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AUTOMATED DETERMINATION OF CARBONYL COMPOUNDS IN ORGANIC SOLVENTS by CONNIE DEE DUNN, B.S., M.S. A THESIS IN CHEMISTRY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE

Approved

Accepted

August, 1992 ACKNOWLEDGEMENTS

Appreciation and credit is due to my advisor, Dr. Purnendu K.

Dasgupta, for his patience, stalwart support, and inspiring guidance in the course of this research and throughout my graduate years at

Texas Tech University. Appreciation is also extended to Dr. Dennis

Shelly and Dr. Heung-Keun Chung for their helpful criticism and encouragement.

I am deeply indebted to the United States Air Force for this opportunity to attend Graduate School at Texas Tech University.

Finally, I am very thankful to my parents and my husband for their moral support throughout my career.

ii TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

LIST OF TABLES vii

LIST OF FIGURES ix

CHAPTER

I. INTRODUCTION 1

1.1 Polymers 1

1.2 3

1. 3 Carbonyls 6

1.4 Significance of Study 9

I I. QUANTITATIVE AND QUALITATIVE MEASUREMENT OF CARBONYL COMPOUNDS 10

2.1 Literature Review 10

2.1.1 Elements of a Flow Injection System 11

2.2 Analytical Methods for Carbonyl Compounds 13

2.2.1 Qualitative Tests 13

2.2.1.1 Melting Points of Derivatives 13

2.2.1.2 Spectroscopic Techniques 13

2.2.1.3 Precipitation Techniques 13

2.2.2 Quantitative Techniques 14

2.2.2.1 Precipitation Techniques 14

2.2.2.2 Techniques with 2,4-dinitrophenylhydrazine 16

2.2.2.3 Spectrophotometric Techniques Not Involving 2,4-DNPH ...... 18

iii 2.2.2.4 Gas Chromatography and Titration Techniques 22

2.2.2.5 Thin-Layer Chromatographic Techniques . . . . . • 24

2.2.2.6 Voltammetric Techniques 25

2.2.2.7 High Performance Liquid Chromatography (HPLC) 25

2.2.2.8 Enzymatic System 26

III. EXPERIMENTAL SECTION 32

3.1 Initial Considerations 32

3.2 Reagents 32

3.3 Preliminary Experiments 33

3.4 Apparatus 33

3.4.1 Pump Conduits 33

3.4.2 Reaction Coils 33

3.4.3 Detection Systems 34

3.4.4 Detector Cells 35

3.4.4.1 Cell 1 35

3.4.4.2 Cell 2 35

3.4.4.3 Cell 3 36

3.4.5 Water Bath 36

3.5 System 1 37

3.5.1 Experiments with System 1 37

3.6 System 2: Introductory Automated Systems 38

3.7 System 3 38

3.8 System 4 39

iv 3.9 System 5 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 39

3o9o1 Experiments with System 5 . 0 0 0 . 0 0 . . 0 40

3o10 System 6 0 . 0 0 . 0 0 . 0 0 0 0 0 0 . 0 0 . 0 0 0 0 40

3o10o1 Experiments with System 6 0 0 0 0 0 0 . 0 . 0 41

3o11 System 7 ...... 0 . 0 0 0 0 0 . . 0 . . . . . 0 41

3 o11. 1 Experiments with System 7 0 0 . 0 . 0 0 0 0 . 42

3.12 System 8 0 . . . . 0 . . 0 0 0 0 . . . 0 . 0 0 43

IV. RESULTS AND DISCUSSION 0 . 0 . . . . . 0 . 0 . . . . 64

4o1 Preliminary Results 0 . . . 0 0 . 0 . 0 . 0 . . . . 0 64

4 .1.1 Determination of Method Viability 64

4 ol.2 Confirmation of 2,4-DNPH 0 0 0 . . . 0 0 . 64

4ol.3 Confirmation of Absorbance Maximum 64

4ol. 4 Determination of Optimal Reagent Solvent 65

4o2 Results with System 2: Introductory Automated

Systems 0 0 . 0 0 . . . 0 0 0 . 0 0 . . . . 0 . 0 67

4.3 Results with System 3 0 0 . . . 0 . . 0 0 . . 67

4o4 Results with System 4 0 . . . . 0 . . 0 . . . . 0 . 0 67

4o5 Results with System 5 . . . . 0 0 . 0 0 . 0 . . . 68

4o6 Results with System 6: Manual Optimization Studies 68

4o6.1 Determination of Minimum Standard Time 68

4o6o2 Determination of Optimum Concentrations of Acid and Base in Reagents 68

4o6o3 Determination of Minimum Reaction Times 69

4o6o4 Determination of Optimum Temperatures 0 0 0 0 70

4.6o5 Determination of Minimum KOH Volumes 70

4o6o6 Determination of Optimum Hydroxide source 70

v 4.7 Results with System 7 71

4.8 Results with System 8 72

V. CONCLUSION 141

REFERENCES 142

vi LIST OF TABLES

2.1 Amax for determinations with 2-hydrazinobenzothiazole and potassium ferricyanide . . . • ...... 28

2.2 Amax for determinations with 3-methylbenzothiazolin-2-one hydrazone and ferric chloride . . . . • . . • . • . . 29

2.3 Amax for determinations with dimethyl-p-phenylenediamine oxalate ...... 30

2.4 A max for determinations with diethyl acetonedicarboxylate and ammonia ...... 31

4.1 Comparison of solvents as blanks ...... 74

4.2 System 2, data set 1 ...... 75

4.3 System 3, data set 1 (5 min./5 min. incubations) . . . . . 76

4.4 System 3, data set 2 (5 min./5 min. incubations) . . . . . 77

4.5 System 3, data set 3 ...... 78

4.6 System 3, data set 4 ...... 79

4.7 System 5, data set 2 (Kratos 0.1 AUFS) ...... 80

4.8 System 5, data set 1 (3 min./3 min. incubation periods) 81

4.9 Minimum incubation time study for first incubation reaction ...... 82

4.10 Study of minimum volume of KOH ...... 83

4.11 Results of experiments with different sources and concentrations of hydroxide for reaction 84

4.12 System 7, data set 1 (Kratos 0.5 AUFS) ...... 85

4.13 System 7, data set 2 (Kratos 0.5 AUFS, room temperature 25°C) ...... 86

4.14 System 7, data set 3 (Kratos 0.5 AUFS, 55°C) ...... 87

4.15 System 7, data set 4 (Kratos 0.5 AUFS, 60°C) ...... 88

4.16 System 7, data set 5 (Kratos 0.5 AUFS, 55°C) ...... 89

vii 4.17 System 7, data set 6 (Kratos 0.05 AUFS) . . 0 0 . 0 0 0 0 90

4.18 System 7, data set 7 (Kratos detector 0.02 AUFS) 0 0 0 0 0 91

4.19 System 7, data set 9 (Kratos detector 0.02 AUFS) 92

4o20 System 7, data set 11 93

4.21 System 7, data set 16 . 0 0 0 94

4.22 System 7, data set 17 95

4.23 System 7, data set 18 0 0 0 0 96

4.24 System 7, data set 19 97

4.25 System 8 response 0 0 0 0 98

4o26 System 8, data set 5 . . . 0 . . . 0 . 0 0 . 99

viii LIST OF FIGURES

3.1 Mini LED detector 46

3.2 First custom cell for Kratos 757 detector 47

3.3 Second custom cell for Kratos 757 detector 48

3.4 Cell for mini LED detector 49

3.5 Introductory system A to test component durability and function • . . • . • ...... 50

3.6 Introductory system B to test FIA applicability to ASTM method ...... • . . . . 51

3.7 System 3 52

3.8 System 4 53

3.9 System 5 54

3.10 Wiring diagram for VIC! digital valve sequence programmer in system 7 55

3.11 System 7 ...... 56

3. 12 System 8 . . • ...... 57

3.13 Wiring diagram for VIC! digital valve 58

3.14 System 8 photos ...... 59

4.1 Determination of maximum absorbance wavelengths 100

4.2 Acetonitrile experiments ...... 102

4.3 Processed data from system 2 ...... 110

4.4 Typical chart output from system 3, demonstrating clear reproducible peaks ...... 111

4.5 Data set 1 from system 3 ...... 112

4.6 Data set 2 from system 3 ...... 113

4.7 Data set 3 from system 3 ...... 114

4.8 Data set 1 from system 5 ...... 115

ix 4.9 Data set 2 from system 5 ...... 116

4.10 Comparison of blanks . . . . . 117

4.11 Change order of introducing reagents ...... 118

4.12 Observations of second incubation period . . . . 119

4.13 Comparison of peaks for 25 ppm C=O (MIBK source) using A (ASTM-recommended KOH) versus B (concentrated KOH) 122

4.14 A ghost peak ...... 123

4.15 Chart output after ghost peak removal . . . . 124

4.16 Comparison of typical chart output of Kratos 757 detector versus the mini LED detector . . . . 125

4.17 Baseline shift in system 7 126

4.18 Data set 1 from system 7 . . . . . 127

4.19 Data set 2 from system 7 128

4.20 Data set 3 from system 7 . . . . 129

4.21 Data set 4 from system 7 . . . . 130

4.22 Data set 5 from system 7 ...... 131

4.23 Data set 6 from system 7 ...... 132

4.24 Data set 8 from system 7 ...... 133

4.25 Data set 9 from system 7 . . . . 134

4.26 Data set 11 from system 7 ...... 135

4.27 Data set 13 from system 7 . . . . . 136

4.28 Data set 18 from system 7 ...... 137

4.29 Data set 19 from system 7 . . . . 138

4.30 Stabilized baseline in system 8 . . . . 139

4.31 Typical chart output of system 8 . . . . . 140

X CHAPTER I

INTRODUCTION

1.1 Polymers

A polymer is composed of macromolecules. The molecules have similar molecular weight and structure. Together, as a polymer, they demonstrate one average characteristic property (Dean, 1987).

Polymers are in every facet of life, from the genetically encoded deoxyribonucleic acid (DNA), which determines who and what we will be, to almost everything we touch. Polymers, in the form of plastics, are used in "every major industry" (Leslie, 1968, p. 33). Plastics have been made into materials to imitate gold, silver, or polished marble

in decorative pieces. Commonplace fabrics, such as Nylon and Orlon, are examples of woven plastic fibers. Special plastics are utilized

for their strength, flexibility, and unique properties of resistance to chemicals, heat, or nuclear radiation. Industrial uses of plastic can include smooth-running gears in machinery that need little or no oiling, lubricants, anti-evaporatives, adhesives, and nuclear reactor shields. The building industry frequently utilizes plastic as insulation, home building material, and plumbing pipe. Plastics are useful in countless ways in medicine because they are not affected by the body's chemicals and are not considered harmful to the body.

Artificial limbs, teeth, and joints can be fashioned from plastics with exact detail and put to use with little difficulty. Plastic screws, rivets, and joints are used in orthopedic procedures (DuBois,

1970).

1 2

In more recent times, plastics are being utilized in hospitals as disposable items to prevent transmission of disease. It is less expensive to simply replace many items than it is to sterilize them.

The cost of hiring a large enough workforce to do this sterilizing is high, and the cost of insuring those personnel against job hazards is even more prohibitive (Lang, 1991a, 199lb). Plastics are used in coatings, inks, paints, seed coatings, and beauty aids, as well as

such complicated processes as protein purification or isolation of different proteins from aqueous mixed protein samples. Because the plastic materials we use today are cheaper, more plentiful, and

frequently better suited to their function than their natural material

counterparts, modern man has become dependent on these plastics. This

is not surprising, as polymer plastics were devised in response to a

shortage of an expensive, natural product--ivory. In 1868, John

Wesley Hyatt devised the first plastic made in the United States. He was a printer and inventor who had heard a New York billiard ball company had offered a prize of $10,000 to anyone who could invent a

substitute for ivory. The billiard ball material he devised was cellulose nitrate, or Celluloid. Celluloid was soon popular for making other things, such as photographic film, false teeth, handles, and buttons. It was the only popular man-made plastic until 1909 when

Leo Baekeland discovered phenol-formaldehyde, the first thermosetting plastic, which sets or hardens after heating. The material was called

Bakelite. It was popular and useful in making telephones and insulators for wireless sets. The steady growth in the plastics 3 industry boomed during World War II when metals and other materials were in short supply (Leslie, 1968).

In 1968, the United States was the world's largest producer of plastics when 10 billion pounds, or $6 billion worth, were produced

(Leslie, 1968). In 1991, the U.S. production of plastics was estimated at 82 billion pounds. The production of major thermoplastics was 33 billion pounds for the first three quarters of

1991. Of these, 4.73 billion pounds of exports alone were worth over

$11 billion, according to a report by the Society of the Plastics

Industry's Committee on Resin Statistics (Storck, 1991).

There are 25 major families of plastic materials. Seven of these depend explicitly on or formaldehyde for manufacture.

Many of the others depend on other alcohols, either directly or indirectly (Dean, 1987).

1.2 Alcohols

There are many alcohols commonly produced by industry. An is defined as R-OH, any compound with a carbinol or C-0-H group where the carbon has three other attachments, filled by carbon or hydrogen atoms (Strong, 1970).

Alcohol is further defined as a broad class of hydroxyl- containing organic compounds occurring naturally in plants and made synthetically from petroleum derivatives, such as ethylene. Sax and

Lewis (1987) summarized alcohols into four large groups:

I. Monohydric (1 OH group) 1. Aliphatic a. paraffinic () b. olefinic () 4

2. Alicyclic () 3. Aromatic (phenol, ) 4. Heterocyclic () 5. Polycyclic (sterols) II. Dihydric (2 OH groups): glycols and derivatives (diols) III. Trihydric (3 OH groups): and derivatives IV. Polyhydric (polyols) (3 or more OH groups). (p. 31)

The carbon chain may be as short as one carbon, or it may be, in theory, infinitely long. Other schemes for classifying alcohols divide the group into three categories: primary, secondary, and tertiary. Primary alcohols have no less than two hydrogens attached to the carbon atom holding the hydroxyl group. Secondary alcohols have only one hydrogen on that carbon atom, and tertiary alcohols have none.

The two most common alcohols are methyl and ethyl alcohol

(methanol and ethanol), also known as wood alcohol and drinking alcohol, respectively. Methanol was first described in 1661 by Robert

Boyle as it occurred in the destructive distillation of wood. Until the 20th Century, this was the only way it could be obtained; hence, the name "wood alcohol." A more modern approach to methanol manufacture is the following:

The reaction is conducted under 270-350 atmospheres, 300-375°C, and a catalyst made of oxides of zinc, chromium, manganese, or aluminum.

Similarly, carbon dioxide and hydrogen may be used to produce methanol

(and water) (Snell & Hilton, 1967): 5

Methanol may be sold for any number of purposes. One of the most common purposes is to make it into formaldehyde. The formaldehyde is used to make phenol-formaldehyde polymers. Methanol also is used in methyl chloride production and as a solvent in dyes, plastics, fats, , soaps, resins, gums, paints, cosmetics, adhesives, and lubricants. Methanol may be used as an anti­ freeze/anti-gum additive in fuel systems and it may be added to certain fuel systems because of its hot, smokeless burning properties

(Snell & Hilton, 1967).

