i/3f* JSf*\£3# i

b STATES TMENT OF IMERCE

ICATION NBS TECHNICAL NOTE 587

Organic Chemistry

Section:

Summary of Activities,

July 1970 to June 1971
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards1 was established by an act of Congress March 3, 1901. The Bureau's overall goal is to strengthen and advance the Nation's science and

technology and facilitate their effective application for public benefit. To this end, the Bureau conducts research and provides: (1) a basis for the Nation's physical measure- ment system, (2) scientific and technological services for industry and government, (3) a technical basis for equity in trade, and (4) technical services to promote public safety.

The Bureau consists of the Institute for Basic Standards, the Institute for Materials

Research, the Institute for Applied Technology, the Center for Computer Sciences and

Technology, and the Office for Information Programs.

THE INSTITUTE FOR BASIC STANDARDS provides the central basis within the United States of a complete and consistent system of physical measurement; coordinates

that system with measurement systems of other nations; and furnishes essential services leading to accurate and uniform physical measurements throughout the Nation's scientific community, industry, and commerce. The Institute consists of a Center for Radiation Research, an Office of Measurement Services and the following divisions: Applied Mathematics—Electricity—Heat—Mechanics—Optical Physics—Linac

Radiation2—Nuclear Radiation2—Applied Radiation2—Quantum Electronics3—

Electromagnetics1—Time and Frequency3—Laboratory Astrophysics3—Cryogenics3

. THE INSTITUTE FOR MATERIALS RESEARCH conducts materials research lead- ing to improved methods of measurement, standards, and data on the properties of

well-characterized materials needed by industry, commerce, educational institutions, and

Government; provides advisory and research services to other Government agencies;

and develops, produces, and distributes standard reference materials. The Institute consists of the Office of Standard Reference Materials and the following divisions: Analytical Chemistry—Polymers—Metallurgy—Inorganic Materials—Reactor

Radiation—Physical Chemistry.

THE INSTITUTE FOR APPLIED TECHNOLOGY provides technical services to pro- mote the use of available technology and to facilitate technological innovation in industry and Government; cooperates with public and private organizations leading to the development of technological standards (including mandatory safety standards), codes

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provides liaison between NBS and national and international engineering standards

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THE CENTER FOR COMPUTER SCIENCES AND TECHNOLOGY conducts re- search and provides technical services designed to aid Government agencies in improving cost effectiveness in the conduct of their programs through the selection, acquisition,

and effective utilization of automatic data processing equipment; and serves as the principal focus within the executive branch for the development of Federal standards for automatic data processing equipment, techniques, and computer languages. The Center

consists of the following offices and divisions: Information Processing Standards—Computer Information—Computer Services —Systems Development—Information Processing Technology.

THE OFFICE FOR INFORMATION PROGRAMS promotes optimum dissemination

and accessibility of scientific information generated within NBS and other agencies of

the Federal Government; promotes the development of the National Standard Reference

Data System and a system of information analysis centers dealing with the broader

aspects of the National Measurement System; provides appropriate services to ensure

that the NBS staff has optimum accessibility to the scientific information of the world,

and directs the public information activities of the Bureau. The Office consists of the following organizational units: Office of Standard Reference Data—Office of Technical Information and

Publications—Library—Office of International Relations.

1 Headquarters and Laboratories at Gaithersburg, Maryland, unless otherwise noted; mailing address Washington, D.C. 20234.

2 Part of the Center for Radiation Research.

3 Located at Boulder, Colorado 80302.
ii&o Organic Chemistry Section:

Summary of Activities, July 1970 to June 1971

<\i>

Robert Schaffer, Editor

Organic Chemistry Section

Analytical Chemistry Division

Institute for Materials Research

(J. 5 > National Bureau of Standards

Washington, D.C. 20234

U.S. DEPARTMENT OF COMMERCE, Peter G. Peterson, Secretary

NATIONAL BUREAU OF STANDARDS, Lawrence M. Kushner, Acting Director,

Issued August 1972
National Bureau of Standards Technical Note 587

Nat. Bur. Stand. (U.S.), Tech. Note 587, 131 pages (Aug. 1972) CODEN: NBTNAE

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 (Order by SD Catalog No. C13.46:587). Price $L 25.
FOREWORD

The Analytical Chemistry Division was established as a

separate division at the National Bureau of Standards on

September 1, 1963, and became part of the Institute for

j Materials Research in the February 1, 1964, reorganization.

! It consists at present of. nine sections and about 100

i technical personnel encompassing some 60 different

analytical competences from activation analysis and atomic

absorption to vacuum fusion and x-ray spectroscopy. These

competences, and in turn the sections which they comprise,

are charged with research at the forefront of analysis as

well as awareness of the practical sample, be it standard

reference material or service analysis . In addition it is

their responsibility to inform others of their efforts

Formal publication in scientific periodicals is a • highly important output of our laboratories. In addition,

however, it has been our experience that informal, annual

summaries of progress describing efforts of the past year

can be very valuable in disseminating information about

our programs. A word is perhaps in order about the

philosophy of these yearly progress reports. In any

research program a large amount of information is obtained

and techniques developed which never find their way into

the literature. This includes the "negative results" which

are so disappointing and unspectacular but which can often

save others considerable work. Of importance also are the

numerous small items which are often explored in a few days

and which are not important enough to warrant publication

yet can be of great interest and use to specialists in a

given area. Finally, there are the experimental techniques

and procedures, the designs and modifications of equipment,

etc., which often require months to perfect and yet all too

often must be covered in only a line or two of a journal

article.

iii
Thus our progress reports endeavor to present this

information which we have struggled to obtain and which we

feel might be of some help to others . Certain areas which

it appears will not be treated fully in regular publications

are considered in some detail here. Other results which are

being written up for publication in the journal literature

are covered in a much more abbreviated form.

At the National Bureau of Standards, publications such

as these fit logically into the category of a Technical

Note. We plan to issue these summaries for all our sections

The following is the seventh annual report on progress of

the Organic Chemistry Section.

W. Wayne Meinke, Chief

Analytical Chemistry Division

IV
PREFACE

This is the seventh annual progress report of the

Organic Chemistry Section of the Analytical Chemistry

Division, and it describes the activities of the Section

from July 1, 1970 to June 30, 1971. Unlike the formal,

journal publications that emanate from the Section from

time to time, this report summarizes the activities of

the entire Organic Chemistry Section during that period.

Following the established pattern within this

Division the programs of the Section have both a research

and a sample aspect : about half of the total effort of

the Section has been applied to research, and the remainder

to Standard Reference Materials (SRMs). The Section must

concern itself with many subjects in organic chemistry in

supporting the National Bureau of Standards T overall mission,

which is to contribute, where possible, to the solution of

nationwide problems through the enhancement of meaningful

measurement. To that end, in the past year, much of the

Section's effort was directed to developing SRMs needed

in clinical chemistry laboratories . This has entailed

working with organic compounds that fall into a number

of classical areas of organic chemistry. To maintain

overall unity in the Section's program, considerable

attention is given to one major area of organic chemistry,

the carbohydrates, developing therein the Section's competences in organic analysis, characterization, and

synthesis. These competences are utilized to the maximum

extent for the development of SRMs. Our efforts in serving

clinical chemistry have been fostered by the National

Institute of General Medical Services of the National

Institutes of Health.
In this report, in order to describe procedures

adequately, it has occasionally been necessary to identify

commercial equipment. In no case does such identification

imply NBS recommendation or endorsement, nor does it imply

that the equipment identified is necessarily the best

available for the purpose.

Robert Schaffer, Chief

Organic Chemistry Section

vi
TABLE OF CONTENTS

PAGE

CLINICAL STANDARDS 2

Bilirubin 2

A Crystalline Bilirubin SRM for

Clinical Analysis 2

Pink-f luorescing Impurity (366-nm Irradiation) Present in Bilirubins ^ Attempted Isolation of the Pinkfluorescing Impurity from Commercial

Bilirubin by Preparative T.L.C. on

Cellulose 9

Attempted Isolation of the Pink-f luorescing

Impurity by Column Chromatography 10

Preparation of a Reference Sample of

Bilirubin; Removal of the Pink-f luorescing

Impurity 11

Standard Solutions of Bilirubin for

Clinical Analysis 20

Cholesterol 29

Thin-layer Chromatography of Cholesterol

Samples 29

Post-issuance Studies 32

Thin-layer Chromatography 32

Gas —liquid Chromatography .... 32

Ultraviolet Spectrophotometry ... 33

Differential Scanning Calorimetry

(DSC) 33

Phase Solubility Analysis 34

Volatile Matter in SRM Cholesterol

by Mass Spectrometry 34

Cortisol 35

Testing for Homogeneity 35

Elemental Composition 36

Behavior on Melting 36

Spectrophotometric Data 36

Loss on Drying . . 37

Insoluble Matter 38

D-Glucose (Clinical) 38

Analysis of D-Glucose by P.m.r.

Spectroscopy at 90 MHz 39

vii
TABLE OF CONTENTS (CONT'D)

PAGE

Polarimetric Analysis of Crystalline

and Melted g--Glucose Samples 4o

Synthesis of PL- 4-Hydroxy- 3-methoxymandelic

Acid (g^- Vanillylmandellc Acid, VMA) . . . ... 43

An Improved Procedure for Synthesis of VMA 43

g-Mannitol (Clinical) 44

Synthesis of g-Mannitol for Use as a Reference Sample Within These Laboratories 44

Determination of Purity by Phase

Solubility Analysis 45

Determination of Purity by

Differential Scanning Calorimetry 46

Optical Rotation of Samples of g-Mannitol . 46

Thin-layer Chromatography (T.L.C.) of

g-Mannitol 50

Paper Chromatography of g-Mannitol 52

Ultraviolet Spectrometry of g-Mannitol. . . 52

Infrared Spectroscopy of g-Mannitol .... 53

Concentration, by Recrystallization, of

Impurities in SRM D-Mannitol 54

Loss of Weight of SRM g-Mannitol on Drying. 55

8-Nicotinamide Adenine Dinucleotide (8 -NAD) and

its 1,4-Reduced Form (8-NADH) 56

Ultraviolet Spectral Data for Commercial

Samples of 8-NADH 56

Paper Chromatography 58

Loss of Weight on Drying, and Elemental

Analysis 60

Stability of 8-NADH under Conditions for

Enzymic Analysis 6l

Inhibitor(s) in 8-NADH 6l

Chromatographic Studies of 8-NADH

Treated with Moisture 6l

Rate of Formation of Inhibitor as

Determined by Paper Chromatography . . 66

Column Chromatography 66

Purification of Synthetic Dyes for Use as

Spectrophotometric Standards 67

Neolan BDack 67

Cibalan Black 68

Alizarine Light Gray 2BLWN 68

viii
TABLE OF CONTENTS (CONT'D)

PAGE

CARBOHYDRATE SYNTHESIS, STRUCTURE, AND

CHARACTERIZATION: NOVEL RESEARCH MATERIALS

AND MODEL COMPOUNDS 70

Enolization of Hexodiulose Acetals . A

Synthesis of Derivatives of U-Psicose by

Reduction of a Hex-3-enulopyranose 70

Application of Homonuclear , Internuclear

,

Double-resonance Techniques to Carbohydrates.

Detection of Small Coupling-constants 75

Introduction 75

Results and Discussion 75

Spectroscopic Studies of Derivatives of

6-Amino-6-deoxy-D-glucose-6- x 5 N 80

Introduction 80

Infrared Spectroscopy 80

Mass Spectrometry 8l

Carbon-13 Magnetic Resonance Studies

by Continuous-wave and Fouriertransform Methods 82

Heteronuclear , Internuclear, Doubleand Triple-resonance Experiments 84

Cyclic Polyhydroxy Compounds 87

(p_-Bromophenyl)osotriazoles from Inositols. 87

GENERAL ORGANIC CHEMISTRY 89

Oxidative Cleavage of Aromatic Azines

with Periodic Acid: E.S.R. Evidence for

the Liberation of Nitrogen Dioxide 89

Evidence for Adsorption as the First Step

in the Solid-state Oxidation of Benzenehexol

with Active Manganese Dioxide 95

Possible Structure of the Precipitated

Manganese Dioxide 95

Adsorption of Benzenehexol by Manganese

Dioxide 100

Reaction Mechanisms Involved in Oxidation

of Benzenehexol to Croconic Acid by

Manganese Dioxide 101

IX
TABLE OF CONTENTS (CONT'D)

PAGE

Stable Radical-ions Derived from

Glyoxal Bis(phenylhydrazones) 102

4. PERSONNEL AND ACTIVITIES 10

8

Personnel Listing 108

Publications 108

Manuscripts in Process of Publication . . . 109

Lectures 110

5. ACKNOWLEDGMENTS 112

6. REFERENCES 113

LIST OF FIGURES

FIGURE NO .

1. Stability of several bilirubin—human

albumin solutions prepared with (a)

methyl sulfoxide (closed symbols) or

(b) KCN-F (open symbols), as determined

by the Jendrassik—Grof diazo test 22

2. Stability of several bilirubin—human

albumin solutions prepared with (a)

methyl sulfoxide (closed symbols) or

(b) KCN-F (open symbols), as determined

by direct spectrophotometry 23

3. Calibration curves for solutions of human

albumin at pH 8.5 enriched with bilirubin

in methyl sulfoxide. The upper curve is

for the Jendrassik—Grof diazo test, and

the lower is for direct spectrophotometry . 25

4. Calibration curves for solutions prepared

enriching human albumin at pH 7.4 with

bilirubin in methyl sulfoxide. The upper

curve is for the Jendrassik—Grof diazo

test, and the lower is for direct

spectrophotometry 27
LIST OF FIGURES (CONT'D)

FIGURE NO. PAGE

5. P.m.r. spectra (16 scans) of the anomerichydroxyl proton region of solutions of

g-glucose in methyl sulfoxide-de at 90

MHz; (a) crystalline a-g-glucose , (b)

crystalline 3-D-glucose7 (c) melted

a-g-glucose, and (d) melted 8-D-glucose . . 4l

6. Proton—proton, internuclear , double-resonance

experiments on 1-d

2 with f 2 decreasing

(spectra a—d) or increasing (e—h) at

various rates (fi constant at peak Al of

H-l, except for single-resonance spectrum

1) - 77

7. Indor spectra obtained from 1-d

2 at 90 MHz

monitoring peak Al (H-l) with f

attenuation, 38) while H-2 was perturbed

by f_2 (attenuation, 44) either decreasing

(spectra a—c) or increasing (spectra

d—f) at various rates. Transient

nutations are apparent in spectra a, b, d,

and e 78

8. Proton-proton indor experiments on 1-d

2 at

90 MHz with peak Fl (H3C-6) monitored by

a constant f 1 , and H-2-H-5 perturbed by

a decreasing f 2 and applied at various

attenuations: (a) 28, (b) 32, (c) 36, (d)

40 dB (down from 0.5 watt), (e) f_i swept

and f 2 off 79

9. Fourier-transform, 13 C n.m.r. spectra of

solutions in chloroform-d, at 25.2 MHz

with proton decoupling: (a) 6-deoxy-l,2:

3 ,5-di-0-isopropylidene-6-phthalimido-aD-glucofuranose 1, (b) 6-deoxy-l,2 : 3,5-

6!i-0-isopropylidene-6-phthalimido-a-Dglucofuranose-6^- 1

N (1- 15 N) . . . ,~f . . . . 83

10. 2 5 N-related n.m.r. spectra of 6-deoxy T l,2 : 3,

5-di-0-isopropylidene-6-(trifluoroac<?tamido)-

a-g-glucofuranose-6- 1

N (4- 15 N) in chloroform-d: (a) computed, theoretical, * 5 N

n.m.r. spectrum; (b) Is N n.m.r. spectrum

obtained indirectly from heteronuclear,

xi
LIST OP FIGURES (CONT'D)

FIGURE NO . PAGE

intripler experiment in which a

19 F

decoupled NH proton peak was monitored by

fi, at ^90 MHz, while the * 5 N frequency

Tf 3 ) was swept; (c_) the same, with fi at

lower power 86

11. Proposed structure of the association complex

between periodate dianion and a resonance

structure of azine la 91

12. X-Ray powder diffraction pattern of manganese

, dioxides (Cu-Ka radiation): A-l, precipitated manganese dioxide dried in vacuo at

room temperature; B-l, sample A-l dried

in an oven at 125 °C; C-l, manganese dioxide

prepared in deuterium oxide as described

in the text (dried in vacuo at 100 °C);

D-l, sample C-l exposed to air for 3 months . 97

13. X-Ray powder diffraction pattern ( Cr-Ka

radiation) of aliquots, on mixing of 1:6

(w/w) benzenehexol (1) and manganese dioxide.

(The 1:6 ratio is for substrate to oxidant.)

A, precipitated (amorphous) manganese

dioxide; B, 30 sec, structure shown is

primarily due to 1; C, 50 min, ^5% of 1

adsorbed; D, 100 min, ^91% of 1 adsorbed,

complex D; and E, manganese croconate

trihydrate, purified product extracted from

D 99

14. Radical-anions from glyoxal bis (phenylhydrazones): A, from la (3-2 mM, in Me 2 S0-5#

aqueous KOH) ; B, from la (2.8 mM, in

Me 2 S0-K0But; c, from 2,5-dichloro

derivative of la (3 mM, in Me 2 S0-K0But )

;

D, from la completely deuterated on the

benzene ring (5 mM, in Me 2 S0—KOBu^ )

; and

E, from phenylglyoxal bis(phenylhydrazone) • 106

xn
LIST OP TABLES

TABLE NO . PAGE

1. Some data showing levels of impurity

extractable with 5% aqueous sodium

bicarbonate from samples of bilirubin

dissolved in chloroform 4

2. Values of e from testing of bilirubin—

methyl sulfoxide enrichments of different

commercial samples of human serum albumin. . 29

3. Two-dimensional t.l.c. of freezer-stored

cholesterol on Silica Gel G 31

4. Reproducibility of the purity of SRM

cholesterol as determined by differential

scanning calorimetry 34

5. Specific optical rotations and millimolar

extinction coefficients of SRM Cortisol . . 37

6. Volatile matter in SRM Cortisol 38

7. Compositions of a- and S-D-glucose and their

supercooled melts, determined by digital

integration of time-averaged, p.m.r.

spectra 40

8. Purity values obtained for g-mannitol by

differential scanning calorimetry (DSC)

and phase solubility analysis (PSA) 47

9. Purity values obtained for g-mannitol by

differential scanning calorimetry, with

slow scanning begun at various temperatures (T) 48

10. Specific rotations, at various wavelengths,

of solutions of various samples of gmannitol in acidified ammonium molybdate

solution 49

11. Specific rotations, at various wavelengths,

of" solutions of samples of g-mannitol in

water 50

12. Loss in weight of SRM g-mannitol on drying

at 110 °C 55

Xlll
LIST OF TABLES (CONT'D)

TABLE NO. PAGE

13. Ultraviolet spectral characteristics for

different commercial samples of 3-NADH

in 0.1 M Tris buffer, pH ^10 57

14. Ultraviolet spectral data for 3-NADH (lot

no. 50C-6420) in 0.1 M Tris buffer, pH

^10.4 58

15. Paper chromatography of commercial samples of

3-NADH. . 59

16. Elemental analysis of a dried sample of

3-NADH, lot no. 50C-6420 60

17. Stability 3-NADH (lot no. 065003) under

conditions for an enzymic assay (after

4 days of drying at 20 °C under high

vacuum); uv spectral results 62

18. Elemental analysis of the modified preparation of Alizarine Light Gray, dried at

20 °C and 1 torr 70

xiv
ORGANIC CHEMISTRY SECTION: SUMMARY OF ACTIVITIES,

JULY 1970 TO JUNE 1971

Edited by Robert Schaffer

This report of the Organic Chemistry Section of the

National Bureau of Standard's Institute for Materials

Research provides a summary of a year's scientific activity;

as such, it covers both work that was completed and some

that is still in progress. The development of Standard

Reference Materials (SRMs) and their certification, and

journal publications describing aspects of that work, as

well as other research activities, are the output of the

Section.

In the SRM category, the major subjects of this report

are bilirubin, cholesterol, Cortisol, D-glucose, 4-hydroxy3-methoxy-DL-mandelic acid (VMA), D-mannitol, and the reduced

form of nicotinamide adenine dinucleotide (NADH)

Studies concerning carbohydrates include synthetic

work with D-psicose, n.m.r. employing internuclear doubleresonance, and mass spectroscopy of 6-deoxy-l,2 : 3,5-di-Oisopropylidene-a-g-glucose, spectroscopic studies of

derivatives of 6-amino-6-deoxy-D-glucose-6_- 1

N (employing

mass spectrometry, Fourier-transform, 13 C-n.m.r. and heteronuclear, 1 5 N indor techniques), and p-bromophenylosotriazoles of inositols

Other research utilized e.s.r. and involved periodic

acid oxidation of azines, solid-state oxidation of

benzenehexol on active manganese dioxide, and the formation

of stable radical-anions from glyoxal bis(phenylhydrazone)

Key words: Bilirubin; cholesterol; e.s.r.; g-mannitol;

NADH; n.m.r.; purity; Standard Reference Materials; SRM; VMA.
1. CLINICAL STANDARDS

A . Bilirubin

1. A Crystalline Bilirubin SRM for Clinical Analysis

Improved analyses for bilirubin in blood are being

pursued by Standards Committees of the American Association

of Clinical Chemists, the College of American Pathologists,

the National Committee for Clinical Laboratory Standards,

and the International Federation of Clinical Chemists. The

National Bureau of Standards, as a part of its effort with

the overall problem, has developed a supply of bilirubin

as a Standard Reference Material (SRM)

.

In 1962, in the absence of a method for characterizing the purity of bilirubin preparations, it was recommended

that the crystalline bilirubin used for preparing needed

standard solutions [1] should show an absorptivity (±2 SD)

of 60,700 ±1,600 liter* cm

-1 •mol~ 1 at 453 nra in chloroform

at 25 °C. Bilirubin can be purified to meet this requirement

by the procedure of Henry, Jacobs, and Chiamori [2]. Neither

a modification of that procedure by Fog [3] nor other spectrophotometry measurements on preparations reported since [4]

suggest that the absorptivity of bilirubin might actually

exceed the value recommended.

After crystallizing bilirubin preparations, it is common

practice to remove adhering solvent by a vacuum treatment

at room temperature in the presence of a desiccant. We

found some commercial bilirubin preparations having low

spectrophotometric absorptivities would lose weight on

drying, but losses were insufficient to improve their molar

absorptivities materially; samples having high absorptivity

lost very little weight [2]. Work at NBS has shown that

bilirubin crystallized from chloroform retains a significant

proportion of this solvent which is not easily volatilized.

