portion remaining in the stationary phase, causing a longitudinal expansion of the solute zone within the system. (Figure 14) This is the predominant kinetic cause of band broadening.
Figure 14. The kinetics of mass transfer lead to band broading As noted earlier most applications deal with analytes with a K D value that is concentration dependent. This shift from an ideal condition further influences the shape of the eluting peak. Consider a compound with a type B isotherm (K D decreases as the concentration increases). We know that solute advancement through a zone is dependent on its concentration within that zone, and that the concentration of the solute is not constant throughout its region of occupation. Figure 15 depicts such a peak under normal conditions.
22
Figure 15. Different zones of peak exhibit pseudo - K Ds Solute in the mobile phase will be advancing ahead of that retained by the stationary phase, represented in the figure by the dashed line. If we focus on discrete bands within various portions of the peak, we find that the changes in concentration caused by this shift of analyte influences the shape of the peak. In zone 1, for example, the concentration of solute in the mobile phase is actually lower than in a zone immediately downstream. Thus, the K D of the solute in zone 1 is higher than what might be expected. Since the K D at this point is higher, we note that the solute in this portion of the peak will tend to favor the stationary phase and, thus, will lag behind solute contained in the eluent. As the solutes concentration increases, the distribution coefficient will shift to allow more solute in the mobile phase, where it is pushed ahead due to eluent flow.
The peak shape of a compound is affected by its isotherm. Most compounds exhibit a type B isotherm. The opposite effect is observed once we have passed the peak maximum, with the solute tending to lag behind the eluent front as its concentration
23
diminishes. For a compound with a type A isotherm (where K D is constant), zones 1 and 2 would simply drift farther apart, thereby broadening while maintaining a Gaussian profile. For type B compounds, however, the changes in solute mobility caused by fluctuations of K D result in a skewing of the peak, with a more abrupt decline in the tailing shoulder compared with what we would expect from a Guassian distribution. A solute with a type C isotherm exhibits the opposite behavior, with a sharper leading edge. (Figure 16)
Figure 16. Effects Isotherm on Peak shape

2.2.2.6 Effects of Stationary Phase on Efficiency
Particle size and the uniformity of packing also influence a columns efficiency. The molecules in the mobile phase contribute to the progress of the solute through the system and that molecules retained in the stationary phase will lag behind the peaks center of mass. This creates a non-equilibrium distribution of solute that is proportional to the rate of eluent flow and which leads to further broadening of the peak. Dispersion of the peak can be minimized by choosing conditions such that the equilibrium conditions are maintained and that the rate of mass transfer between the two stages is maximized.
24
2.2.2.7 Particle Size
A molecules travel through the column can be considered as a series of steps at which it must make a decision on which path to follow through the system. (Figure 17) Although net movement will be in the direction of eluent flow, at some junctures a molecule may choose a lateral path, resulting in a loss of forward motion.
Figure 17. Flow of molecules through column packing
Decreasing the particle size increases the number of decision steps. If particle size is decreased without reducing the volume of the stationary phase, there will be more decisions against forward progress. This event is repeated over many stages.
The molecules will tend to re-bunch around the center of mass of the peak. Increasing the number of particles generates a greater surface area of interaction between solute and the stationary phase, resulting in less dispersion due to eluent flow kinetics. Inconsistency in the size of column packing can also lead to loss of efficiency. Molecules travel through the column at a rate
25
determined by the eluent flow and the size of the column packing. Solute molecules will progress through the column at different rates depending on the size of the particles they encounter in their zone of travel, leading to a dispersion of molecules away from the center of mass.
2.2.3
Summary The goal of chromatography is the separation, or resolution, of the

individual components of a mixture of analytes. This is achieved through the unequal partitioning between mobile and stationary phases under the influence of eluent flow.
The thermodynamic factors that influence peak resolution are the capacity factor and selectivity. These factors describe the phenomena associated with the establishment of an analytes equilibrium distribution between the mobile and stationary phases, and determine the differential migration of solutes through a given system. The predominant kinetic factors associated with resolution are those which contribute to the efficiency of separation, or, the ability of a column to retain a component without spreading it out. Efficiency is determined mainly by the physical components of a system such as the size of the stationary phase particle, uniformity of column packing, and the flow rate of the eluent.
26

