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SUPERCRITICAL FLUID CHROMATOGRAPHY OF NITROGEN·CONTAININGCOMPOUNDS ON PACKED COLUMNS

bvMehdi AshrafZKhorassani

Dissertation submitted to the Faculty of the

Virginia Polytechnic Institute and State University

in partial Fulfillment oF the requirements For the degree of

Doctor oF Philosophy‘ in

Chemistry

APPROVED:. A ,/7

L. äflaylor, Chairmäöi

¤ .

J. G. Mason 7 H. M. McNair

A1 - M · \7 ll. C. Dorn G. L. Long/

_—

September, 1988

Blacksburg, Virginia

SUPERCITICAL FLUID CHROMATOGRAPHY OF N1TROGEN·CONTANING

COMPOUNDS ON PACKED COLUMNS

by

Mehdi Ashraf·Khorassani

L. T. Taylor, Chairman

Chemisty

(ABSTRACT)

YSThe separation of basic nitrogen-containing compounds has been investigated via

supercritical CO; and 1% methanol modified CO;. Packed colurnns with the following

stationary phases were employed: silica, octadecyl (C18), propylamino (NH;), and

polystyrene-divinyl benzene (PRP). Without modifier the range ofbasicities which could

be eluted increased in the order of silica < PRP < C18 = NH;. Chromatographic peak

shapes and selectivity were much better with propylamino column. DifTerent aromatic

amines and azaarenes were successfully separated on both analytical scale and microbore

propylamino bonded phase packed colurnns with 100% supercritical CO;. Separation

is compared with both reversed phase and normal phase high performance liquid

chromatography (HPLC). The retention mechanism study for these aromatic amines

and azaarenes shows that the elution order not only depends on basicity and steric hin-

drance, but also on the solubility of the solute in CO;.

New cross-linked cyanopropyl and phenyl bonded phases are studied as stationary

phases for packed column SFC, as well as for separation of nitrogen-containing com-

pounds. The cross-linked bonded phase impedes access to uncapped silanol sites,

thereby giving rise to better peak shapes, and more rapid elution without the necessity

of a polar modifier in the mobile phase. Experiments both at elevated temperature and

in the presence of methanol modifier revealed that there is no short or long term dele-

terious effect on the column.

The separation of model mixtures of nitrated diphenylamine and nitrated anilines

via SFC employing cyanopropyl packed and capillary columns is described. Peak iden-

tification and peak purity were performed by on-line Fourier transform infrared

spectrometric detection. Supercritical CO2 is employed with cyanopropyl packed col-

umns for separation of non-polymeric components in double-base rocket propellants.

Both supercritical CO2 and CH2Cl2 were compared as a solvent for extraction of non-

polymeric components in "good" and "bad" double-base propellant.

Finally twenty four phenylthiohydantion amino acids (PTH—AA) have been rapidly

and efficiently separated on a cyanopropyl packed column by gradient elution of super-

critical CO2 and tetramethyl arnmonium hydroxide-modified methanol. Complete or

partial resolution of 22 derivatives is observed with only valine co-eluting with

norleucine and lysine co-eluting with asparagine. No modifier was required for elution

ofneutral PTH-AA's from the cross·linked stationary phase. The addition of base plays

a major role in elution of acidic and basic PTH-AA's.

This dissertation is dedicated to my loving parents for their confidence

and encouragement throughout the downs and ups ofmy career.

Acknowledgements

The challenges presented in obtaining an advanced degree are often met with sup-

port and aids of others. One occasion which is provided for offering thanks to others

for their help comes in these acknowledgments. I am especially grateful and appreciative

to professor Larry T. Taylor for his suggestion and help in my research and writing. I

also thank him for pushing me along the "learning curve" and for his never ending work

in obtaining funds needed for equipment and to support his graduate students. I would

like to thank my advisory comrnittee Dr. J. G. Mason, Dr. H. C. Dorn, Dr. G. L. Long,

and especially Dr. H. M. McNair for being an excellent professor in chromatography.

My thanks are extended to our hyphenated research group: M. G. Fessahaie, S.

Shah, W. C. Saunders, J. L. Hedrick, and L. H. Mulcahey. My special thanks to Dr.

John William Hellgeth for his advice and help during my graduate work at Virginia

Tech. Also, thanks to those who were not directly involve in this research, but who

helped create the cooperative atmosphere which was so conductive.

Thanks to all friends I have made during my stay in VPI, too numerous to mention

individually, for making the graduate life easier and more enjoyable. Special thanks to

Dr. Abbass Kamalizad for all of his advice and discussions in my last year of the grad-

Acknowledgcments v

uate school. Thanks to two of my best friends, Dr. Kamaruzaman Mohammad and

Mas Rosemal Hakim (future doctor in chemistry), who have made my life cheerful at the

times I felt down.

Financial support of the chemistry department in the form of a graduate teaching

assistanship (1984-1985), and research assistanship from funds supplied by Department

of Energy (1985-1987), and graduate project assistanship from funds supplied by Naval

Surface Warfare Center (1987-1988) is gratefully acknowledge. Also thanks to Dr.

Terry Berger and Dr. Jerry Deye from Hewlett Packard, for sharing their expertise and

their knowledge with me during my stay at Hewlett Packard. Thanks to Suprex coop-

eration for providing us with an instrument which made the SFC research much easier.

Special thanks to Dr. R. A. Henry, president of Keystone Scientific Inc., for his gener-

osity in providing us different columns, and for his helpful discussions concerning the

chemistry and the nature of stationary phases.

Last but most certainly not least, l would like to thanks my parents, sister and, es-

pecially my brother, , for encouraging me to continue the graduate study. Their love,

guidance, and support have made everything 1 achieved possible. Without them, I could

not appreciate the value of dedication and the satisfaction of achievement.

Acknowledgements‘

vi

Table of Contents

Chapter 1: Introduction and Literature Review ................................... 1

Introduction ............................................................ 2

Viscosity ............................................................. 2

Solute Diffusivity ....................................................... 3

Density .............................................................. 3

Research Objectives ...................................................... 4

Literature Review ........................................................ 5

Chapter 2: Comparison of Different Stationary Phases for Separation of' Basic Compounds Via

SFC ................................................................ 14

Introduction ........................................................... 15

Experimental .......................................................... 16

Results and Discussion ................................................... 18

Column Comparison for Elution of Basic Compounds .......................... 21

PRP—1 Column................................................21

Silica Colunm ................................................24

Octadecyl Column .............................................25

Table of Contents vii

Propylamino Column .,.........................................28

Separation of Basic Nitrogen Models ....................................... 40

Effect of Restrictor Size (i.d.) on Chromatography .....................43

Effect of Density on Chromatography ..............................45

Retention Mechanism .................................................. 46

Chapter 3: Enhancement of Packed Column SFC Via Deactivated Stationary Phase: ...... 57

Introduction ........................................................... 58

Experimental .......................................................... 60

Results and Discussion ................................................... 62

DELTABONDTM Cyanopropyl Column Evaluation ........................... 62

Thermal Stability of DELTABONDTM Cyanopropyl ....................67

Effect of' Modifier on DELTABONDTM Cyanopropyl ...................75

DELTABONDTM Phenyl Column Evaluation ................................ 81

Chapter 4: SFE/SFC of Double-Base Propellant Using PTIR as a Detector ............. 88

Introduction ........................................................... 89

Experimental .......................................................... 92

Result and Discussion .................................................... 94

SFC of Nitrated Diphenylamines and Nitrated Anilines .......................... 94

SFC of Double base Propellant .......................................... 106

Soxhlet Extraction ............................................106

Supercritical Fluid Extraction (SFE) ...............................116

Chapter 5: Gradient Separation of PTH-Amino Acids Employing Supercritical CO; and

Modifiers ........................................................... 125

Introduction .......................................................... 126

Table of Contents viii

Experimental ......................................................... 128

Results and Discussion .................................................. 129

Development of the Separation .......................................... 129

The Role of Base ..................................,................. 135

Column Preparation .......................................'........... 138

Mobile Phase Considerations ............................................ 140

Resolution ......................................................... 140

Chapter 6: Conclusions .................................................. 142

Conclusion ........................................................... 193

References ........................................................... 146

Vita ................................................................ l52

Table of Contents ix

List of Illustrations

Figure 1. Elution Behavior of Basic Compounds from PRP-1 Column .........23

Figure 2. Elution Behavior of Basic Compounds from Silica Column..........27

Figure 3. Elution Behavior of Basic Compounds from C1; .................29

Figure 4. Elution Behavior of 2,6-Lutidine From C1; .....................30

Figure 5. Elution Behavior of Basic Compounds from NH; Column ..........34

Figure 6. Separation of Methylated Pyridine on Various Columns ............37

Figure 7. Separation of Benzoquinoline Isomers on Various Columns .........38

Figure 8. Effect of Modifiers on Retention of Azaarenes ...................39

Figure 9. Isobaric Separation of Model Mixture .........................41

Figure 10. Pressure Programming Separation of Model Mixture ..............42

Figure 11. Effect of Restrictor Size on Separation ........................44

Figure 12. Plot of Log k' Versus Density for Basic Compounds ..............47

Figure 13. Reversed Phase HPLC of Model Mixture ......................49

Figure 14. Normal Phase HPLC of Model Mixture .......................50

Figure 15. Separation of Benzoquinoline Isomer with NH; Column ...........54

Figure 16. Proposed Surface of DELTABONDTM Packing Materials ..........63

Figure 17. Comparison of DELTABONDTM and Conventional Cyanopropyl forActivity ................................................65

Figure 18. Separation of Caffeine on Cyanopropyl DELTABONDTM ..........66

Figure 19. Separation ofAliphatic Amines with DELTABONDTM ............68

List of illustmions x

Figure 20. TGA of DELTABONDTM Cyanopropyl Packing Materials .........70

Figure 21. Pyrolysis TGA-MS of DELTABONDTM Cyanopropyl Packing Material 71

Figure 22. Effect of Temperature on Cyanopropyl DELTABONDTM Colurrm ....72

Figure 23. Effectof“

Temperature on Hypersil Cyanopropyl Column ...........73

Figure 24. Effect of Temperature on Conventional Endcapped Cyanopropyl Column 74

Figure 25. Effect of" Methanol on Conventional Cyanopropyl Column .........78

Figure 26. Effect of Methanol on DELTABONDTM Cyanopropyl Column ......79

Figure 27. Separation of Basic Compounds on DELTABONDTM Cyanopropyl ViaModified CO2 ...........................................80

Figure 28. Comparison of DELTABONDTM and Conventional Phenyl for Activity 83

Figure 29. Separation of' Polyaromatic Hydrocarbons with phenyl Column ......84

Figure 30. TGA of DELTABONDTM Phenyl Packing Materials ..............85

Figure 31. Effect ofTemperature on DELTABONDTM Phenyl Column ........86

Figure 32. Separation of Model Nitrated Diphenylamine Mixture with ConventionalPacked Column ..........................................96

Figure 33. Separation of Model Nitrated Aniline Mixture with Conventional PackedColumn................................................97

Figure 34. Separation of Nitrated_Il)1\iFhenylamine Mixture with Capillary andPacked DELTABOND Colurnns ..........................99

Figure 35. GSR of Model Nitrated Diphenylamine Mixture ................101

Figure 36. On-line SFC/FTIR Spectra of N-nitrosodiphenylamine and2,4-dinitrodiphenylamine ..................................102

Figure 37. On-line SFC/FTIR Spectraof’

Diphenylarnine and 2-nitrodiphenylamine 103

Figure 38. On-line SFC/FTIR Spectra of 2,4,6-trinitrodiphenylamine and2,4'-dinitrodiphenylamine ..................................104

Figure 39. On-line SFC/FTIR Spectra of 4-nitrodiphenylamine ............. 105

Figure 40. Separation of Model Nitrated Aniline Mixture with Capillary andPacked DELTABONDTM Columns. ........................107

Figure 41. GSR of Model Nitrated Aniline Mixture ......................108

List or Illustration: ‘xi

Figure 42. On-line SFC/FTIR Spectra of 2,4-dinitrotoluene andN-ethyl-2,4—dinitroaniline ..................................109

Figure 43. On-line SFC/FTIR Spectra ofN·methyl-2,4,6-trinitroaniline and N-methylariiline ...........................................1 10

Figure 44. Separation of CHZCIZ Extract of "Good" and "Bad" Double-BasePropellants with Supercritical CO2 ..............................

Figure 45. Comparison of' GSRs for Cl-12Cl; Extract of" "Good" and "Bad"Double-Base Propellants ..................................113

Figure 46. On-line SFC/FTIR Spectrum of DNPA, TA, and 2-NDPA ........114

Figure 47. On-line SFC/FTIR Spectrum of Nitroglycerine .................115

Figure 48. Separation of Supercritical CO2 Extract of' "Good" and "Bad" Double-Base Propellant with Supercritical CO2 .......................117

Figure 49. Comparison of GSRs for Separation of Supercritical CO2 Extract of"Good" and "Bad" Double-Base Propellant .................... 121

Figure 50. Co-added File Spectrum of Peak W ..........................122

Figure 51. Co-added File Spectrum of Peak X ..........................123

Figure 52. Co-added File Spectrum of" Peak Eluting at Approximately 7 min in theSeparation of "Bad" Propellant .............................124

Figure 53. Schematic of" Flow Gradient SFC/UV lnstrumentation............130

Figure 54. Isocratic Separation of Selected PTH·AA’s on DELTABONDTMCyanopropyl Colunm ....................................132

Figure 55. Gradient Separation of 24 PTI-I-AA’s on Zorbax Cyanopropyl Column 136

Figure 56. Effect of Different Flow Rate on Analysis Time and Resolution . . . 137

Figure 57. Effect of Base on Separation of Basic and Acidic PTH-AA’s .......139

List of lllustrations xii

List of Tables

Table l. Physical Properties of Gases, supercritical Fluids and Liquids .........2

Table 2. Basicity and Solubility of Model Basic Compounds in 100% CO2 .....19

Table 3. Basicity and Structure of Model Basic Compounds ................20

Table 4. Chromatographic Data on PRP-1 Column ......................22

Table 5. Chromatographic Data on Silica Column .......................26

Table 6. Chromatographic Data on Octadecyl Column ....................31

Table 7. Chromatographic Data on Propylamino Column .................33

Table 8. Elution Order Comparison of NP·I~lPLC and RP-HPLC to SFC ......51

Table 9. Elution Order of Basic Compound at Different Densities ............56

Table 10. Reproducibility of Cyanopropyl DELTABONDTM Column .........76

Table 11. Amino Acids Group Separation .............................133

List er Tables xaaa

Chapter 1

Chapter 1: Introduction and Literature Review

Chapter I: Introduction and Literature Review I

Introduction

Supercritical fluid chromatography (SFC) describes chromatographic methods in-

volving a mobile phase at supercritical conditions. These conditions are achieved in

circumstances in which the operating temperature is higher than the critical temperature

of the mobile phase and the operating pressure is above critical pressure. Pressures

typically used vary from about one to several times the critical pressure of the mobile

phase.

Supercritical fluids possess physical properties (viscosity, diffusion, and density) that

are intermediate between those mobile phases for gas chromatography (GC), and liquid

chromatography (LC). Therefore it is reasonable to expect SFC to bridge the

chromatographic extremes represented by GC and LC. Table 1 illustrates approximate

values for physical properties characteristic of gases, liquids and supercritical fluids.

Table I. Physical Properties of Gases, supercritical Fluids and Liquids

Property Units Gases Liquids Fluids

Wscosity g/cm-s 10** 10** 10**

Dißusion coeßicient cm'/s 10** 5xI0*‘ 10**

Density g/mL 10** 1 0.3-0.8

Viscosity

Supercritical fluids have viscosities that are similar to those mobile phases in GC.

However, the mobile phases in LC have viscosities between ten to one hundred times

Chapter I: Introduction and Literature Review z

greater than gases or supercritical fluids. One must recognize that the mobile phase

viscosity determines the pressure drop across the column. For example liquids with high

viscosity can not be used with capillary columns, and they cause the high pressure drop

(about hundred times) across the packed columns, compared to gases and supercritical

fluids. Capillary and packed columns can be used with both gas and supercritical fluid

mobile phases, and pressure drops are about the same for both.

Solute Diffusivity

Solute diffusivity is approximately two order of magnitudes higher in supercritical

fluids than in the liquid phases, but is lower in supercritical fluid than in the gases. So

when SFC is compared with high performance LC (HPLC), high analyte diffusivity in

SFC causes narrower chromatographic peaks, which results in increased detector sensi-

tivity. Higher analyte diffusivity also results in higher optimum average linear velocity.

As a result of this, optimum mobile phase flow rates are highest for gases, lowest for

liquids, and intermediate for supercritical fluids. Speed of analysis is therefore expected

to increase in the sequence: liquid chromatography, supercritical fluid chromatography,

and gas chromatography.

Density

The physical and chemical properties of supercritical fluids, such as solvating power,

diffusion and viscosity, are a function ofdensity. Increasing the density of a supercritical

fluid increases its ability to dissolve larger molecules. By controlling the density of the

Chapter I: Introduction and Literature ReviewV

3

supercritical fluid through pressure control, one can adjust the solvating ability of the

mobile phase. Progressively incrcasing the mobile phase density throughout a

chromatographic run causes higher molecular weight material to be eluted. This is

similar in many respects to temperature programming in gas chromatography and sol-

vent gradient programming in LC.

Further discussion about the physical properties of supercritical fluids and SFC in

general can be found in references 1-9.

Research Objectives

The research was carried out with the following objectives in mind:

l. To determine effectiveness of several packed colunm stationary phases for sepa-

ration of nitrogen-containing compounds of different basicity.

2. To provide possible mechanisms for elution behavior of these nitrogen-containing

compounds.

3. To compare conventional packed colurrms with highly deactivated columns for

elution behavior of nitrogen-containing compounds.

4. To efliciently separate different complex mixtures, containing nitrogen groups, via

SFC, and identify the peaks using Fourier transform infrared spectrometry (FTIR)

as a detector.

Chapter 1: Introduction and Literature Review 4

Literature Review

Analysis of nitrogen·containing compounds are becoming increasingly important in

studying the effects of chemical structure on the properties of pharmaceuticals, food

products, pesticides, drugs, coal derived products, etc. For example, nitrogen-containing

compounds constitute a major fraction of coal liquids‘° and are of particular concern for

several reasons. Nitrogemcontaining compounds adversely affect catalytic refining

processes“, which contribute to the instability of stored fue1s‘*·‘° . Exposure of plant

workers to synfuel process streams and products that contain nitrogen, and also forma-

tion of similar compounds during combustion of nitrogen-rich fuels pose both occupa-

tional and environmental concerns, because many of these compounds are known

mutagens and/or carcinogens"·". On the other hand, nitrogen containing compounds

are the source of pharmaceuticals, pesticides and herbicides‘°. Since most of these

compounds appear in a complex matrix, various methods ofchromatography have been

performed for isolating specific nitrogen~containing compounds.

Reversed phase HPLC with Cg and C18 columns has been utilized with mobile

phases of water and methanol, isopropanol or acetonitrile for separation of azaarenes

and aromatic amines"·*°. However, Bidlingmeyer2‘ employed a normal phase silica gel

column with reversed phase eluents of acetonitrile and water for the analysis of organic

amines. An ion-pairing chromatographic studyzz suggested that heterocyclic nitrogen

compounds were retained as a group to a greater extent than aromatic amines. Cation

exchange chromatography was found to separate primary arnines from azaarenes".

Thin layer chromatography (TLC) on silica gel“, aluminazs, and magnesium

hydroxide2‘ adsorbents has also been employed for these types of compounds. GC has

also been employed to separate volatile basic nitrogen compounds. Anilines, pyridines,

Chapter I: Introduction and Literature Review S

quinolines, and benzoquinolines have been chromatographed on base deactivated gas

capillary columns"‘” using conventional GC detectors (thermal conductivity [TCD], and

flame ionization [FID]). Each technique has its limitation, especially as the need arises

to separate more complicated mixtures of broader polarity and molecular weight range.

For example, HPLC can deal with compounds having wide ranges of polarity, but the

analysis time and chromatographic resolution become limiting factors given the current

state-of- the-art. TLC is a relatively simple and sensitive method, but it has disadvan-

tages: (1) poor quantitation; (2) several developers; and visualization methods have to

be used which are time consuming, and (3) exact composition of developers and spray

mixtures is important’°. Capillary GC yields reasonable results, but derivatization is

often necessary because many nitrogen compounds are either not volatile or are

thermally labile at temperatures required for GC analysis".

In recent years there has been a great deal of interest in analysis of complex mix-

tures using SFC. It is known that supercritical fluids exhibit properties, the values of

which are intermediate to those of liquids and gases, and thus one would expect SFC to

have chromatographic properties intermediate to those of GC and HPLC. Moderate

operating temperature of SFC currently offers an alternative for the separation of a

complex mixture containing nitrogen compounds which are thermally labile or non-

volatile.

A large portion of the literature on SFC is devoted to instrument development”·”,

detection methods"*, and analysis of relatively non-polar materials such as polymers and

natural fuels samples. However, there are not too many reports on the analysis of polar

compounds via SFC. The potential of the technique for analysis of polar compounds

was shown at an early stage. Historically the first reported use of SFC dealt with the

separation of metal porphyrins. Klesper‘ demonstrated the potential of different

halomethanes as supercritical fluid mobile phases with a solid supported liquid station-

Chapter I: Introduction and Literature Reviewi

6

ary phase (33% carbowax 20M on 60/80 mesh Chromosorb W). In this initial report,

pressure control and mobile phase flow rate could not be maintained adequately and

component detection was obtained via X- ray analysis of the isolated colored fractions.

Separations of etioporphyrin Il metal chelates were detailed in subsequent articles”·“ .

Packed gas-liquid chromatography columns and supercritical CCl2F2 were employed.

