peter regier seminar - ic

7/30/2019 Peter Regier Seminar - IC

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Development of an Environmental Sample Analysis

Method Using Ion-Exchange Chromatography

Peter Regier

Bethel College

Advisor: Dr. Gary Histand


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Table of Contents

I. AbstractII. Introduction

III. Ion-Exchange Chromatography TheoryIV. Methods

a. Development of Operation Procedureb. Instrumentationc. Reagentsd. Calibratione. Testing an Unknownf. Sample Preparation

V. ResultsVI. Discussion

VII. ConclusionVIII. Acknowledgements

IX. ReferencesX. Appendices A-K


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I. Abstract

Ion Chromatography is a rapidly expanding analytical technique, used for simultaneous

determination of ions. Bethel College recently purchased a Metrohm Ion Chromatograph (IC) and the

successful development of proper operating conditions and techniques was met through completion of

this seminar. All aspects for creating a precise, accurate and repeatable analysis environment weretaken into account. As IC analysis detects ionic compounds on the sub-ppm (parts per million) level,

great care was taken in all steps, from cleaning of glassware to general maintenance and creation of

necessary solutions. The method was verified through use of standard anion solution*. Soil samples

were taken from a tallgrass prairie restoration plot at Bethel College to provide environmental samples.

As nitrogen is one of the most important nutrients for a healthy, functioning prairie[1] and is often the

limiting nutrient in terrestrial environments[2], nitrate and nitrite were analyzed. Substantial research

into ionic species in aqueous environments already exists[3][4][5]

but less for environmental investigation

of ionic concentrations in soil, especially cationic compounds[6]

. As there is concern of the health of the

restoration plot, nitrate and nitrite levels are key to understanding what is wrong and possible remedies

to restore the health of the plot.

*See Sec. IVc: Reagents for solution details


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II. Introduction

Chromatography is a common and diverse method of separation where a mobile phase flows

over a stationary phase. Ion Chromatography is a branch of Liquid Chromatography that has become

increasingly popular in recent years. Ion Chromatography separates ionic compounds in solution based

on ionic bond strength ability[6]

. As all ions have different bond strengths, each individual compound is

retained inside a column packed with a stationary phase coated in an ion-exchange resin for a certain

amount of time (stronger ionic bonds result in longer retention times). Samples to be separated and

analyzed are injected into a continuous stream of eluent (also referred to as the mobile phase) in a

sharp plug via sample loop on an injection valve. One of the major advantages of this process is the

ability to simultaneously determine multiple ionic species at sub-ppm (parts per million) concentrations.

In addition, IC has become common in environmental analysis, being used to determine ionic

concentrations in a wide variety of samples, from sea water to polar ice to food products[7]

. Indeed, the

ability of IC to handle samples from air, soil and water accounts for the rapid growth of the IC as an

accurate tool for environmental monitoring[6]

. Ion chromatographic methods can be applied to a variety

of ionic solutes, and can separate anions (negatively charged ions, eg. Br

-

) or cations (positively charged

ions, eg. Ca+). This paper focuses on quantitative determination of anionic compounds and qualitative

discussion of environmental samples. Of particular interest is a series of prairie restoration plots which

have received experimental treatments. One plot is currently severely damaged with nitrogen

deficiency being a possible culprit.

Nitrogen is the most important nutrient to a healthily functioning prairie[8]

. Nitrate and nitrate

are the two most prevalent anionic compounds found in soil. Nitrogen is deposited into soil via

Eluent: solvent used to carry extracted ions through chromatograph

[10][11]


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atmospheric deposition or anthropological sources and doesnt adsorb with soil particles, meaning it

stays in soil until used by plants or transported by groundwater[9]

. During the growing season, nitrate is

absorbed into plant material. Unless this growth is removed from the area, such as harvesting of crops,

the nitrate concentration will mostly return to the soil through decomposing plant material[9]. During

the winter, nitrate concentrations tend to remain fairly constant due to limited use by plants.

Substantial research into analyzing ionic species in water already exists[3][4][5]

but less for

environmental investigation of ionic concentrations in soil[6]

. The purpose of this research is to develop

and test a method for analyzing nitrate and nitrite concentrations in tallgrass prairie soil samples using

Ion Chromatography. In order to accomplish this, a good operating environment must be established,

then proper calibration must be demonstrated. When these steps are complete, known and unknown

samples will be analyzed, including environmental samples from the prairie.

III. Ion-Exchange Chromatography Theory

Note: IC methods can be used for separation of cations (C+) or anions (A

-). However, for simplicity, the

following theory details anions separated on a positively charged column, as this was the primary area ofresearch. For cationic separations, all charges would be switched (C

+ A

-, A

- C

+)

The theory of Ion Exchange Chromatography is relatively simple. There is a stationary phase and

a mobile phase. The stationary phase is a packed column which is coated with positively charged ions.

A sample is injected into the column, separated out in the column and recorded by the detector. The

sample flow path through the instrument is shown in Fig. 1

For a more detailed flow path, seeAppendix K: Ion Chromatograph Flow Diagram (detailed)

EluentPum In ector

Sam le

Column Su ressor Detector

Figure 1: Flow Path


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The sample is injected from the sample loop into a constant flow of charged mobile phase

(eluent). As the anions enter the column, they are attracted to the positively charged ions on the

column and must compete for binding locations. All ions have different binding strength based on the

equilibrium constant for the exchange reaction (Kex.)[12][13]

. Ions are sent into the column in a sharp plug

and separate along the column based on binding strength. When the samples anions are flushed from

the column, they come out separated into species and pass through the detector.

