National Bureau of Standard*

Library E-G^ NBS TECHNICAL NOT

457

Organic Chemistry

Section:

Summary of Activities

July 1967 to June 1968

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U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards1 was established by an act of Congress March 3,

1901. Today, in addition to serving as the Nation's central measurement laboratory,

the Bureau is a principal focal point in the Federal Government for assuring maximum application of the physical and engineering sciences to the advancement of technology in industry and commerce. To this end the Bureau conducts research and

provides central national services in three broad program areas and provides central national services in a fourth. These are: (1) basic measurements and standards,

(2) materials measurements and standards, (3) technological measurements and

standards, and (4) transfer of technology.

The Bureau comprises the Institute for Basic Standards, the Institute for Materials

Research, the Institute for Applied Technology, and the Center for Radiation Research.

THE INSTITUTE FOR BASIC STANDARDS provides the central basis within the

United States of a complete and consistent system of physical measurement, coor- dinates that system with the measurement systems of other nations, and furnishes

essential services leading to accurate and uniform physical measurements throughout

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of an Office of Standard Reference Data and a group of divisions organized by the

following areas of science and engineering

Applied Mathematics—Electricity—Metrology—Mechanics—Heat—Atomic Physics—Cryogenics2—Radio Physics2—Radio Engineering2—Astrophysics2—Time

and Frequency.2 THE INSTITUTE FOR MATERIALS RESEARCH conducts materials research leading to methods, standards of measurement, and data needed by industry, commerce,

educational institutions, and government. The Institute also provides advisory and

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areas of materials research:

Analytical Chemistry—Polymers—Metallurgy — Inorganic Materials — Physical

Chemistry.

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of the Institute may be broadly classified as programs relating to technological meas- urements and standards and techniques for the transfer of technology. The Institute

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UNITED STATES DEPARTMENT OF COMMERCE

C. R. Smith, Secretary

NATIONAL BUREAU OF STANDARDS • A. V. Astin, Director

NBS TECHNICAL NOTE 457

ISSUED SEPTEMBER 1968

Organic Chemistry

Section:

Summary of Activities

July 1967 to June 1968

Edited by Robert Schaffer

Organic Chemistry Section

Analytical Chemistry Division

Institute for Materials Research

NBS Technical Notes are designed to supplement the Bureau's regular publications program. They provide a means for making available scientific data that are of transient or limited interest. Technical Notes may be

listed or referred to in the open literature.

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C., 20402

Price $1.25 cents.
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

Materials Research In the February 1, 1964, reorganization.

It consists at present of nine sections and about 100 technical personnel encompassing some 57 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

must be covered in only a line or two of a journal article.

li
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 abbreviated form.

At the National Bureau of Standards publications such

as these fit logically into the category of a Technical Note

In 1968 we plan to issue these summaries for all of our

sections. The following is the fourth annual report on

progress of the Organic Chemistry Section.

W. Wayne Meinke, Chief

Analytical Chemistry Division

111
PREFACE

This is the fourth in a series of NBS Technical Notes

which are prepared annually to summarize the technical activities of the Organic Chemistry Section of the Analytical

Chemistry Division. The present report, which covers the period from July 1, 1967 to June 30, 1968, describes much work

that is still in progress and also provides accounts of studies

that were completed during the year and are, or are about to

be, published. In line with a uniform policy of the Division,

the Section's programs have both a research and a sample aspect. About 60 percent of the total effort has been devoted

to research and the remainder to Standard Materials

For a great many years, because of their exceptional importance as natural products (to biochemical science and to

industry) and as research materials, the chemistry of the

carbohydrates has been a subject of central interest in the

Section. The competences that have been developed in studying carbohydrate synthesis and reaction mechanisms, in developing methods of separation and purification, and in the

characterization of molecular structure and conformation, are

skills that are applied to all areas of organic chemistry

where, within the mission of the National Bureau of Standards,

the Section can uniquely serve the scientific needs of the

Nation, e.g. , with Standard Reference Materials for research

and analysis, or conjointly with other research organizations,

attack pressing national problems, e.g. , air pollution, and

clinical analysis

Indeed, this year's report shows much of the Section's

effort newly directed to the certification of organic Standard Reference Materials for use in clinical analysis, thereby

helping clinical laboratories throughout the Nation to improve capabilities for performing their vital analytical tasks.

In addition to continuing to provide needed clinical standards, a long-range research and development program is being

iv
undertaken to investigate the clinically significant, organic

compounds present in clinical samples, with the objective of

developing better means for their characterization and determination . Recent technological developments in starch hydrolysis

have resulted in the industrial production of many new syrup

types for which previously developed physical property relationships, currently in use in the industry and by consumers,

are not entirely satisfactory. To remedy this, a Research

Associate Program has been initiated, in cooperation with the

Corn Industries Research Foundation, for restudying syrup properties by many of the most modern techniques. Dr. Daniel W.

Vomhof and Mr. James Thomas are engaged in this program.

During the year, the staff of the Section was augmented

with the appointment of Miss Barbara F. West, Dr. Robert F.

Brady, Jr., and Dr. Bruce Coxon.

Robert Schaffer, Chief

Organic Chemistry Section

v
TABLE OF CONTENTS

PAGE

CARBOHYDRATE SYNTHESIS, STRUCTURE, AND CHARACTERIZATION: NOVEL RESEARCH MATERIALS AND MODEL

COMPOUNDS

Reaction of Sulfonic Esters of Alditols with

Methoxide

Polyhydroxy Cyclic Compounds

Acylation of Tetrahydroxy-p_-benzoquinone . . Acetic Anhydride—Phosphoric Acid as an

Acetylating Agent

Bromine Oxidation of Inositols for Preparation of Inosose Phenylhydrazones and

Phenylosazones

Formation of Arylazocyclohexene Derivatives on Acylation of Certain Inosose

Phenylhydrazones

Determination of the Conformations of

1,2-0-Benzylidene-a-D-glucofuranose Derivatives by Nuclear Magnetic Resonance Spectroscopy

Chemical Shifts and Coupling Constants. . . Conformations

Reactions, Conformations, and Acidity of

Bis (ethylsulfonyl)- 3-g- ribopyrano sylme thane

and Related Derivatives

Disulfones Derived from D-Allose and

D-Altrose 7

Deuterium Exchange Experiments

Rare Carbohydrate Standard Reference

Materials

Standards Issued During the Past Year . . . 2 , 3-0-Isopropy lidene-3-D-thre_opentulofuranose . . . ."

1,2:3, 4-Di-O-isopropylidene-D- erythro - pentulofuranose 7

Studies of g-Psicose Derivatives

CLINICAL CHEMISTRY

Creatinine

Some Physical Properties of Creatinine. . . Preparation of Purified Creatinine for

Comparison

Loss of Weight on Drying

vi

6

6

8

9

11

12

15

21

24

25

27

32

32

32

39

41

49

50

51

51

52
TABLE OF CONTENTS (CONT'D)

PAGE

Chromatography of Creatinine 52

Spectrophotometric Methods 54

Stability of Creatinine 55

Quantitative Determinations of Creatinine 57

Creatinine as a Standard Reference

Material 59

Uric Acid 59

Some Physical Properties of Uric Acid ... 59

Preparation of Purified Uric Acid for

Comparison 60

Loss of Weight on Drying 6l

Chromatography of Uric Acid 6l

Spectrophotometric Methods 62

Stability of Uric Acid 64

Uric Acid as a Standard Reference

Material 64

Urea 65

Thin-layer Chromatography 65

Paper Chromatography 6 8

Spectrophotometric Examination of

Biuret—Copper Complex 69

Determination of the Purity of Urea by

Differential Scanning Calorimetry 70

Cholesterol 73

Source of the Material 73

Some Physical Properties of Cholesterol . . 74

Spectroscopic Properties 75

Chromatographic Methods 77

Selenium Dioxide Test for Lathosterol ... 83

Liebermann—Burchard Analysis 83

Issuance of the Cholesterol as a Provisional Standard 84

Post-issuance Studies of the Standard

Cholesterol 85

PROPERTIES OF POLYCLIC AIR-POLLUTANTS 91

Periodic Acid, A Novel Oxidant of Polycyclic , Aromatic Hydrocarbons 91

Vll
TABLE OP CONTENTS (CONT'D)

PAGE

4. OTHER STANDARD REFERENCE MATERIALS PROCESSED

THROUGH THE ORGANIC CHEMISTRY SECTION ....

