IC Troubleshooting

7/28/2019 IC Troubleshooting

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Principles and Troubleshooting

Techniques in

ION CHROMATOGRAPHY

2002 Dionex Corporation

Document No. 034461January 2002


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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


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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


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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 mobilephase, usually a liquid or gas, is used to transport the

analytes through the stationary phase.

The matrix, orstationaryphase, 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 compoundscontained 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.


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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


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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 itcame to be known, allowed low levels of common inorganic ions to be separated

and detected using a standard ionic mobile phase.


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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 InjectionValve

Column Suppressor DetectorData

Collection


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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 canbe affected by altering eluent composition and/or using different

formulations of stationary phase (Figures 3 and 4).


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Comparison of Anion Analysis With Varying Eluent Concentrations

Figure 3. Effect of Eluent Concentration on an AS14A separation


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Comparison of Anion Analysis with Varying Stationary Phase

Eluent: 1.8 mM Na2CO3/1.7 mM NaH CO3Flow Rate: 2.0 ml/min

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

(a) AS4A-SC

(b) AS4A


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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)(T2 - T1)

(W1 + W2)

where: T1 = retention time of peak 1

T2 = retention time of peak 2

W1 = peak Width at baselie of peak 1W2 = peak width at baseline of peak 2


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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


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Figure 6b. Partial Resolution

Figure 6c. Poor Resolution

Bulanesulfonate

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

PentanesulfonatePropanesulfonate


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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 (KD)

The flow rate of the eluent and the distribution of the solute between the

mobile and stationary phases determine a solutes retention time. In asystem without flow, a solute will achieve equilibrium between the two

phases. This equilibrium can be described as the distributioncoefficient

KD and is defined by the equation:


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KD = CS/CM

where : CS = the concentration of solute in the stationary phase

CM = 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 KD is more likely to be found

associated with the stationary phase at any given moment. A a low KD

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 KDs is the basis for the differential migration of various

components.

An analyte with a relatively low KD 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 KD 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 KD 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 KD will vary over a range of

solute concentrations. The relationship between the KD 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 mobileand stationary phases.


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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 KD 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 KD 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.


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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 = (Ve-Vm)/Vm

where: Ve = the elution volume of the solute

Vm= 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.

The capacity factor gives us a measure of the time the analytes

spends in the stationary phase versus the mobile phase.

K = (Te - T0)/T0


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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)/ (T1-T0)

If = 1, is equal to one there is no resolution between the analytes.

Increasing values of indicate analytes that would be morethoroughly 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).


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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


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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 KDs

(Figure 11).

Figure 11. Separation in an ideal system

1. In actuality it is observed that the concentration of an analyte variesthroughout 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)


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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 eluentflow 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 oftheoretical 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)


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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 KD.

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.


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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 BicarbonateStandard 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.


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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 KD 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 (KD decreases as the

concentration increases). We know that solute advancement through azone 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.


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diminishes. For a compound with a type A isotherm (where KD 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 KD

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.


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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 lessdispersion 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


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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.


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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.


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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


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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 11.320.0

0.01

0.02

0.03

0.04

0.05

0.065

S

Minutes17249


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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

Minutes

S

17270


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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 Divinylbenzeneto 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 theentire column volume.


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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 HeadspaceHeadspace 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 shortperiod of time. This results in a phenomenon known as peak tailing.

(Figure 24)

sulfonation (or amination) of

inert bead substrate

surface of activated

substrate


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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 formationof 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)


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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, causingblockages 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.


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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


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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


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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.


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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 withthe 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.


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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

HSO4-

Cation-ExchangeMembrane

Waste

Na+ HSO4-

Cation-ExchangeMembrane

Waste

Na+ HSO4-

Na+HSO4-

H+ HSO4-

Regenerant

H+ HSO4-

Regenerant

Na+OH

-

Na+

H+ OH- H+

Na+ A-

H+ AH2O

-A-


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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-ExchangeMembrane

Eluent

Anode

CathodePerforatedIon-Exchange

Material

Regenerant

Regenerant

2H2O 2H+ +1/2 O

2+ 2e-

2H2O + 2e- 2OH- + H

2 ConductivityCell

16180

Eluent OutEluent In

Regen Out

Ion Exchange MonoDisc

Electrode Electrode

Regen In


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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 OutRegen Out Regen In

_ +

RegenChamber

EluentChamber

RegenChamber

16771

Flow

DistributorDisc

Disk


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Selecting a Suppressor

Figure 31. Choosing the optimum suppressor for your application

SuppressorRegeneration

Mode

Optional

RequirementsCapacity Benefits

Applications

Anions Cations

SRS-ULTRA

2-mm & 4-mm

Formats

15 and 50L void

volume

Electrolyic or

chemical

All ex isting

systems except

DX-80

Anion :

200 mN

at 1.0 mL/min

Cation:

110 mN

at 1.0 mL/min

Ease of use

Moderat ely Low

Noise

Versatile

Use wi th

carbonate and

hydroxide eluents

For solve nt

applications, use

external water or

chemical

regeneration

Columns: Al l anion

columns

Use wit h

methanesulfonic

acid and sulfuric

acid eluents

For sol vent

applications use

external water or

chemicalregeneration. for

eluents containing

chloride or nitrate,

use chemicalregeneration

Column s: allcation 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 orexisting systems

with SC20 Power

supply

Anion a nd Catio n:

25 mN

at 1.0 mL/min

Ease of use with

DCR

Low Noise

Fastest S tart-up

Use wi th

carbonate eluents

Use wit h

methanesulfonic

acid and sulfuric

acid eluents

No solvents

Columns : CS12,CS12A, CS14

MMS III

2-mm & 4-mm

formats

Chemical All existing

systems

Required forDX-80

Anion :

150mNat 1.0 mL/min

Cation:

70 mNat 1.0 mL/min

Ease of use with

DCR

Lowest Nois e

Fastest S tart-up

Use wi th

carbonate andhydroxide eluents

and for eluents

containing

solvents

Columns: Al l anion

columns

Use wit h

methanesulfonicacid and sulfuric

acid eluents

containing

solvents, chloride

or nitrate

Column s: all

cation columns


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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 manualon 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 = 2.94Anion factor = 2.47

Concentration = mN


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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 _

Minutes

S

17303


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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


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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 bubblesobtained 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

S

17307

S

0.150

0.155

0.160

0.165

0.170

40 44 48 52 56 60Minutes

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


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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


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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.


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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


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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 ispossible 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.


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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 thisprocess.

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 typeof 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 exchangechromatography. Two other commonly used separation methodologies are ion

exclusion and reversed phase chromatography.


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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, areprevented 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

RC00H

H20

C1

C1


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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 orconcentration 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 towhether 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 ofdifferent 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


62/64



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). Thebackground 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 byusing 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 byinvestigating 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


63/64



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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IC Troubleshooting