Ethanol is known as drinking alcohol, due to its long history of being the intoxicating component of fermented beverages. After 1947, over 80% of the ethanol produced in this country was via synthetic means. It is used as a solvent or extractant in the production of cleansers, plastic nitrocellulose, smokeless powder, disinfectants, antiseptics, pharmaceuticals, dyes, lacquers, stains, and varnishes.

Ethanol is also used as raw material for ethyl esters, chloroform, and ethyl ether manufacture. Synthetic ethanol manufactured for these uses is produced several ways, the simplest of which involves direct hydration of ethylene over a phosphoric acid catalyst. In using alcohols, the purity is important according to the intended use (Snell

& Hilton, 1967).

Some alcohols, if not stored carefully in a tightly sealed bottle, can become impure from oxidation. Consequently, a converts into an aldehyde and, finally, a carboxylic acid.

The most familiar occurrence is when table wine acquires a vinegar smell and taste. The ethanol in the wine has oxidized to acetic acid. 6

Similarly, secondary alcohols may convert this way into ketones and then to carboxylic acids. This deterioration represents impurity of the alcohol and a decrease in value for sale or industrial use.

Often, the presence of the carbonyl compound impurity is a consequence of the alcohol manufacturing process. The carbonyl content is a critical parameter in some of the end uses of alcohols and must meet specifications. The carbonyl content, therefore, must be determined by a general carbonyl detection scheme.

1.3 Carbonyls

A carbonyl compound is one having a carbonyl group. A carbonyl is the divalent group >C=O, which occurs in a wide range of chemical compounds. The group consists of a carbon double bonded to an oxygen.

This bond has one pair of exposed pi electrons. The oxygen has two pairs of unshared electrons. The carbonyl group is polar and may be attacked by nucleophiles or electrophiles. The carbonyl functionality is seen most frequently in three major classes of organic compounds: aldehydes, ketones, and carboxylic acids (Fessenden & Fessenden,

1986). Carboxylic acid derivatives, such as esters, acid halides, and amides, are also considered carbonyl compounds (Snell & Ettre, 1969).

An amazing assortment of products are produced from carbonyl compound intermediates. Carbonyl compounds, like acetone, are used as solvents in paints, lacquers, and polishes. Others are used in drugs, perfumes, germicides, food flavorings, rubber chemicals, and preservatives. 7

Among the carbonyls, formaldehyde is used most frequently in synthetic chemistry. This is due to its high reactivity (Snell &

Ettre, 1969). Consequently, 5.2 billion pounds of formaldehyde were consumed in 1983 for manufacturing purposes (Levin, Andersson,

Lindahl, & Nilsson, 1985).

Until 1940, when Otto Roelen discovered hydroformylation and launched modern catalysis of carbonyl forming reactions, carbonyl compound production was difficult. His process was carried out thus:

HCo(C0) 4

Due to the comparatively mild conditions and stable catalysts, hydroformylation is superior to the processes used prior to its discovery. Those processes were difficult to carry to completion because they required high temperatures and high pressures in the presence of unstable catalysts (Colquhoun, Thompson, & Twigg, 1991).

Since 1940, literally hundreds of new processes have been devised with milder conditions and excellent yields of carbonyl products.

Aldehydes and ketones, as shown above, may be prepared by direct oxidation of corresponding alcohols. Aldehydes may be readily oxidized further to their corresponding acids via oxidation.

It is convenient that most aldehydes and ketones react similarly with reagents that condense with the carbonyl group. This reactivity has been used on numerous occasions as a carbonyl detection process.

The most frequently mentioned method involves reaction of the carbonyl group with 2,4-dinitrophenylhydrazine (2,4-DNPH) to form a

2,4-dinitrophenylhydrazone. In 1920, W. E. Mathewson discussed using 8 it for acetone determination in water-soluble samples. Six years later, Brady and Elsmie (1926) discussed the 2,4-DNPH reaction with carbonyl compounds. Lappin and Clark (1951) determined that 2,4-DNPH could be used for trace determination and quantitation of aldehydes or ketones in organic solvents, organic reaction products, or water.

Since that time, many variations and applications of this have been proposed and used for the detection of the carbonyl group.

Jones, Holmes, and Seligman (1956) did ultraviolet and visible spectrophotometric studies of some 2,4-dinitrophenylhydrazones and proposed that the rate of temporal decrease in absorbance is characteristic of the parent carbonyl compound and would aid in its identification.

A "completely stable" product for carbonyl detection with

2,4-DNPH was developed in 1958 by Lohman (p. 972). He proposed that solutions carrying 3 to 300 parts per million (ppm) carbonyl could be quantitated via condensation of the carbonyl group with 2,4-DNPH. The yellow complex formed was then separated from excess reagent by hexane extraction. Next, the absorbance of the extract was measured spectrophotometrically at 340 nm and corresponded to the concentration of the carbonyl group. By 1969, the reaction was so common it was included in the Encyclopedia of Industrial Chemical Analysis as one of

" ••. the most important reactions of aldehydes and ketones (also used in their determination) ••. " (Snell & Ettre, 1969, p. 333).

Ariga (1972) used a thin-layer chromatography system to determine 2,4-dinitrophenylhydrazone products of keto-acids in cellular metabolism studies. Wakelyn (1974) used 9

2,4-dinitrophenylhydrazine to confirm aflatoxin B1 via derivatization and subsequent thin-layer chromatography. Methyl glyoxal was studied spectrophotometrically by observing and recording the absorbance of its product with 2,4-dinitrophenylhydrazine (Gilbert & Brandt, 1975).

Nanogram amounts of carbonyls could be determined by

2,4-dinitrophenylhydrazine derivatization and high performance liquid chromatography (Fung & Grosjean, 1981). Airborne formaldehyde and other carbonyl compounds were reacted with 2,4-dinitrophenylhydrazine by Grosjean and Fung (1982). The hydrazone products were separated and quantified by high performance liquid chromatography to determine parts per billion quantities of carbonyl compounds in air. In 1985,

Levin et al. used the 2,4-dinitrophenylhydrazine reaction with carbonyl compounds as the basis of their passive samplers for monitoring workers' exposure to formaldehyde.

1.4 Significance of Study

As evidenced previously, carbonyl groups may be detected qualitatively or determined quantitatively by any of several methods.

None of these methods was particularly fast or convenient. The purpose of this study was to devise an automated system that would quantitate carbonyl group concentration at parts per million levels in alcohol samples. It was desired that this system would be precise, accurate, and faster than the standard method from the American

Society for Testing and Materials (ASTM, 1986). CHAPTER II

QUANTITATIVE AND QUALITATIVE MEASUREMENT

OF CARBONYL COMPOUNDS

2.1 Literature Review

Flow injection analysis (FIA) is considered a relative of continuous flow analysis. Numerous reviews of FIA, its applications, and its theory have been written (Betteridge, 1978; den Boef, 1986;

Karlberg, 1988; Painton & Mottola, 1983; Patton & Crouch, 1986; Patton

& Wade, 1990; Ranger, 1981; Ruzicka & Hansen, 1975, 1980, 1981, 1986,

1987; Snyder, 1980; Stewart, 1981; Valcarcel & deCastro, 1987;

Van der Linden, 1986) since its inception in the 1970s. While some authors have argued that pioneering experiments in FIA were conducted as early as the 1950s (for example, by Skeggs (1957) while designing his bubble segregating analyzer), the scientists credited with

formally recognizing and applying the principles behind FIA are

Stewart and, simultaneously, Ruzicka and Hansen in 1974 (Ruzicka &

Hansen, 1975; Stewart, Beecher, & Hare, 1976).

The predecessor and main commercial competitor to FIA is continuous flow analysis (CFA). CFA is "in its broadest context

.•. any process in which the concentration of analyte is measured uninterruptedly in a stream of liquid (or gas)" (Ruzicka & Hansen,

1981, p. 3).

In 1957, Leonard T. Skeggs devised a continuous flow analyzer in which the samples were segregated by air bubbles. Technicon

Corporation supported the idea and successfully developed it for a

10 11 growing commercial market. This initial system, marketed as the

Technicon AutoAnalyzer, was cumbersome and incurred error due to an excess of 10% sample interaction, but it was a tremendous step in automating manual test methods. Since that time, the AutoAnalyzer has matured from the one-channel general purpose system through four generations to an instrument that performs over 30 tests on a given sample without carryover problems (Patton & Wade, 1990).

FIA is simpler in terms of equipment needed for a system.

Without bubbles of air or wash solution being injected in FIA, as in the segmented CFA systems, neither a bubble injector nor a "debubbler" is required (Snyder, 1980).

CFA is considered a steady state analysis system where the reaction between analyte and reagent is expected to reach a plateau-­ the plateau height is measured. Ruzicka and Hansen (1980) rejected the necessity for achieving a plateau and demonstrated that if (in the case of FIA) the sample is examined at a reproducible time period, the reaction does not have to proceed until completion. In this way, FIA can produce test results faster and do so with less reagent being used

(Patton & Wade, 1990).

2.1.1 Elements of a Flow Injection System

The essential elements of CFA, according to Valcarcel and deCastro (1987, pp. 1-57), are:

1. A fixed time or fixed volume sampling system.

a. Fixed-time sampling system; for a fixed period

of time, sample is taken into the system. 12

b. Fixed-volume sampling system; electrodes detect

passage of the bubble and signal the sampler to

move to the next sample cup and withdraw another

sample. This has the best reproducibility.

2. Propulsion system--usually a peristaltic pump.

3. Separation system to separate analyte fraction from

sample. Some separation systems are:

a. A dialyzer to separate interferents from the

sample.

b. Liquid extractor to improve selectivity.

c. Filtration system.

4. Debubbler.

5. Detection system.

The essential elements of the FIA system, according to Valcarcel and deCastro (1987, pp. 101-102), are:

1. Lack of segmentation.

2. Samples are directly inserted or injected into a

flow--not aspirated into it.

3. The sample slug is transported in the system and, in

addition to transport, the analytical reactions

occur.

4. Dispersion or dilution of analyte controlled by

hydrodynamic and geometric factors.

5. Signal noted by continuous sensing system. 13

6. System may or may not be at equilibrium at the point

of signal detection, thus reproducible timing is

essential.

2.2 Analytical Methods for Carbonyl Compounds

2.2.1 Qualitative Tests

2.2.1.1 Melting Points of Derivatives

The presence of a carbonyl group containing compounds may be determined by any of several tests. One method involves formation of a carbazone from the reaction of the carbonyl group and a semicarbazide or a thiocarbazide. The melting point of the resulting carbazone is compared to known melting points for identification of the carbonyl compound present.

2.2.1.2 Spectroscopic Techniques

Spectroscopic techniques may be used for direct detection of the carbonyl group. The carbonyl group absorption band occurs between

1 1900 and 1600 cm· • In ultraviolet spectroscopy, a unique absorbance maximum is exhibited by carbonyl compounds if other components present in the sample do not absorb similarly. Nuclear magnetic resonance spectroscopy can demonstrate the presence of carbonyl groups in a sample, but these components must be present at a relatively high concentration (Snell & Ettre, 1969, pp. 345-348).

2.2.1.3 Precipitation Techniques

The iodoform test is positive for formaldehyde, acetone, and acetaldehyde. Hypoiodite in an alkaline solution oxidizes a 14 carbonyl-containing compound; for example, formaldehyde is oxidized to

, sodium formate. A positive result is the formation of CHI 3 a yellow precipitate (Stone, 1956).

Another technique involves sodium bisulfite. The sample may be added to a saturated ethanol solution of sodium bisulfite. Aldehydes or methyl ketones are indicated if a precipitate forms.

OH >C=O + NaHS03 ~ >C< S03 Na

(Stone, 1956)

2.2.2 Quantitative Techniques

2.2.2.1 Precipitation Techniques

Carbonyl compounds can be determined quantitatively by precipitation methods. These methods are not popular because they are slow and because of the effort required (beyond forming the product) to dry and weigh the product for quantitative analysis. They are also insensitive (Stone, 1956).

The iodoform test mentioned previously in the qualitative, precipitation techniques section, may be modified for quantitative use. The hypoiodite in the alkaline medium oxidizes the formaldehyde, acetaldehyde, or acetone.

0 0 II ll R-CH + 3NaOH + 12 ~ HCONa + 2Nai + 2H20

After the oxidation, the solution is acidified and the excess iodine is titrated with sodium thiosulfate (Stone, 1956). 15

Other precipitation methods include using semicarbazide,

p-nitrophenylhydrazine, phenylhydrazine, 2,4-dinitrophenylhydrazine,

or dimedone. Of these, the dimedone and 2,4-dinitrophenylhydrazine

methods are most practical in terms of precipitation completeness,

rate, and product formed (Stone, 1956).

Dimedone, 5,5-dimethylcyclohexane-1, 3-dione, or

dimethyldihydroresoreinol precipitation relies on the methylene group

between the two carbonyl groups condensing with an aldehyde.

This reaction is only useful for aldehyde detection; ketones do not

condense (Stone, 1956).

Another preferred precipitating agent is

2,4-dinitrophenylhydrazine. The reaction is as follows:

The 2,4-dinitrophenylhydrazine reagent has a higher molecular weight

than other reagents; thus, the product is less soluble and easier to

recover. The reagent is somewhat insoluble; therefore, a dilute

reagent solution (2.5 to 5 g/L) is necessary. Large amounts of alcohol dissolve the precipitate and some aromatic aldehydes do not 16 precipitate well. For these reasons, it is not a universal method

(Stone, 1956).

2.2.2.2 Techniques with 2,4-dinitrophenylhydrazine

For detecting smaller quantities (0.5-50 ~g) of carbonyl groups, a 2,4-dinitrophenylhydrazine procedure may be used. According to

Lappin and Clark (1951), the sample is reacted with an acidic solution of 2,4-dinitrophenylhydrazine. The hydrazone is reacted with potassium hydroxide to form a wine-red color, presumably due to a resonating quinoidal ion.

Lappin and Clark ( 1951) claimed that traces of ( 10· 4 to 10.6M) aldehydes or ketones in water could be determined with good sensitivity. The only interferences they reported were nitroaromatic groups. NOa ~ base R-CIJ = X-NH-L_)-N0 2 ~ NOz 0- 1 / R-CII =N-N =C> = N"-. I 0

Lohman (1958) developed a method where

2,4-dinitrophenylhydrazine reagent reacts with the carbonyl compounds to form 2,4-dinitrophenylhydrazones. The hydrazones are extracted into hexane (to remove excess reagent) and the resulting stable yellow color of the hexane solution quantitated by measuring absorbance at a wavelength of 340 nm. The procedure involves several extractions prior to the spectroscopic determination. Net sample absorbance 17 divided by the average molar absorptivity yields the molar concentration of the carbonyl group.