For example, on heating a high-absorptivity sample at 110 °C

for 9 h at 0.3 torr, only a slight weight-loss resulted

2
(^0.1%), even though by analysis this material was found to

contain chloroform in excess of 1.5%-

The SRM bilirubin contains 0.8$ of chloroform which

needs 4 h at 250 °C and 0.3 torr to be volatilized. (Such

heating destroys the sample.) At 180 °C, with like amounts

of time and vacuum, only about half of the chloroform is

removed. Numerous drying experiments and chloride

determinations showed that the chloroform ^as homogeneously

distributed in the SRM. G.l.c. [5] and n.m.r. measurements

assured that chloroform was present and was being volatilized

In the purification process of Henry et al . [2], brown

pigment is removed from a chloroform solution of bilirubin

by extracting the solution with 5% aqueous sodium bicarbonate

We have quantitated the extraction procedure by determining

the weights of (a) the unextracted and (b) the bicarbonateextracted material, which contains little bilirubin. The

latter is recovered by neutralizing the bicarbonate extracts

and extracting with chloroform. The absorbance maximum of

these chloroform extracts of recovered bilirubin occurs

usually at ^449 nm. The weight of material in the extract

is determined by drying. The overall recovery of material

is almost quantitative. By this technique, some commercial

preparations showed upwards of 10JB of extractable impurity;

the better of the commonly available bilirubin preparations

we examined contained only as little as 2-3$ . The SRM has

none; see table 1.

The values cited in the literature for the molar

absorptivity of bilirubin in chloroform were calculated on

the assumption that the measurements had been made on

100/S-pure materials. Judged on that basis for comparison,

the SRM bilirubin (as supplied) shows (measurements by Dr.

R. W. Burke and E. R. Deardorff of Section 310.04) a molar

absorptivity (±2 SD) of 61,100 ±400 liter* cm

-1 •mol" 1 at

453 nm.
Table 1. Some data showing levels of impurity extractable

with 5% aqueous sodium bicarbonate from samples

of bilirubin dissolved in chloroform

Recovery

Mfr. T s lot no.

Sample

(mg)

Unextract

(mg)

ed Extracteda

(mg)

5310 107.20 95.10 11.88

39148A 99.63 90.65 9.39

7322 106.97 100.35 6.85

8162 105.5^ 102.88 2.48

SRM 95.61 95.34 0.23

heights include about ^0.2 mg of bilirubin.

2. Pink-fluorescing Impurity (366-nm Irradiation)

Present in Bilirubins

a. Commercial Bilirubins

(A. J. Fatiadi)

In May 1968, by use of thin-layer chromatography (t.l.c.)

[activated Silica Gel G overloaded as a spot with bilirubin

(^50 ug) and developed with 8:1:1 (v/v) benzene-fl,N-dimethylformamide—acetic acid], we observed with bilirubin samples

from four different commercial suppliers an impurity that

fluoresced under 366-nm light. Such thin-layer chromatograms

showed (a) at the origin, a yellow-orange spot (non-migrated

bilirubin) , (b) extending from the origin, a faint yelloworange streak (bilirubin), (c) at R

f 0.64, a green spot

(presumed due to biliverdine) , (d) at R

f 0.8l, a faintly

visible, yellow spot that fluoresced under 366-nm irradiation,

and (e) a yellow-orange spot at R

f 0.86 (bilirubin). The

bilirubin spots were identified by isolation and spectrophotometry, but the fluorescent material could not be evaluated.

4
However, further attention was given to the fluorescent

component after the presence of a pink-fluorescing impurity

in SRM bilirubin was reported to us by Drs . D. C. Nicholson

and M. Stoll [5], who used t.l.c. on polyamide plates, with

3:1 (v/v) methanol—aqueous ammonium hydroxide (3.3$) as the

developer [6]. At about that time, we had found that, by

oxidation of bilirubin with periodic acid, there resulted a

biliverdine—iodine charge-transfer complex, and that reduction

of the isolated material with sodium borohydride regenerated

bilirubin [7]. This product was found to be free of fluorescent impurity by this t.l.c; hence, this fluorescence

was not an artifact of the chromatography

b. SRM Bilirubin

(B. F. West and A. Cohen)

On a polyamide thin-layer plate heavily loaded as a

spot with SRM bilirubin (^75 ug) and developed with the

methanol—aqueous ammonium hydroxide, the bilirubin was found

as an elongated spot at R

f ^0.57, and a minor, second

component was found at R ~0.7^. The latter was barely

visible as a yellow spot7 but, when illuminated with 366-nm

light for about 2 min, a somewhat stable, pink fluorescence

developed in its place, and after this exposure to uv, the

color of this spot under ordinary light was found changed to

a very pale gray. Two-dimensional chromatography did not

separate a second pink-fluorescing material from the bilirubin.

The fluorescent component in SRM bilirubin was also seen

by t.l.c. on cellulose. On either commercially precoated

plates or hand-spread layers of microcrystalline cellulose,

development with methanol, which did not move the bilirubin

from the origin, separated a component having pink fluorescence (under 366-nm irradiation). It was found near the

solvent front. Its fluorescence is visible immediately on

exposure to uv light, and persists with further uv

5
U. S. DepartiSrejrf' of Commerce

MauricqsJH. Stans

dkrttfttate of ^nal)|sts

Standard Reference Material 916

Bilirubin

i

i

This standard reference material is certified as a chemical of known purity. It is intended

primarily for use in the calibration and standardization of procedures employed in clinical analysis

and for routine critical evaluation of the daily working standards used in these procedures.

Purity (bilirubin content) 99.0 percent

Chloroform 0.8 percent

Insoluble (in chloroform) less than 0.01 percent

Ash 0.01 percent

The value of the bilirubin content has a possible estimated inaccuracy of two percent. (See

section on thin-layer chromatography).

The material in chloroform (ACS reagent containing ethanol) aK25.0\\C \cjhc^shows at 453 nm an

absorptivity (± 1 SD) of 104.6 ± 0.2 liter cm 1 g- 1. In methyl si*^oxi)j£^jZ5^C, the absorptivity

(± 1 SD) of the material at 453 nm is 107.7 ± 0.6 liter cm 1

It is recommended that bilirubin and its solutions ne haVdl^ only under low-intensity incandes- cent light. The bilirubin should be stored undef^on/hn^nj/fnat totally exclude light. Once removed

from the ampoule, the vial should be rerJa^cNfia^to^age in an air-tight container and stored at low

temperature. The vial must be allowed tdlreturn^o-oom temperature before opening.

The bilirubin used for thu^standarasreference material was obtained from Pfanstiehl Laboratories, Inc., of Waukegarf^llinois^lAnalyses were performed by R. F. Brady, Jr., R. W. Burke, A.

Cohen, B. Coxon, J£RVU*«t$60^; Enagonio, A. J. Fatiadi, D. H. Freeman, J. M. Ives, B. A. Johnson, E. C. Kuehner^^AY^Paulson, T.C. Rains, R. Schaffer, W. P. Schmidt, J. T. Sterling, J. K.

Taylor, B. F. West, wN^L Zielinski, Jr.

The overall direction and coordination of technical measurements leading to the certification were under the chairmanship of R. Schaffer.

The technical and support aspects concerning the preparation, certification, and issuance of this standard reference material were coordinated through the Office of Standard Reference Materials

by T. W. Mears.

Washington, D. C. 20234

March 10, 1971

J. Paul Cali, Chief

Office of Standard Reference Materials

(over)

6
The standard reference material was prepared from material that was isolated from hog bile and

crystallized as the acid. It was purified further by treatment in chloroform solution with sodium

sulfate according to Fog [1] and recrystallization from chloroform.

Ash was determined on 100-mg samples heated overnight at 250 °C and then for 3 hrs at 650 °C.

Flame photometry and atomic absorption measurements on a solution of the ash obtained from

0.275 g of the material showed, in /ig/g (ppm) of metal: <0.05 lithium, 7.3 sodium, 1.6 potassium,

2.1 calcium, and 2.3 magnesium.

Elemental composition found for the material was: C, 67.36; H, 6.24; N, 9.42; CI, 0.73. Required for a bilirubin preparation containing 0.8 percent of chloroform: C, 67.25; H, 6.17; N,

9.50; CI, 0.71. In these analyses, nitrogen was determined by the Kjeldahl technique; and chlorine was determined by Carius digestion followed by gravimetric determination as silver chloride. By the

latter method and recalculated as chloroform, the average (± 1 SD) of ten 25-mg samples, was 0.83

± 0.04 percent. The chloroform content was also obtained by heating at 250 °C at 0.5 torr for 4 hrs; the average (± 1 SD) of six 100-mg samples was 0.79 ± 0.02 percent. (Only about one-half or one-tenth of the chloroform was removed at 180 °C and at 110 °C, respectively.) During gradual

51 heating of the material in a quadrupole mass spectrometer, only ion-fragments characteristic of

chloroform were observed until the sample was heated above 300 °C, whereupon water and carbon

dioxide were detected, and at a somewhat higher temperature, the mass spectrum typical of

bilirubin was obtained. Gas chromatography and nuclear magnetic resonance spectroscopy with the

material dissolved in methyl sulfoxide confirmed the presence of chloroform Because the chloroform is so firmly retained and homogeneously dispersed, it is recommended that the material be

used as supplied.

Thermogravimetric analysis with the sample heated in a dry nitrogen atmosphere at 5 °C/min

showed the initiation of the loss of a large proportion of sample weight at temperatures between

319 and 323 °C; determined in air, however, comparable weight-loss began between 288 and

292 °C.

For determining the absorptivity of the standard material, absorbances were recorded with a Cary 16 spectrophotometer on solutions using a capped 1.000 ± 0.001-cm path-length cuvette in a

cell-holder thermostated at 25.0 ±0.1 °C. Solution preparation and handling were performed under

low intensity, incandescent light; at other times, solutions were totally protected from light. To

preclude any change in concentration of chloroform solutions due to evaporation of the solvent

during solution transfer, nitrogen gas was used to force the solution from the flask through Teflon

tubing into the cuvette and, to effect rinsing of the cuvette, from the latter to an overflow.

Solutions in methyl sulfoxide were transferred to the cuvette by pipet.

For the determination of chloroform-insoluble matter, sample (0.1 g) and solvent (200 ml),

protected from light, were agitated in an ultrasonic bath, heated to 60 °C briefly, and then cooled

and filtered, using pressure, (with 50 ml of the solvent for washing) through a tared 0.4 jum-porosity

membrane.

Extraction by Bicarbonate. Yellow extracts were obtained by shaking a solution of the standard

reference material (0.010 g) in chloroform (40 ml) with 10-ml volumes of 0.6 molar aqueous

sodium bicarbonate. Their absorption spectra, with maxima in the range 415--430 nm, showed

absorbance values that ranged from 0.035 to 0.030 in successive extracts using 1-cm cells. The

qualitative and quantitative compositions of such extracts were examined using the filtered chloroform solutions containing 0.1-g quantities of the standard reference material that were prepared in the tests for chloroform insolubles: Extractions twice with 50-ml volumes of the bicarbonate

solution and with water, and neutralization of the combined aqueous extracts followed by extrac- tion into chloroform, yielded pigment (dry weight approximately 0.2 mg) that was shown by its spectrum and specific absorptivity to be typical of bilirubin in chloroform. Thus, bicarbonate

extraction provided no evidence of impurities more acidic than bilirubin.
Extraction by Carbonate. Extraction of a 200-ml chloroform solution containing 0.1 g of standard with 100-ml volumes of 0.1 molar sodium carbonate reduced the 1-cm absorbance value of the

chloroform layer to approximately 0.22 after the second extraction, and to 0.08 and then 0.01 with

the third and fourth extractions, respectively. On washing with water and then evaporating the

extracted chloroform layer to dryness, residue weights were typically about 0.01 mg. Hence, this extraction process showed a negligible proportion of nonacidic impurities.

Thin-layer Chromatography. An impurity in the standard material may be detected by thin-layer

chromatography with polyamide as the absorbent and 3:1 (v/v) methanol-aqueous ammonium

hydroxide (3.3 percent) as the developer [2]. Thus, 0.05 mg of the material (spotted from a chloroform solution) provides, on development, an elongated, yellow-orange streak preceded (and

usually separate) by a faintly visible, yellow spot. Under 366-nm irradiation, a pink fluorescence

develops very quickly at the location of this spot. The material responsible for the pink fluorescence

and its precursor have not been characterized. A means for obtaining a reliable estimate of this contribution to overall impurity has not been ascertained, but is under study. Repurification of the

standard material does not affect this behavior.

References

1 J. Fog,Sccmd. /. Clin. Lab. Invest, 16, 49 (1964).

2 Z. J. Petryka and C. J. Watson, J. Chromatog., 37, 76 (1968).
exposure. After uv exposure, the spot exhibits a purpleto-mauve color when viewed under ordinary light . Triple

development in the same direction with methanol (but each

to a distance 1 cm less than before so that a new solvent

front can be distinguished after each development) revealed

by the second development a much diminished quantity of

separated pink-fluorescing component, and, by the third

development, essentially none. (The chromatogram is irradiated only after the third development.)

Evidence for a green pigment, as was found by use of

t.l.c. on silica gel was not observed by t.l.c. on either

the polyamide or the cellulose.

3. Attempted Isolation of the Pink-fluorescing

Impurity from Commercial Bilirubin by Preparative

T.L.C. on Cellulose

(A. J. Patiadi)

Microcrystalline cellulose (50 g) was stirred in 5:1

(v/v) methanol—chloroform (200 ml) at room temperature for

30 min, filtered, and washed with methanol (100 ml); after

it had dried at room temperature, the material was used to

prepare a thin-layer plate (20 X 20 cm X 2 mm). The plate

was dried for 2 h at room temperature.

Under dim incandescent light, a solution of bilirubin

(50 mg) in a mixture of chloroform (1 ml) and diethylamine

(0.1 ml) was transferred to the t.l.c. plate as a long streak

(nitrogen flushing) about 2 cm from an edge. Chromatography

was begun with methanol used as developer, but, as soon as

the orange-red band had moved about 3 cm from the origin,

the plate was removed from the developer, sprayed with 1%

acetic acid in methanol (to convert the complex into free

bilirubin), and dried (nitrogen flushing). Then the chromatogram was developed further with methanol. Development was

stopped when the solvent front had moved to within 1 cm of

9
top (45—65 min) . After drying (nitrogen flushing), the

slightly yellowish band near the top of the plate was scraped

off, and extracted with methanol. The extract was concentrated, and the residue (^0.8 mg) showed, on cellulose t.l.c,

a distinct pink fluorescence (366-nm irradiation).

The orange band, on extraction with chloroform and

concentration of the extract, gave bilirubin (28 mg) almost

free from the pink impurity (t.l.c. on cellulose).

The experiment was repeated with 200 mg of bilirubin

on a t.l.c. plate of similar dimensions. The light-yellow

band was scraped off, and extracted with methanol (75 ml).

Rechromatography of this extract on a cellulose plate

(5 cm X 20 cm X 2 mm), followed by extraction with methanol,

and concentration, gave a residue (^1.2 mg) containing

much pink-f luorescing impurity (t.l.c. on cellulose).

4. Attempted Isolation of the Pink-f luorescing

Impurity by Column Chromatography

(B. F. West)

To the top of a column of methanol-washed, microcrystalline cellulose (30 X 3 cm) protected from light was added

a methanolic slurry of microcrystalline cellulose (10 g) that

had been coated with SRM bilirubin (90 mg) by evaporating

a solution of the bilirubin in chloroform on it. Fractions

of effluent were collected using methanol as the developer,

and they were monitored for the impurity by t.l.c. on cellulose. When partly concentrated, each of the first six

100-ml fractions of effluent gave similar uv—visible

spectra: a broad maximum at 448—450 nm, a sharp doublet at

275—285 nm, and a strong maximum (cut off) at 224 nm. Further

concentration of the methanolic solutions led to the deposition of bilirubin. Total concentration followed by extraction

of the residue with methanol and filtration gave a green

solution, the spectrum of which showed a weak band at

10

<P
650 nm but none at 370 nm. (For biliverdine, the 370-nm band

is stronger than that at 650 nm.) A strong peak was now

found at 435 nm, and the doublet at 275-285 nm and the peak

(cut-off) at 225 nm were still present. There was no absorption between 550 and 600 nm.

Although we carried out these procedures under dim,

incandescent illumination in attempts to isolate and quantitate the fluorescing component in the SRM bilirubin, our

efforts were hampered by the instability of the substance.

As an alternative, we turned to attempts to prepare a quantity

of bilirubin that would be free from this impurity, so that

we might compare its properties with those of the SRM bilirubin

5 . Preparation of a Reference Sample of Bilirubin;

Removal of the Pink-f luorescing Impurity

a. Comments on Alkylamine Complexes of Bilirubin

(R. S. Tipson and A. J. Fatiadi)

We observed that complexes of bilirubin with secondary

and tertiary alkylamines are formed in chloroform. This

ability of bilirubin to form adducts with alkylamines

enabled us to prepare concentrated solutions of bilirubin in

very small volumes of chloroform. The structures of these

alkylamine complexes have not yet been thoroughly studied;

however, their solubility in chloroform or dichloromethane

suggests that they are of the acceptor—donor type. Other

| solvents were found useful for preparing complexes with

alkylamines : N ,N -dimethy Iformamide (DMF) , N-methylpyrrolidinone, N ,N -dimethylacetamide , f ormamide, hexamethylphosphoramide and methyl sulfoxide. Of these, N,N-dimethylacetamide was found very efficient, and next best were

N-methylpyrrolidinone , and hexamethylphosphoramide

Formamide containing triethylamine dissolves bilirubin

very effectively, but, on attempting crystallization,

appreciable oxidation of the bilirubin occurs; the same

11
was found with methyl sulfoxide. Ethylenediamine in DMF

dissolves much bilirubin (only a trace of this strong organic

base was needed) ; however, no crystals of bilirubin were

recovered after prolonged concentration (120 h) of

solutions in a vacuum desiccator.

b . Preparation of Bilirubin-—Diethylamine Complex

(A. J. Fatiadi)

Commercial bilirubin (200 mg) was suspended in

chloroform (1 ml) and, while being stirred, the suspension

was treated with diethylamine (0.2—0.3 ml) until all of the

solid had dissolved; sometimes, slight warming at 45 °C

for 2 to 5 sec was necessary. The deep-red solution was

then cooled in an ice-bath, and ethyl ether was added

dropwise to faint turbidity (3 to 4 ml). Dense,

brown-orange, micro-crystalline material separated after

5 to 10 min of additional stirring in the ice-bath. The

product was filtered off, washed with 1:1 (v/v) chloroformethyl ether, and then with ether, and dried in a vacuum

desiccator, to give crop I (55 mg) , and, after concentration

and cooling, crop II (85—88 mg)

.

The complex was quite soluble in chloroform,

dichloromethane, and water, but sparingly soluble in carbon

tetrachloride or ethyl ether. Slight warming of the

complex in ethyl ether (water bath) caused dissociation,

and yielded orange-colored bilirubin. Acidification (acetic : acid or hydrochloric acid) of an aqueous solution of the

complex regenerated bilirubin in high yield. The complex is,

however, very difficult to purify by recrystallization;

usually, recrystallization was performed at room temperature

by dissolution of the complex in a minimum volume of

chloroform containing a small proportion of diethylamine,

followed by precipitation of the complex by addition of

ethyl ether or ethyl ether—heptane . Its elemental composi12
tion conformed closely to that of a 1:1 complex, but the

proportions are not ideal.

Anal. Calc. for C^H-gN^Og .C^H-^N: C, 67.6; H, 7-2; N,

10.6 Pound: C, 66.6; H, 7.8; N, 10. 7.

T.l.c. on cellulose [methanol or 1:1 (v/v)

methanol—acetone] revealed the presence of a second

component, possibly the 1:2 complex of bilirubin with

diethylamine . The visible spectrum in chloroform

CHC1

(^mav 3 453 nm) corresponded with that of bilirubin; the

max jj q

u.v. and visible spectra * in water [A 2 224 (sh), 280, and

max 3 3 435 nm] were typical of a bilirubinate salt.

c . Preparation of the Bis( triethylamine) Salt

of Bilirubin

(R. S. Tipson)

Triethylamine (2.0 ml/g) was added to a suspension of

bilirubin in N ,N-dimethylformamide (20 ml/g), with stirring;

addition of an excess of triethylamine (a further 5 ml/g)

caused precipitation of a yellow compound. This was

filtered off, washed successively with DMP and dry ether,

and dried. Its elementary analysis agreed with that

calculated for C„H-gN|,0g .2CgH, ,-N, and its infrared

spectrum (potassium bromide pellet) differed from that

of bilirubin.

The salt is not very soluble in methanol, ethanol,

acetone, ethyl acetate, dichloromethane , or benzene, but

is fairly soluble in chloroform. It is readily soluble in

water to give concentrated solutions; on preparation of

dilute solutions, dissociation occurs and part of the

bilirubin separates out, but addition of a trace of

triethylamine causes it to redissolve. B. A. Johnson

found that a solution of the salt (0.6 mg) in water (1 ml)

had pH 8.83; on standing, the clear solution became cloudy.

13
d. Growth of Crystals of Bilirubin

(R. S. Tipson)

By dropwise addition of t pie thy 1 amine (^2.5 ml/g) to

a stirred suspension of bilirubin in DMF (80 ml/g) until a

clear, brown solution is obtained, followed by slow

evaporation in a vacuum desiccator at 25 °C and 25 torr,

discrete crystals of bilirubin are readily obtained. The

red crystals were examined by C. P. Say lor, who described

them as "blades" (platelets), parallelogram-shaped, about

0.25 X 0.05 mm. Single crystals are pale brown, but a mass

of the crystals is bright carmine red. By slower

evaporation, larger crystals, suitable for X-ray crystal

study, could undoubtedly be grown.

e

.

Purification of Bilirubin by Recrystallization

(R. S. Tipson)

A solution of bilirubin in DMF containing triethylamine

was prepared; although it appeared to be perfectly clear,

it was (as a precautionary measure) filtered by gravity

through hard filters-paper. The filtrate was evaporated in

a dish in a vacuum desiccator at 25 °C and 25 torr for 1 h, - and then at 25 °C and 1 torr. Crystals grew on the bottom and

sides of the dish, and a dark-green crust formed at the top of

the crystal layer on the walls. Without disturbing the green

crust, the suspension of red crystals was removed, by means

of a bulb pipet fitted with a rubber bulb, and filtered with

suction. The crystals were successively washed with

DMF, 1:1 (v/v) DMF-water, water, and absolute ethanol,

and dried in a vacuum desiccator at 25 °C and 0.01 torr.

Mass-spectrometric examination (by E. E. Hughes) showed

that the crystals were devoid of chloroform (a contaminant

in the original bilirubin), N ,N-dimethy If ormamide , or

triethylamine. The infrared spectrum was identical with

that of the original bilirubin. T.l.c. on polvamide with

14

:

3
9:2:1 (v/v) methanol—10% ammonia^water (by B. F. West)

showed that it contained very much less of the pinkfluorescing impurity than did the original sample of

bilirubin.