Chromatographic behaviors related to component gas-liquid partitioning, solubility, and

stability were examined as a function of liquid stationary phase. At times, the addition

of a polar modifier to the supercritical mobile phase was necessary to elute certain

compounds from the different stationary phases. Small percentages of liquid o-

dichlorobenzene or pyridine were used. Analysis of more polar compounds such as

amino acids, nucleosides, nucleotides, purines, terpenes, proteins, etc., was performed

by Giddings et al?. They demonstrated the enhanced solubility and different migration

of these compounds in supercritical CO2 at 40°C and 70 to 2000 atmospheres and in

supercritical NH; at l40°C and 100 to 340 atmospheres. It was concluded that NH; is

most effective in eluting highly polar compounds such as amino acids, while CO2 is best

suited for non-polar systems, particularly unsaturated molecules. However, there was

considerable overlap with many quite polar molecules soluble in CO2, and many non-

polar molecules dissolving in NH;. This work appeared very promising at the time, but

was soon overshadowed by rapid advances in capillary GC and HPLC.

During the l970's few reports were published regarding SFC. Most of the literature

was devoted to instrumental development and analysis of relatively non-polar material

such as polymers"·‘°. In 1978 Randall reported elution of azobenzene using supercritical

fluid CO2 and ethano1‘*‘. Emphasis in this study was placed upon detection of com-

pounds rather than the optimization of the separation. In 1982, the introduction of a

modified commercial HPLC system by Hewlett-Packard was a development which had

a profound impact on the general acceptance of SFC as an analytical technique. The

Chapter 1: Introduction and Literature Review 7

availability of a commercial instrument not only gave the opportunity for many labora-

tories to use SFC, but it also provided an instrumental design on which applications

could be standardized. The initial works of Lauer et al.‘*2, Gere etal.“

and Randall“

showed the effects of various mobile phases for the separation of caffeine in beverages.

Also they were able to elute theophylline, theobrornine, xanthine, 1-

aminoanthraquinone, 2-aminoanthraquinione, valmorin, 1- nitronaphthalene and p-

nitrophenylacetonitrile with supercritical CO; modified up to 10 percent with different

modifiers such as 2-methoxyethanol, 2-propanol, chloroform and methylene chloride.

Lauer also demonstrated the use of supercritical NH; as a mobile phase on polymeric

packings such as PRP-1 for separation of caffeine, theophyllin and nicotine. Blilie and

co-workers'“·“ used a CP-spher C18 column with different percentages of methanol to

separate nitrated polycyclic aromatic hydrocarbon. Later, Levy and Ritchey" evaluated

supercritical CO8 modified with different organic compounds such as hexanol,

2-methoxyethanol, 1-propanol and methanol to separate aromatic amines. In this study

different packed columns, C18, Diol and Cyano, were employed. However there was no

consensus as to which packed column stationary phase was more effective for separation

of nitrogen-containing compounds. Schmitz et al.“, studied the separation of N-

vinylcarbazole oligomers with a silica packed column. They used pentane as a super-

critical fluid and 1,4·dioxane as a modifier. In order to improve the separation of

N~vinylcarbazole oligomers, pressure programming from 50 to 150 atmospheres, and a

modifier gradient from 5 to 40 percent at 270°C was applied.

Since the development of capillary SFC‘°, a number of studies have been conducted

regarding the separation of nitrogen-containing compounds with supercritical CO;. lni

1983, Fjeldsted, Konig and Leeö demonstrated that by using CO; as the mobile phase

with a capillary SFC, certain labile solutes such as azo compounds and peroxides could

be successfully analyzed. Wright et al. expanded this application of capillary SFC to

Chapter i: Introduction and Literature Review’

8

marine diesel fuel’° and middle distillated fuelsl. In these studies they found supercrit-

ical CO; to be an excellent mobile phase for elution of nitrogen-containing compounds

in the fuel. More applications of SFC techniques for separation of labile compounds

were introduced in 1985. Wright and Smithsz showed the separation of four carbamate

pesticides in 90 seconds using a short capillary column (90 cm) with a small inner di-

ameter (25 pm). The analysis of these thermally labile pesticides was of interest since

operating temperature above 75°C was found to cause sample decomposition. Later a

short capillary column was used to separate additional carbamates which are also

thermally labile".

Richter et al."‘ used supercritical CO2 to separate various drugs to determine the

suitability of SFC for analysis. The drugs used in this study included

tetrahydrocannabinol and its metabolites, cocaine, phencyclodine, phenobarbital and

emthandone. The low temperature of the analysis was found to be helpful in preventing

decomposition and/or rearrangement of the compounds studied. Lee etal.“

showed the

capillary separation of benzylpeniciline—1-ethoxy— carbonyloxyether ester. Supercritical

CO2 was used as the mobile phase to elute the solute from a 10 m x 50 um fused silica

column coated with 25 percent biphenyl polysiloxane. Jackson and Later" subse-

quently showed the capillary SFC separation of commercial azo, aniline and

anthroquinone dyes. These polar dye samples were chromatographed using UV detector

and pentane as the mobile phase. These preliminary results suggested that SFC may

become an important tool for dye analysis. More recently Holzer etal.’7

reported the

application of capillary SFC for the separation of pyrrolizidine alkaloids. Most of these

compounds are powerful toxins. SFC proved to be an excellent method for analysis of

pyrrolizidine alkaloids of the retronecine and otonecine family. The operating temper·

ature was mild enough to prevent thermal degradation of the alkaloids during analysis.

Chapter 1: inuoduezion and Literature Review 9

Detection of nitrogen-containing compounds using both selective and high infor-

mation detectors with SFC has become another active area of research. Fjeldsted et al.‘

V for the first time reported the usage of a nitrogen phosphorous detector (NPD) with

supercritical CO2 for detection of nitrogen heterocyclics. In this study, both FID and

NPD were used for detection of standards. Excellent selectivity of the NPD was ob-

served for detection of nitrogen-containing compounds. A thermoionic detector was

also described by West and Leess for capillary SFC. Several modes of thermoionic de-

tector were evaluated. Detection limits in the range of nanogram to picogram were ob-

tained for nitro-polycyclic aromatic compounds. Markides et al.” used a modified

dual-flame photometeric detector with a capillary SFC system. The detection limit of

0.5 to 25 ng was found for several pesticides which contain sulfur, phosphoreus and ni-

trogen.

ln 1978, Randall et al." were the first to report use of mass spectrometry (MS) as

a detector for SFC. Mass spectra of several compounds such as azobenzene,

anthracene, phenanthrene and triphenylbenzene were obtained. However, some prob-

lems with the interface orifice were reported. More recent developments in SFC/MS are

detailed in subsequent articles°°·‘*. Henion eta1.‘°·“

used both a benchtop (HP 5970)

mass selective detector (MSD) and a MS (HP 5985B) to detect several polar compounds

with both capillary and packed columns. Supercritical CO2 was used to elute caffeine

from the capillary column. Excellent signal to noise ratios were obtained with MSD for

detection of 30 ng of caffeine. However, for elution of compounds with higher polarities

such as codeine, cocaine, phenylbutazone and methocarbamol an amino packed column

with supercritical CO2 modified with 10-20 percent methanol was employed. Berry,

Games and Perkins°‘, also used an arnino-bonded phase silica column with CO2 modi-

fied with 10-15 percent methanol or methoxyethanol to study the separation of a mixture

of xanthines, carbamates, sulphonamide and ergot alkaloids. The chromatograph was

Chapter 1: iritreduetieu and Literature Review 10

coupled to a Finnigan 4000 MS which was equipped with a 4500 EI/CI source and

moving belt HPLC/MS interface. lnterfacing of the chromatograph with the moving

belt interface was effected using a Finnigan MAT thermospray deposition device.

In 1981, Novotny et al/*9 predicted that SFC with Fourier transform infrared

spectrometry (FTIR) could be a viable technique to analyze non-volatile organic mix-

tures. In the following year, supercritical CO2 was successfully employed" in this

combination to detect and obtain an identifiable spectrum of 3 pg of nitrobenzene. In

1984 two different interfaces were reported for SFC/FTIR. The first interface for

microbore SFC was a diffuse reflectance infrared Fourier transform (DRIFT) interface

which involved deposition of the sample on powdered KCI followed by evaporation of

the solvent and analysis of the component". The second approach involved the use of

a high pressure flow cell". In this method, chromatography was limited in such a

fashion that constant supercritical CO2 conditions had to be employed since the infrared

absorption spectrum of CO2 changes with density. Jordan etal.‘°

showed separation

of basic nitrogen-containing compounds on a propylamino bonded phase silica employ-

ing CO; at constant density using FTIR as an on-line detector. Emphasis in this study

was placed upon the detection of material rather than optimization of the separation.

Recently Wieboldt and l—lanna’° reported a method for removing undesirable features

in chromatograms generated from FTIR data by using Gram-Schmidt orthonalization.

The procedure involves adding vectors containing the undesired information to the basis

set and recalculating the chromatogram with the augmented basis set. The technique

allows density programming with no alteration of the original spectral data. Employing

a prototype high pressure flow cell (1.4uL volume), the on-line detection of four

chromatographed pesticides has been demonstratedl‘.

The main objectives of the research described in the following chapters were to ex-

tend analysis ofpolar compounds, especially nitrogen-containing materials, using packed

Chapter 1: Introduction and Literature Review ll

columns with supercritical CO2 and modified supercritical CO2 as mobile phases.

Packed columns have several advantages over capillary colurrms:

l. more plates per unit time,

2. direct injection,

3. wider range of linear velocity, and

4. shorter analysis time.

However, packed colurrms stationary phases have the disadvantage of being more

active than the capillary colunms. It has been shown’2 that the concentration of free

silanol groups on packed columns is much higher than capillary columns. Keeping this

in mind, several silica base packed columns with different bonded stationary phases have

been studied to address the problems associated with the SFC for analysis of polar

compounds, especially nitrogen-containing compounds.

In chapter 2, packed columns (4.6 mm i.d.) with the following stationary phases

were evaluated: silica, octadecyl, propylamino, and polystyrene-divinyl benzene. They

all were employed for the separation of basic nitrogen-containing compounds with

pK,'s ranging from -4 to + ll. Both supercritical CO2 and methanol-modified CO2 were

investigated. The retention mechanism for the elution of basic nitrogen-containing

compounds with supercritical fluid CO; is compared with both reversed phase and

normal phase liquid chromatography.

The application of SFC with packed columns led to the development of new and

well deactivated stationary phases. The methodology which has been proven successful

with GC columns, such as immobilization of various kinds of monomers and oligomers

in the stationary liquid by cross-linking or thermal decomposition, has also been applied

on packed columns for SFC. Well deactivated cross-linked bonded phase columns

Chapter 1: Introduction and Literature Review I2

(DELTABONDTM) as opposed to conventional (non- crosslinked) packed columns and

endcapped deactivated columns were studied for the separation of nitrogen-containing

compounds via supercritical fluid CO2. Chapter three expands the results that obtained

from these cross-linked bonded stationary phases and shows the effect of both the tem-

perature and the modifier on stability and activity of columns.

Chapter 4 describes the application of SFC for analysis of non-polymeric materials

(e.g. nitrated esters, stabilizers and plasticizers) in "good" and "bad" double-base

propellants. Methylene chloride and supercritical CO2 were employed to extract these

non-polymeric compounds. A highly deactivated packed column (DELTABONDTM)and

well deactivated capillary column were used for the separation of explosive stabilizers

and extracted components. Results regarding the separation of extracted materials are

presented. Identification of the separated components was achieved with on-line FTIR

and FID detectors.

Packed column SFC using supercritical CO2 as a mobile phase, and

tetramethylammonia hydroxide (TMAOH) in methanol as a modifier, was used to sep-

arate 24 PTH-AA's. Gradient elution of methanol and base was used to obtain satis-

factory resolution. The effect of flow rate on resolution, role of base on separation,

column preparation, and mobile phase consideration are described in chapter 5.

Chapter 1: intreduerieu and Literature Review 'I3

Chapter 2

Chapter 2: Comparison of Different Stationary

Phases for Separation of Basic Compounds Via SFC

Chapter 2: Comparison of Different Stationary Phases for Separation of Basic Compounds Via SFC I4

Introduction

Analysis of non-volatile and thermally labile components (especially those contain-

ing nitrogen) in a complex mixture has been one of the most demanding tasks for the

analytical chemist. Approaches to these analyses have been facilitated by increasing

technological advances in chromatography. High performance liquid chromatography

(HPLC) has partially solved this problem by the development ofmore efficient columns,

and by the employment of a variety ofhighly specific detectors. However, the problems

with HPLC, such as poor efficiency, slow method development, and lack of a universal

detector, has limited this technology.

Currently SFC offers an attractive alternative for the separation of these polar

non-volatile or thermally labile components. The greater diffusivity and lower viscosity

afforded by a supercritical fluid relative to a liquid has been demonstrated to yield faster,

more efficient separations; while "liquid like" densities enable many thermally inaccessi-

ble components to be solubilized. Specific advantages of SFC over HPLC include’°·":

1. resolution per unit time is shorter,

2. total analysis time is less,

3. methods development is more rapid, and

4. interface to a mass spectrometer is simpler.

Applications of SFC using both packed and capillary columns, which utilize selec-

tive as well as universal detectors for analysis of nitrogen-containing compounds, were

described in chapter one. However, no exhaustive study has appeared specifically re-

garding the optimization of the separation of basic nitrogen-containing compounds via

Chapter 2: Comparison nr binären: Stationary Phases rer Separation er Basie Compounds vin src is

SFC. Jordan and Taylor‘° showed the separation of different nitrogen-containing

compounds with FTIR as an on-line detector rather than optimization of the sepa-

ration. Levey et al."7 compared supercritical CO; with different modifiers and different

packed colunms for the separation of nitro-aromatic amines. Again, in this study, the

emphasis was placed on the usage and effect of modifiers on packed columns rather than

the optimjzation of the separation. The effectiveness of different packed column sta-

tionary phases with nitrogen-containing compounds is not known.

In this chapter, the results which evolved from the evaluation of several packed

columns (silica, propylamine, octadecyl, PRP-1) for the separation of basic compounds

using supercritical CO2 and methanol modified CO2 is discussed. Samples of numerous

azaarenes, aromatic amines, and aliphatic amines with different basicity have been eluted

and separated on the colunms.

Experimental

A Hewlett-Packard (Avondale, PA) model 1082B liquid chromatograph modified for

use with supercritical fluids was utilized for studies with analytical scale (4.6 mm i.d.)

columns. A variable wavelength UV detector (1-lewlett—Packard model 79875) equipped

with a high pressure 1 cm pathlength (8 aL internal volume) flow cell was employed.

Samples were injected onto the packed column via a Rheodyne model 7125 six port in-

jection valve having a 10 ,aL loop. In order to provide a uniform evaluation of each

colunm, supercritical CO2 parameters (pressure at 350 atm and temperature at 50°C)

were kept constant.

Chapter 2: Comparison of' Different Stationary Phases for Separation of Basic Compounds Via SFC I6

A Suprex (Pittsburgh, PA) 200A supercritical fluid chromatograph which has both

capability for density and pressure programming was utilized with microbore packed

columns (1.0 mm i.d.). The unit was equipped with a flame ionization detector (FID).

Restrictors were drawn from short fused silica capillary (25-50 um i.d.) tubing using a

reproducible procedure similar to that described by Chester". The restrictor was con-

nected to the microbore packed column via a zero dead volume adapter (Anspec Co.

Ann Arbor, MI). The tip of the restrictor (7-12 um i.d.) was positioned approximately

0.5-2.0 cm below the hydrogen/air flame of the FID, and the temperature was set at

300°C to allow expansion of the effluent jet at the tip of restrictor. A Valco injection

valve with 0.1 ;rL rotor volume was employed for sample introduction to the small bore

column.

Analytical scale silica and bonded phase (propylamino and octadecyl) silica·based

columns (25 cm length, 4.6 mm i.d., 5 um particle diameter) were obtained from Alltech.

The percent coverage provided by the octadecyl phase was 12% according to the man-

ufacturer and was stated to also be endcapped. The polystyrene-divinylbenzene (PRP-1)

stationary phase column (15 cm length, 4.6 mm i.d., 5 um particle diameter) was pur-

chased from Hamilton. A microbore bonded phase propylarnino silica based packed

column (25 cm length, 1.0 mm i.d., 5 um particle diameter) was obtained from Anspec

Co. (Ann Arbor, MI).

Model nitrogen·containing compounds employed in this study were obtained from

either Aldrich Chemical Co. (Madison, WI) or Fisher Scientific Co. (Richmond, VA).

Each sample was dissolved in HPLC grade methylene chloride (Fisher Scientific Co.)

prior to the introduction onto the column. The concentration of samples per component

for both UV and FID detection (1.0 mm i.d. column) ranged from 2 ug/;rL for early

eluting components, to 3 ug/;rL for late eluting components. Typically 2 ,uL of each

solution were applied to the column for UV detection, 0.1 pL for FID detection. A flow

Chapter 2: Comparison of Different Stationary Phases ro: Separation or Basic compounds via src 17

rate of 2-3 mL/min was employed for analytical scale columns. For small bore columns

flow rates varied with the restrictor intemal diameter, and were applicable to the pres-

sure programming routine. Carbon dioxide and methanol-modified CO; were obtained

from Scott Specialty Gases (Plumsteadville, PA).

Results und Discussion

The first objective of this research was to compare the elution behavior of

nitrogen-containing compounds of different basicity on packed columns containing a

variety of stationary phases via both supercritical CO; and methanol-modified super-

critical CO; mobile phases. Different factors in addition to basicity such as steric hin-

drance of the nitrogen lone pair and solubility of the compounds in supercritical CO;

were studied in order to infer the retention mechanism. Thus, by matching the chemical

and physical properties of certain compounds with the optimized stationary phase, we

expect to achieve the best separation. A discussion of our results with four stationary

phases (silica, C18, NH; and PRP—l) follows. The model nitrogen-containing com-

pounds employed along with their aqueous pK, values and solubility in supercritical

CO; (where known) are listed in Table 2. The second part of this research describes the

separation of a model mixture of nitrogen-containing compounds, Table 3, via SFC

employing both 4.6 mm and 1.0 mm i.d. packed colunms. We also compared normal

phase and reversed phase HPLC of this model mixture with our optimized SFC sepa-

ration, and attempted to rationalize any variation in retention behavior.

Chapter 2: Comparison of Different Stationary Phases for Separation of Basic Compounds Via SFC l8

Table 2. Basicity and Solubility of Model Basic Compounds in 100% CO;

Comgound pK§ Solubi1ity·(25 °C, 950 gsi)

Pyrrole - 3.8 NA‘°

Quinoxaline 0.56 NA

Pyrazine 0.65 NA

Diphenylamine 0.79 1 %

Aniline 4.63 3%

N-Methylaniline 4.83 20%

N,N-Dimethylaniline 5. 15 M

Pyridine 5.25 M

2,6-Lutidine NA NA

N,N-Dimethylbenzylamine > 10 NA

Triethylamine 1 1.01 NA

* A. W. Francis, J. Phys. Chem., 58, 1099( 1954).

° NA = Data not available, M = Miscible

= Data (aqueous) taken from "CRC Handbook of Chemisty and Physics", 60th Ed.,CRC Press, Inc., Boca Raton, FL (1980).

Chapter 2: Comparison of Different Stationary Phases for Separation of Basic Compounds Via SFC 19

Table 3. Baaleity and Structure er Model Basie Compounds

Comgound pK, Structure

· H CH,N-Methylaniline 4.85 ä7,8·Beuz0quir1o1ir1e4.211,2,3,4—Tetrahydroquino1ir1e5.03 @32-Benzylpyridine 5.13 H Hz

Quinaldiuc 5.83 cH3 Q4-Azafluorene ·-- BQuinoline4.905,6-Benzoquinoline5.112,3—Cyclohexenopyridi¤e

-·-Aniline

4.633-Picolinc5.68 @CH:Pyridinc 5.25 fin:}

chapter 2: Comparison of Different Stationary Phases nn separazlen er Basic Compounds via src 20

Column Comparison for Elution of Basic Compounds

PRP-1 Column

A11 separations were conducted at a fixed pressure (350 atm) and temperature

(50°C). Two supercritical mobile phases were commonly employed (100% CO2 and

99% CO2/1% CH3OH). Under these conditions some of our model compounds failed

to elute from the PRP-1 column. Only materials with pK, less than 1.0 could be re-

moved with 100% CO2, provided no steric hindrance of the nitrogen lone pair electrons

was evident. For example, quinoxaline, pyrazine, diphenylamine, and pyrrole with pK,

values of less than 1.0 were eluted from the column, while pyridine and aniline with

higher pK,’s were not eluted. On the other hand, N,N-dimethylaniline possessing a

pK,= 5.15 but a sterically hindered nitrogen lone pair was eluted withk’=

1.63. In like

manner 2,6-lutidine was also eluted (Table 4). The single methyl group in N-

methylaniline apparently does not present sufficient steric hindrance since this com-

pound does not elute (Figure 1). lncorporation of 1% methanol into the supercritical

CO2 resulted in the elution of all components except aliphatic amines (pK,> 10). In

addition, chromatographic peak shapes were much improved relative to the situation

with 100% CO2. Decrease in the selectivity of the column was however noted, but this

no doubt could be minimized had we chosen to change pressure-temperature parameters

in going from CO2 to modified CO2.

The role of the methanol modifier with the PRP-1 column is puzzling. lt may pro-

vide (although it is unexpected at this low modifier level) a greater solvating power to

the mobile phase. The stationary phase is expected to exhibit no reactive groups which

might otherwise be deactivated by the methanol. However, if impurities reside on the

Chapter 2: Comparison er Dinbrenr Stationary Phases rar Separation ar Basic campaimda via src zi

Table 4. Chromatographic Data on PRP-1 Column

Capacity Factor jk')

Comgound 100% CO; 99% CO2 + 1% CH;OH

Pyrrolc 1.54 0.59

Quinoxaline 5.77 1.26

Pyrazine 5.76 0.1 1

Diphenylaminc 7.26 5.25

Anilirxc NE* 1.84

N·Methy1anilim-: NE 1.30

N,N-Dimethylaniline 1.63 0.98

Pyridine NE 0.45

2,6-Lutidine 2.25 0.48

N,N-Dimethylbenzylamine NE NE

Triethylamine NE NE

‘ NE = Not Eluted: 350 atmospherc, 50 °C.

Chapter 2: Comparison of Different Stationary Phases for Scparation of Basic Compounds Via SFC 22

DIPHENYLAMINE N-METHVLANILINE

100%ÜO2J.;

0 5 10 15 20 0 E Nmin. mln.