Sucrose, Standard Reference Material 17a;

Reissuance of the Standard

Dextrose, Standard Reference Material 4la;

Reissuance of the Standard

Nicotinic Acid, Standard Reference

Material 148

Reissuance of Metallo-organic Standard

Reference Materials

5. ANALYTICAL CHEMISTRY OP CORN SYRUPS AND

RELATED PRODUCTS

The Near-Infrared Method

Thermogravimetric Analysis

Other Methods

6. PERSONNEL AND ACTIVITIES

Personnel Listing

Publications

Manuscripts in Process of Publication . .

7. ACKNOWLEDGMENTS

8. REFERENCES

LIST OF FIGURES

FIGURE NO.

1. P.m.r. spectrum of 1 ,2 :

, 5-di-0-benzylidene6-0-methyl-a-g-glucofuranose (2) in chloroform-d at 100 MHz. The signals' of the

methyl and phenyl groups are not shown . .

99

99

99

99

99

103

104

105

105

106

106

106

107

109

110

16

Vlll
LIST OP FIGURES (CONT'D)

FIGURE NO. PAGE

2. P.m.r. spectrum of 3 ,5 ,6-tri-O-benzoy 1-1,2-

O-benzylidene-ct-D-glucofuranose (j5) in

chloroform-d at 100 MHz. The phenyl resonances are omitted 17

3. P.m.r. spectra in chloroform-d at 60 MHz,

with the methyl resonances at diminished

amplitude: (a) bis (ethylsulfonyl)-( 2 , 3 , 4-

tri-O-acetyl-3-jD-ribopyranosyl) me thane

(,12), (b) bis(ethylsulfonyl)-(2,3,4-tri0-acetyl-3-D-ribopyranosyl)methane-d (13)

plus one-third of a molecular equivalent

of trimethylamine 31

4. Infrared absorption spectra of creatinine

and uric acid, each in a potassium bromide

pellet 63

5. Differential scanning calorimeter having a

sample-holder assembly and multi-range,

bidirectional, linear temperature programmer with eight speeds 71

6. (A) The electron-spin resonance spectrum of

pyrene radical prepared by treatment of

pyrene (in acetic acid) with periodic acid,

H 5 I0 6 , (In water) at room temperature, g

= 2.0041.

(B) Anthracene (in N,N-dimethylformamide

)

on treatment with periodic acid in water.

Sample B seen under comparable conditions. . 92

7. The e.s.r. spectrum (A) of the solid radical

prepared by treatment of azulene (in acetic

acid) with periodic acid, H 5 I0 6 , (in water)

at room temperature, g = 2.003^+0.0002; the

e.s.r. spectrum (B) of the solid radical

obtained on treatment of azulene (in acetic

acid) with cone, nitric acid at room temperature; g = 2.0087±0.001 97

IX
LIST OF TABLES

TABLE NO. PAGE

1. Chemical shifts (x values) of 1,2-0-

benzylidene-a-D-glucofuranose derivatives ~ 18

2. First-order, proton coupling-constants (Hz)

of 1 , 2-0-benzy lidene-a-D-glucofuranose

derivatives at 100 MHz 7 19

3. pKa values of substituted bis ( alkylsulfonyl)-

methane derivatives 26

4. Comparison of yield of diulose acetal (2)

obtained with various molar proportions of

acetic anhydride and methyl sulfoxide, per

molar proportion of acetal 1 43

5. Molar proportions of acetic anhydride and

methyl sulfoxide, per molar proportion of

the alcohol, employed in oxidations at

25-30°C 44

6. R_ values in thin-layer chromatography. ... 52 —

_

7. Analysis of creatinine by titration 57

8. Gas-chromatographic behavior of the standard

cholesterol 77

9. T.l.c. data on SRM cholesterol 79

10. T.l.c. of a degraded sample of SRM

cholesterol 80

11. Liebermann—Bur chard results with the

standard cholesterol, six months after

issuance 88

12. DSC results with the standard cholesterol,

six months after issuance 90

x
ORGANIC CHEMISTRY SECTION: SUMMARY OF ACTIVITIES

JULY 196 7 TO JUNE 196 8

Edited by R. Schaffer

ABSTRACT

This report describes work in progress in the Organic

Chemistry Section of the Analytical Chemistry Division of the

NBS Institute for Materials Research. The following research

areas are discussed:

Carbohydrate Synthesis, Structure, and Characterization : Novel Research Materials and Model Compounds . The reaction

of sulfonic esters of D-mannitol with methoxide has been examined, and a new dianhydrohexitol has been isolated, and

identified as 2 ,3

^, 5-dianhydro-D-iditol . Methods for the

esterification of tetrahydroxy-p_-benzoquinone and benzenepentol, and the disproportionation of the former compound during

acetylation have been studied. A novel method for acylation

in 100% phosphoric acid has been developed. The usefulness

of bromine for oxidizing cyclohexanehexols into inososes and

diketoinositols was investigated. Acylations of certain

inosose phenylhydrazones was proved to provide arylazocyclohexene derivatives, whereas previous workers had reported

otherwise. NMR spectroscopy was used to determine the conformations of several bi- and tri-cyclic carbohydrate derivatives in solution, and the influence of substituents and

of dipolar interactions on conformation was analyzed. The

factors that determine the rates of reaction of the cyclic

disulfones that are intermediates in the MacDonald—Fischer

degradation of sugars were evaluated by pKa measurements

and NMR analysis . Syntheses of "D-ribulose" and g-psicose

have been carried out.

Clinical Chemistry . Extensive examinations of the properties

of high-purity samples of creatinine, uric acid, and urea

have been made, as a preliminary stage in the development

of Standard Reference Materials for these compounds. The

xi
application of phase solubility analysis and differential

scanning calorimetry has been studied where appropriate

for evaluations of purity. The properties and methods for

determining the purity of SRM cholesterol were studied.

Properties of Polycyclic Air-Pollutants . The oxidation of

a variety of polycyclic, aromatic compounds by periodic acid

was studied and found to proceed by (1) a free-radical

mechanism that gives rise to coupling products, or (2) a two-equivalent oxidation mechanism that leads to quinonoid

derivatives, or both. On treatment with periodic acid,

azulene, a non-benzenoid, aromatic compound, gave a freeradical-containing, polymeric material having ionic properties . Analytical Chemistry of Corn Sugars and Related Products . As a preliminary but basic step in a program for developing

relationships between the compositions and physical properties of corn syrups prepared by various processes, a variety

of methods have been explored for assaying the water content

in syrups

In order to describe experimental procedures adequately,

It has occasionally been necessary to identify commercial

materials and equipment in this report. In no case does

such identification imply recommendation or endorsement by

the National Bureau of Standards, nor does it imply that the

material or equipment identified is necessarily the best

available for the purpose.

Key Words

Carbohydrate structure and characterization, sulfonic

esters of alditols, polyhydroxy cyclic compounds, acylation,

n.m.r. spectroscopy, standard reference materials, 1,2:3,4-

di-Q-isopropylidene-D- erythro-pentulofuranose , D-psicose,

clinical chemistry, creatinine, uric acid, urea, cholesterol,

polycyclic air-pollutants, periodic acid, sucrose, dextrose,

nicotinic acid, metallo-organic standard reference materials,

analysis of corn syrups

xii
1. CARBOHYDRATE SYNTHESIS, STRUCTURE, AND CHARACTERIZATION:

NOVEL RESEARCH MATERIALS AND MODEL COMPOUNDS

As one of the major categories of annually renewable, naturally occurring, organic materials, the carbohydrates are

basic to many industries. Interest in the carbohydrates at

NBS dates back to the early 1900 's, when the Bureau first verified the calibration of polariscopes and other apparatus used

by the Customs Bureau in levying duties on imported sugar.

Shortly thereafter, sucrose was Issued as a Standard Reference

Material (SRM) for polariscopic measurements, and this was

followed later by SRM dextrose used as a standard for reducingsugar determinations. The active study of analytical and

polarimetric methods continued for many years, during which

time the Bureau became the "greatest repository of sugar technology in the country." The recovery of valuable chemicals

from molasses and waste waters of sugar manufacture, the

development of commercially feasible processes for the manufacture of sugars and derivatives, and the development of

economically valuable products (gums, sugars, cellulose products) from farm waste-materials were additional activities

pursued.