Snell and Ettre (1969) stated that 2,4-dinitrophenylhydrazine is

"the most effective in detecting carbonyl groups; a colored reaction product is formed" (p. 342). Yellow products indicate aliphatic carbonyl compounds, red products indicate aromatic carbonyl compounds , and orange products are indeterminate.

There are other spectroscopic techniques involving

2,4-dinitrophenylhydrazone formation. Pesez and Bartos (1974) recommended using a solution of 0.1% 2,4-dinitrophenylhydrazine and

0.5% hydrochloric acid in glacial acetic acid. A yellow-orange color indicates the presence of carbonyl compounds. If the procedure is conducted in a nonaqueous medium, the product will be red-violet. The product is read spectrophotometrically at 412 nm, and the absorbance is compared to known calibration curves for quantitation of the concentration of the carbonyl group in the sample.

Gilbert and Brandt (1975) were only interested in studying methyl glyoxal, a part of enzymatic processes in several organisms .

The reaction, using 2,4-dinitrophenylhydrazine as R, in the following diagram is:

R R I I HN HN I I 0 0 N N II II II II R - N - NH 2 + CH3 - C - CH ~ CH3 - C - CH I H 18

They used a 2,4-dinitrophenylhydrazine solution in ethanol containing

12% (v/v) HCl. The product absorbance was read spectrophotometrically at 432 nm wavelength.

Finally, the ASTM (1986) recommends a specific variation of the

Lappin and Clark procedure. The reaction is specific for the carbonyl group. After the initial incubation period, a lime-yellow solution results. Potassium hydroxide is then added and changes the color of the solution from lime-yellow to black. This color rapidly fades to a red color that is measured at 480 nm.

2.2.2.3 Spectrophotometric Techniques Not Involving 2,4-DNPH

One test involves detection of aliphatic aldehydes by formation of a benzothiazole hydrazone and development as a formazan with diazonium salt generated in the medium via oxidation of excess

2-hydrazinobenzothiazole. The result is a blue color that is read spectrophotometrically. See Table 2.1 for the Amax of the product with different carbonyl compounds. 19 N C-NH-N=CH-A "I OC 5

(Pesez & Bartos, 1974)

Aliphatic aldehydes may also be determined by spectrophotometric means after reaction with 3-methylbenzothiazolin-2-one hydrazone and ferric chloride. Initially, a condensation with

3-methylbenzothiazolin-2-one hydrazone occurs. A d i azonium salt is generated in the reaction medium by oxidation of the excess reagent and development as a formazan occurs, resulting in a blue-green or green color. See Table 2.2 for the Ama x of the product with different carbonyl compounds. With a modification of the procedure, glyoxal may be detected as well and quantitated spectrophotometrically at 625 nm.

The difference in Amax reflects a bathochromic shift due to extended conjugation in the product molecule. This shifting may cause complications in spectrophotometric measurements made without the benefit of a scanning detector that would compensate for these shifts .

Aromatic aldehydes react variably and, reportedly, do not always follow Beer's law. 20

+

(Pesez & Bartos, 1974 )

Dimethyl-p-phenylenediamine oxalate in a glacial acetic acid solution is the reagent used for determining aliphatic aldehydes as shown by the following:

The reagent and sample are cooled to a temperature just above freezing, then they are mixed. The resulting yellow or orange color is read at the appropriate wavelength. See Table 2. 3 for the Amax of the product with different carbonyl compounds. Some simple ketones, such as acetone or methyl ethyl ketone, can react in this scheme, but the method is insensitive (Pesez & Bartos, 1974).

Aliphatic aldehydes may also be determined after reaction with diethyl acetonedicarboxylate and ammonia. A pale yellow reaction product, assumed to be a 4-substituted

3,5-dicarbethoxy-1,4-dihydro-2,6-pyridinediacetic acid diethyl ester, is formed. The absorption maximum is in the ultraviolet range. 21

Formaldehyde, furfural, acetaldehyde, and cinnamaldehyde sample

reaction products are read at 344 nm; and the reaction product for

propionaldehyde is read at 340 nm. See Table 2. 4 for Ama x of products

with different carbonyl compounds. Citra! and aromatic aldehydes do

not follow Beer's law. Ketones do not react.

(Pesez & Bartos, 1974)

Other colorimetric techniques are applicable as well. The

oxalhydrazide and cupric acetate method utilizes the following

reaction:

)c=o + H2N-NH-CO-CO-NH-NH2 ----i )c:N-NH-CO-CO-NH-NH2

)c=N-N=~-C-NH-NH I \\ 2 0 .0 'c.:; 2

The reagents are Teorell-Stenhagen buffer for pH control and a 1:1

mixture of 0.25% aqueous solution of oxalhydrazide and a 0.0156%

aqueous solution of cupric acetate monohydrate.

The resulting absorbance is read at 590-610 nm, depending on the

sample. Cyclohexanone and acetone are read at 590 nm; streptomycin

and isobutyraldehyde are read at 600 nm; and formaldehyde, acetaldehyde, propionaldehyde, and glyoxylic acid at 610 nm. Excess oxalhydrazide yields this blue complex: 22

)c=N-N=C-C-NH-NH2 I \\ 0~ ,.0 'C:U 2

Excess carbonyl yields this violet complex:

OH I 'c=N-N=C-C / I \\ 0 .N-N=C/ 'ci.i ' 2

(Pesez & Bartos, 1974)

2.2.2.4 Gas Chromatography and Titration Techniques

Gas chromatography is useful but rather cumbersome as different

columns must be used for optimal separation of ketones versus

aldehydes. Carbonyl compounds may be determined by any of three major

variations of a reaction between the carbonyl group and hydroxylamine

or its salts. In one case, the carbonyl group reacts with

hydroxylamine (a strong base), then excess reagent is back titrated.

In a variation, the carbonyl reacts with hydroxylamine hydrochloride

or hydroxylamine sulfate. The mineral acid formed is then titrated.

Finally, since both of these reactions produce water, the water formed

can be determined by the Karl Fischer method. The sample is titrated

with Karl Fischer reagent until the color matches that of an anhydrous

blank (Snell & Ettre, 1969).

The reaction with hydroxylamine hydrochloride is the most popular, but it has a shortcoming. It is too slow--especially for 23

ketones. For some aldehydes, the oximation reaction stops at

equilibrium. These problems can be eliminated by adding an acid

acceptor (pyridine) to force oxime formation to completion. Visual

titration with bromophenol blue is acceptable, but potentiometric

titration is recommended (Bryant & Smith, 1935).

The carbonyl group on the aldehyde or ketone is detected by its

reactions with sodium bisulfite.

OH

>C=O + NaHS03 ~ >C< S03Na

This reaction has an unfavorable equilibrium constant; therefore, a

large excess of reagent is necessary to drive the reaction to

completion. The bisulfite may be removed by oxidation with iodine;

however, this allows the product to revert. Stone (1956) reported,

" .•. the measurement destroys the quantitative nature of the reaction"

(p. 61). To counter this problem, Siggia and Maxcy (1947) modified

this approach by using a large amount of sulfite, then adding a known

amount of acid. In this way, bisulfite was produced as needed and

little product dissociation occurred. There was small risk of an

equilibrium reaction. Methyl ketones interfere and any acids or bases

present must be determined separately and test results corrected

(Stone, 1956).

Stone (1956) suggested oxidizing the carbonyl group into a carboxylic acid, then titrating the acid formed. The method only demonstrated the determination of aldehydes. Prior to testing, any free acid in the sample must be determined separately. Some esters may become aldehydes during the reaction. For this, the esters must 24 be determined separately by saponification after an aldehyde-aldoxime conversion. An overall error of 0.3% is expected.

2.2.2.5 Thin-Layer Chromatographic Techniques

Thin-layer chromatography is useful if the sample components are nonvolatile at room temperature. Lighter carbonyls must be derivatized. This is usually done with 2,4-dinitrophenylhydrazine.

This method is not quantitatively accurate, but qualitatively the method is acceptable (Snell & Ettre, 1969).

Ariga (1972) discussed thin-layer chromatography of keto acid

2,4-dinitropheny1hydrazine in the form of its sodium salt. He stated that clear separation of the DNPH derivative of keto acids could be achieved. This method required 40 minutes time for activation of the chromatoplate and 2.5 hours for development after the sample was applied to the plate and atmosphere saturated with solvent vapor for an hour. The thin-layer chromatography method was useful for

"identification, determination, and preparation of keto acid DNPH"

(p. 439), but it is a time-consuming process and not a quantitative measure of all carbonyl compounds present.

In 1974, Wakelyn similarly used 2,4-dinitrophenylhydrazine and thin-layer chromatography. However, the object of the study was to

detect and confirm the presence of aflatoxin 8 1 derivative. Again, in comparison to more general methods, the method was time consuming

(time for formation, elution, drying 30 minutes, spraying, incubating

60-90 minutes minimum), and nonquantitative. For confirmation of the 25

identity of aflatoxin 8 1 , it was "convenient, fast, and reliable"

(p. 483) compared to earlier methods.

2.2.2.6 Voltammetric Techniques

Afghan, Kulkarni, and Ryan (1975) discussed twin cell potential sweep voltammetry to determine and differentiate carbonyl compounds in waters. This process involved the formation of azomethine derivatives to determine carbonyls electrochemically without separation or preconcentration of the sample. The derivatives are reduced at the dropping mercury electrode.

2.2.2.7 High Performance Liquid Chromatography

The method recommended by Fung and Grosjean (1981) was to use high performance liquid chromatography to separate and quantify

2,4-dinitrophenylhydrazones of trace levels of carbonyl compounds found in air samples. The formation of 2,4-dinitrophenylhydrazone proceeds by "nucleophilic addition on the carbonyl followed by

1,2-elimination of water ... Because DNPH is a weak nucleophile, the coupling reaction is carried out in the presence of acid which promotes the protonation of the carbonyl" (p. 168). The separation of the different hydrazones was performed by HPLC with quantitation by spectrophotometry at 360 nm.

In another variation of this method, during air sampling, the

DNPH reagent is coated on 20-mesh, HF-etched glass beads. The DNPH reagent consists of a H3P04-saturated DNPH solution in . The beads are immersed in the reagent, and the water 26 evaporated off. The ethylene glycol promotes film formation on the beads. The cartridges are filled with the beads and plugged with glass wool, sealed with plastic, and refrigerated until use. Air is sampled through the cartridges, and the cartridge washings are then analyzed by HPLC as described (Grosjean & Fung, 1982).

Levin et al. (1985) employed 2,4-dinitrophenylhydrazine in personal dosimeters to monitor formaldehyde levels in air. The glass fiber filters were impregnated with 2,4-dinitrophenylhydrazine and phosphoric acid. The hydrazone formed is extracted from the filter with acetonitrile and determined by high-performance liquid chromatography using ultraviolet detection at 365 nm.

2.2.2.8 Enzymatic System

Almuaibed and Townshend (1987) described a flow injection system that utilized an enzyme to determine acetaldehyde. They proposed two methods involving the enzyme aldehyde dehydrogenase (AlDH). Both systems were based on flow injection, but whereas one system used the merging zones method and considerable amounts of expensive reagent, the second system utilized an immobilized enzyme reactor for greater economy. Both systems were simple, sensitive, and reproducible. The analytical reaction is:

CH3CHO + NAD+ + oa· # CH3 coo· + a• + NADH.

The NADH product was monitored spectrophotometrically at 340 nm. 27

Pyrophosphate 1.0 ml/min R 1 AIDH 25 ° C R2 buffer pH 9.0 ..... NAo• w

KCI 25 ° C II

Pyrophosphate 0.8 ml/min sample 25 C R3 buffer pH 9.5 ° NAD+ 2-mercaptoethanol t-+----<.,_...,..-f D w

KCI ..____ immobilized enzyme According to the authors, ethanol up to 5% (v/v) does not interfere.

This system is specific only for acetaldehyde due to its reliance on an enzymatic reaction. For the same reason, it is very expensive. 28

Table 2.1. Amax for determinations with 2-hydrazinobenzothiazole and potassium ferricyanide.

Sample Amax I nm

Acetaldehyde 576

Propionaldehyde 577

Formaldehyde 582 29

Table 2.2. lmax for determinations with 3-methylbenzothiazolin-2-one hydrazone and ferric chloride.

Sample Amax I nm

Acetaldehyde 610

Butyraldehyde 610

Propionaldehyde 620

Caprylic aldehyde (in isopropanol) 635

Formaldehyde (in water) 635

Lauric aldehyde 635

Citral 640

Citronellal (in isopropanol) 640 30

Table 2.3. 1max for determinations with dimethyl-p-phenylenediamine oxalate.

Sample 1max, nm

Isobutyraldehyde 370

w-Hydroxyvaleraldehyde 385

Acetaldehyde 390

Butyraldehyde 390

Isovaleraldehyde 390

2-Ethylcaproic aldehyde 390

Citronella! 390

2,2-Dimethyl-3-hydroxypropionaldehyde 390

Enanthaldehyde 400

Lauric aldehyde 400

Glyceraldehyde 410

Propionaldehyde 430

Caprylic aldehyde 435

Citra! 445

Crotonaldehyde 450

p-Nitrobenzaldehyde 450

p-Tolualdehyde 460

p-Anisaldehyde 465

Benzaldehyde 465

Protocatechualdehyde 465

Salicylaldehyde 465

Cinnamaldehyde 490 31

Table 2.4. Amax for determinations with diethyl acetonedicarboxylate and ammonia.

Sample Amax, nm

Propionaldehyde 340

Acetaldehyde 344

Cinnamaldehyde 344

Formaldehyde 344

Furfural 344 CHAPTER III

EXPERIMENTAL SECTION

FIA is a well-established technique for automating wet analysis.

This study intended to automate the determination of carbonyl compounds by applying the FIA technique to a standard manual method.

3.1 Initial Considerations

The samples in this study were alcohols containing carbonyl compound impurities at parts per million levels. Any system chosen

for quantifying the carbonyl compounds must be compatible with alcohol

samples.

3.2 Reagents

Reagent grade 2,4-dinitrophenylhydrazine (Aldrich Chemical

Company, Milwaukee, Wisconsin) was used to prepare the

2,4-dinitrophenylhydrazine reagent. The dry 2,4-DNPH was dissolved in distilled (vide infra) methanol acidified with HCl and diluted with deionized water.

Solvent reagents were distilled in 4-L amounts after the addition of 20 g 2,4-DNPH and 2 mL HCl (cone.). The distillation protocol used a 2-hour reflux before distilling over. Only 75% of the total amount could be distilled over due to the volatile nature of the

2,4-DNPH itself. Potassium hydroxide solutions were prepared by dissolving potassium hydroxide pellets in water and diluting with methanol or u.s.P. ethanol, as noted.

32 33

3.3 Preliminary Experiments

Preliminary experiments were carried out manually to verify the feasibility of applying certain detection methods to the alcohol samples. The ASTM method was determined to be satisfactory. Systems to automate this method were designed. This method was modified to work in automated systems to produce results comparable to those of the manual procedure.