Figure 2: Four steps to ion exchange within the column. The column is coated with positive charges, represented

by plus signs. The smaller, thinner minus signs represent the anions in the eluent, while the thicker, bigger minus

signs are the anions in the sample being analyzed.

+++++++++++++++++++++

+++++++++++++++++++++

Flow Direction

A B

+++++++++++++++++++++

+++++++++++++++++++++

Flow Direction

+++++++++++++++++++++

+++++++++++++++++++++

Flow Direction

D

+++++++++++++++++++++

+++++++++++++++++++++

Flow Direction

C

Fig. 2


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In Fig. 2, A is the column before injection of the sample. B is at the moment of sample injection

where the strong anion from the sample is attracted to the wall of the column. C shows the sample

anion bonded to the positively charged wall of the column after kicking the weaker eluent anion off the

bonding site. D shows the sample anion moving down the column after its bond is broken with the

cation by an eluent anion. The exchange equilibrium for the column used in this experiment isxNR3+-

OCO2H-+ Ax

- [NR3

+]x-A

x- + OCO2H-where NR3

+is the stationary phase, OCO2H

-is the mobile phase and

Ax- is the anionic compounds in the injection sample to be analyzed[14].

IV. Methods

a. Development of Operation Procedure:The initial phase of research included the development of a proper operating environment

for the Ion Chromatograph as well as clear and precise instructions for running the machine and

maintenance. During this process, a cleaned stock of glassware was assembled, procedures for

mixing of solutions were developed, several trouble-shooting sessions resolved issues with

software, hardware and interfacing and general maintenance was conducted. To document this

information for future operators, all important procedures were recorded to facilitate continued

operation of the IC. These procedures are attached asAppendices (A-H). Appendix J:

Troubleshooting provides a table which details problems that were encountered in the

development of the instrument method and the indications and steps taken to fix the issue.

b. Instrumentation:All samples were analyzed using a Metrohm (Herisau, Switzerland) 820 IC Separation Center

equipped with a Metrohm 819 IC detector for conductivity detection. Samples were placed in


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Metrohm 11 mL sample vials and automatically drawn into the column using a Metrohm 838

Advanced Sample Processor. A Metrosep A Supp 5 - 50/4.0mm anion separation column was

used for separation with a mobile-phase eluent of 3.2mM Na2CO3 / 1.0 mM NaHCO2. All

samples were run under standard operating conditions of flow rate: 0.70 mL/min, pressure: 7.0

MPa, column temperature: . The sample was injected by a 20 L injection loop mounted on

a 6-port rotary injector valve (flow path rotation detailed in Fig. 3). In the fill position (3A),

sample ran through the sample loop until the injector valve rotated to the inject position (3B).

In inject position, contents of the sample loop were injected as a sharp plug into the mobile

phase and enters the column.

c. Reagents:All water used for washing glassware, dilutions and making standards was filtered with a

Barnstead (Dubuque, Iowa) EASYpure line-fed ultra-pure water (UPW) system equipped with a

0.2 m irradiated filter producing de-ionized UPW. All water used for this experiment had a

Fig. 3A: Valve in Fill Position Fig. 3B: Valve in Inject Position

Fig. 3


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resistivity between 17.8 and 18.2 M-cm. Anion standards were mixed from Metrohm MCal

Custom Anion Mix consisting of the ions at the concentrations presented in Table 1:

Table 1

Concentration

(mg/L)

Anion

Name

Anion

Formula

10 Bromide Br-

10 Nitrate NO3-

10 Phosphate PO4-3

10 Sulphate SO4-2

5 Chloride Cl-

5 Nitrite NO2-

2 Fluoride F-

Four concentrations of standards were then diluted from the MCal Anion Mix using a 100-

10000.5% L micro-pipette and 50.000.05 mL volumetric flasks filled with UPW. All

concentrations were based on the relative concentration of nitrate in each standard in mg/L and

are presented in Table 2:

Table 2

Level Dilution Concentration

NO3-(mg/L)

1 1:50 0.2

2 1:20 0.5

3 1:10 1

4 1:5 2

d. Calibration:The four standards were run at the beginning of every sample queue and then batch-

reprocessed to achieve a best-fit calibration curve. An example of a calibration curve is

presented in Fig. 4.

As solutions are mostly water, which has a density of 1 kg/L, 1 mg/L 1 part per million


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e. Unknown TestingOnce a satisfactory multi-point calibration curve had been produced, a sample of the 1:10

dilution standard was added to a sample queue but with the level set to 0, meaning that it was

treated as a sample, not a standard (seeAppendix B: Setting up a Sample Queue for detailed

information). The resulting 1:10 dilution sample chromatogram was compared with the 1:10

dilution standard to check that the machine could accurately analyze an unknown sample.

f. Environmental Sample Preparation:All soil samples were taken using a T-sampler at the experimental tallgrass prairie restoration

plots of Kauffman Museum on the grounds of Bethel College (North Newton, KS) on October 24,

2011. Three plots were examined, labeled Plot 0, Plot 1 and Plot 2 for this experiment. Plot 0

and Plot 2 were older sections of prairie that were relatively undisturbed, while Plot 1 had been

recently seeded and was less healthy than the other two plots. To obtain each core, the T-

sampler was pushed vertically eight inches into the soil and then pulled vertically back up. For

each plot, 6 core samples were taken, each for a randomly selected location within the plot.