Research in the chemistry of rare sugars was stimulated

when World War I cut off German sources of these and other

organic compounds. The chemistry associated with the proximity and multiplicity of reactive substituent groups in each

molecule, the multiple forms that each sugar may assume when

dissolved, and the structural interrelationship of members

of the carbohydrate family that make them ideally suited for

use as model compounds for studying the fine details of organic reactions have been subjects of the Section's numerous publications . With this extensive experience in carbohydrate chemistry,

an intensive program in method development and synthesis of

radioactive, position-labeled carbohydrates was undertaken in
the Section when carbon-14 and tritium became available and

no effective sources existed that were able to provide the

position-tagged sugars much needed for research in numerous,

primarily biochemical, laboratories. Until commercial and

other outside laboratories undertook to use the procedures

developed in this Section, the Bureau served as the sole supplier of position-labeled sugars. The isotopic methods of

analysis utilized in developing optimal syntheses of the labeled

compounds were subsequently adapted and expanded for studying

(a) complex reactions and their mechanisms, and (b) isotope

effects; some aspects of these studies related closely to the

biochemistry of the sugars

The carbohydrates have been and continue to be of immense

scientific importance, and the development of the deepest

possible understanding of their chemistry is a subject at the

forefront of organic chemical research. The work described

in this year's report concerns (i) novel methods for the introduction of functional groupings, whence they provide new materials, new derivatives, or better procedures for synthesizing

those known previously, and (ii) some intense applications of

nuclear magnetic resonance spectroscopy and other techniques

for examining fundamental chemical properties of model compounds and for determining the structure of previously unknown

compounds. As an outgrowth of research activities pursued

earlier, four rare-carbohydrate SRM's were issued during the

past year. In addition, g- erythro-pentulose ( "D-ribulose") and

g-psicose syntheses were carefully evaluated, and as a consequence, in the near future, stable derivatives of these two ketoses also will be made available as research standards, since

there is no commercial source for either of these rare sugars.
A Reaction of Sulfonic Esters of Aldltols with Methoxide

( R. S. Tipson and A. Cohen)

In Technical Note 427, we described the reaction of

methanolic barium methoxide with the 3 , 4-dimethanesulfonate

(la) or 3 j 4-di-p_-toluenesuIfonate (lb) of D-mannitol to give

a dianhydrohexitol. We showed that the structure of this

dianhydrohexitol (A) was not that of the 1, 4 : 3

, 6-dianhydride

(2) or of the 1, 4 : 2 , 3-dianhydride (3), but that it was one

of the following: the 2 ,3 : 4 , 5-dianhydride (4), the 1,3:4,6-

dianhydride (5), or the 2 , 3 : 4, 6-dianhydride (6)

Compound A was found to have two free hydroxyl groups,

because, on acetylation with acetic anhydride—pyridine (160

min at 25°C), it afforded a crystalline compound having an

elementary composition agreeing with that calculated for a di-O-acetyl-dianhydrohexitol; yield 97%- A crystalline

dibenzoate was also prepared; its properties differed from

those expected for the 2 ,5-dibenzoate of 2 on the basis of

those of the dibenzoate of the enantiomorph of 2 (a sample of

which was kindly supplied by Dr. H. G. Fletcher, Jr., of the

National Institutes of Health, Bethesda, Maryland 20014).

The n.m.r. spectrum of compound A, measured in methyl

sulfoxide-d_6 , showed signals of the hydroxyl protons as a 2-proton, apparent triplet, and hence the two hydroxyl groups

are both primary [1]. Hence, structures 5 and 6 are excluded

from further consideration. This conclusion was confirmed by

the observation that compound A, found to be stable in aqueous

solution at 25°C, is unaffected by sodium metaperiodate in

water during 24 hours at 25°C, showing that the two hydroxyl

groups of compound A are not vicinal. As a further confirmation, the n.m.r. spectra of the diacetate and dibenzoate,

measured in chloroform-d, showed a downfield shift of the

signals of four protons. Consequently, the two anhydro rings

of compound A must involve carbon atoms 2, 3, 4, and 5.

Unless rearrangement occurred after the initial reaction, a
HOCH

HOCH

ROCH

HCOR

HCOH

H

2 COH

la, R = Ms

lb, R = Ts

— CH

HOCH

HCO-^

-OCH

HCOH

H

2 C

'

1,4:3,6-

DIANHYDROD-IDITOL

-CH

•OCH

HC-*

^OCH

HCOH

CH OH

1,4:2,3-

DIANHYDROD-IDITOL

CH

2 OH

OCH

HC

•CH

j

HCO

CH OH

2

| HOCH

HCO-*

-OCH

HCOH

' CH„

CH o 0H

I 2

•OCH

i HC

rocH

I HCOH

-CH„

2,3:4,5-

DIANHYDROD-IDITOL

1,3:4,6-

DIANHYDROD-IDITOL

2,3:4,6-

DIANHYDROD-IDITOL
2,5 :

s 4-dianhydride is ruled out because of the method of

preparation, suggesting that compound A is the 2,3:4,5- or

2 , 4 : 3,5-dianhydride . The n.m.r. spectra of compound A and

its 1,6-diacetate and 1, 6-dibenzoate were consistent with

either of these structures (and the respective esters thereof),

The n.m.r. spectrum of compound A showed only four main groups

of signals, and, for some compounds, this situation could be

attributed to chemical-shift averaging caused by completely

free rotation at the C-l-C-2, C-3-C-4, and C-5-C-6 bonds.

However, inspection of molecular models of the 2,3:4,5- and

2 ,4 : 3,5-dianhydrides of D-iditol reveals that, with suitable

orientation of the hydroxymethyl groups, each molecule can

have a simple, two-fold axis of symmetry vertical to the

C-3—C-4 bond. As compound A and its two diesters are optically active (and therefore lack a plane of symmetry), the

optical asymmetry at C-2 differs from that at C-5 3 and that

at C-3 differs from that at C-4. Consequently, it seems

reasonable to attribute the observed chemical-shift equivalence of H-l with H-6 or H-6 ' , of H-l' with H-6 ' or H-6 , of

H-2 with H-5, and of H-3 with H-4, to the presence of the

two-fold axis of symmetry. Such symmetry, which does not

make the molecule optically inactive, is nevertheless sufficient to give chemical-shift equivalence of protons located

similarly on each half of the chain of carbon atoms. Because

the 2 , 4 : 3 , 5-dianhydride structure has two oxetan rings, seldom

encountered in sugar chemistry, we have assigned the 2,3:4,5-

4

dianhydride structure (£) to compound A. We thank Mr. W. D.

Dorko (National Bureau of Standards) and Mrs. Margaret Lake

(Food and Drug Administration) for recording the n.m.r.
spectra, and Dr. B. Coxon of this Section for measuring and

interpreting them.

These results have been incorporated in an article which

has been accepted for publication [2]

B . Polyhydroxy Cyclic Compounds

(A. J. Fatiadi

1. Acylation of Tetrahydroxy -p-benzoquinone

The acetates of a simple enediol compound, the first

oxidation product of benzenehexol (hexahydroxybenzene) 3a } namely, tetrahydroxy-p_-benzoquinone ( la) , were not described

in recent reports by Verter and coworkers [3,4] on the preparation of other fatty acid esters of la. In order to fill

this gap, we have prepared and characterized [5] the di- and

tetra-acetates (compounds Zb and lb, respectively) of la.

II

OR HO

a.b.

c

d.

e .

R = H

R = CH 3 COR = CH 3 CH2 COR = CH 3 CH 2 CH2 COR = (CH 3 ) 2 CHCOWe attempted unsuccessfully to prepare diacetate 2Jd by

the method [3] used by these authors for the preparation of

the dipropionate 2£; however, a procedure employing simultaneous acetylation and hydrolysis ("hydrolytic acetylation"

)

was successful in providing 2b in a yield of 85%. A mixture

of tetrahydroxy-p_-benzoquinone (,1a) (3 g, 17.4 mmole) in

acetone (50 ml) and acetic anhydride (20 ml) containing 20
drops of concentrated sulfuric acid was placed in a water

bath at 50°C and stirred until the solid had dissolved (5 min)

.