3.4 Apparatus

3.4.1 Pump Conduits

One peristaltic pump (Gilson Minipuls-2, 10 roller, 4-channel head, Gilson International, Middleton, WI) was used for all pumping in the systems. Masterflex Viton tubing (Cole-Parmer Instrument Company,

Chicago, IL) (size 13, 0.7 rnm inner diameter (i.d.) x 4 rnm outer diameter (o.d.) and size 14, 1.5 rnm i.d. x 4.8 rnm o.d.) was used. In some systems, a second pump was used (Lab Pump Junior, Model RHSY,

Fluid Metering, Inc., Oyster Bay, NJ). Standard polytetrafluoroethylene (PTFE) tubing was used throughout the system in various inner diameters as noted in the respective figures.

Coiled 30-gauge tubing (0.3 rnm i.d.) of 1-meter length was used after the detector in the systems to act as a restriction coil. This coil imposes a restriction to inhibit bubble formation in the detector.

3.4.2 Reaction Coils

The reaction coil device was heated by two flexible heaters

(HOTWATT, Inc., 120V, 30W) sandwiching PTFE tubing woven on a 34 perforated board (Radio Shack, Catalog No. 276-1396). The board had to be drilled with a size 48 drill bit (1.93 mm) in order to enlarge the holes to accommodate the tubing. The weave was Serpentine II, as described by Curtis and Shahwan (1988). The heaters were soldered in parallel to a power cord and plugged into a Powerstat Variable

Autotransformer (3PN116C, The Superior Electric Company, Bristol, CT)

so that the temperature could be controlled.

The second generation reaction coils were constructed of two

Watlow 120V, SOW (Watlow, St. Louis, MO) flexible heaters sandwiching

2 meters 30-gauge tubing (0.30 mm i.d., 0.75 mm o.d) similarly wound

around a core of 1 em diameter. The heaters were powered by a variable voltage source as above.

3.4.3 Detection Systems

Initial manual experiments were performed using a Hewlett

Packard 8451A spectrophotometer. Initial automated experiments utilized the Kratos 757 Spectroflow spectrophotometer. Modified flow­ through optical cells were used with the latter.

Subsequent automated experiments utilized a detector developed in-house for this project. The detector was housed in a 3" x 5" x 2" project box (P/N 270-238, Radio Shack). The light source was a 470 nm wavelength light emitting diode (L200CWB5, LEDtronics, Torrance, CA) seated in a PEEK 1/4-28 threaded female union (Dionex). In front of the light emitting diode, a hole was drilled through to hold the cell in place at the top. The cell was a 3.5 mm o.d. x 2.1 mm i.d. glass tube connected to 0.5 mm i.d. teflon tubing by heat shrink tubing 35 encasing silicon tubing. (See Figure 3.1.) Final manual spectroscopic experiments were performed on a Perkin Elmer Model 559 spectrophotometer.

3.4.4 Detector Cells

Manual experiments utilized quartz 1 em x 1 em x 4 em square spectrophotometer cell. Automated experiments were performed utilizing a variety of cells as detailed below.

3.4.4.1 Cell 1

The first custom cell for the Kratos 757 detector was made of quartz optical tubing 3 mm x 3 mm x 30 mm. Conduits to the cell at either end were 0.5 mm i.d. Tefzel tubing. The cell was placed in a custom plastic cell holder devised for the Kratos 757. The tubing was held in place by silicone pump tubing swollen by chloroform. The silicone tubing was crimped onto the cell by 32-gauge (0.202 mm) wire.

(See Figure 3.2.)

3.4.4.2 Cell 2

Another custom cell for the Kratos detector was devised. A new cell holder was constructed as well. The new holder's cell aperture was too large for the cell installed. The aperature diameter was reduced by inserting two 13.5-gauge (1.77 mm i.d.) tubular stainless steel pieces 4 mm in length inserted in 10.5-gauge (2.5 mm i.d.) tubular stainless steel pieces. These were glued with silicon household glue to the cell holder, one tube pair on each side of the holder. The axis of the cell was perpendicular to the axes of the 36 steel tube pairs. The steel tube pairs' lengths were such that they were useful in stabilizing the flow cell in the holder. The flow cell was connected to the rest of the system by inserting PTFE tubing in either end. This was held in place with polyethylene heat shrink tubing which was, in turn, secured again by 32-gauge nichrome

(0.202 mm) crimping wire. (See Figure 3.3.)

3.4.4.3 Cell 3

A cell for the miniature light emitting diode (LED) detector was

constructed of a l-inch length of 2.1 mm i.d. glass tubing. The

system was linked to the flow cell by teflon tubing (0.5 mm i.d.).

The tips of this green tubing were covered by small heat shrink tubing. The tips then were inserted in the ends of the glass tube.

The glass tube was inserted through the LED holder. The ends of the glass tube were covered by large heat shrink tubing over the glass and teflon tubes. (See Figure 3.4.)

3.4.5 Water Bath

Up to seven 25-mL volumetric flasks were incubated simultaneously in a 2000-mL beaker of water. Aluminum foil was wrapped around the necks of the flasks to keep them from tipping over. seven lengths of wire were prepared with identification labels on one end of each wire. The other end of each wire was wrapped around the neck of its respective flask. The labelled end could then be looped over the beaker neck to act not only as an extra means of identification, but as an extra means of keeping the flask from turning over. The beaker was partially filled with water and loaded 37 with the flasks as described, then placed over an electric hot plate.

A thermometer was inserted in the bath to monitor temperature.

3.5 System 1

The first set of studies consisted of a series of manual experiments. The results were monitored on a Hewlett Packard model

8451A spectrophotometer.

3.5.1 Experiments with System 1

The 2,4-DNPH on the shelf was nearly 30 years old. Its usefulness was questionable. It was tested by preparing and determining a reagent blank containing 0.1% 2,4-DNPH and 0.5% HCl in glacial acetic acid.

Several carbonyl compounds were diluted in methanol. The ASTM method for testing was followed. Two mL of each sample (methanol blank, formaldehyde, butyraldehyde, 1-benzoylacetone, acetone,

2,4-pentanedione) were mixed and incubated in separate 25-mL volumetric flasks for 30 minutes with 2 mL of the 2,4-DNPH reagent.

The flasks were filled to the mark with potassium hydroxide solution and incubated for 12 minutes. Each resulting solution was poured into a quartz cell and scanned from 310 to 820 nm wavelengths to determine the wavelength of maximum absorbance.

The manual method was performed using acetonitrile instead of methanol. It was expected that the acetonitrile would not have the carbonyl impurities expected in the undistilled methanol. 38

3.6 System 2: Introductory Automated Systems

Several introductory automated systems were designed. The first automated systems were rudimentary investigations of the function of potential components, including the pumps and valve. The 8-way valve

(Dionex inert valve, dual stack air actuated, four passages per stack,

P/N 035914, Dionex Corp., Sunnyvale, CA), the Gibson Minipuls and Lab

Pump Junior (Lab Pump Junior, Model RHSY, Fluid Metering, Inc., Oyster

Bay, NJ) were examined for durability in the first system illustrated

(Figure 3.5). Other introductory systems (such as that illustrated in

Figure 3.6) were designed to investigate the use of FIA in automation of the ASTM method. The sample, carrier, and

2,4-dinitrophenylhydrazine reagent were put into the system by the peristaltic pump. The Dionex loop injection (described above as the

8-way valve) injected 40 ~L volumes. The sample, or carrier, met the

2,4-dinitrophenylhydrazine at a T-intersection. They united and mixed in a reaction coil as they proceeded to another T-intersection. At that point, potassium hydroxide (KOH), pumped in by a second pump, the

Lab Pump Junior, united with the mixture. This new solution proceeded through a final mixing coil to the detector, Kratos Spectroflow 757.

Results were monitored for reproducibility.

3.7 System 3

This system was designed to totally automate the ASTM method and produce results faster than the manual method. In this system, one

Gilson 4-channel Minipuls peristaltic pump was utilized for all pumping needs. Reagent and carrier were pumped and the sample was 39 aspirated through the previously mentioned Dionex 8-way valve. The sample was injected by the valve into a carrier stream of the DNPH.

The stream then passed into a heated reaction/mixing coil (1.5 m,

24-gauge, 0.56 mm i.d., PTFE tubing, Zeus Industrial Products,

Orangeburg, SC) and then merged with a stream of KOH. The final mixture passed to a second heated reaction and mixing coil (1.5 m,

20-gauge, 0.86 mm i.d., PTFE tubing, Zeus) and to the detector (Kratos

757 Spectroflow). Timing for the various reaction periods was provided by a VIC! Digital Valve Sequence Programmer (DVSP-4, Valco

Instruments Company, Inc., Houston, TX). (See Figure 3.7.)

3.8 System 4

System 4 utilized only the peristaltic pump, poly(vinylchloride) peristaltic pump tubing, and two clean 100-mL volumetric flasks. (The flasks were washed in accordance with ASTM E400-70 glassware washing procedure.) The first flask (Flask A) was filled with a solution containing 4-mL HCl (cone.), 50-mL methanol, and 46-mL deionized water. The solution pumped at a rate of 0.17 mL/min to the dry flask

(Flask B). Solution A was compared to Solution 8 visually and spectrophotometrically. (See Figure 3.8.) This automated system was designed to determine whether or not ordinary peristaltic pump tubing could be used as opposed to the more expensive Viton pump tubing

(Cole-Parmer Instrument Company, Chicago, IL).

3.9 System 5

This system was constructed using the Gilson minipuls peristaltic pump. By using 0.7 mm and 1.5 mm i.d. Viton tubing, it 40 was possible to send methanol carrier, 2,4-DNPH reagent, and sample at

0.17 mL/min and KOH at 0.7 mL/min, respectively. The sample was injected into the methanol carrier by the Dionex valve and the carrier stream was then merged to the 2,4-DNPH reagent stream. The mixture then passed through a PTFE reaction coil and merged at a second

T-intersection with a KOH solution stream. This mixture proceeded through the final mixing coil to the Kratos detector. Timing was controlled by the VIC! programmer. (See Figure 3.9.)

3.9.1 Experiments with System 5

Experiments with the amount of time allowed for mixing, stopping, and flowing were done by adjusting the VIC! Digital Valve

Sequence Programmer. The results were monitored. Responses from standard solutions of different carbonyl compounds in alcohol samples were determined by monitoring peak heights on a strip chart recorder.

3.10 System 6

The manual method was performed, in accordance with ASTM specifications, such that a 2-mL sample was put in a 25-mL volumetric flask. Then, 2 mL of the 2,4-dinitrophenylhydrazine reagent was added. This mixture was incubated at room temperature for 30 minutes.

After the incubation, potassium hydroxide was added to the mark. The flask was capped and shaken. It was then incubated for 12 minutes at room temperature. At the conclusion of the second incubation, the solution was poured into a 1-cm pathlength quartz cell and the absorbance at 480 nm wavelength was measured on a Hewlett-Packard spectrometer. In a variation of this experiment, incubation at a 41 higher temperature was carried out by putting the volumetric flasks in a water bath, as described in Section 3.4.5.

3.10.1 Experiments with System 6

These manual experiments were designed to determine the

following:

Whether the ASTM method worked as described.

Amount of time needed for manual method.

Optimum concentrations of acid and base in the reagents.

Minimum amount of time needed for incubations.

Optimum temperature for reaction.

Minimum necessary volumes.

Optimum hydroxide source.

3.11 System 7

System 7 utilized the peristaltic pump with 0.7 mm i.d. and

1.5 mm i.d. Viton pump tubing. The pump was run at its speed 250.

This yields an output of 0.17 mL/min for the smaller pump tubing

(carrier, sample, and 2,4-DNPH reagent) and 0.7 mL/min for the larger tubing (KOH). The sample (40 ~L) was injected by the Dionex valve into the carrier. After a travelling 4.5 em from the valve through a

0.5 mm i.d. PTFE tube, the carrier stream meets the

2,4-dinitrophenylhydrazine reagent at a tee. The mixed stream proceeds through a 1.5-m 24-gauge (0.56 mm i.d.) woven, heated mixing coil. The temperature of the heated mixing coil was monitored by one arm of the FLUKE 52K/J thermocouple thermometer (John Fluke

Manufacturing Co., Inc., Everett, WA). Afterwards, at a second 42

T-intersection, the stream merges with the potassium hydroxide reagent and enters a second heated mixing coil (1.5 m long, 20-gauge, 0.86 mm i.d., woven on drilled perforated project board). The temperature in this coil was monitored by the other flexible arm of the FLUKE thermometer. This final mixture then continues 14 em farther through a 0.5 mm i.d. PTFE tube to the detector. The timing for the

incubation and flow periods was controlled by the vrcr programmer.

The incubation coils were heated by the HOTWATT coil device previously described. (See Figures 3.10 and 3.11.)

3.11.1 Experiments with System 7

The first experiment was designed to find a way to eliminate a double peak phenomenon. The heating protocol of the incubation coils was changed from heating only the first coil to heating both coils.

A second experiment was designed to speed up the reaction in the

system by increasing the temperature of the incubation coils. The temperature was raised from 55-60°C used previously to a range of

70-81°C. This is higher than the boiling point of methanol. Methanol was replaced with distilled propanol for this system. Distilled propanol was used as the carrier and as the solvent for the 2,4-DNPH reagent. Ethanol was used to replace methanol in the potassium hydroxide solution.

several detector cells were devised and used with this system.

A new miniature LED detector was devised. The design and fabrication of this detector were carried out by Dasgupta, Bellamy, Liu, Lopez,

Loree, Morris, Petersen, and Mir (in press). Response of the new 43 detector was compared to the Kratos 757 Spectroflow spectrometer detector with the new detector being connected in series following the

Kratos.

Various lengths of coiled tubing were substituted and/or added to the system. The sample loop size was changed by cutting it in half. The timing was changed by changing the valve sequencer programming. The objective was to attain a stable baseline for the system.

3.12 System 8

The system is shown schematically in Figure 3.12. One peristaltic pump was utilized. The sample was injected via an electropneumatically driven 6-way rotary valve (V1, type 5701,

Rheodyne, Cotati, CA). The injection loop consists of a 15.5-cm

length of 28-gauge (0.38 mm i.d.) tubing (Zeus). The sample size thus

injected was 20 ~1. A 28-gauge (0.38 mm i.d.) tubing, 19 mm in

length, connected V1 to the T-intersection where the

2,4-dinitrophenylhydrazine reagent is merged to the carrier. From the

T-intersection to the common port, V2 (a 3-way valve, LFYA 1203032H,

The Lee Company, Westbrook, CT), the connection was made by 10-cm

length of 30-gauge (0.30 mm i.d.) standard wall PTFE tubing (Zeus).