The cores were placed in a clean bucket and broken by hand into small aggregates, forming a

homogenous mixture[7]. Approximately five grams of this mixture were placed in a clean paper

bag and allowed to air dry for 72 hours[6]. The following is the extraction process for one

sample. This process was repeated for all analyzed samples:

Ion extraction: The sample was ground by mortar and pestle into a fine matrix and

0.2500 grams were measured out on a Ohaus (Pine Brook, NG) AV264 Analytical Balance

(capacity of 260.0000g, readability of 0.0001g) and transferred gravimetrically into a

sterile Corning (Corning, NY) 15 mL polypropylene centrifuge tube. The weigh boat was


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weighed both before and after sample transfer to give a net weight of soil transferred,

presented in Table 3:

Table 3

Sample Full Weight (g) Empty Weight (g) Soil Weight (g)

Plot 0: South 1 0.2518 0.0038 0.2480

Plot 0: South 2 0.2493 0.0125 0.2368

Plot 0: North 1 0.2488 0.0089 0.2399

Plot 0: North 2 0.2503 0.0003 0.2500

Plot 1: East 0.2504 0.0007 0.2497

Plot 1: West 0.2493 0.0025 0.2468

Plot 2: East 0.2493 0.0015 0.2478

Plot 2: West 10.2507 0.0025 0.2482

Plot 2: West 2 0.2518 0.0034 0.2484

A 10.000.04 mL volumetric flask was used to measure 10 mL of UPW into the sample

tube which was capped and agitated on a rotary shaker for 1 hour[8]

. UPW was used

instead of eluent (as suggested[8]

), because of the possibility of solute dispersion which

reduces efficiency of the method[9]

. At the end of this time period, the sample was

centrifuged for 5 minutes. Approximately 2 mL of the resulting solution were decanted

and filtered through a Whatman Uniprep 0.2 m PTFE membrane syringeless filter

device and 1 mL of this filtered solution was transferred via micropipette to a clean 10

mL volumetric flask which was filled to the line with UPW, capped and shaken. The

solution was then poured into a clean Metrohm 11 mL sample vial.

After the samples were run, they were reprocessed with the standards using the best-fit

calibration curve obtained in the Calibration section. During the reprocessing, retention times

for samples were automatically adjusted to adhere to the retention times of the calibration


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standards. Concentration values in mg/L were assigned to each sample based upon the values

obtained from the calibration curve.


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V. Results

The four anion standards were run as shown below:

1:20 Dilution

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 1 4 15 1 6 17 1 8 19 mi

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

mV

ch1

fluoride

0.1

chloride

0.

nitrite

0.2

43

brom

ide

0.5

00

nitrate

0.5

01

sulp

hate

0.5

04

phosphate

0.5

01

1:50 Dilution

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

mV

ch1

fluoride

0.

chloride

0

nitrite

0.1

20

brom

ide

0.1

nitrate

0.1

sulphate

0.1

81

phosphate

0.1


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1:5 Dilution

0 1 2 3 4 5 6 7 8 9 10 1 1 1 2 13 1 4 15 1 6 17 1 8 1 9 mi

0

2

4

6

8

10

12

14

16

18

mV

ch1

1

fluoride

0.4

03

chloride

1.

nitrite1.0

01

brom

ide

2.0

09

nitra

te

2.0

51

sulphate

2.0

16

phosphat

e

2.0

09

1:10 Dilution

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 1 4 15 1 6 17 1 8 19 mi

0

1

2

3

4

5

6

7

8

9

mV

ch1

fluoride

0.2

02

2

chloride

0.

nitrite

0.4

94

brom

ide

1.0

03

ni

trate

1.0

06

sulphate

1.0

07

phosp

hate

1.0

01


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The standard solutions were then batch-reprocessed**

and multi-point calibration curves were

created for each anion based upon the four concentration values obtained for each standard dilution

level. Fig. 4 shows an example calibration curve where each points number cooresponds to its

calibration level:

The peak area for each standard level for a specific anion is plotted against the concentration

given by the method. This data was fitted to a best-fit linear formula with a y-intercept of 0 and the

process was repeated for all anions. The correlation in this case is 0.99998 with a residual standard

deviation (RSD) of 2.494%. The results for each anion tested are presented below inTable 4:

Table 4

Anion RSD (%) Correlation

Retention

(min) Concentration (mg/L)

F- 2.494 0.99998 4.184 0.403

Cl- 6.675 0.99996 6.149 1.017

NO2- 2.655 0.99969 7.189 1.001

Br- 1.216 0.99997 8.997 2.009

NO3- 1.548 0.99996 9.977 2.051

SO4-2

2.443 0.99996 13.754 2.016PO4

-3 1.177 0.99997 17.357 2.009

MeanSD 2.6011.901 0.999930.00011

**SeeAppendix E: Batch Reprocessing for procedure

Fig. 4


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Once a standard curve was successfully developed, an unknown sample was run along with the

standard solutions. The unknown solution was a repeat of one of the standard solutions but the level

was not entered in the sample queue, making its anion concentrations unknown to the instrument. As

ambient conditions and variation of parameters in the instrument environment can significantly affect

chromatographic output, the 4-point standard curve was generated for every sample queue. The

unknown sample run was 1:10 dilution, and this is compared with the 1:10 standard run in Table 5. As

the concentrations are sub-ppm, the data is presented with units of parts per billion (ppb):

Table 5

Anion (ppb)

Sample F-

Cl-

NO2-

Br-

NO3-

SO4-2

PO4-3

1:10 Standard 190 211 544 1002 984 994 988

1:10 Sample 189 203 543 1003 988 975 996

Difference 1 8 1 1 4 19 8

Once the method was developed and an unknown sample was tested and experimentally

proved the reproducibility and accuracy of the method, environmental samples were taken. However,

the soil samples were run using water from the UPW machine that wasnt properly cleaned due to worn

out filters. The samples run with this water saturated the column with ions which were subsequently

flushed by making new solution and running many blank samples of clean UPW. At completion of the

project, the column was not completely cleaned and so repeat extraction and analysis of the soil

samples was not possible. As the eluent was more conductive due to excess ions, the two most diluted

standards run for the environmental sample queue were removed due to excessive peak interference.