The rather dark solution was filtered through carbon, which

was then washed with 10 ml of acetone. The filtrate and

washing were combined, and concentrated to about 25 ml, and

the solution was poured into a mixture of 50 ml of concentrated

hydrochloric acid and 75 g of crushed ice. The mixture was

stirred for 15 min. at room temperature, and the resulting product was filtered off, washed with ice-cold hydrochloric acid

( 6 M )

, and dried in a vacuum desiccator over potassium hydroxide; the yield of lustrous, gold-orange plates of diacetate

(2b) was 2.5 g. Cooling of the filtrate (ice-bath) for 3-4 hr.

gave an additional crop (1.3 g) ; total yield 3.8 g (85%), mp

246-248°C. The product was recrystallized quickly from warm,

glacial acetic acid (prolonged heating causes decomposition)

or from hot nitromethane , mp 250-251°C (with effervescence);

lit. [6] mp 205°C.

By the same method, dipropionate 2£ was prepared in a yield of over 60%; however, neither the dibutyrate 2d nor the

diisobutyrate 2je was obtainable by this type of procedure.

a. Tetraacetate of Tetrahydroxy-p-benzoquinone

Refluxing of tetrahydroxy-p_-benzoquinone (la) with

acetic anhydride for 3 minutes gave tetraacetate lb in 75-80%

yield; similarly, tetrapropionate (,1c) was obtained in 70-75%

yield; tetrabutyrate Id (66-70% yield) and tetraisobutyrate

le (78-81% yield) were also obtained.

b

.

Disproportionation Reactions of Tetrahydroxy-pbenzoquinone

In contrast, treatment of la with acetic anhydride and

pyridine at 60-65°C gave benzenehexol hexaacetate 3b and

rhodizonic acid (£) in nearly equal proportions (due presumably to a disproportionation) . The other fatty acid esters

of 3a have been prepared by an analogous procedure; compounds

3c, 3d, and 3e have been obtained in 48, 46, and 47% yields,

respectively

.

7
c . Esters of Benzenepentol

One-step aromatization of m^jo- inositol (5) (a cyclohexanehexol) , following treatment with methyl sulfoxide—acetic

anhydride in the presence of pyridine, has been reported

previously [7]; the product, namely, pentaacetoxybenzene (,6b)

was isolated in about 50$ yield.

HO OH

a. R = H

b. R = CH 3 COc. R = CH3CH2COd. R = CH 3 CH 2 CH 2 COe. R = (CH 3 ) 2 CHCOIn order to test the general applicability of this procedure [7] 5 compound 5 was separately treated with propionic,

butyric, and isobutyric anhydrides [8]. As expected, compound

5 was converted into the corresponding fatty acid esters of

benzenepentol (,6a); namely, pentapropionate (6c) in 25-30$

yield; pentabutyrate (6d) in 23-26$ yield; and pentaisobutyrate

(6e) in 21-23$ yield. Hydrolysis of pentaacetoxybenzene (6bJ

with concentrated hydrochloric acid in methanol gave benzenepentol (6a) in 77-81$ yield.

2 . Acetic Anhydride—Phosphoric Acid as an Acetylating

Agent

A satisfactory method was needed for acetylating the

enolic compound, croconic acid. Attempts with all known

acetylation procedures were incapable of providing an isolable

acetate of that compound. However, when a reagent was used
that consisted of 100% syrupy phosphoric acid and acetic

anhydride (mole ratio, about 1:1.7) the crystalline diacetate

of croconic acid was obtained. The new reagent was found [9]

to be effective for the acetylation of other difficultly and

easily esterifiable hydroxylated compounds. These include

carbohydrates, cyclitols, enols, phenols, sterically hindered

secondary alcohols, and tertiary alcohols.

The active actylating species in the acetic anhydridephosphoric acid reagent is believed to be monoacetic phosphoric anhydride. Use of 100% phosphoric acid in conjunction

with other aliphatic acid anhydrides was found to be an effective means for the preparation of the propionate, butyrate,

and isobutyrate of myo-inositol

.

3 . Bromine Oxidation of Inositols for Preparation of

Inosose Phenylhydrazones and Phenylosazones

The methods usually employed for the preparation of the

dicarbonyl derivatives of cyclohexanetetrols involve prolonged

oxidation of inositols by Acetobacter suboxydans [10,11] or

oxidation of inososes or their phenylhydrazones in the presence

of phenylhydrazine and acetic acid [12]. The dicarbonyl

derivatives are isolated as phenylosazones. The application

of sodium hypobromite for oxidation of cyclitols has also been

reported [13]; for example [14], myo-inositol was oxidized

with bromine in the presence of a sodium carbonate buffer,

and the mixture was then treated with phenylhydrazine to give

gL-myo-inosose-1 phenylosazone (yield, about 7%). Similarly,

phenylosazones have been obtained by oxidation of (+)- proto - quercitol [15] with aqueous bromine, and from oxidation of

(-)-viburnitol with sodium hypobromite [16]; however, for the

last two reactions, the yields were not stated.

Except for a general statement by Posternak [13] that

hypobromite oxidation of inositols provides unsatisfactory

yields of inososes, there appears to be no literature report

on the subject of the oxidation of inositols by bromine to
provide monoketoinositols . The bromine oxidation of cyclitols was studied in order to clarify whether this oxidation

to inosose might be of special usefulness, in that the

inososes obtained might be different from those resulting

from the stereospecific catalytic and biological oxidations.

The results were the subject of a paper [17] that has been

accepted for publication.

It has been found that myo- inositol treated with bromine

in a sodium acetate buffer, gave, on addition of phenylhydrazine, the usual phenylosazone , DL-my_o-inosose-l phenylosazone,

in 22-30% yield. ^-Inositol, on the other hand, gave g-myo- inosose-1 phenylhydrazone , a monoketone phenylhydrazone,

(12% yield) as well as g-myo-inosose-1 phenylosazone, the

expected diketone phenylosazone (28% yield). The corresponding

enantiomorphs were obtained in 8 and 29% yields, respectively,

from D-inositol. The oxidation reaction was also applied to

pinitol and quebrachitol, the naturally occurring, monomethyl

ethers of D- and L-inositol. The previously unknown diketone

that results from quebrachitol ( 1-0-methyl- levo-inositol)

was isolated as the phenylosazone .1 (29% yield), but no monoketone was isolated. From pinitol ( 5-O-methyl- dextro-inositol)

,

an inosose derivative was not found either; although here too,

a new diketone phenylosazone (2) was isolated (10-15% yield).

Interestingly, compound 2 shows a rapid mutarotation in 1:1

(v/v) ethanol—p_-dioxane . In the course of this study, a new

phenylosazone 3 was prepared from gj^- epi- inosose- 2 phenylhydrazone; (+)- proto-quercitol (from acorns) was converted into its

phenylosazone in 20% yield (a yield has not heretofore been

reported) , and myo- inositol was converted in low yield by bromine oxidation into the known compound rjL-xylo-pentahydroxy-2-

cyclohexen-1-one

10
PhHNN OMe

PhHNN =3

NNHPh

=NNHPh

plus its enantiomer

NNHPh

=NNHPh

4 . Formation of Arylazocyclohexene Derivatives on

Acylation of Certain Inosose Phenylhydrazones

Recent communications by Wolfrom and coworkers [18] describing the preparation of arylazoethylene derivatives from

aldose phenylhydrazones prompted our article [19] on related

findings for inosose derivatives , in which were considered

the products that result from acylation of myo-inosose-2

phenylhydrazone (la) and gL- epi - inosose- 2 phenylhydrazone

(,2a) in the presence of pyridine. Angyal and Matheson [20]

had reported earlier that, on treating a solution of penta0-acetyl-myo- inosose-2 (Id,) in warm acetic acid with phenylhydrazine, they obtained a pale-yellow, crystalline compound

(mp 112-113°C) to which they assigned the phenylhydrazone

structure lb. In our laboratory, it has been found that,

under these conditions, compound Id actually loses the elements of acetic acid, and yields the elimination product

6-phenylazo-5-cyclohexene-gL-ido-l> 2,3,4-tetrol tetraacetate

(3a) instead of the phenylhydrazone lb.

11
The structure assigned to 3a was supported Dy its elemental and spectral analyses: u.v. and visible spectra

^MeOH 222>5 (sh) (£ . 12} 300); 228 (e -13,700); 233 (e -12,800);

max Tv.