In a second recrystallization, no green crust formed,

indicating that the green component was an impurity, or

was derived from an impurity, in the original bilirubin.

Twice recrystallized in this way, the bilirubin contained

none of the pink-fluorescing material (t.l.c.).

Bicarbonate extracts of a chloroform solution of this

bilirubin showed 0.04 absorbance units, whereas SRM

bilirubin showed 0.03 (by R. P. Brady, Jr.). The z y. of this bilirubin in chloroform was 60.7-

f . Procedure for Removal of the Fluorescent

Impurity and Other Impurities from

Commercial Bilirubin

(A. J. Patiadi)

In this procedure, two alkylamines, namely,

triethylamine and diethylamine , are used in the purifying

of bilirubin from its fluorescence-forming impurity. Redissolution of the purified bilirubin in a specified amount of

N,N-dimethylformamide (DMF) and triethylamine (NEt-J can be

a troublesome operation, because of the ease of formation

of the crystalline adduct . The addition of diethylamine

(EtpNH) overcomes this difficulty. The purification

involving the recrystallization of bilirubin from

i DMF—NEt- removes a considerable proportion of the

fluorescent impurity, and repetition lowers it to a

negligible proportion. However, the recrystallized

material still contains other impurities. Hence, the

following procedure for the purification of bilirubin

includes crystallizations with DMF-NEt^-Et^H, extraction

with 5$ aqueous bicarbonate [2] and, finally, column

chromatography on sodium sulfate [3].

15
Ill

Commercial bilirubin (500 mg) was suspended in DMP

(10 ml) and stirred magnetically (3 min) to give a

homogeneous suspension. About 0.5 ml of NEt- was introduced

rapidly (30 sec), and the suspension was stirred for about

3 min. Then, slowly (2 to 3 min), more NEt_ was added.

There was a change in color, and a gradual disappearance of

the solid. With the further addition of 0.4 to 0.5 ml of

NEt~, a deep, orange-red solution resulted. The solution

was filtered (either through glass wool or through "rapid"

.filter paper) , and the filtrate was kept in a porcelain dish

over concentrated sulfuric acid in a vacuum desiccator at

10 torr. After 36 h, the deep-red crystals of bilirubin

that had formed were carefully filtered (avoiding removal

of the brown-green impurity in the crust), and washed

successively with DMF and ether; yield 360—380 mg.

Once-recrystallized bilirubin (500 mg) in 10 ml of

DMP was gently stirred with NEt_ (1.1 ml); if the solid

did not dissolve within 4 to 5 min, or if the complex

had crystallized out (recognized by its light yellow-orange

color in comparison to the orange-red of bilirubin)

,

dropwise addition of EtpNH (up to 0.35 —0.5 ml) caused

the solid to dissolve. The orange-red solution (after

filtration was transferred to a dish, and concentrated

in a vacuum desiccator (10 torr) for 36 h. The orange-red

crystals were carefully separated, washed with DMF and

ether, and dried; yield, 370—385 mg. The product thus

obtained was essentially devoid of the pink-fluorescing

impurity, as shown by t.l.c. on cellulose; however, it

still contained impurities extractable with 5% aqueous sodium

bicarbonate

.

Bilirubin ( twice-recrystallized, 350 mg) was dissolved

in chloroform (550 ml) at 55 °C by stirring in the dark.

The solution was cooled to room temperature, successively

washed with freshly prepared, 5% aqueous sodium bicarbonate

solution (4 X 100 ml) and water (5 X 150 ml), and dried with

16
sodium sulfate (75 g) with vigorous stirring while slowly

adding the desiccant (30 to 45 min) . The suspension was

filtered through a layer of microcrystalline cellulose mixed

with acid-washed carbon, and the filtrate concentrated

at 40 °C to incipient crystallization (^110 ml) . Addition

of ether (30—40 ml) and cooling overnight gave crop I,

200 mg; crop II, 55-60 mg (total 255-260 mg) . It showed

an e mM of 60.8.

This bilirubin (250 mg) was dissolved in chloroform

(350 ml) at 50 °C, cooled to 30 °C, and introduced onto

a column of anhydrous sodium sulfate (135 g) protected

from light by wrappings of aluminum foil. Complete elution

land washing (250 ml of chloroform) required 12 h. The

effluent was filtered, and concentrated to incipient

crystallization (^100 ml) ; addition of diethyl ether and

cooling (18 h) gave an orange-red, microcrystalline product;

yield 155 mg. Dilution of the mother liquor with some

ether, and cooling, gave crop II, 70 mg; total 225 mg. The

product was devoid of the pink-f luorescing impurity

(t.l.c), and showed e^C1

3 6l.l. It contained 0.8$ of

chloroform by chlorine analysis.

g. Sodium Borohydride Procedure : Alternative

Method for Removal of the Fluorescent Impurity

from Commercial Bilirubin

(A. J. Patiadi)

This procedure depends on (a) the dissolution of

bilirubin in a methanol solution of sodium borohydride,

(b) the relatively low pH of the solution after the

addition of water, and (c_) selective extraction by

chloroform of the impurity responsible for the fluorescence.

Recovery of the bilirubin from the alkaline solution by

acidification is followed, without intermediate recrystallizations, by the bicarbonate extraction and sodium sulfate

17
I

treatments. The best results in this procedure were

obtained with 0.1 to 0.2 g of bilirubin, although the

procedure is described for 1.0 g of material.

Sodium borohydride (1.0 g) was quickly added to a

stirred suspension of bilirubin (1.0 g) in methanol (250 ml,

precooled to ^10 °C) . The solids dissolved, sometimes

with effervesence , in 30 to 45 sec, to give an orange-red

solution. The stirred solution was diluted with water

(250 ml; the solution showed pH ^8.0), and filtered

(Millipore) . The filtrate was extracted with chloroform

(10 X 100 ml). The aqueous methanol layer was transferred

to a large separatory funnel containing 1.2 liters of fresh

chloroform, and acidified (to Congo Red paper) with 2.5 M

hydrochloric acid (12 to 15 ml). By shaking, the

precipitated bilirubin was dissolved in the chloroform

layer. After a second extraction of the aqueous layer with

chloroform (300 ml), the chloroform extracts were combined,

washed with 5% aqueous sodium bicarbonate (6 X 250 ml),

water (4 X 500 ml), and then dried by stirring vigorously

with small additions (30—45 min) of sodium sulfate (total

100 g) . The suspension was then filtered through a precoated

layer of microcrystalline cellulose (10.0 g) mixed with

acid-washed decolorizing carbon (2.0 g) . This filtrate was

concentrated under diminished pressure to incipient

crystallization (^100 ml) , saturated with ethyl ether

(40—50 ml), and placed in a refrigerator for 18 h, to give

a crop I (500 mg) . Further concentration and cooling gave

crop II (150 mg) , total yield, 650 mg . Further purification

by extraction with sodium bicarbonate and passage through

a column of sodium sulfate was performed as described in the

preceding procedure. T.l.c. analysis showed only a trace

of the pink-f luorescing impurity (366-nm irradiation).

18
h . Evidence for Presence of Two Additional

Fluorescent Substances

(A. J. Fatiadi)

The presence of additional fluorescing substances was

observed on examining the chloroform extracts obtained from

the purification of 2.0 g of commercial bilirubin by the

borohydride method. Bilirubin (2.0 g) in methanol (300 ml)

at 10 °C was treated with sodium borohydride (1.2 g) ; the

deep-orange solution was diluted with water (300 ml),

filtered, and extracted with chloroform (6 X 150 ml).

The combined extract was washed with water (3 X 150 ml),

dried by stirring with 140 g of sodium sulfate for 30 min,

filtered, and concentrated to about 10 ml; addition of a

few ml of ether and slight cooling gave crude bilirubin

(^8 mg) which was filtered off. The yellowish-orange

filtrate was concentrated to ^5 ml and examined by t.l.c.

Spotting of 10- to 50 \il of solution on a cellulose

plate and developing with methanol revealed three

fluorescent spots: one at R

f ^0.95 (strong orange-red

fluorescence under 366-nm irradiation), one at R

f 0.90

(pink fluorescence), and one at the origin (a strong,

light-blue fluorescence). The one at the origin can be

readily moved by developing with chloroform. (The light blue

fluorescent spot moves with the solvent front, and leaves a

yellow, non-fluorescent residue at the origin.)

i. Isomeric Forms of Bilirubin

The effort to estimate the proportion of the fluorescent

impurity in the SRM from comparisons of the spectrophotometric

properties of the SRM with those of SRM freed from this

impurity, assumed that the bilirubin IXa from natural sources

remains bilirubin IXa on purification. However, in the latter

part of June 1971 we learned from Dr. A. F. McDonagh

(University of California, San Francisco) that he and F. Assisi

19
were preparing to publish the discovery of the Ilia and XHIa

isomers in commercial preparations of bilirubin. They used

t.l.c. on silica gel with 1:99 (v/v) acetic acid-chloroform.

They found the highest proportions of these unexpected isomers

in bilirubin from Pfanstiehl, the supplier of the bilirubin

that is certified as the SRM. As the spectrophotometric

properties McDonagh and Assisi had found for each bilirubin

isomer were somewhat different , further work toward estimating

the fluorescent impurity in the SRM would have to take into

account the proportions of the isomers and their properties.

6 . Standard Solutions of Bilirubin for

Clinical Analysis

(B. Johnson and B. West)

In NBS Technical Note 547, we described our efforts

to that time to find an optimal procedure by which to

prepare standard solutions of the SRM bilirubin. Results

which related mainly to the use of pooled human serum as

the matrix for the standard solutions, were discussed at a

meeting of the Expert Panel on Bilirubin of the International Federation of Clinical Chemists in Aarhus , Denmark,

in August, 1970.

Some suggestions were raised at that meeting. These

included (a) examination of the use of 5$ solution (w/v) of

human serum albumin (in place of the pooled serum), (b) use

of tris(hydroxymethyl)aminomethane (Tris) buffer at pH 8.5

for dissolving the protein, (c) combination of the bilirubin

concentrate and the protein solution at that pH, and (d)

lowering the ratio of bilirubin to albumin to 1:10 (which

would limit the maximum obtainable concentration of bilirubin) .- These suggestions became the basis for our further work.

20
a. Stability of Solutions of Bilirubin in

Albumin—Tris Buffer at pH 8.5; Comparison

with Two Enriching Solutions

A 5% solution of human serum albumin in pH 8.5 Tris

buffer was divided into several portions, and each was

enriched with SRM bilirubin that was freshly dissolved in

either 0.1 M potassium cyanide—formamide (KCN—P) [8] or

in methyl sulfoxide (Me-SO) [9]. Albumin from only one

commercial lot was used, and enrichments were made on

different days. As usual in our work, the volume of

bilirubin solution added was limited to 2% of the final

volume of the enriched albumin solution. Each enriched

solution was tested (with albumin used as the blank) at

the time of enrichment and during the next several days

by direct spectrophotometry and by diazo coupling [10].

The results showed that the solutions prepared with

bilirubin in 0.1 M KCN—P were not stable. However, the

j solutions enriched with bilirubin in MepSO were much more

j stable. The latter solutions also exhibited higher

absorbances in both tests (see figs. 1 and 2). Solutions

with KCN—F were not studied further.

b

Effect of Storage Conditions on th e Stability of Bilirubin—Albumin Solutions

A fresh solution of albumin in pH 8.5 Tris buffer was

enriched using bilirubin—MepSO and divided as small

aliquots into three groups for storage under different

conditions. One group of aliquots was stored at + 2 °C

in a refrigerator, a second at -20 °C in a freezer, being

cooled from room temperature to -20 °C in the freezer,

and the third group was quick-frozen by immersion in

Dry lee—acetone, and immediately transferred to the freezer

at -20 °C.

21
Days after

10

enrichment

15

Figure 1. Stability of several bilirubin—human albumin

solutions prepared with (a) methyl sulfoxide

(closed symbols) or (b) KCN-F (open symbols),

as determined by the Jendrassik—Grof diazo test

22
K) X! o5 P

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CD

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n

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X2 Sh rH O • 03 >5 ^ u C to P

CO i-H CD c

01 SOS 3 ,0 O 2 E

JS E P 1 >j o u C W£

•H ft

wc JD 13

O

0) 3 CD

m

m CO P l_ •H O O 4) rH rH CD

«+» •H

O ft o £3 s^ w H CD P

CO T3

O

Q

m -H CD >o CD X U > O -H Cm T3

COH3 >»

On

W XJ

O

H TJ

>s >s CD P £ C

•H P «H H CD S

•H S Ih ,q CD 03^ P o P CO CD CO 1^ ^ Wwa C\J

CDM

bO

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23
The results from direct spectrophotometry and diazo

testing indicated that samples stored under all three

conditions underwent very similar rates of change over

the 7-day period of the study. Absorbances by direct

spectrophotometry declined by 1.5$ and those from the diazo

test by 1.1$ during the 7-day test period. Newly prepared

samples were stored thereafter at -20 °C in a freezer.

c

.

Calibration Curves for Bilirubin—Albumin

Solutions; Enrichment at pH 8.5

Bilirubin—albumin solutions having different

concentrations of bilirubin were prepared as follows : A

5$ solution of albumin in pH 8.5 Tris buffer was enriched

with bilirubin in Me

2

S0 to a level of 6.4 mg/100 ml, and

aliquots of this solution were diluted 1:2, 1:4, and 1:8

with additional albumin solution. The solutions were tested

on the day of preparation only . Results by direct spectrophotometry and diazo coupling gave fairly consistent values

for the millimolar absorptivity (e „) ; by direct spectrophotometry the average was 52.7; by the diazo test, 72.4.

The data, illustrated in fig. 3, showed that the standard

solutions would provide satisfactory calibration curves.

d. Bilirubin—-Methyl Sulfoxide Enrichments

of Albumin in pH 7.4 Tris Buffer

The properties of solutions prepared with albumin

dissolved in pH 7-4 Tris buffer and enriched with bilirubin—MepSO were next examined. Analysis of stored

aliquots showed that samples prepared at this pH are

stable for at least six days (the duration of the

experiment) . Enrichment at this pH had no effect on

the "azobilirubin" millimolar absorptivity. However, it

was found that, if samples are diluted with pH 7.4 Tris

buffer for direct spectrophotometric measurement, the e M

is only 48.7; but, if pH 8.5 buffer is used as the diluent,

24
2 4

Bilirubin, mg/lOOml of solution

Figure 3. Calibration curves for solutions of human albumin

at pH 8.5 enriched with bilirubin in methyl

sulfoxide. The upper curve is for the

Jendrassik—Grof diazo test, and the lower is

for direct spectrophotometry.

25
the e -. has the more "normal" value of 52.0. Presumably,

the binding on the albumin is more uniform at the higher pH

.

e

.

Stability of Enriched Albumin Solutions (1)

Enriched at pH 8,5, and (2) Neutralized to

pH 7.H Before Storage

Two enrichments of 5% solutions of human serum albumin

in pH 8.5 Tris buffer were made with bilirubin—Me

p

S0

.

Before storing at -20 °C, one of the solutions was

neutralized to pH J.k with M hydrochloric acid; the other

was kept at pH 8.5. For both, pH 8.5 Tris buffer was used

as the diluent before direct spectrophotometry. Over the

21-day test-period, the neutralized solution was found to

be essentially stable while the unneutralized solution

showed a total decrease in e M

(by both direct spectrophotometry and diazo test) of 3-3$.

f

.

Calibration Curves for Bilirubin—Albumin

Solution; Enrichment at pH 7-4

A 5% albumin solution at pH 7-4 was enriched to a

level of 17.50 mg of bilirubin per 100 ml of enriched

albumin solution. Aliquots of this solution were diluted"

with additional 5$ albumin (pH 7.4) to make a series of

bilirubin—albumin standards . The solutions were stored

overnight at -20 °C before the analyses were performed.

Dilutions for direct spectrophotometry employed pH 8.5

Tris buffer. Plots of absorbance versus concentration

of standard for both direct spectrophotometric and diazo

tests gave straight lines (see fig. 4). The average e M

as determined by direct spectrophotometry was 52.2 and by

diazo test was 71.3.

g. Comparison of Commercial, Human Albumin

Preparations

It was of interest to determine whether lyophilized

human albumin (fraction V) from different commercial

26
8 16

Bilirubin, mg/lOO ml of solution

Figure 4. Calibration curves for solutions prepared byenriching human albumin at pH 7*4 with bilirubin

in methyl sulfoxide. The upper cUrve is for

the Jendrassik—Grof diazo test, and the lower

is for direct spectrophotometry.

27
sources, or different lots from the same source, might

significantly affect the values of e „ obtained in our

tests . Six commercial samples and one sample supplied to

us by Dr. A. Hainline (Center for Disease Control) were

utilized.

There were variations in the physical appearance of

the different albumin preparations . The more discolored,

yellow materials were flaky in texture; one sample was

gray -white and granular; and other samples were pale to

off-white in color and fine textured. The more colored

solids gave straw-colored solutions, and the lighter samples

gave almost colorless solutions. A spectrum from 700 to

250 nm of the most yellow sample as a 5% solution showed an

absorbance of 0.15 at 450 nm and of 0.30 at 410 nm. At a

1:5 dilution (the concentration used in direct spectrometry

and diazo coupling of bilirubin) , these absorbances fell to

0.03 and 0.06, respectively. The spectrum profile was a

slowly increasing value of absorbance from 700 to 300 nm

and then a more rapid increase in absorbance at shorter

wavelength

.

With some of these albumin samples, only one solution

was tested. The results are summarized in table 2. Direct

spectrophotometry showed the e M varying from 52.0 to 55.6,

with the X ranging between 46*0 to 465 nm. Diazo testing

max

showed e .. varying from 72.6 to 76.8, with the X ranging mM " ° 3 max

from 595 to 600 nm. Although not directly apparent from

these data, the use of lighter colored albumins led to

somewhat higher values of e M

and, for direct spectrophotometry, to the shifting of the X to longer wavelengths. max

28
Table 2. Values of e „ from testing of bilirubin—methyl

sulfoxide enrichments of different commercial

samples of human serum albumin

Albumin Bilirubin

lot number (mg/100 ml)

27

132

133

401X

1849

none

CDC

16

4

4

16

16

4

3

Direct

spectrophotometry

e .. Xmax mM

53.6 450

55.6 465

52.0 460

53.6 463

52.5 463

54.3 465

54.7 464

Diazo testing

e mM Xmax

74.6 595

75-1 598

73.4 598-600

75.8 597

72.6 597

75.4 597

76.8 599

B. Cholesterol

(R. Schaffer, A. Cohen, and R. P. Brady, Jr.)

In Technical Notes 457, 507, and 547, the analysis and

shelf-life stability of cholesterol were reported. With the

present data, it can be concluded that, after 3 years, the

bulk SRM cholesterol continues to maintain its stability.

1. Thin-layer Chromatography of Cholesterol Samples

(A. Cohen)

Unidimensional chromatography on activated Silica Gel G

plates with 1:1 (v/v) ethyl acetate—heptane was described in

Technical Note 457 [11].

29
a. Differentiation Between Impurities and

Artifacts by Two-dimensional, Thin-layer

Chromatography

The impurities in the cholesterol, and artifacts formed

during chromatography in the above were differentiated by

two-dimensional t.l.c. Cholesterol (160 ug) was spotted

from a solution in chloroform, on an activated plate

(250-um thick), developed for a distance of 10 cm in a

saturated tank, dried rapidly with gaseous nitrogen, and

then redeveloped in the second direction with the same

developer. After spraying with 5:1 (v/v) methanol—sulfuric

acid and charring, three spots were visible at R„ 0.6l

(first), 0.56 (second); at R

f 0.44, 0.06; and at~Rf 0.44,

0.48. The first of these spots is an impurity. The second

is an artifact; it conforms in shape and has the R

f value

found after unidimensional development of an equal amount

of cholesterol with the same developer. In the direction

from this artifact to the intense spot at R

f 0.44, 0.48,

no other spots were detected, even under 366""-nm light.

Other impurities on the two-dimensional chromatogram were

detected by their faint fluorescence, observed with both

reflected and transmitted 366-nm light, along a line from

the origin (R f 0.0) through the intense spot at R

f 0.44,

0.48. The results are summarized in table 3.

Thus, the spots detected on unidimensional

chromatography, other than those of the cholesterol and

the artifact, the spot at the origin, are considered to

be those of impurities.

30
Table 3. Two-dimensional t.l.c. of freezer-stored

cholesterol on Silica Gel G

Light

R

f value3" used for,

detection

1 2

0.61 0.56 vis

0.44 0.06 vis

0,44 0.48 vis

0.29 0.32 uv

0.24 0.26 uv

0.12 0.15 uv

0.00 0.00 uv

Remarks Identification

weak U

faint A

strong C

faint U

faint U

d trace U

faint U

?Key: 1, first direction; 2, second direction.

vis, visible; uv, visible under 366-nm light.

JCKey: A, artifact; C, cholesterol; U, unknown.

One spot, or two.

b. On Aluminum Oxide G—Silver Nitrate

T.l.c. was also performed on chromatoplates (250-um

thick) of 33:10 (w/>) aluminum oxide G-silver nitrate [13].

Freshly prepared plates were allowed to dry overnight,

activated for 2 h at 120 °C, and stored over phosphorus

pentaoxide . Chloroform was used as the developer. The

bulk, frozen cholesterol was examined after spotting 20- to

160-ug quantities from a chloroform solution. The chromatoplate was developed 10 cm, dried, sprayed with methanolsulfuric acid, and charred for 15 min at 120 °C. Elongated,

yellow spots appeared whose length was related to the

amount of cholesterol applied; these spots fluoresced

yellow. On further spraying with methanolic sulfuric acid

and charring for an additional 15 min, the spots became

brown and fluoresced pink. All spots tailed to the origin,

where a faint spot was present; a separate experiment,

31
employing two-dimensional chromatography, suggested that

this was an artifact. There were no indications of other

components

.

2 . Post-issuance Studies

a. Thin-layer Chromatography

(A. Cohen)

Samples of the bulk supply of cholesterol that had

been stored in a freezer for 35 months showed no significant

evidence of change, but samples in loosely capped, brown

bottles that had been exposed to air at room temperature

in the laboratory for 30 months were found to be less pure.

Cholesterol that had been packaged under nitrogen, sealed,

and stored at room temperature for 22 months appeared

almost comparable to the freezer-stored cholesterol.

b

.

Gas —liquid Chromatography

(A. Cohen)

G.l.c. was performed with a Varian Aerograph Series

2100 gas chromatograph equipped with a flame-ionization

detector and a glass column (180 X 0.2 cm i.d.) packed with

Gas Chrom Q (60-80 mesh, pretreated with 3% of OV-17) as

the stationary phase, heated to 240 °C, with the injector

and detector set at 260 °C. Chloroform solutions of

cholesterol (^10 ug/ul) were injected.

