Ic Principle

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AN INTRODUCTION

TO

ION CHROMATOGRAPHY

Basic Principles of Chromatography

Chromatography is an experimental technique used for analyzing and/or

separating mixtures of chemical substances. There are many different

kinds of chromatography, among them paper chromatography, gas

chromatography, liquid chromatography, and ion-exchange

chromatography. An ion chromatograph is an example of the latter kind of

chromatography.All chromatographic methods share the same basic principles and

mode of operation. In every case, a sample of the mixture to be analyzed

(the analyte) is applied to some stationary fixed material (the adsorbent)

and then a second material (the eluent) is passed through or over the

stationary phase. The compounds contained in the analyte are then

partitioned between the stationary adsorbent and the moving eluent. The

success of the method depends on the fact that different materials adhere

to the adsorbent with different forces. Some adhere to the adsorbent more

strongly than others and are therefore moved through the adsorbent more

slowly as the eluent flows over them. Other components of the analyte are

less strongly adsorbed on the stationary phase and are moved along more


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The number of peaks corresponds to the minimum number of different

substances (compounds or ions) contained in the analyte. If the analyte is

found to display only a single peak, it is an indication that it is composed of

only a single component, i.e., it is pure, although rigorous confirmation of

purity may require additional testing.Ion ChromatographyA. The Injector and the Eluent

Commercial ion chromatography systems have become available

within the past ten to fifteen years. The basic components of such a system

are shown in the cartoon below:

For anion chromatography the eluent is a dilute aqueous solution of

sodium bicarbonate and sodium carbonate, each about 1-4 mM in

concentration. The water used for the preparation of the eluent is of the

highest purity - Type I water which is virtually free from any contaminating

ions. The sample is injected into the instrument through the injection

system. The eluent, which the pump draws from the eluent jar and forces

through the system, then carries the analyte onto the ion exchange column

under a pressure of about 1000 psi (ca 65 atm).


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B. The Ion Exchange ColumnThe ion exchange column (sometimes called the analytical column)

is tightly packed with the stationary adsorbent. This adsorbent is usually

composed of tiny polymer beads which have positive charge centers

(actually ammonium type cations) attached to their surfaces with covalent

chemical bonds. If we could magnify the column packing, we imagine that

it would look something like the cartoon which follows:

When just the pure eluent is flowing through the column these

positive centers on the polymer surface attract the negatively charged

bicarbonate and carbonate ions in the eluent. But as the anions contained

in the analyte sample begin to enter the column, these anions, which are

also attracted to the positive centers on the polymer surface, may replace

(i.e., they exchange with) the bicarbonate and carbonate ions stuck on the

polymer surface. The analyte anions and the eluent anions compete with

each other for the positive centers on the polymer surface. As a result of

these interactions a rather complex equilibrium is set up as the analyte is

carried through the column by the eluent. This equilibrium can be

described by the cartoon below:


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How tightly the competing anions stick (i.e., how strongly they are

attracted) to the surface of the polymer beads depends on the size of the

anion and its charge. Usually, the greater the charge on the anion the more

strongly it is attracted to the surface of the polymer beads (so, for example,

sulfate ions are more tightly held to the polymer beads than chloride ions).

Also, larger anions generally move more slowly through the column than

smaller anions (e.g., bromide ions usually are more tightly bound to the

polymer beads than chloride ions).

So now we have a situation in which the eluent is trying to move the

anions of the analyte forward through the column, but the polymer beads

are trying to hold the anions in place by attracting them to the positive

charge centers. Obviously, anions can only move forward through the

column when they are in solution. So the speed at which a given type of

anion moves through the column is dependent on the relative amount of

time the ion is in solution. If an ion is only weakly attracted to the positive

centers on the surface of the polymer beads, it will spend relatively more of

its time in the solution phase than an anion that is strongly attracted to the

polymer surface. So these anions move through the column more quickly.

The more strongly the anion is attracted to the surface of the polymer

beads, the more slowly it moves through the column.