302 (e -23,000), and 436 nm ( e -400); i.r. spectrum v luoroluDe

rn.a,x 1658 (C=C), 1584 (N=N), 1430 cm

-1 (N=N); n.m.r. spectrum

(p. p.m., CDCI3), the olefinic proton at H-5 appears as a

PhlM = N

a,b,

c

d

R = H, R

T = NNHPh a

R = Ac, R' = NNHPh b

R = OCEt, R' = NNHPh c R = Ac, R' =

2

R = H

R = Ac

R = OCEt

3

a. R = Ac

b. R = OCEt

quartet centered at 6.95, due to vicinal coupling with H-4

(Jif,5 ~2.5 Hz) and long-range coupling with H-l (Ji,s ~1.0 Hz)

Acylation of D^- epi -inosose-2 phenylhydrazone (2ja) gave

pentaacetate 2b (mp 134-135°C), and pentapropionate 2c, (mp

112-113°C) . C . Determination of the Conformations of 1,2 -0-Benzylideneq-g-glucofuranose Derivatives by Nuclear Magnetic Resonance

Spectroscopy

1. Introduction

(B. Coxon)

The fundamental importance of the conformational analysis

of carbohydrate molecules and, indeed, of organic molecules in

general, has been reviewed in Technical Note 405 [21]. During

the past ten years, nuclear magnetic resonance spectroscopy

has provided a powerful experimental method for the determina12
tion of the conformations of organic molecules in_ solution , thereby eliminating much of the speculation that was apparent

in earlier attempts at dealing with molecular topography.

Additionally j this type of spectroscopy furnishes important

new physical parameters, namely, chemical shifts, and nuclear

spin—spin coupling-constants and their signs, which have

proved to be extremely useful in the analysis and characterization of the structure of chemical compounds. Although the

conformations are of considerable importance to an understanding of the kinetic and thermodynamic behavior of organic

molecules in reaction, of even more profound implication is

the characterization of the intramolecular and intermolecular

interactions, both steric and dipolar, that govern the distribution of conformations and their relative stability. With

greater knowledge of such interactions, it will be possible

to predict favored conformations more accurately. This type

of information is, for example, of basic importance in comparisons of the chemical reactivity of structurally related

organic molecules

.

The cylic acetals have always been valuable as intermediates in synthetic carbohydrate chemistry. The conformational

influence of cyclic acetal substituents fused to furanose and

pyranose rings is important, both from this point of view and

as an aid to understanding the factors that determine the conformation that is adopted. In this study, the conformations

of a number of bicyclic and tricyclic 1,2-0-benzylidene-a-Dglucofuranoses have been determined by proton magnetic resonance (p.m.r.) spectroscopy of their solutions at a radiofrequency of 100 MHz. The compounds [22] examined were

1,2 : 3,5-di-O-benzylidene-a-D-glucofuranose (1) and its 6-0-

methyl, 6-0-acetyl, and 6-0-benzoyl derivatives (2, 3, and 4,) , and 1,2-0-benzylidene-a-D-glucofuranose (5) and its 3 3 5,6-

tribenzoate (6) . These derivatives were kindly supplied by

Dr. H. G. Fletcher, Jr., of the National Institutes of Health.

13
ROCH

1 R= H

2 R - Me

3 R s AC

4 R =Bz

5 R * H

6 R = Bz

Compounds 5 and 6 afforded the opportunity for comparison

of the conformational effect of the fusion of a 1, 2-benzylidene

group to a furanose ring with that of the fusion in a 1,2-0-

isopropylidene-a-D-xylo-hexofuranose derivative [23]. As is

well known, reactions of carbohydrates with aldehydes and

ketones yield different types of ring systems for both the

sugar moiety and the acetal moiety according to the particular

catalysts and carbonyl compounds that are employed, and the

conformational stability of the benzylidene acetals may be

expected to have some bearing on this problem.

In order to assign a definite conformation to a carbohydrate ring system, it is desirable to measure as many as

possible of the spin—spin coupling constants of the ring

protons. Consequently, it is usually necessary that at least

some of the signals of these protons be fully resolved. With

molecules as complex as the benzylidene acetals mentioned,

it is therefore necessary to study their proton magnetic

resonance at a high magnetic-field strength (23,000 gauss or

greater) in order to secure spectra that are amenable to a

reasonably simple analysis. At this field strength, the

chemical shifts of the nuclei may be sufficiently large to

14
ensure separation of several of the multiplets . Accordingly,

the p.m.r. spectra of compounds l-j5 were recorded on a

commercial, high-field, n.m.r. spectrometer at the National

Physical Laboratory, Teddington, Middlesex, England, and the

analysis and interpretation of the spectra were conducted at

the National Bureau of Standards

.

2 . Chemical Shifts and Coupling Constants

In some of the p.m.r. spectra of 1-J5, the proton multiplets overlapped one another in a complex manner. Hence,

the spectral assignments were confirmed by frequency-swept,

double-irradiation experiments, and by the chemical-shift

differences (solvent shifts) between spectra measured in

solutions in chloroform-d and in pyridine. Examples of the

spectra (for compounds 2 and 6,) are shown in figures 1 and

2, respectively. The spectra were, in most cases, analyzed

by first-order methods, and the chemical-shift and couplingconstant parameters obtained in this way are listed in

tables 1 and 2. The conformational significance of the

proton—proton coupling-constants (J) was assessed by calculating approximate, vicinal proton—proton, dihedral angles

((J)) from the theoretically derived equation of Karplus C 24

]"

J = 4.22 - 0.5 coscf> + 4.5 cos 2<j>,

which may be rewritten as

J = 9 cos

2

cf) - 0.5 cos<|> - 0.28.

If this quadratic equation in cos <J> is solved in the usual

way, there is obtained

= cos" 1 0.5±[0.25 + 36(J + 0.28)] 2

. (1)

18

This equation represents a convenient, explicit form for the

calculation of approximate, theoretical, dihedral angles from

vicinal-proton coupling-constants

.

It often happens that a pair of vicinal protons in an organic molecule show an apparent zero coupling-constant, that

is, the multiplets in the spectrum shown no resolvable split15
o

St

to

St

C\J CO

stmo

st10

ro

ro

in

rCDO

st

CDroO

cd

ro ^ CD C\J*5h w cd

CD tQom OC

cd

Qh rH o >»C 3 CD

bo

a i QlrXS 1 C 3 cd I H H

>i >s

-C Xi

-P -P

CD CD B S

OlCD I .C VO +3 I CD <m C O

CD t3 ra •H H H cd >a C N bO C -H

CD CQ £3I CD O.I.C I Eh HI • ens

C\J o

"O

rH

H

<M P O cd S T3| 3 I M g

-P

M

O

O

CD ^ • a o g W M ^ o o • rH XI m XI 03 • O S po

PL,

C

M

50

•H 16
o 7\ C\J \\ &-A^^p X X \^^ o —u — o o isl N

DQ OQ

CMI X

Ooro

CM

iri

<£> O ^

roI X

V "^

CM

moC

cti U

Oh • O T3 O CD 3 P H -P hO-H l g

quo i a co i u

0) Cti C

CD CO T3 CD •H O H C

>3 Cti N C C o

CD CQ £2 CD I U

OlI iH

CM >5 » c

rH CD i .c

in

a

>jON X! C Eh

CD £)I • oIn

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g

-P

OI o

MO

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LPv -P

•> Cti on a I Cm I O g g o ^ O P M o o

CD rH

CQ

O • c M -H

Ph MD(

CO

00

ro CM

CM CD CO M

ro 3

•H

Ph 17
co

CD>

•H

-P

ctf >

•HU

CD d

CDCOOC

ctf !h CmOO

rH&0I OilI 3I

(I) C

cd d

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>3Nc

CD

iiI CM

CmO

co

CD H

ctf >

to p

CmH

s:

CO

ctf oHB

cd H

0) H

X3

ctf Eh o

VOI KI ffil lp o

on pl, I Kl

CM

O

H Pm

VOI

VOI

LPIi

onI

CMI

ffi

ctf I P O

coo o o a

hOLP

i—

hO

CMLP o CM CO

o>o B

oo

vo 6oo

•H 11 S

CMON OO

LP 6

CT\ VO

B

on

rH

•H 11 OO on vo VO r— CO CM CM 1 rH on CM CM 1 H o o CO

CM o^ voH

LP

CO II 11 111 ^3" on -=r on -=3" on ^3" on l 1 1 VO

o>

rH LO

OA

CMr— on

vo

LP CM CO

on on on on on on on on

m

m cn

LP

VO

•H 11 So

r—

•H 11 6o•H 11 6

CO

VO 6oo

•H 11 S

CM

H 11 B

VO &

en

•H 11 • 1 • 1 • 1 • • 1 • 1 • • 1 CM 1 CM 1 CM 1 CM CM 1 H 1 CM CM 1

rH

Cm

a1 OO

CO o^3"