99% CO2+1% MGOH

0 5 ,0minmln. °

lFigure l. Elution Behavior of Basic Compounds from PRP-1 Column: Elution behavior ofdiphenylamine and N-methylaniline from PRP-1 column with 100% supercritical CO; and

methanol- modified supcrcritical CO2 (99/1.0). UV detection, 254 nm.


polystyrene packing material, methanol may serve to deactivate these contaminants.

Clearly a better understanding of the PRP-l stationary phase is required before an ac-

curate assessment of this phenomenon can be presented.

Silica Column

Basic nitrogen-containing compounds such as those shown in Table 2 can be ade-

quately separated on silica via HPLC by varying the polarity of the mobile phase.

However, with non- polar supercritical CO2 as the mobile phase competition with the

analyte for the stationary phase is not very great. Consequently, most of the materials

did not elute from the silica column. Specifically, only pyrrole with pK, < 0 and highly

sterically hindered diphenylamine and N,N- dimethylaniline eluted with 100% CO2.

Addition of 1% CHBOH to the mobile phase had a marked effect on the elution

(Table 5). In this situation only the highly basic aliphatic amines failed to be eluted.

It again appears doubtful that the small amount of CH3OH added to the mobile phase

could promote a significant increase in solubility ofsolute in mobile phase. On the other

· hand, there could be sufficient CH3OH present to interact with and effectively neutralize

(deactivate) the numerous accessible, free silanol sites via hydrogen bonding with either

the silanol oxygen (A) or silanol hydrogen (B). In other words, the silica colunm would

become less active in the presence of polar modifier, and the moderately basic analytes

would be less retained. The improvement in chromatographic peak shape shown in

Figure 2 with methanol-modified CO2 also supports this contention.

Chapter 2: Comparison er Different Stationary Phases rer Separation er Basic Compounds via SFC 24

H

Octadecyl Column

Employing 100% CO;, the C18 column permitted all material with pK„< 6 to eluteregardless of steric hindrance. However, the chromatographic peak shapes suggest inmost cases an irreversible interaction between analyte and stationary phase (Figure 3).Most compounds (e.g. pyrazine, aniline, N-methylaniline, 2,6-Iutidine) eluted with lowefliciency and large tailing except for the weakly basic or sterically hindered analytes

(e.g.pyrrole, N,N-dimethylaniline, and diphenylamine). It was originally believed that

the nitrogen-containing compounds would exhibit little aflinity for the C18 phase since

the material had been endcapped following 12% bonded phase coverage. In other

words, any analyte-column interaction would be via the accessible unreacted silanol

groups to the analytes rather than via partitioning with the octadecyl bonded phase.

Since a fair degree of selectivity was realized with C18 and 100% CO; as the mobile

I phase, we believe such an interaction to be a valid one. The accessibility of these

uncapped silanols may be poor since all peaks are broadened, suggesting that the inter-

action is not reversible. Evidence for this point was obtained when 2,6-lutidine

(Figure 4) and pyridine were individually injected three times each onto the column.

Chapter 2: Comparison of Dif1‘crentStationary Phases ro: sepamion or Basic compounds via src 25

Table S. Chromatographic Data on Siliea Column

Cagacity Factor gk]


Pyrrole 0.75 0.50

Quinoxaline NE* 3.55

Pyrazine NE s 2.23

Diphenylamine 1 . 12 0.42

Anilinc NE 1.32

N·Methy1ani1ix1e NE 0.66

N,N-Dimethylaniline 3.50 0.41

Pyridine NE 4.57

2,6-Lutidine NE 6.25

N,N-Dimcthylbcnzylaminc NE NE

Triethylaminc NE NE

* NE = Not clutedz 350 atmosphcrc, 50 °C.

Chapter 2: Comparison of' Different Stationary Phascs for Separation of Basic Compounds Via SFC 26

DIPHENYLAIMNE N -M£THYLANlL|NE 4

100% CO2

J...„....0 _s 10 0 s 10mm. min.

99%+ CO21% MQOH

0 5 ß 0 E 10mim min.

’Figure 2. Elution Behavior of Basic Compounds from Silica Columnz Elution behavior ofdiphenylamine and N-methylaniline from silica column with 100% supercritical CO; andmethanol- modified supercritical CO; (99/1.0). UV detection, 254 nm.

Chapter 2: Comparison of Different Stationary Phase: for Separation of Basic Compounds Via SFC 27

Retention times for each peak decreased as the number of injections to the column were

increased. This decrease in retention and large peak tailing indicates that the stationary

phases may be becoming saturated by irreversible adsorption of basic lutidine (or

pyridine) to the column (i.e. free silanol sites). Addition of 2% CH;OI~l to the poorly

solvating CO2 phase caused the adsorbed lutidine (no sample injectcd) to be

spontaneously stripped from the column.

As expected chromatographic separation with l"/o Cl·l3Oll modified CO2 elutcd all

components except the aliphatic amines when the mixture was injectcd on a rejuvenated

column. Efficiency was increased, however selectivity was extremely poor relative to theU

situation when l00% supercritical CO2 was used (Table 6).

Propylamino Columnl

All the model compounds studied here except the aliphatic amine were elutcd with

l00°/o CO2 under the standard pressure (350 atm) and temperature (50°C) conditions

(Table 7). The relatively weak solvating power of supercritical CO2 was again appar-

ently unable to disrupt the strong interaction between the two aliphatic amines and res-

idual silanols even though the amines which were injectcd were highly hindered. Peak

shape for the elutcd compounds was much improved over analogous results with any

of the other stationary phases (Figure 5). ln comparison with the C I g column, the same

number of components were eluted, but they were elutcd as much sharper peaks from

the amino column, even though this stationary phase had not been endcapped. lm-

proved column performance relative to C18 may be attributed to several factors: (I)

greater bonded phase coverage for the propylamino; (2) internal hydrogen bonding of

propylamino groups with accessible silanol hydroxyls, (C); and (3) derivatization of the

propylamino group by reaction with CO2 to yield a "urea—like" functionality (D). While

Chapter 2: Comparison or Different stationary Phases for separation or Basic Compounds via src zs

OWHENYLAMINE M-METHYLANILIME

100% CO2.

0 g 10 0 s 10~ mln. min.

100 % CO2+

1% MeOI-I

Q 5 D 0 E I0min. mi"-

Figure 3. Elution Behavior of Basic Compounds from C13: Elution behavior of diphenylamine andN-mcthylaniline from a C„ column with 100% supercritical CO; and methanol-modifiedsupercritical CO; (99/1.0). UV detection, 254 nm.


0510152025303540 0510152025303540min. min.

Addition ot 21 M•OH#3 INJECTION to mobiI• •>h•••

NO INJECTION

6 5 IO 15 20 25 30min.

0 5 . E 15min.

Figure 4. Elution Behavior of 2,6·Lutidine From Cu: Three consecutive injections of 2,6-lutidine“onto octadecyl column with 100% supercritical CO; as the mobile phase followed by addi~_ tion of 2% methanol to the mobile phase. UV detection, 254 nm.

Chapter 2: Comparison of Dilferent Stationary Phases for Separation of Basic Compounds Via SFC 30

Table 6. Chromatographic Data on Octadecyl Column

Capacity Factor gk]


Pyrrole 0.09 0.02

Quinoxaljnc 4.39 0.37

Pyraziue 9.79 0. 17

Diphenylamine 0.40 0.37

Aniline 1.80 0.16

N-Methylanilinc 1.50 0.17

N,N-Dimethylanilinc 0.58 0.22

Pyridine 8.36 1.07

2,6·Lutidinc 10.7 3.22

N,N-Dimethylbenzylanaine NE' NE

Triethylamine NE NE

' NE = Not clutcd: 350 atmosphere, 50 °C.

Chapter 2: Comparison of Dilferent Stationary Phases for Separation of Basic Compounds Via SFC 31

the relative extent of silica coverage by the propylamino and octadecyl bonded phase isnot known, we do not believe that the difference is significant. Deactivation of residualsilanols via interaction with the polar bonded phase, however, appears to be an attrac-tive rationale for the improved chromatographic performance of propylamino with100% CO2 relative to octadecyl, which does not contain a polar bonded phasefunctionality. This, likewisc, would be the case for silica, which contains no bondedphase. Reaction of the stationary phase with CO2, thereby converting the aminobonded phase to an amide or urea phase, structure D, cannot be discounted, especiallyin light of the fact that primary unhindered aliphatic amincs readily react with CO2 ina non-polar solvent. ln order to determine whether the stationary phase had reactedwith CO2, both diffuse reflectance Fourier transform infrared spectrometery and X·rayphotoelectron spectroscopy were applied to the packing material both before and afterexposure to supercritical CO2. Apparently there was no reaction, since nospectrometeric changes were noted between these two materials. Structure C, whichwould afford the nitroger: less nucleophilicity, no doubt could account for this surprisinglack of reactivity with CO2.

H S? .

Q :Rü R

H sein0 0 ’Ä;I. I. :_E: S: US: S:

C 0The use of 1% methanol in the supercritical CO2 failed to elute the aliphatic arnines

from the amino column. A modifier effect was, nevertheless, observed for the othercomponents as evidenced by the higher capacity factors obtained with 100% CO2. In

Chapter 2: Comparison of ¤am:::„« Stationary rhoso: ro: Scparation of Basic Compounds vao src sz

Table 7. Chromatographic Data on Propylamino Column

Capacity Factor gk]


Pyrrolc 1.36 0.83

Quinoxaline 2.40 0.29

Pyrazine 1.14 0. 16

Diphenylamine 1 .88 0.74

Aniline 5.00 1.12

N-Methylaniline 2.29 0.34

N,N-Dimethylanilinc 0.45 0. 16

Pyridine 4. 13 0.45

2,6—Lutidine 3.42 0.54

N,N~Dimethy1benzylamincNE•

NE

Triethylamine NE NE

* NE = Not eluted: 350 atmosphere, 50 °C.


DIPHENYLAMINE NJIETHYLANILINE

100% CO2

0 6 10 o 5 10mm. mlm

99% CO2+

1% Me0H

0 5 % 5 E 10mm mtn.

‘Figure 5. Elution Behavior of Basic Compounds from NH; Column: Elution behavior ofdiphenylamine and N-methylaniline from a propylamino column with 100% supercriticalCO; and methano1~modi1ied supercritical CO; (99/1.0). UV detection, 254 nm.

Chapter Z: Comparison of Different Stationary 1'hases for Separation of Basic Compounds Via SFC 34

contrast to the results obtained with the octadecyl phase and modifier where all analytes

essentially eluted together, selectivity was in part retained with the propylamino phaseand modifier.

It therefore appears that with the silica and C18 stationary phases the primary

interaction of the basic nitrogen-containing compounds (100% CO;) is via active silanol

groups. The amino phase gives the best results with 100% CO; relative to silica and

C18 since the number of free silanol groups is reduced by both silanol derivatization and

intemal hydrogen bonding through the propylamino group, structure C.

With 1% methanol the best selectivity is also found with the propylamino bonded

phase. On the other hand, very poor selectivity (i.e. poorer than silica) is achieved with

C18 wherein most components eluted with k' < 0.4. The reason for selectivity being re-

tained may lie in the fact that a slightly different mechanism of interaction is introduced

with the propylamino phase in the presence of methanol. For example, 1% methanol

no doubt deactivates a large fraction of silanols in both C18 and NH; case. The mode

of analyte interaction in the C18 case would have to be via the deactivated silanols, E.

For the amino situation, F, the analyte may choose to interact with either the polar

bonded phase or the deactivated silanols.

R R J Häl.. .5

R Ezgx 51 JRIHQQ =° t·f·QQ S{·

Q E FThe superiority of amino relative to silica and C18 stationary phases is apparent

even with 1% methanol, as revealed in Figure 6 where the separation of various

Chapter 2. Comparison or nslroroor Stationary Phascs vor sopararsoo of Basso corrspoorsus vaa src ss

methylated pyridines is shown for the four stationary phases. Only the PRP-l and

propylamino columns eluted all four compounds. 2,4,6-Collidine has a pK, = 7.43

which explains its failure to be eluted from the more activated silica and octadecyl col-

umns. Pyridine and the lutidines with pK, < 6 were easily removed from these columns

with 1% methanol-modified CO2. A measure of the goodness of propylamino relative

to silica, octadecyl and PRP-l is further shown in Figure 7, where the separation of

isomeric benzoquinolines is compared. Methanol-modified CO2 is required for elution

of all four components from silica, C18 and PRP-1; whereas, the same materials are re-

moved from the propylamino column with only 100% supercritical CO2. The more ac-

tivated silica column demonstrated longer retention times than the C1; colurrm as would

have been expected.

Even though the propylamino functionality is expected to deactivate a fraction of

the silanols, the presence of methanol in the supercritical CO2 has a profound modifier

effect. A number of azaarenes were successively separated (Figure 8) with 100% CO2,

99.9% CO2/0.1% CH3OH, 99.8% CO2/0.2% CH;OH, and 98%CO2/2% CH;OH. The

reduction in retention times and improvement in chromatographic peak shapes with in-

creasing percentages of methanol are dramatic. Holding pressure and temperature con-

stant, it would appear that 0.2% CH3OH is the optimized modilier content for this

column and these compounds. It is also interesting to note that the order of elution is

not maintained with increasing modifier content (e.g., 7,8·benzoquinoline, quinoline and

quinazoline are interchanged).

Chapter 2: Comparison er Ditrerem Szaziohary Phase; re: separation or Basie Compounds via SFC 36

UI •••••.1 I ‘snucn

’ 3

4

2

S 1 2 3 4 1 o 2 4 • I E E"min. min.

I

1nemo

°°*

2,4

2

24

6“i‘8 I I I I E 3mm. mn

_ Figure 6. Separation of Methylated Pyridine on Various Columns: lsobaric separation (350 atm.,50°C, 99% CO;/1% CH;OH) of methylated pyridines on various stationary phases. UVdetection, 254 nm. I = pyridine, 2= 2,6-lutidinc, 3= 2,4,6- collidine, 4= 3,5~lutidine.


I

ISILICA ODS

I

3.4

2 2

6'“ä_¥·*•‘?»7• 6"s—.a—aw.*„;—;T1. Illu. U1. III.

‘2

3

PII-! MI:I•4

•

Q A I 12 0 10 20 50TIM!. mtu. TIMI. mm.

Figure 7. Separation of' Benzoquinoline Isomers on Various Columnsz lsobaric separation (350 atm.,55°C) of benzoquinoline isomers on various stationary phases. Silica (99% CO2/1 %CH,OH); ODS or Ci; (99% CO.,/1% CH,OH); PRP-1 (99% CO,/1% CH„OH); Amino(100% CO;). UV detection, 254 nm. l == 7,8-benzoquinoline, 2= 2,3- benzoquinoline, 3 =3,4·benzoquino|ine, 4= 5,6-benzoquinoline.


2*l ° msn. ’

' " '¤.2¤•••o••

I

I

1

5

I

6 ä I I I 5 E IYIHIJIII'

;•_ •

1 I gn I. IGJ! HIGHI

-

1

I

I

° „JZ.„„„ °° °° *···¤···••••

Figure 8. Effect of' Modifiers on Retention of' Azaarenes: lsobaric separation (350 atm., 50°C) ofazaarenes on propylamino column (250 mm x 4.6 mm, i.d., 5 pm) with variable amountof modifier. UV detection, 254 nm, 2 mL/min, l= quinoxaline, 2= pyridine, 3=7,8-benzoquinoline, 4= quinoline, 5= quinazoline, 6= acridine, 7= phenanthridine, 8=5,6- benzoquinoline.

Chapter 2: Comparison of Different Stationary Phases for Separation of' Basic Compounds Via SFC 39

Separation of Basic Nitrogen Models

The specific basic nitrogen-containing compounds ( Table 3) selected for study were

those previously separated via both normal and reversed phase HPLC"". An optimized

SFC separation was initially developed with a 4.6 mm, i.d. amino column employing UV

(254 nm) detection. Figure 9 illustrates the type of isobaric separation of these polar

materials that one can expect with supercritical fluid CO;. Only two of the 12 compo-

nents are found to co-elute with other components during the approximately 40 minutes

separation (i.e. pyridine co-eluted with 7,8- benzoquinoline and 5,6-benzoquinoline co-

eluted with 2,3- cyclohexenopyridine). For this mixture, peak identification could be

accomplished by comparing single compound injection retention times since each com-

ponent was known to exhibit UV absorption at 254 nm.

A similar study was conducted with these same nitrogen-containing compounds on

a microbore (1.0 mm, i.d.) column. Since the SFC unit used for this analysis had pres-

sure programming capability, higher selectivity and efliciency was expected. In addition,

the more universal FID detector could be employed, since the volume of decompressed

CO2 was considerably reduced relative to the 4.6 mm column. Figure 10 illustrates the

separation of this mixture on an amino bonded-phase column (25 cm, 1.0 mm i.d.). A11

I2 components could be resolved in-part with supercritical fluid CO2 as the mobile

phase. Advantagesof‘

pressure (density) programming in order to achieve a better re-

solution are apparent by comparing Figures 9 and 10.

Chapter 2: Comparison of DilTerent Stationary Phases for Separation ol' Basic Compounds Via SFC 40

‘2

°·‘11,12

95

6

7

8

10

S

0 10 20 35 40 55TIME, min.

Figure 9. lsobaric Separation of Model Mixture: SFC separation of nitrogemcontaining compoundson an amino propyl bonded-phase packed column (250 mm x 4.6 mm i.d.) employingisoconfertic CO2 (350 atm., 50°C) as the mobile phase and UV (254 nm) detection, 3mL/min. l= N- metlxylaniline, 2= l,2,3,4-tetrahydroquinoline, 3= pyridine, 4=7,8-benzoquinoline, 5= 3-picoline, 6= quinoline, 7= quinaldine, 8= aniline, 9=·2-benzylpyridine, l0== 4-azafluorene, 11 = 2,3- cyclohexenopyridine, l2=5,6 = bezoquinoline.

Chapter 2: Comparison of' Different Stationary Phases for Separation ol' Basic Compounds Via SFC 4l

_ FID RESPONSEä ¤

UI

N

U

- "l

O 0

u _- °g oe

·¤“‘ u

,2 Q gI 8 3 N 0in

5· Ot

?' 6

°E3

OlU!

0Q .0

Figure I0. Pressure Programming Separation of' Model Mixture: SFC separation of nitrogen-containing compounds on an amino propyl bonded-phase silica packed column (250 mmx 1.0 mm i.d.) employing pressure programmed supercritical CO; at 50°C as the mobilephase and FID detection. See Figure 9 for identity of numbered peaks.

Chapter 2: Comparison of' Different Stationary Phases for Separation of' Basic Compounds Via SFC 42

Effect of Restrictor Size (i.d.) on Chromatography

In addition to varying the stationary phase, the effect of different size restrictor

internal diameters on the quality of the separation was determined with the amino col-

umn. Diameters were estimated from a scanning electron micrograph of each restrictor

orifice. Figure ll demonstrates separation of the nitrogen compounds in Table 3 under

identical pressure programming and temperature conditions with restrictors of 7 um (A)

and 10 um (B) i.d. The 10 pm restrictor is found to naturally yield a shorter time sepa-

ration, since mobile phase linear velocity should be approximately 2.2 times greater than

with the 7 um i.d. restrictor. What may be surprising from these data is that the degree

of resolution is equal if not better with the 10 um restrictor (i.e. faster separation) than

that obtained with the 7 pm restrictor. This may no doubt be due to the fact that with

the 7 um restrictor the benefits of pressure programming were nullified by the lower

linear velocity (i.e. the pressure ramp employed was too steep). The greater pressure

drop across the column with the l0 um restrictor as opposed to the 7 um restrictor does

not appear to be detrimental to the separation. One, therefore, must be cautious in

comparing separations from other laboratories, or in situations when a single

chromatographic parameter has been changed, unless there is some assurance that the

flow rates (i.e. restrictors in this case) are comparable throughout the separations. In

other words, an optimum separation in SFC will have a characteristic density

program/restrictor i.d. (linear velocity) combination. If one chooses to perform pressure

rather than density programming, temperature also becomes a necessary characteristic

of this optimum separation. A partial solution to this problem has recently been com-

municated wherein flow rate was controlled both for isobaric and pressure programmed

operations using two pumps and two restrictorsw.


e III mcnon

7 mcnou

5 ig 25 35 40 53 GS 70

TIME, mln.

ufo ago als assPRESSURE, atm.

Figure ll. Effect of Restrictor Size on Separation: SFC scparation of nitrogen-containing com-pounds on an amino propyl bonded~phase silica column (250 mm x 1.0 mm i.d.) with 7pm i.d. restrictor and 10 um restrictor.


Effect ofDensity on Chromatography

While mobile phase density programming may improve the overall quality of the

separation, it may also change the elution order of a series of components. This is es-

pecially true if the components have limited solubility in the mobile phase. Rawdon and

Norris°° have shown the effect of mobile phase density on elution of olefins and

aromatics. They demonstrated that by varying the column temperature and keeping

pressure constant, or altematively by changing pressure at a given temperature, the

separation of these two classes of compounds can be altered considerably. Furthermore,

Eckert et al.°‘ showed the changes in solubility of naphthalene in carbon dioxide as a

function of pressure at different temperatures. In both studies it was concluded that at

low pressure and high temperature (low density) the solubility is deterrnined by the vapor

pressure of compounds, however, at high pressure and low temperature (high density)

the solubility increases due to a decrease in the intermolecular mean distance, and an

increase in the specific interaction between solvent and solute. Figure I2 compares log

k' values for pyrrole, quinoline and aniline at various mobile phase densities. As might

have been expected elution time decreases with an increase in density at constant tem-

perature for each of the compounds. This observation reflects greater solubilizing powerT

at greater density by the mobile phase for each component. The solubility coeflicient

of pyrrole, quinoline and aniline in supercritical CO2 probably differs; therefore, for

closely eluting species such as these, their relative elution order may change. This effect

is illustrated in Figure 12 with a propylamino bonded-phase silica column. At low

densities aniline elutes before quinoline; whereas, at high densities the reverse situation

occurs. No doubt the increase in solubility per unit increases in supercritical CO2 den-

sity is greater for quinoline than aniline. This hypothesis naturally assumes that the re-

tention mechanism for these two compounds does not change with a density change, and


that the solubility of one of these analytes in the stationary phase does not markedly

change relative to the other.