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The result is that what was injected as a homogeneous solution of analyte

anions at the top of the column, separates into bands of different kinds of

ions as they travel through the column. For example, if we could see what

happened to a solution of chloride, bromide and sulfate ions as they

traveled through the ion exchange column, after sometime the column

might look some thing like this:

While the details of what goes on inside the column is more

complicated than described above, perhaps this description will provide

you with a qualitative understanding of how different anions can be

separated on such a column.


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Detection and the Suppresser ColumnThe detector that is most commonly used in ion chromatography is

a conductivity cell which measures the conductance of the solution

passing through it. The conductance of a solution is proportional to the

concentration of the ions dissolved in the solution. Because we use

conductivity measurements to detect the different anions as they emerge

from the column, it is essential to pass the eluent through a suppresser

column before entering the detector. Heres why. The eluent contains

relatively high concentrations (1-5 mM) of sodium bicarbonate and sodium

carbonate - both strong electrolytes. If such a solution were passed directly

into a conductivity detector (which measures the electrical conductance of

the eluent solution), a large signal would be obtained just from the

presence of these eluent ions. The actual concentration of the analyte

anions of interest is usually only a tiny fraction of the concentration of the

bicarbonate and carbonate ions contributed by the eluent itself. Usually the

analyte anions of interest are present in the parts per million level or less;

however the bicarbonate and carbonate ions on the eluent are present in

much greater concentration approximately 100 times as large as the

anions of interest. Thus the bicarbonate and carbonate ions impart a large

conductivity to the solution entering the detector and the additional

conductivity imparted to the solution of the eluent by the presence of a tiny

amount of an anion of interest coming from the sample is extremely small.

That is, we would have a very hard time measuring the difference in the

conductivity of the pure eluent itself and the conductivity of the eluentcontaining a tiny amount of an anion of interest. In other words, the

sensitivity of the measurement would be too low to be of value.


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To get around this problem, after emerging from the column and before

entering the conductivity detector, we pass the solution through a

suppresser column which effectively removes all the bicarbonate and

carbonate ions from the solution, leaving only the analyte ions of interest

dissolved in the solution. When the resulting solution is now passed

through the conductivity cell, we can readily detect the signal due to the

presence of a small amount of the analyte ion, because in this case the

reference signal we are comparing it to is that of pure water which has an

exceedingly low conductivity. So by removing the large quantities of the

eluent anions, bicarbonate and carbonate, we greatly increase thesensitivity of the measurement. Indeed, using suppression, it is possible to

detect ion concentrations as low as parts per billion in the analyte sample. The suppresser column essentially is another ion exchange column.

Although there are different designs for such columns, we may think of

these columns also as being packed with small polymer beads, but these

beads carry acidic protons (H+1) on their surface:


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As the solution from the analytical column flows into the suppresser

column, the carbonate and bicarbonate ions, both of which are basic,

combine chemically with the protons on the polymer surface forming

carbonic acid which , being unstable in aqueous solution, decomposes to

carbon dioxide gas and water. In this way the carbonate and bicarbonate

ions are removed from the solution:HCO3

-1 + H+1 ----> [H2CO3] ---> H2O + CO2

CO3-2 + 2 H+1 ----> [H2CO3] ---> H2O + CO2

The spaces that are left on the polymer surface by the departure of the

protons are filled by sodium ions provided by the eluent solution (i.e.,

sodium ions undergo exchange with protons on the surface of the polymer

beads). This process essentially removes all of the anions (bicarbonate and

carbonate) that were present in the eluent solution but leaves the anions

that were contained in the analyte dissolved in the solution. The reason

that the analyte anions are not removed from the solution by the

suppresser column is that anions like chloride, bromide, nitrate, sulfate,

etc., are non-basic and do not combine with protons as they pass through

the suppresser column.When only pure eluent is entering the suppresser column, the

bicarbonate and carbonate anions are removed, the sodium ions are

retained on the column, and what emerges from the end of the suppresser

column is essentially pure water which has exceedingly low conductivity.

