Supplementary Material for Online Publication

Supplementary Material for Online Publication

Laboratory documentation

Exercise in Quality Assurance

Lab summary: Attached

Written directions: Attached is a copy of the preliminary standard operation procedure

(SOP) available in the laboratory to the students at third year bachelor level. The

experiment is performed in groups of two-three students. A report with equal

contributions from all students is produced containing a final revised version of the SOP

and the results are presented orally at a colloquium of the final lesson. All solutions and a

sample of unknown concentration are provided by the laboratory.

Instructor’s notes

Scope. This experiment allows the students to perform the basics of quality assurance and

they will understand the importance of many repetitions to instrumental analysis. The

students may determine iron in other types of samples such as blood samples of animals

where the method can be applied for the determination of the haematocrite value. The

experiment is suitable for instrumental analysis, quality assurance, spectrophotometry,

clinical chemistry, forensic analysis and environmental analysis.

1
 Supplementary Material for Online Publication

Qualifications. The students need to know basic statistics including methods for testing,

and they need basic training in the handling of solutions, safety and waste disposal. The

students are expected to be familiar with spreadsheet calculations, e.g. MSExcel for data

processing and statistical testing. They also need basic training in spectrophotometry,

software and preparation of solutions.

Techniques. Since the students are supposed to spend much time of data treatment and

data interpretations by the aid of spreadsheets, most of the solutions were prepared by the

laboratory assistant before start of experiment. The equipment with the FIA manifold was

also constructed in advance of the exercise, so the work could be focused on an excellent

performance of the experiments and on processing of data.

Concepts taught. In this experiment, it is demonstrated that quality assurance is an

important tool for decision makingin real experiments, and that it is possible to achieve

correspondence between predicted and measured uncertainties. The students learn to

perform experiments carefully, as to ensure a high degree of repeatability. Construction

of calibration lines, determination of unknowns, writing of a standard operation

procedure (SOP) and the construction of an uncertainty budget are integrated in the

exercise.

Chemicals and instrumentation. The experiments are supposed to be uncomplicated for

the students but the preparation of solutions and construction of the FIA manifold are

2
 Supplementary Material for Online Publication

more time consuming. For the construction of the FIA manifold, the following items are

required:

• Spectrophotometer (460 nm)

• Injection valve (Tubings ID 0.5 mm)

• Cuvettes (1 cm) for batch measurements

• Flow-through cell (1 cm, ID 1 mm)

• MicrolineTM tubings

• Peristaltic pump (Ismatec Reglo, 20 rpm)

• Tube connectors

• Beaker

• Bottle for waste disposals

Time requirements. An introduction of 45 min. duration to the subject of QA is given by

the teacher. Four hours are allocated to the measurements and to collection of data. The

data processing and testing of data, writing the SOP, constructing the uncertainty budget,

writing the report and preparation of the colloquium requires 8 hours. The colloquium

requires a presentation of 15 min. from each group and 30 min. for evaluation of the

results.

Expected results. Some of the groups produce many measurements and many repetitions,

and thus found a good basis for statistical interpretations and for testing. Other groups

may not obtain a sufficient number of repetitions, which may prevent them from making

a reliable estimate of the concentration of the unknown. It is expected that most of the

3
 Supplementary Material for Online Publication

groups obtain results that are incompatible, as judged by the analysis of the t-test. They

will find that their results of batch and FIA deviate significantly, and they will also find

that their results deviate significantly from those of the other groups, which may be

deduced during the colloquium. Finally, by merging all results, it is demonstrated that the

correct value of the unknown is determined at the expense of a higher RSD however,

which is the main pedagogical issue of the exercise.