Retention Mechanism

Very little information has been published regarding the elution behavior of polar

components in SFC and how their behavior differs from HPLC. Recently, a unified

theory ofchromatography encompassing gas, liquid and supercritical fluid mobile phases

has been presentedsz. General equations applicable to all three phases were obtained

for the equilibrium composition of the stationary phase and for solute retention in

fluid-liquid chromatography. It was shown that replacing a "poor" solvent by a

"good" solvent in HPLC is formally equivalent to replacing empty space by molecules

in SFC with a single-component mobile phase through increasing the density of the

supercritical fluid carrier. Based upon these findings such a comparison of elution in

HPLC and SFC seems justified.

Previously the separation of the model mixture under study here was reported using

reversed phase and normal phase HPLC”·". Figures 13 and 14 show the individual

chromatographic conditions and corresponding separations. Twelve components are

common to the NP-HPLC, RP-HPLC and SFC. The HPLC separations contain a

thirteenth component (i.e. 0-toluidine, peak x). Obviously, the RP-HPLC separation -employs a different stationary phase than NP-HPLC and SFC. Column dimensions

differ, but the detector (e.g. UV) is the same for all three separations. The superiority

of the RP-HPLC separation in terms of speed of analysis is obvious. For approximately

equivalent resolution, the reversed phase separation requires essentially one-third of the

time. The general incompatibility of RP-HPLC with high information on-line detectors,

Chapter 2: Comparison er Dinerem stationary Phase; ra: Separation er Basic cempauhds via SFCg

46

10 E K°=•

P *‘r·

~P en en —-‘¤ ,

Bul

9~•

Li¤ GII

¤ rnä äa„‘;‘

Pu

:2;E E §

l

E Z :0z ¤ 0Q m E P. z Man |'|I

.UI

Figure I2. Plot of Log k' Vcrsus Density for Basic Compounds: Log capacity factor versus CO;mobile phase density at 50 "C on an amino propyl bonded-phase silica packed column(250 mm x 1.0 mm i.d.) for pyrrole, quinoline and aniline.

Chapter 2: Comparison of Difl'erent Stationary Phases for Separation of Basic Compounds Via SFC 47

such as mass spectrometery and FTIR, is nevertheless a major disadvantage. Sepa-

rations employing NP-HPLC and SFC require approximately the same time, but the

greater degree of resolution in SFC is apparent (Figure 9) and predicted even without

density programming. The greatly different elution order in the two cases is, however,

unexpected.

From previous studies, several mechanisms have been suggested regarding the

elution order of basic nitrogen-containing material in both RP-HPLC and NP-HPLC.

Frei” assumed that in NP- HPLC separation occurs via variable nitrogen

donor/stationary phases acceptor complexing such that retention is a function ofanalyte

basicity. More recently, this correlation was observed for NP-HPLC separation of

chloroanilines and nitroanilines8‘*. Steric effects, however, were thought to markedly

influence the extent of donor/acceptor complexing. Guiochon and co- workersss con-

cluded in their work, using RP-HPLC, that retention time was shorter for the more basic

analytes which were expected to interact more strongly with the polar mobile phase.

They also concluded that the accessibility of the nitrogen lone pair of electrons influ-

enced retention time. Similar conclusions have been reached by other workers“*"

wherein basicity and steric hindrance both influence resolution. In general these trends

are observed in our HPLC separation (Table 8). Decreased basicity and/or increased

steric hindrance favors elution in the normal phase case, suggesting that the nitrogen

lone pair interaction with the stationary phase is important. increased basicity (greater

analyte interaction with the mobile phase) and/or increased steric hindrance (less analyte

interaction with the stationary phase) favor elution in the reversed phase case.

The elution order observed via SFC matches neither the normal nor the reversed

phase modes. For example, pyridine, which elutes last in the normal phase case and first

in the reversed phase case, elutes third in the SFC case. Intuitively one would have ex-

pected an elution order mimicking normal phase (i.e. the more basic the analyte, the

Chapter 2: Comparison of Diüerent Stationary Phases for Separation of Basic Compounds Via SFC 48

1, ‘ ,10

4

3 8•5 1

. 6 11 27

Rsrsmiou mas, MINUTES

Figure I3. Reversed Phase HPLC of Model Mixture: Reversed phase HPLC of nitrogen-containingcompounds on an octadecyl bonded-phase silica column (250 mm x 4.6 mm i.d.) withCH,CN/H20 (60:40 plus 0.02% triethylaminc) as the mobile phase. UV detection (254nm) and flow rate = I.0 mL/min.”

Chapter 2: Comparison of DiITerent Stationary Phases for Separation of Basie Compounds Via SFC 49

21 4

x1/2,0 6,11,12

X

1 9 7

~ X2

0369121äI82I2427ZX)33639FIETENTION TIME, MINUTES

Figure 14. Normal Phase HPLC of Model Mixture: Normal phase HPLC of nitrogen containingcompounds on an amino propyl bonded·phase silica column (I00 cm x 1.0 mm i.d.) withCDC};/CC}, (70/30) plus 0.02% triethylamine as the mobile phase. UV detection (254 nm)and flow rate of 30 ;.1L/min."

Chapter 2: Comparison of Different Stationary Phases for Separation of' Basic Compounds Via SFC 50

Table 8. Elution Order Comparison of NP-HPLC and RP—HPLC to SFC

Elution 0rder*

Compound pK, NP-HPLC RP-HPLC ß

N-Methylaniline 4.85 1 7 1

7,8-Benzoquinoline 4.21 1 10 3

1,2,3,4-Tetrahydroquinoline 5.03 1 9 2

0-Toluidine --- 2 4 ---2-Benzylpyridine 5. 1 3 3 7 8

Quinaldine 5.83 4 5 6

4-Azafluorene --- 5 7 9

Quinoline 4.90 6 3 5

5,6-Benzoquinoline 5.1 1 7 8 10

2,3-Cyclohexenopyridine 8 6 10

Aniline 4.63 9 2 7

3-Picoline 5.68 10 2 4

Pyridine 5.25 11 1 3

* Number indicates the peak in which the component(s) e1ute(s). For example inNP-HPLC, N-methylaniline, 7,8 benzoquinoline and 1,2,3,4-tetrahydroquinolineco-elute in the first peak.

Chapter 2: Comparison of Different Stationary Phases for Separation or Basic Compounds via SFC 51

longer its retention, given no severe steric hindrance). Support for this ideavisevidenced

in part by pyridine (pK, = 5.25) eluting earlier than 3-picoline (pK, 5.68). If on the

other hand, the nitrogen lone pair is sterically hindered, the more basic (but hindered

analyte) may elute first. Compare the stronger base N- methylaniline (pK, = 4.85)

which elutes earlier than aniline (pK, = 4.63).

To further amplify this point a series of isomeric benzoquinolines were studied

(Figure 15). On the basis of basicity, 7,8—benz0quino1ine (pK, = 4.21) would be ex-

pected to elute prior to any other isomers, because it has the lowest pK,, and nitrogen

lone pair is not as accessible as it is with the other isomers. The nitrogen sites in

3,4-benzoquinoline (pK, = 5.58) and 5,6- benzoquinoline (pK„ = 5.15) have essentially

the same steric environment, but due to resonance effects their pK,’s

differ. Therefore,

5,6-benzoquinoline should elute sooner than 3,4-benzoquinoline. Acridine (2,3-

benzoquinoline) with same pK, would be predicted to co-elute last with

3,4-benzoquinoline, however, due to slight steric hindrance by the fused ring at the

2,3-position, acridine elutes much earlier.

2,3·Benz0quir10Iim2. 3,4-Benzoquinoline

Chapter 2: Comparison of Different Stationary Phase; for Separarion of Basie Compounds via SFC 52

5,6-Benzoquinoline 7,8—Benz0quin0Iine

A third factor that appears to be significant in determining the retention behaviorof analytes in SFC (as alluded to earlier with the quinoline and aniline discussion) issolubility in the supercritical fluid medium. In order to show that solubility of analytein supercritical CO; has a direct effect on elution order, several substituted anilines withknown solubilityss were studied at two different densities (Table 9). Discounting sterichindrance for the moment the elution order considering only basicity should bediphenylamine (DPA); N·methy1aniline (NMA); N,N-dimethylaniline (DMA) and

N,N·diethylani1ine (DEA). At relatively high CO2 density the order observed is DEA-

DMA-DPA-NMA. Based on steric grounds it is not surprising that the di-substituted

anilines are observed to elute earlier than monosubstituted anilines even though thetertiary amines are stronger bases. On going to a lower mobile phase density (2000 psi,

35°C), the miscible DMA now elutes before the less soluble (17%) DEA. In otherwords, both analytes experience a longer retention time; however, the increase is greaterfor DEA (96%) than DMA (77%). For more strongly retained analytes (NMA, DPA)

the increase in retention is approximately 3 fold rather than 2 fold (DMA, DEA) on

going from high to low densities. Again the increased retention is greater for the lowersoluble analyte such that NMA (20% solubility) elutes before DPA (1% solubility) at

lower density, even though DPA is a much weaker base. In summary, at the lower


. 1 2

3

4

4

4

0 10 20 30TIME, min.

Figure I5. Separation of Benzoquinoline lsomer with NH; Column: SFC separation of7,8—benzoquinoline (1); acridine (2); 3,4-benzoquinoline (3) and 5,6-benzoquinoiine (4) onan amino propyl bonded-phase silica column (250 mm x 4.6 mm, i.d.) employingisoconfertic CO2 (350 atm., 55°C) as the mobile phase and UV (254 nm) detection,2mL/min.

Chapter 2: Comparison of' Different Stationary Phases for Separation of Basic Compounds Via SFC 54

density, components of similar chemical structure (i.e. secondary aromatic amines) hav-

ing higher solubility are eluted before their less soluble counterparts; whereas, at the

higher density the elution order is reversed. This can be explained by the notion that

when analyte solubility is not limited, separation is govemed by basicity and steric hin-

drance. Limited analyte solubility (i.e. reduced analyte-mobile phase interaction) ap-

parently can override the traditional mechanism at low density.

In summary, it has been demonstrated that basic compounds are able to beI

chromatographed with packed coluxrms via SFC. However, the excess number of silanol

sites appears to enhance the modifler effect and dominate the separation mechanism in

the absence of modifier. This study, has shown that the propylarnino phase deactivates

the free silanol sites by internal hydrogen bonding. Furthermore, the propylarnino phase

yielded the best results compared to silica, octadecyl and PRP-l in terms of both selec-

tivity and efliciency. A retention mechanism study of basic compounds with SFC

showed that the elution order not only depends on basicity and steric hindrance, but also

on the solubility of solute in supercritical CO2.

Chapter 2: Comparison of Different Stationary Phases for Separation of' Basic Compounds Via SFC S5

Table 9. Elution Order of Basic Compound at Different Densities

Retention Time (min)5000 psi 2000 psi

Compound pK„ So1ubility• 50 °C 35 °C

N-Methylaniline 4.85 20% 5.57 14.50

N,N-Dimethylaniline 5.15 M 2.88 5.09

Dipheuylamine 0.79 1% 5.38 15. 19

N,N-Diethylaniline 6.61 17% 2.76 5.40

• 25 °C, 950 psi, CO; (from reference 88).

Chapter 2: Comparison of Different Stationary Phases f‘or Separation of' Basic Compounds Via SFC 56

Chapter 3

Chapter 3: Enhancement of Packed Column SFC

Via Deactivated Stationary Phases

Chapter 3: Enhancement of Packed Column SFC Via Deactivated Stationary Phases 57

Introduction

Although efficient sample introduction devices, sensitive detectors, sophisticated

electronically controlled pumps and ovens, high speed recorders and integrators, and

other devices are essential components in SFC systems, the column remains the heart

of the analytical instrument. The ultimate quality of any separation cannot be any bet-

ter than the column itselli The growth and widespread use ofboth packed and capillary

columns in SFC has paralleled the development of column technology. While the virtues

and problems of packed and capillary technology have been espoused for years, the use

of both packed and capillary columns has become more widespread, and it would seem

logical that an even greater demand will be placed on their ability to perform more

complex and dillicult separations. To realize the full potential available for the resol-

ution of complex mixtures into individual components, the preparation of highly elll-

cient, well deactivated and thermally stable columns is imperative.

The development of improved stationary phases for packed columns, wherein basic

organic analytes are selectively and reversibly partitioned, is greatly desired. Currently,

commercially available stationary phases sulfer from insuflicient deactivation of the

support which leads to tailing and incomplete elution due to irreversible adsorption. The

elution behavior of basic compounds ranging in pK, from -3.0 to +11.0 has been de-

scribed in chapter two via SFC with silica, octadecyl-silica, propylamino silica, and

polystyrene-divinyl benzene as stationary phases and supercritical CO2 as the mobile

phase. The chromatographic data suggested that silanol-analyte interaction dominated

the mechanism of separation, regardless of stationary phase. This finding was not too

surprising since it has been known for some time that residual silanols are a particular

problem in the separation of basic compounds via liquid chromatography.

Chapter 3: Enhanccment or Packed Column SFC via Doaotivmd Smiomuy Phases S8 _

Griebrokk and Blilie"° briefly compared results obtained on a traditional reversed

phase octadecyl colunm and a C,„ column that had been specifically treated by the

manufacturer to remove silanol groups. Polyaromatic hydrocarbons and their nitrated

analogues showed much more symrnetric peak shapes with the "endcapped" column in

the absence of a modifier. Several modiliers were exarnined (e.g. methanol, 1-hexanol,

tetrahydrofuran and methyl—t-butyl ether) with the two colurrms with the result that the

non-capped column demonstrated a greater modifier effect.

Kohler and Kirkland8°, however, have noted that only isolated or unbonded acidic

SiOH surface groups are largely responsible for the irreversible adsorption of basic

molecules in HPLC. They discovered that fully hydroxlated silicas exhibited a larger

number of associated silanols and markedly lower adsorptivity for basic compounds.

Octyl and octadecyl silica-bonded phases were demonstrated to show a similar effect.

ln a related study°° a dual retention mechanism that postulates analyte retention as a

result of both solvophobic (hydrophobic) and silanophilic interactions was proposed.

Surface silanol groups were shown in this investigation to be masked by increasing either

the water concentration of the eluent or by the addition of a suitable amine to the eluent.

In this way regular retention behavior was observed in the separation of both crown

ethers and peptides.

While more complete silanization reactions may eliminate a significant fraction of

isolated silanols, steric considerations would never allow all silanols to be reacted.

Schomburg etal.°‘

have tried to increase surface coverage by employing a more reactiveh

silanization reagent, and by improving access of the reagent to the inside of the silica

pores. Irnmobilization of various kinds of monomers and oligomers in the stationary

liquid by cross·linking or thermal peroxide decomposition has also been performed using

methods which have proven successful with GC columns. The application of this kind

of technology to SFC has been applied to a limited degree.


This chapter expands on this approach by noting the improved column performance

afforded by cross-linking the bonded phase as opposed to non-crosslinking. These new

materials which are referred to as DELTABONDTM colunms are believed to shield any

remaining silanol sites from involvement in the separation mechanism. Compounds of

varying polarity have been separated via two silica·bonded phases (cyanopropyl, and

phenyl) employing supercritical CO2

Experimental

A Suprex 200A (Pittsburgh, PA) supercritical fluid chromatograph which has both

capability for density and pressure programming was utilized with l mm i.d. packed

columns. Restrictors were drawn from a short 50 am i.d. fused silica capillary tubing.

The restrictor was connected to the packed column via a zero dead volume adapter

(Anspec Co., Ann Arbor, MI). The tip of the restrictor was placed approximately

0.5-2.0 cm below the hydrogen/air flow of a flame ionization detector, and the temper-

ature was set at 385°C to allow expansion of the effluent jet at the tip of the restrictor.

A Valco injection valve with 0.1 pL rotor volume was employed for sample introduction

to the small bore column. Supercritical fluid CO; (Scott Specialty Gases,

Plumsteadville, PA) was pressurized with 104 atm helium to fill the 250 mL syringe

pump.

Model compounds employed in this study were obtained from Aldrich Chemical

Company (Madison, WI) and Sigma Chemical Company (St. Louis, MO). Samples

were dissolved in HPLC grade methylene chloride (Fisher Scientific Co., Richmond, VA)

prior to injection onto the column. The concentration of samples per component ranged

Chapter 3: Enhanccment or Packen Column SFC via Demivmd Smionuy Phases 60

from 2-4 pg/pL. The stationary phases that were used in this study are cross-linked silica

based cyanopropyl, phenyl polysiloxane (DELTABONDTM-Keystone Scientific Co.,

State College, PA), and regular (Hypersil) cyanopropyl, phenyl and

trimethylchlorosilane endcapped cyanopropyl. The dimension of the colurrms were 250

mm x 1.0 mm i.d. and 5 um particle size diameter for all cyanopropyl colurnns and 100

mm x 1.0 mm i.d. and 5 um particle size diameter for phenyl columns.

Thermogravimetric analysis (TGA) was carried out with a Perkin- Elmer TGS-2 instru-

ment to study the thermal stability of each cross-linked bonded phase silica packing

material. Samples (z 10 mg) were heated at rate of 50 °C/min from 50°C to 800° C in

a stream of helium (5 mL/min). Pyrolysis TGA-mass spectrometry (TGA-MS) was

performed on-line on a apparatus constructed in this laboratory. The pyrolysis products

were pumped out of the TGA to the MS ion source using 100 um i.d. fused silica as a

transfer line. The tip of the fused silica was placed approximately 2 to 3 mm above the

TGA pan, while the other end of the transfer line was pointed about 2 to3 mm apart

from the ion source of MS. The length of the transfer line was approximately 1 meter

long. The surrounding temperature of the first half of the transfer line from the TGA

to the GC oven was operated at room temperature (22°C). However, the other half of

the transfer line was fed from the injection port of the GC to the oven and then to the

ion source of MS. The operating temperature of the oven was set to 200°C. Mass-

spectral data were collected with a Hewlett-Packard (HP 5970) benchtop mass selective

detector. The MS source pressure was in the range of 1-5 x 10-* torr.

Chapter 3: Enhancement of Pocken column SFC via Deactivated Stationary Phases 61

Results and Discussion

The object of this research was to study the activity and stability of different silica-

based cross-linked bonded stationary phases (DELTABONDTM) withlsupercritical CO2

as the mobile phase. A surface coating technique similar to the one previously devel-

oped for the purpose of capillary GC was empl0yed°2.' To achieve a less activated sur-

face the appropriate polysiloxane chains were subsequently linked together by action of

a cross-linking agent, thereby effectively masking most of the silanol groups (Figure 16).

It should be noted here that capillary SFC colurnns are predicted to be still more inert

when prepared by this procedure due to substantial differences in the surface areas of the

two colurrms.

DELTABONDTM Cyanopropyl Column Evaluation

The silica based cyanopropyl (DELTABONDTM) packed column was tested first to

compare its efficiency and activity against a regular cyanopropyl column. Second, the

stability of this stationary phase was studied with respect to heat and added methanol.

In order to study the activity of the column, a mixture of organic compounds with a

wide range of polarity was chosen. The separation of the mixture (e.g. 4 ug/;,tL/each

Cl, hydrocarbon, phenylacetate, acetophenone, 2,6- dirnethylaniline and phenol) on both

a regular Hypersil cyanopropyl and a cross-linked cyanopropyl column are shown in

Figure 17. Peak shapes were much improved for the cross-linked phase, especially for

2,6-dimethylaniline, the most basic component. All polar solutes exhibited some tailing

on the regular cyanopropyl column which is probably due to the presence of accessible

Chapter 3: Enhancement or Packen Column SFC via Deactivated Stationary Phases 62

R R R RFI R FI

Io II /O\9 9 9H O

—Sl-O-S|—O—S|·O—S|-O—S|-O-Sl-0-éI-

Figure I6. Proposed Surface of DELTABONDTM Packingruaterials: Proposed surface of cross-linked bonded stationary phase (DELTABOND‘

) packed column, R = cyanopropyl,phenyl, etc.

Chapter 3: Enhanccment of Packed Column SFC Via Deactivatcd Stationary Phase; 63

non-associated silanol groups or water absorbed on the silica surface. This type of

chromatographic behavior was not observed with the DELTABONDTM column, sug-

gesting that silanol groups are much less accessible and that the partitioning is with the

bonded phase. With the regular cyanopropyl column a mixed retention mechanism may

be operating, although the retention order is maintained. The difference in t„ between

the two separations can be attributed to the fact that the silica base, pore size and pore

size distribution are somewhat different for the two columns.

While peak shapes were improved, it was of interest to determine if more polar or

basic analytes could be chromatographed with the DELTABONDTM cyanopropyl col-

umn. The separation of caffeine has previously been reported”·“ employing a capillary

column and 100% CO2 at 100°C. This same separation on a conventional packed col-

umn without a modifier at l60°C was accomplished with poor efliciency and peak tail-

ing. The separation, however, could be improved with a packed column by going to

5.5% methanol-modified supercritical CO2 at 75°C°". Figure 18 shows the separation

of caffeine on the cyanopropyl DELTABONDTM with 100% CO2 at moderate pressure.

Excellent efliciency and minimum peak tailing were observed in less than 10 minutes at

both 60°C and 100°C.

In chapter two it was indicated that tertiary aliphatic amines could not be eluted

with supercritical CO2 from a packed column, regardless of the density and temperature

of the mobile phase. Primary and secondary amines naturally would react with CO2,

therefore, they were not attempted. Employing the cross-linked cyanopropyl stationary

phase, however, we have been able to elute both triethylamine and

N,N-dimethylbenzylamine in less than four minutes with 100% CO2 at 160°C, Figure

19. The significant peak tailing accompanying the elution of each amine suggests that

the inaccessibility of silanol sites is not complete in this stationary phase. Elution of

these two tertiary amines from a regular cyanopropyl column was only possible at

Chapter 3: Ennonoomom or Pocken column SFC Vio Deactivated Stationary Phases 64

I

‘2 5

A

3

4

S6

I. 2 4

3

8”

~

.Till. mm.ä ' ' ' ' E I I I II!