The resulting sample chromatograms are presented inAppendix I: Sample Chromatograms and the

values for the calibration curve are given in Table 6:


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Table 6

Anion RSD (%) Correlation Retention (min) Concentration (mg/L)

F- 3.628 1.00000 4.184 0.405

Cl- 46.473 1.00000 5.811 1.058

NO2-

6.677 1.00000 6.805 0.975

Br- 0.149 1.00000 8.470 1.999

NO3- 1.163 1.00000 9.632 2.008

SO4-2

0.482 1.00000 12.336 2.003

PO4-3

0.912 1.00000 15.089 2.006

Using the data from Table 4 and Table 6, the differences between the standard curve concentrations

were determined and are presented in Table 7:

Table 7

Anion Actual conc. (ppb) |Actual - Table 4| (ppb) |Actual - Table 6| (ppb)

F- 400 3 5

Cl- 1000 17 58

NO2- 1000 1 25

Br- 2000 9 1

NO3-

2000 51 8SO4

-2 2000 16 3

PO4-3

2000 9 6

Finally, the nitrate and nitrite concentrations of all environmental samples are shown in Table 8:

Table 8

Sample NO2-(ppm) NO3

-(ppm)

Plot 0: South 1 0.014 0.204

Plot 0: South 2 0.016 0.107

Plot 0: North 1 0.009 0.049

Plot 0: North 2 0.008 0.008

Plot 1: East 0.012 0.209

Plot 1: West 0.013 0.098


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Plot 2: East 0.007 0.090

Plot 2: West 1 NA 0.015

Plot 2: West 2 NA 0.091

Mean 0.011 0.097

VI. Discussion

Initial results for running a standard curve are very precise. The chromatograms all have strong,

sharp peaks with limited broadening and no strong tails. Metrohm states that a value for RSD of less

than 5% and a Correlation Value of 0.999 or better is expected for a strong standard curve[15]

. The

samples provide good RSD values for each anion except chloride, with a RSD of 6.675%. Though this is

close to the target value of 5%, it must be noted that this is a possible point of concern. When observing

the chromatograms a negative peak is observed (most strongly in the weak dilutions) on the end of the

nitrite peak. A negative peak generally means that something less conductive than the eluent is running

through the detector. It is possible that some sort of contaminant in small quantities with a similar

retention time to nitrite caused this peak.

The chromatograms for the samples run are much more concerning than the chromatograms

run simply for the standard. This is due in part to the nature of environmental samples. It is relatively

easy to get strong, even peaks when the sample is a standard anion solution of certified concentration

diluted with UPW. However, environmental samples will inevitably have competing anionic and cationic

compounds and a more complex matrix filled with other compounds that can interfere with accurate

detection. In addition, the sample runs coincided with the failure of the filters on the UPW machine,

leading to even the tested standards giving poor results. The large negative peaks present on each

chromatogram are most likely the result of an eluent with higher ionic activity than desired. This

presence of extra ions increases the general conductivity of the eluent. As the conductivity is increased,


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the baseline increases as well and the bottoms of peaks are either less clear or partially cut off. Despite

these difficulties, the two strongest standard dilutions built a curve detailed in Table 6 that is acceptable

except chloride. The correlation values are all at the maximum possible value as there are only two

points on which the curve was based around, and therefore, correlation data are ignored. The resulting

RSD values show that all anions except chloride and nitrite have under 5% residual standard deviation.

The first standard multi-point calibration curve (data presented in Table 4) was assumed to be precise

and accurate based on using all four points for calibration and virtually all requirements for a decent

curve having been successfully met. Based on strong evidence against the precision of the

environmental standard method, the difference between the curves detailed inTable 4 and Table 6 and

the actual concentration value were calculated in Table 7. As the concentrations of anions are known,

the difference between the actual values and the values provided inTable 4 and Table 6 were

calculated. This provided a comparison of the two sample curve concentrations using a shared

reference point. Based upon the evidence against the strength of the two-point curve, it was used to

batch-reprocess all of the environmental samples to label peaks and assign concentrations, but the data

are considered to be inaccurate. Therefore, the data are analyzed qualitatively based on relative shape

and peak strength rather than quantitatively.

Most noticeable of all the anion peaks in the environmental samples is the chloride peak. While

it is possible that there is contamination from the problems with UPW, the chloride peaks do not seem

to be spiked in the standards. Therefore, it may be inferred that the chloride levels in the prairie plots

appear to be high. It must be noted that the field duplicate for Plot 0: North shows a dramatic

difference between samples in terms of chloride level. As it is a duplicate, it is assumed that both

samples will have the same concentration, while the chloride differs by 212 ppb. Chloride is generally

anthropologically deposited and in this case, it is quite plausible that irrigation of grass in close proximity


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to the prairie plots has led to a marked increase in chloride concentration within the plots over time.