Cm

a1 CO

on

d

co

Cm

VO

Cm

CMLP

a1 o^3"

Cm

W

t—

CO

Cm

a1 o

^3"

a1

CM VO un vo vo LP LP LP LP LP LP LP a1 COO

Cm

d o1

CM

Cm

ry

-=3" CM

Cmt—

Cm

do

CM

Cm&

CM

a1 CM

CT\

Cm£

on

a1 VOo

Cm

CO

VD lp vo VO

50

•>

LP LP LP ^3" LP LP -^T

CmCO

Cm

^3"

50p

LP, r—

CmP

on

LP

-=3" VO

b

OO

on

Cm6

CO

CmS

vo

Cm

B

CM

CmS

VO

CM

CmsH

O^

lp LP LP LP LP LP LP LPe

LP -^r on

Cmlp

CO

Cmon

^3"

a1

co

a1 CM VO

d

CO

Cm

d

COVO CO

Cm

d

LP

CmS

cn

Cm

a1

CM

Cm

CM CO

lp LP LP LP LP LP LP LP LP LP -=r

'OS LP

-=JCmdo dO

d

CMCM

dO

LP

d

CM

d

rH

d

on

rH

Cmd

COo d

LP

CM

d

lp LP LP LP LP LP LP LP LP -^r on d

CO

CM

Cmd

CM

T3on

on

OS VO

d

CMon

d

en

d

CM

do

Cmd

ooo do doo m lp LP LP LP LP LP LP LP LP LP

0)d

OO

d

LP, vo

do d

CO

VO

d

CO

d

VO

vo

13 vo

CO

d

rH VO

d

r—

vo

d

ON

OO

d

LP

on en on en on en on on on

m on

CQ

d

r-ll < CQ

•<-3CMi

CQ CQ

M

ml %>

PQ

LP^ VO?

CQ

18
Table 2. First-order, proton coupling-constants (Hz)

of 1,2-0-benzylidene-a-D-glucofuranose derivatives at 100 MHz

Com- Sol- TTTT T b T b T pound vent

a -1,2 -2,3 -3,4 -4,5 ~5,6 ^,6' ^6,6'

l

c,d

A 3.6 <0.4 2.3 5.2 5.1 11.4

(5.32) q (5.005) q

B 3.6 <0.4 ^4.9

2

J

' A 3.7 <0. 4 2.5 1.5 3.7

B 3-7 <0.4 2.4 1.2 4.3

3

k

A 3-6 <0.4 2.4 1.35 6.1

B 3-6 <0.4 2.4 1.2 ^7-7

4

m

A 3-6 <0.4 2.4 1.5 8.1

B 3-6 <0.4 2.2 1.2 7.4

5

n

B 3.4 ^3-3 2.9

6 P A 3-6 <0.4 3.4 8.9 2.5

B 3-6 <0.4 3.3 ^9.0 2.5

^5.7 ^11.5

4.3 10.3

4.8 10.3

3.5 9.7

3.1 9.9

4.3 13.1

4.0 10.7

^4.9 ^11.7

5.7 12.3

6.4 12.2

Footnotes to Tables 1 and 2

aSolvent: A is CDC1 ; B is C.-H.-N.

d 5 5

H-6 is the methylene proton at lower field; H-6 ' is that at

higher field.

c R-3, R-5, and R-6 refer to the O-substituents at C-3, C-5,

and C-6,. respectively.

dR-6 = H.

Q Signal multiplicities are indicated by d(doublet),

m(multiplet ) , q(quartet), or t(triplet).

f Incompletely resolved multiplet.

°Broad band.

19
Assignment confirmed by deuterium exchange.

Obscured by solvent signals

j'r-6 = Me.

kR-6 = Ac.

m

R-6 = Bz.

nR-3 = R-5 = R-6 = H.

p R-3 = R-5 = R-6 = Bz.

The figures in parentheses were obtained from an ABX analysis

of the H-6 (A) and H-6 ' (B) signals, with H-5 as the X nucleus.

ting, due to the mutual coupling of the protons. For most of

the modern, high-resolution, n.m.r. spectrometers, the physical

significance of this observation is that the coupling constant

is less than 0.4 or 0.5 Hz, that is, less than the resolving

power of the instrument. Although it has often been suggested

in the literature that the non-observation of a vicinal coupling

implies a proton dihedral angle of 90°, our re-examination of

this situation, using equation 1, shows that this conclusion is

only very approximately true. For a positive coupling in the

range of to 0.4 Hz, two dihedral angles of about 72-78° or

99-104° may be calculated, whereas, for a negative coupling in

the range to -0.2869 Hz, equation 1 implies that <J> is about

78-88° or 88-99°. The value -0.2869 represents the minimum

value of J that will allow a real solution for equation 1,

corresponding to <j> = 88°. The latter two values are also obtained

by equating to zero the derivative

dJ/dcf) = 0.5 sine}) - 18 sinc|)cos<})

In compounds 1-4, and 6,, the coupling constant (J 2 ,3)

between the protons on C-2 and C-3 was not resolved, and, as

its sign therefore cannot be determined, it can only be

speculated from equation 1 that <p 2)3 lies in the approximate

range of 72-104°. These values represent the limits of the

20
dihedral angle for an "apparently zero" coupling constant

and a finite reolution afforded by the spectrometer.

3 . Conformations

In order that H-2 and H-3 may achieve a dihedral angle

of 72-104° j it is necessary that the furanoid ring exist in

one or more of those nonplanar conformations in which C-3

is above a plane containing C-l, C-2, and C-5 or the ring

oxygen atom (0-4) or both (assuming that the ring is viewed

in the usual orientation with 0-4 at the back and C-l on

the right). Compound (5 displayed a large value (8.9-9.0 Hz)

for J 4 5 , indicating that the favored rotamer about the

C-4—C-5 bond is 7,j i n which <K , 5 is 180°, and in which C-3

and C-6, and C-4 and C-5, are also in a true trans relationship. From consideration of all of the measured coupling

constants of 6, its conformation was deduced to be that

depicted in 8.

H-5

OBz

„v^l%

H-4

/CH2OBz

( 8 Ph

The coupling constants (see table 2) of the dibenzylidene

acetals 1-4 indicated conformations intermediate between 9

and 10, -and it was therefore inferred that fusion of a 3,5-

benzylidene ring to the l,2-0-benzylidene-a-r)-glucofuranose

system has the effect of distorting the symmetric, twist

conformation of the furanoid ring (as in 8 and 9) towards

the envelope conformation 10.

21
ROCH2

H v ^Ph

ROH2 C —

H Ph

10

Because the m-dioxane ring in 1-4 is cis-fused to C-3

and C-4 of the furanoid ring, it can exist in either of two

chair conformations, as in 9 (or 10) and 11. However, the /Vrf ******* /NJ*>rf "* small measured value of J\ 5 in 1-4 is inconsistent with

the large dihedral angle of H-4 and H-5 in 11, and, hence,

its contribution to any conformational equilibrium can only

be small.

The skew form 12 provides a possible alternative to

the chair forms 9 and 1J3 of the m-dioxane ring, as, in this

conformation, H-4 and H-5 are also both equatorial and would

be expected to display a small coupling-constant. This

possibility was, however, discounted by analysis of the geometrical implications of the variation of J 3,4 on introduction

of the 3 j5-benzylidene group.

Prom the increased values of J 5 6 in the esters 3 and 4,

it was deduced that solutions of these compounds contain a greater population of rotamer(s) about the C-5—C-6 bond, in

which H-5 and H-6 are trans . As the H-6 and H-6' resonances

cannot be individually assigned, this rotamer could be either

22
ROH 2C

H Ph

HGhROCH.

II 12

1^ or 1^, in which the acyloxy group at C-6 is trans to

either C-5 or 0-5, respectively. In the less-favored rotamer

l^i in which ^^ = $5,6' > this acyloxy group is in gauche

relationship with both C-4 and 0-5.

13

H-6

H-5

0-5^ —

\H-6

C—

4

15

0-5^ — ^C-4

H-6

14

23

1
In view of the fact that hydroxymethyl, methoxymethyl,

and acyloxymethyl substituents at C-5 of a pyranoid ring

normally show a strong tendency to assume the equatorial

orientation [25-29], it is surprising that the favored conformation (related to 9 and 10) of the dibenzylidene acetals

contains C-6 in axial orientation of the m-dioxane ring.