Objectives
Discuss the functions of each component of the chromatography system Overview basic troubleshooting of each component
Introduction Although there are several configurations of ion and liquid chromatographs, they share many components including:
Eluent Pump Injection valve Column Suppressor (ion chromatography) Detector
3.1
Eluent
3.1.1 Function of the Eluent
The function of the eluent in a chromatography system includes:
Establishing the basic ionic condition of the separation environment. Stabilizing the sample in solution. Promoting progression of the analytes through the system. There are several characteristics of the eluent which affect its interactions with the column and analyte.
Counter ions in the eluent will preferentially elute sample ions of

the same valence.
The selectivity of a column for the counter ion in the eluent will
affect the equilibrium distribution of sample ions in the system. Counter ions with a high affinity for the stationary phase and to the active sites, resulting in a loss of retention of the sample.
27
Counter ion valence and selectivity are affected by the pH of the

eluent.
3.1.2
Preparation of Eluent
Eluent Preparation
Use reagent grade chemicals and at least 18 M deionized

water
Thoroughly degas eluent (for hydroxide eluents degas

water prior to adding sodium hydroxide)
Dilute running strength eluents from concentrated stock

solutions
3.1.3
Troubleshooting Eluents
Problems associated with the eluent may manifest in a shift in analyte retention time due to changes in eluent concentration.
Eluents should be made from dry, high purity reagents using the highest quality 18 m or higher deionized water. Eluents should be made in a consistent manner, preferably from a concentrated stock solution. Eluent reservoirs and lines should be kept clean and free of contamination or particulate matter. Ionic contaminants in the eluent may also generate analysis problems. Since eluent is continually flowing through the system it is constantly generating a background signal. Any contamination in the eluent is subject to the same separation process as the sample and will generate a signal response. Because the eluent flow is constant, ionic contamination usually results in series of random peaks or an increase in background conductivity.
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Eluent Troubleshooting
Changes in retention time Loss of peak efficiency Loss of sensitivity Increased background conductivity
3.2
Pump
Different types of pumps include isocratic and gradient versions of serial and parallel pumps. A pulse-free pump is essential for optimum chromatography. Inconsistencies in flow rate and pressure may result in noisy baseline, retention time changes, and/or irregular peak shapes. Changes in retention times can also occur when the eluent proportioning valves used in gradient analysis malfunction. Routine pump flushing and maintenance, especially when running high salt eluents, is recommended to help ensure continuous smooth operation.
0.065
0.05 0.04 S 0.03 0.02 0.01 0.0 4.88
8 Minutes
10
11.32
17249
Figure 18. Pump Noise
29
3.3
Injection Valve
Following the pump, eluent flows through an injection device, usually consisting of a two-position valve. The valve serves as a means of directing eluent flow and introducing sample into the system. A malfunctioning injection valve may lead to:
Reduced peak height No response Excessive pressure Poor reproducibility (Figure 19) Sample carryover between runs (Figure 21)
45
30
S 20
10
-5 0.13
0.20
0.30
0.40 0.50 Minutes
0.60
0.75
17270
Figure 19. Poor run-to-run reproducibility
30
50 40 S 30 20 10
-5 0.00
0.20
0.40
0.60 Minutes
0.80
1.00
1.25
17269
Figure 20. Excellent Reproducibility (overlay of 6 runs)
Figure 21. Sample Carryover
31
3.4
Columns
The flow path continues from the injection valve to the column(s). In general, the column packing is constructed of an inert core composed of polystyrene molecules that have been cross-linked with divinylbenzene to form a bead of uniform size. The beads are then modified with an ionic moiety that provides the appropriate functionality for separation. (Figure 22 and 23)
Figure 22. Polymerization of Polystyrene and Divinylbenzene to form Column Substrate By varying the amounts of cross-linker and/or modifier used in the formulation, it is possible to generate and optimize stationary phases for a wide range of analytes under diverse conditions. Columns are the site of chemical activity in the separation process. Anything that alters the structural or chemical makeup of the stationary phase (column) has the capacity to affect resolution. Structural changes in the column packing generally result in changes in the shape of the analyte peaks. When packing a column, great care is taken to ensure that the particles are distributed uniformly throughout the entire column volume.
32
sulfonation (or amination) of inert bead substrate
surface of activated substrate
Figure 23. Modification of Column Substrate to Generate Active Sites This ensures that the analytes will have consistent chemical and physical interactions with the stationary phase as they migrate through the column.
Two predominant changes that can occur within the column packing are the generation of headspace and the formation of channels.
3.4.1
Headspace
Headspace occurs when a gap is formed between the column bed support and the column packing. Under normal circumstances the volume of mobile phase before the column packing is negligible and the sample is transferred into the column as a slug of fairly uniform concentration (variation in concentration resulting from laminar flow through the tubing will not significantly affect peak shape). The formation of headspace creates a small void volume of mobile phase that allows the sample to diffuse before it enters the stationary phase. This causes the concentration of the latter portions of the slug to be less than the leading portions and, in effect, broadening the peak by prolonging the introduction of sample in continually decreasing amounts over a short period of time. This results in a phenomenon known as peak tailing. (Figure 24)
33
Figure 24. Peak Tailing resulting from Headspace
Headspace is generally caused by excessive back pressure or by mishandling the column during routine maintenance. A small amount may occur under normal operating conditions due to compaction of the column matrix over a long period of time. This does not usually affect peak shape as long as the direction of eluent flow is not changed.
3.4.2
Channels
Channels are tiny void spaces within the column packing. The formation of channels can occur following excessive spikes in pressure, changes in the direction of eluent flow, or as a result of the column packing drying out. As an analyte passes through a region containing a channel, a small portion of the band will pass out of the solid phase and into the void space. It is then carried by the mobile phase to the end of the void where it re-enters the solid phase. This results in the advancement of a small amount of sample ahead of the rest of the peak. The effects of a channel, which can range from a slight amount of peak fronting to the appearance of ghost peaks before each analyte, depend on its severity and location in the column. (Figure 25)
34
Figure 25. Peak Disruption due to Column Channeling
3.4.3
Contamination The prevalent cause of loss of column performance is contamination.

