IC - Principles and Troubleshooting Dionex

Principles and Troubleshooting
Techniques in
ION CHROMATOGRAPHY
2002 Dionex Corporation
Document No. 034461
January 2002
2002 by Dionex Corporation
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Contents
Principles of Ion Chromatography 1/2002 iii
Table of Contents
1 Introduction
What is Chromatography? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 The Process of Ion Chromatography
2.1 Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1.1 Functions of the Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2 The Chromatographic Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2.1 Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2.2 Thermodynamic Factors of Chromatography . . . . . . . . . . . . . 12
2.2.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3 The Chromatography System
3.1 Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.1.1 Function of the Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.1.2 Preparation of Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.1.3 Troubleshooting Eluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2 Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.3 Injection Valve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Contents
Principles of Ion Chromatography 1/2002 iv
3.4 Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.4.1 Headspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.4.2 Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.4.3 Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.4.4 Column Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.5 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.5.1 Conductivity Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.5.2 Amperometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.5.3 Absorbance Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.5.4 Fluorescence Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.5.5 Other Detection Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4 Method Development
4.1 Define Goals of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.2 Selecting the Appropriate Separation Mode . . . . . . . . . . . . . . . . . . . . 55
4.2.1 Ion Exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.2.2 Reverse Phase Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 57
4.2.3 Column Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.3 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Principles of Ion Chromatography 1/2002 1
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
2 Principles of Ion Chromatography 1/2002
Figure 1. Separation resulting from differential migration of compounds
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
Chromatography is the separation of the components
of a mixture by differential partitioning between a mobile and
stationary phase
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
Figure 2. Components of an Ion Chromatograph
Eluent Pump
Injection
Valve
Column Suppressor Detector
Data
Collection
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 Na
2
CO
3
/1.7 mM NaH CO
3
Flow Rate: 2.0 ml/min
Figure 4. Effect of varying column (stationary phase) on anion separation
(a) AS4A-SC
(b) AS4A
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:
where: V = the elution volume of the peak
W = the width of the peak at the baseline
Figure 5. Resolution
R = (2)(flowrate)(T
2
- T
1
)
(W
1
+ W
2
)
where: T
1
= retention time of peak 1
T
2
= retention time of peak 2
W
1
= peak Width at baselie of peak 1
W
2
= peak width at baseline of peak 2
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
Figure 6b. Partial Resolution
Figure 6c. Poor Resolution
Bulanesulfonate
Total Eluent 12.56mM Carbonate Ratio: 90.4%, Resolution 0.090
Pentanesulfonate Propanesulfonate
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:
K
D
= C
S
/C
M
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.
Given a particular combination of mobile and stationary phases, any two
analytes will generally have distinct distribution coefficients. This
difference in K
D
s 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 K
D