There were two 3-way valves in the system (V2 and V3) connected to each other by two 2-meter lengths of 30-gauge (0.30 mm i.d.) standard wall PTFE tubing. The normally closed (NC) ports of V2 and V3 are connected. The normally open (NO) ports of the two valves were

similarly connected. The tubes connecting the 3-way valves were 44 coiled and held in a 60°C heating chamber. The chamber was constructed of flat heaters (Watlow, 02005097C, 120V, SOW, 7726ILS,

St. Louis, MO) housed in an insulated, covered drinking cup (12-ounce,

Shell Friend's Convenience Store). The temperatures of the coils were monitored by the FLUKE 52K/J thermometer. The common port of V3 was connected by a 17.5-cm length of 30-gauge (0.30 mm i.d.) PTFE tube

(Zeus) to a second tee where potassium hydroxide was fed into the system. The new mixture flowed through a 2.5 m x 0.38 mm i.d. reaction coil and entered the detector. This detector was the light emitting diode flow cell detector designed especially for this project. The system terminated with a 1-meter length of 30-gauge coiled PTFE tubing to act as a restriction that inhibits bubble formation in the system.

This system was a hybrid stop-flow and continuous flow system.

The pump operated continuously. Sample was fed into one of the heated incubation coils and sealed inside for six minutes. During this time, carrier flowed through the other coil. Upon completion of the incubation period, the sealed tube was reopened and the incubated sample and reagent mixture flowed to the potassium hydroxide intersection. The opening and closing of valves in this system was controlled by the VICI programmer. See Figure 3.13 for the wiring diagram for the VICI programmer.

This system was designed to eliminate the shifting baseline observed in previous stop-flow systems. The baseline was expected to be held at one place by the washing action of the continuously flowing potassium hydroxide and the carrier flowing for six minutes between 45 successive sample injections (the 6-minute sample incubation period).

Figure 3.14 shows photographs of the system. 46

Outside of Miniature LED Detector

Signal cable LED and Reference cable

Inside of Detector

Cell

Tefzel tubing to system from system

Figure 3.1. Mini LED detector. 47

<-..T~e"'t""'z-eTI-t~""u..,b~,.-. n-g-t~""o_s_y_s_tr-e-m-" >

Quartz cell

swollen after soaking in chloroform

Plastic cell holder

Complete cell sealed in holder

Figure 3.2. First custom cell for Kratos 757 detector. 48

.":. ..-:.": .... :...-: . .-: ... :.<-<-":.':.<.':. .-: . .-: ....:./. . .-: • .;-_.'><·" <"",,,,,,, ...... " ...... X)

Tefzel tubing Small heatshrink tubing c D

Large heatshrink tubing Glass tube cell

13.5 ga 10.5 ga

Stainless steel tubing

Plastic cell holder

Complete cell

Figure 3.3. Second custom cell for Kratos 757 detector. 49

L: Light Emitting Diode (LED) LC: LED Cable G: Shrink tubing SC: Signal cable N: Nuts s D: Detector photocell S: Screw s H: Hole LL: Solder lug R: Reference photocell B: Body HS: Heat shrink tubing P: Partition T: Threaded hole for screw SL: Slot GT: Glass tubing

Figure 3.4. Cell for mini LED detector. 50

1 m, 0.5 mm i.d. sample~ 0.5mm i.d . . 17 ml min D

1 ml/min 2,4-DNPH ·reagent ~ KOH

Figure 3.5. Introductory system A to test component durability and function. 51

sample 0.17 ml/min 20 cm,0.5 mm l.d.

17 ml/mi 1m, 0.5mm i.d.

2,4-DNPH .17 ml/mi w 1m, 0.5mm i.d.

1 ml/min

0

w

Figure 3.6. Introductory system 8 to test FIA applicability to ASTM method. 52

0.17 ml/min w+------+------P----

.17 ml/min i.~.

2,4-DNPHLJ .17 ml/mi ~----~------~--~ 1 ml/min

1m, 0.5mm l.d. KOH

D

w

Figure 3.7. System 3. 53

0.17 ml/min

'

A B

Figure 3.8. System 4. 54

0.17 ml/min

0.7 ml/min

0.17 ml/min

0.17 ml/min w

Figure 3.9. System 5. 55

F NC :l...... H...... == NC 'm;~;~...... ;· ~: ..... :~ . NO ·······::::::: .- ·········:·:·:·:······· NO ~~HH~ :~m~m c: ~~~ii~!t=====~~~;t::::::~tt~:~:.;:~;::~::r:::~··l,c~------~~~~~---i!ltltf~f----i~~~~~------~r ·······::::::: .········::~:·:· NC ·······::::::: ········:::::::: NC ······· ········ ·······'•'• '•' r.- ········ NO ·······::::::: ~...." !tL ········::::::::•'•'• '•' NO ·······::::::: .,...... ········:::::::: .•••••••...... ~I...... ~ ~ ...... •••••••• . c ·······::::::: - ~ - ········:::::::: c ······· ········ NC ·······::::::: ········:::::::: NC 1!::::::······· ········:::::::: NO ·······::::::: ········:::::::: NO I!••• •e• • • • • • • • • ·=·=·=·~···... .········... ' ... c 1:::::::I ······ ········:::::::: c 1 ~=~:.:-,;:.:,i:.: .HHHH...... NC .:::::: :::::::: NC 1t:;·;~;· l1:;:;:; NO NO. ~~~··:·: ~~ · c cal ~ :::~?-: ~~:: 12 ;:>":::::.::: :·: @~ GND :~~~ NEUT C<~~

Wires A, B, C, F to solenoid valves. Wire E to pump. Wire D to external power input.

Figure 3.10. Wiring diagram for VIC! digital valve sequence programmer in system 7. 56

4 0'.17 ml/min 1 sample

2,4-DNPH

0.17 ml/min 2

carrier

0.7 ml/min

5

0.17 ml/min r----+------4-~ w

7

W+---1 D

Figure 3.11 . System 7. 57

0.7 ml/min

0.17 ml/min

0.17 ml/min

0.17 ml/min

5 6 w D

(1) 19 em, 28 ga ( 2 ) 10 em, 3 0 g a (3) 2 m, 30 ga (4) 17.5 em, 30 ga (5) 1 m, 28 ga (6) 6.1 m, 30 ga

Figure 3.12. System 8. 58

NC . NC ... NO ...... :::.. NO ········:::::::: c ········...... c ········...... :::::::: NC ········ NC .········...... NO c

NC NC

NO NO c c

NC NC

NO NO c

12

GND NEUT

Wire A to valves. Wire B to 12v power source. Wire c to one solenoid valve. Wire D to other solenoid valve. Wires E and F are paired to the solenoid valves with wires C and D, respectively.

Figure 3.13. Wiring diagram for VICI digital valve . 59

Overall picture

Figure 3.14. System 8 photos. 60

(A) KOH (B) Sample (C) 2,4-DNPH reagent (D) Peristaltic pump (E) Waste collector (F) Auto transformer (G) Chart recorder (H) Reaction coil for KOH reaction

Figure 3.14. (continued) 61

(A) Sample loop (B) 6-way valve, V1 (C) 3-way valve, V2 (D) 3-way valve, V3 (E) Reaction coil for KOH reaction

Figure 3.14. (continued) 62

(A) Solenoid valves (B) VIC! valve sequence programmer (C) Insulated cup containing reaction coils (D) Mini LED detector (E) Chart recorder

Figure 3.14. (continued) 63

(A) Fluke thermometer (B) Heater sandwiching reaction coils (C) Reaction coil for KOH reaction

Figure 3.14. (continued) CHAPTER IV

RESULTS AND DISCUSSION

4.1 Preliminary Results

2,4-dinitrophenylhydrazine has a of

(N02 )2C6H3NHNH 2 and a molar mass of 198. The optimum wavelength for the detection of the final product after addition of KOH was 480 nm.

4.1.1 Determination of Method Viability

Initial results with the manual standard method using 30-year­ old 2,4-dinitrophenylhydrazine reagent were unsatisfactory because of high blank values. Results obtained with freshly obtained 2,4-DNPH reagent showed low blanks, as predicted by the ASTM manual. Fresh

2,4-dinitrophenylhydrazine was thus found to be essential for the desired results.

4.1.2 Confirmation of 2,4-DNPH

The 2,4-DNPH on the shelf was nearly 30 years old. It was tested as detailed in Chapter III. A "nearly colorless" (Pesez &

Bartos, 1974, p. 257) blank was expected. The resulting blank was as an intense lime-green color as the sample. Consequently, fresh

2,4-DNPH was ordered and utilized.

4.1.3 Confirmation of Absorbance Maximum

Several carbonyl compounds were diluted in methanol and used as samples to test the ASTM manual method described in Chapter III. The maximum absorbance in the range of 300-820 nm wavelength was sought. some representative spectra are shown in Figure 4.1. The reference

64 65 solution for the spectra was the same solution as the measurement solution, except that the reference solution lacked carbonyl sample

(it contained methanol alone rather than a carbonyl compound diluted in methanol). In the wavelength range of 300-450 nm, a significant amount of absorbance occurred due to the yellow-orange color of the

2,4-DNPH reagent. Subtraction of the reference solution from the sample solution absorbance signals resulted in the apparent noise in that portion of the spectra. The optimal wavelength was determined from experiments like this. A wavelength of 480 nm was chosen as the optimum although a range of 430-520 nm may have provided satisfactory results.

4.1.4 Determination of Optimal Reagent Solvent

This ASTM procedure calls for distilled methanol solvent. The methanol is distilled so as to remove any carbonyl compounds. The procedure calls for 20 g of 2,4-dinitrophenylhydrazine and 2 mL of hydrochloric acid (HCl, sp.gr. 1.19) to be added to 4 liters methanol.

This is refluxed for two hours and then distilled using a 2-to-3-foot fractionating column. The first 200 mL are discarded and the distillation is only allowed to continue until 75% has distilled over, as the dry residue will violently decompose. The distillation is inconvenient as it requires a full workday to complete. To avoid the distillation problem, the use of other solvents was investigated.

Longer carbon-chain alcohols are less easily oxidized to carbonyl compounds. If another alcohol or some other suitable solvent having no significant concentration of carbonyl compounds could have been 66 found, it would have precluded the need for distillation. Another motivation to find a replacement for methanol was that higher alcohols have higher boiling points. Solvents with higher boiling points allow the reaction to be conducted at higher temperatures. At these temperatures, the reactions may be accelerated. The proposed solvents were tested as samples in the standard ASTM manual method against distilled methanol to determine whether the level of carbonyl impurities present were adequately low. If the solvent passed this test, it was then used to make the reagents needed. The reagents were tested against methanol reagents for performance. Experiments were done to compare acetonitrile to undistilled methanol as a solvent.

The expectation was that the acetonitrile would be free of carbonyl impurities expected in the methanol. Representative data are reported in Figure 4.2. Ordinarily, the standard ASTM protocol results in a lime-yellow colored solution after the first incubation. After the

KOH is introduced, the solution turns black. The black color quickly fades to a stable wine-red to brown color during the second incubation. Once this occurs, the solution's absorbance is read. By replacing methanol as the solvent in the 2,4-DNPH reagent with acetonitrile, a temporal increase in baseline absorbance is observed instead. This disqualified acetonitrile as a possible methanol replacement. Other experiments revealed that ethanol can be used in the 2,4-dinitrophenylhydrazine reagent and the potassium hydroxide reagent. 1-propanol formed an immiscible layer with the aqueous potassium hydroxide phase. Other solvents, such as 67

N,N-dimethylforamamide, acetonitrile, 2-methoxyethanol, and ethylene glycol, were found similarly inapplicable. (See Table 4.1.)

4.2 Results with System 2: Introductory Automated Systems

One of the introductory systems was designed to simply establish the good function and durability of the various component parts; this was accomplished. A different introductory system was devised later

for establishing compatibility of FIA and the ASTM method. Results were monitored for reproducibility. Peak groups had standard

deviations of 0.29 and variance of 0.083. Data is presented in

Table 4.2. Processed data is charted in Figure 4.3.

4.3 Results with System 3

This system yielded good results. A typical chart output is

shown in Figure 4.4. The jagged appearance of some peaks was

attributed to poor mixing of the solutions or pump noise. Some processed data are illustrated in Figures 4.5 through 4.7. Detector cells for the Kratos 757 leaked or were broken during use and were replaced as discussed previously. The second pump (the KOH pump) had a flaw in the casing and had to be returned to the manufacturer, as it leaked the caustic solution. Data are presented in Tables 4.3 through

4.6.

4.4 Results with System 4

White precipitate was visible in Flask B. This was confirmed by spectroscopic examination. The tubing deteriorated after constant 68

contact with the challenge solution. Viton tubing was, therefore,

necessary and could not be replaced with ordinary tubing.

4.5 Results with System 5

The VICI Digital Valve Sequence Programmer allowed for

experimentation with the time periods allowed for mixing, stopping,

and flowing of material in the system. No substantial differences in

results were noted between the 3-minute and the 4-minute reaction

periods. See Table 4.7. Experimentation also revealed that the

source of the carbonyl group was important. For example, the

absorbance (A.U.)/ppm C=O for formaldehyde was calculated to be

5.85E-4, whereas that for butyraldehyde was 2.79E-3. See Table 4.8.

See Figures 4.8 and 4.9 for typical processed data.

4.6 Results with System 6: Manual Optimization Studies

4.6.1 Determination of Minimum Standard Time

The standard manual ASTM method clearly states necessary

incubation times. The amount of time needed for the manual method was

determined by performing the testing and timing it with a stopwatch.

The minimum amount of analysis time needed for eight samples (while

following the ASTM protocol) is 55 minutes.

4.6.2 Determination of Optimum Concentrations of Acid and Base in Reagents

one idea to cut the amount of time needed for satisfactory

completion of the entire reaction was to delete the KOH reaction.

This idea was only explored briefly. It was abandoned when it was 69 observed that test solutions were all strongly absorbing lime colored solutions. The KOH reaction is essential to quantification.

Another experiment devised to cut reaction time by varying pH was to increase the amount of acid (HCl) in the 2,4-DNPH reagent.

This was compared to the normal ASTM method blank of distilled methanol sample. Other experiments involved using excess base. This moved the peak maximum to unsatisfactory ranges (wavelengths not useable on the Kratos 757 detector). (See Figure 4.10.) By changing the order of introducing reagents, the amount of time needed for reaction was not meaningfully affected. The spectra are shown in

Figure 4.11.

4.6.3 Determination of Minimum Reaction Times

The first incubation per ASTM instructions is 30 minutes long.

The reaction temperature was raised to 60°C--just below the boiling point of methanol. The results, using a dodecanal sample set, are shown in Table 4.9. The minimum incubation time yielding acceptable performance is 5 minutes.

The second incubation period per ASTM instructions is 12 minutes as previously noted. The minimum time for this incubation was determined by observing the absorbance at 1480 nrn. This was essentially stable by 6 minutes at room temperature. Representative spectra are presented in Figure 4.12. 70

4.6.4 Determination of Optimum Temperatures

The optimum temperature for the reaction was 60°C. This was determined to be the highest temperature at which the methanol-based reagents do not boil.