The same possibility applies to the fluoride peaks, though they are much less pronounced than the

chloride values.

The discrepancies in Plot 0: North samples regarding chloride raise serious questions about the

reproducibility of the soil analysis method. However, in general, the duplicates appear to be reasonably

similar in terms of present peaks and their relative concentrations. Plot 0: South, Plot 0: North and Plot

2: West samples all have relatively similar concentration patterns. It is also noted that Plot 0 has no

sulfate peaks recognized while the other two plots samples all have sulfate peaks. As Plot 0 and Plot 2

are supposed to be similar, this is surprising, though not alarming as the concentration of sulfate is

uniformly low across all samples.

However, of all the anions, nitrogenous compounds are of the most interest for this study.

Nitrate is present in all samples with an average concentration of 97 ppb. Nitrite is present in all

samples except Plot 2: West and its field duplicate and had an average of 11 ppb. As nitrite is located at

the beginning of a negative peak on all chromatograms, its likely that the contamination causing the

negative spike also interfered with the resolution of the nitrite peak. However, nitrate peaks are

consistently taller and clearer than nitrite, even at the beginning of the peak where nitrite would be

unaffected by the negative peak on its tail. This leads to the tentative conclusion that nitrate is either

more common in higher concentrations in the prairie soil, or that the extraction method or instrumental

procedure retain or detect nitrate better than nitrite. As the original concern was that nitrogen species

may be low in the experimental Plot 1, the data are compared between plots. It is observed that, when

the nitrate and nitrite values are summed for each plot and then divided by the number of plots, the

experimental plot has the highest average nitrogenous species concentration. This is opposite of the

initial prediction that nitrogen deficiency is causing ecological problems in the plot. However, as the


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concentrations of both compounds are so small, so varied, and the operating state of the machine is in

question, these observations do not hold statistical significance.

VII. Conclusion

The development of the method was successful as far as it was tested. A standard curve with

strong correlation was created, demonstrating an ability to accurately get reproducible results.

Furthermore, the ability to identify an unlabeled standard via a batch reprocess proves that the curve

can be used to accurately calculate ionic concentrations in an unknown. Soil samples were run with

limited success due to interference from poorly filtered UPW, but the developed extraction method is

ready to be tested. Using the given operating protocols and research, accurate analysis of

environmental samples using Ion Chromatography should be quite easily reached with limited further

study, possibly leading to data that can be used to diagnose problems in the Kauffman prairie plots.

VIII. Acknowledgments

Dr. Gary Histand

Dr. Richard Zerger

Derrick Law

Carrie Shultz

Martin Olson

Matthew Carda

Charles Mayer


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IX. References

1. P.G. Risser, W.J. Parton, Ecosystem Analysis of the Tallgrass Prairie: Nitrogen Cycle, Ecology, 63,1342-1351 (1982).

2. P.M. Vitousek, R.W. Howarth, Nitrogen limitation on land and in the sea: how can it occur?Biogeochem. 13, 87115 (1991).

3. A. Eaton, M. Carter, A. Fitchett, J. Oppenheimer, M. Bollinger, J. Sepikas, Comparability of IonChromatography and Conventional Methods for Drinking Water Analysis in Proc. AWWA

Water Qual. Tech. Conf., 175-188, (1984).

4. USEPA Method 300.1, The Determination of Inorganic Anions in Water by Ion Chromatography,USEPA, Cincinnati, OH, (1997).

5. R. Michalski, I. Kurzyca, 'Determination of Nitrogen Species (Nitrate, Nitrite and Ammonia Ions)in Environmental Samples by Ion Chromatography', Polish Journal of Environmental Studies,

15, 5-18 (2006)

6. P.E. Jackson, Ion Chromatography in Environmental Analysis in Encyclopedia of AnalyticalChemistry, ed. R.A. Meyers, John Wiley & Sons Ltd, Chichester, (2000).

7. B. Lpez-Ruiz, Advances in the determination of inorganic anions by ion chromatography,Journal of chromatography, A, 881-1, 607 (2000)

8. P.G. Risser, W.J. Parton, Ecosystem Analysis of the Tallgrass Prairie: Nitrogen Cycle, EcologicalSociety of America, 63-5, 1342-1351 (1982).

9. A. Wild, Soils and the Environment, Cambridge University Press, New York (1993).10.H P.E. Jackson, Ion Chromatography in Environmental Analaysis in Encyclopedia of Analytical

Chemistry, ed. R.A. Meyers, John Wiley & Sons Ltd, Chichester, (2000).

11.Chemistry Dictionary, Definition of eluent, ChemiCool, viewed November 7, 2011, (2011).

12. J.S. Fritz 1999, Analytical Solid-Phase Extraction, Wiley-VCH, New York.13.Metrohm, IC Theory, Metrohm IC Tutorials, Herisau, Switzerland, CD-ROM, (2011).14.D. Skoog, F.J. Holler, S.R. Crouch, Principles of Instrumental Analysis, 6th edn., Brooks Cole

(2006).

15.Metrohm, Multipoint Calibration, Metrohm IC Tutorials, Herisau, Switzerland, CD-ROM,(2011).