Contaminants may consist of strongly retained ions that do not elute under normal operating conditions or non-ionic molecules or particles that lead to column blockage.
Particulate matter and larger non-ionic contaminants may collect in the bed supports located at the upstream end of the columns, causing blockages and high system back pressure. Bed supports can be changed although care must be taken not to compromise the integrity of the column packing. Columns are frequently contaminated with ionic components that bind so strongly to the stationary phase that they can not be released under normal operating conditions. This type of contamination, which can result from either organic or inorganic species, primarily affects the capacity of the column. Large, polyvalent ions and metals are frequent culprits and may come from impurities in chemicals used to make up the mobile phase or may arise from the sample itself. As the level of contamination increases, fewer active sites are available for analyte separation, thus shortening retention times and decreasing peak resolution.
35
It is possible for certain contaminants to preferentially affect particular ions by inhibiting their passage through the column. This may result in loss of efficiency or in loss of recovery. In anion chromatography, for example, iron contamination will tend to decrease phosphate recovery before changes in the other analytes are noticed. Similarly, aluminum contamination may cause lower recoveries of phosphate and sulfate but will leave monovalent anions relatively unaffected.
Sample matrices often contain a wide range of contaminants, many of which can be reduced or eliminated by various methods of sample pre-treatment
Various forms of contamination may also cause loss of efficiency. Some contaminants, after associating with the stationary phase, retain the capacity to bind to the analytes of interest, in effect serving as alternate active sites. These pseudo-sites function with the same thermodynamic and kinetic principles as the actual sites, and, thus, we can expect different effects on the elution of the sample. Since the contaminants do not initially reside throughout the full length of the column, a sample analyte will, in effect, pass through a stationary phase for which it exhibits different capacity factors. This can cause either fronting or tailing of the peaks, depending on the nature and amount of the contamination. If the source of contamination continues over time the entire column will become affected, with efficiency steadily growing steadily worse. Indications of Contamination Changes in retention time High back pressure Irregular peak shapes (fronting or tailing) Loss of efficiency Loss of sample recovery Changes in analyte selectivity
36
Even if all necessary care is taken to ensure that all reagents used are of high purity, contaminants are often introduced via the sample matrix. For this reason it is strongly recommended that a guard column be placed ahead of the analytical column. A guard column generally has the same or similar composition as its associated analytical column but is shorter and less expensive. As the sample passes through the guard column, non-ionic contaminants and monovalent ions will be retained, leaving the sample analytes to pass through to the separator. The guard column also accounts for a certain portion of chromatographic separation and can therefore be used as an indicator of contamination by monitoring changes in analyte retention over time.
3.4.4
Column Cleaning
It is often possible to clean a column that has become contaminated. A thorough cleaning protocol will generally involve washing the columns with specific solutions for removing contaminants with different properties (i.e. acid or base-soluble, organic ions, etc.). It may not be necessary to use multiple cleaning solutions if the nature of the contamination is known. Columns should be cleaned when analyte retention times shift by 10 percent Column matrices come in a variety of structural and chemical formulations and can respond quite differently to different mixtures of eluents and/or solvents. Acetonitrile, for example, is a useful solvent for removing hydrophobic moieties from some columns, but can cause excessive swelling in the resin beds of others, thereby increasing the likelihood of headspace or channel formation. Other solutions may be capable of chemically modifying the column packing or active site, possibly causing irreversible damage. Therefore, when selecting an appropriate cleaning medium, it is necessary to select a solution that will effectively dissociate the contaminant from the column without adversely affecting the physical structure of the stationary phase or the nature of the active site. An extensive list of cleaning solutions for Dionex columns is provided in the Dionex Consumables CD-ROM (p/n 053891).