4.6.5 Determination of Minimum KOH Volumes

A study was undertaken to determine the minimum KOH volumes needed. The minimum volume of KOH was determined to be 9 mL versus the 21 mL. The representative data are shown in Table 4.10.

4.6.6 Determination of Optimum Hydroxide Source

Another study regarding the hydroxide portion of the reaction was undertaken. The standard method calls for a potassium hydroxide solution of 100 grams potassium hydroxide dissolved in 200 mL of water. This is cooled and diluted to 1 liter with methanol. It would be easier to use a commercially-prepared source of hydroxide, such as

50% NaOH or SN NaOH. These were compared to the ASTM-recommended potassium hydroxide solution. By a comparison to the standard manual method, rather than the standard 21 mL KOH for a 12-minute period, 1 mL of SN NaOH was added to the 2 mL sample/2 mL 2,4-DNPH reagent mixture for 12 minutes. At the end of this time period, the flask was diluted to the mark with water and the absorbance measured. The same procedure was used with the 50% NaOH, but 5 mL of the NaOH was used.

Data from this experimentation is shown in Table 4.11. Presumably, the alcohol component of the KOH is essential to the reaction and the hydroxide should not be concentrated. A later experiment using a KOH 71 solution composed of 100 g KOH dissolved in 100 mL water, cooled and diluted to 500 mL with methanol was performed on an automated system.

Peak height was significantly lowered, as demonstrated in the representative example in Figure 4.13. The performance of the ASTM potassium hydroxide solution was superior.

4.7 Results with System 7

Where a single peak was expected, a double peak occurred

(Figure 4.14). This meaningless second peak (ghost peak) was removed

by heating both of the incubation/mixing coils. A representative

chart output is shown in Figure 4.15.

The initial results appeared to be uniform and proportional to

the concentration of the carbonyl groups present in the sample

solutions. Tables 4.12 through 4.20 reflect typical data from

system 7. Where manually 8 samples can be tested in 55 minutes, this

system provides results for 16 samples in the same period of time.

Detector cell leaks or breakages were dealt with by replacing the cells, as discussed previously. The new detector, the mini LED detector, was implemented. Plotting the absorbance (x-axis) and the

concentration (y-axis) of a calibration set of samples reveals a line whose slope may be referred to as the calibration sensitivity. A high calibration sensitivity is desirable as it reflects a detector's heightened ability to detect and display sample concentration differences in smaller increments. The superior performance of the mini LED detector was readily evidenced by its having higher calibration sensitivities than the Kratos 757 detector. These 72 calibration sensitivities are noted in Tables 4.21 through 4.24. It worked as well or better than the Kratos 757. (See Figure 4.16.) The new detector was put into full-time operation.

This system did not show significant baseline drift. However, this was a stop-flow system. It is necessary to have the stop and flow periods adjusted perfectly to prevent a portion of the sample from remaining in the detector cell. That remaining portion would cause the signal to not return to the baseline, thus an apparent shift of the baseline would be observed. Although the absolute values of successive injections did not change, the baseline shift (the extent of which varied with injected concentration) was aesthetically unsatisfactory. Changing the sample size by cutting the sample loop

in half did not solve the shift problem. Changing the timing of the stop and flow periods did not really solve the problem because it was not possible to achieve a perfect adjustment as required. A new system would need to be designed and built to solve the shift problem.

(See Figures 4.17 through 4.29.)

4.8 Results with System 8

The new system, by virtue of its smaller overall volume, required less reagent and sample than previous systems. The baseline was effectively stabilized in this system. (See Figures 4.30 and

4.31.) Results were satisfactory. Representative data are presented in Tables 4.25 and 4.26. The absorbance (A.U.)/carbonyl concentration

(ppm c=O) varies among different carbonyl group sources. This is probably due to shifts brought about by different degrees of 73 conjugation in reaction product molecules. Some of the samples produced the same absorbance readings no matter what the carbonyl group concentration (for example, benzaldehyde, Table 4.26). This

apparent lack of response may be the result of little or no reaction or it may be because the reaction products formed do not absorb maximally at this wavelength. Most of the samples tested, however, do

react in such a way that the carbonyl group concentration may be

quantitated. A calibration curve must be devised for any substance

tested due to unique responses per carbonyl group source. In this

way, satisfactory quantitation of carbonyl group concentration in

samples may be achieved more quickly and with less reagent cost than

by the ASTM manual method. 74

Table 4.1. Comparison of solvents as blanks.*

Absorbance Solvent A., nm Reading for "Blank"

Undistilled methanol 430 0.341 480 0.116 520 0.095

Distilled methanol 430 0.292 480 0.077 520 0.052

Distilled 2-methoxyethanol 430 2.864 480 2.266 520 2.659

Distilled 2-methoxyethanol** 430 2.797 480 2.228 520 2.630

2-Propanol (spectroscopy grade) 430 1.098 480 0.502 520 0.532

2-Propanol 430 1.800 480 0.872 520 0.937

Ethylene glycol 430 0.384 480 0.092 520 0.058

* Recommended absorbance for blank at 248 nm is less than or equal to 0.08. ** Replicate. 75

Table 4.2. System 2, data set 1.

C=O cone. Absorbance C=O Source (ppm) (A.U.) A.U.-Methanol A.U.

MIBK 5 0.0565 0.0135 15 0.0725 0.0295 25 0.0995 0.0565 25 0.0956 0.0526 so 0.1648 0.1218

Methanol 0.0430

MIBK: y = 2.46E-3x + 0.04, r2 = 0.9896. 76

Table 4.3. System 3, data set 1 (S min./S min. incubations).

C=O cone. Absorbance C=O Source (ppm) (A.U.) A.U.-Methanol A.U.

Formaldehyde s 0.0280 0.01 1S 0.023S 0.01 2S 0.033S 0.02 so o.osso 0.04

Methanol 0.018S

MIBK 2S 0.018S 0.01 so 0.1140 0.10

Formaldehyde: y = 6.7SE-4x + 1.90E-2, r 2 = 0.8732. Formaldehyde-methanol: y = 7.2E-4x + 3.02E-3, r 2 = 0.9S3S. 77

Table 4.4. System 3, data set 2 (S min./S min. incubations).

C=O cone. Absorbance C=O Source (ppm) (A.U.)

MIBK so 0.148S so 0.128S

Formaldehyde s 0.0420 1S 0.0430 2S O.OS80 so 0.084S 2SO 0.261S

Formaldehyde: y 9.07E-4x + 0.04, r 2 = 0.9984. 78

Table 4.5. System 3, data set 3.

C=O cone. Absorbance C=O Source (ppm) (A.U.)

2,4-Pentanedione 15 0.0325 25 0.0381 50 0.0345

2,4-Pentanedione: y = 1.85E-Sx + 0.03, r 2 = 0.0138. 79

Table 4.6. System 3, data set 4.

C=O cone. Absorbance C=O Source (ppm) (A. U. ) A.U.-Methanol A.U.

Glyoxal 5 0.0365 0.02 15 0.0645 0.04 25 0.0940 0.07 50 0.1610 0.14 50 0.1460 0.12

Butyraldehyde 5 0.0360 0.01 15 0.0755 0.05 25 0.1093 0.09 50 0.2306 0.21 250 0.8263 0.81

MIBK 50 0.1175 0.10

Methanol 0.0213

Butyraldehyde via AU: y = 3.18E-3x + 0.04, r 2 = 0.9962. Butyraldehyde via AU-methanol: y = 3.22E-3x + 1.21E-2, r 2 = 0.9957. Glyoxal via AU: y = 2.57E-3x + 0.03, r 2 = 0.9880. Glyoxal via AU-methanol: y = 2.48E-3x + 6.11E-3, r 2 = 0.9957. 80

Table 4.7. System 5, data set 2 (Kratos 0.1 AUFS).

C=O cone. Absorbance C=O Source (ppm) (A.U.)

4 min./4 min. incubations

Butyraldehyde 50 0.0083 250 0.0374

p-Dimethylaminobenzaldehyde 25 0.0176 50 0.0342 250 0.0845

Glyoxal 50 0.0023

Formaldehyde 50 0.0023 250 0.0097

2,4-Pentanedione 50 250 0.0026

1-Benzoylacetone 50 0.0008 250 0.0034

3 min./3 min. incubations

MIBK 50 0.0038 250 0.0093

Glyoxal 250 0.0228 250 0.0266

Formaldehyde 50 0.0020 250 0.0096

2,4-Pentanedione 50 0.0004 250 0.0026

1-Benzoylacetone 50 0.0008 250 0.0032 81

Table 4.8. System 5, data set 1 (3 min./3 min. incubation periods).

C=O cone. Absorbance C=O Source (ppm) (A.U.)

Kratos 0.2 AUFS

Butyraldehyde 15 0.0432 25 0.0680 50 0.1324

MIBK 50 0.0538 250 0.1400

2,4-Pentanedione 50 0.0048 250 0.0252

Formaldehyde 50 0.0298 250 0.1436

Glyoxal 50 0.1212 250 0.3505

p-Dimethylaminobenzaldehyde 50 0.3520

Kratos 2.0 AUFS

p-Dimethylaminobenzaldehyde 25 0.2780 50 0.7490 250 1.5600

Glyoxal 250 0.3880

MIBK 5 0.0600 5 0.1000

Butyraldehyde 250 0.5880

Using A.U.s, the following calculations were done:

Butyraldehyde: y = 2.55E-3x + 4.58E-3, r 2 = 0.9999. p-Dimethylaminobenzaldehyde: y = 5.07E-3x + 3.13, r 2 = 0.9288. MIBK: y = 3.01E-4x + 6.06E-2, r 2 = 0.7914. 82

Table 4.9. Minimum incubation time study for first incubation reaction.*

Readings at A480 nm

30 Minutes 20 Minutes 10 Minutes 5 Minutes Sample

0.074 0.069 0.073 0.070 blank

0.273 0.281 0.279 0.279 15.4 ppm c = 0 MIBK

0.409 0.421 0.408 0.402 25.7 ppm c = 0 MIBK

0.083 0.074 0.075 0.079 blank

0.493 0.504 0.548 0.566 15.1 ppm c = 0 dodecanal

0.801 0.800 0.847 0.890 25.1 ppm c = 0 dodecanal

* Reaction held at 60°C, times varied from 30-minute standard method as noted. 83

Table 4.10. Study of minimum volume of KOH.

Absorbance mL KOH ppm C 0 Dodecanal Reading, A-480

21 mL blank 0.107 21 mL 14.4 0.352 21 mL 24.0 0.600

10 mL blank 0.096 10 mL 4.8 0.174 10 mL 14.4 0.305 10 mL 24.0 0.456 10 mL 48.0 0.627

9 mL blank 0.221 9 mL 4.8 0.173 9 mL 14.4 0.352 9 mL 24.0 0.474 9 mL 48.0 0.644

8 mL blank 0.099 8 mL 4.8 0.143 8 mL 14.4 0.248 8 mL 24.0 0.311 8 mL 48.0 0.472

4 mL blank 0.097 4 mL 4.8 0.110 4 mL 14.4 0.153 4 mL 24.0 0.218 4 mL 48.0 0.333 84

Table 4.11. Results of experiments with different sources and concentrations of hydroxide for reaction.

Carbonyl Source Hydroxide Source (Four Concentrations) Slope

Standard method MIBK 0.335 Dodecanal 0.327 Acetaldehyde 0.212 Acetone 0.314

1mL SN NaOH MIBK 0.329 MIBK 0.296 MIBK 0.300 MIBK 0.296 Dodecanal 0.019 Dodecanal 0.008 Acetaldehyde 0.170 Acetone 0.296

5mL 50% NaOH MIBK 0.109 Dodecanal 0.108 Acetaldehyde 0.051

Comparison:

Standard method: 21 mL KOH, wait 12 minutes, read. 5N NaOH: 1 mL SN NaOH, wait 12 minutes, dilute to mark with water, read. 50% NaOH: 5 mL 50% NaOH, wait 12 minutes, dilute to mark with water, read. 85

Table 4.12. System 7, data set 1 (Kratos 0.5 AUFS).

C=O cone. Absorbance C=O Source (ppm) (A.U.)

MIBK 5.14 0.093 15.70 0.097 51.40 0.175 258.00 0.670

MIBK: y = 2.33E-3x + 6.62E-2, r2 = 0.9983. 86

Table 4.13. System 7, data set 2 (Kratos 0.5 AUFS, room temperature 25°C).

C=O cone. Absorbance C=O Source (ppm) (A.U.)

MIBK 5.14 0.1140 25.00 0.1160 51.40 0.1515 257.00 0.3625 257.00 0.3600

MIBK: y = 1.02E-3x + 9.98E-2, r 2 = 0.9975. 87

Table 4.14. System 7, data set 3 (Kratos 0.5 AUFS, 55°C).

C=O cone. Absorbance C=O Source (ppm) (A.U.)

MIBK 5.14 0.1100 15.40 0.1125 15.40 0.1280 25.70 0.1150 51.40 0.1725 257.00 0.4000

MIBK: y = 1.17E-3x + 0.10, r 2 = 0.9919. 88

Table 4.15. System 7, data set 4 (Kratos 0.5 AUFS, 60°C).

C=O cone. Absorbance C=O Source (ppm) (A.U.)

MIBK 5.14 0.0100 15.4 0.0400 25.7 0.0800 51.4 0.1570 51.4 0.2900

Dodecanal 5.02 0.0100 15.1 0.0370 25.1 0.0600 50.2 0.1240

MIBK: y = 4.81E-3x + -2.81E-2, r 2 = 0.8142. Dodecanal: y = 2.51E-3x + -2.13E-3, r 2 = 0.9996. 89

Table 4.16. System 7, data set 5 (Kratos 0.5 AUFS, 55°C).

C=O cone. Absorbance C=O Source (ppm) (A.U.)

MIBK 5 0.0100 25 0.0975 50 0.2075

Dodecanal 5 0.0100 25 0.0925 50 0.1825 50 0.1915 250 0.5625

MIBK: y = 4.39E-3x + -0.01, r 2 = 1.000. Dodecanal: y = 2.11E-3x + 4.77E-2, r 2 = 0.9728. 90

Table 4.17. System 7, data set 6 (Kratos O.OS AUFS).

C=O cone. Absorbance C=O Source (ppm) (A. U. )

MIBK s o.ooos 2S 0.0033 so 0.006S 2SO 0.0249

Dodecanal s o.ooos 2S 0.0031 so 0.0064 2SO 0.0137

MIBK: y = 1.0E-4x + 7.9E-4, r 2 = 0.9962. Dodecanal: y = S.OE-Sx + 1.91E-3, r 2 = 0.9276. 91

Table 4.18. System 7, data set 7 (Kratos detector 0.02 AUFS).

C=O cone. Absorbance C=O Source (ppm) (A.U.)

MIBK 2S 0.0024 so 0.0040

Dodecanal s 0.0004 2S 0.0033 so 0.0066

Dodecanal: y = 1.4E-4x + -2.3, r 2 = 0.9993. 92

Table 4.19. System 7, data set 9 (Kratos detector 0.02 AUFS).