16.Metrohm, IC Flow Path, Metrohm IC Tutorials, Herisau, Switzerland, CD-ROM, (2011).
http://www.chemicool.com/definition/eluent.htmlhttp://www.chemicool.com/definition/eluent.html


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X. Appendices:

Appendix A: Startup and Run the IC

Appendix B: Setting up a Sample Queue

Appendix C: Setting up Standard Concentrations

Appendix D: Peak Labeling

Appendix E: Batch Reprocessing

Appendix F: Preparation of Anion Standard

Appendix G: Preparation of Sulfuric acid standard

Appendix H: Replenishing Ultrapure H2O

Appendix I: Sample Chromatograms

Appendix J: Troubleshooting

Appendix KIon Chromatograph Flow Diagram (detailed)


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Appendix A: Startup and Run the IC

1. Double-click IC Net 2.3.exe on desktop2. Enter user-name and password (located at the bottom of the monitor) into the dialog box. Click

Log In or hit Return.

3. Right-Click on the graphic and select Z to make it disappear4. Open System: File > Open > System Select the Bethel folder, then either anions.smt for

the anion column, or cations.smt for the cation column. It is important to select the right one,

as it controls which parts of the machine operate. IMPORTANT. A box with cute little drawings

of the instruments parts will show up. This is your control panel.

Note: if the control panel window is closed, it is easy to bring it back by simply clicking

right button on the toolbar. It will be the farthest right of three buttons showing three

green boxes.

5. Start up the instrumenta. Turn the power switch on behind the instrument. This will turn on all components

EXCEPT the 830 IC interface. Check to make sure all component lights come on.

b. Turn on the power switch located at the back of the 830 IC Interface and check the lighton the front. If it is blinking rapidly, turn 830 off and then turn it back on. It should blink

twice slowly then stay on if the interface is talking to the computer.

6. Interface the software and instrument: in the control panel, click Control > Connect ToWorkplace. A dialog box in the lower left corner of the screen will show On-line [830 ICInterface] in gray. If it does not, check that the light on the 830 component isnt blinking.

7. Start hardware: in the control panel, click Control > Connect Hardware (Measure Baseline). Adialog box in the lower left corner of the screen will show the status of each component. Red

means a component is not connected, yellow means it is connecting, and gray means its

connected. Wait until everything is ready (gray) before proceeding to the next step.

8. Prepare peristaltic pumps: before starting the pumps, the clamps for each need to be attached.The two lines that need to be tight for measuring the baseline are the acid and water lines.

Thee pump and lines are located on the front right of the 833 IC Liquid Handling Unit. Make

sure that each line (yellow and black) have their colored tabs in the cutouts on the left-hand side

of the plastic clamp and are lined up. Then, make sure that the lines on the other side of the

pump are in between the two arms for each clamp. These steps are important to make sure

that the lines arent damaged during the tightening process. Carefully slip the arms on the right-

hand side down past the metal bar until the indention lines up and snaps into place. Next,


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Regier 25

tighten the clamps down using the circular gears on top of each one. Make them hand-tight but

DO NOT OVERTIGHTEN. It would be a good idea to have an instructor demonstrate proper

tightening technique before you do this. The end result should be that the lines each run in the

middle of their respective clamps (not pinched anywhere) and are tightened against the rollers

of the peristaltic pump.

Note: To make sure that the clamps are tightened and that pumps are drawing correctly,

watch for any bubbles moving through the lines. If there is a bubble that is sitting in the

line and not moving, no liquid is moving through. Additionally, check the waste lines to

make sure that they are dripping constantly.

9. Measure baseline: in the control panel, click Control > Connect Hardware (Measure Baseline)again. This time, a chromatogram window will pop up and the 833 pump will engage.

Additionally, you will hear a strange sucking sound from inside the machine. Thats okay. Its

supposed to happen. A status window will pop up displaying the conductivity, pump pressure

and column temperature. The column temperature will be red until it is close to 35 degrees. No

samples should be run until the column reaches prime operating temperature. Let the baseline

run for about half an hour remembering to step the injector every 10 minutes or so (see next

step)

10.Check acid and water lines with litmus paper. Acid should produce red on blue and water lineshould produce nothing on either.

11.Step injector (as needed): in the control panel, double-click on the 820 IC Separation Centericon. This will bring up a dialog box. Under the Manual tab, click STEP. You will here the

injector valve rotate inside the machine and this is supposed to be a fairly strong peak

associated with a STEP event. This step should only be done during the measuring of the

baseline. DO NOT do this any time you are running a sample as you will RUIN IT FOREVER.

12.While your baseline is running, make sure to take off all lids for any samples that need to be runand prepare, label and place your samples in the autosampler. You may need to move the

autosampler manually to get access to the rinse bottles or slots you need. To do this:

a. In the control panel, double-click the 838 Advanced Sample Processorb. In the dialog box the pops up, select the Autosampler tabc. In the drop-down box in the Move column, select the position that you wish to move tod. Click Start

13.Once your baseline has leveled out, around 16-20 mV, check your Method (see Setting up aMethod), load your Sample Queue (see Setting up a Sample Queue), tighten the peristaltic

pump on the auto-sampler (see Step 8 for instructions) and run your samples.