PIIESSURI. nun.

Figure I7. Comparison of DELTABONDTM and Conventional Cyanopropyl for Activity: Separationof polarity tes}„“mixture on (A) conventional cyanopropyl and (B) cyanopropyl' DELTABOND ' column with 100% CO; and flame ionization detection at 60°C. l =n- pentadecane, 2 = phenyl acetate, 3 = acctophenone, 4 = 2,6- dimethylaniline and 5= phenol.

Chapter 3: Enhancement of Packed Column SFC Via Deactivated Stationary Phase: 65

PRESSURE.••«•. PRESSlRE,•u••.

Figure 18. Separation of Calfcine on Cüapiopropyl DELTABONDTM: Separation of caffeine oncyanopropyl DELTABOND‘

column with 100% CO2 and llame ionization detectionat 60°C and 100°C.


modilier levels greater than 10% methanol. Triplicate injections of these materials

yielded identical retention times and peak shapes at l60°C, which testiües to the good

thermal stability of the cross-linked phase. ln order to promote the stability and

reproducibility of the DELTABONDTM cyanopropyl column compared to Hypersil

cyanopropyl packed column, further experiments were followed.I

Thermal Stability ofDEL TABONDTM Cyanopropyl

This part of our study was concerned with determining the temperature stability and

chromatographic reproducibility of the cross-linked cyanopropyl silica based

(DELTABONDTM) packing. Thermal stability of the DELTABONDTM cyanopropyl

packing materials was tested utilizing TGA (Figure 20). DELTABONDTM cyanopropyl

stationary phase was stable toward weight lost up to 220°C. The degradation which

begins at temperature above 220°C represents the start of the degradation of the bonded

stationary phase. The molecules leaving the stationary phase from 220°C up to 770°C

were studied by pyrolysis TGA-MS analyzer. The degradation products from bonded

cross-linked stationary phase cyanopropyl (DELTABONDTM) can thus be seen in Total

Ion Current (TIC), Figure 2l A. Most of the molecular ions are the characteristic for

the fragmentation of the propyl groups with m/z 15 (CH;*), 27 (C;H;+), 28 (C2 H4), and

41 (C;H5), Figure 21 B. The molecular ion which corresponds to the fragmentation of

cyanopropyl group with m/z 40 (CH2CN+), and 41 (CHZCNH) has also been observed.

However there is no reason why the m/z 54 (CZHACN) and 68 (C;H6CN) that are

characteristic series for cleavage of C-C bond in cyanopropyl functional group has not

been observed. The total percent weight lost of cyanopropyl bonded stationary phase

from DELTABONDTM packing materials was :4.75%. Several factors have been pre-

dicted for the low percent weight coverage of bonded stationary phase on silica, e.g. 1)

Chapter 3: Enlmncemenr of Packed column SFC via Deactivated Stationary Phases 67

BA

0 I 2 3 I 50 1 5 5 IT"*E·m‘“· rms, man.

Figure 19. Separation of Aliphatic Amines with DELTABONDTM Columnz lsobaric (150 atm)separation of tpqhylamine (A) and N,N- dimcthylbenzylamine (13) on cyanopropylDELTABOND

‘column with 100% CO; and flame ionization detection at l60°C.


molecular weight of bonded stationary phases, 2) steric hindrance, and 3) the reactivity

of stationary phases in a solvent with silica packing materials°‘.

Further corroboration for the stability of the cyanopropyl DELTABONDTM column

is seen in the separation of diphenylamine, indole, 7,8-benzoquinoline and carbazole first

at 60°C (A), then at l60°C (B) followed again by 60°C (C). Identical pressure programs

were employed in each case which accounts for the different separation features at the

two temperatures (Figure 22). lt is noteworthy that the separation improved upon go-

ing to the higher temperature, and that both separations at 60 °C were essentially iden-

tical. What is even more significant is the results of similar experiments with a regular

cyanopropyl column. With the same pressure programming, all four components could

be separated at 60°C, albeit with considerable peak tailing, especially for the most basic

component, 7,8- benzoquinoline (Figure 23). Furthermore, while all peak shapes appear

similar for the two runs at 60°C (i.e. before and after heating) the retention time of the

7,8-benzoquinoline decreased by approximately 0.5 minute after heating, in contrast to

the other components. The decrease in retention of the most basic compound in the

mixture is probably due to the loss of absorbed water at high temperature and further

deactivation of the cyanopropyl stationary phase.

The cross-linked cyanopropyl "chemistry" seems to be preferable to conventional

endcapping chemistry for deactivating silanol groups based upon the following exper-

iment. The regular cyanopropyl column was endcapped with trimethylchlorosilane by

the manufacturer. The four component mixture was separated in the same manner as

previously described (i.e. 60°C and l60°C, identical pressure programming, same amount

injected), Figure 24. The initial 60°C and l60°C runs yielded results which were similar

in selectivity to the cross-linked column results. Upon decreasing to 60°C the column

performance was similar to the 60°C runs with the regular column. lt therefore appears

that the elevated temperature caused the trimethylsilyl phase to be removed.

Chapter 3: Enlmnoomone or Packed column SFC vin Denozlvnred Stationary Phascs 69

U 7: WEIGHTZ 8 8 81.*• 8 8 8

i..l..—4—.—_.. .- ,.,. -..._+_.l___,i_

2?_

·· IU-!B 8 8 1x1 1A —• N xlx ¤ ,..¤ __"

___I°' 6 gs.: - 1

Q SI 1„ 2 ¤ I-1 0 I 12 *" 2 é21 IQ g > I2 ä ¤2 2 8 I2 PI ISE 1

„1 T6I 1-1 S; 5Q ’I2 .I I„ zu BT $1 I

Z SI ' 1c ... ‘3 I s¤ IUI 1,„ 2 ä 11*1 S -1 §\} „

I ’'ID .

§I 2 II —· I1 L1 11 1xa! 1Q? 8 II2I

3 . ¤- Ixl •°

I$1 1-1 Y °° I¤ ;....-.„..„..-----..--„---...---__-.._.--..:I_I

Figure 20. TGA of DELTABONDTM Cyanopropyl Packing Materials: TGA of DELTABONDTMegyanopropyl äonded staüonary phase silica. Sample was heated at rate to 50°C/min form0°C to 800" .


TIC of DHTR:Ill737CN.Dl.¤ES

9.¢E4 r

8.@E4 ‘I

7 . QE4 II ,

,„ 8. E4 ’ ’I,Q T rR III! I , „gr A**4 5.ÜE4 « wr rp, ,‘°

-2 r ,,0; rT3 4. 0:4 ' rQ « 0 ‘ Il. ',t•·‘rT'

2.0E4

ißßaß

Ü2 4 6 8 IQ 12 I4 IS

Trme (mrn.)

I 'au Sega II]? Kil„¢B5 MIR] ¤¢ D9T'Hr|l!?27CN.D I

°;; R ¤r.1¤·r·¤¤c1·:¤ ¤c•=•¤.:¤R gg 2/7

° rar:‘ sa a°xr Z( sa 4 1z¢ xsan zan1 ua

B(S aa

M35):/CharEQ as 4a

Figure ZI. Pyrolysis TGA·MS of DELTABONDTM Cya¤0pr0pyI{;acking Material: (A) Total ioncurrent obtained from dcgradation of DELTABOND‘

cyanopropyl packing materials.(B) Mass spectrum of gascs from pyrolysis of stationary phases from cyanopropylDELTABONDTM silica base.

Chapter 3: Enhancement of Packed Column SFC Via Deactivated Stationary Phases 7l

2,3

.

1A 60°c

3

4

B 2 2 160 °c

2, 2

4

1

60 °cC

6 ' { ' Z 3 Z 10TIME, min.

260PRESSURE, atm-

Figure 22. Effect of Tcmperature on Cyanoprfqélyl DELTABONDTM Column: Effect of temperatureon cyanopropyl DELTABOND‘

column. Sequenüal separation of four componentmixture at 60°C (A), 160°C (B), and 60°C (C) with 100% CO; and flame ionization de-tection. 1 = diphenylamine, 2 = indole, 3 = 7,8-benzoquinoline, 4 = carbazole.

Chapter 3: Enhancement 0I'1‘acked Column SFC Via Deactivated Stationary Phases 72

S

~

A 21

3 6o°c

S

B 3 4, 2 160 °c

S

V4

21

C3

60 °c

TIME. mh.

15 150 ähPRESSURE. atm.

Figure 23. Effect of Temperature on Hypersil Cyanopropyl Column: Effect of temperature on con-ventional Hypersil cyanopropyl column. See Figure 22 for details of separation.

Chapter 3: Enhancement of Packed Column SFC Via Dcactivated Stationary Phases 73

4- 2

1

A ’60 °c

4B

‘2

„ 160 °c

21

C 60°c:1

0 5 I • • 10mes. min.

160 160 260PRESSURE. atm.

Figure 24. Effect of Temperature on Conventional Endcapped Cyanopropyl Column: Effect of tem-perature on cyanopropyl column endcapped with trimethylchlorosilane. See Figure 22 fordetails of separation.

Chapter 3: Enhancemcnt of Packcd Column SFC Via Deactivated Stutionary Phase: 74

In order to study the reproducibility of cyanopropyl colurrm, seven replicate in-

jections of our polarity test rnixture at 60°C were made, followed by calculation of rela-

tive standard deviations for both retention time and peak area. The column was then

heated to l50°C for 12 hours with a flow of CO2. The column was brought back to

original operating temperature, and replicate injections were repeated. The results

which are found in Table 10 demonstrate very good reproducibility in retention time and

peak area both before and after heating for each component. These data suggest that

the cross·linked column could be reliably used at relatively high temperature.

Effect ofModüier on DEL TABONDTM Cyanopropyl

The stability of DELTABONDTM cyanopropyl to methanol was next established.

Previously in our and other workers' laboratories it was reported that a methanol mod-

ifier leaves a permanent or memory effect on conventional packed column stationary

phases, which is most noticeable during the separation of basic analytes. Only after ex-

tensive equilibration with CO2 (24-48 hours) could the colunm be retumed to its original

state after methanol exposure. Equilibration times of 30-45 minutes have been noted in

reference 46, but these analytes were polyaromatic hydrocarbons and their nitrated an-

alogues not basic materials. An illustration of this phenomenon is provided in Figure

25. A regular Hypersil cyanopropyl column was washed with approximately 30 mL of

methanol, followed by equilibration with supercritical CO2 for two hours. Employing

the same pressure program again and the same four component rnixture as previously

described, 7,8- benzoquinoline could, surprisingly, not be eluted (Figure 25, A). The

same mixture when injected at l60°C now revealed four peaks, wherein

7,8-benzoquinoline exhibited much peak tailing (B). Upon retuming the column to

Chapter 3: Enhancement or Packed Cauumh SFC via Deaerivaied Stationary Phase; 75

Table 10. Reproduclbility of Cyanopropyl DELTABONDTM Column

BEFORE HEATING

SymmetryComgound t;·• (% RSD) E Factor % RSD Area

C15 Hydrocarbon 7.29 (0.3) 2.96 1.3 5.3

Phenylacetate 7.52 (0.3) 3.09 2.1 3.6

Acetophenone 7.94 (0.3) 3.32 2.4 2.6

2,6-Dimethylaniline 8.55 (0.4) 3.65 3.6 2.9

Phenol 10.34 (0.4) 4.62 4.5 3.6

AFTER HEATING

C15 Hydrocarbon 7.21 (0.2) 2.92 1.0 4.9

Phenylacetate 7.45 (0.2) 3.05 1.5 5.9

Acetophenone 7.85 (0.1) 3.27 1.8 2.7

2,6-Dimethylaniline 8.45 (0.1) 3.59 2.5 4.2

Phenol 10.21 (0.3) 4.52 3.7 1.2

* Average Retention Time (7 injections) in Minutes.


60°C. the efficiency for the most basic compound had changed, as evidenced by the ap-

pearance of a peak ascribable to 7,8-benzoquinoline which originally at 60°C did not

elute (C). An identical experiment with the cross—linked cyanopropyl phase (i.e. 30 mL

methanol, 2 hr., CO2 equilibration) showed no varying effect due to methanol pretreat-

ment (Figure 26). The results were identical to those previously shown in Figure 22.

In the case of the regular column, the limited methanol pretreatment apparently acti-

vated the stationary phase by converting siloxane units to silanols which after CO;

equilibration are more numerous than before methanol pretreatment. Heating the col-

umn no doubt removes a fraction of the silanol units thereby creating a less active col-

umn which facilitates the elution of 7,8-benzoquinoline. With cyanopropyl

DELTABONDTM these reactive siloxane groups are not accessed by the liquid methanol,

and consequently the activity of the column is unchanged. These results should not be

contrasted to mean that the cyanopropyl DELTABONDTM colurrm is without a modifier

effect. An optimized separation of the same four components was accomplished em-

ploying 100% CO2 (Figure 27, A) followed by identical pressure programming but with

mobile phase of either 99% CO2/1% CH;OH (B) or 98% CO2/2% CH;OH (C). The

three least basic nitrogen-containing compounds eluted with a small change in retention

time regardless of the mobile phase. The presence of methanol caused

7,8-benzoquinoline to elute at shorter retention times. In fact with 2% methanol,

7,8-benzoquinoline eluted before indole. From these results one can conclude that there

are still some free silanol groups, which only interact with strong bases, and the addition

of methanol can cause the deactivation of these free silanol groups.

Chapter 3: Enhancement of Packed column SFC via Deactivated Smionury Plurses 77

-2

1

4

„ 160 Y:B‘

23

4

2

60 ·Y:1

C

:1

TIME, min.

V E fg '"'rnsssuns , nm. am

Figure 25. Effect of Methanol on Conventional Cyanopmpyl Column: Effect of methanol preueat-ment on conventional Hypersil cyanopropyl column at 60°C and l60°C. See Figure22 fordetails of separation.

Chapter 3: Enhancement of' Packed Column SFC Via Deactivated Stationary Phases 78

S2.3

41

A_ ‘ u

60%:

s

3

.”

B

1 2 _ 160C

‘2.: .

41

C , ,60C

0 I Z •X ° 10 °r nmz, min. _

up IO0 2%0PRESSUIE, atm. .

Figure 26. Effect ol' Methanol on DELTAQONDTM Cyanopropyl Column: Effect of methanol pre·treatment on DELTABOND

‘cyanopropyl column at 60°C and l60"C. See Figure 22

for details of separation.

Chapter 3: Enhancement of' Packed Column SFC Via Deactivated Stationary Phases 79

43

1 2 ¢

98/ 2 % CO2/M•o•~•

3

1 2 ‘

99/1 76 ¢°2/ M•OH

3

1z 4

100 16 co,

öiääzgziäaßnTIME, min.

ä 150 210 At

PRESSURE, atm.

Figure 27. Separation of' Basic Compounds on DELTABONDTM Cyanopropyl Via ModifiedCO;: Eiiect of' difierent percentage of methanoi on separation of basic compounds. Se-quential separation of' basic compounds with 100% CO; (A) 99%CO;/1% CH;,OH (B)and 98%CO2/2% CH;,OH (C) with UV detector (254 nm) at 60°C. See Figure 22 for peakidentification.


DELTABONDTM Phenyl Column Evaluation

The cross~linked phenyl silica based (DELTABONDTM) packed column was tested

for activity, efiiciency and selectivity against a regular Hypersil phenyl packed column.

In order to study the activity and etliciency of the column in a single chromatographic

run, a mixture of 3 components, C,, hydrocarbon, naphthalene and acetophenone was

chosen (it is important to note that none of the nitrogen·containing compounds were

eluted from neither DELTABONDTM phenyl nor Hypersil phenyl columns with 100%

CO;.) The components in the test mixture were selected in a manner that

chromatographic data on C,, hydrocarbon and naphthalene show the efliciency of the

column, and at the same time acetophenone shows the activity of the column. Sepa-

rations of the mixture on both DELTABONDTM phenyl (A) and Hypersil phenyl (B)

columns are shown ir1 Figure 28. Acetophenone, the most polar compound in the mix-

ture, and naphthalene were eluted with less tailing and better efliciency from the

DELTABONDTM phenyl column rather than the Hypersil phenyl. DELTABONDTM

phenyl column showed a lower activity for elution of polar compounds. The significant

peak tailing accompanying the elution of acetophenone and naphthalene from the

Hypersil phenyl column suggests greater accessibility of silanol groups on the surface

of the column. Both columns showed reasonably nice symmetrical peaks for elution of

non-polar C15 hydrocarbon. But in terms of efliciency, the number of theoretical plates

for 10 cm DELTABONDTM phenyl column was =2x larger than the number of theore-

tical plates in 10 cm Hypersil phenyl column.

Next to the efficiency and activity of the DELTABONDTM phenyl column, it was

also interesting to study the influence of cross-linking on selectivity of packed columns

for the separation of several polyaromatic hydrocarbons (m-xylene, naphthalene,

Chapter 3: Enhancement or Packed Column src via Deactivated Stationary Phases st

2,3-dirnethyl naphthalene, bibenzyl, fluorene, pyrene, and benzopyrene). Figure 29

shows the optimized separation of the mixture on each column. The DELTABONDTM

phenyl column presented a higher selectivity, and separated all the components in the

mixture. However, with the Hypersil phenyl colurrm, co-elution ofbibenzyl and fluorene

occurred (peak 4). This higher selectivity of DELTABONDTM can oe attributed to the

higher interaction of solute with phenyl functional groups on the surface of the station-

ary phase and/or fewer active silanol groups or water on the surface of silica.

The next part of our study was concemed with establishing the thermal stability of

the DELTABONDTM phenyl column. The DELTABONDTM phenyl stationary phase

showed reasonably good stability to temperatures up to 250°C. From 250°C to 800°C

degradation of the stationary phase was observed (Figure 30 A). At 800°C the total

percent weight lost of the stationary phase from silica was :6%. The mass spectral data

obtained for the DELTABONDIM phenyl column from pyrolysis TGA-MS is shown in

Figure 30 B. The peak with m/z = 18 corresponds to H20 while m/z = 78 acknowl-

edges the loss of phenyl groups. Further information on the thermal stability of the

DELTABONDTM phenyl column is observed in the separation of polyaromatic

hydrocarbons mixtures. The separation was first conducted at 60°C. The column was

heated then to l50°C, held for 12 hours, and again cooled to 60°C. Under the same

conditions the separation was repeated, Figure 31. The results indicate excellent thermal

stability of the column in terms of both peak areas and retention times.

In summary, the results from DELTABONDTM cyanopropyl column showed basic

compounds can be eluted with good efliciency and selectivity from the column with

100% CO2. However, the basic compounds with high basicity (pK,> 10) were eluted

with some tailing from the column. The DELTABONDTM cyanopropyl column showed

much better selectivity and thermal stability than the endcapped cyanopropyl or the

Hypersil cyanopropyl packed column. The DELTABONDTM phenyl column also

Chapter 3: Enhancement or Packed column SFC via Deaerivaied Stationary Phase; sz

S ,2

3

1

B

S

2

A

1

3

TI|IE,m1n.V 0 5 *0

,,,,13 PRESSURE.atm.100 100 150 309

Figure 28. Comparison of DELTABONDTM and Conventional Phenyl for Activity: Separationrgfpolarity test mixture on (A) conventional Hypersil phenyl and (B) DELTABOND‘

phenyl column with 100% CO; and flame ionization detection at 60°C. 1 = naphthalene,2 = I'1·p€I1Iad€C8H¢, 3 = aC¢!0ph¢l’10|1¢

Chapter 3: Enhancement of Packed Column SFC Via Dcactivated Stationary Phases 83

S 7

4 s1

A 32

(7

6

4B 2 3 5 6

TlME,min.° 0 4 8 12 16

PRESSURE, atm.80 95 140 400

Figure 29. Separation of Polyaromatic Hydrocarbons with phenyl Column: Separation ofpolyaromatic lpxfilrocarbons with (A) conventional Hypersil phenyl and (B)DELTABOND‘

phenyl column with 100% CO; and flame ionization detection at 60°C.1 = m-xylcne, 2 = naphthalene, 3 = 2,3-dimethyl naphthalene, 4 = bibenzyl, 5 =fluorene, 6 = anthracene, 7 = pyrene, 8 = benzopyrene

Chapter 3: Enhancement of Packed Column SFC Via Deactivated Stationary Phase; 84

11 1739 D-PHENYL SECOND im 12.2145 ng RAT:. 50. 00 6-9/nen

IIIILIII 1....II F8 81. 18T¤• 18.7I, n x num:} s.s• °"‘”I

IEIL1 ‘1^ 2**** 1“II

I

II

I

mn1ü"—“äEI—Y1E'I

““aö.äi1"‘— '_ÜT—Eü?1h¤’7F"IFfm MG]- M TEMPERATURE <c> TG

DATE 8/D4/D5 TIME 18• 28

Bann 1360 K 13.637 men} av ¤¤T¤¤ 111729DP. D1 QB

QLIITRPCTED SCRLEDID

I 3 „. <··: sa¢ saI: 4 H

· ·

IIan’an1 a

ra" " „.2‘Z„¤„.„

’.“"

’°

_Figure30. TGA of DELTABONDTM Phenyl Packing Materials: A) TGA of DELTABONDTMphenyl bonded staüonary phase silica base. l}[)‘}l\«lass spectrum of gases from pyrolysis ofstationary phase from phenyl DELTABOND‘

silica base.

_ Chapter 3: Enhancemcnt of Packed Column SFC Via Deactivated Stationary Phases 85

7

_ 6

4B 2 3 51 8

7

6

14

2 3 5 6A

miß-‘ 0 4 8 12 16

PRESSURE, atm.80 95 140 400

Figure 3l. Effect of Tempefuure on DELTABONDTM Phenyl Column: Elfect of temperature onDELTABOND‘

phenyl column. Sequential separation of polycyclic aromatic at 60°C(A) and 60°C (B) after being heated for 12 hours at l50°C. See Figure 29 for detail ofseparauon.


showcd its superiority over the Hypersil phenyl column in selectivity and thermal sta-

bility for the separation of polar and polycyclic aromatic compounds, but did not show

any promise for elution of nitrogen-containing compounds. Although, cross-linking of

the bonded stationary phases has shown very good results, it is predicted that some free

silanol groups are still present on the surface which can be a problem in the elution of

highly basic analytcs.