37
3.4.4.1 General Column Cleaning Procedure
Determine the nature of contamination (if possible). Select appropriate cleaning solution(s) refer to Dionex Consumables CD ROM to specify column. Re-plumb the column set by placing the guard column after the analytical column in the flow path WITHOUT changing the flow direction. Pump cleaning solution through the column at the appropriate flow rate for 45-60 minutes (some columns may need to be rinsed with deionized water before and after exposure to cleaning solutions) Repeat with additional cleaning solutions if necessary. Replace columns in their proper order and equilibrate for 30 minutes with standard eluent.
NOTE -- Cleaning procedures for some columns may vary. Always consult a column care manual before proceeding with cleanup.
Regardless of the amount of care given to a column, over time the column packing will begin to deteriorate. This is evidenced by the irrecoverable loss of capacity or efficiency or by abnormal operating pressure. Under these circumstances the column must be replaced.
38
3.5
Detection
The three common modes of detection used in chromatography include:
Conductivity Amperometry Absorbance
When selecting the mode of detection for the application: The detector must have an adequate dynamic range for the solute concentration. It is preferable that the output signal over this range vary linearly with the concentration of the analyte being measured. The detector must have sufficient sensitivity to detect low levels of analyte.
3.5.1
Conductivity Detection
Ionic species, by nature, will dissociate into their constituent components when dissolved into a solvent with a high dielectric constant. These components have the capacity to conduct an electric current when placed between two electrodes with opposite polarity. Ohms law states that the voltage of a circuit is a product of the current and the resistance across two points, or V=IR Conductance, measured in Siemens, is the reciprocal of the resistance. At the concentrations routinely encountered in ion chromatography, conductivity is found to be directly proportional to the concentration of an ion in solution.
39
Factors Affecting Conductivity
Temperature Thermal stabilization is important for low noise, minimum drift, and consistent retention times. Flow A well maintained, pulse free pumping system will help ensure smooth baselines
3.5.1.1 Suppression
For many years the use of conductivity detection was not considered to be practical for ion chromatography due to the previously mentioned restrictions involved with bulk detection. In order to achieve lower background levels, it was determined that the conductivity contributed by the mobile phase would have to be eliminated entirely or reduced to an acceptable level. This became possible with the advent of suppression technology in the 1970s. Advances in suppression technology have led to modern units utilizing ion exchange membranes with neutralizing ions supplied by a chemical solution or through the electrolysis of water. Suppression increases the efficiency of analysis not only through reduction of background signal, but also by converting analyte ions into their acid forms, thereby enhancing their conductance as much as 3 to 5 fold.
Typical IC Setup
Detector Cell Eluent SRSULTRA Waste
Waste Chemical Regenerant
Figure 26. The flowpath of a typical ion chromatograph using chemical suppression. A Dionex Micromembrane Suppressor (MMS) or Self Regenerating Suppressor (SRS) may be used in this configuration.
40
Waste
CationExchange Membrane
CationExchange Membrane
Waste
Na+ HSO-4 Na + A Na+ OH Na+ - OH H+ A H 2O HSO4H+
Na + HSO-4
HSO4H+ H + HSO4-
Na + A-
H+ HSO 4 -
Regenerant
To Detector
Regenerant
Figure 27. Anion Eluent Suppression
SRS Self Regenerating Suppressor
The SRS may be used in 3 modes:
Chemical Suppression Recycle Mode External Water Mode