Potential student problems. A number of the students might be inexperienced or have lost

their routine of laboratory work. Thus, a careful instruction is required before analysis, as

to avoid gross errors. In the batch measurements, they must make sure that the cuvette is

free from air bubbles and that the cuvette is filled with liquid. The same is true for the

FIA measurements but air bubbles outside the flow cell may also pose a problem because

they may suddenly be released and enter the flow cell together with the injected sample

during measurement. However, irregularities of this kind may be identified by spikes on

the FIA peak. The pump rate must be constant during the measurements and may be

monitored by a measuring glass and, at the exit to the waste bottle, measuring the volume

added during, e.g. one minute. Make sure never to empty the beaker with reagent (Figure

A).

CAS Registry Numbers: KSCN [333-20-0], NaOH [1310-73-2], HNO3 [7697-37-2]

Safety: MSDS for KSCN, NaOH and HNO3

4
 Supplementary Material for Online Publication

In the laboratory: (Handout)

Exercise in Quality Assurance

Background

Iron is present everywhere in our surroundings, and the iron(II)/iron(III) couple are

important to living organisms in general. The element is life supporting, and wherever

iron is found in the oceans, you also find life. Iron may thus be determined in food and in

the environment:

• Vitamins

• Wine

• Meat

• Sea water and ocean water

• Drinking water

• Blood and serum

• Soil

The daily intake of humans is approx. 7 mg from food, and the body contains approx.

35 mg/kg body weight deposited in various proteins.

Determination of iron is straightforward by modern instrumental methods of analysis.

In clinical analysis, a very high number of samples are analyzed for iron every day, and

the methods are also used to determine the blood percentage or the heamatocrite value

5
 Supplementary Material for Online Publication

for, e.g. monitoring of doping in sports. However, in order to perform and report

reliability of results, quality assurance (QA) is imperative to the methodology. Since the

number of analytical results is growing, there is also a demand for QA in the work

performed at professional laboratories, which is also monitored by the Food and Drug

Administration (FDA). Accordingly, the students of analytical chemistry must be

introduced to some of the basic tools of QA.

Introduction

Iron is most frequently determined by spectrophotometric methods, atomic

absorption spectrometry or by mass spectrometry. In the present method, iron is

determined spectrophotometrically by formation of a red-colored complex of iron that

absorb electromagnetic radiation at a wavelength of 460 nm. The red complex is formed

by complexation with thiocyante in alkaline solution:

3
6 6 (1)

The number of ligands in eq. 1 may vary from zero to six, as dependant on the ratio of

concentrations but the complex remains colored red. The iron content may be determined

by:

• Batch

• Flow Injection Analysis

6
 Supplementary Material for Online Publication

Both methods are based on the same principle of detection where the complex of iron in

eq. 1 is measured at 460 nm in batch experiments and monitored at the same wavelength

in the FIA measurements (FIA).

The aim of the work is to:

1. Determine the concentration of an unknown (Supplied by the laboratory assistant)

2. Write and SOP for each of the two experiments, batch and FIA

3. Work out an uncertainty budget for the two methods

4. Decide by statistical tests, which one of the two methods exhibits the better

performance in terms of sensitivity, LOD and uncertainty.

5. Write a report,make a presentation and discuss the results at a colloquium

The concentration of the unknown was revealed to the students at the end of the

colloquium that was organised after writing the report and after the final presentation.

The SOP is the basis for the construction of the uncertainty budget. The SOP is written

when students have received the required instructions from the supervisor about how to

operate the instrument. Details of the methodology may be found in D.C. Harris,

Quantitative Chemical Analysis, 2007, W.H. Freeman and Company. The uncertainty

budget may be constructed by the instructions given in the ‘Guide to the Expression of

Uncertainty in Measurement’, 1st ed., International Organization for Standardization,

Genéva, Switzerland (1995) at http://www.citac.cc/

7
 Supplementary Material for Online Publication

Solutions

All standards and a sample denoted as the ‘unknown’ were prepared by dilution

of a certified standard solution of iron(III)nitrate in nitric acid (999 ± 2 mg/L, Merck