In earlier studies of the SRM, g.l.c. was performed

with a Glowall Corporation Chromolab 320 Gas Chromatograph

fitted with a column (180 X 0.2 cm i.d.) with the same

phases, but operated at 240 °C. Solutions of cholesterol in

ethyl acetate were injected [12].

With the Varian chromatograph, two peaks were detected

having the same retention times, 0.5 and 1.8, relative

to cholesterol as revealed with the Glowall instrument.

This pattern was found with samples of the bulk cholesterol

32
kept in a freezer for 35 months, with a packaged sample

stored at 20 °C for 22 months, and with an unpackaged sample

exposed to air and light for 32 months . There were no

peaks at 1.2 and 1.6, as had been observed with the

Glowall instrument, but a distortion in the tailing of

the cholesterol peak was noticeable at 1.2.

c

Ultraviolet Spectrophotometry

(A. Cohen)

Cholesterol exposed to air for 36 months at 20 °C in

a loosely capped, brown bottle was examined as a 1% solution

in dichloromethane ; it showed very substantial absorption,

with a shoulder at ^245 nm that could be observed only after

dilution. In contrast, a 1.0% solution of SRM cholesterol

stored for 39 months in the freezer showed a simple

spectrum similar to that originally observed.

d. Differential Scanning Calorimetry (DSC)

(R. F. Brady, Jr.)

Five separate samples of the freezer-stored SRM

cholesterol (39 months after the first issuance of the

Standard Reference Material) were tested. The meltingpoint curves were processed in the usual way [14]. The

results are given in table 4, where AH is the molar heat

of fusion, T° is the melting point in kelvins, and AT is

the melting-point depression. The purity values obtained

are like those previously obtained with the SRM.

33
Table 4. Reproducibility of the purity of SRM cholesterol

as determined by differential scanning calorimetry

AH T° AT

(cal/mol) L (J/mol) (K) (K)

79^0 33,200 419.67 0.07

7870 32,900 419.76 0.10

8020 33,600 419.77 0.10

7750 32,400 419.56 0.03

8190 34,300 419.76 0.13

Purity

(%)

99 .85

99 .78

99 .77

99 .93

99 .69

e

.

Phase Solubility Analysis

(R. F. Brady, Jr.)

Solubility analyses of three samples of freezer-stored

cholesterol were made 34 months after the first issuance

of the standard Reference Material. For a description of

the technique used, see reference [15]. The cholesterol

was found to 'be 99-80, 99-68, and 99-64 weight-percent pure . in the three determinations

.

f

.

Volatile Matter in SRM Cholesterol by

Mass Spectrometry

A Bell and Howell-Consolidated Electrodynamics

Corporation (CEC), double-focusing, mass spectrometer

Model 21-103 was used. The ionization potential was

70 eV, and the ion-source temperature was 250 °C. A sample

(223 mg) of SRM cholesterol that had been stored in the

freezer for ^43 months was examined (by J. M. Ives of Section

310.05) for impurities by heating from ^80 to 170 °C, and

analyzing for any material that vaporized. Only water in a

34
very small proportion was released from the cholesterol under

these conditions. No fragments indicative of decomposition

of the cholesterol were found.

C. Cortisol

(R. P. Brady, Jr.)

In Technical Note 5^7, some of the early survey work

done on the Cortisol standard was described. Since that

report, Cortisol suitable for use as a Standard Reference

Material has been obtained, and work leading to its certification was begun.

1. Testing for Homogeneity

Nine samples were withdrawn from the bulk material

according to a statistical plan, and analyzed by use of

a commercial carbon-hydrogen analyzer. The standard

deviation for nine analyses was 0.16$ for carbon, and 0.13$

for hydrogen; these values are well within the 0.3$ limit

to the precision of the analytical method, and thus, the

standard material is homogeneous in respect to carbon and

hydrogen.

35
The specific optical rotation at five wavelengths, and

the ultraviolet spectrum were measured in 95$ ethanol for each

of the nine samples. The data are given in table 5. The

uniformity of these data is indicative of the homogeneity of

the sample.

2. Elemental Composition

The analysis for carbon and hydrogen was made on four

large samples of the standard material, and showed (±2 SD)

carbon, 69.^9 ±0.21$, and hydrogen, 8.39 ±0.09$. The

theoretical values are: carbon, 69-58$, and hydrogen, 8.3^$.

3. Behavior on Melting

The compound melted at 219.0 to 220.5 °C (corr.) when

heated in an open capillary tube at 0.5 °C/min, and produced

a pale-yellow melt that did not resolidify. When sealed

under vacuum in a capillary tube, the material melted at

220.5 to 221.5 °C without yellowing, and did not

resolidify. Thermogravimetric analysis of the sample

heated in a dry nitrogen atmosphere at 2 °C/min showed,

at about 221 °C (uncorrected), the initiation of the loss

of a large proportion of sample weight; in air, however,

the loss of weight began at 204 °C. Attempted use of

differential scanning calorimetry showed that thermal

curves obtained under nitrogen were not independent of

heating rate or total heating time.

4. Spectrometry Data

The infrared and nuclear magnetic resonance spectra of

the sample were identical with those reported earlier [16,

17]; neither technique gave evidence of impurity.

36
Table 5. Specific optical rotations and millimolar

extinction coefficients of SRM Cortisol

Wavelength (nm)

589 578 546 436 365 242

Sample

No.

1 +167.1

Specific

+175.2

optical rotation

(degrees

)

+205.7 +390.5 +576.2

e mM at

Xmax

16.2

2 +167.0 +175.4 +204.7 +388.4 +573.7 16.1

3 +167.1 +175.1 +205.4 +389.1 +575.6 16.1

4 +166.9 +175.3 +204.1 +389.7 +574.8 16.1

5 +167.1 +174.9 +204.9 +388.8 +575.9 16.2

6 +167.0 +175.4 +205.1 +380.0 +576.1 16.1

7 +167.2 +175.2 +205.5 +389.3 +575.7 16.1

8 +167.2 +175.2 +205.5 +389.3 +575.7 16.1

9 +166.9

e+167.0

+174.8

+175.1

+204.4

+205.2

+388.7

+389.3

+575.1

+575.3

16.1

Averag 16.1

1 SD 0.1 0.2 0.5 0.7 0.6 0.020

5 . Loss on Drying

Three portions of Cortisol were heated at 110 °C and 0.1

torr for three 24-hour periods. An average of 0.083$ of the

sample weight was lost in the first 24-hour period, and

virtually no weight loss occurred on further heating. The

data are given in table 6.

37
Table 6. Volatile matter in SRM Cortisol

Weight

(g) 1

Sample No.

2 3

Initial 2.814093 2.719680 2.747191

After 24 h

% loss

2.812008

0.074

2.717195

0.091

2.744883

0.084

After 48 h

% loss

2.811995

0.075

2.717186

0.092

2.744868

0.084

After 72 h

% loss

2.813986

0.075

2.717180

0.092

2.744856

0.085

6. Insoluble Matter

Solutions of Cortisol (1.8—2.0 g) in prefiltered

methyl sulfoxide (9—20 ml) were filtered through tared

Millipore filters having a pore size of 1.5 ym. Reweighing

of the filters after drying indicated a gain in weight

corresponding to 0.001$ of the weight of Cortisol used.

The stability of the filter membrane and the solvent blank

were determined by passing prefiltered methyl sulfoxide

through preweighed membranes . The average gain in weight

of the three membranes was 1 pg.

D. D- Glucose (Clinical) ~ (B. Coxon)

g-Glucose has now been issued as Standard Reference

Material 917, but some work in addition to that described

in previous reports [19, 20] has been done towards (a) the

certification of this material, and (b) confirmation of the

anomerization noted during previous analyses [20 ] by

differential scanning calorimetry

.

Gas—liquid chromatography of trimethylsilyl derivatives

prepared from the crystalline Standard Reference Material

indicated [20] that it contained 99-5 percent of a-B-glucose

38
and 0.5 percent of the 3 anomer. However, similar analyses

of material that had been partially or completely melted

under the conditions of determination of purity by differential scanning calorimetry demonstrated that anomerization of the initial preponderance of a anomer occurs

rapidly to give a mixture containing a slight excess of $

anomer [20]. In the present work, the proportions of a- and

g-g-glucose in the syrups prepared by melting them have been

determined by p.m.r. spectroscopy at 90 MHz and correlated

with the results from polarimetry of solutions of these

materials at five different wavelengths. These techniques

were performed by using solutions of the D-glucose materials

in either methyl sulfoxide or methyl sulfoxide-^ y for which

our work showed that neither a-g-glucose nor 3-D-glucose

(nor mixtures of the two anomers) undergoes significant

anomerization during the first 1.5 h following dissolution.

1 . Analysis of g- Glucose by P.m.r. Spectroscopy

at 90 MHz

The most 'useful signals for the analysis of a- and 3-

D-glucose are those of the anomeric hydroxyl proton, which

are well separated at lowest field. The Bruker n.m.r.

spectrometer used in this work was sufficiently sensitive

to allow proportions of the minor anomer as small as 0.9

percent to be detected in a single scan of the spectrum.

However, signal averaging (16 scans) was employed to improve

the precision of the data. For solutions in pure, dry

methyl sulfoxide-^ , the signals of the anomeric hydroxyl

protons occur as doublets, because of spin coupling with

the proton (H-l) bound to C-l of the sugar. Under these

conditions, anomerization and rapid chemical exchange of

the hydroxyl protons are suppressed by strong hydrogenbonding with the solvent. The partial spectra obtained

from solutions of crystalline a- and 3-D- glucose are shown

39
in figure 5 (a and b) . The spectra of solutions of melted

a- and 3-g-glucose in figure 5 (c and d) are practically

identical, and provide direct confirmation that, not only

do both a-g-glucose and 3-g-gliicose anomerize during

melting, but they do so to give equilibrated mixtures of

anomers . The quantitative proportions of anomers , obtained

by digital integration of all of these spectra within 4096

channels of a Fabritek instrument computer, are shown in

table 7. The proportions of anomers thus determined for

the melts are similar to those determined earlier [20] by

g.l.c. analysis of small quantities of a-g-glucose that

had been melted completely in a differential scanning

calorimeter.

Table 7. Compositions of a- and B-g-glucose and their

supercooled melts, determined by digital

integration of time-averaged, p.m.r. spectra

Content of

g-Glucose a-g-glucose 3-g-glucose

{%) {%)

Crystalline a- 99.14 0.86

Crystalline 3- 0.97 99.03

Melted a- 47.4 52.6

Melted 3- 47.9 52.1

16 scans were accumulated for a solution of each material

in methyl sulfoxide-d 6 . Standard deviation of five

individual determinations, 0.11.

2 . Polarimetric Analysis of Crystalline and

Melted D- Glucose Samples

Further confirmation of anomerization during melting,

to give an equilibrium mixture, was obtained from

40
6.56 6.20 6 (RRMJ

Figure 5. P.m.r. spectra (16 scans) of the anomerichydroxyl proton region of solutions of Dglucose in methyl sulfoxide-d 6 at 90 MHz;

(a) crystalline a-D-glucose, (b) crystalline

$-D-glucose 3 (c) melted a-D-glucose, and (d)

melted 3-D-glucose.

41
polarimetric studies of solutions, in methyl sulfoxide,

of crystalline and melted g-glucose samples; it was found

that the optical rotation values of melted a- and 3-D-glucose

samples are essentially the same for each of five different

wavelengths, and also that these values are intermediate

between the values for the unmelted crystalline anomers

.

The mole fractions of anomers determined by p.m.r.

spectroscopy for the crystalline a- and 3-B-glucose were

then substituted in simultaneous equations connecting mole

fractions and optical rotations, and these were used to

calculate the optical rotations of theoretically pure aand 3-D-glucose for each of five wavelenghts . These

rotations were then used in other simultaneous, mo le-r fraction

equations connecting the specific rotations of the melted

samples, so as to permit calculation of the mole-fractions

of anomers present in these samples. For five different

wavelengths, the mean mole-fraction of a anomer calculated

to be present in the melted a-g-glucose was 0.4324, and for

the melted 3-D-glucose, this mole-fraction was 0.4320.

These fractions are quite close to, but do not agree

precisely with, the contents of a anomer in the melts

determined directly by p.m.r. (see table 7), probably

because of the assumption (used in the polarimetric

calculations) that the melts contain only the a and 3

anomers. In fact, a small amount of caramelization of

the sugars occurred during melting, and this may slightly

modify the optical rotations observed for the melts

.

In summary, the anomerization of crystalline a- and

3-D-glucose on melting, to give the same equilibrated

mixture of these anomers, has been confirmed by both p.m.r.

spectroscopy and polarimetry

.

Pull experimental details of the characterization of

the D-glucose SRM, and relevant numerical data and

discussion, are given in a paper that has been published [21],

42
E. Synthesis of DL- 4-Hydroxy-3-methoxymandelic Acid

(DL-Vanillylmandelic Acid, VMA)

(A. J. Fatiadi)

In connection with a program on clinical standards

being conducted at the NBS , there was need for a simple,

practical method for the large-scale preparation of pure

VMA in high yield.

A literature survey revealed that VMA had been prepared

in 19 [22] to 45 [23] percent yield by a multiple-step

procedure from vanillin via the cyanohydrin intermediate.

1. An Improved Procedure for Synthesis of VMA

An alternative route for the synthesis of VMA (4) is

based on the procedure of Goodman and coworkers [24 ]

, who

made use of the reaction previously employed in the synthesis

of vanillin [25], in which guaiacol (1) is condensed with

glyoxylic acid (2).

COOH

+ Ic=o

OCH3 H

COONa

CHOH

HCL

OCHCOOH

CHOH

Our procedure for preparation of 4 entails slow

addition of an ice-cold, aqueous solution of 2 to an

ice-cold alkaline solution of 1, with efficient mechanical

stirring. This one-step condensation provides VMA in

68—75 percent yield. The procedure has been described

in a paper ready for publication.

43
F. D-Mannitol (Clinical)

(B. Coxon)

This work was directed towards certification of a

D-mannitol SRM for use in the clinical analysis of

triglycerides present in sera. The g-mannitol serves

the function of providing, on periodate oxidation, a

standard source of formaldehyde with which can be compared

the formaldehyde produced by similar oxidation of the

glycerol liberated on saponification of the triglycerides

.

A preliminary screening of four commercial samples

of g-mannitol by thin-layer chromatography, paper

chromatography, and optical rotation studies was described

in Technical Note 547 [26].

1 . Synthesis of g-Mannitol for Use as a Reference

Sample Within These Laboratories

Most commercial g-mannitol is prepared either by

electrolytic or catalytic reduction of alkaline solutions

of D-glucose, and, before purification, the g-mannitol may

contain [27] as much as 1.5—2.5$ of sorbitol (g-glucitol)

.

Thus, we anticipated that, on a small scale, the reductionof g-mannose with sodium borohydride might more quickly

provide a pure sample of g-mannitol than many recrystallizations of commercial D-mannitol.

To a cooled solution of recrystallized g-mannose (30 g)

in water (90 ml) was added dropwise, with magnetic stirring,

a solution of sodium borohydride (10 g) in water (150 ml)

during 0.5 h. The mixture was stirred for a further 0.75 h;

a negative test for reducing sugar was then obtained with

boiling Fehling solution. Washed, regenerated, Amberlite

IR-120 (H ) ion-exchange resin was then gradually added and,

when the effervescence had ceased, ^200 ml of this resin was

added, followed by Norit. The suspension was stirred for

1 h at room temperature, and was then filtered through Celite

that had been thoroughly prewashed with water. The filtrate

44
vas concentrated to a syrupy suspension that was evaporated

ander diminished pressure with three 400-ml portions of

methanol, and then twice at atmospheric pressure with 500 ml

of methanol, to remove boric acid as methyl borate; a

negative flame-test for methyl borate was then obtained.

The preparation, evaporated to dryness, was dissolved in

hot water (50 ml), hot ethanol (500 ml) was added, and the

product was allowed to crystallize at room temperature and

then in a refrigerator. The crystals of g-mannitol were

filtered off, washed twice with ethanol, and dried under

vacuum at 60 °C; yield 26.5 g (87%), mp 166.9-167-9 °C.

Three recrystallizations from 5:1 (v/v) ethanol^water

(600 ml) gave g-mannitol* as globular clusters of colorless

needles, wt . 19.2 g, mp 167. 1-168.0 °C.

D-Glucitol was prepared in a similar way by reduction

of SRM g-glucose. After two recrystallizations from

ethanol, and drying under vacuum at 55 °C, the g-glucitol

showed premelting at 87-89 °C, and melted at 95—96 °C.

2. Determination of Purity by Phase Solubility Analysis

(E. Hohertz)

Pyrex test-tubes ( 2 X 15 cm) were cleaned by successive

washing with soap solution, tap water, distilled water, and

acetone. After being dried, the tubes were constricted near

the open end by drawing them out in a natural gas—oxygen

flame. Samples of g-mannitol (0.1, 0.3, 0.7, 1, 2, 3, and

4 g) were placed in each of 7 tubes, together with 1:1 (v/v)

methanol—water (10 ml). The tubes were sealed in a

gas-oxygen flame, shaken in a bath at 26 °C for six days,

and then opened and centrifuged.

*Referred to hereinafter as reference D-mannitol.

45
Aliquots (2.0 ml) were removed from each supernatant

solution and allowed to evaporate at room temperature in

tared, aluminum pans. The pans and contents were then dried

under vacuum at 50 °C. From the weights of these contents

and the initial sample-weights, a phase-solubility diagram

was plotted. The results for purity are compared in

table 8 with those derived by differential scanning

calorimetry

.

3

.

Determination of Purity by Differential Scanning

Calorimetry [28J

Typically, each sample of D-mannitol (^0.5 mg) was

weighed in an aluminum pan which was then hermetically

sealed and heated in a Perkin—Elmer Model DSC-1B

differential scanning calorimeter from 380 to 436 K at

80 K/min, and then from 436 to 442 K (beyond melting) at

0.625 K/min, by use of the maximum sensitivity (range 1)

of the calorimeter. The areas of the thermograms of the

g-mannitol studied for certification as a Standard Reference

Material required a correction of only 1.5—3% in order to

linearize the plot of temperature versus the reciprocal of

the fraction melted. The purity values obtained by DSC are

compared in table 8 with those measured by phase solubility

analysis. The data shown in table 9 indicate that the

purity values obtained for g-mannitol by DSC are not

significantly dependent on the temperature (T) at which

slow scanning is begun.

4. Optical Rotation of Samples of g-Mannitol

g-Mannitol (0.4 g) was dissolved in freshly prepared,

aqueous, 5% ammonium molybdate tetrahydrate solution

(20.0 ml); the solution was diluted with 0.5 M sulfuric

acid [29] to 25.0 ml at 20 °C.

46
Table 8. Purity values obtained for D-mannitol by

differential scanning calorfmetry (DSC) and

phase solubility analysis (PSA)

(B. Coxon, R. P. Brady, Jr., J. H. Thomas, and E.

Hohertz)

Manufacturer

Purity,

mole % (DSC)

Purity, . weight-^ (PSA) D

1 99.60 99.73

2 99.74 99.62

3 99.60, 99.63 99.67

4 99.68 99.78

4, experimental

sample

4, test sample

4, SRM test sample99-91

d

see table 12

99.91, 99.91

4, SRM sample

e

f

Reference

D-mannitol

99.94

99.94

99.92

99.84

99.78

99.74, 99.85

[1:1 (v/v)

ethanol—water]

99.95

These values should not be considered to be equally

reliable. The crystals were extracted with 1:1 (v/v)

methanol-water , unless otherwise stated. These samples

of g-mannitol were prepared by reduction ofog-mannose with

sodium borohydride. From top of supply,

supply. Prom bottom of supply.

e Prom middle of

47
Table 9. Purity values obtained for B-mannitol by

differential scanning calorimetry, with slow

scanning begun at various temperatures (T)

(R. F. Brady, Jr., and E. C. Douglass)

nufacturer Purity , mole % T (K)

4 99 .88, 98.80 431

99 .91, 99.91 433

99 -75, 98.34

99.98

434

435

99 .65, 98.90

99.94

436

438

4, experimental 99.80 429

i a sample 99.75

99.86

99.91

430

433

436

Reference sample of

g-mannitoia

99.92

99.89

99.92

99.98

431

435

436

438

These samples of g-mannitol were prepared by reduction of

g-mannose with sodium borohydride.

48
Measurements were made on freshly prepared solutions

at 20 °C by use of a thermostated, 1-dm cell in a Perkin—Elmer Model l4l polarimeter. The values of specific

rotation obtained from various samples are shown in

table 10.

Table 10

Specific rotations, at various wavelengths , of

solutions of various samples of g-mannitol in

acidified ammonium molybdate solution

2 o [a], (degrees) of

Wavelength

(A) , in nm

SRM

test sample l

a

SRM samples

2b 3C

589 (Na578

546

436

365

Concn.

-D)

d +138.7, 138.9

145.2, 145.2

166.3, 165.0

297.1, 296.9

f f

1.60, 1.62

Recrystalli zed

SRM sample6

H39

145

166

298

1

+139-4 +139.0 (139.0)

8 145.2 146.0

3 166.8 166.1

,6 299.0 298.2

,60 1.60 1.61 (1.60)

Mother liquors

from the

recrystalli zat ion

489 (Na578

546

436

365

Concn.

-D)

d +139.4

145.8

165.5

298.0

f

1.60

+139.0

145.3

166.2

297.8

f

1.60

,From top of supply.

Prom middle of supply.

,From bottom of supply.

^Literature value [30], [a.]* +139.2 ±0.3° (c 1.60).

The value in parentheses is the mean of 10 readings measured

fby the use of a Rudolph, high-precision, manual polarimeter.

Values were not obtained at this wavelength, because of

strong absorption by the solution.

sRecrystallized from 1:1 (v/v) methanol-water

49
Small values of optical rotation were obtained for

solutions of various samples of g-mannitol in water

(see table 11)

.

Table 11. Specific rotations, at various wavelengths, of

solutions of samples of g-mannitol in water

2 Wavelength

( A ) , in nm

of SRM

test sample

[a], (degrees)

of SRM

sample 2

Literature

value

389 (Na--D) -0.4b -0.3 C -0.2 to -0.5

578 -0.5 -0.2 —

546 -0.5 -0.5 —

436 -1.3 -1.1 —

365 -3.1 -2.8 —

Concn. 10.03 16.03 7 to 16.6

D-Mannitol from middle of supply.

c Unchanged after 16 h at 20 °C.

Mean of 10 readings

.