C=O cone. Absorbance C=O Source (ppm) (A.U.)

MIBK 5 0.0040 25 0.0028 25 0.0024 50 0.0042 50 0.0040

Dodecanal 5 0.0005 25 0.0028 50 0.0046 50 0.0050

MIBK: y = 1.0E-5x + 3.05E-3, r 2 = 0.1074. Dodecanal: y = 9E-5x + 1.9E-4, r 2 = 0.9852. 93

Table 4.20. System 7, data set 11.

Kratos Reading C=O cone. 0.02 AUFS C=O Source (ppm) (A. U. )

MIBK 5 0.0006 25 0.0032 50 0.0063 250 0.0211

Dodecanal 5 0.0005 25 0.0025 25 0.0024 50 0.0049 250 0.0117

Kratos - MIBK: y = 8.07E-5x + 1.14E-3, r 2 = 0.9913. Dodecanal: y = 4.20E-5x + 1.41E-3, r 2 = 0.9786. 94

Table 4.21. System 7, data set 16.

C=O cone. Absorbance (A.U.) Absorbance (A.U.) Time C=O Source (ppm) Kratos (1.0 AUFS) Mini LED (1 volt) (sec)

MIBK so 0.16S O.lSO

Dodecanal so 0.170 0.160 50 0.220 0.230 90 100 0.3SO 0.310 73 0.340 0.347 71 0.440 0.4SO 72 95

Table 4.22. System 7, data set 17.

Absorbance (A.U.) C=O cone. Absorbance (A.U.) Mini LED C=O Source (ppm) Kratos (0.2 AUFS) (0.25 V FS)

MIBK 5 0.006 0.0075 15 0.034 0.0350 50 0.032 0.0363

Dodecanal 5 0.020 0.0225 25 0.110 0.1175

Kratos - MIBK: y = 4.1E-4x + 1.44E-2, r2 = 0.3883. Mini LED - MIBK: y = 4.8E-4x + 1. SE-2, r2 = 0.4914. 96

Table 4.23. System 7, data set 18.

Absorbance (A.U.) C=O cone. Absorbance (A.U.) Mini LED C=O Source (ppm) Kratos (1 AUFS) (1 V FS)

MIBK 5 0.0075 0.0075 25 0.0200 0.0240 50 0.0400 0.0500

Dodecanal 5 0.0050 0.0050 25 0.0200 0.0300 50 0.0300 0.0500

Kratos - MIBK: y = 7.3E-4x + 3.16E-3, r 2 = 0.9953. Kratos - Dodecanal: y = 5.5E-4x + 3.69E-3, r 2 = 0.9683. Mini LED - MIBK: y = 9.SE-4x + 1.88E-3, r 2 = 0.9979. Mini LED - Dodecanal: y = 9.9E-4x + 1.89E-3, r 2 = 0.9837. 97

Table 4.24. System 7, data set 19.

Absorbance (A.U.) C=O cone. Absorbance (A.U.) Mini LED C=O Source (ppm) Kratos ( 1 AUFS) (1 V FS)

MIBK 25 0.0243 0.0300 25 0.0150 0.0200 50 0.0510 0.0580 50 0.0306 0.0525

Dodecanal 25 0.0245 0.0275 50 0.0550 0.0575 98

Table 4.25. System 8 carbonyl group response.

>C=O Source Response Slopejppm>C=O

MIBK 1.67

Dodecanal 0.70

Benzoyl acetone no response

Acetone 2.56

Formaldehyde 1.37

Butyraldehyde 3.42

Glyoxal 0.61

Isovaleraldehyde 6.00

Acetophenone 2.30

trans-Cinnamaldehyde 6.00

Benzaldehyde 0.45

p-Dimethylaninobenzaldehyde 8.48 99

Table 4.26. System 8, data set s.

C=O cone. Absorbance C=O Source (ppm) (A.U.) A. U. /ppm C=O

MIBK 25.1 0.038 l.SE-3 50.2 0.08 l.SE-3

Dodecanal 25 0.1 4.0E-3 so 0.1775 3.6E-3

Benzoyl acetone 34.9 0.01 2.9E-4 69.8 0.01 1.4E-4

Acetone 26.4 0.0575 2.1E-3 26.4 0.075 2.8E-3 52.8 0.125 2.4E-3

Formaldehyde 25.5 0.03 1.2E-3 51 0.066 1.3E-3

Butyraldehyde 25.4 0.088 3.SE-3 50.8 0.175 3.SE-3

Glyoxal 23.9 0.0925 3.9E-3 47.8 0.107 2.2E-3

p-Dimethylaminobenzaldehyde 25.1 0.289 1. 2E-2 50.2 0.501 1. OE-2

Isovaleraldehyde 26.1 0.21 8.0E-3 52.2 0.3675 7.0E-3

6.SE-3 Acetophenone 25.1 0.31 50.2 0.163 6.2E-3

6.0E-3 trans-Cinnamaldehyde 25.1 0.15 50.2 0.30 6.0E-3

7.7E-3 Benzaldehyde 22.2 0.17 44.4 0.18 4.0E-3 100

. ·- C\1 ., ,1 I I I··' \I I I I Abs. I ',I I I I I \ I I I 'I I •. •.. '·. ~~~lt./fr11~\~~~~... 1 l).r/ - It). I 1 300 500 700 .A., nm

A) Acetone sample

Abs.

300 500 700 J.' nm

B) Methylisobutylketone (MIBK) sample

Figure 4.1. Determination of maximum absorbance wavelengths. 101 -

Abs. I . r--. / l

·~--~--~--~----~~--~--~--~-- -- 300 500 700 J.., nm

C) Butyraldehyde sample

Figure 4.1. (continued) 102

... -.

0~--~---L--~----~--~--~----~------J 400 500 600

A, run

(A) Time (t) = 0 seconds (B) Time (t) = 30 seconds (c) Time (t) = 1 minute (D) Time (t) = 1 minute 30 seconds (E) Time (t) = 2 minutes (F) Time ( t) = 2 minutes 30 seconds

A) Using acetonitrile instead of methanol in mixture with 2,4-DNPH and KOH. Absorbance measured every 30 seconds.

Figure 4.2. Acetonitrile experiments. 103

Abs.

0~--~--~--~--~~~--~--~~~~~~ 400 500 600

A., nm

(A) Time ( t) = 0 seconds (B) Time (t) = 30 seconds (C) Time ( t) = 1 minute (D) Time ( t) = 1 minute 30 seconds (E) Time (t) = 2 minutes (F) Time (t) = 2 minutes 30 seconds (G) Time ( t) = 3 minutes (H) Time ( t) = 3 minutes 30 seconds (I) Time (t) = 4 minutes

B) Comparison of methanol blank with absorbance measured every 30 seconds.

Figure 4.2. (continued) 104

Abs.

.A., run

(A) Time ( t) 0 seconds (B) Time (t) = 30 seconds (c) Time ( t) = 1 minute (D) Time ( t) 1 minute 30 seconds (E) Time (t) = 2 minutes (F) Time ( t) = 2 minutes 30 seconds (G) Time ( t) = 3 minutes (H) Time ( t) = 3 minutes 30 seconds (I) Time (t) 4 minutes ( J) Time ( t) = 4 minutes 30 seconds (K) Time ( t) = 5 minutes

C) Acetonitrile is substituted for methanol in 2,4-DNPH reagent; KOH is added. MIBK is added as a sample; absorbance increases every 30 seconds.

Figure 4.2. (continued) 105

Abs.

Q ·4~=oo~--~--._--~~--~--~------so~o

A., nm

(A) Time (t) = 4 minutes (B) Time (t) = 4 minutes 30 seconds (C) Time (t) = 5 minutes (D) Time (t) = 5 minutes 30 seconds (E) Time ( t) = 6 minutes

D) 2,4-DNPH made with acetonitrile; sample MIBK. Absorbance measured every 30 seconds.

Figure 4.2. (continued) 106

Abs.

600 .A., nm

(A) Time (t) = 0 seconds (B) Time (t) = 12 minutes

E) Acetonitrile solvent 2,4-DNPH reagent, add KOH, and measure absorbance.

Figure 4.2. (continued) 107 ...

Abs.

I J 190 A., nm 820

F) Use methanol 2,4-DNPH reagent and acetonitrile as sample. Absorbance measured every 12 minutes .

.-r------,------,

.. ·r-. I ,.····' ', / I I I r I Abs. 1 i \ I I r I i I I I I I __. ------.. _ .• ...... _...... , .. - "'·-......

OL-L-----~----~-----~---.~---­ ·--..-- 190 400

A.' nm

G) Use methanol 2,4-DNPH reagent and acetonitrile as sample. Absorbance measured every 12 minutes

Figure 4.2. (continued) 108

Abs.

,.

c. ~--~--_. ____._ __ _.____ ~ __ .___ ~----~--~~~ 400 600

.l.., nm

(A) Time ( t) = 40 seconds (B) Time ( t) = 1 minute (c) Time (t) = 1 minute 20 seconds (D) Time (t) = 1 minute 40 seconds (E) Time (t) = 2 minutes (F) Time ( t) = 2 minutes 20 seconds (G) Time (t) = 2 minutes 40 seconds (H) Time ( t) = 3 minutes (I) Time (t) = 3 minutes 20 seconds ( J) Time ( t) = 3 minutes 40 seconds (K) Time (t) = 4 minutes (L) Time ( t) = 4 minutes 20 seconds

H) 2,4-DNPH reagent made with acetonitrile. Absorbance measured every 20 seconds.

Figure 4.2. (continued) 109

-

Abs.

0~4-0~0----~------~--~~~~--~--~6~0~0

A., run

(A) Time (t) = 30 seconds (B) Time (t) 1 minute (c) Time ( t) 1 minute 30 seconds (D) Time (t) = 2 minutes (E) Time (t) = 2 minutes 30 seconds (F) Time ( t) 3 minutes (G) Time ( t) 3 minutes 30 seconds (H) Time ( t) = 4 minutes

I) 2,4-DNPH reagent made with methanol. Stabilization with the methanol reagent occurs sooner. Absorbance measured every 30 seconds.

Figure 4.2. (continued) 110

0.200

,...... ,_. 0.160 ***** MIBK * ::> <( .._., 0.120 Q) u c _g 0.080 L 0 (f) _() <{ 0.040

0.000 0 20 40 60 C=O Concentration (ppm)

MIBK: y = 2.46E-3x + 0.04, r 2 = 0.9896.

Figure 4.3. Processed data from system 2. 111

1------. - ~ ...- I------______--_ -_-:._-.::..·...::===:..::...::==--=--=-=-::--_:_-::_·-:..::- _=:.:.._ ------· ______..:..:..._::""'-:::.:.:::_::: . .. ------·: =~--~=~~ -~.= -~-==.=-= --~ __ : _-_ ~- ~:~~ - ----~~-:

~------~---- ~- _ ------~=---.::..- ~-~-=~~~==--==.~~=~~- ~- -~-

Abs.

------·--·- . . ···- - ·- _... _ ------·-- ~-----~

6 mm/min.

(A) 5.14 ppm C=O (B) 25.7 ppm C=O MIBK

Figure 4.4. Typical chart output from system 3, demonstrating clear reproducible peaks. 112

0.120

***** MIBK ***** Formaldehyde ,.-...... :=> 0.080 ...... _,

Q) () c 0 _!) ~ 0.040 en ...0 <(

0.000 0 20 40 60 C=O Concentration (ppm)

Formaldehyde: y = 0.00067486x + 0.0189721, r 2 = 0.8632 .

Figure 4.5. Data set 1 from system 3. 113

0.300

***** MIBK 0.250 ***** Formaldehyde ,.--.... :::> <( 0.200 ""--""'

Q) (.) 0. 150 c * 0 ...0 * ~ 0.100 C/) _() <( 0.050

0.000 100 200 300 C=O Concentration (ppm)

2 Formaldehyde: y = 0.000907155x + 0.0352063, r = 0.9984.

Figure 4.6. Data set 2 from system 3. 114

1.000

***** Butyraldehyde *****1 MIBK ,..-..... 0.800 • • " ' Glyoxal ::) <( ...._,0.600 Q) u c _g 0.400 '- 0 (f) ..!) <( 0.200

0.000 0 100 200 300 C=O Concentration (ppm)

Butyraldehyde: y = 0.0318483x + 0.0144464, r 2 = 0.9962 .

Glyoxal: y = 0.00257461x + 0.00465629, r 2 = 0.9880.

Figure 4.7. Data set 3 from system 3. 115

0.400 I I I I t Butyraldehyde xxxxx MIBK ***** 2.4-pentanedione ,.,.,.,.,. Formaldehyde -;o.3oo ~Glyoxal ...... _...<{

Q) u 0.200 c 0 ...!) L 0 (/) ...0 0.100 <{

0.000 0 100 200 300 C=O Concentration (ppm)

Butyraldehyde: y = 2.55E-3x + 4.58E-3, r 2 = 0.9999.

MIBK: y = 3.01E-4x + 6.06E-2, r 2 = 0.7914.

Figure 4.8. Data set 1 from system 5. 116

0.120 l}l}l}l}l} Butyraldehyde I I I I I p-Dimethylaminobenzaldehyde xxxxx Glyoxal *****Formaldehyde ...,-..... ***** 2,4-pentanedione ~ =:> >PPPP~ 1-benzoylacetone ~"»"»"»~ Ml BK ...... _....

Q) () c 0 _!) ~ 0.040 (/) _!) y <{ >< + * 0.000 0 100 200 300 C=O Concentration (ppm)

Figure 4.9. Data set 2 from system 5. 117

Abs.

0~--~--~--~------~--~--~--~--~--~ 300 500 A, run

Abs.

A, run

(1) basic, 30% KOH (2) strongly acidified, 30% HCl (3) acidified, 4% HCl (ASTM-recommended)

Figure 4.10. Comparison of blanks. 118

Abs.

A., run

Starting with the KOH, the sample, then the 2,4-DNPH were added. The sample absorbance at A-480 run was almost as low as that of the blank.

Figure 4.11. Change order of introducing reagents. 119

.---r--T----.-----.,.--- · r---r-] I ~ j Abs. I j

.A., run

(A) Time (t) = 0 seconds (B) Time (t) = 30 seconds (C) Time (5) = 1 minute

A) Sample from 0 to 1 minute. Absorbance measured every 30 seconds.

Figure 4.12. Observations of second incubation period. 120

Abs.

5 0

A, run

(A) Time (t) = 0 seconds (B) Time ( t) = 30 seconds (c) Time (t) = 1 minute (D) Time (t) = 1 minute 30 seconds (E) Time (t) = 2 minutes (F) Time ( t) = 2 minutes 30 seconds (G) Time (t) = 3 minutes (H) Time (t) = 3 minutes 30 seconds (I) Time (t) = 4 minutes

B) Same sample, 4 minutes. Absorbance measured every 30 seconds.