1.19781.0500). The concentration of the unknown was chosen at approx. 40 mg/L and

the precise value was determined by flame atomic absorption spectrometry (FAAS). The

laboratory value is not a true value but should be regarded as another value that adds to

the pool of results that was determined by the students. Standards for the batch

experiments were prepared in 0.5 mM thiocyanate in 0.1 M sodium hydroxide at

concentrations of 0, 5, 10, 20, 40, 50, 75 and 100 mg/L. The sample of the unknown was

prepared from this stock solution as well, as to provide a signal that corresponded to an

absorbance approx. at the middle absorbance value of the calibration line of batch

experiments. The reagent of FIA determinations contained 1 mM thiocyanate in 0.2 M

sodium hydroxide, and it was prepared by dissolving solid KSCN (99% p.a., Merck prod.

no. 848 A299625) and adding sodium hydroxide (Merck, Titrisol 109956) in doubly

distilled water.

Safety

The exercise must be performed in a chemical laboratory equipped with proper

installations for suction and handling of chemicals including procedures for waste

disposal. The operator must wear a laboratory coat, eyewear and safety gloves.

8
 Supplementary Material for Online Publication

• Solution of thiocyante (KSCN) should be handled with care. Poisonous gases may

evolve from the solution when acidified. The preparation of solutions must be

performed in a fume hood.

• Sodium hydroxide is a strongly-etching chemical, and skin contact must be

avoided. In case of accident, the area of incident must be flushed with tap water

for at least ten minutes.

• Nitric acid is a strongly-etching chemical, and skin contact must be avoided. In

case of accident, flush with tap water.

Procedure

Batch

The freshly prepared standards at the table adjacent to the HP8452A

spectrophotometer are poured into the cuvette that is positioned in the cell holder.

Remove any air bubbles from the cuvette, tighten the cuvette in the holder and measure

the absorbance at 460 nm, and follow the instruction of the supervisor. Remove the

cuvette, and pour the solution into the bottle for waste disposals. Flush carefully the

cuvette with water, flush with new solution, add new solution, and make next

measurement. Continue until all solutions have been measured at least twice. Note any

detail of the procedure in the lab journal, which is used for writing the SOP and for

construction of the uncertainty budget.

9
 Supplementary Material for Online Publication

FIA

The FIA manifold is constructed according to the diagram of fig. A.

Figure A. Schematic representation of the manifold of the FIA measurements. The

reagent was propelled at a flow rate of 3 ml/min and the length of the loop was 20 cm.

Follow the instructions of the supervisor, and make notes on every detail of the

instrument settings. Write an SOP and construct the uncertainty budget including

uncertainty on pump rate. Before start of analysis, make sure that the system operates

well and that it is in perfect condition without any air bubbles in the tubings and cell. Add

reagent to a 250 ml beaker, and start the pump. Inject a sample and note the peak height

of the signal. Repeat injection until the peak height does not change significantly. When

the system provides reliable measurements, perform the analysis of blanks, standards and

samples.

10
 Supplementary Material for Online Publication

Analysis

Batch

The absorbances of the blanks, standards and unknown are recorded and copied

into an MSExcel spreadsheet. A calibration line is plotted (not shown), average values

and standard deviations are calculated followed by testing the results for potential

deviations.

FIA

The characteristic peaks of FIA measurements are shown in figure B. The peak

height is used to construct a calibration line. Plot the results together with uncertainties in

a spreadsheet.

11
 Supplementary Material for Online Publication

Figure B. Characteristic peaks of the FIA measurements after injection of iron(III)

at four different concentrations. Overlay plot.

Statistical testing

The uncertainties may be tested for differences by F-testing and the average

values may be tested for differences using Student’s-test.

Colloquium

Make a t-test of your own results and compare to the results presented during the

presentations of the other groups. Although it may cause distraction from the

presentations, it provides an immediate response to the work presented, and thus makes

the session more interesting. Discuss the results, and obtain from the teacher the

laboratory value of the unknown. Analyze by statistical means if your result differed from

the laboratory value.

12