It is possible that dipolar interaction between the C-2—0-2

and C-3—0-3 bonds causes the 0-2—0-3 dihedral angle to be

maximized, as in conformations 8, 9, and 10 (170-180°).

In the unfavored conformation 11, this angle is considerably

smaller (85°) • However, 11 also has some of the characteristics of an unstable, "H-inside" conformation [30], since

H-2, H-5, and H-3,5 occupy endo positions with respect to

the fused furanoid and m-dioxane rings. Conversely, 9 and

10 resemble a stable "0-inside" conformation [35], because

0-3, 0-4, and 0-5 are endo with respect to the C-3—C-4 ring

junction

These results elucidate the observation that reactions

of g-glucose with acetone (in the presence of zinc chloride,

phosphoric acid, or other condensing agents) [31] do not give

an appreciable yield of 1 ,2 : 3,5-di-O-isopropylidene-a-Dglucofuranose , because, if this compound exists in the same

conformation (resembling 9 or _]_£) as the 1, 2 : 3 ,5-di-benzylidene

acetals, one of the methyl groups in its 3 ,5-isopropylidene

moiety would be In syn- axial relationship with the R0CH 2 substituent on C-5.

D . Reactions, Conformations, and Acidity of Bis (ethylsulfonyl)-

g-g- ribopyranosylmethane and Related Derivatives

(B. Coxon)

1. Introduction

One of the more useful methods for the conversion of an

aldose (or a ketose) into its next lower homolog is the

MacDonald—Fischer sulfone degradation [32]. This process

involves the oxidation of a sugar dialkyl dithioacetal with

hydrogen peroxide or a peroxy acid, to give a disulfone deri24
vative which is then degraded to the next lower sugar and

bis (ethylsulfonyl)methane by treatment with a dilute base.

The oxidation is complicated by the fact that three possible

types of disulfone structure may be obtained. The initial

product is, undoubtedly, an acyclic, saturated disulfone,

formed merely by addition of four oxygen atoms to the diethyl

dithioacetal . This type of product is, however, unstable

and may suffer elimination of the elements of water from C-l

and C-2 to give an acyclic, unsaturated, disulfone derivative.

Depending on the conditions of the reaction and the structure

of the sugar, this alkene can then cyclize to give a third

type of disulfone, which usually contains a pyranoid ring.

Thus, cyclic disulfones are often the stable end-products of

the oxidation reactions.

The rates of reaction of these various disulfones with

dilute base differ widely, for, although the acyclic, saturated disulfones are degraded almost instantaneously to the

next lower sugar, the unsaturated and cyclic disulfones, once

formed, may require from two to fifteen days for complete reaction. Thus, a knowledge of the factors that influence the

rate of degradation of the cyclic disulfones is important

from the point of view of the preparative application of the

reaction.

2 . Disulfones Derived from D- Allose and D- Altrose

As part of a previous study of the general chemical and

physical properties of cyclic disulfones, the oxidation by

peroxypropionic acid of several diethyl dithioacetals having

the D- allo and D- altro configurations was investigated [33].

Oxidation of 3-acetamido-3-deoxy-D-allose and -D-altrose

diethyl dithioacetals gave ( 2-acetamido-2-deoxy-3-p= - ribopyranosyl)bis (ethylsulfonyl)methane (1), and oxidation

of the parent D-allose and D-altrose diethyl dithioacetals

gave bis (ethylsulfonyl)-3-D-ribopyranosylmethane (2). The

rate of reaction of 1 with dilute ammonium hydroxide to give

2-acetamido-2-deoxy-g-ribose was found to be much lower than

25
that of 2 to give D-ribose., and it became apparent that the

rates of reaction of cyclic disulfones could be influenced

by the inductive effect of the substituent at C-2. It had

been discovered earlier [34] that certain other cyclic

disulfones are weakly acidic , due to the electron-withdrawing

character of the ethylsulfonyl groups and of the pyranoid

ring [33]. The relationship of these acidic properties to

the rates of degradation of the disulfones became obvious,

and potentiometric titrations of the cyclic disulfones gave

characteristic pKa values. Activity corrections were applied

by using the Debye—Huckel theory as outlined next . The

results are presented in table 3, with literature values for

two related sulfones.

Table 3- pKa Values of substituted bis (alkylsulfonyl)methane

derivatives

Compound pKa Reference

Bis (methylsulfonyl)methane 14.0 35

Bis (ethylsulfonyl)-a-D-lyxopyranosylmethane 10.1

Bis (ethylsulfonyl)-3-D-ribopyranosylmethane (2 ) 9.9

Bis(ethylsulfonyl)-(2-0-methyl-a-Darabinopyranosyl) methane 9.6

(2-Amino-2-deoxy-3-D-ribopyranosyl)-

bis (ethylsulf onyl)methane hydrochloride 9.5

5.9

( 2-Acetamido-2-deoxy-S-D-ribopyranosyl)-

bis (ethylsulfonyl)methane (1) 9.4

Tris (ethylsulfonyl)methane <1.0 36(a)

The titration curve for each of the cyclic carbohydrate

disulfones listed in table 3 displayed a recognizable inflection at the neutralization point, whereas that for bis(ethylsulfonyl)methane did not. A pKa value for the latter compound

26
was not available in the literature, although that of its methyl

homolog has been reported [35] to be 1,4. The pKa values of

the disulfones were determined, from the half-neutralization

point on the titration curve, by means of the equation [36(b)]

T u : i [HA] , 0.50 5 /I

P V

= pH + log [A=T

-+ i + 1.6 n

where I is the ionic strength at half-neutralization of either

the bis (ethylsulf onyl)pentopyranosylmethyl anion (positive

activity term) , or the pentopyranosylammonium cation (negative activity term) . The carbohydrate disulfones showed pKa

values in the range of 9.4 to 10.1, and the development of

appreciable acidity in them is due to the introduction of

the glycopyranosyl substituent, which is electron-attracting.

The main influence must be the inductive effect of the ringoxygen atom, as this electronegative atom is only three bonds

removed from the ionizing hydrogen atom on C-l. It is apparent that the inductive effect of the substituent at C-2

also influences the pKa value of the disulfone, although to

only a minor extent. There is, therefore, some correlation

of these values with the rates of degradation of the disulfones.

For example, compound 1, which is degraded very slowly, is the

most acidic (pKa 9.4) of the disulfones examined. It must

therefore be inferred that an electron-withdrawing substituent

at C-2 tends to prevent recession of the electrons in the

C-l—C-3 bond, and, hence, the cleavage of this bond, thus

inhibiting the degradation.

3 . Deuterium Exchange Experiments

a . Bis (ethylsulfonyl) -3 -D- ribopyranosylme thane ( 2

)

Although the reaction of compound 2 with dilute ammonium

hydroxide had previously been analyzed by polarimetry and

paper chromatography [33], this reaction was examined further

by means of proton magnetic resonance spectroscopy in order

to gain information on the acidity of the disulfone and on

the nature of the exchange processes involved in its reaction

27
with base. In ammonium hydroxide-d 5 at pD 12 and 42°C,

cleavage of 2 was rapid. The complex series of changes observed in the p.m.r. spectrum of the solution is best interpreted in terms of the reaction sequence 2->3 + 4->ll involving,

initially, rapid duterium exchange of H-3 in 2, followed by

a comparatively fast cleavage to deuterated D-ribose (3) and

bis (ethylsulf onyl)methane-d 2 (5); and then a slower, but progressive, deuterium exchange of all a-protons of the ethyl

groups, finally giving bis (ethylsulfonyl)methane-d 6 (11).

The latter material was isolated, and characterized by its

melting point and infrared and proton magnetic resonance

spectra

b . Bis(ethylsulfonyl)-(2,3 , 4-tri -0- acetyl-g -gribopyranosyl) methane

In the previous work [33] 3 evidence for the configuration

and conformation of £ had been obtained from measurements of

optical rotation and from periodate oxidation studies. Because

cyclization of an intermediate 1 ,1-bis (ethylsulfonyl)-hex-l-ene2 s 3 , 4 j 5 , 6-pentol can theoretically product two diastereoisomers,

due to the introduction of new asymmetry at C-2, it was necessary, in each case, to prove the configuration at this carbon

atom. Direct confirmatory evidence for the configuration and

conformation of 2 has now been obtained from the p.m.r. spectrum

(see fig. 3a) of its triacetate (12) measured at 60 MHz. Acetyl

methyl signals at t 7.82, 7-98, and 7-99 suggested the presence

of one axial and two equatorial acetoxy substituents s respectively [37,38], and the spectral assignments snown in fig. 3a

yielded first-order coupling-constants J 1 o 1.4, J 1 2 10.8,

J 2 ,3 2.3, J 3 ,^ 2.7, J^se 5.8, Ji/,sa 10.8, and J 5 e, 5 a 11 . 4 Hz.