An analyte with a higher K
D
favors distribution towards the
stationary phase. This analyte elutes at a slower rate.
The K
D
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.
An analytes retention time is determined by the eluent flow
rate and by the distribution of solute between the mobile
and stationary phases.
Figure 8. Isotherms
Figure 8 depicts the three types of isotherms with K
D
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.
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.
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 = (V
e
-V
m
)/V
m
where: V
e
= 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 = (T
e
-T
0
)/T
0
where: T
0
= the time needed to flush one column volume (this
is the duration of time from the injection to the water dip).
T
e
= 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.
The capacity factor gives us a measure of the time the analytes
spends in the stationary phase versus the mobile phase.
K = (T
e
- T
0
)/T
0
2.2.2.3 Selectivity
Selectivity is described as the ratio of two analytes capacity factors.
The selectivity factor, , is defined by the equation:
= (T
2
-T
0
)/ (T
1
-T
0
)
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).
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
Elution order most likely results from a combination of analyte
hydration energy and electrostatic attraction
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
D
s
(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 (K
D
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)
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)
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.
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 3.5 mM Carbonate 3.5 mM Bicarbonate
Standard AS4A
Eluent
Fluoride 1480 2520 1050
Chloride 2220 3060 2850
Nitrate 1900 3120 3130
Phosphate 2930 2660 2130
Sulfate 3960 3140 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.
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.
Figure 15. Different zones of peak exhibit pseudo - K
D
s
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 opposite effect is observed once we have passed the peak maximum,
with the solute tending to lag behind the eluent front as its concentration
The peak shape of a compound is affected by its isotherm. Most
compounds exhibit a type B isotherm.
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.
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
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.
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.
Counter ion valence and selectivity are affected by the pH of the
eluent.
3.1.2 Preparation of Eluent
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 18m 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.
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
3.2 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.
Figure 18. Pump Noise
Eluent Troubleshooting
Changes in retention time
Loss of peak efficiency
Loss of sensitivity
Increased background conductivity
4.88 6 7 8 9 10 11.32
0.0
0.01
0.02
0.03
0.04
0.05
0.065
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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)
Figure 19. Poor run-to-run reproducibility
0.13 0.20 0.30 0.40 0.50 0.60 0.75
-5
10
20
30
45
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17270
Figure 20. Excellent Reproducibility (overlay of 6 runs)
Figure 21. Sample Carryover
0.00 0.20 0.40 0.60 0.80 1.00 1.25
-5
10
20
30
40
50
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17269
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.
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)
sulfonation (or amination) of
inert bead substrate
surface of activated
substrate
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)
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.
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.
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.
Sample matrices often contain a wide range of contaminants, many
of which can be reduced or eliminated by various methods of
sample pre-treatment
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
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.
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).
Columns should be cleaned when analyte
retention times shift by 10 percent
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.
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.
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.
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.
Typical IC Setup
Eluent
Waste
SRS-
ULTRA
Detector Cell
Chemical
Regenerant
Waste
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.
To Detector
HSO
4
-
Cation-
Exchange
Membrane
Waste
Na
+
HSO
4
-
Cation-
Exchange
Membrane
Waste
Na
+
HSO
4
-
Na
+
HSO
4
-
H
+
HSO
4
-
Regenerant
H
+
HSO
4
-
Regenerant
Na+
OH
-
Na
+
H
+
OH
-
H
+
Na
+
A
-
H
+
A
H
2
O
-
A
-
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.
Figure 29. The Atlas Electrolytic Suppressor.
16209
Suppressed Eluent
Ion-Exchange
Membrane
Eluent
Anode
Cathode
Perforated
Ion-Exchange
Material
Regenerant
Regenerant
2H
2
O 2H
+
+1/2 O
2
+ 2e
-
2H
2
O + 2e
-
2OH
-
+ H
2
Conductivity
Cell
Atlas Electrolytic Suppressor
16180
Eluent Out Eluent In
Regen Out
Ion Exchange MonoDisc
Electrode Electrode
Regen In
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.
Figure 30. The Flow of the Eluent through the Monodisk of the Atlas Suppressor
Eluent
In
Cation-Exchange
Monolith
Cation-Exchange
Membrane
Eluent Out
Regen Out Regen In
_
+
Regen
Chamber
Eluent
Chamber
Regen
Chamber
16771
Flow
Distributor
Disc
Disk
Selecting a Suppressor
Figure 31. Choosing the optimum suppressor for your application
Suppressor
Regeneration
Mode
Optional
Requirements
Capacity Benefits
Applications
Anions Cations
SRS-ULTRA
2-mm & 4-mm
Formats
15 and 50L void
volume
Electrolyic or
chemical
All existing
systems except
DX-80
Anion:
200 mN
at 1.0 mL/min
Cation:
110 mN
at 1.0 mL/min
Ease of use
Moderately Low
Noise
Versatile
Use with
carbonate and
hydroxide eluents
For solvent
applications, use
external water or
chemical
regeneration
Columns: All anion
columns
Use with
methanesulfonic
acid and sulfuric
acid eluents
For solvent
applications use
external water or
chemical
regeneration. for
eluents containing
chloride or nitrate,
use chemical
regeneration
Columns: all
cation columns
Atlas
1 format for 2-, 3-
& 4-mm formats
35 L
void volume
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
Cation:
70 mN
at 1.0 mL/min
Ease of use with
DCR
Lowest Noise
Fastest Start-up
Use with
carbonate and
hydroxide eluents
and for eluents
containing
solvents
Columns: All anion
columns
Use with
methanesulfonic
acid and sulfuric
acid eluents
containing
solvents, chloride
or nitrate
Columns: all
cation columns
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
Spikes
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
0.1 5 10 15 20 25 28
-0.07
0.00
0.05
0.10
0.15
ECD_1
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Noisy Baselines
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
Conditions: GP50 pump, 4-mm AS14 column, 3.5 mM Na2CO3/1.7 mM NaHCO3,
1.2 mL/min, 32 mA, recycle mode.
20 30 40 50 60 70 80
Minutes
16.530
16.550
16.570
16.590
16.610
16.630
S
17305
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.
Figure 35. An example of noisy chromatography baseline due to trapped bubbles
obtained using a Cation Atlas Suppressor
Conditions: GP40 pump, 4-mm AS9HC column, 9.0 mM Na2CO3 1.0 mL/min, 60 mA, recycle mode.
Burping the DS3 cell
550 600 650 700 750 800 850 900
23.58
23.59
23.60
23.61
23.62
23.63
Minutes
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17307
S
0.150
0.155
0.160
0.165
0.170
40 44 48 52 56 60
Minutes
Average noise: 4.96 nS
Before burping the DS3 cell
Minutes
0.1230
0.1235
0.1240
40 44 48 52 56 60
Average noise: 0.12 nS
After burping the DS3 cell
Conditions: GS50 pump, 3-mm CS12A column, 20 mM MSA,
0.5 mL/min, cation Atlas, 33 mA, recycle mode.
17309
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.
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
820 825 830 835 840
Minutes
16.780
16.782
16.784
16.786
16.788
16.790
820 825 830 835 840
Minutes
16.610
16.612
16.614
16.616
16.618
16.620

400 psi 100 psi
Conditions: GP50 pump, 4-mm AS14 column, 3.5 mM Na2CO3/1.7 mM NaHCO3,
1.2 mL/min, 32 mA, recycle mode.
S S
17310
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.
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
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)
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.
Filter/Grating
Photodetector
Flow
Cell
Reference
Beam
Splitter
Light Source
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.
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.
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.
Figure 39. Separation in ion exclusion
Donnan Membrane
R
C00H
H
2
0
C
1
C
1
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 non-
polar 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
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
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.

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