Chapter 4

Chapter 4: SFE/SFC of Double-Base Propellant

Using FTIR as a Detector

Chapter 4: SFE/SFC of Double-Base Propellant Using FTIR as a Detector 88

Introduction

The columns that were evaluated with supercritical CO2 and methanol modified

CO2 in chapter 2 and 3 have been shown to be effective in the separation of nitrogen-

containing compounds. These columns have been employed for separation of non-

polymeric components in double-base propellant (chapter 4) and different PTH-amino

acids (chapter 5). This Chapter deal with analysis of different stabilizers and non-

polymeric components in double-base propellant using FTIR and FID simultaneously

as a detection system.

Double-base propellants consist of' nitrocellulose and another nitrated ester, usually

nitroglycerin. In addition, a number of additives such as stabilizers, plasticizers,

oxidizers, and metals, which seek to stabilize the propellant and control its buming, are

incorporated. However, the nitroglycerin and stabilizers at a certain temperature, after

a period of time, are degraded. The determination of nitroglycerin and stabilizers in the

presence of degradation products, such as lower nitrated esters of nitroglycerin and

nitrated stabilizers in "bad" double-base propellant, has been a problem for sometime.

Traditional methods for analysis of non-polymeric components in propellant sys-

tems have involved the extraction of more readily soluble components via Soxhlet ex-

traction or by means of filteration after refluxing in an appropriate solvent°‘. After

extraction of the sample for 12 to 72 hours (depending on solvent), extracted compounds

are analyzed. Today chromatography has been used routinely for analysis of soluble

components in double-base propellants. Yasuda" and Volk" used thin layer

chromatography (TLC) to separate and analyze different types of stabilizer. Macke”

has used a combination of TLC and spectrometry for the quantitative analysis of

stabilizer in double-base propellant. A UV detector was used to detect and measure 2-

Chapter 4: SFE/SFC or Double-Base Propellant Using FTIR as a Detector 89

nitrodiphenylamine and resorcinal, however, the propellant plasticizers were measured

via IR spectroscopy. Ammana and co- workers‘°° used high performance TLC

(HPTLC) as well as column chromatography in order to separate and identify different

synthetically prepared mixtures of ethyl-centralites (i.e. another stabilizer component)

and its decomposition products. Different developing mode of HPTLC (e.g. linear as-

cending, linear horizontal, circular mode and anticircular mode) were used to separate

diphenylamine reaction products. Although TLC is a relatively simple and sensitive

method, it has some limitations. For example (l) several developers and visualizations

have to be used which are time consuming and the exact composition of developer and

spray mixture is important‘°‘, (2) some of the nitroso-derivatives are photochernically

unstable on the silica gel layer’°, (3) quantitation is difIicult‘°2, and (4) retention

reproducibility and spot color depends on the thickness of the thin layer.

Gas chromatography is another method which has been used for the analysis of

propellant stabilizers. This technique has the advantage of being compatible with both

universal (FID) and selective (NPD) detectors. GC is most suited for analysis of

nitrated aniline and toluene mixtures‘°’. However, analysis of the most important

propellant stabilizers (e.g. diphenylamine and isomers of nitrosodiphenylamine and

nitrated diphenylamine) is not possible, due to the fact these materials are easily

thermally decomposed‘°". A typical example to this phenomenon is the transformation

ofN-nitrosodiphenylamine into diphenylamine at high temperature. Sporanetti et al.‘°‘

have used heptafluorobutyric acid anhydride to derivatize diphenylamine into a com-

pound which has a different elution time compared to nitrosodiphenylamine. In this

fashion both quantification and qualitative results were obtained. However, this

derivatization procedure is relatively long and complicated. An altemative method to

TLC and GC is high performance liquid chromatography (HPLC). Since HPLC has

several advantages compared to TLC and GC, mainly the ability to obtain exact quan-

Chapter 4: SFE/SFC or Double-Base Propellant Using Frm as a Detector 90

titative information and sample derivatization is not necessary, it has become the

method of choice for this type of analysis. Several workers“"·‘°’ have used both reversed

phase and normal phase HPLC to perform the analysis. One of the major difficulties

with this technique, however, is its lack of efficiency to separate a extracted complex

mixture ofnon-polymeric components even when gradient elution has been used. Also,

the need for a sensitive and universal detector has limited the capability of HPLC.

Supercritical fluids offer several attractive features as a solvent for both extraction

and chromatography. lt has been demonstrated that greater diffusivity and lower

viscosity afforded by supercritical fluid yield more efficient extraction and faster

chromatography with higher efliciency. Many supercritical fluids are inert, non-toxic,

pure, inexpensive, readily available and are easily removed from the resulting extract.

Another advantage of supercritical fluid as a solvent is the ability to control solvent

power by applying different pressure and temperature. Many authors have reported the

extraction of one compound out of a matrix using low density fluid, while retaining the

compound of interest for later extraction using a more dense fluid‘°°. Supercritical flu-

ids with "liquid like" densities, are also able to solubilize many thermally inaccessible

components. Several articles have been reported regarding analysis of thermally labile

compounds via supercritical CO2 using both capillary and packed colunms with different

type of detectionsystems.‘·”

With the demonstration of supercritical fluids for both extraction and

chromatography, the desirable extension would be to extract the compounds of interest

into the supercritical fluid before analysis with SFC. This would be analogous to the

case in HPLC where the mobile phase solvent is comrnonly used for dissolving the

sample. The solvent for sample introduction in SFC is usually different from the mobile

phase. Therefore solvent peaks and any associated impurities are always present. Se-

veral articles have appeared regarding on·line supercritical fluid extraction (SFE) of

Chapter 4: SFE/SFC of D0ub|e·Base Propellant Using FTIR as a Detector 9l

complex mixtures followed by the separation of cxtracted materials using SFC‘°’.

The

advantagcs of' this on-line simultaneous extraction and chromatography are: 1) sample

preparation and the chance for sample contamination are minimized, 2) solvent

absorbances that inhibit spectral interpretation when higher information detectors are

used can be avoided, 3) column life would be extended, because only fsolubilized materi-

als are placed on the column. In this study we describe the separation ofmodel mixtures

of nitrated diphenylamine and nitrated aniline compounds via SFC employing both

capillary and packed columns. Also the results obtained from the analysis of non-

polymeric materials in "good" and "bad" double-base propellants will be discussed. The

efficiency of methylene chloride (CHZCI;) and supercritical CO2 are compared as sol-

vents for extraction of double-base propellants. Detection and identification of the

separated components were obtained with UV, FID and FTIIÄ, respectively.

Experimental V

Lee Scientific 501 (Salt Lake City, UT) and Suprex 200A (Pittsburgh, PA) super-

critical fluid chromatograms were utilized with packed and capillary columns. A

restrictor was drawn from fused silica capillary (50 am i.d.) tubing. A Valco injection

valve with 0.1 ;,¢L total volume was used to introduce the CHZCIZ extract ofdouble-base

propellant into the column. However, an on-line Rheodyne model 7125 injection valve

with 10 ;,tL sample loop was used to inject, directly, a supercritical CO; extract of

double-base propellant on to the column. A Hewlett-Packard (Avondale, PA) model

1082B liquid chromatogram modified for use with supercritical fluids was utilized for

study with and analytical scale (4.6 mm i.d.) column. A variable wavelength UV detec-

Chapter 4: SFE/SFC or Double-Base Propellant Using FTIR as a Detector 92

tor (1-Iewlett-packard model 79875) equipped with a high pressure 1 cm pathlength flow

cell was employed. Sample were injected onto the 4.6 mm i.d. column via a Rheodyne

Model 7125 injection valve having a 10 pL loop.

SFC/FTIR data were collected with Nicolet (Madison, WI) 5SXC spectrometer

equipped with a prototype 0.6 mm i.d. x 5 mm pathlength high pressure flow through

cell. The temperature of the flow cell (1.4 ;1L) was maintained at 32°C. Spectral data

at 8cm·‘

resolution were acquired at 4 scan/file to yield a time resolution of 1 file/sec.

Model compounds and propellants employed in this study were provided by Ann

Richardson and Georg Nauflett of the Naval Surface Warfare Center, Indian Head,

MD. Model compounds were all dissolved in pesticide grade methylene chloride (T. J.

Baker Chemical Co.) prior to introduction onto the column. The concentration of

sample per component ranged from 200-500 ng/pL. A DELTABONDTM packed column

(Keystone Scientific), 25 cm x 1.0 mm i.d., and 10 cm x 1.0 rnrn i.d., 5 pm particle size,

a propylarnino bonded phase silica column (Alltech) 25 cm x 4.6 mm i.d., 5 pm particle

size and an SB-cyanopropyl-25 bonded fused silica column (Lee Scientific) 10 m x 100

um i.d. with 0.25 um film thickness were employed for chromatographic separations.

Carbon dioxide and methanol-modified CO2 were obtained from Scott Specialty Gases

(Plumbsteadville, PA).

Non-polymeric components of two different propellants ("bad", RAD84F 002-008,

and "good", RAD87B 004-003) were isolated via two extraction methods. In the first

method, samples were extracted by methylene chloride. A soxhlet extractor with 300

mL extraction flask was employed to extract 2 grams of each propellant. The recom-

mended extraction time for each sample was 72 hours. After extraction, 20 mL of ex-

tract was evaporated to 1 mL by aeration, and the concentrated extract was

chromatographed. In the second method samples were extracted with supercritical

CO2. Milton Roy (Redondo Beach, FL) sample preparation accessory equipped with

Chapter 4: SFE/SFC er Double-Base Pmpeuam Using FTIR as a Damn: 93

extraction bomb and recirculating pump was used to extract 100 mg of the propellant

for 12 hours. The extraction parameters were set at 275 atm and 60°C.

Result and Discussion

Supercritical fluid chromatography (SFC) has been conducted on different samples

containing nitrogen. ln the first part of this study two mixtures of various nitrated

diphenylamincs and nitrated anilines were separated using both capillary and packed

colunms via both supercritical CO2 and methanol-modified supercritical CO2 as a mo-

bile phases. In the second part, separation of non-polymeric material in "good" and

"bad" double-base propellants was obtained with a cross-linked packed column using

supercritical CO2. Also the efliciency of CH2Cl2 and supercritical CO2 are compared

as solvents for extraction of these double-base propellants.

SFC of Nitrated Diphenylamines and Nitrated Anilines

The selected compounds used in this part of study are those which either are com-

monly used a propellant stabilizers or are found in propellant as decomposition products

of stabilizers. The separation of these two mixtures was initially developed using a

conventional propylamino packed column via 100% CO2. Most of the later eluting

compounds in each mixture were eluted as broad peaks with long retention times under

these chromatographic conditions. With the addition of 2% methanol as modiiiers to

supercritical CO2, however, all the components were eluted with good efliciency, selec-


tivity and within a reasonable elution time. Figure 32 shows the resulting separation of

nitrated diphenylamines. Separation of all seven compounds was obtained. Under the

same conditions, separation of the mixture of nitrated anilines was obtained in less than

eight minutes (Figure 33). A baseline separation was, however, not obtained here. N-

nitroso-N-methylaniline co-eluted with p—nitro N- methylaniline and

N-methyl-2,4-dinitroaniline co-eluted with N- methyl N-nitroso-2,4-dinitroaniline. Nei-

ther decreasing the percentage of methanol nor the CO2 pressure significantly improve

the chromatographic resolution. The mechanism of separation for these compounds on

this conventional packed column is believed to be based on both adsorption and parti-

tion as has been previously discussed in chapter 2.

Identification of the components in a simple mixture usually is carried out by single

injections of a series of known compounds. However, for a complex mixture, a more

specific detector such as Fourier transform infrared spectroscopy (FTIR) or mass

spectrometry (MS) is required for unequivocal identification. On-line FTIR detection

of components with a mobile phase such a methanol-modified CO2 is not possible due

to the incompatibility of methanol with FTIR. Previous studies in our laboratory have

indicted that polar compounds (i.e. basic compounds, steroids, etc.) cannot be eluted

via 100% supercritical CO2 from conventional packed columns due to an excess of free

silanol groups. However, the same components have been shown to be eluted from

more deactivated columns, such as capillary or DELTABONDTM packed columns.

Therefore, a similar study was conducted with the same nitrated diphenylarnine and

nitrated aniline mixtures on a small bore (1.0 mm i.d.) DELTABONDTM cyanopropyl

column and a capillary cyanopropyl (100 um i.d.) colurrm. Figure 34 shows the sepa-

ration of nitrated diphenylamines on both the DELTABONDTM and capillary column

using flame ionization detection. A11 of the seven components were eluted with 100%

CO2 from both columns. Different selectivity was exhibited by each column, although

Chapter 4: SFE/SFC or Double—Ba.se Propellant Using FTIR as a Detector 95

1 2

4

3

44

44

*1 6

7

0 2 4 6 .8 10 12 14TIME, min.

Figure 32. Separation of Model Nitrated Diphenylamine Mixture with Conventional PackedColumn: Separation of model nitrated diphenylamine mixture with UV (254 nm) de-tection; propylamino column (25 cm x 4.6 mm, i.d., 5 pm); 55°C; 98/2 CO;/CHJOH (inlctpressure = 3300 psi); 2 mL/min; 1 = Nmitrosodiphcnylamine, 2 = 2-nitrodiphenylamine, 3 = diphenylamine. 4 = 2,4-dinitrodiphenylamine, 5 =2,4,6-trinitrodiphenylamine, 6 = 2,4’-dinitrodiphenylamine, 7 = 4·nit.rodiphenylamine.


1 2,3

6 ,7

p .n

54

1 " 8

S

0 1 2 3 4 5 6 7 8 9TIME , min.

Figure 33. Separation of Model Nitrated Aniline Mixture with Conventional Packed Columnz Sepa-ration of model nitrated aniline mixture with UV (254 nm) detection. Conditions are de-scribed in Figure 31. 1 = dinitrotoluene, 2 = N·niu·oso-N-methylaniline, 3 =para~nitro-N- mcthylaniline, 4 = N-ethyl-2,4-dinitroaniline, 5 = N·methyl-2,4,6~trinitroaniline, 6 = N·methyl-2,4-dinitroaniline, 7 = N-methyl-N-nitroso~2,4·dinitroaniline, 8 = N-methylaniline.


both contained the same bonded phase. For example, with the DELTABONDTM packed

column, N-nitrosodiphenylamine co—eluted with diphenylamine, but partial separation

of 2,4’- dinitrodiphenylamine from 2,4,6-trinitrodiphenylamine was observed. On the

other hand, with the capillary column separation of N-nitrosodiphenylamine and

diphenylamine was obtained, but co—e1ution of 2,4’-dinitrodiphenylamine and 2,4,6-

trinitrodiphenylamine occurred. These slight difference in resolution probably can be

accounted for on the basis of different column efficiencies. Each separation was also

carried out at 100°C with different (from above) pressure programming on both col-

umns. At 100°C, the DELTABONDTM colunm showed a better resolution for compo-

nents 5 and 6. However, the capillary separation at 100°C became worse in that N-

nitrosodiphenylamine co-eluted with diphenylamine. The general elution order of all

seven compounds was the same on both colunms.

The separation of nitrated diphenylarnines was next performed with on-line FTIR

detection employing the capillary column at 70 °C with pressure programming. The

Gram Schmidt Reconstruction (GSR) for this experiment is shown in Figure 35. The

match with the FID trace in Figure 34b is excellent. In order to prove the identity of

each peak, on-line FTIR spectra obtained during the chromatographic run were com-

pared with spectra obtained via SFC/FTIR of individual component injections. Figure

36-39 show FTIR spectra of the neat components in comparison with on-line FTIR

spectra obtained during the separation of the mixture. The spectrum of the component

eluting ir1 peak #1 apparently is N-nitrosodiphenylamine because its spectrum matches

very favorably with that of the spectrum of authentic N- nitrosodiphenylamine. Similar

observations can be made regarding the identification of peaks #2-4 and #6. Evidence

for the partial co-elution of 2,4’-dinitrodiphenylamine and 2,4,6- trinitrodiphenylamine

in peak #5 is provided by noting the difference in FTIR spectra (Figure 38) of material

eluting in the peak (PKSF) relative to material eluting in the back of the peak (PKSB).

Chapter 4: SFE/SFC or Double-Base Propellant Using FTIR as a Detmo: 98

1.Z

3

A

74 5 8

0 5 10 15 Z? ttME,mIn.

80 100 130 310 PRE$$u1\E,•tm

32

1B

5,64

7

0 5 10 15 22 2; QO 32 *nM!.m•n.

120 120 2 0 415 415 PRESSUREMM

Figure 34. Separation of Nitrated Diphenylamine Mixture with Capillary and Packed DELTABONDTMColumnsz Separation of' model nitrated diphcnylamine pkiixture at 70°C with flameionization detection and 100% CO;. (A) DELTABOND

‘cyanopropyl column (100

mm x 1.0 mm, i.d., Sym). (B) Capillary cyanopropyl column (10 m x 100 ym, i.d., filmthickness = 0.25 ym). See Figure 32 for peak identification.

Chapter 4: SFE/SFC of Double-Base Propellant Using FT1R4as a Detector 99

It would appears in this case that 2,4,6- trinitrodiphenylamine slightly precedes the

elution of 2,4’- dinitrodiphenylamine.

The group of nitrated anilines was next separated on both the capillary and the

DELTABONDTM packed columns (Figure 40). An identical elution order of nitrated

anilines was anticipated for both the capillary and the packed column, based on the

elution behavior of nitrated diphenylamine. However, this was not the case. With the

DELTABONDTM column N-methyl-2,4,6-trinitroaniline and N-methylaniline were

eluted, respectively, as the fourth and sixth peak, but with the capillary column the

elution order of these two compounds was reserved (i.e. N- methylaniline eluted as the

fourth peak and N-methyl-2,4,6- trinitroaniline eluted as the sixth peak). Regardless of

the chromatographic conditions employed N-methyl-2,4-dinitroaniline and

N-methyl-N-nitroso-2,4-dinitroaniline co-eluted as was the case when the separation was

performed with the aminopropyl column. With FID and UV detection of these com-

pounds can naturally not be distinguished from one another. Employing FTIR as an

on-line detector with cyanopropyl capillary column we hoped to spectrometrically re-

solve these pairs of components. The GSR for this experiment is shown in Figure 4l.

FTIR revealed that these pairs of compounds are in fact identical and not different as

originally thought. The spectra of the front and back of peak #2 are identical; while a

similar situation exists for the front and back of peak #5. After cbtaining the SFC/FTIR

of individual components provided by the Naval Surface Warfare Center, results re-

vealed that the nitroso aniline derivatives were indeed lacking integrity. The compounds

stated to be N-nitroso-N-methylaniline and p-nitro-N- methylaniline were both in fact

the latter as revealed by a comparison with the Aldrich reference SpCCtrUm. A similar

observation was made concerning N-methyl-N-nitroso-2,4- dinitroaniline in that it

tumed Out to be really N-methyl-2,4- dinitroaniline. In retrospect it is not too surpris-

ing that these chemical transformations would have taken place given the known insta-

Chapter 4: SFE/SFC or Double-Base Propellant Using FTIR as a Damm: 100

DETECTUH S I GNHL , VOLT5/SCFIN.0023 .0088 .0113 .0158 .0203 .02't8 .0293NG

(DO

Ui

•¤—lD1]G ~1F•1am ‘U)

N

a UGG

ACJ

Ui•·F

N

N•F

Figure 35. GSR of Model Nitrated Diphenylamine Mixture: GSR of model nitrated diphenylaminemixture on capillary column described in Figure 34. See Figure 32 for peak identification.

Chapter 4: SFE/SFC of Double-Base Propellant Using FTIR as a Detector l0l

J'

5 1. N-nItrosod|phanyIamIn• ‘•1

tutxt3 ° <¤ 5m Q lu ·[E 0:0

‘OU-) U]

°° EIS S<£'210 1 00 1 90 80 2010 1 00 1190 B0NHVENUMBEH NHVENUMBEH

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0 tn Ou: . E OIw 3

ÜQ O9 0O 0I'Z 10 l ÜÜ 1 90 80 •2 10 [ gg 1 gg B0WRVENUMBEH ' wavsuumasn

Figure 36. On-line SFC/FTIR Spectra of Nmitrosodiphenylamine and2,4-dinitrodiphenylaminez Comparison of on-line SFC/FTIR spcctra of N-nitrosodiphenylamine and 2,4-dinitrodiphenylamine eluting during capillary separation ofmodel nitratcd diphenylamine mixture with spectra obtaincd during elution of singly in-jected components. See Figure 34 for chromatographic conditions.

Chapter 4: SFE/SFC of D0ub|e·Base Propellant Using FTIR as a Detector 102

2. 3. 2-nItr¤dIph•nyI•mIn•Q n

3 Üz FE - z Qää ¤ ä ¤C UI

.C) C)tn in(D (T') (D U')C ·—·

C OO OQ ¤I'20 10 1600 1 90 780 * 2010 1 00 1 90 80

NHVENUMBEH HFWENUMBEH

Q LD•-I(T)

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UJ .(L55 U LDZQgQ cn Q

Q CQ C)Q U7CD .1·QC3*.2

10 1 00 1 90 80 *2 10 1 00 1 90 80NHVENUMBEH NHVENUMBEH

Figure 37. On·|ine SFC/FTIR Spectra of Diphenylamine and 2-nitrodiphenylamine: Comparison ofon-line SFC/FTIR spectra of diphenylamine and 2—nitrodiphcny1amine eluting duringcapillary separation of model nitratcd diphenylamine mixture with spectra obtained duringelution of singly injected components. See Figure 34 for chromatographic conditions.