5. Thin-layer Chromatography (T.L.C.) of D-Mannitol

a. Partition T.L.C. on Layers of Silica Gel G

T.l.c. of the test sample, SRM test sample, and SRM

samples of manufacturer 4, and of the reference D-mannitol

(0.5—2.0 ul of 10$ solution in water) was conducted on

either 20 X 20 cm layers, or on 20 X 40 cm layers of Silica

Gel G developed in the 40-cm direction for 13-16 h. The

following solvent mixtures were used for development:

ethyl acetate—isopropyl alcohol—water (1:1:1, 1:2:1, 1:2:2,

1:3:1, 1:2:3, and 6:2:1 v/v) , butyl alcohol equilibrated

with 1:1:1 (v/v), 3 M ammonium hydroxide^nethanol-water

and 1:1 (v/v), methanol-saturated sodium tetraborate

50
solution. Although slight separation of g-glucitol from

D-mannitol was achieved with some of these solvent

mixtures, mixtures of these alditols containing 1% or 5%

of g-glucitol were not usually reproducibly resolved.

However, these experiments served to confirm the absence

of significant amounts of organic impurities having

mobilities different from those of D-mannitol and

fj-glucitol. Spraying of the t.l.c. plates with 5% sulfuric

acid and then heating at 120 °C revealed g-mannitol as a brown spot, and B-glucitol as a black spot.

b . Adsorption T.L.C. on Layers of Calcium Sulfate

Initially, 0.2—2 yl of aqueous, 10% solutions of the

SRM g-mannitol samples, of g-glucitol, and of g-mannitol

containing 1, 5, and 50% of g-glucitol were applied to

layers (5 X 20 cm) of calcium sulfate (for t.l.c.) that

had been activated at 110 °C for 0.5 h. Activation at

higher temperatures or for longer times caused cracking

or peeling of the layer from the glass plate. On the other

|hand, the use of non-activated layers gave poor results,

with much streaking.

The plates were developed with 1:1:3 (v/v) acetic

acid—benzene—methanol . However, development of spots of

0-mannitol that had been applied from aqueous solution

resulted in the appearance of a separate, artifactual spot

that remained at the origin. This spot was virtually

absent when solutions of the g-mannitol in methanol were

applied to the plate and then developed. The appearance

of an artifactual spot after spotting of aqueous solutions

is apparently due to rehydration of the calcium sulfate

layer (CaSCu-0.5 H 2

-> CaSCU • 2H 2 0) , and subsequent partition

of the D-mannitol between the water remaining at the origin

(not removed by drying at 40 °C under vacuum) and g-mannitol

adsorbed on the layer and developed by the solvent. The use

of methanol as solvent is not particularly convenient,

51
because of the low solubility of g-mannitol in it (<0.2#);

this necessitates multiple spotting in order to deposit

sufficient material on the plate.

By the use of this technique, proportions of g-glucitol'

in the D-mannitol at the 50% level could be detected, but not

at the 1% and 5% levels. Evidently, this phenomenon is due

to the fact that a slower-moving, large amount of g-glucitol

causes a slightly faster-moving, large amount of g-mannitol

to move farther along the plate than if none, or only a small amount, of the g-glucitol is present, i.e. , saturation

of the adsorption capacity of the layer.

6

.

Paper Chromatography of g-Mannitol

This technique was performed as described previously

[26], except that larger volumes (20 ul) of aqueous 10$

solution were also applied to the paper. No impurities

were detected in the samples of SRM D-mannitol by this

technique, although D-glucitol was not well resolved

under these conditions.

7

.

Ultraviolet Spectrophotometry of g- Mannitol

(B. A. Johnson and B. Coxon)

Samples (2 g) of the SRM g-mannitol from the top,

middle, and bottom of the supply, of the recrystallized SRM

g-mannitol, and of the solid obtained by evaporation of the

mother liquor of this recrys tallization (see later) were

each made up to a volume of 10.0 ml with distilled water.

The ultraviolet spectra of the solutions of SRM samples

showed only a low background absorbance (0.01—0.02) in the

region of 380-240 nm. However, between 219 and 200 nm, the

absorbance increased very rapidly to a strong, end

absorption. The spectrum of the recrystallized SRM

D-mannitol was similar, both as to shape and as to

absorbance values . The spectrum of the solution of motherliquor material showed in many regions more absorbance than

52
could be accounted for by the assumed enrichment-factor of 5

(see later) . For example, at 290 nm, the mother liquors

showed an absorbance of 0.1, whereas the SRM and recrystallized SRM samples displayed an absorbance of only ^0.01.

It seems probable that the mother liquors might have gained

some absorbing material from the methanol or filter paper

used in the recrystallization . In any case, no impurities

were detected in the SRM fj-mannitol by this method.

8 . Infrared Spectroscopy of D- Mannitol

(B. Coxon, B. A. Johnson, and E. Hohertz)

The D-mannitol samples were examined as pellets

prepared by pressing with potassium chloride. The samples

of manufacturers 1—4, the SRM samples, the mother liquor

material from recrystallization of the SRM sample, and the

experimental sample from manufacturer 4 after recrystallization, all showed very similar infrared spectra, with

a very strong, broad, hydroxyl absorption in the region of

3000—3500 cm , C—H stretching absorptions of medium

intensity in the 2900—3000-cm~ region, and a relatively

complex "fingerprint" region. In contrast, the spectra

of the experimental sample from manufacturer 4 (prepared

from B-mannose) and the SRM g-mannitol that had been

recrystallized from aqueous methanol were similar to each

other, but different from the aforementioned spectra,

particularly In the C—H stretching and fingerprint regions

In further contrast, the spectra of the reference

g-mannitol (long, fine needles), which had been recrystallized from aqueous ethanol, and of the sample of

manufacturer 4 after recrystallization from the same

solvent mixture, were similar to each other, but markedly

different, with respect to band intensities and positions

in the C—H and fingerprint regions, from all of the

aforementioned spectra.

53

mantm
These results indicate that at least three polymorphic

forms of D-mannitol have been encountered in the present

work, but neither infrared spectroscopy nor proton or

carbon-13 magnetic resonance spectroscopy indicated the

presence of any impurities in the samples of SRM Q-mannitol

The infrared spectra of these samples were very

similar to a published spectrum of D-mannitol [30]

.

9 . Concentration, by Recrystallization, of Impurities

in SRM g-Mannitol

The SRM g-mannitol (100.0 g of material taken in

approximately equal amounts from the top, middle, and

bottom of the supply) was dissolved in distilled water

(400 ml) by warming to 34 °C 3 and methanol (400 ml) was

added to the solution. After nucleation with g-mannitol,

the solution was allowed to cool slowly from 34 °C to

room temperature, and was kept at this temperature for

several hours, and then in a refrigerator for three days.

The large, crystalline clusters of colorless slivers

obtained were washed with methanol, and dried at 60 °C

in vacuo; yield 80.8 g. The mother liquor and washings

were combined, and evaporated to a colorless solid

(19.2 g) . Thus, the maximum possible enrichment factor

for impurities transferred from the SRM material to the

mother liquor is approximately five

.

The recrystallized material and the mother-liquor

material were subjected to analysis by infrared spectroscopy, ultraviolet spectrophotometry, optical rotations

in acidified ammonium molybdate solution, and gas—liquid

chromatography of hexakis-0-( trifluoroacetyl) derivatives.

54
10 . Loss of Weight of SRM D-Mannitol on Drying

a. At 110 °C

(B. A. Johnson)

The samples of SRM g-mannitol (50 g, accurately

weighed) in tared, aluminum-foil pans were dried in an

oven at 110 °C and r^3 torr, and were periodically removed

from the oven and allowed to cool for one hour over

phosphorus pentoxide in a desiccator before being weighed.

The weights lost are shown in table 12.

Table 12. Loss in weight of SRM D-mannitol on drying

at 110 °C

Loss in weight (mg) of samples

Drying time Top sample Bottom sample

(h) (49,98700 g) (49.51548 g)

17 4.14 3.53

37 6.11 5.90

57 7.26 7.14

80 10.88 11.01

Total weight lost, % 0.022 0.022

b. At 70 °C

On drying at 70 °C for 24 h, 7-g samples from the top,

middle, and bottom of the SRM supply lost 0.002, 0.001, and

0.002%, respectively.

The ash content of 25-g samples was found to be less

than 0.001%, and therefore no determinations of trace

inorganic constituents were made.

55
The material in the SRM g-mannitol (25-g samples)

that was insoluble in water was found to be 0.0006,

0.0001, and 0.003% for the top, middle, and bottom samples,

respectively

.

Satisfactory carbon and hydrogen analyses were obtained

for these samples

.

G. g-Nicotinamide Adenine Dinucleotide (3-NAD) and its

1,4-Reduced Form (g-NADH)

(A. Cohen)

In Technical Note 547, some initial studies were

described on the characterization of g-NAD, a compound

relatively more stable than g-NADH. With that work as

a background, we began a study of g-NADH, desired for

issuance as a standard reference material.

1. ultraviolet Spectral Data for Commercial Samples

of g-NADH

The NRC values [31] of e for g-NADH at pH 10 are

14.4 at 259 nm and 6 . 2 at 340 nm; A 26 o/A 3 m 2.32. It is

generally accepted that low values of A 2

6

i/A 3 4 are

indicative of purer g-NADH.

All samples were dried for at least 24 h under high

vacuum at ^20 °C. Lot 065003 was dried for 4 days. Two

samples of lot 2549 were used; one was dried for 24 h and

the other for 48 h. Lot 678-9 was obtained as a preweighed

vial, and the weight given on the label ("5-0 mg") was used

in the calculation of the millimolar absorptivity (e M

) of

the sample. The results are summarized in table 13.

a. g-NADH, Lot 50C-6420

Two samples of powdered g-NADH were obtained from the

commercial source that supplied lot 678-9. The data sheet

supplied with lot 50C-6420 indicated that the sample had a molecular weight of 791; this includes solvation by ^3-5

56
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57
molecules of H20/molecule and 0.4 molecule of ethanol/

molecule. Although we examined the samples after they had

been dried under high vacuum, the molecular weight of 7 91

was used to calculate £ M

. The sample weights used were 3.178

and 3-741 nig. The data obtained are given in table 14.

Table 14. Ultraviolet spectral data for 3-NADH (lot no.

50C-6420) in 0.1 M Tris buffer, pH ^10.4

w aveleng

(nm)

235

259

260

290

340

A 2 6 0/A3 it

A 2 5 0/A2 6

A2 8 0/A2 6

£ mM

Manufacturer's

data

6.86

14.9

15.0

1.57

6.96

15.1

15.1

1.62

6.20 6.00 6.10

Abso rbance ratio

2.3 2.49 2.48

0.81 0.79 0.81

0.23 0.25 0.26

2 . Paper Chromatography

From our previous chromatographic studies, the

fluorescent intensity of a solution of 3-NADH in 0.1 M Tris

buffer (pH ^10) kept at ^4 °C for 38 days appeared to be only

slightly greater than that of an unbuffered NADH solution in

25$ ethanol. All samples of NADH were therefore dissolved in

0.1 M Tris that contained 25$ of ethanol. The ethanol facilitated evaporation (with nitrogen gas) of solvent from the

58
spots applied, and also permitted the preparation of morecompact spots . Each sample (300—320 yg) was spotted on Whatman No. 1

filter paper, and developed with 2:1 (v/v) pyridine—water

[32] by descending chromatography. The R

f values and

manufacturer's lot numbers are listed in table 15.

Identifications of NADPH and AMP were based only on

the similarity of their R

f values to those of reference

samples used as standards . Lots 065003 and V3837 seemed

to contain more NADPH (based on a higher fluorescence

intensity with 366-nm light) than the other samples.

Lot 678-9 did not appear to show any evidence of NADPH

'for the amount spotted, but it had an impurity at R 0.35.

"Adenosine diphosphoribose" sodium salt (ADPR) at R

f 0.73

and a-NADH at R f 0.71 could not be distinguished from

g-NADH, possibly because of the amount of 3-NADH spotted.

Table 15. Paper chromatography of commercial samples of

3-NADH

R- values of components

Detection by

fluorescence

under after

of 1 ot no

366-nm

light

KOH

spray

Identifi

cation

065003 901.76 V3837 2549 678-9 [33]

0.68 0.67 0.66 0.66 0.66 blue 3-NADH

0.57 0.56 0.56 0.54 0.55 none blue 3-NAD

0.47w 0.46w 0.46w 0.45w blue NADPH

0.43 0.43 0.42 0.42 0.42 none none AMP C

__ ._ — — — . 35vvw

blue unknown

0.0 0.0 0.0 0.0 0.0 pale

whitish unknown

,Key: v, very; w, weak.

Reduced nicotinamide adenine dinucleotide phosphate,

sodium salt.

5'-Adenylic acid.

59"
In the chromatographic analysis for impurities given in

table 15, the inspection was performed after the chromatographic sheets had been allowed to dry. To assure valid

observation of properties of these impurities (loss of

fluorescence on drying of the paper might lead to an

incorrect identification or characterization) , the

chromatography was repeated, and the wet sheets were examined

and compared to the air-dried sheet. No differences were found

3 . Loss of Weight on Drying, and Elemental Analysis

Approximately 200 mg of 3-NADH from lot 50C-6420 was

weighed accurately into an all-glass weighing container,

and dried under high vacuum at ^20 °C to constant weight.

The loss amounted to 2.8%. The dried sample was analyzed

for its elementary composition. The results are shown in

table 16,

Table 16. Elemental analysis of a dried sample of 3-NADH,

lot 50C-6420

Calcu lated

For C H N P Na

C 2 1H2 9 N7O 1 i+P 2 37.9 4.4 14.7 9-3

C 2 iH27N 7 Na 2 0i i»P 2 35.6 3.8 13.8 8.7 6.5

2 H 2 33.8 4.2 13.2 8.3 6.2

3 H 2 33.0 4.4 12.8 8.1 6.0

3.5 H 2 32.7 4.4 12.7 8.0 6.0

4 H 2 32.3 4.5 12.6 7.9 5.9

Found 31.6 4.1 12.2 8.1 6.40; 6.22

60
4. Stability of 3-NADH under Conditions for Enzymic

Analysis

The reaction occurs as follows:

NADH 2 t CH 3 CHO^=^ NAD + CH3CH2OH

The method of Ciotti and Kaplan [34] was followed

I with use of the following reagents: 1:40 dilution of

I alcohol dehydrogenase, 0.5 M acetaldehyde , and 0.1

: M potassium phosphate buffer (pH J .5) • The quantities

used were increased by a factor of 33 for greater precision;

hence, 100 ml of total solution was prepared. The change in

absorbance at 340 nm was measured. The results are summarized

in table 17.

Thus, the study of the enzymic analysis revealed possible

sources of error, for example, the acetaldehyde should have

, been buffered before combination with the 3-NADH. If a number

of portions are to be taken from a container of dry 3-NADH,

this must be done in air having a low relative humidity, and

the remaining 3-NADH should be re-examined after additional

drying under high vacuum. The slight difference of A260/A31+0

in the two samples of the 21-day, freezer-stored sample cannot

be explained.

5. Inhibitor(s) in 3-NADH

Samples of 3-NADH may contain an inhibitor that affects

kinetic studies with some enzymes . The inhibitor is stated

to be produced when 3-NADH is in contact with moisture.

a . Chromatographic Studies of 3-NADH Treated

with Moisture

i . Paper Chromatography

A sample of 3-NADH from lot 065003 was exposed to

moist air for 48 h; then it was dissolved in Tris buffer

and chromatographed against unexposed 3-NADH from lot 065003

and lot 678-9 . The chromatographic characterization of these

untreated commercial samples of 3-NADH was reported in table 15.

61
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62
Footnotes to table 17

Key and comments: I. Duplicate samples of the dried material

were reexamined after storage for an additional 21 days in a

freezer. The samples appeared to be stable.

II. The required volume of acetaldehyde solution was

premixed with some of the buffer. This solution was used to

transfer the weighed 3-NADH to the 100-ml volumetric flask.

The spectral values were obtained within 20 min- the value of

A 2 6o/A 3 «fO indicated stability of the solution for at least

20 minutes.

III. First, the required amount of acetaldehyde solution

was added to the volumetric flask, and then the weighed sample

of 3-NADH was transferred with the aid of the phosphate buffer.

The high value of A 2 6o/A 3 ^o and the low value at 340 nm may

have resulted from uncontrolled contact with the unbuffered

acetaldehyde, which contained (according to the manufacturer)

0.01$ of acetic acid; 3-NADH is known to be unstable under

acidic conditions. From the 100 ml of solution, 9.97 ml

| (calibrated pipet) was withdrawn, and combined with 0.33 ml

; (calibrated pipet) of the diluted enzyme solution. (The 1:40

j dilution of the enzyme was used to avoid oxidation of any NADPH

that might be present.) There was a rapid decrease in

I absorbance at 340 nm, and the absorbance appeared to be stable

after 0.5 h. The residual material in the solution was fluo-

; rescent under 366-nm light.

IV. Acetaldehyde was not present in experiment IV. The

higher absorptivity at 340 nm and the lower A260/A340 compared

to III, suggests that the unbuffered acetaldehyde solution

initially put into the flask in III was destructive to the

3-NADH added.

Comparison of the results listed in columns II and IV

suggested that the dried 3-NADH was undergoing some change,

possibly caused by uptake of moisture from the air (from

opening the sample vial several times for sampling during

63
a 12-day period) . The vial had been stored in the dark in a

desiccator, but not redried between samplings.

Comparison of Tris with phosphate buffer (in columns IV

and V) indicates that, in the time used for the measurement,

the phosphate-buffered solution was not a source of decomposition of the NADH. In IV and V, the samples were weighed out

on the same day, but dissolved several days later for spectral

examination.

In method VI, the sample was taken from the vial and

dissolved in the buffer on the same day, and, in comparison

with IV and V, higher values at 340 nm and 260 nm were

obtained.

VII. The main vial of NADH from which the previous

samples had been taken was redried under high vacuum (over

phosphorus pentaoxide); the sample then taken had higher

spectral values at 340 nm and 260 nm than by method VI.

b 0.1 M Tris buffer, pH ^10.4.

^Day when sample was weighed.

Day when sample was dissolved for analysis.

eBased on disodium salt of 3-NADH»3H 2 0; molecular

-.weight, 763-64.

Calculated.

^Enzymic analysis.

Horse-liver alcohol dehydrogenase, 213 U/mg [35].

The chromatogram of the exposed 3-NADH (chromatography

in 2:1 (v/v) pyridine-^water) was examined under 366-nm

light, and showed a highly fluorescent, blue zone from the

vicinity corresponding to the R

f of the 3-NAD to that of

NADPH. It also showed a weak fluorescent impurity having

an R

f corresponding to that in untreated lot 678-9 . Treatment of the chromatographic sheet with alcoholic

potassium hydroxide showed a large increase in a spot

having the R

f of 3-NAD (3-NAD or a ternary nicotinamide

type of compound) . The use of potassium hydroxide as an

indicating spray was mentioned in table 15.

64
A 33-day old solution of moisture-exposed NADH was

further examined in other chromatographic systems, with

ultraviolet light and the alcoholic potassium hydroxide spray

ii. PEI-Cellulose

By use of 0.2 M Tris buffer (pH 9.0) 3 no resolution

occurred, except for 3-NAD.

iii. PEI-Bakerflex

With 0.2 M Tris buffer (pH 9-0) as developer, only

resolution of 3-NAD resulted.

iv. Cellulose, Eastman Chromagram

With 2:1 (v/v) pyridine^water as the developer, NADPH

Is not resolved, but exposed 3-NADH, as well as a fresh

solution of 3-NADH, was resolved into two additional

fluorescent spots ( 366-nm light).

v. DEAE-Cellulose (Bakerflex)

With 0.13 M aqueous ammonium hydrogen carbonate,

'traces of a fluorescent impurity (366-nm light) and of a quenched impurity (254-nm light) emerged from both the

exposed and fresh solutions of 3-NADH. There appeared

to be a substantial increase in 3-NAD in the moistureexposed material, indicating that this is not due alone

to the paper chromatography, which required 24 h. There

was no resolution of a major fluorescent spot such as has

been attributed to the inhibitor. A fresh solution of

lot 678-9 NADH that had been exposed for 19.5 h, examined

in this system, behaved essentially like the older sample.

However, the quenched spot (254 nm) was somewhat more

evident and may be related to ADPR.

The best resolution seemed to be obtained by use of 2:1

, (v/v) pyridine-^water on Whatman No. 1 filter paper or t. I.e.

ion cellulose.

65
b . Rate of Formation of Inhibitor as Determined

by Chromatography

3-NADH samples were exposed to moisture during 1, 2, 3,

4, and 5 h, and their interaction was quenched by dissolving

the material in 0.1 M Tris buffer (pH ^10). These solutions

were then compared with an untreated, fresh control.

With DEAE-cellulose (Bakerflex) and 0.13 M ammonium

hydrogen carbonate solution as the developer, no apparent

differences could be observed.

By t.l.c. on cellulose, or overnight paper chromatography,

with 2:1 (v/v) pyridine-water , no apparent differences were

observed.

c . Column Chromatography

With a column of DEAE-cellulose pretreated with 1 mM

ammonium hydrogen carbonate, 3-NADH exposed for 8 days to

moisture was applied as a solution in, and developed with, the

same solvent and then, with 10 mM ammonium hydrogen carbonate.

Only four well-separated, nonf luorescent fractions that did

not appear to absorb at 3^0 nm were eluted.

Chromatography of three of the separate eluted materials',

on PEI-cellulose with 0.2 M Tris buffer (pH 9), with subsequent

examination by uv light (254 and 366 nm) and alcoholic potassium hydroxide spray, indicated that one of the fractions might

consist of a,3-NAD, another might be nicotinamide; the third

was not identified. Prom their R_ values, these fractions were

not ADPR, NADP, or nicotinamide mononucleotide (NMN).

With the alcoholic potassium hydroxide spray NMN

fluoresces, further confirming the usefulness of the KOH spray

for compounds of the ternary nicotinamide type, and as a

replacement for the potassium cyanide spray that has been

used to detect such compounds.

66
H . Purification of Synthetic Dyes for Use as Spectrophotometric Standards

The Analytical Coordination Chemistry Section is

working to develop liquid filters for use in checking the

photometric scales of spectrophotometers. We are cooperating

by developing procedures for purifying organic dyes to their

specifications .

(A. J. Fatiadi)

Samples of the purified, synthetic dyes were needed

as the reference material for the spectrophotometric

standards. Three commercial dyes were checked, namely,

Neolan Black (Ciba), Cibalan Black (Ciba), and Alizarine

Light Gray (Allied)

T.l.c. studies on non-activiated silica gel plates

with 3:1 or 5:1 (v/v) isopropyl alcohol—acetone showed

a considerable proportion of trailing spots caused by

impurities. Solubility studies showed the presence of

a considerable proportion of neutral, inorganic components.

All three dyes were readily soluble in water, and moderately

to difficultly soluble in ethanol, acetone, or ethyl acetate.