The best mode to use depend on the application. Figure 28 shows the SRS used in the recycle mode.
41
Ion-Exchange Membrane
2H 2O
2H+ +1/2 O 2 + 2eAnode Regenerant Suppressed Eluent Regenerant
Eluent
Perforated Ion-Exchange Material
Cathode 2H2O + 2e2OH- + H2 Conductivity Cell

16209
Figure 28. Anion Eluent Suppression Diagram for Chemical Regeneration
Atlas Electrolytic Suppressor
The Atlas Electrolyic Suppressor (AES) is designed for use in the recycle mode.
Atlas Electrolytic Suppressor

Ion Exchange MonoDisc
Regen Out
Regen In
Eluent In Eluent Out Electrode Electrode

16180
Figure 29. The Atlas Electrolytic Suppressor.
42
AES Flow Path
Figure 30 shows the flowpath of the eluent and regenerant through the anion AES. Resonance time is increased as the eluent is routed around flow distribution disks. The strong monodisc suppression bed enhances the suppressors ability to withstand backpressure.
Regen Chamber
Regen Out Eluent In
Eluent Chamber
Cation-Exchange Monolith
Regen Chamber
Eluent Out Regen In
+
Cation-Exchange Membrane Flow Distributor Disk Disc
16771
Figure 30. The Flow of the Eluent through the Monodisk of the Atlas Suppressor
43
Selecting a Suppressor
Suppressor Regeneration Mode Electrolyic or chemical Optional Requirements All existing systems except DX-80 Capacity Anion: 200 mN at 1.0 mL/min Cation: 110 mN at 1.0 mL/min Benefits Anions Ease of use Moderately Low Noise Versatile Use with carbonate and hydroxide eluents For solvent applications, use external water or chemical regeneration Applications Cations Use with methanesulfonic acid and sulfuric acid eluents
SRS-ULTRA
2-mm & 4-mm Formats 15 and 50 L void volume
For solvent applications use external water or chemical regeneration. for Columns: All anion eluents containing columns chloride or nitrate, use chemical regeneration Columns: all cation columns
Atlas
1 format for 2-, 3& 4-mm formats 35 void volume L
Electrolyic
Requires PeakNet 6.2 and DX-600 Series A detectors or existing systems with SC20 Power supply
Anion and Cation: 25 mN at 1.0 mL/min
Ease of use with DCR Low Noise Fastest Start-up
Use with carbonate eluents
Use with methanesulfonic acid and sulfuric acid eluents No solvents Columns: CS12, CS12A, CS14
MMS III
2-mm & 4-mm formats
Chemical
All existing systems Required for DX-80
Anion: 150mN at 1.0 mL/min
Ease of use with DCR Lowest Noise Fastest Start-up
Cation: 70 mN at 1.0 mL/min
Use with carbonate and hydroxide eluents and for eluents containing solvents
Columns: All anion columns Columns: all cation columns
Use with methanesulfonic acid and sulfuric acid eluents containing solvents, chloride or nitrate
Figure 31. Choosing the optimum suppressor for your application
44
3.5.1.2 Suppressor Troubleshooting
Symptoms of a suppressor failures may include:
Alarms Spikes Increased Noise High Background Conductivity

Alarms
Common causes of suppressor alarms include:
Needs hydrating The suppressor may have dried out due to a prolonged period
without flow.
Hydrate and quick-start. These procedures are described in

the appropriate suppressor manual on the Dionex Consumables CD Rom.
Contamination Samples may have contaminated the suppressor Clean and quick start. Cleaning procedures and
recommended solutions are listed in the suppressor manual on the Dionex Consumables CD Rom.
Overcurrenting Running applications at the appropriate current setting will

help increase the suppressor lifetime. Running at higher settings will shorten its lifetime.
Equation for calculating suppressor current settings: SRS current settings = flow rate x eluent concentration x
factor Cation factor = 2.94 Anion factor = 2.47 Concentration = mN
45
Spikes
0.15 ECD_1
0.10
S 0.05
0.00
-0.07 0.1
10
15 Minutes
20
25
28
17303
Figure 32. Spiking
Spiking can indicate contamination Spiking can indicate running at too high of a suppressor setting Calculate appropriate suppressor setting using the equations
given in Alarms section
Hydration and quick-start may help eliminate spikes Cleaning may eliminate spikes
46
Noisy Baselines
Conditions: GP50 pump, 4-mm AS14 column, 3.5 mM Na2CO3/1.7 mM NaHCO3, 1.2 mL/min, 32 mA, recycle mode. 16.630 16.610 S 16.590 16.570 16.550 16.530 20 30 40 50 Minutes 60 70 80
17305
Figure 33. Noisy Baseline
Possible cause:
Bubbles in the conductivity cell or a tubing connection

Troubleshooting:
Burping the conductivity cell Disconnect the backpressure coil from the suppressor
REGEN IN port
Turn pump on and create a pressure difference momentarily

by plugging and unplugging the outlet of the tubing (3 seconds)
Repeat 2-3 times Turn pump off and reconnect backpressure coil to REGEN
IN port
47
Conditions: GP40 pump, 4-mm AS9HC column, 9.0 mM Na2CO3 1.0 mL/min, 60 mA, recycle mode.
23.63 23.62 23.61 S 23.60 23.59 Burping the DS3 cell 23.58 550 600 650 700 Minutes
17307
750
800
850
900
Figure 34. Noisy Baseline
Effects of Trapped Bubbles on Baseline Noise
The baseline noise was reduced from 4.96 ns to 0.12 ns after removing the trapped air.
Conditions: GS50 pump, 3-mm CS12A column, 20 mM MSA, 0.5 mL/min, cation Atlas, 33 mA, recycle mode. Before burping the DS3 cell
0.170 0.1240
After burping the DS3 cell
0.165 S 0.160
0.1235
0.155 Average noise: 4.96 nS 0.150 40 44 48 52 Minutes 56 60 0.1230 40 44 Average noise: 0.12 nS 48 52 Minutes 56 60
17309
Figure 35. An example of noisy chromatography baseline due to trapped bubbles obtained using a Cation Atlas Suppressor
48
Effects of Backpressure on Baseline Noise
The baseline noise was significantly reduced when excessive backpressure was removed from the suppressor. It is important to check for plugs in the backpressure tubing and fittings after the suppressor to ensure appropriate pressure is being applied to the suppressor.
Conditions: GP50 pump, 4-mm AS14 column, 3.5 mM Na2CO3/1.7 mM NaHCO3, 1.2 mL/min, 32 mA, recycle mode.
16.790 16.788 16.786