Chapter 4: SFE/SFC of Double-Base Propellant Using FTIR as a Detcctor l03

,.. mQ 5. 2,4,l·trInItr¤dlph•nyI•mIn• g 0. 2.4'·dInItr¤dIph•ny1•mIn•

' . ' I.

ui ” **—**2* <¤ ‘2’ :3 2a: 9. g 0E 9 <= '0 ° OU) U1

Qcc 0Q CE·20l0 1 00 1 190 780 *2010 1800 1190 780' NHVENUMBEH NHVENUMBEF1

PxsaQ 0tu_ *-* r~ Ü mä N zÖ Q: QÜ (D QIE •

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O 0**1 umLD O gg ,.,

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Figure 38. On-line SFC/FTIR Spectra of 2,4,6-trinitrodiphenylamine and2,4’-dinitrodiphcnylaminez Comparison of on-line SFC/FTIR spectra of 2,4,6~trinitrodiphcnylamine and 2,4’-dinitrodiphcnylaminc eluting during capillary separaüon ofmodel nitrated diphenylamine mixture with spectra obtaining during elution of singly in-jected components. See Figure 34 for chromatographic conditions.

Chapter 4: SFE/SFC of Double·Base Propellant Using FTIR as a Dctcctor 104

rt;7. 4-nttrodIph•ny|•mIn•

O

6Z 3gg 0¤: Q0tnrg ...Q O

OO"2010 1600 1 190 760

HHVENUMBEF1

$9O PK6O

6Z 3gg 0u: QOU1

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Figure 39. On~line SFC/FTIR Spectra of 4-nitrodiphenylamine: Comparison of on·line SFC/FTIRspectrum of 4- nitrodiphcnylamine eluting during capillary separation of model nitrateddiphenylamine mixture with spectrum obtaining during clution of singly injected com-pound. See Figure 34 for chromatographic conditions.


bility ofN·nitroso derivatives to light and heat. Peak #l, #4, #8 and #5 could be readilyassigned to 2,4- dinitroaniline, N-ethyl-2,4-dinitroaniline, N·mcthylaniline andN-methyl-2,4,6-trinitroaniline (Figure 42, 43).

SFC of Double base Propellant

Soxhlet Extraction

Supercritical fluid chromatography (SFC) has been conducted on both "good"(RAD87B 004-003) and "bad" (RAD84F 002-008) double-based propellant using apacked column with on-line sequential detection via FTIR and FID. The componentsextracted into the CHZCIZ are generally believed to be di-n-propyl adipate (DNPA),triacetain (TA), nitroglycerin (NG) and 2-nitrodiphenylamine (2-NDPA).

0II

0 c-c—cn3C O ( I IHHcH -3 VOC (CH2)4COC3H7ca -0-c-cn2, H 3

0DNPA.............................................................TA

cu·O•N&O

2 \O0

lN \ _\c

&ocn -0-2 Nxo

NG..............................................................2-NDPA

Chapter 4: SFE/SFC of Double-Base Propellant Using FTIR as a Detector l06

Packed

2,:1

1 6.7.’ ¤

0 5 10 15gg80

100 150 400 v¤sssu¤:,••«•.

S

capillary

2,3

1 Q 6,7

4 S

0ä 12 12 20 2E 50, TIME ,mIn.

120 120 1 0 , 415 415 messunmntm.

Figure 40. Separation of Model Nitrated Aniline Mixture with Capillary and Packed DELTABONDTMColumns.: Separation of' model nitrated aniline mixture with flame ionization detectionand 100% CO; on packed (70°C) and capillary (100°C) columns. See Figure 34 forchromatographic condition and Figure 33 for peak identification.

Chapter 4: SFE/SFC of Double-Base Propellant Using FT[R as a Dctcctor 107

N OO O

CDO

OO

JPO

IJIT'!

Fi O0 S Z':> ‘*C>-1 E CDJ) — ZE _

2 g E ‘-1 mf-" „ <ua O O"° 20 --2

N *"' -

° EI-!(D

OO 0

•F

° 9iO

N O

OO

Figure 4l. GSR ol' Model Nitrated Aniline Mixture: GSR of model niuated aniline mixture oncapillary column described in Figure 40. See Figure 33 for peak identification.

Chapter 4: SFE/SFC of Double-Base Propellant Using FTlR as a Detector l08

=•·to1. 2,4-DINITROTOLUENE 4. N·ETHYL·2,4-DINITROANILINE

tu2 ·~ l§·¤ä ¤z „‘ä¤2E ¤:O 0W tn¢¤ O . m 0G: 0 · GI 0Q 0

2 10 1 00 1 90 80 2 10 1 00 1 90 80HFWVENUMBEF1 NHVENUMBEF1

S SO PK1 ___ PK3•O

S S. z Ü z 3g 0 g 0u: ' tt CZ0 0tn in ·D •-•

D (T) .c 0 <t: 0 ·° 8•2 10 1 00 1 90 80 I·2 10 1 00 1 90 80

NHVENUMBEH NHVENUMBEF1

Figure 42. On-line SFC/FTIR Spectra of 2,4-dinitrotolucne and N-ethyl-2,4-dinitroaniline: Compar-ison of on~line SFC/FTIR spectra of 2,4- dinitrotoluene and N-ethyl-2,4-dinitroanilineeluting during capillary separation of model nitrated aniline mixture with spectra obtainedduring elution of singly injected components. See Figure 40 for chromatographic condi-tions.


ä s. N·METHYL—2,4,6·TR|N|TROAN|LINE é B- N-MET**Y*-^N*L*NE

B‘

Ef29 2%%m CZ m Q .tx: nt ·0 0cn snm m m CJa; Q az CJ0 0*2 10 1 00 1 90 80 2 10 1 00 1190 80HHVENUMBEH · NHVENUMBEH

r~E PK6 PK*e

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ii,T

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Q 0*2 10 1 ÜÜ l 90 80 :2 10 1 gg [ gg ggNHVENUHBEH NgVENUMgEg

Figure 43. On-line SFC/FTIR Spectra of N-methyl-2,4,6-trinitroaniline and N·methy|aniIine: Com-parison ofon-line SFC/FTIR spectra of N—methyl-2,4,6-trinitroaniline and N-methylanilineeluting during capillary separation of model nitrated aniline mixture with spectra obtainedduring elution of singly injected components. See Figure 40 for chromatographic condi-tions.

Chapter 4: SFE/SFC of Double-Base Propellant Using FTIR as a Detector ll0

Other components which result from the breakdown of TA, 2-NDPA, NG, and DNPA

are also expected.

Initially, separation of three standards (TA, DNPA, and 2-NDPA) was developed

with 100% CO;. Next, under similar conditions, the separation of each CH;C1; extract

sample was obtained, with both flame ionization and FTIR detection, Figures 44 and

45. No major difference was observed in chromatograms of "good" and "bad"

propellant with the same detector. Each of the four major components was separated

and subsequently identified using FTIR as a detector. Several minor components were

observed in FID traces of both sample extracts, but due to the lower concentration the

identification of these components via FTIR was not achieved. Infrared spectra of

components eluting in the first three peaks of each separation coincided to spectra that

we obtained on the three standards (i.e. DNPA, TA, and 2·NDPA, respectively, Figures

46a-c). A fourth major peak which has a relatively weak response to FID but high

absorbance in the IR region was determined to be NG (Figure 47). An authentic sample

of NG was not available, but the spectrum corresponds quite well to that previously

reported for the NG in the solid state‘·‘°. Bands at 1276 and 1669cm·‘

correspond to

the asymmetrical and syrnmetrical stretching vibrational modes of the NO; group. Also,

several low intensity bands were observed in the aliphatic C-H stretching region (2900

cm·‘). The 1017cm*‘

bands is probably due to the C·O stretch. A fifth peak (detected

via FTIR) was eluted just after the solvent but was not observed in FID trace. This

co-elution of solvent and fifth peak in FID trace may be attributed to band broadening

in the transfer line since the FTIR preceded the FID. The IR spectrum of the peak

component (labeled A in Figure 45) was found to be due to acetone which was used to

wash-out the Soxhlet extractor even though the extractor was heat dried prior to use.

Chapter 4: SFE/SFC or Double-Base Propellam Using FTIR as a Dezmor in

TA ZNDPA

N0

rnz0inQE RAD87B 004-003u.

WPA TA 2NOPA

NG

SSaen2E RAD84F 002-008u.

b

0 5 10 15 ZQ Q? TIM!1 5 125 I50 200 400 PR!SSUH!

Figure 44. Separation of CH;Cl; Extract ol' "Good" and "Bad" Double-Base Propellants with Super-critical CO; : Separaüon CI-I;Cl; extract of both "good" (RAD87) and "bad"(RAD84) double-base propellants via 100% CO; at 60°C with DELTABONDTM (25 cmx 1.0 mm i.d., 5 pm particle size) packed column using FID as a dctector.

Chapter 4: SFE/SFC of Double-Base Propcllant Using FTIR as a Dctector IIZ

S. 42

1 3 GOOD. 00 . 00 10. 00 15. 00 20. 00

RETENTION TIME (MIN)20 320 620 920 1220 1520 1620DHTR P0 I NTS

$ A 2 4

13 am

RETENTION TIME (MIN)

DHTF1 PUINTS

Figure 45. Comparison of GSRs for CHICI; Extract of "Good" and "Bad" Double-BasePropellantsz Comparison of GSRs for CHZCI2 extract of "good" and "bad" doub1e·basepropellant using 100% CO;. S = C1;C1;, A = aceton, 1 = DNPA, 2 = TA, 3 =2-NDPA, 4 = NG

Chapter 4: SFE/SFC of Double-Base Propellant Using FTIR as a Deteetor 113

HBSÜHBHNCE HBSÜHBHNCE-.00% .022 .0%6 &.008 .023 .05%w „

0Z ; § CJ 0D ;_,< z< m ;¤m zz rv C Nc 3 O3 OCD2 ° m °I

~Pl

IJ0

8 0HBSOBBHNCE HBSUHBHNCE

.._gg ,09 ,09 -.00 .03 .07U) w _ii_,_30Z 8 2 g O ;·D io <ä mz rv ä N8 C x 02·= 2°08 0

HBSOBBHNCE HBSOHBHNCE- 003 .016 .039 - .002 .030 .062ux ' N0 O M§ ¤ R — § ¤ [ g< ~· 2 2. rx: 8 N0 3 O2¤ 2°:¤ _ N

08 0

Figure 46. On-line SFC/FTIR Spectrum of DNPA, TA, and 2-NDPA: Comparison of on·lineSFC/FTIR spectra of components eluting during separation of CH;Cl; extract of double-base propellant with spectra obtained during elution of singly injected components. SeeFigure 44 for chromatographic conditions.

Chapter 4: SFE/SFC of Double-Base Propcllant Using FTlR· as a Detcctor ll4

NM g

"'_'

PK. 4i .;.•<

6*8°°2*2<II

(IJg •-I

gf) .:t·•III ° r~ -(E §

NCJ

*3210 2720 2230 17*10 1250 760NHVENUMBEH

Figure 47. On-line SFC/FTIR Spectrum of Nitroglycerinez On-line SFC/FTIR spectra of peak 4from separation of double-base propellant. For chromatographic conditions see Figure44.

Chapter 4: SFE/SFC of Double-Base Propellant Using FTIR as a Detector H5

Supercritical Fluid Extraction (SFE)

Next, on-line SFE was performed on the same propellants followed by SFC em-

ploying the same conditions as the previous separations shown in Figure 44 and 45.

FID traces of both the separations are shown in Figure 48. A noticeable difference ex-

ists between chromatograms of CH2Cl2 extract and SFE. It would appear that either

the supercritical extract is more concentrated than the CH2Cl2 extract or the former

process has solubilized a larger number of components since at least twice as many ma-

jor components are detected via FID of the SFE as was detected for the CH2Cl2 extract.

No major difference was observed between FID traces obtained from the SFE/SFC of

"good" and "bad" double-base propellant. The differences in retention times between

RAD84 and RAD87 reflect slightly different restrictor internal diameters. Although

flame ionization is a very sensitive mode of detection, the identification of peak compo-

nents can be questionable and may be difficult. lt should also be noted that since no

extraneous solvent is require in SFE/SFC experiment, all components detected via FID

originate from the propellant (i.e. no solvent peak). Based on retention time compar-

isons DNPA and TA could be accounted for; whereas, 2-NDPA apparently was not

extracted since no chromatographic peak matched the retention of 2-NDPA. This ob-

servation was quit surprising since 2-NDPA elutes from a column under similar condi-

tions employed for extraction. This type of behavior has been observed previously for

extraction and separation of caffeine. For example, it has been shown that caffeine can

be eluted from a column with 100% supercritical CO2, but can not be extracted, unless

the supercritical CO2 contains a small percentage of water"‘. It is possible that 2-

NDPA binds to the propellant and is not released, unless a modifier is added to the

supercritical CO2. Pairs of peaks labeled Y and Z are not identifiable since none of our

standards exhibited these retention times.

Chapter 4: SFE/SFC of Double-Base Pmpcllam Using FT111 as a Detector 116

1 2 4 x Y Z ·Z

W

RAOMF1

2 4 x Y 1 ZW

RA¤•1¤ ooe-oo:

25 10 15 E 2: 30 Tlll

125 1 s 160 zoo eoo eoo mum:

Figure 48. Separation of Supercritical CO; Extract of "Good" and "Bad" Double·Base Propellant withSupercritical CO;: Separation of 10 ;¢L supcrcritical extract ofgood and bad double-basepropellant. See Figure 44 for chromatographic conditions and experimental section forextraction conditions. 1 = DNPA, 2 = TA, 4 = NG, W = Unknown, X = Unknown,Y = Unknown, Z = Unknown

Chapter 4: SFE/SFC of Double-Base Propellant Using FTIR as a Detector ll7

Figure 49 like Figure 48, shows the separations of "good" and "bad" double-base

propellants but now with FTIR detection. As expected fewer components are observed

via FTIR, since it is a less sensitive detector than the flame ionization detector. In order

to observe several minor components, the detector detector response was expanded such

that some peaks were off scale. Individual file spectra were examined in order to gain

information about eluting materials. Three major components, DNPA, TA, and NG

were easily identified by their retention time (see Figure 49) and also from side-by-side

· comparisons of the file spectra with the spectra of the individual components. Even with

more specific detection (FTIR), no evidence for the presence of 2-NDPA could be as-

certained in the SFE. Again, difference in retention time for the same components found

in the "good" and "bad" propellant reflect different flow restrictor intemal diameters

employed in the two separations. Since FTIR and FID were accomplished sequentially

(i.e. one injection) valid comparison can be made between these two traces insofar as

retention times are concerned. In that regard, peak Y and Z demonstrated much less

infrared response than flame ionization, such that no identifiable spectra could be ob-

tained on these components. The large FID signal, however, reflects eluting materials

with much hydrocarbon character.

File spectra of two common peaks in both "good" and "bad" propellant separations

were studied in hope of identifying those compounds. The IR spectra of the component

leading to peak W in both "good" and "bad" propellant were identified (Figure 50). The

bands at 1663, 1617 and 8llcm*‘ are typical stretching vibrational modes for nitrite

functional groupsuz. Also weak C·H stretching bands at 2951 cm·‘ indicate the pres-

ence ofaliphatic carbon. It is conceivable that a reduction ofnitrate ester to nitrite ester

has taken place in both types of propellant. This early eluting component, of course,

would not be seen in the separation of the CH2Cl; extract since it would have been ob-

scured by the solvent peak given identical chromatographic parameters. Unfortunately,

Chapter 4: SFE/SFC of DoubIe—Base Propellant Using FTIR as a Detector ll8

we do not have appropriate model compounds to test this hypothesis. Individual files

taken throughout the peak indicate the elution of only one component. The Spectra of

co-added files across peak X (Figure 51) are strikingly similar to the spectrum of NG

(Figure 47). A small difference in the position of bands, compared to the spectrum of

NG, was observed. Specifically, the bands at 1669cm·‘

was shifted to lower

wavenumber and the bands at 835 cm·‘ was shifted to 844cm*‘.

This compound is be-

lieved to be a lower nitrated ester of NG such as 1,3-dintiroglycerine. The absence of

an authentic sample, however, makes an absolute assignment tenuous.

The separation of the "bad" double-base propellant yielded a unique peak (t, = 7

min) with a large tailing which was detected only with the FTIR, not FID. At first, it

was believed this compound to be highly nitrated because it is known that FID has low

response to nitrated compounds‘ *3, whereas, nitrated functional groups have strong in-

frared absorption bands. However, after looking at the co-added spectrum across this

peak (Figure 52), several unique bands were observed that cannot be attributed to

nitrate ester. The spectrum has two bands at 1273cm·‘

and 1384cm·‘

and several weak

bands in the 2000cm·‘

region. Also a strong absorption band at 1608cm·‘

was ob-

served. After studying of the spectrum closer, it was reasoned that all of these bands

are fundamental frequencies for C02 and H20. As water from the "bad" propellant

elutes from the column, it apparently adducts with C02. This interaction alters the force

constant of both molecules; thus, the exact frequencies of both C02 stretching and

bending vibrations are slightly altered **2. Being slightly shifted the weaker bands due

to C02 at 2052, 1944, 1385 and 1274cm·‘

are not totally compensated for in the back-

ground subtraction routine. The intense absorbance for C02 observed at both the low

and high energy side of the totally absorbing regions (3900-3400 cm·‘; 2600-2000 cm·‘)

also supports this notion. The 1608 cm·‘ band can be confidently assigned to the H20

deformation mode. Spectra of the front and back of this broad chromatographic peak

Chapter 4: SFE/SFC or Double-Base Propcllant Using FTIR ns a Detector 119

were examined and no difference was observed. The absence ofan FID response further

corroborates the presence of H20 for this chromatographic signal.

In conclusion, since some of the non-polymeric materials in double-base propellant

are thermally labile (i.e. nitroglycerin, 2-NDPA) SFC is a more suitable method for

separation of these components. Alos the results suggest that for detection and iden-

tification of separated compounds, both FID and FTIR are desirable. However, for the

detection of highly nitrated compounds FTIR or another specific detector such as mass

spectrometer would be preferred. lt was further demonstrated that SFE double-base

propellants has several advantages over the conventional liquid solvent extraction. It

therefore appears that SFE/SFC provides an easy and efficient method for both ex-

traction and separation ofnon-polymeric materials in double-base propellant.

Chapter 4: SFE/SFC er Deubln-Bun Pmpeltam Using FTIR as e Deinem- 120

1 2 4 X

W

GOOD

5.00 0.00 15.00 20.00 25.00RETENTIÜN TIME (MIN)

0 3%0 660 1020 1360 1700 20&0DHTH POINT6

1 2 4 x

I I IIW I

IBAD

.00 5.00 10.00 15.00 20.00 25.00RETENTIÜN TIME (MIN)

"“120 350 060 _1010 ;3%0 1670 2000

_ ÜQTH FOINT3

Figure 49. Comparison of' GSRs for Separation of Supercritical CO; Extract of °‘Good" and “Bad"Double-Base Propcllantz Comparison of' GSRs for supercritical CO; extract of' "good"and "bad" double-base propeliant via 100% CO;. See Figure 44 for chromatographicconditions. See Figure 48 for peak identification.

Chapter 4: SFE/SFC of Doub|e·Base Propellant Using FTIR as a Detector 12l

ä(D' PK . W

MBCO $,3* .

LU(3 N-

äLJ OZ

•

[I LDC) U')cn 0 E(D Ü E(K ° {ii 2 °3 : E

LO(\l

S_ *3210 2720 2230 17110 1250 760

' wavsmumßam

Figure 50. Co-added File Spectrum ol' Peak W: Co-added file spectrum of peak W


or~E-| PK · X

CJLD

LU ·-• ·LJ I

Zv~6%u: =** ,O 1* 5gf) Ü

I

U1

N §(DG

'3210 2720 2230 1 7*10 1250 760HHVENUMBEFK

Figure 51. C0·added File Spectrum of Peak X: Co-added üle spectrum of peak X

Chapter 4: SFE./SFC of Double-Base Propellant Using FTIR as a Detector 123

LI') __3.1** ‘

¤ Q6’•~

II20-{Ig6Ttu cu QL.) C) §E ' „a

0 §LDM *2Q 0

·

M Il\00

0zu3-400 390 3180 270 260150 l4013O 20' wavsmumasn

Figure S2. Co-added File Spectrum of Peak Eluting at Approximately 7 min in the Separation of"Bad" Propellant: Co-added file specu-um of' peak eluting at approximately 7 min in Lheseparation of' "bad" pmpellant. See Figure 49.

Chapter 4: SFB/SFC of Double-Base Propellant Using FTIR as a Detector l24

Chapter 5

Chapter 5: Gradient Separation of PTH-Amino

Acids Employing Supercritical CO2 and Modiüers

Chapter 5: Gradient Separation of PTH-Amino Acids Employing Supercritical CO; and Modiüers I25

Introduction

The most commonly used fluids in SFC are all relatively non-polar. Carbon dioxide,

the most widely used fluid, is no more polar thanhexane“‘·“‘

, even at high densities.

Solute polarity should be between that of the stationary phase and the mobile phase in

order to have a well-behaved separation. Few-world samples contain only non-polar

solutes, so a major objective of research into SFC has been directed toward increasing

the range of solute polarity that can be handled by the technique.

Pure CO2 can elute some rather polar molecules, such as underivatized fatty acids

from capillary columns. Many biological molecules, pharmaceuticals and environ-

mentally important chemicals tend to be both polar and present in very low concen-

trations. Separation of such compounds on capillary may be feasible, but detection is

likely to be a problem due to the low sample capacity of such columns. Packed columns

offer huge increases in sample capacity, but it is difficult to elute even alcohols from

conventional packed columns with pure supercritical fluids. Polar modifiers added to the

fluid are required to elute these and more polar species. Even with modifiers, however,

SFC has not been very successful in eluting highly polar species.

The interactions between polar solutes, modifiers, supercritical fluids and stationary

phases are poorly understood. Practical methods for using SFC with polar species are

only now emerging. This work attempts to provide further insight into interactions oc-

curring on the colunm and practical aspects, such as column preparation needed to ob-

tain reproducible results. The application to amino acids was chosen both because of

its inherent importance and because it offers a family of compounds with a very wide

range of solute polarity, including free acids, bases and amides which have not previously

been separated by SFC.

Chapter S: Gradient Separation of PTH-Amino Acids Employing Supcrcritical CO; and Modiüers l26

Numerous HPLC methods"‘·‘*° have been proposed during the last three years for

the analysis of PTH derivatives formed during the Edman degradation. Most of these

methods prescribe the inclusion of a nonvolatile buffer of low ionic strength and a pH

value in the range 3.5-5.5 as part of the composition of at least one of the solvents.