Note: make sure that this one is tight or it wont properly. Check the sample waste line

to make sure sample is running


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Regier 26

14.To shut down the machine:a. In control panel, click Control > Shut down Hardware. The pump should stop runningb. Click Control > Disconnect from Workplacec. Close all open windows in ICNet and the program, saving if promptedd. Turn off IC Detector unite. Turn off power stripf. Release all peristaltic clamps. This is IMPORTANT to keep the tubing in good shape

without indentions

Appendix B: Setting up a Sample Queue

1. Refer to Startup and Run the ICfor proper instrument setup.2. Once these steps are completed, go to File > Open> Sample Queue3. Navigate to the current folder for Sample Queues (currently C:\Program Files\Metrohm\IC Net

2.3\IC Net\Systems\Bethel)

4. Enter the name of your sample queue, leaving the .que5. In the dialog box that pops up, click edit to open the Queue Editor6. Under System for all samples, type the name of the system being used (currently

anions.smt)

7. Under Ident type a short name for each sample8. Under Vial enter the location of the sample vial on the autosampler rack9. All other categories EXCEPTLevel can be copied down for all samples10.For level, all samples will be 0 except for standards being used to build a standard curve. For 4

standards, the lowest concentration is 1, the highest is 4. If you are not sure if your standardsare correct in the method, check Setting up Standard Concentrations and follow the

instructions for adjusting the concentrations of each level

Appendix C: Setting up Standard Concentrations

1. Refer to Startup and Run the ICfor proper instrument setup.2. Once these steps are completed, go to File > Open> Chromatogram3. Choose a chromatogram that was created using the method whose standard concentrations you

want to change

4. With the chromatogram window active, go to Method > Calibration > Concentrations which willopen a Concentrations dialog box

5. For any levels that you wish to change, double-click each cell and change the concentration6. If all levels are to be changed, simply click a cell within a level and click the Delete button.

Then, click add and add a blank level column

7. If prompted to save, make sure to save every change. Add appropriate comments as prompted.


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Regier 27

Appendix D: Peak Labeling

1. Refer to Startup and Run the ICfor proper instrument setup.2. Once these steps are completed, go to File > Open> Chromatogram3. Choose the chromatogram that you wish to edit and double-click8. With the chromatogram window active, go to Method > Calibration > Components which will

open a table below the chromatogram.

4. All anions will be listed but may be assigned to the wrong peak, or no peak at all. Leave peakassignments that are correct.

5. Under the Peak column, double-click the anion row that you wish to change. Enter the correctpeak number and press Return.

6. Click the OK button and exit the chromatogram7. If prompted to save, make sure to save every change. Add appropriate comments as prompted.

Appendix E: Batch Reprocessing

1. Refer to Startup and Run the ICfor proper instrument setup.2. Refer to Setting up Standard Concentrations and Peak Labeling to make sure that all standard

chromatograms to be processed have been properly labeled and are using the same standard

concentration levels for calibration (sample chromatograms will be automatically labeled during

the batch reprocessing)

3. Once these steps are completed, go to File > Open> Chromatogram4. Hold and click on each chromatogram to be included in the batch reprocess, including all

standards and samples and click To Batch on the right-hand side of the dialog box

5. Give the batch a name and press OK6. Click the Edit sample table button to view the chromatograms selected. Make sure that all

samples to be reprocessed are included and that the levels are correct (1-4 for standards, 0 for

all samples)

7. Select Reintegrate (Recalibrate is automatically selected as well)8. Press the Reprocess button9. If prompted to save, make sure to save every change. Add appropriate comments as prompted.10.To double-check, select a chromatogram and check the calibration table and that components

are properly labeled


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Regier 28

Appendix F: Preparation of Anion Standard

Empty eluent bottle of any remaining eluent (but do not rinse)

Obtain 1 L clean volumetric flask labeled IC Eluent only (located near Ultrapure machine)

Remove parafilm, dump out contents

Rinse 3 times with UltraPure (UP), shaking vigorously when dumpingFill the volumetric half full of UP.

Make sure that the eluent snip (located on the south shelf in the instrument room) has all liquid

at bottom. If not, try inverting or flicking the tube, as complete volumetric transfer is key.

Cut off the top of the tube and dump contents into the volumetric flask

Rinse the tube three times and dump this into the flask as well

Fill Volumetric to line and cover with parafilm

Invert 10 times

Dump into open eluent bottle, and cap the bottle

Rinse the flask out 3 times, swirling it, with UP

Fill to top with UP and cap with parafilm

Caution:

Do NOT touch any part of the rim or inside of the flask with anything other than solution and

clean parafilm. Contamination from your hands, gloves or parafilm can effect the eluent!

This is a very dilute, very specific solution. Take great care.

Dont rinse the eluent bottle. The extra water will mess up the concentration

Dont let the tube inside touch anything, make sure it is resting on Kimwipes, not the machine

Make sure that the UP machine reads between 17.8-18.2 M-cm. If it goes below this

conductivity, give it some time to get back into this zone.ALWAYS use Ultrapure. NEVER use distilled or tap water, as they will contaminate the lines.

Appendix G: Preparation of Sulfuric acid standard

Obtain 1L clean volumetric flask labeled IC Sulfuric Acid (located near Ultrapure machine)

Remove parafilm, dump out contents

Rinse 3 times with UltraPure (UP), shaking vigorously when dumping

Measure out 5.55 mL (5.5 is good enough) of 18M Sulfuric Acid into graduated cylinder

Add sulfuric acid to empty flask

SLOWLY add a small amount of water and allow the flask to sit until it cools down

Repeat the above step with increasing amounts of water, making sure the flask stays cool

Fill volumetric to line and cover with parafilm

Invert 10 times

Dump into open acid bottle and cap bottle


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Regier 29

Rinse the flask out 3 times, swirling it, with UP

Fill to top with UP and cap with parafilm

Caution:


clean parafilm.

Dont let the tube inside touch anything. Make sure it is resting on Kimwipes, not the machine


conductivity, give it some time to get back into this zone.