S
400 psi
16.620 16.618 16.616

S
100 psi
16.784 16.782 16.780 820
16.614 16.612 16.610 820
825
830 Minutes
835
840
825
830 835 Minutes
840
17310
Figure 36. Effects of backpressure on the chromatographic baseline obtained using an Atlas Suppressor
Possible cause:
Backpressure coil for conductivity cell generating incorrect

backpressure Troubleshooting:
Determine the pressure drop across the backpressure coil Replace or adjust length according to recommended values
49
High Background Conductivity
Possible cause:
No current is applied to the suppressor Wrong current setting is applied to the suppressor Eluent is not properly prepared for the target application.
Troubleshooting:
Be sure the correct suppressor type is selected on detector front

panel
Apply the correct current setting for the application. Confirm eluent concentration is correct for intended application. Confirm eluent preparation is to the correct concentration with
chemicals of the required purity.
Ensure the correct current is applied for the concentration and

flow rate of the eluent.
Suppressor Summary
Choose the most appropriate suppressor for your application. MMS provides the fastest start-up, is compatible with most
solvents, and operates in the chemical regeneration mode.
SRS is the most versatile, and may be operated in chemical or

electrolytic moses. Solvent use is limited.
Atlas gives low baseline noise, increased ruggedness, and fast

equilibration in its more limited applications.
Minimize current settings, keep suppressors clean and hydrated to

increase suppressor life.
50
3.5.2
Amperometry
Not all analytes separated by ion chromatography are amenable to conductivity detection. Amperometric detection takes advantage of some analytes capacity to undergo chemical reactions when subjected to an applied potential. An amperometric cell is composed of a small-volume channel flowing between a pair of electrodes. A potential is applied across these electrodes and causes either the oxidation or reduction of the analyte, thereby rendering it capable of conducting an electrical current. This current is referenced to a separate electrode and the result is compared to a standardized value to generate a viable measurement. For some applications, the use of a fixed potential may result in poor reproducibility and loss of sensitivity due to the plating of the electrodes with contaminants generated from the sample itself. By cycling the electrodes through a repeating sequence of potentials over a set period of time it is possible to shift the redox state of the sample, resulting in the electrochemical cleaning of the electrode surfaces. (Figure 37) This technique, called pulsed amperometry, leads to better reproducibility and allows the detection of a broader range of analytes than fixed potential amperometry.
Figure 37. Example of Amperometric Waveform
51
In the course of normal usage these electrodes may become plated with contaminants which will result in the loss of sensitivity. This can usually be remedied by polishing with a special polishing compound or a pencil eraser.
3.5.3
Absorbance Detection
Absorbance detection relies on the ability of molecular bonds within an analyte to absorb electromagnetic radiation at specific wavelengths. This radiation, usually in the visible or ultraviolet spectrum, causes the promotion of outer valence electrons in certain bonds within the sample analyte to a higher energy state. This results in a change in energy, or intensity, of the applied radiation, which can then be detected with a photometer. (Figure 38)
Filter/Grating
Photodetector
Beam Splitter Light Source Reference
Flow Cell
Figure 38. Schematic of an Absorbance Detector Beers Law states that, in a fixed cell path, the absorbance of a solution will be proportional to its concentration. Although deviations from this law do occur, for most analytes measured by this method, it is possible to determine an effective range of linear response. This method can be extremely advantageous in that it is often possible to choose a wavelength that is not readily absorbed by the sample matrix or mobile phase, thereby significantly increasing sensitivity.
52
3.5.4
Fluorescence Detection
Not all spectrophotometric methods rely solely on the absorption of radiation for sample detection. Following excitation to a higher energy state, the electrons of some molecular bonds will relax, shifting to a lower energy level and releasing a portion of the energy at a different wavelength than that which was absorbed. The intensity of this fluorescence is proportional to the concentration of absorbing species in the sample.
3.5.5
Other Detection Techniques

Some molecules do not inherently exhibit an absorptive capacity. It is possible in some cases to treat such a compound with a reagent that can confer the ability to absorb light at various wavelengths. The analyte is mixed with a chemical reagent, usually following separation, leading to a stable reaction product that passes into the detector cell. This is a common method used in the analysis of transition metals and various amines.
53
54
Objective
To provide basic guidelines to aid in method development Much work has been done in liquid chromatography over the last several decades. When trying to develop a method, a thorough search of chromatography literature will, in most cases, either yield complete protocols or at least enough information to provide a significant head start. Occasionally, however, it will be necessary for the analyst to spend some time and effort in the development of a suitable method for the analysis in question. This section will provide some basic steps that are useful in this process.
4.1
Define Goals of Analysis