It was difficult to develop a general procedure for

purification of all three dyes; hence, a specially designed

procedure was necessary for each individual dye

1. Neolan Black

Crude Neolan Black (1 g) was extracted in a Soxhlet

apparatus with ethyl acetate (200 ml) for 3 h, and the

residue was recrystallized from hot water (250 ml).

Concentration of the aqueous filtrate under vacuum was

difficult, because of foaming, even after addition of an

antifoaming agent such as octyl alcohol. Addition of

alcohol or acetone to the filtrate gave an amorphous

product containing inorganic salts. The filtrate was

concentrated to incipient crystallization at 85—90 °C

67
(water bath) under a stream of nitrogen, and left to

crystallize at room temperature. Washing with water, and

drying in a vacuum desiccator, gave dark-blue, lustrous

crystals (yield 50 percent) . The product was homogeneous

by t .I.e.

2. Cibalan Black

A sample of the crude dye (1 g) was extracted in a

Soxhlet apparatus with acetone (1 liter) for 15 h; the

extract was filtered, and the filtrate concentrated almost

to dryness, to give lustrous crystals ; yield 550 mg . The

product was recrystallized from acetone (with concentration)

and submitted for spectrophotometric testing; it was found

satisfactory as to absorption, and stability in aqueous

solution

,

3

.

Alizarine Light Gray 2BLWN

The Color Index [36] generic name and number of this

dye are Acid Black 48, C.I. 65005.

(S03 Na)x

The crude dye had the following analysis: Na, 8.8; S, 3-9$.

Initially, purification was attempted by extraction with hot

water and concentration of the filtrate at 80 °C under a

68
stream of nitrogen to incipient crystallization. However,

the lustrous product that crystallized out was difficult to

filter off, requiring 10 to 12 h for a 1-g batch., and analyses

for sulfur and sodium were not reproducible. The following

procedure was therefore devised.

Commercial dye (8 g) was suspended in N,N-dimethyIformamide (DMF) (70 ml) and stirred at 45 °C (water bath)

for 20 min. The suspension was filtered, and the solids

were washed with DMF (30 ml) to give a dark-blue solution.

The filtrate and washings were combined, cooled to 20 °C,

and treated, with stirring, with ethyl ether (25—30 ml);

the precipitate (dark-blue microcrystals ) was washed with

ether, and dried in a vacuum desiccator at 78 °C/0.1 torr;

yield 250 mg. The crystals had the following analysis.

Found: Na, 4.4; S, 5-3%.

The filtrate was diluted with another portion of

ether (60-80 ml), stirred at room temperature for 10 min,

and precipitate 2 was obtained; yield 1.6-2.0 g, dried at

78 °C and 0.1 torr. Found: Na, 3-9; S, 5.5*. The filtrate was

treated with more ether (60 ml), to give a third precipitate

(0.2 g)

(A. Cohen)

A second portion of the crude dye (24 g) was purified

by the procedure using DMF and ether. However, after the

first precipitate had been collected, the mother liquor,

I without further addition of ethyl ether, was stored in a

refrigerator, and a crop of crystalline dye deposited. Its

spectrum in the visible range was judged superior to that

of any other crop examined. The infrared spectrum of this

crop of dye lacked a band at 6 urn that was found in all

other crops; by elemental analysis, the preparation most

closely corresponds in composition to a hydrated dye; see

table 18.

6 9
Table 18. Elemental analysis of the modified preparation

of Alizarine Light Gray, dried at 20 °C and 1 torr

Percent calculated for C 2 sHi 6 N 3 Na0 7 S

C H N Na S

anhydrous 59,9 2.9 7.5 4.1 20.0 5-7

1 H 2 58.0 3.1 7.3 4.0 22.1 5.5

1.5 H 2 57.1 3.3 7.1 3-9 23.1 5.5

2 H 2 56.3 3.4 7.0 3.9 24.1 5.4

2.5 H 2 55.5 3.5 6.9 3.8 25.1 5.3

3 H 2 54.6 3.6 6.8 3.7

Found

26.0 5.2

55.8 3.7 6.4 3.9 5.0

2. CARBOHYDRATE SYNTHESIS, STRUCTURE, AND CHARACTERIZATION:

NOVEL RESEARCH MATERIALS AND MODEL COMPOUNDS

A . Enolization of Hexodiulose Acetals . Synthesis of

Derivatives of g-Psicose by Reduction of a

Hex-3-enulopyranose

(R. F. Brady, Jr.)

Previous papers from this laboratory have reported the

synthesis of isopropylidene acetals of D- ery thro-pentulose

[373, D-threo -pent ulose [37], g-fructose [38], and g-psicose

[39], and have demonstrated their utility in the synthesis

and purification of rare ketoses. This work describes an

attempt to synthesize similar acetals of g-tagatose

(g- lyxo -hexulose)

.

1,2:4 , 5-Di-O-isopropy lidene-$-g-ery_fchro-2 , 3-hexodiulo2,6-pyranose l s an intermediate previously used [39

]

3 was

70
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I ii

oo

i~

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71
treated with an acetic anhydride—triethylamine reagent [40

]

for 3 days at 50 °C, and gave 46$ of a new, crystalline

compound whose i.r. spectrum showed bands at 1770 and

1746 cm" 1

, indicative of a 0-C=C-0-C=0 system [40], and

whose elemental analysis was consistent with the formula

CiitH 2 o07. The n.m.r. spectrum showed the presence of two

isopropylidene acetal groups and one acetyl group; two

1-proton doublets for the protons on C-l, and a well-defined,

eleven-line, AMX pattern representing the protons on C-5

and C-6 were also readily identifiable. These data, and

the fact that diulose 1 has a proton on C-4 but not on C-2,

established that the product is 3-0-acety 1-1,2 : 4 ,5-

di-0-isopropylidene-g-g- glycero-hex-3-enulopyranose (2)

.

Attempts to prepare the enediol acetate 2 by treating

diulose 1 with acetic anhydride and pyridine (a) at room

temperature for 16 h, (b) under prolonged heating at 80 or

125 °C, or (c_) under reflux for 1 h, were unsuccessful.

Treatment of diulose 1 in carbon tetrachloride solution

with an acetic anhydride—perchloric acid reagent [4l] did

not give the desired product 2 . Treatment of 1 in

tetrahydrofuran solution with an acetic anhydride—sulfuric - acid reagent [42], or slow distillation of the solvent from

a solution of diulose 1 in acetic anhydride containing

p-toluenesulfonic acid [43], also failed to produce enediol

acetate 2. Earlier attempts, using acidic conditions, to

prepare an enediol acetate from a precursor ketone having

acid-labile blocking groups were also unsuccessful [40 ]

.

On reduction of 2, cis -addition of hydrogen at C-3

and at C-4 might result in formation of the corresponding

derivative of g-tagatose or g-psicose, or both. Inspection

of a molecular model of compound 2 revealed that the

molecule could assume either an h£ conformation or that

sofa conformation [44] having the oxygen atom of the

pyranoid ring exoplanar to the plane of the carbon atoms

72
of this ring. Reduction of 2 at either side of either

conformation appeared possible.

One possible reduction product, namely, 3-0-acetyll,2:4,5-di-0-isopropylidene-e-g-psicopyranose (3), would be

formed by cis-addition of hydrogen at that side of the plane,

defined by C-2-C-3-C-4-C-5 , on which 0-2 is located. By

analogy with the conformation [45] of 1 ,2 : 4 ,5-di-0-

isopropylidene-6-g-fructopyranose . compound 3 may be expected

to assume a conformation, intermediate between 1C(D) and

H*(g), in which that part of the molecule containing C-4,

p-5, and C-6 is slightly flattened from the ideal chair

conformation because of the presence of the 1,3-dioxolane

ring involving C-4 and C-5 . The unacetylated compound 4

has been prepared and found to be stable [39].

The other possible reduction product, namely,

3-0-acetyl-l,2 : 4 ,5-di-0-isopropy lidene-3-g-tagatopyranose (5)

,

would be formed by cis -addition of hydrogen to that side

of the plane on which C-l is located, Diacetal 5 would

contain an isopropylidene group trans -fused to the equatorially

attached hydroxy 1 groups on C-4 and C-5, Although several

hexulopyranose derivatives containing a trans -fused

isopropylidene group have been reported [46], isopropylidene

groups trans -fused to six-membered rings generally cause

steric strain [47] in these rings, and are thus disfavored.

Neither diacetal 5 nor the unacetylated compound 6 has yet

been reported. In view of the steric strain in compound

5, similar strain in the transition state in the reduction

of 2 to 5 could also be anticipated.

Experimentally, it was found that reduction of 2 is

istereospecific , and that only diacetal 3 is formed.

^Hydrogenation of compound 2 at atmospheric pressure in the

jpresence of 5% palladium-on-carbon catalyst gave a syrup that

was shown to be 3-0-acetyl-l ,2 : 4 ,5-di-0-isopropylidene-$-

g-psicopyranose 3). Acetate 3 was deacetylated with methanolic

barium methoxide [48] to afford crystalline 1, 2 : 4 ,5-di-0-

73
isopropylidene-B-g-psicopyranose (4). Diacetal 4 was

converted into a furanose derivative by treatment with

acetone containing 0.5% of sulfuric acid to effect

rearrangement, giving a chromatographically homogeneous,

levorotatory , crystalline compound, identified as

1,2 : 3,4-di-O-isopropylidene-g-psicofuranose (7). The

corresponding derivative of g-tagatofuranose (8) is a

strongly dextrorotatory, crystalline solid [49].

Reduction of the enediol acetate 2 with sodium

borohydride in ethanolic solution is complete after 18 h,

and affords diacetal 4 in high yield. This reaction

evidently proceeds by reductive deacetylation of 2, with

intermediate formation of the enolate anion; this ketonizes, : and is protonated by the alcoholic solvent, forming diulose

1, which is subsequently reduced to diacetal 4. Compound 4

has also been obtained [39 J by direct reduction of diulose

1 with borohydride. Although the reduction of 2 to 4 with

sodium borohydride is much slower than similar borohydride

reduction of an enediol acetate prepared by Horton and Just

[40], the product obtained in both cases is the same as that

obtained when the diulose is reduced by borohydride directly.

This suggests that the reduction proceeds through the

diulose in each case.

As a synthetic route to isopropylidene acetals of

g-psicose, the reduction of compound 2 is not preferred,

because direct reduction of diulose 1 with borohydride

gives diacetal 4 in 99$ yield [39]. However, this synthesis

of derivatives of g-psicose may prove valuable for the

synthesis of acetals made with other aldehydes and ketones,

in which the reduction of the diulose analogous to 1 would

not be stereospecific because of bulkier alkylidene acetal

groups

.

74
B . Application of Homonuclear, Internuclear , Doubleresonance Techniques to Carbohydrates. Detection

of Small Coupling-constants

(B. Coxon)

1. Introduction

In previous reports [19, 20, 50 ], iterative analyses

of the proton magnetic resonance spectra of a variety of

derivatives of carbohydrates have been described. Such

analyses are necessary for full characterization of these

derivatives by n.m.r. spectroscopy, and also for the

measurement of accurate coupling-constants and chemical

shifts to be used in the assignment of conformations and

conformational equilibria.

An important step in the iterative analysis of an

experimental spectrum is the assignment of peaks in this

spectrum to the theoretical transitions generated by the

computer program for the analysis . From the point of view

of the validity of the analysis, it is preferable if

additional experimental evidence for these assignments is

obtained from some type of multiple-resonance experiment.

2

Results and Discussion

In the present work, homonuclear, internuclear , doubleresonance (indor) techniques [51] have been employed to

confirm the spectral assignments for 6-deoxy-l,2 : 3,5-di-Oisopropylidene-a-D-glucofuranose-di 2 (l-di 2 ) • The iterative

analysis of this compound by magnetic-equivalence factoring

has been described in a previous report [20]. The essential

experimental requirement for an indor experiment in the

frequency-sweep mode is that the measuring frequency (fi) be

held constant at the peak of a resonance while the doubleresonance frequency (£2) is swept through the frequencies

of connected transitions. For example, in figure 6i is

shown the complete, high-resolution, p.m.r. spectrum of

75
1-di 2 i and in fig. 6a—6h are given the indor spectra obtained

by monitoring the low-field peak of H-l with f i while tz was

swept through the resonance frequencies of H-2—H-5 3 either

with f_2 decreasing (fig. 6a—6d) or increasing (fig. 6e—6h) . Under favorable conditions, the positive and negative

responses [52] in the indor spectra (for example, those at

the resonance frequencies of H-2) indicate the progressive

or regressive connectivity of the transitions that can be

correlated with the lists of progressively and regressively

connected transitions provided by the computer program.

The positive responses observed at the resonance

frequencies of H-3 (see fig. 6a—6h) indicate the presence

of a small, long-range coupling between H-l and H-3, and,

in fact, a more detailed study of the spectrum of fig. 6i

has revealed parameters additional to those described in

our previous report [20], namely, Ji 3 0.45, J 2 3 0.4l, and

J 2 ^ 0.42 Hz.

In this work, a detailed investigation of the influence

on homonuclear, indor spectra of the sweep direction and

sweep rate of f ^ , and of the power levels of fi and f 2 , has

been made. Under certain conditions of sweep rate and power

levels, transient nutations [53] of the net, macroscopic,

magnetization vector may be observed (see fig. 7). The

initial direction of the oscillation also indicates progressiv<

or regressive connectivity, and the rate of decay of the

oscillation gives [53] a measure of relaxation times, since

the time constant of the decay is equal to the harmonic mean

of these times, i.e. , (2TiT 2 )/(Ti + T 2 ).

In other experiments (see fig. 8), the crossover from

general Overhauser effects to spin-tickling effects in

indor spectra was demonstrated. This crossover occurs

because the mechanism by which indor spectra are generated

depends on the power level of f 2 . Very low power-levels

(see fig. 8c and 8d) cause only transfers of spin population

76

1
5.46 5.83 5.92 6.39 \—

Figure 6. Proton—proton, internuclear , double resonance

experiments on 1-d 1 2 with fj. decreasing

(spectra a-d) or increasing (e—h) at various

rates (fi constant at peak Al of H-l, except

for single-resonance spectrum i).

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K^/

E3

EIE2 E4E5

Figure 8. Proton—proton indor experiments on 1-d 1 2 at 90

MHz with peak Fl (H 3 C-6) monitored bya constant

f i

, and H-2—H-5 perturbed by a decreasing f 2 applied at various attenuations: (a) 28, T"b) 32,

(c) 36, (d) 40 dB (down from 0.5 watt), (e) fi

swept and f 2 off.

79
between energy levels that result in positive or negative

increments to the intensity of the monitored line (general

Overhauser effects), whereas higher power-levels (as for

fig. 8a and 8b) result in swamping of these effects by

spin-tickling effects (negative responses only) due to

mixing of spin states

.

The structures of compound 1-d

i

2 and its non-deuterated

precursor 1 have been confirmed by mass spectroscopy. A

detailed description of the fragmentation pathways, and of

their confirmation by means of the isotopic substitution,

together with a review of multiple-resonance techniques and

other indor experiments , has been given in a full report

which has been accepted for publication [54].

C . Spectroscopic Studies of Derivatives of 6-Amino-6-

deoxy -g- glucose -6- * 5 N

(B. Coxon)

1 . Introduction

The synthesis of derivatives of 6-amino-6-deoxyD-glucose-6- 1

5

N in high yield by reaction of the 6-0-ptolylsulfonyl or 6-deoxy-6-iodo derivative of l,2:3 3 5-di0-isopropylidene-2-glucofuranose with potassium phthalimide- 1 °N

has been outlined in previous reports [ 19, 55], as has also

the iterative analysis of the p.m.r. spectra (measured at

220 MHz) of the 6-deoxy-6-phthalimido derivatives produced

in these reactions [20]. This program for the synthesis and

characterization of isotopically labeled, carbohydrate

reference-materials of biomedical interest has been continued

along the pathways of infrared spectroscopy, mass spectrometry,

and heteronuclear , magnetic resonance studies.

2

.

Infrared Spectroscopy

A brief investigation was made of the isotope effect

of the nitrogen-15 isotope on the C—N stretching frequency

of 6-deoxy-l,2 : 3 ,5-di-0-isopropylidene-6-phthalimido-a-D80

1 5-
glucofuranose (1). This compound shows a strong absorption

band at 1400 cm" (mean of five determinations), whereas its

15 N-labeled analog (1- 15 N) shows it at 1388 cm" 1

. The

isotopic shift of 12 cm is considerably smaller than the

value (47 cm ) calculated from the relationship

v(C- 15 N) = v(C- 11+ N)/(lV15) , which is based on Hooke's law

and which depends on the assumption that the force constant

for each type of carbon—nitrogen oond is the same [56]-

3 . Mass Spectrometry

Previous studies of the mass spectra of unlabeled

amino sugars have been concentrated on their N-acetyl, and

N-acetyl-d.3 derivatives. For the present work, the

availability both of l ^N compounds and 15 N-labeled derivatives

permitted confirmation of pathways of fragmentation by a method alternative to that of deuteration. Thus, molecular

fragments that retained the nitrogen-15 atom could be

expected to appear at mass units (m/e_) one unit greater than

those containing the nitrogen-l4 atom. This approach was

applied to the N,N-phthaloyl derivatives 1 and 1- 15 N and to

the amines 2 and 2- 15 N derived from them by reaction with

hydrazine. The mass spectra of these amines indicate at

least four different routes of fragmentation, the most

characteristic of which involves scission of the C-5—C-6

bond to give the stable CH 2 =NH 2 ion. Other series of ions

were produced from both the amines and their N,N-phthaloyl

derivatives by loss of methyl radicals, ketene, acetone,

acetic acid, 2-methyl-m-dioxolenium ion, or various

combinations of these moieties . The phthalimido group of

these derivatives displays its own characteristic fragmentation

patterns, including the formation of benzoyl and cyanobenzoyl

ions, a dehydro-benzoyl radical cation, a protonated phthalimidomethylene radical ion formed by a McLafferty rearrangement,

and a radical io:

benzyne ion [57]

and a radical ion C 6 Hit

' that has been interpreted as a

81
4 . Carbon-13 Magnetic Resonance Studies by Continuouswave and Fourier- trans form Methods

Although our previous studies [19,55] of the proton

spectra of 6-amino-6-deoxy-g-glucose-6_- 1

5

N derivatives at

100 MHz had yielded a selection of 1 H—15 N coupling-constants

that could be expected to help define the applicability

of 15 N magnetic resonance parameters to the structural and

conformational analysis of amino sugars, the measurement of

the 13 C—15 N coupling-constants of these molecules requires

direct observation of either 13 C or 15 N spectra. At the

present time, greater progress has been made with 13 C n.m.r.

than , with 15 N n.m.r., and so compound 1 and its 15 N-labeled

analog 1- 15 N have been studied by both continuous-wave (CW)

and Fourier-transform (FT) 13 C techniques ' at 25.2 MHz in order

to assist our evaluation of Fourier-transform methods.*

13 C spectra of solutions of 1 and 1- 15 N in benzene were

accumulated by CW methods** in 1,024 channels of a

signal-averaging computer, whereas spectra of solutions of

these compounds in chloroform-d were obtained by FT methods***

in which 4,096 signal-averaged points in the time-domain

spectrum were transformed into 2,048 points in the frequency

domain. In each method, noise-modulated decoupling of protons

was employed to simplify the spectra and provide an enhancement of signal intensity by means of the nuclear Overhauser

effect.

By using the FT technique, complete 13 C spectra of 1 and

1- 15 N (see fig. 9) were obtained in one experiment with ^800

seconds of pulsing, whereas the CW technique required the

*By courtesy of L. F. Johnson of Varian Associates.

**Varian HA-100 spectrometer and C-1024 time-averaging

computer.

***Varian XL-100-15 spectrometer and 620i computer.

82
C=0 AROMATIC C O-C-0 C-2-C-5 C-6 4 Me Me.Si

( 1 I

'

1 4

(•)

V^j^^^K^V^W^jVA^^V [^L^yJ

c-i;r C-1 CDCI,

~\ WVW"^ u

Me TMn °

Ma

V V^^vw^A/vvv^rwi

(b)

I I I I I I L vM.^*/^ ^J^Wa«viJv,

J I I L J 1 I I I

' ' 200 ISO 6C (wj.)

100 50

Figure 9 Fourier-transform, 13 C n.m.r. spectra of

solutions in chloroform-d, at 25.2 MHz with

proton decoupling: (a) F-deoxy-1, 2 : 3, 5-di-Oisopropylidene-6-phthalimido-a-D-glucofuranose

(1), (b) 6-deoxy-l,2: 3>5-di-0-isopropylidene-6-

pnthalimido-a-D-glucofuranose-6- N (l- 1N)

83
division of the 13

C spectral region into four zones of

50 p. p.m. each, followed by separate accumulations of 16—289

scans of each zone. For the same accumulation time

(^800 sec) and approximately the same weight of compound

(^0.4 g) , the FT technique afforded a signal:noise ratio ovei!

six times that obtained by the CW method, corresponding to a

time-saving factor of about 40 . However, correction of

this factor on the basis of division of the CW spectrum into

four zones gives a total time-saving factor of 160

.

From fig. 9, it may be seen that three of the carbon-13

resonances appear as singlets for compound 1, but doublets

for 1- 15 N, indicating the coupling constants 1 Ji3p_r i5 N 9.9,

1Jl3 C=0 , 15 N

12 ' 9 and 2 ^13 C-lV 5 N

=

2 -13 C-6V 5 N 11,4

Hz, where C-l"* and C-6' refer to the aromatic ring.

For the N ,N-phthaloyl derivative 1- 15 N and its N-acetyl

and N-(trif luoroacetyl) analogs, the ^-^N and 13 C- 15 N

coupling-constants measured in these studies have been used

to assess the degree of s-character in the bonds between thes

nuclei by means of the empirical relationships developed by

Roberts and co-workers [59]. The results indicated that

the polarized, canonical forms of the amide structures of

these compounds are generally more important than the

nonpolarized forms; that is, the 15 N atoms of these amides

are closer to sp

2 than to sp3 hybridization.

5 . Heteronuclear, Internuclear , Double- and Tripleresonance Experiments

During the course of the synthetic studies, crystalline

N-acetyl and N-trifluoroacetyl derivatives (3- 15 N and 4- 15 N,

respectively) of the amine (2- 15 N) were prepared, in addition

to the corresponding l 4 N analogs (3 and 4). The N-(trifluoroacetyl) derivatives are of special importance, because of

(a) current interest in the use of trif luoroacetyl derivatives

of sugars instead of acetyl or trimethylsilyl derivatives

for gas—liquid chromatography, and (b) the fundamental

84
importance of measurements of the magnetic interactions

between 15 N and 19 F.