In this condition the conductivity detector is zeroed. Then when eluent

carrying a band of analyte anions enters the suppresser column, the

bicarbonate and carbonate anions are again removed as before, but the

analyte anions emerge from the suppresser column unscathed. When they

enter the conductivity cell of the detector, the conductance of the solution


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increases and this increase is measured. The greater the concentration of

the analyte anions the greater the conductivity and the larger is the peak

that is recorded in the printout.Qualitative Identification of Anions

The identification of the anions present in an analyte mixture is

achieved by comparing the results of the analysis with results obtained by

analyzing samples of known substances. The key to such identification

rests on the fact that under a fixed set of analytical conditions (type of

analytical column packing, concentration of eluent electrolytes, etc.) a

given anion will always take the same amount of time to travel through the

analytical column. This time is called the retention time (RT) see diagram

following:

. Each anion has its own characteristic retention time under a given

set of conditions. Thus, for example, if we find that a known solution

containing only sodium chloride, takes 2.76 min to emerge from the

column, then finding a peak in the chromatogram of an unknown analyte

which contains a peak at 2.76 minutes allows us to conclude that the

sample contains chloride ion. In all cases identification of an anion needs

to be done by comparison of the sample results with the results obtained

by analysis of known solutions.


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Quantitative Identification of AnionsOne can also quite readily obtain a measurement of the

concentration of a given anion in an unknown sample. This measurement

rests on the fact that the conductivity that a solution displays is directly

proportional to the concentration of ions it contains. So the larger the peak

for a given ion in a chromatogram the greater is its concentration, that is,

the area of the chromatogram peak for a given ion is proportional to the

concentration of the ion in solution. To determine the concentration of an

ion in an unknown solution one needs to compare the peak area of the ion

in the unknown solution with the peak area of that ion is some solution(s)

in which the concentration of the ion is known, i.e., a standard solution.

Typically quantitative results are achieved by using external or internal

standards. The use of external standards is more common.

Suppose one wants to determine the precise concentration of

chloride in an unknown solution. First, a set of standard solutions

(prepared by dissolving pure sodium chloride in pure water) is made in

which the precise concentration of the chloride ion is known. Typically a

minimum of three standards are used - say a 100 ppm, 50 ppm and 10 ppm

solution (ppm = parts per million). Then each of these solutions is analyzed

on the ion chromatograph and from the results a standard curve is

obtained. Such a curve typically is obtained by plotting peak height (or

peak area) in the chromatogram Vs concentration of chloride ion which

usually produces close to a straight-line graph. A typical result would look

something like that shown below:


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Finally, the unknown solution is analyzed and, by measuring

the peak area for the chloride ion (identified by retention time), and

comparing it with the standard curve, the concentration of chloride ion in

the unknown solution can be calculated.The Ion Chromatography ExperimentA. Sample Preparation and Analysis

The actual experiment is very simple. All that needs to be done is to

prepare the sample by gravity filtering a small portion (10 mL) of the water

sample into an ion chromatography analysis tube, label the tube and placeit into the ion chromatography auto-sampler. An identifying name for the

sample needs to be entered on the computer. After all samples are loaded

into the auto-sampler and entered into the computer, the analysis begins

and needs no further attention. The results will be distributed next week


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B. Analysis of ResultsTo analyze the results of the ion chromatography experiment, we

will pool the results that were obtained for water samples that were

collected from the same source. So there will be different sets of results -

one each for samples collected from each of the different ponds. For each

of these locations we will prepare a spreadsheet using Excel or some other

spreadsheet program. Each student should enter his or her data on the

relevant spreadsheet. The spreadsheet should contain columns for theconcentrations of each of the ions analyzed for - chloride, nitrate,

phosphate, and sulfate and for the Acid Neutralizing Capacity (ANC) of the

water sample (you measured this by titration last week in lab). After all the

individual results are entered, we will calculate an average concentration

for each ion and a standard deviation for the measurement of each ion

concentration.This data will then become part of the data entered into the Calvin

Environmental Assessment Program (CEAP), a program which seeks to

monitor how human activities on the campus and surrounding

neighborhoods impact the natural environment

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