The first step in the development of a method is to define realistic goals for the analysis. Although this may seem basic, it is essential to consider the different aspects involved in identifying and/or quantifying analytes in a sample mixture. It is helpful to have as much information as possible about the analyte(s) in question. For instance, is the chemical structure known? Are the molecules inorganic or organic? In what types of solutions and at what pHs are the molecules soluble and in their ionic form? In many cases this information will be readily available to the analyst and all that is necessary is to determine which type of column and mobile phase to use. Some research applications may require the analysis of compounds for which little information has been gathered. In these circumstances it may be necessary to perform separate qualitative tests in order to determine how to proceed with chromatographic separation.
4.2
Selecting the Appropriate Separation Mode

Once enough information has been gleaned regarding the sample, it is necessary to select an appropriate column and mobile phase. In choosing a column it is first necessary to determine what style of separation must be used for a given analyte or sample matrix. Some sample types, such as organic acids or hydrophobic molecules, may not be suitable for separation using ion exchange chromatography. Two other commonly used separation methodologies are ion exclusion and reversed phase chromatography.
55
Principles and Troubleshooting Techniques of Ion Chomatography
4.2.1
Ion Exclusion
Ion exclusion chromatography is useful in the separation of weak organic acids from strongly dissociated ions. In ion exchange, separation is achieved through differential interactions between the sample and stationary phase. Active sites on the column packing are readily available to all sample ions. An ion exclusion column is highly sulfonated throughout the resin structure. By using a dilute solution of a strong acid as the mobile phase, a perimeter of water molecules will be established a short distance from the surface of the stationary phase. This perimeter, known as the Donnan membrane, will be slightly polarized with a partial negative charge oriented away from the exchange resin. (Figure 39) Strong acids in the sample, which remain negatively charged, are prevented from passing through the Donnan membrane and are eluted in the void volume. Weak acids become protonated and, in their neutral state, are allowed access to the active sites on the stationary phase. Separation in ion exclusion is achieved by a combination of Donnan exclusion, steric exclusion, and classic exchange partitioning.
Donnan Membrane
R C00H
H 20
C 1
C 1
Figure 39. Separation in ion exclusion
56
4.2.2
Reverse Phase Chromatography

Whereas ion exchange chromatography exploits the polar characteristics of various compounds to bring about separation, reverse phase chromatography separates compounds based on their relative hydrophobicity. Column packings are generally composed of a porous, non-polar core that is capable of hydrophobic interactions with organic compounds. Organic ions may also be analyzed by a technique known as ion pairing. In ion paring, a hydrophobic ion of an opposite charge to the analyte of interest is added to the mobile phase and forms a complex with the analyte. This complex is then able to associate more readily with the non-polar stationary phase. It is possible to alter the capacity of this system, and thus optimize separation, by changing the type or concentration of pairing agent, or by increasing the percentage of nonpolar solvents in the mobile phase. For some applications it may be necessary to incorporate elements of normal and reverse phase chromatography. Columns capable of operating in mixed mode are able to accept ionic mobile phases containing higher levels of solvents than normal exchange columns. This facilitates the separation of samples containing mixtures of neutral and ionic hydrophobic analytes.
4.2.3
Column Selection
Once the type of chromatography to use has been determined you must choose a specific column set. The best source of information as to whether or not a particular column will be useful for a given application is the literature provided by the manufacturer. Many column manuals provide example applications with various types of samples commonly run on a given column. If there is no information pertaining to a specific analyte, it can be helpful to choose a column that is compatible with a similar compound for which a method has been developed. Some analytes, common anions or cations for example, may be easily separated on several different columns. It is then necessary to consider what type of mobile phase is to be used. Most columns are formulated for particular applications with a specific mobile phase. Issues to consider when choosing a mobile phase include sample solubility, the valences of different compounds in the sample, and detection requirements. We know that, in general, eluent salts will preferentially elute solutes of like charge. If a sample mixture has a strong contingent of divalent anions, for example, it would be beneficial to use an eluent that also contained
57
bivalent ions, such as sodium carbonate. Additionally, changes in eluent salts can alter the capacity factors for a column. Thus, although both are monovalent salts, sodium hydroxide and sodium bicarbonate may not be equally suitable for the separation of various anions on a given column. Detection requirements also factor in eluent suitability. In suppressed conductivity it is desirable to reduce the conductivity contributed by the mobile phase as much as possible in order to maximize the sample detection limits. Sodium hydroxide is commonly used in place of a sodium carbonate/sodium bicarbonate eluent in low level analysis of anions. By choosing sodium hydroxide as for the mobile phase, background conductivity can be reduced to negligible levels (consider the suppression products of hydroxide and carbonate/bicarbonate). The background signal from suppressed carbonate/bicarbonate systems will usually be 15 to 20-fold higher than that of sodium hydroxide.
4.3
Detection
Additional care must be taken to combine a detection scheme that is compatible with the appropriate separation parameters. Few difficulties are encountered for analysts performing common ion analysis in that interferences arising from the mobile phases used with standard columns can be eliminated before the detection process. In some situations, however, it is necessary to use a certain mobile phase composition that is not compatible with various forms of detection. For example, the separation of carbohydrates can be achieved on an anion exchange column by using a sodium hydroxide/sodium acetate mobile phase with integrated amperometry as a detection method. While this might be a suitable process for separation and quantitation, the detection method is not capable of allowing the identification of a compound. More qualitative information could be obtained by using mass spectrometry for detection. This raises a dilemma in that significant interferences will arise from the high salt concentration in the mobile phase (most liquid chromatography/mass spectrometry methods (LC/MS) utilize reverse phase columns with solvent/water mobile phases). While there are some mass spectrometers that can remedy this interference, in most cases the analyst is faced with having to sacrifice functionality in either separation or detection. In many situations the analyst will be able to select an appropriate system by investigating what others have used to analyze the same or a similar molecule. Even in these cases there may be significant difficulties in obtaining useful results. While a given system may be capable of separating a variety of closely eluting organic acids, the conditions used in the analysis of such acids in wine
58
may be quite distinct from those used in the analysis of beer. Indeed, with the myriad analytes and sample matrices available for investigation come a wide range of possible interferences that must be dealt with in order to acquire suitable data. Often, the only way to optimize a particular separation is through a process of trial and error with minor variations in separation conditions (i.e. gradient profiles, modifiers, temperature, etc.). Careful consideration of the theory behind this separation, as well as an understanding of the strengths and limitation of a given system, will help the chromatographer to determine the appropriate conditions for their particular separation.
59
60