The p.m.r. spectrum of 4 in chloroform-d at 90 MHz

showed the NH signal as a broad hump having no obvious

fine structure, whereas 4- 15 N showed two, complex, submultiplets separated by Ji5 N_H 92.5 Hz. A detailed analysis

of this complexity showed that it was due to a combination

of long-range coupling (^0.7 Hz) between the fluorine nuclei

and the NH , second-order effects produced by strong coupling

of H-5, H-6, and H-6', and, for compound 4, broadening of

the NH resonance by the lk N quadrupole

The 19 P n.m.r. spectra of compounds 4, 4-d, 4- 15 N, and

4- 15 N-d at 84.7 MHz all showed for the trif luoromethyl group

quite narrow resonances that indicated J„ i 5 „ <0.6 Hz. This —r , IN coupling is unexpectedly small, but resembles the values

3 J„ „ <1 Hz of pentafluoroethyl derivatives for which it has

been supposed [60] that the "through bond" contribution to

the coupling is attenuated by the (electronegative)

fluorine atoms

The 'long-range coupling between the fluorine nuclei

and NH was confirmed by (a) irradiation of the fluorine

nuclei at a fixed frequency (^84.7 MHz), whereupon the NH

multiplets of 4- 15 N sharpened up, and (b) indor experiments

in which f 2 was swept through the fluorine region while one

of the stronger lines of the NH multiplet was monitored

by f i

. In order to assist the analysis of these complex

spectra, other indor experiments were conducted in which

f_2 was swept through the 15 N resonance frequency at %9-12

MHz; and also, internuclear triple-resonance (intripler)

experiments in which f 3 was swept at ^9.12 MHz while a fixed f 2 was applied at ^84.7 MHz in order to decouple

the fluorine nuclei from the NH proton. For most of the

power levels of fi and f_2 or f_3 investigated, positive

responses of the NH peak were observed (see fig. 10b) as

85
(a)

10 H3

(b)

\aA|/\A^vV

(c)

9.120390 9.120659 f,(MHz)

Figure 10. 1 5 N-related n.m.r. spectra of 6-deoxy-l,2: 3, 5-

di-0-isopropylidene-6-(trifluoroacetamido)-aD-glucofuranose-^1 5 N (4- 15 N) in chloroform-d:

Xa) computed, theoretical, 15 N n.m.r. spectrum;

(b) x 5 N n.m.r. spectrum obtained indirectly

from heteronuclear , intripler experiment in

which a

1 9 F decoupled NH proton peak was

monitored by f i , at ^90 MHz, while the * 5 N

frequency (f 3 T was swept; (c_) the same, with

fi at lower power.

86
f 2 or f 3 was swept through the resonance frequencies of each

group of 15 N transitions. The advantage of the intripler

experiment was that positive responses having 12 percent

greater amplitude (on average) were obtained in the presence

of 19 F decoupling than in its absence, due, apparently, to

the fact that the decoupling gave a sharper NH peak to be

monitored by f i . It may be seen that the intripler spectrum

shown in fig. 10b is a replica of the theoretical, 15 N

spectrum computed for 4- 15 N (see fig. 10a). These results

are- unusual, and are in agreement neither with the negative

and positive responses expected from considerations of connectivity as the 15 N frequency was swept from low to high

values, nor with the negative peaks expected from

spin-tickling effects. It seems that, for most of the

conditions employed, general Overhauser and spin-tickling

effects are dominated by a positive, intramolecular, nuclear

;0verhauser effect due to magnetic dipole—dipole interaction

between the 15 N and proton nuclei. Full numerical data and

details of the experimental methods are given in a paper

that has been accepted for publication [61].

D. Cyclic Polyhydroxy Compounds

(A. J. Fatiadi)

1. (p -Bromophenyl) osotriazoles from Inositols

In continuation of studies on cyclitols, the products

resulting from treatment of three inosose phenylosazones

with aqueous bromine have now been examined. As expected,

the products were (p-bromophenyl) osotriazoles (1, 2, and 3),

albeit they were isolated in only 28—42 percent yield.

87
—Br Br—</ \> —N N

N=<

— Br

L

2

In addition to preparation of their acetates or benzoates,

the phenylosotriazoles were oxidized with periodate a to

afford new dialdehydes . A note on this work has been

accepted for publication [62],

88 .
3. GENERAL ORGANIC CHEMISTRY

(A. J. Patiadi)

A. Oxidative Cleavage of Aromatic Azlnes with Periodic

Acid E.S.R. Evidence for the Liberation of

Nitrogen Dioxide

As part of a continuing interest in non-Malapradian

reactions of periodic acid, a number of aromatic and

heterocyclic azines (la-le) have been treated with periodic

acid at room temperature. Brisk evolution of nitrogen was

observed in all cases, indicating the lability of the

hydrazino moiety ( C=N-N=C^) of the azines towards this

reagent. Extraction of the reaction mixture gave the

parent aldehyde (or ketone) in over 95 percent yield,

indicating that the oxidative cleavage was complete.

R —

R'

I C = N

R'

I N = C-R

1 a, R= -^\ , R'=H

MeC!

b,R= -£}, R'=H

,R= -IsqJ , R'=H

d, R = , R'=H

e, R= -^3 • R

' =Me

89
In a typical experiment, a solution of benzalazine

(l,4-diphenyl-2,3-diazabuta-l,3-diene) (la) (1.04 g 3 5

mmol) in glacial acetic acid or N ,N- dimethy Iformamide

(15 ml) was stirred with an aqueous solution of paraperiodic acid (2.3 g, 3 ml, 10 mmol) at room temperature

for 15 min. An exothermic reaction ensued, and brisk

evolution of nitrogen commenced immediately after mixing.

The mixture was treated with a few drops of 10 percent

aqueous sodium hydrogen sulfite to remove iodine (and

any excess of the oxidant), acidified with hydrochloric

acid if necessary, and extracted with ethyl ether (4 X 10 ml),

to give benzaldehyde in 96 percent yield.

Similarly, by the use of a 1:2 molar ratio of substrate

to oxidant, azines lb-le were converted into the parent

aldehydes or ketones in over 95 percent yield.

The formation of nitrogen may arise through collapse

of an association complex initially formed between periodate

dianion and a resonance structure of the azine la. The

structure of the complex and the pathway of formation of

nitrogen are shown in fig. 11.

Examination of the association complex (fig. 11) showed

that the azine had already suffered a two-electron oxidation

(formation of two bonds), and the question was whether to

depict a transition complex with iodine(VII) or with iodine(V)

Our statement that the formation of a complex (fig. 11) is a

symmetry-allowed reaction, as predicted from MO symmetry ruleswas based on the assumption that iodate ion I(V) should be

depicted. However, the e.s.r. evidence regarding the action

of both oxidants (H 5 I06 and HI0 3 ) on azines, particularly

a low-field peak at 1.864 kG due to a binuclear, tripletstate species that is observed only for periodic acid, made

it difficult to conceive the existence of a dimer species

for the iodic acid (or its ion) in solution. Consequently,

the formation of such a complex may involve, as a single step , the addition of 10^- (or its hydrate) across the conjugated

90
N == N

HO —I—OH

H

C C6 Hn 5

H

H 5C N = N

H

CcH6 n 5

Figure 11. Proposed structure of the association complex

between periodate dianion and a resonance

structure of azine la.

91
azine system, to give an iodate I(V) complex; this view is

in agreement with that of another worker [63].

The molecular-orbital overlap in the transition state,

particularly in the formation of the complex (fig. 11) can

be treated either by symmetry or topology. The symmetry

rules of Woodward and Hoffmann [64] require knowledge of

the molecular orbitals of the reagents or products; the

alternative treatment by Zimmerman [65], using a topological

approach to the orbital levels of the transition state, gives

rise to the same predictions as the Woodward—Hoffmann

treatment, but it does not require a correlation of the

molecular orbitals of the reactants with those of the

products

.

The Zimmerman method [65] requires the classification

of the transition state as either "Hiickel-like , " in which

all overlapping pairs of reacting orbitals are bonded, or

"MSbius-like, " in which one, or an odd number of, anti-state

bonding overlap(s) must occur. In a Hiickel transition state,

4n + 2 electrons form a closed shell (giving it stability),

whereas 4n electrons are required for stable, closed shells

in a Mobius state. It will be noted that 4n electrons are

involved in the azine oxidation with periodic acid (fig. 11)

Thus, an "allowed" reaction may have either a Hiickel or Mobius

type of transition state, provided that the correct number

of electrons is available to stabilize that transition state

[66].

One possible way for an "allowed" reaction that should

include an allowed decomposition of iodine(V) in the iodate

I(V) analog of the complex (fig. 11) is depicted in scheme 1.

The immediate difficulty, however, of imagining an addition

step involving iodate ion I(V) to benzalazine to give the

complex (fig. 11) can be overcome by the possible pathway

shown in scheme 2. A communication on this topic has been

published. [67].

92
CO

<T

uizo

en< o oo ^ cc

i- o X UJ

h- 0- 5 £ + <M OH

io_o.

CM OH

CO< + CM UJ OH

CO<

UJ

CCh-O3

or

h-co

UJ<

COUJI Q

oroooI

UJ

CDUJ

ccou.

UJccUJ

LU

LUXo

en

mX

cj° uCC

UJ CO > 3 < 2 xo

UJ

r)CO

CO<

CO <

UI

cc

\>\

—

H + CM UJ o x H h93
M

//c\\

^ N

I^ : N

wH R n = 2, MOBIUS-TYPE OVERLAP

<F

p- REACTING ORBITAL

"NONREACTING ORBITAL"

ON 3- FOLD AXIS OF IODATE

TWO sp3

d LONE PAIRS

ON EQUATOR OF TRIGONAL

BIPYRAMID (AS BrF3

)

FOLLOWED BY REACTION WITH I (VII) TO REGENERATE I (V)

AS REQUIRED FOR SCHEME I SCHEME 2

94
B . Evidence for Adsorption as the First Step in the

Solid-state Oxidation of Benzenehexol with Active

Manganese Dioxide

Evidence has been acquired of an unusual adsorption

on the surface of manganese dioxide, as the first step in

the solid-state oxidation of benzenehexol (1) to give 4

(see scheme 3); a possible pathway in the conversion of 1

into 4 is also discussed and a structure for the solid

oxidant is suggested.

1. Possible Structure of the Precipitated Manganese

Dioxide

The adsorptive ability of manganese dioxide has been

thoroughly studied; for example, its adsorption of water or

benzene vapors, hydrogen, organic substrates from solution,

and inorganic cations (Ba 2+

, Mn 2+, or Na+

); an example of

adsorption of an organic solid on a solid is now described.

The mechanism of oxidations with manganese dioxide

undoubtedly involves a process typical of heterogeneous

reactions, and the difficulties encountered in the study of

these reactions may involve the structure of the oxidant,

particularly the relationship of its surface-active sites

toward a substrate (quadrupole—dipole electrostatic

interaction [68]). The amorphous character of the precipitated

manganese dioxide is shown in the X-ray diffraction spectra

A-l and B-l (fig. 12), and this character is in accordance

with earlier observations [69,70]. To our surprise, however,

the oxidant prepared in deuterium oxide instead of water

MnS0<» + KMnCU + D 2 (acidic, 80 °C) -» Mn0 2 was a dark, crystalline product having the pattern shown in

C-l and D-l (fig. 12). The structure of this compound

resembled that of y-Mn02, as shown by comparison of their

interplanar spacings and intensities. In order to shed more

light on its structure, a sample of active manganese dioxide

(300 mg) was decomposed by stirring it with aqueous

95
==o

/\> o o o ro / \ o o

-<

°K >

=o o

-<o I I I X oy o

-i i r \ X

°AJt

-o o o I X

==o

<0

xo

:=0

CO Oc

0>

00

:=o o h*

Xo

(M Xo

CO

96
A-

%ulv**^^^^ '"wv'vA*r^^

Figure 12. X-Ray powder diffraction pattern of manganese

dioxides (CuKa radiation): A-l, precipitated

manganese dioxide dried in vacuo at room

temperature; B-l, sample A-l dried in an oven

at 125 °C; C-l, manganese dioxide prepared in

deuterium oxide as described in the text (dried

in vacuo at 100 °C); D-l, sample C-l exposed to

air for 3 months.

97
hydrochloric acid (3 M, 20 ml) at room temperature for 2k h

In a hood (evolution of chlorine); the dark residue

(50—60 mg) that resisted decomposition was crystalline.

The isolated oxide contained manganese of the lower valency,

and its interplanar spacings resembled [71] that of Mn0i #8 8»

Moreover, its infrared spectrum showed little or no adsorption

in the region for hydroxyl bands, in comparison to that of

the initial manganese dioxide prepared in water (OH band

at 3380 cm" water of hydration at 1625 cm" ) or the

manganese dioxide prepared in deuterium oxide (OD band at

3350 cm" , deuterium oxide of "hydration" at 1535 cm" )

.

A crystalline oxide containing manganese of lower valency

was also obtained on partial decomposition of the active

manganese dioxide with aqueous hydrogen peroxide at room

temperature

.

The e.s.r. measurements of the oxidant (A, fig. 13)

and of the complex (D, fig. 13) showed, for each, a broad,

one-line peak which can be explained as being due to a

dipole—dipole interaction between polycrystalline manganese

dioxide aggregates. Alternatively, the line broadening

observed may be due to interaction of an unpaired electron

with a proton of a neighboring water molecule, as has been

reported [72] for certain coordination complexes (in this

case, precipitated manganese dioxide). The situation is

comparable to the change in structure of the oxidant from

amorphous to crystalline when hydrogen is replaced by

deuterium (see fig. 12), and lessening in the line width

for the deuterated oxidant is expected. On the basis of

this evidence and the e.s.r. spectrum of the oxidant,

precipitated manganese dioxide may contain a locked, waterassociated chain as depicted; this structure provides

important active sites of low electron-density on the surface

of the solid oxidant [68] and potentially labile hydroxyl

groups

.

98
!

MiVH*w**)^^^

III j; H jj NWr

Figure 13. X-Ray powder diffraction pattern (Cr-Ka radiation)

of aliquots, on mixing of 1:6 (w/w) benzenehexol

(1) and manganese dioxide. (The 1:6 ratio is

for substrate to oxidant.) A, precipitated

(amorphous) manganese dioxide; B, 30 sec,

structure shown is primarily due to 1; C, 50 min,

^45$ of 1 adsorbed; D, 100 min, ^97%~of 1 adsorbed,

complex D; and E, manganese croconate trihydrate,

purified product extracted from D.

99
0—H —Ck 0— H--0

%. .. y \

^

0—H— 0— H — ^

t

2. Adsorption of Benzenehexol (1) by Manganese Dioxide

A 1:6 (w/w) mixture of finely ground 1 [73] and

manganese dioxide was mechanically shaken at room temperature,

and the rate of disappearance of 1 was monitored by use of

an X-ray powder diffractometer . Fig. 13 shows the background

spectrum A (amorphous manganese dioxide); the decrease in

the concentration of 1 with time is seen in spectrum B

(30 sec; the structure shown is primarily due to 1) and in

spectrum C (50 min; ^45% of 1 adsorbed); in spectrum D,

at 100 min, ^91% of 1 is adsorbed. The high affinity of

manganese dioxide for 1, which seems to be an example of an

irreversible adsorption (none of 1 could be extracted from

the complex), is typical of a chemisorption [7*0; also, the

adsorption can partially be explained as being due to the

formation of a ir-complex between 1 and the oxidant [75].

The formation of an ionic bond (-0-Mn-0-) is, however, evident

from the isolation of 4.

The amorphous pattern shown in spectrum D (fig. 13) was

almost identical with that of manganese dioxide (spectrum A);

however, an electron-diffraction probe of the complex at

this stage showed indeed, a crystalline patt-ern, thus proving

that adsorption of 1 by the oxidant is exclusively a surfaceinteraction process

.

Extraction of this complex with water, and gradual

concentration of the extract, yielded, first, a small

quantity of white crystals, identified as manganese oxalate

100
by X^-ray diffraction, and then greenish crystals of manganese

croconate (4) (E, fig. 13).

The measurements of magnetic susceptibility of a 1:6

(w/w) mechanical mixture of 1 and manganese dioxide at room

temperature showed little appreciable change (from 2254 to

2262 x 10" cmVmol) after 5, 30, 60, 100, and 1,000 min;

the final value observed is comparable to that reported for

precipitated manganese dioxide (2280 x 10~ cm 3 /mol)

3 . Reaction Mechanisms Involved in Oxidation of

Benzenehexol (1) to Croconic acid (4) by

Manganese Dioxide

Possible chemical transformations of 1 into 4 are

shown in scheme 3; this conversion may involve the

manganese ester intermediate 5 3 an oxidation process to

give intermediates 6 and 7, and benzilic acid-type rearrangements of intermediates 8 and 9 to give, via carbonium ion

10, product 4. Whereas conversion of 7 into 4 requires

participation of a strong nucleophile (hydroxyl ions) (see

also, the correlation of increase in yield of 4 in alkaline

media) , the steps from 1 to 7 and the presence of manganese

oxalate in the product (probably arising from carbon—carbon

cleavage of 1) can only be reconciled by invoking radical

participation.

Refluxing (60 min) of a mixture of 1 (1 g) , manganese

dioxide [76] (12 g) , and dry benzene (200 ml, Dean-Stark

trap) gave 0.25-0.40 ml of water. Crude 4 (250 mg) was

obtained by extraction of the reaction mixture with water.

After the reaction, analysis of the benzene (by g.l.c.)

revealed the presence of a trace of phenol, which could have

been formed by collision of hydroxyl radicals (derived from

manganese dioxide) with the solvent; also, the trapped water

indicates the participation of hydroxyl radicals [77].

Assuming that our hypothesis is correct, consumption

of one equivalent of active oxygen present in manganese

101
dioxide corresponds to two hydroxyl radicals, as shown for

a certain hydrated form of it; and this pathway would provide

MnO(OH) 2

-> MnO + 2«0H > 2 0* + H 2 + Mn 2+

+ 2 e species that could either (a) abstract hydrogen atoms or (b_)

donate hydroxyl radicals . This mechanistic approach is

analogous to those proposed for the oxidation of alcohols

by nickel peroxide [78] 3 with the difference that any paramagnetic species formed on the surface of manganese dioxide

is probably consumed immediately , as, otherwise, any species

having free spins would have been detected by the balance

used in magnetic-susceptibility measurements . In this

alternative aspect, trapping of free radicals in solution

may furnish additional evidence to clarify the mechanism

of the oxidation.

Thus, conversion of 1 into 4 by manganese dioxide

probably proceeds by a concerted mechanism involving ionic

and free-radical pathways. This work has been published [79].

C . Stable Radical-anions Derived from Glyoxal

Bis (phenylhydrazones

)

In the series of stable hydrazyl radicals, a neutral

radical, namely, l,l-diphenyl-2-picrylhydrazyl (DPPH) , and

its analogs are the best known and the most thoroughly

studied [80,8lJ; recently, e.s.r. studies established the

existence of stable, hydrazinium radical-cations in colored

solutions of tetraphenylhydrazine dissolved in strong acids

[82]. The preparation and some of the properties of some

new, stable, hydrazyl radical-anions are now described.

Treatment of a solution of glyoxal bis (phenylhydrazone)

(la) in methyl sulfoxide (Me 2 S0) (10~ 2

to 10" 3 M, x Me2S0

~ ~ — max

382 nm, e „ ^49) with a strong base (5% aqueous potassium

hydroxide or potassium tert -butoxide in Me 2 S0) and a trace

of oxygen produced a deep violet-purple solution exhibiting

bands at A 455 and 562 nm having rapidly changing

max

intensity. E.s.r. examination of the solution revealed that

102
CJ

CM O

z

1E

ft.

ii I CD CC

ii il m X or cr I

II m m n

>» * QC (J I cr

ii II ii ii

CCL (T cr cr ^_

^

,^^

o .£> o D

CM CVJ CM OJ

0) Q.O

CO CM o

o

z

i ^ O u z E

I

II X

it X

II cr cr cr

II X I I i X i

II ii 11 n n ii X.cr cr cr X X cr

ii ii n ii 11 ii cr cr cr cr cr cr o .o o -D Q> «+-

103
the deep-purple color, and, consequently, a bathochromic

shift in the visible spectrum, is partly associated with

the presence of a paramagnetic species having the hyperfine

structure shown in formula 1 (A and B)

.

Cooling and rapid filtration of the concentrated,

alkaline solution from la yielded a somewhat air-sensitive,

purple-violet, paramagnetic dipotassium salt (2** 2a). Prom

the fact that neither the N-methyl nor the N-benzoyl derivative

derivative of glyoxal bis (phenylhydrazone) (lb and lc,

respectively) showed either a color change or the presence

of paramagnetic species following treatment with base, it

was concluded that the purple radical-anion from la must

arise following the abstraction of acidic imino protons

(X in la) by base. The dianion character of the radical

anion (2<+2a) was deduced from the fact that methylation

or benzoylation of the latter gave the di-N-methyl (lb) or

di-N-benzoyl (lc) derivative of glyoxal bis (phenylhydrazone)

in 45 and 60% yield, respectively.

The contribution of the resonance form (2>o2a) to the

stability of the radical anion from la was deduced from a study of radical anions derived from substituted bis(phenylhydrazones). The most stable radical-anions were obtained

from (a) the unsubstituted glyoxal bis (phenylhydrazone) la,

(b) those having the para position in the benzene ring

unsubstituted (for example, the 2,5-dichloro derivative, 2b),

or (c_) those in which the example, para position was substituted with an electron-withdrawing group, for example, the

P_-nitro derivative le . Derivatives having electron-donating

groups on the phenylhydrazino group (for example, the p-chloro

or p_-bromo derivatives Id) produced, on treatment with base,

little or no bathochromic shift and gave little or no e.s.r.

signal

.

These observations are in agreement with the substituent

effect proposed by Walter [83], in which it was considered

that electron-withdrawing groups ( e.g . , p_-NC>2) enhance the

104
stability of the hydrazyl radical by derealization of an

unpaired electron. It should be mentioned, however, that,

in contrast to neutral and cationic hydrazyl radicals having

p-Cl or jD-Br substituents [ 80] , the corresponding anionradical of type 2c was not observed following treatment of

the glyoxal p_-Cl- or p-Br-substituted bis (phenylhydrazones)

Id with base. Formation of a radical anion (2«*2a) from the

bis(phenylhydrazone) la in the presence of oxygen and base

appears to be a straightforward example of electron transfer;

similar electron transfer occurs in the formation of radical

anions from formazans [84]. The bis (phenylhydrazone) la

gave the e.s.r. spectra shown in fig. 14 . The 15 lines in

spectra A and A-l (aqueous potasbium hydroxide) had intensities

of ^I:2:6:10:l4:l8:22:24:22:l8:l4:10:6:2:l. Structure A is

a centrosymmetric , resonance-stabilized, radical anion having

two different pairs of nitrogen atoms (N=CH and N-Ph) ; e.s.r.

spectrum (A and A-l, fig. 14) can be explained by assuming

interaction of the unpaired electron with two pairs of

equivalent nitrogen atoms; this spectrum is consistent with

aN

= 4.95 and 4.95 G, aH

= 4.95, 4.95, 2.35, and 2.35 G.