Figure 4.12. (continued) 121

Abs.

A., run

(A) Time ( t) = 0 seconds (B) Time (t) 30 seconds (c) Time (t) 1 minute (D) Time ( t) = 1 minute 30 seconds (E) Time (t) = 2 minutes (F) Time ( t) = 2 minutes 30 seconds (G) Time ( t) 3 minutes (H) Time ( t) = 3 minutes 30 seconds (I) Time ( t) = 4 minutes ( J) Time ( t) 4 minutes 30 seconds (K) Time (t) 5 minutes (L) Time ( t) = 5 minutes 30 seconds (M) Time ( t) = 6 minutes ( N) Time (t) 7 minutes (0) Time (t) = 8 minutes (P) Time (t) = 9 minutes (Q) Time (t) = 10 minutes (R) Time (t) = 11 minutes ( s) Time ( t) = 12 minutes

C) Same sample at 12 minutes. Stabilization at A.480 nm occurred at six minutes. Observations repeated every 30 seconds until time equaled six minutes. After that, measurements were repeated every minute. Reaction was held at room temperature.

Figure 4.12. (continued) 122

------~ -- --- . ------..,------~ · ~====:::=::·: -=· : -__ :-· ------·- - -::--:::--- ._ . .- 1~-m..~------. _-__- -====- :-.=.:._ :_- ·=--=-~...:.:::

______A .-.-~-- --=-~A:-_ . ---f-t------· --- -=~ =1 B -:_-_ _- -··u-- -·- --··-'------./\~ -- -·- ·------6-::. Abs. /- . ~ -- - ·_=-;,.-·- : ·==-=--=--==:::::::::.--=-=-:.. ------. t--· / . x-_. _ :~-._....;..-=-;;;;;...- - x-·- : = ~- -~ . :-.:--" _=\-_- ..-- _. -·_ --. --·- · ------­' ·

---·- ··------­ -- ---·-- - ~ . . .. - · ----- .. -- ·------~ -- · · ·· - · -- · . ------·-· - ·------. --.

75 mrn/min.

Figure 4.13. Comparison of peaks for 25 ppm C=O (MIBK source) using A (ASTM-recommended KOH) versus B (concentrated KOH). 123

----- ·------.;. ______._ _ __ ------· 10mm -.

·--- · --· ·- - - - ··· - · ·- ---· ···- ---~- - -

Abs.

·---·· - ----­ ------~ ------·------·-· -----· -- --

11 mrn/min.

sample illustrated had C=O concentration of 15.4 ppm. Carbonyl source was MIBK.

Figure 4.14. A ghost peak. 124

----·------··- -·------. . ------· ----- .. ------·------...--- - .... - ... ------.. ------·- -·- - - -~ . ------. - - · - -- . --- ..1 - ··· - -:_. - :.:..-:=:"7":~-·.:=-~-==~-=::::_------·----- · ·· - -· ·- - . - -- ·------_-_: :::· ·.-:::-.-::.-=---:-:::=-:=~------:::-::.-: : -

--.----_-:::-_--. ---· -- . . f--· ------·-·-----

---..- _·-.-.-.--.·::: -I- - ~:_:::~ ~ -~:.·.- _· _·_ _...:_ _.

Abs. ------. 11· ------·--- .. ·· ._: __ · :~- - .. ~--- ····· V_- · ------..._"';_:J _:__L=-:===:=· =· =·==· ::''!._· ~· ~====:::::::::==~~~ -··· ------·- ·· --· ------·------·--·-·--

-- '(

11 mm/min.

Figure 4.15. Chart output after ghost peak removal. 125

-~ .: · ------_____=..::.--.:...=- ·-----

---- ·· -· ~- - . -- -· ------·· -- .. ---· - -- . -----·-· ------·------~_:__ __ :...::.,_ __ ·:.. - ':..:":"""':..': ... -- .------. _ --- -- . -- .. ····---·- Abs. . - ----. ------:::::::&:. _· ==·=- --= --=.:. Kra""los : -~------=-===- . - ~ ··::: ,------+11-'=--.._-- . -_.--.-- --=~- 1=-f---. --.-=-· ~-=----=----~~-_-._~_-___ ----·- . -- --- v v v _ - ...... :..:. . --- --=- ., ...:...:..:.::.. ---

30 mm/min.

Figure 4.16. Comparison of typical chart output of Kratos 757 detector versus the mini LED detector. 126

·--I • . I t- ... - ' - .. • .:=:: ·~'---tt=:. .. :1=: ::-~ ; _ ~ _: -~: ~ -l -:r-- :---L- .·.· --=-- -·-. 1--- r- t=_~ -----4 - · '--t-- · I · • - I- · I -1 ~ -· , •• • 'I - - _1---· -r--- I- _,_ . . -. - I' - ... ' --- • - I -- . .• -r-- _j -t---4. - --.. : ·__ : __ .- '; __ : _-_-: -- . . "'!:'=.! -- ··- _: -.---

- .. - -· _ _===: _:==:=::.:::.::. :__ .:..-=::=. :== : = ~: -=: · =: -=-- ·=·!::-.: -. - -· ..:_- .. ~------· . --.-- _------,------· _.. __ . _-_-_ -_._--_·----- :;___ -_·=-tt- _ ---.-~=--i =-<~- -_:. __:__ :_ __ : ~ ~.... -~ -:-~:. _;: _ __ P i - -:~ -: -: __ · r-~=~ :~~ ::?----

Abs.

:__ -__ ;.= -- ~ :.: __:~ ;I ~==:-:_:~ ~-;-~ :._ ~ - --. ~ __:__ -- - · ----4+---tt---+r-- -- -<~------~-~--~J\- - --- ·- .. - - -- I -- :/ :L:;_}_ L ::·_ _-_ :- . -~ -- -__c _ - - - ~: ... --- 1 . L....;:...j Li r ;__ . : ~ = -s --_- ~ :- :. -__: ·_=-_·-- --+1f---+1f---+l-----fl---- ·_- ----,-1-----tt-- ___ -L------~=~~ : -- - - -~ --;;_: ~~- :=E~ ==-· _:: ~=- _,_ ---.--- - -1\ ·----t

6 mm/min.

(A) 250 ppm C=O (B) 5 ppm C=O (C) 50 ppm C=O (D) 25 ppm C=O

(Carbonyl source, MIBK)

Figure 4.17. Baseline shift in system 7. 127

0.800 ***** MIBK

,--.... :) 0.600 . ....__,<(

Q) 0 0.400 c 0 ...0 L 0 (/) ..0 0.200 <(

0.000 0 100 200 300 C=O Concentration (ppm)

MIBK: y = 2.33E-3x + 6.629E-2, r 2 = 0.9983.

Figure 4.18. Data set 1 from system 7. 128

0.400

***** MIBK

-;0.300 ...... _..,<(

(l) (.) 0.200 c 0 .0 L 0 U) .0 0.100 <( .

0.000 0 100 200 300 C=O Concentration (ppm)

MIBK: y = 1.02E-3x + 9.984E-2, r 2 = 0.9975.

Figure 4.19. Data set 2 from system 7. 129

0.500 x x x X* MIBK

....--.... 0.400 • ::). <( ...... _,0.300 Q) () c _g 0.200 L 0 (/) ..0 <( 0.100

0.000 0 100 200 300 C=O Concentration (ppm)

MIBK: y = 1.17E-3x + 0.10, r 2 = 0.9919.

Figure 4.20. Data set 3 from system 7. 130

0.300 ***** MIBK ***** Dodecanal .....-.,.. :J <( 0.200 ......

Q) u c * ' 0 ..0 ~ 0.100 en ..0 <{

0.000 0 20 40 60 C=O Concentration (ppm)

MIBK: y = 4.81E-3x + -2.81E-2, r 2 = 0.8142.

Dodecanal: y = 2.51E-3x + -2.13E-3, r 2 = 0.9996.

Figure 4.21. Data set 4 from system 7. 131

0.600 ***** MIBK ****!f-

...--...... => .....__....

Q) u c 0 ..0 0 0.200 (/) ..0 <(

0.000 0 100 200 300 C=O Concentration (ppm)

MIBK: y = 4.39E-3x + =-0.01, r 2 = 1.000~

Dodecanal: y = 2.11E-3x + 4.77E-2, r 2 = 0.9728.

Figure 4.22. Data set 5 from system 7. 132

0.008 ***** Dodecanal ***** MIBK -;o.oo6 . ..._..,<(

Q) () 0.004 c 0 ..0 L. 0 Ul ..0 0.002 <{

0.000 0 20 40 60 C=O Concentration (ppm)

Propanol 2,4-DNPH reagent, system at 55°C.

Dodecanal: y = 5.0E-5x + 1.91E-3, r 2 = 0.9276.

Figure 4.23. Data set 6 from system 7. 133

0.020 l}l}Rl}l} Dodecanal ~~>?>?>? MIBK

,.--..... => 0.015 . <{ '--"'

Q.) () 0.010 c 0 _Q L. 0 rn ...0 0.005 <1::

0.000 0 100 200 300 C=O Concentration (ppm)

MIBK: y = 5.88E-Sx + 1.14E-3, r 2 =0.9998.

Dodecanal: y = 4.20E-Sx + 1.41E-3, r 2 = 0.9675.

Reaction coil 1 at 69°C; coil 2 at 78°C.

Figure 4.24. Data set 8 from system 7. 134

0.030 ***** MIBK ***** Dodecanal

=> <( ..___.... 0.020

Q) u c 0 ..0 0 0.010 en ..0 <(

0.000 0 100 200 300 C=O Concentration (ppm)

MIBK: y = 1.0E-5x + 3.05E-3, r 2 = 0.1074.

Dodeconal: y = 9E-5x + 1.9E-4, r 2 = 0.9852.

System held at 52°C; standard methanol reagents, new cell.

Figure 4.25. Data set 9 from system 7. 135

40

J}l}l}l}l} Dodecanal >¢>¢>¢>¢>¢ MIBK

~30 ::>...... _.,<(

Q) u 20 c 0 _Q L 0 _2 10 <(

o~~~~~~~~~~~~~~~~~~ 0 20 40 60 C=O Concentration -(ppm)

MIBK: y = 8E-Sx + l.lSE-3, r 2 = 0.9913.

Dodecanal: y = 4E-Sx + 1.4E-3, r 2 = 0.9786.

Instrument held at 55°C; propanol 2,4-DNPH reagent.

Figure 4.26. Data set 11 from system 7. 136

0.500 ***** Dodec.a.nol >¢>QPP> MIBK ,-..... 0.400 :::::>. <( "-" 0.300 (l) (.) c _g 0.200 L 0 Ul ...0 <( 0.100

0.000 0 100 200 300 C=O Concentration (ppm)

MIBK: y = 0.00162977x + 0.021923, r 2 = 1.000.

Figure 4.27. Data set 13 from system 7. 137

0.060 xxxxx Mini Dodecanal ***** Mini MIBK ,.,.,..,..,.. Kratos Dodecanal >¢>¢>¢>¢>¢ Kratos MIBK ,-...... * :::> <( 0.040 ...... _....

Q) u c 0 ..0 ~ 0.020 U) ..0

0.000 0 20 40 60 C=O Concentration (ppm)

Kratos--MIBK: y = -5.06x + 25.2, r 2 = 0.2500.

Kratos--Dodecanal: y = 5.49E-4x + 3.69E-3, r 2 = 0.2500.

Mini LED--MIBK: y = 9.48E-4x + 1.88E-3, r 2 = 1.0000.

Mini LED--Dodecanal: y = 9.92E-4x + 1.89E-3, r 2 = 0.9800.

Figure 4.28. Data set 18 from system 7. 138

0.060 xxxxx Mini Dodecanal ***** Mini MIBK ***** Kratos Dodecanal 0.050 ~~~~~ Kratos MIBK

~ <( 0.040 "--""'

Q) (.) 0.030 c 0 ...!) 0 0.020 (/) * ...0 <( 0.010

0.000 10 20 30 40 50 60 C=O Concentration (ppm)

Figure 4.29. Data set 19 from system 7. 139

======::::~=~=~===:=::.:::==-...... ------= ·--==- ·-·------··· -·------·------·-----·-·- ----· ------··------·. ·------.-----

______- -·- --, - ­

1_QI'J1m ·--·-- -

- ~· .._;;;__ ; ____:___:_ ___...::...__...::..._ __~ _..:.._ ..:..__...:: ______-=...·-=-_- _ -:;~-----_ · ~--- - Abs. ______,_, ··· -----.. - -···· - · - .

------~------1. _ _. ____ -----:--=-- ... ~.... ------·- - ... _,_ - /. • • • ..J' --· _, . --· . ~ ·: ._ ~ - f .. - ~ - - :: ;_ . ~ . :I ·-- -- - ·---~ .-.- . _.... _ .~,-· --- -"-=., . -=-=-- ~ ~ - ~ -~:~ = -= ~ ~~ _;;;'_-_-· ~~- '__ ___ -~-:~--- ~~ -~ - - . --:- - · -- . . -- .... ~-~--~ -:_;.=·-· --::= ==-=·~ ~:: :· J_ -_ ::,_::_::_ ·-_ - · .:= 7-·:=:~ t·-__::.~ ~--:: ,._=~40_~= ~~---~_.-_J_- __

--· . -'

6 rnmjmin.

(A) 50 ppm C=O (B) 25 ppm C=O

(C=O source, butyraldehyde)

Figure 4.30. Stabilized baseline in system 8. 140

------'

------· .. - ·---

------r· -- ·---·- ·-··- ----· ,--~------~ .:..------'-I ------~-

- -·· ----, ;. ·---' · <-- --­ ._ _---· ...... ------_------

------p -- -~------~--===--- =====-_;__·======---=. - ~- . ... /'. ------·- ---·------·' ______- -•: __ ------·· . --~- -. - :------' ------. _: __ ------...... ------·---~----r--~ . -· - -·------·--. -·------Y------+------,, ·· --- --....- -- -- · -- ___ L __ ·--- - __ Abs. ------t--_ __;; ______,_+------____.....______, ---;-?~ ------. __......

_. a:

----- 1 ~------I 1------· ------

6 mm/min.

(A) 50 ppm C=O (B) 25 ppm C=O

(C=O source, butyraldehyde)

Figure 4. 31. Typical chart output of system 8. CHAPTER V

CONCLUSION

This work demonstrates that the ASTM method of carbonyl quantitation may be automated with a FIA system. Because the response pattern of different carbonyl-containing compounds is reproducible, quantitation of carbonyl compounds in the samples may be performed as

long as the specific type of carbonyl compound is known. This quantitation is substantially faster and easier in this automated mode than by the manual method. The amount of reagent and sample required by this method is much smaller (several microliters versus several milliliters). While is was hoped that a uniform response for a

carbonyl group, regardless of its source, could be obtained, this has

not yet proved feasible. A calibration curve for individual compounds must be used. Further work may be necessary with the variation in the

concentration of the dinitrophenylhydrazine reagent to see if this

parameter can affect the uniformity of response.

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