ALWAYS use Ultrapure. NEVER use distilled or tap water, as they will contaminate the lines.

Use caution, Sulfuric acid is nasty stuff

Appendix H: Replenishing Ultrapure H2O

Fill bottle labeled Ultrapure H2O at Millipore machine

Caution:


clean parafilm.

Dont let the tube inside touch anything. Make sure it is resting on Kimwipes, not the machine


conductivity, give it some time to get back into this zone.

ALWAYS use Ultrapure. NEVER use distilled or tap water, as they will contaminate the lines.


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Regier 30

Appendix I: Sample Chromatograms

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 1 4 15 1 6 17 1 8 19 mi

0

2

4

6

8

10

12

14

16

mV

ch1

1

fluoride

0.

chloride

1.

nitrite

0.9

75

5

brom

ide

1.

nitrate

2.0

sulphate

2.0

03

phosphat

e

2

1:5 Standard Dilution

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 1 4 15 16 17 18 19 min

-1

0

1

2

3

4

5

6

7

8

mV

ch1

1

fluoride

0.1

chloride

0.

211

4

nitrite

0.5

44

6

bromide

1.

nitrat

e

0.9

sulphate

0.9

94

phosph

ate

1:10 Standard Dilution


31/37

Regier 31

Plot 0: South 2

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 1 4 15 1 6 17 1 8 19 min

-1

0

1

2

3

4

5

mV

ch1

1 2flu

oride

0.0

23

chlor

ide

0.

5 ni

trite

0.0

16

78 br

om

ide

0.0

23

nitrate

0.1

07 p

hosphate

0.4

32

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 1 4 15 1 6 17 1 8 19 min

0

5

10

15

20

25

mV

ch1

1

fluoride

0.0

84

3

chl

oride

1.

nitrite

0.0

14

6 7 bro

m

ide

0.0

09

nitrate

0.2

04

10p

hosphate

0.2

84

Plot 0: South 1


32/37

Regier 32

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 14 1 5 1 6 17 18 19 min

-1

0

1

2

3

4

mV

ch1

1 2fluoride

0.0

12

chloride

0.0

87

5 nitrite

0.0

08

7

8 brom

ide

0.0

17

10 nitrate

0.0

08

phosphate

0

13

Plot 0: North 2

Plot 0: North 1

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 14 15 16 17 1 8 19 min

-1

0

1

2

3

4

5

mV

ch1

1 2fl

uoride

0.0

15

4

chlo

ride

0.

6 ni

trite

0.0

09

89 br

om

ide

0.0

19

n

itrate

0.

049

pho

sphate

13


33/37

Regier 33

Plot 1: West

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 1 4 15 1 6 17 1 8 19 min

0

2

4

6

8

10

12

14

16

mV

ch1

fluoride

0.0

89

chlor

ide

1.

nitrit

e

0.0

13

4 brom

ide

0.0

26

nitrate

0.0

98

sulph

ate

0.0

92

ph

osphate

0.2

72

Plot 1: East

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 14 1 5 1 6 17 1 8 19 min

-1

0

1

2

3

4

5

6

7

8

mV

ch1

1 2 fluo

ride

0.0

20

chloride

0.

nitrite

0.0

12

6 brom

ide

0.0

25

nitrate

0.2

09

sulphate

0.0

88

phosphate

0.5

55

11


34/37

Regier 34

1:10 Sample

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 1 4 15 1 6 17 1 8 19 min

-1

0

1

2

3

4

5

6

7

8

mV

ch1

1

fluoride

0.1

3

chloride

0.2

03

5

nitrite

0.5

43

7

brom

id

e

1.

nitrate

0.9

sulphate

0.9

75

phospha

te

0

Plot 2: East

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 14 1 5 1 6 17 1 8 19 min

0

2

4

6

8

10

12

mV

ch1

fluoride

0.0

85

chloride

0.

34

brom

ide

0.0

07

nit

rate

0.0

90

7 sulp

hate

0.0

98

p

hosphate

0.2

00


35/37

Regier 35

Plot 2: West 2

0 1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 1 4 15 1 6 17 1 8 19 min

0

5

10

15

20

mV

ch1

f

luoride

0.0

84

c

hloride

1.

3 4 brom

ide

0.0

13

6nitrate

0.0

91

sulp

hate

0.0

44

phosphate

0.4

15

10

Plot 2: West 1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 min

0

5

10

15

20

mV

ch1

1

fl

uoride

0.0

80

chlorid

e

1.

4 5 brom

ide

0.0

12

nitra

te

0.0

15

sulph

ate

0.0

99

ph

osphate

0.3

40

10


36/37

Regier 36

Appendix J - Troubleshooting

Problem Diagnosis Action Result

Peaks slipping off the

end of the

Chromatogram

Bad Eluent Mixed New Eluent Shorter Retention

Rates

Peaks unclear/blend in

with baseline

Need more

sensitivity Full Scale

for 819 Detector

Change Control Panel >

819 IC Detector > Method

Parameter > Full Scale

Stronger peaks

Baseline decreases at

beginning of

chromatogram

Acid and Water lines

are switched

Test with litmus. Blue

litmus should turn red for

acid

No acid interfering

with the beginning of a

chromatogram

Weak peaks on lower

dilutions

Need stronger

standards

Mixed new standards with

higher concentration

Stronger, clearer peaks

but more

contamination from

residual ions left in

column


37/37

Appendix K Ion Chromatograph Flow Diagram (detailedi)

iImage from IC Flow Path

[16]

peter regier seminar - ic

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