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Principles and Troubleshooting Techniques in ION CHROMATOGRAPHY

2002 Dionex Corporation

Document No. 034461 January 2002

DISCLAIMER OF WARRANTY AND LIMITED WARRANTY

2 The Process of Ion Chromatography

The Chromatographic Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 2.2.2 2.2.3

Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Thermodynamic Factors of Chromatography . . . . . . . . . . . . . 12 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3 The Chromatography System

Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Injection Valve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Principles of Ion Chromatography 1/2002

Principles of Ion Chromatography 1/2002

Principles of Ion Chromatography 1/2002

Principles and Troubleshooting Techniques of Ion Chromatography

Principles of Ion Chromatography 1/2002

Principles of Ion Chromatography 1/2002

Principles and Troubleshooting Techniques of Ion Chromatography

Principles of Ion Chromatography 1/2002

2 The Process of Ion Chromatography

Figure 2. Components of an Ion Chromatograph

Principles of Ion Chromatography 12/2001

Principles and Troubleshooting Techniques of Ion Chromatography

Principles of Ion Chromatography 12/2001

2 The Process of Ion Chromatography

Comparison of Anion Analysis With Varying Eluent Concentrations

Figure 3. Effect of Eluent Concentration on an AS14A separation

Principles of Ion Chromatography 12/2001

Principles and Troubleshooting Techniques of Ion Chromatography

Comparison of Anion Analysis with Varying Stationary Phase

Figure 4. Effect of varying column (stationary phase) on anion separation

Principles of Ion Chromatography 12/2001

2 The Process of Ion Chromatography

The Chromatographic Separation

Principles of Ion Chromatography 12/2001

Principles and Troubleshooting Techniques of Ion Chromatography

Figure 6a. Complete Resolution

Principles of Ion Chromatography 12/2001

2 The Process of Ion Chromatography

Figure 6b. Partial Resolution

Total Eluent 12.56mM Carbonate Ratio: 90.4%, Resolution 0.090

Figure 6c. Poor Resolution

Principles of Ion Chromatography 12/2001

Principles and Troubleshooting Techniques of Ion Chromatography

Figure 7. Thermodynamic and Kinetic Factors determining resolution

Thermodynamic Factors of Chromatography

Principles of Ion Chromatography 12/2001

2 The Process of Ion Chromatography

Principles of Ion Chromatography 12/2001

Principles and Troubleshooting Techniques of Ion Chromatography

Isotherm A represents an ideal state where K D remains constant

Isotherm B is a more accurate representation of most molecules in ion

Isotherm C is a less common situation in which lower concentrations of

Principles of Ion Chromatography 12/2001

2 The Process of Ion Chromatography

2.2.2.2 Capacity Factor

Principles of Ion Chromatography 12/2001

Principles and Troubleshooting Techniques of Ion Chromatography

Figure 10. Selectivity determines analyte elution order

Principles of Ion Chromatography 12/2001

2 The Process of Ion Chromatography

Factors Controlling Selectivity:

Principles of Ion Chromatography 12/2001

Principles and Troubleshooting Techniques of Ion Chromatography

For solutes with a type A isotherm (KD is constant throughout the