Chromatography is often performed in a gradient mode at temperature of 30-40°C, al-

though one recent report has involved isocratic elution of PTH- AA’s at 60°C **9.

Stationary phases have generally been silicas which have been derivatized with either

octadecyl, cyanopropyl or phenyl functionality. Separation times vary between 20 and

40 minutes depending upon the number (and type) of PTH-AA in the rnixture and the

chromatographic parameters. The report of Kolbe et al.‘2° detailed the separation of

25 amino acids including cysteic acid, 4-hydroxyproline methionine sulfone, S-

carboxymethylcysteine and S-methylcysteine. Commonly in the hydrolysis step, termi-

nal residue removal is incomplete, and the efficiency of removal is dependent on the

residue identity. This produces increasingly complex chromatograms as sequencing

proceeds down a peptide chain. Baseline or near baseline resolution of all present amino

acids is essential. Unresolved pairs of peak produce numerous points of uncertainty in

the sequence, making the overall result only partially useful.

SFC offers the potential of both increased speed of analysis and the possible use of

more sensitive GC detectors, such as the FID, NPD or FPD. There is also the possi-

bility that SFC/MS may be more straightforward than LC/MS, yielding confirmation

of identity. Very little attention has been given to the separation of amino acids via

supercritical fluid chromatography. Giddings et al. briefly noted many years ago that

amino acids migrated readily from a column with dense NH; gas as a mobile phase"‘.

Underivatized amino acids apparently exhibited no solubility in liquid or supercritical

CO;. Certain derivatives of amino acids, however, have been demonstrated to be

mobilized by modified supercritical CO;. The rapid optical resolution of racemic N-

Chapter 5: Gradient Separation of PTH-Amino Acids Employing Supercritical CO; and Modifiers 127

acetyl·amino acid tert- butyl esters on chiral (N-fromayl-L~valylamino) propylsilica with

supercritical CO2 modified with either methanol, acetonitrile or diethyl ether has been

reported"2. More recently chiral SFC separations of amino acid is0propylester-3,5-

dinitrobenzamides have been noted‘” employing an open tubular capillary column

wherein a methylpolysiloxane stationary phase which has been substituted by chiral

moieties of the "pirk1e type" has been immobilized on the fused silica surface. In none

of these studies was the amino acid very complex.

The first reported separation of 13 PTH-AA's"‘* employed CO2/methanol gradients

on Licosorb silica. The mix, however, only contained PTH-AA’s with hydrocarbon and

the slightly more polar alcohol and amide side chains. The highly polar side chains

which contain carboxylic acids and thiols did not elute. This chapter will describe the

details separation of 24 PTH·AA’s on a conventional packed cyanopropyl column using

a gradient of supercritical CO2 and tetramethylammonia hydroxide in methanol. Also,

this study will point out potential application of this gradient approach.

Experimental

A Hewlett Packard (Avondale, PA) 1082B liquid chromatograph modified for

supercritical fluids and equipped with an HP 79875 variable wavelength ultraviolet de-

tector was used to deliver CO2 to the column. Suprex (Pittsburgh, PA) and applied

Biosystem (santa Clara, CA) micro LC syringe pumps were individually used as pro-

gramrnable flow (5-2000 aL/min) liquid modifier pump. Modifier and supercritical

CO2 were dynamically mixed via a T~mixing chamber (Lee Co., West Brook, CT) at

40°C. The modified CO2 was then immediately passed to the column, also maintained

Chapter 5: Gradient Separation of PTH-Amino Acids Employing Supercritical CO2 and Modiliers l28

at 40°C, UV detector and finally back-pressure regulator fixed at 3500 psi as outlined in

Figure 53. A zorbax Dupont, Wilmigton, DE) cyanopropyl column (25 cm x 4.6 mm

i.d., 5 um particle diameter) and DELTABONDTM (Keystone Scientific, State College,

PA) cross-liked cyanopropyl column (25 cm xl.0 mm i.d., 5 um particle size) were em-

ployed for the separation of PTH- AA’s. Carbon dioxide was obtained form Scott Spe-

cialty Gases (Plumsteadville, PA). Methanol containing 0.00lM tetramethylarnmonium

hydroxide (J. T. Baker, Phillipsburg, NJ) served as a modifier. A kit of 30 PTH-AA’s

was obtained from Sigma Chemical Co. (St. Louis, MO).

Results and Discussion

Development of the Separation

The major objective of this research was to efficiently separate PTH-AA’s ir1 less

than 20 minutes via SFC. Initially, a highly cross-linked cyanopropyl stationary phase

(DELTABONDTM) at 70°C was used with pressure programmed pure CO2 as the mo-

bile phase. Results, as in Figure 54, indicate many of the PTH-AA’s can be eluted from

such a packed column without modifiers and detected with an FID. The sample solvent

peak requires more than 10 minutes to elute, and the separation, as presented, is much

too long to attain our goal of 20 minute separations. The first group of peaks ending

at 29 minutes all represent PTH-AA’s with hydrocarbon side chains (Table ll). These

peaks are relatively symmetrical and exhibited reasonable column efliciency. The last

three peaks are PTH- threonine, PTH~serine and PTH-tryptophan each ofwhich contain

Chapter 5: Gradient scpcmicn cr PTH-Amino Acid; Empieyiiig Supercritical co, and Mcdiiici; l29

HP 10828

SUPREX„„•„. rc „.„„„Eback ¤r•••u

ugulator

. EXIT

. Figure 53. Schematic of Flow Gradient SFC/UV lnstrumentation:

Chapter 5: Gradient Separation of PTll-Amino Acids Employing Supercritical CO; and Modifiers l30

a moderately polar hydroxyl group in the side chain. Peak shapes are very poor, and

severe tailing is evident. More polar compounds, such as PTH-AA’s with carboxylic

acid—containing side are not eluted. lt was felt that further development of this approach

was unlikely to produce the desired separation.

An altemative approach was then developed using more traditional LC packed col-

umns and modifier added to the supercritical fluid. Preliminary results indicated that at

least 28 individual PTH-AA’s could be eluted from a Zorbax-CN column. Three of the

less commonly occurring PTH-AA’s were eliminated from further consideration. These

included S-phenylthio-carbamyl- cysteine, amino caprylic acid and S-phenylthio-

carbamyl~lysine. Two important PTH-AA’s not included in the original list gave ques-

tionable results. Cysteine gave a small broad peak. There was some question as to

whether this peak was in fact cysteine. Argenine also presented a problem in that mul-

tiple peaks were typically detected. As is often the case in LC, there is a reasonable

probability that this PTH-AA is unstable in the chromatographic system. These

questions were not resolved and these two compounds were also not included in this

study. Development of the present work into a viable AA analysis technique could re-

quire resolution of the problem with these two PTH-AA’s and their inclusion in the

separation scheme.

Elution times and conditions naturally separated the remaining PTl-l-AA's into

three elution groups, as indicated in Table ll. The fact that these groups each contain

similar polarity side chains was satisfying. Group 1 contained only similar polarity side

chains PTH-AA’s, all of which elute easily with approximately 3.2% methanol. Group

2 contains four hydroxyl-substituted hydrocarbon side chains, a phenyl side chain, an

amide and a sulfone. Methanol concentration was increased to approximately 15% and

the base, tetramethylammonia hydroxide (TMAOH), was added to the mobile phase.

Group 3 contains carboxylic acids, amines, an amide and aicdic sulfur side chain. This

Chapter 5: Gradient Separation of PTH-Amino Acids Employing Supercritical CO; and Moditiers 131

A,0

FL

I

F

lv

VI

S

T

°4 • u 1• z• za 2• u :••

ee M M sz“"‘·"""‘

«l ‘“| ‘|“OO! OO! PIISSURI, dn.

Figure 54. Isocratic Separation of Selected PTH~AA's on DELTABONDTM CyanopropylColumnz Isocratic separation of selected PTH-AA's on DELTAB0NDTMcyanopropylcolumn (250 mm x 1.0 mm, i.d., 5 pm) employing supercritical CO; at 70°C and flameionization detection. See Table 11 for peak identification.

Chapter 5: Gradient Separation of PTH-Amino Acids Employing Supercritical CO, and Modiliers |32

Table ll. Amino Acids Group Separation

GROUPI

P Prolinc Nv NorvalincL Lcucinc Aba a-Aminobutyric AcidV Valinc A AlanincI Isolcuciric M McthionincO Norlcucinc F PhcnylalanincAib oz-Aminoisobutyric Acid G Glycinc

GROUP 2

Hyp 4-Hydroxyprolinc Q GlutamincMs Mcthionine Sulfonc Y TryptophanT Thrconinc W TryptophanS Scriuc

GROUP 3 ·

K Lysinc E Glutamic AcidN Asparagirie Ca Cystic AcidH Histidinc D Aspartic acid

Chapter 5: Gradient Separation of PTH-Amino Acids Employing Supercritical CO, and Modiüers l33

group required larger amount of modifier and base to yield reasonable peak shapes in

short times.

Preliminary isocratics separations were developed to resolve each of three groups.

Most of the effort went into resolving the severely overlapped region in group l, since

changes in conditions for this region impact the later eluting PTH-AA’s. Base was added

to the group l separation to make it more compatible with group 2 and 3 separations

and avoid requiring three pumping channels. Step changes in modifier were then em-

ployed to progressively elute groups 1, 2 and 3 from a single injection. Finally a multiple

ramp rate gradient elution profile was developed, as shown in Figure 55. For this sepa-

ration a fixed flow of supercritical CO2 (4.5 mL/rnin) was maintained for 12 minutes with

a steadily increasing flow of modifier. This procedure eluted both group l and 2 com-

ponents as well as PTH- lysine and PTH-asparagine. The noise in the chromatographic

trace at 12.5 min. is due to the surge of a much greater percentage of modifier brought

on by both a further increase in flow of modifier and a simultaneous decrease on flow

of CO2 to 3.0 mL/min. PTH-glutarnic acid was found to elute at this point under these

conditions but as a broad peak more poorly defined than PTH-histidine. For this reason

the PTH-glutamic acid was excluded from the mixture, however, it can be efiiciently

eluted under slightly different conditions, vide infra. Note that while the time objective

has been substantially met, two unresolved pairs still exist. Other work on a different

cyanopropyl column allows partial resolution of PTH-valine and PTH-norleucine (V,O)

which indicates they can likely be resolved with further work. Likewise asparagine and

lysine (N,K) were shown to be resolved isocratically. The 24 PTH-AA's could be

reproducibility chromatographed employing the multiple gradient profile provided

equilibration time between runs was sufficient to allow the Zorbax colunm to return to

its original condition. Equilibration could be accomplished by flowing supercritical

Chapter 5: Gradient Separation cr PTH-Amino Acid; Empicying Supercritical co, and Mcdinci; [34

(3500 psi) CO; containing 0.1% TMAOH/CH;OH, V/V, through the column for 20-30

minutes.

One of the striking features of this work and a factor which has led to the success

of this method has been the observation that increased modified CO2 flow rates have

little deleterious effect on resolution. lt appears that the Van Deemter plot for these

chromatographic parameters has an exceedingly broad minimum allowing for rather

high linear velocities without sacrificing very much in colunm efficiency. This phe-

nomenon is dramatically shown in Figure 56 where the separation of Group 1

PTH-AA's at two CO2 flow rates but identical gradients ofTMAOH modified-methanol

(i.e. fixed time- ratio of CO2-to-CH;OH, V/V) has been performed. Currently, we are

studying the fundamental aspect of this phenomenon in more detail.

The Role of Base

There are apparently several separation mechanism at work during the gradient ex-

periment. The retention of some PTH-AA's is more strongly affected by methanol and

· base concentrations than others. Some components change elution order as well as re-

tention time more radically than others for relatively small changes in mobile phase

composition. We had earlier determined that methanolic solutions of TMAOH (0.005

- 1.0M) when added to CO2 did not cause precipitation or "p1ug" the system. It had

also been found that the apparent basicity of TMAOH was greatly diminished in

TMAOH/MeOI-I/CO; systems compared to TMAOH/MeOH/Freon 13 or

TMAOH/MeOI—I/Freon 23. We, therefore, feel it is highly probable that TMAOH re-

acts with CO2 to from a carbonate or forms an additional complex with CO;.

Chapter 5: Gradient Separation of PTH-Amino Acids Employing Supercritical CO; and Modiliers 135

gut N¤•

7P

- si

äY Y

Y V•‘¤ 8 „. 2 ä

E¤¤si ä s >„ I TI

1: Q' ul '¤ .¤ § e „

i o: s

‘ta ä

ai ä N z

r· Q 2 ‘6

9_)

.°’

¤‘ “

ae S :E 2 2g ==!“

zi 3 ‘+‘ ga $9% g

°

i

9. z{

-|nl {l §Q

II

Figure 55. Gradient Separation of 24 PTH-AA'; on Zorbax Cyanopropyl Column: Separation ofP’TH-AA'; on Zorbax cyanopropyl column (250 mm x 4.6 mm, i.d., S pm) employing amobile phase gradient of supercritical CO; and 0.001 M tetramethylammonium hydroxidein methanol. Initial CO; flow rate = 4.5 mL/min; after 12 minutes flow rate decreasedto 3.0 mL/min. Outlet pressure = 3500 psi, temperature = 40°C. See Table 11 for peakidentification.

Chapter S: Gradicnt Scparation of PTl—1-Amino Acid; Employing Supercritical CO; and Modifier; 136

> tto 6

z!I

”„•

2 =’·· “

Ü " ·‘ _ ää

Q <Z U":1 ° Y‘u

6 6

N 6 ‘

at •~• A

"°

r·

f _ —<

6‘

- UZ 2.

°Ö < V _Q , '

{ )

2_'i E

*·‘

>2

6

1*E „Nä?

Figure 56. Effect of Different Flow Rate on Analysis Time and Resolution: Gradient separation of'selected PTH·AA’s on Zorbax cyanopropyl column (250 mm x 4.6 mm, i.d., 5 pm) inmethanol at various total flow rates but with a fixed time ratio of CO;·to-Cl—l,OH, V/V.CO2 flow rate = 3 mL/min (A) and 5 mL/min (B) outlet pressure = 3500 psi. See Tablell for peak identification.

Chapter S: Gradient Separation of' PTH·Amino Acids Employing Supcrcritical CO, and Modiflers 137

The base also apparently interacts with or blocks active sits on the column to sig-

nificantly improve peak tailing. lt also has a rather profound impact on elution times

of some components, as shown in Figure 57 where the only difference in conditions is

the addition of 0.00lM TMAOH to the methanol. A case in point is the retention time

of PTH-glutamic acid and PTH-asparatic acid which increased over 100% upon intro-

ducing TMAOH to the modifier phase. Naively, one might have expected retention time

to decrease given a less activated column (i.e. conversion of silanol sited to

tetramethylammonia sites) since modifiers are usually thought to be most effective in

promoting the elution of more polar solutes. Altematively, it is likely that there may

be an association ofTMAOH and solute in the mobile phase. Clearly this phenomenon

should be studied more fully. lt should be noted in this regard that no evidence of

PTH-AA decomposition was observed as evidenced by the repeatability of the

chromatography.

C0lllIl1ll Pl’€p3|°3Ü0ll

It has been our experience that preliminary evaluation of new columns without

careful preparation can be rnisleading. Many LC columns are quality-checked before

shipment with aqueous-based solvents and, consequently, water must be removed before

use. Washing ovemight with methanol is partially successful, but we have found that

more severe conditions are often required. We often heat the column to 60-l00°C for

several hours with pure methanol flowing and the back pressure regulator holding se-

veral hundred bar of pressure. Solvents such as tetrahydrofuran and acetonitrile were

also noted to cause problems. Such columns were useless, thereby giving minimal sep-

aration until all solvent was removed via the procedure noted above for removing water.

Chapter 5: Gradient Separation of PTH-Amino Aeids Employing Supereritical CO, and Modiüers 138

G

A••

A0

I

° cn

2 1 Q ! ;10 12 I4 II T1uI.nI••.

I W Ü Ü ·I 20 2 g ~•°“

G

BA1:

Q

0

I

Cu

4 10 12 Il 1 .„.„„...„„. „„„„

Figure 57. Effect of Base on Separation of Basic and Acidic PTH-AA's: Gradient separation of se-lected F'TH·AA's on Zorbax cyanopropyl column (250 mm x 4.6 mm, i.d., 5 pm) em-ploying methanol as a modilier with (A) no TMAOH and (B) TMAOH (0.001M). CO;flow rate = 4mL/min. See Table ll for peak identification.

Chapter 5: Gradient Separation of PTH—Amino Acids Employing Supercritical CO, and Modifiers l39

Mobile Phase Considerations

Some may question whether this application is in fact an SFC separation of

PTH-AA's, since the last group is resolved in a mixture which is 33% MeOI—l. It is our

feeling that separation clearly employs supercritical fluids at the beginning, which may

become a liquid as modifier composition increases. We find the distinction irrelevant in

methods development. There is clearly no transition from one type of behavior to an-

other and no detectable discontinuities.

Others may question whether such high modifier compositions can be reached

without phase separation. We have varied CO2/MeOl—l composition over a much larger

range, and at high pressure have never observed detector noise associated with the

presence of two phases. We have also not observed retention time instabilities which

can be associated with multiple phase systems. This is in contrast with other work we

have performed with Freon 116. Exceeding this solubility immediately produces severs

baseline noise. Any attempts to mix supercritical CO2 with very small amounts ofwater

(< 0.1%) rapidly produced severe noise. Furthermore, attempts to add methanol-

containing water to CO; even in very small amounts also produced severe noise. In

most cases, however, noise does not occur immediately upon addition of an irnmiscible

phase. the column first adsorbs polar modifiers until saturated and when the fluid finally

exits the column, the noise is immediately apparent.

Resolution

Extra-column band broadening is rather severe in this instrumental setup, thereby,

yielding similar peak widths for the earliest eluting components both with and without

Chapter S: Gradient Separation of PTH-Amino Acids Employing Supercritical CO, and Moditiers 140

a colurrm. Maximum measured isocratic efliciencies are not better than 4000 plates from

a colurrm capable of producing grater than 20,000 plates in HPLC. Improvement in the

instrumeritation might significantly improve resolution.

In summary, the results presented here show that many PTH-AA’s can be eluted

using CO2 and modifiers and indicate significant progress toward baseline resolution of

the most important members in less than 20 minutes. The fact that the unresolved pairs

in the gradient method could be resolved isocratically and that the system shows signif-

icant extra column band-broadening gives us confidence that further development will

result in completely meeting our objectives. The use of very low to very high percent

modifiers in this study demonstrates the convergence of SFC with HPLC.

Chapter 5: Gradient Separation of PTH-Amino Acids Employing Supercritical CO, and Modiüers l4l

Chapter 6

Chapter 6: Conclusions

Chapter 6: Conclusions 142

Conclusion

The objectives oF this research have been to extend the analysis oF nitrogen~

containing compounds on packed column containing a variety of stationary phases via

supercritical CO; and modified supercritical CO;. Due to advantages oF SFC, high

diffusivity, low viscosity, and moderate operating temperature, it is believed the tech-

nique to be the method oF choice For analysis 0F thermally labile and non-volatile com-

pounds. This thesis has demonstrated application of packed column SFC For separation

oF basic nitrogen-containing compounds. Also this work has described several Funde.-

mental concepts and mechanisms, which exist between stationary phases, mobile phases,

and analytes in the packed column SFC.

In summary, chapter two concluded nitrogen-containing compounds with different

basicity could be separated via SFC employing conventional packed columns. The re-

sults showed addition oF modifier to supercritical CO; promote elution and improve

peak shapes. It was demonstrated that without modifier the range of basicities which

could be eluted increased in the order 0F silica < PRP < C18 = NH;.

Chromatographic peak shapes were much better For NH; bonded phase packed column.

lt is believe that propylamino stationary phase deactivates the adjacent silanol sites via

internal hydrogen bonding. lt is also believed that this internal hydrogen bonding in-

hibits the reaction oF CO; with propylamino bonded phase. Clearly SFC oF basic com-

pounds on silica-base stationary phases would be enhanced and greater selectivity would

be achieved iF these residual silanol sites could be either permanently capped or made to

be less accessible.

The solution to the elution oF highly basic compounds such as aliphatic amines with

100% CO; From packed columns, is addressed in chapter three. The lower activity of

Chapter 6: Conclusions M3

the new cross-linked (DELTABONDT") cyanopropyl stationary phase showed the su-

periority of this column to the conventional or endcapped deactivated columns for

elution of nitrogen·containing compounds. In addition, the stability of this cross-linked

columns to high temperature and polar modifier were much higher compare to

conventioanl packed columns. Similar results were obtained for elution of polar and

polyaromatic hydrocarbons from DELTABONDTM phenyl and conventional phenyl

packed columns. Again, higher efficiency and selectivity was observed with

DELTABONDTM phenyl column.

Chapter four and five describe applications of SFC for separation of nitrogen-

containing compounds via packed and capillary columns. In chapter four, SFC em-

ploying highly deactivated stationary phases appears to provide an efficient mode for

separating the various decomposition products of gun propellant stabilizer and non-

polymexic component in double-base propellant. The results suggested that the iden-

tification of separated compounds with FTIR is highly desirable. It was demonstrated

with FTIR as a detector that most of nitroso derivative compounds were converted to

nitro derivatives on storage. This chapter also showed advantages of supercritical CO2

over conventional liquid solvent for extraction of non·polymeric components in

double-base propellant. It is clear that SFE/SFC provides an easy and efficient method

for both extraction and separation ofnon·polymeric materials in double-base propellant.

Analysis of polar compounds via SFC was one of the major goals of this research.

Chapter five show separation of 24 PTH-amino acids in less than 20 minutes on packed

column by gradient elution of supercritical CO2 and TMAOH-modified methanol. Po-

larity of pure CO2 is relatively weak for elution of basic or acidic components. Due to

this fact addition of small amount ofmodifier or modifiers to supercritical CO2 enhanced

the elution of these polar compounds. This approach may prove to be widely accepted

Chapter 6: Conclusions ‘I44

for routine analysis of polar compounds via SFC. It is believed this area of research

may hold the most excitement in the near future.

Chapter 6: Conclusions |45

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