Spectrum B (fig. 14) (prepared with anhydrous potassium

tert-butoxide in methyl sulfoxide) is consistent with

a =5-70 and 5.70 G; a = 5-70, 5.70, 1.4l, 1.4l, 0.70, and

0.70 G. The radical intermediate may be represented as a

resonance-stabilized, acyclic structure ( trans-cis structures

S^^a); however, the cyclic 1, 2 , 3, 4-tetrazine structure (4«+4a)

cannot be excluded completely.

A symmetrical radical-anion 2b derived from glyoxal

bis [( 2 ,5-dichlorophenyl)hydrazone] gave the e.s.r. features

shown in fig. 14C; this can also be explained by assuming

interaction of the unpaired electron with two pairs of

M H equivalent nitrogen atoms, a = 6.35 and 6.35 G; a = 6.35,

6.35, 1.65, 1.65, 0.82, and 0.82 G.

A paper describing some properties of these stable

radicals has been accepted for publication.

105
HC = N-N-(3

@-N-N=CH H

HC = N-N-{3

N-N=CH B

5G

CI

CI HC = N-N

D D DJ

D

HC = N-N-H-D Q-C = N-N

D-Q-R-N=CH

DD

DD / N-N=CH

5G

i 1 IOG E I5G

Figure 14. Radical-anions from glyoxal bis (phenylhydrazones)

A, from la (3.2 mM, in Me 2 SO-5$ aqueous KOH); B,

from la T2.8 mM, in Me 2 S0-K0Bu ; C, from 2,5-

dichloro derivative of la (3 mM, in Me2S0-K0Bu );

D, from la completely deuterated on the benzene

ring (5 mM, in MegSO-KOB^ )

; and E, from

phenylglyoxai bis (phenylhydrazone)

.

106
O-N-N-£h ^=/ 2 K"

HC

I HC

N

,N

2\C

N'

(3) TRANS (3a) CIS

HC

I HC

^N

N

N\ + ^R

HC ^N

"%,/»

(4) (4a)

R=C6 H 5

HC —N=N—R

II R-N«N—CH

(5)

107
4. PERSONNEL AND ACTIVITIES

A. Personnel Listing

R. Schaffer, Section Chief

R. S. Tipson

B . Coxon

A. J. Patiadi

R. P. Brady, Jr.

W. H. McCurdy, Jr.

W. J. Rossiter, Jr. to 7-13-70

B. F. West (Mrs. A. M. Tejeda) to 5-21-71

A. Cohen

B. A. Johnson

E. C. Douglass, Guest Worker (1970, 1971)

E. Hohertz, Guest Worker (1970)

J. James, Guest Worker (1970, 1971)

B

.

Publications

NBS Technical Note 5^7. Organic Chemistry Section.

7/1/69 - 6/30/70.

Edited by R. Schaffer (November, 1970).

Isotopic Methods [in Carbohydrate Chemistry]

.

R. Schaffer, in "The Carbohydrates: Chemistry and - Biochemistry," edited by W. Pigman and D. Horton,

Vol. 2B, 1970, pp. 765-776.

The Conformations of 1 ,2 : 4 ,6-Di-O-benzylidene-a-gglucopyranose Derivatives

.

B. Coxon, Carbohyd. Res. 14, 9 (1970).

Cyclic Acetals of Ketoses. Part III. Re-investigation

of the Synthesis of the Isomeric Di-O-isopropylidene-3-

g-fructopyranoses

.

R. F. Brady, Jr., Carbohyd. Res. 15, 35 (1970).

Electron-spin Resonance Study of an Alkaline Solution

of Copper (II) Oxalate—me so -Tart rate Complex (the

Somogyi Reagent) and Related Complexes.

A. J, Fatiadi, J. Res. NBS 74A, 723 (1970).

Determination of Moisture in Starch Hydrolyzates by

Near-Infrared and Infrared Spectrophotometry.

D. W. Vomhof and J. H. Thomas, Anal. Chem. 4_2,

1230 (1970) .

108
Oxidative Cleavage of Aromatic Azines with Periodic Acid:

E.s.r. Evidence for the Liberation of Nitrogen Dioxide.

A. J. Fatiadi, Chem. Ind. (London) 64 (1971).

Cyclic Acetals of Ketoses . Part IV. Re-investigation

of the Oxidation of 1,2 : 4 , 5-Di-O-isopropylidene-g-gfructopyranose with Methyl Sulfoxide-Acetic Anhydride.

R. S. Tipson, R. F. Brady, Jr., and B. F. West,

Carbohyd. Res. 16, 383 (1971).

Determination of Inososes with an Alkaline Solution of

Copper (II) Oxalate-Tartrate Complex (the Somogyi

Reagent) and Reaction Mechanisms Involved.

A. J. Fatiadi, Carbohyd. Res. 17, 419 (1971).

Manuscripts in Process of Publication

Occurence, Properties, and Preparation of Naturally

Occurring Monosaccharides, Including 6-Deoxy Sugars.

R. Schaffer. (Manuscript accepted for "The

Carbohydrates," edited by W. Pigman and D. Horton,

Vol. IA, in press)

Infrared Spectroscopy of Carbohydrates

R. S. Tipson and F. S. Parker. (Manuscript accepted

for "The Carbohydrates," edited by W. Pigman and

D. Horton, Vol. IB, in press)

Conformational Analysis via Nuclear Magnetic Resonance

Spectroscopy . B. Coxon. (Manuscript accepted for "Methods in

Carbohydrate Chemistry," edited by R. L. Whistler

and J. N. BeMiller, Vol. 6, in press)

Review of "The Carbohydrates : Chemistry and

Biochemistry," 2nd edition, Volume IIA, edited by

W. Pigman and D. Horton, Academic Press, New York, 1970.

R, S. Tipson. (Manuscript accepted for publication

in Carbohydrate Research)

Cyclic Acetals of Ketoses . R, F, Brady, Jr. [Manuscript accepted for publication

in Advances in Carbohydrate Chemistry and Biochemistry,

26 (1971)].

Evidence for Adsorption as the First Step in the Solidstate Oxidation of Benzenehexol with Active Manganese

Dioxide

,

A. J. Fatiadi. [Manuscript accepted for publication

in J . Chem. Soc. (B) ] . Enolization of Hexodiulose Acetals. Synthesis of

Derivatives of g-Psicose by Reduction of a Hex-3-

enulopyranose

R. F. Brady, Jr. (Manuscript accepted for publication

in Carbohydrate Research)

109
Application of Internuclear , Double-resonance Techniques '; to Carbohydrates. Detection of Small Coupling-constants.

B. Coxon. (Manuscript accepted for publication in

Carbohydrate Research)

(p_-Bromophenyl) osotriazoles from Inositols.

A. J. Fatiadi . (Manuscript accepted for publication

in Carbohydrate Research)

N.m.r. Spectroscopy of Derivatives of 6-Amino-6-deoxyg-glucose-^- 1

5

N. 13 C Fourier Transform and Internuclear

Double- and Triple-resonance Studies.

B. Coxon and L. F. Johnson. (Manuscript accepted for

publication in Carbohydrate Research)

Stable Radical-anions Derived from Glyoxal

Bis (phenylhydrazones )

.

A. J. Fatiadi. (Manuscript accepted for publication in

J. Res. NBS)

Determination of Moisture in Syrups by Gas—Solid

Chromatography

.

D. W. Vomhof and J. H. Thomas. (Manuscript submitted

for publication in J. Ass. Offic. Anal. Chem.)

Cholesterol: Standard Reference Material 911.

R. Schaffer, A. Cohen, and R. F. Brady, Jr.

(Manuscript submitted for publication in American

Journal of Clinical Pathology)

Periodic Acid as a New Oxidant for the Degradation of

Bile Pigments . Isolation of a Biliverdine Type of

Reaction Intermediate on Oxidation of Bilirubin with

Periodic Acid.

A. J. Fatiadi and R. Schaffer. (Manuscript submitted"

for publication in Experientia)

Characterization and Quantitative Analysis of g-Glucose

for Use in Clinical Analysis.

B. Coxon and R. Schaffer. (Manuscript submitted for

publication in Analytical Chemistry)

Documentation Interest of the National Bureau of

Standards and Collaborating Groups for Communication

of Laboratory Results

.

R. Schaffer, (Manuscript submitted for publication in

Proceedings of a Conference on Clinically Oriented

Documentation of Laboratory Data, Buffalo, N. Y.,

May 10-12, 1971)

D. Lectures

9/18/70 - Programs for Standards Relating to Clinical

Laboratories . National Committee for Clinical

Laboratory Standards, Atlanta, Georgia.

R. Schaffer .

110
10/9/70 - NBS SRMs for Clinical Analysis. Symposium on

Standardization for Meaningful Clinical

Analysis, National Bureau of Standards.

R. Schaffer . 11/24/70 - Studies of Carbohydrates by Internuclear,

Magnetic Double-resonance Techniques.

Department of Chemistry, Villanova

University, Villanova., Pennsylvania. B. Coxon

1/5/71 - Application of Internuclear Double- and

Triple-resonance Techniques to Carbohydrates

School of Molecular Sciences, University of

Sussex, Brighton, England. B. Coxon . 2/4/71 - Enolization of Hexodiulose Derivatives

Synthesis and Reduction of a 3,4-Enediol

Acetate. Sixth Middle Atlantic Regional

Meeting of the American Chemical Society,

Baltimore, Maryland. R. F. Brady, Jr.

by Internuclear,

Techniques

Meeting,

B. Coxon.

4/1/71 - Studies of 6-Deoxy Sugars

Magnetic Double-resonance

American Chemical Society

Los Angeles, California.

4/15/71 - The Use of Differential Scanning Calorimetry

and Phase Solubility Analysis in the

Characterization of Clinical Standard

Reference Materials. Merck, Sharp and Dohme

Research Laboratories, Rahway , New Jersey.

R. F. Brady, Jr.

Clinical Chemical Analysis Tied to NBS SRMs.

State University of New York, Buffalo,

5/12/71

5/16/73

New York. R. Schaffer . The NBS Program to Aid Clinical Chemical

Analysis . American Association of Clinical

Chemists, Wrightsville , North Carolina.

R. Schaffer.

Ill
5 . ACKNOWLEDGMENTS

The editor gratefully acknowledges the cooperation

of his associates in the Organic Chemistry Section in the

preparation of this report. Verna Koshute, Section

Secretary, brought the manuscript to final form. We wish

also to thank Mrs. R. S. Maddock, Administrative Assistant

for the Division of Analytical Chemistry in Charge of

Publications, for assisting in its publication.

112
6 . REFERENCES

[1] Recommendation on a Uniform Bilirubin Standard, Clin.

Chem. 8_, 405 (1962) . [2] Henry, R. J., Jacobs, S. L., Chiamori, N., Clin.

Chem. 6, 529 (I960)

[3] Fog, J., Scand. J. Clin. Lab. Invest. 16, 49 (1964).

[4] With, T. K., Bile Pigments, Academic Press, Inc.,

New York, N. Y., 1968, pp. 22-23-

[5] Nicholson, D. C, Stoll, M. , personal communication.

[6] Petryka, Z. J., Watson, C. J., J. Chromatogr. 37,

76 (1968).

[7] Fatiadi, A. J., Schaffer, R., Experientia, in press.

[8] Dybkaer, R., Hertz, H., Scand. J. Clin. Lab. Invest.

25, 151 (1970)

[9] Gadd, oK. G., J. Clin. Pathol. 19., 300 (i960); Simmons,

N. A.; ibid . 21, 196 (1968); Billing, B. E\ 3 Halsom,

R., Wald, A., Ann. Clin. Biochem. , in press.

10] Michaelson, M., Scand. J. Clin. Lab. Invest. 1_3,

Suppl. 56, 46 (1961)

Cohen, A., NBS Technical Note 457, 77 (1968).

Kammereck, R., Lee, Wen-Hui, Paliokas, A., Schroepfer,

G. J., Jr., J. Lipid Res. 8, 282 (1967).

Brady, R. F., Jr., NBS Technical Note 457, 70 (1968).

Brady, R. P., Jr., NBS Technical Note 507, 97 (1969).

Brady, R. F., Jr., NBS Technical Note 547_, 24 (1970).

Hayden, A. L., Anal. Chem. 27, i486 (1955).

Brady, R. F., Jr., NBS Technical Note 457 , 58, 90

(1968).

Parsons, W. H., Gennaro, A. R., Osol, A., Amer. J.

Pharm. 133, 351 (1961)

Coxon, B., NBS Technical Note 50J_, 28 (1969).

Coxon, B., NBS Technical Note 547, 68, 75, 77 (1970).

Coxon, B., Schaffer, R., Anal. Cbem. , in press.

Gardner, J. A. F., Hibbert, H., J. Amer. Chem. Soc.

66, 607 (1944)

Shaw, K. N., McMillan, A., Armstrong, M. D. J. Org.

Chem. 23, 27 (1958); Recondo, E. F., Rinderknecht

,

H., J. Org. Chem. 25, 2248 (I960).

113
[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

Goodman, I., Olenczak, A. P., Scherrer, J. W., Hiatt,

R. B., Biochim. Biophys . Acta 156, 364 (1968).

Boedecker, P., Volk, H . , U. S. Pat. 2,062,205; Kamlet,

J., U. S. Pat. 2,640,083 [Chem. Abstr. 48_, 5884 (1954)]

Reference 21, p. 38.

J. LeMaistre, personal communication.

Coxon, B., NBS Technical Note 457, 70 (1968).

Richtmyer, N. K., Hudson, C. S., J. Amer. Chem. Soc.

73, 2249 (1951) . C. J. Pouchert, Aldrich Library of Infrared Spectra,

spectrum No . 93c

.

Specifications and Criteria for Biochemical Compounds,

Tipson, R. S.(Ed.), Publication 13^4, National

Academy of Sciences—National Research Council,

Washington, D. C, 1967, CoE-7, p. 207.

[32] Burton, R. M. Pietro, A. S., Arch. Biochem. Biophys.

48, 184 (1954)

.

[33] Cohen, A., NBS Technical Note 547, ^7 (1970).

[34] Ciotti, M. M., Kaplan, N. 0., Methods Enzymol. 3,

890 (1957)

.

[35] Worthington Biochemical Corp., Freehold, New Jersey.

[36] Colour Index, Society of Dyers and Colourists, Second

edition, Vol. 3, 1956, p. 3513-

£37] Tipson, R. S., Brady, R. F., Jr. Carbohyd. Res. 10,

549 (1969)

.

[38] Brady, R. F., Jr., Carbohyd. Res. 15, 35 (1970).

[39] Tipson, R. S., Brady, R. F., Jr., West, B. F.,

Carbohyd. Res. 16, 383 (1971).

[40] Horton, D. Just, E. K., Carbohyd. Res. 9, 129 (1969).

[4l] Barton, D. H. R., Evans, R. M. , Hamlet, J. C, Jones,

P. G., Walker, T., J. Chem. Soc. 7^7 (1954); Barton,

D. H. R., Ives, D. A. J., Thomas, B. R., ibid . 903

(1954)

.

[42] Franchimont, M., Compt . Rend. £2, 1054 (1881).

[43] Marshall, C. W., Kritchevsky, T. H., Lieberman, S.,

Gallagher, T. F., J. Amer. Chem. Soc. 70, 1837 (19W.

[44] Isbell, H. S., Tipson, R. S., J. Res. Nat. Bur. Stand.

64A , 171 (I960) . [45] Takagi, S., Rosenstein, R. D., Acta Crystallogr.

A25 [S3], S197 (1969); Carbohyd. Res. 11, 156 (1969).

114
Maeda, T., Kiyokawa, M. , Tokuyama, K., Bull. Chem.

Soc. Jap. 38, 332 (1965); Maeda, T., Ibid . 4_0,

2122 (1967TT Maeda, T., Tori, W., Satoh, S.,

Tokuyama, K., ibid. 42_, 2635 (1969).

Angyal, S. J., Macdonald, C. G., J. Chem. Soc. 686 (1957)

(1957).

Levene, P. A., Tipson, R. S., J. Biol. Chem.

93, 631 (1931); Tipson, R. S., Cohen, A., Carbohyd.

Res. 7, 232 (1968)

Reichstein, T., Bosshard, W., Helv. Chim. Acta 17,

753 (1934)

Coxon, B., Carbohyd. Res. 12, 313 (1970); 13, 321

(1970) ; 14, 9 (1970)

Baker, E. B., J. Chem. Phys . 37, 911 (1962).

Kaiser, R., J. Chem. Phys. 39, 2435 (1963).

Ferretti, J. A., Freeman, R., J. Chem. Phys. 44 , 2054 (1966).

Coxon, B., Carbohyd. Res., in press.

Coxon, B., Carbohyd. Res., 11, 153 (1969).

Pasto, D. J., Johnson, C. R., Organic Structure

Determination, Prentice-Hall , Inc., Englewood

Cliffs, N. J. (1969), pp. 110 and 137.

Budzikiewicz , H., Djerassi, C., Williams, D. H.,

Mass Spectrometry of Organic Compounds, Holden—Day,

San Francisco, Calif. (1967), p. 225.

Coxon, B., Carbohyd. Res., in press.

Binsch, G., Lambert, J. B., Roberts, B. W., Roberts,

J. D., J. Amer. Chem. Soc. 86, 5564 (1964).

Emsley, J. W., Feeney, J., Sutcliffe, L. H.,

High Resolution Nuclear Magnetic Resonance

Spectroscopy, Vol. 2, Pergamon Press, Oxford (1966),

p. 878.

Coxon, B., Johnson, L. F., Carbohyd. Res., in press.

Fatiadi, A. J., Carbohyd. Res., in press.

Littler, J. S., University of Bristol, England,

personal communication.

Woodward, R. B., Hoffmann, R., Angew . Chem. Intern.

Ed. 8, 781 (1969)

Zimmerman, H. E., Angew. Chem. Intern. Ed. 8^, 1

(1969); see also, Pearson, R. G., Accounts Chem.

Res. 4, 152 (1971)

Littler, J. S., Tetrahedron 27, 81 (1971).

115
[67] Fatiadi, A. J. Chem. Ind. (London) 64 (1970).

[68] Kiselev, A. U., Quart. Rev. (London) 15, 99 (1961).

[69] Evans , R. M. , Quart. Rev. (London) 13, 6l (1959).

[70] Gattow, G., Wendlandt, H. G., Z. Anal. Chem. 174 , 15 (I960)

.

[71] Inorganic Index to Powder Diffraction, File 1969,

Amer. Soc . Testing Materials, Philadelphia, Pa.,

1969, p. 701.

[72] Ayschough, P. B., Electron Spin Resonance in Chemistry,

Methuen, London, 1967> p. 170.

[73] Fatiadi, A. J., Sager, W. F., Org. Synth. 42, 66 (1962)

[7^] Stone, F. S., Advan. Catal. 1, 13 (1952).

[75] Dillimore, D., Tonge , K. H., J. Chem. Soc. (B) 1380

, (1967).

[76] Fatiadi, A. J., Isbell, H. S., Sager, W. F., J. Res.

NBS 67A, 153 (1963)

[7 ] Pratt, E. F., Van de Castle, J. E., J. Org. Chem.

26, 2973 (1961).

[78] Konaka, J., Terabe, S., Kuruma, K., J. Org. Chem.

34> 1334 (1969)

.

[79] Fatiadi, A. J., J. Chem. Soc. (B) 889 (1973).

[80] Buchachenko, A. L., Stable Radicals, Consultants

Bureau, New York, 1965, pp. 100-144.

[8l] Forrester, A. R., Hay, J. M., Thomson, R. H.,

Organic Chemistry of Stable Free Radicals, Academic

Press, New York, 1968, pp. 137-179-

[82] Razuvaev, G. A., Abakumov, G. A., Pestunovich,

V. A., J. Struct. Chem. 5, 278 (1964).

[83] Walter, R. J., J. Amer. Chem. Soc. 88, 1923 (1966).

[84] Maender, 0. W., Russell, G. A., J. Org. Chem. 31,

442 (1966) j Russell, G. A., in Radical Ions, Kaiser,

E, T., Kevan, L. (Eds.), Interscience , New York,

1968, p. 87 ff.

116
form NBS-114A (i-7i)

1. PUBLICATION OR REPORT NO.

NBS IN -587

U.S. DEPT. OF COMM. BIBLIOGRAPHIC DATA

SHEET

2. Gov't Accession No.

3. Recipient's Accession No.

4. TITLE AND SUBTITLE

Organic Chemistry Section;

July 1970 to June 1971

Summary of Activities,

5. Publication Date August 1972

6. Performing Organization Code

7. AUTHOR(S)

Dr. Robert Schaffer

i. Performing Organization

Editor

9. PERFORMING ORGANIZATION NAME AND ADDRESS

• NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

10. Project/Task/ Work Unit No.

11. Contract/Grant No.

12. Sponsoring Organization Name and Address

Same as No. 9

13. Type of Report & Period Covered Interim - July 1970

fn June 1971

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

16. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here.) This report of the Organic Chemistry Section of the National Bureau

of Standard's Institute for Materials Research provides a summary of a year's scientific activity; as such, it covers both work that was

completed and some that is still in progress . The development of

Standard Reference Materials (SRM's) and their certification, and

journal publications describing aspects of that work, as well as other

research activities, are the output of the Section.

In the SRM category, the major subjects of this report are bilirubin, cholesterol, Cortisol, D-glucose, U-hydroxy-S-methoxy-PL-mandelic

acid (VMA), D-mannitol, and the reduced form of nicotinamide adenine

dinucleotide"(NADH)

Studies concerning carbohydrates include synthetic work with

D-psicose, n.m.r. employing internuclear double-resonance, and mass

spectroscopy of 6-deoxy-l ,2:3, 5-di-O-isopropylidene-a-D-glucose

,

spectroscopic studies of derivatives of e-amino-B-deoxy-P-glucose-B- 1

N

13 C-n.mTr. and heterotechniques), and p_-bromophenylosotriazoles of

inositols

Other research utilized e.s.r. and involved periodic acid oxidation

of azines , solid-state oxidation of benzenehexol on active manganese

dioxide, and the formation of stable radical-anions from glyoxal

bis(phenylhydrazone)

(employing mass spectrometry, Fourier-transform,

nuclear, N indor

17. KEY WORDS (Alphabetical order, separated by semicolons)

Bilirubin; cholesterol; Cortisol; e.s.r.; D-mannitol; NADH ; n.m.r.;

purity; Standard Reference Materials; SRM; "VMA.

18. AVAILABILITY STATEMENT

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