These values compare favorably with the values J 1}2 10.1,

J 2 , 3 2.8, J 3 , 4 2.6, Jit j5 e 5.6, and J>,sa 10.5 Hz reported previously [26] for acetamido-(2,3,4-tri-0-acetyl-3-D-ribopyranosyl)<

methane. The large values of J x s 2 and J h

5 show that both H-l

and H-2, and H-4 and H-5a, exist mainly in diaxial relationship

28
CH(S02Et)2

CH/a (S02 Et) 2

HO NHAc HO OH

ND4 0D

H,OD

DO OD

S02CD2 CH3

CDS02CD2CH3 S02CD2 CH 3 S02CHDCH 3 S02CH2 CH 3

CD? < CD? * CD? < CD?

I I I I S02CHDCH 3 S0 2CH2CH 3 S02CH 2 CH3 S0 2CH2CH 3

AcO

S02CD2CH 3 S02CD2 CH 3

S02 Et

AcO

\ S02Et

II 12 R=H

13 R=D

29
[37]. Since the triacetate of the a-D anomer of 2 would be

expected to show a small value of J

3 2

i- n either of its chair

conformations, this result demonstrates that the bis(ethylsulfonyl)methyl derivative has the 3-D anomeric configuration,

and that, in chloroform-d, it exists mainly in the conformation

12. The small value of J 1%ft indicates that the favored rotamer

about the C-l—C-3 bond is one or both of those in which H-l

and H-3 are in gauche orientation [39], as depicted in 12.

In order to confirm the assignments of the H-l and H-3

multiplets in the spectrum (see fig. 3a) of the triacetate

12, the deuterium exchange of its weakly acidic H-3 atom was

investigated. Exchange did not occur readily under uncatalyzed conditions, although, with very vigorous mixing of the

chloroform-d and deuterium oxide layers, H-a in the a-g- lyxo

analog of .12 underwent exchange slowly (half-time 1 hr) . In

the presence of a small proportion of trimethylamine, exchange

of H-3 in ^12 was rapid, as indicated by the disappearance of

its doublet at t 5-73 and the simplification of the H-l signal

to a doublet that no longer contained the small Ji,g coupling

(see fig. 3b). When isolation of the C_-3-deuterated disulfone

(13) was attempted, its deuterium was lost at a surprisingly

high rate, owing, perhaps, to retention of some trimethylamine

by salt formation with the disulfone. Although the spectrum

(fig. 3a) of 12^ shows the methyl protons of the ethyl groups

as nonequivalent triplets, in the spectrum (see fig. 3b) of l._3 measured in chloroform-d with one-third of a molecular equivalent of trimethylamine present, these protons are equivalent.

That this difference is not due to a deuterium isotope-effect

on the chemical shift of the ethyl groups was demonstrated by

the fact that a sample of 13 prepared by equilibration of a chloroform-d solution of 12 with a deuterium oxide solution of

sodium deuteroxide gave a spectrum in which the methyl protons

were still nonequivalent. This result was confirmed by the

observation that treatment of the chloroform-d solution of 12

30
H* H

2 H

4 H H

/3 H5e H 5a + 2CH

2

4.20 4.47 4.91 5.17 5.73 5.96 782 798799 8.56 8.61

(b)

S0

2 CH

2 CH3

S02 CH

2 CH

3

4.12 4.41 4.86 5.1 5.93 7.74 7.81 7.96 7.97 8.57

Figure 3- P.m.r. spectra in chloroform-d at 60MHz, with the

methyl resonances at diminished amplitude: (a)

bis (ethylsulfonyl)-( 2,3,4- tri-O-acetyl- 3-D-ribopyranosyl)methane (12,), (b) bis (ethylsulfonyl)-

(2,3, 4-tri-0-acetyl-3-D-ribopyranosyl)methane-d

(13) plus one-third of~a molecular equivalent of

trimethylamine

.

31
with trimethylamine , alone, or in water, also caused the nonequivalence of the methyl groups to disappear, and, of course,

produced chemical exchange of H-$. It therefore seems possible

that the chemical-shifts effect in chloroform-d solution is due

to some specific association between the nitrogen atom of trimethylamine and H-3 or D-3 in 12 and 13, respectively.

Most of the n.m.r. spectra described herein were recorded

on a Varian Associates Model A-56/6O spectrometer at Southern

Illinois University, Carbondale, Illinois.

E. Rare Carbohydrate Standard Reference Materials

1

Standards Issued During the Past Year

As a result of the work described in our Technical Note

427 [40], the following standards have been issued during the

past year:

(i) 1 ,2-0-isopropylidene-3-L-idofuranose as Standard

Reference Material 1591;

(ii) 2 ,3-0-isopropylidene-g-rj- threo-pentulose as SRM

1592;

(iii) levo-inositol as SRM 1593; and

(iv) quebrachitol as SRM 159 4.

Reproductions of the individual Certificates of Analysis

are given on the following pages

2. 2 ,

-Q- Isopropylidene -3-D-threo-pentulofuranose

(R. S. Tipson)

In Technical Note 427 [40], the preparation of 2,3-0-

isopropylidene-3-D- threo-pentulofuranose (13) for use as a standard reference material was described. Further improvements in its preparation have now been devised.

In 1936, Levene and Tipson [4l] reported that the syrupy

mixture of sugars obtained by boiling a solution of D-xylose

(1) in dry pyridine for 4.5 hours, followed by removal of the

pyridine and as much as possible of unchanged 1, could be

resolved to a considerable extent by acetonation followed by

fractional distillation of the resulting mixture of mono- and

di-isopropylidene acetals . They thus isolated four acetals,

32
U. S. Departrt*i|a$£y of Commerce

Alexander IMtrowbridge,

Secretary'

National I urWtfXt^oi A. V. S4stii,

3fi

Standards

rector Certificate of gnalpsitsi

Standard Reference Material 1591

l,2-(9-Isopropylidene-/?-L-idofuranose

This Standard Reference Material is certified regarding its identity

and has the following properties:

Melting Point 114°- 115°C

Specific Rotation [a] ^° -27.8° (c2; water)

Neither of the enantiomorphs of idose has been reported as a crystalline material, and the syrupy sugar is unstable, isomerizing to a ketose or losing water to form an anhydro-sugar. Reference samples of

this sugar are obtained by hydrolysis of this Standard Reference Material

with acetic acid. Thus, 2 mg of the Standard Reference Material dissolved in 0.5 ml of 70-percent acetic acid and warmed at 70°C for 2.5

hr provide L—idose free of the starting material and containing only a

trace of impurity, possibly 1,6—anhydro—L—idopyranose.

Washington, D. C. 20234 W. Wayne Meinke, Chief

January 18, 1968 Office of Standard Reference Materials

33
U. S. Departr%i$jifof Commerce

Alexander E&jTrowbridge,

Secr^ry

National I ur§Wfc=9£ A. V.

2ite

S^stji^plrector

Standards

Certificate of lUnalpsfts;

Standard Reference Material 1592

2,3-0-Isopropylidene-/?- d -fhreO-pentulose

This Standard Reference Material is certified regarding identity and

has the following properties:

Melting Point 70°-71°C

Specific Rotation [a] 22 +1.6° (£4; acetone)

This compound is also known by the synonyms monoacetone—D—

lyxoketose, monoacetone—D—lyxulose, monoacetone—D—xyloketose, and

monoacetone—D—xylulose.

To obtain the free pentulose (the crystalline form of this sugar has

never been reported) dissolve 2 mg of the Standard Reference Material in

1 ml of water, add 1.25 mg of oxalic acid dihydrate and heat the solution

to 65°C for eight hours. Cool, add finely powdered calcium carbonate,

with stirring, until the solution is neutral to congo red. Filter and wash

the calcium salts with water. Combine the filtrate and washings and

evaporate to dryness at 30°C at 25 mm Hg. Dissolve the syrup in absolute ethanol, filter, and evaporate the filtrate to dryness.

Washington, D. C. 20234 W. Wayne Meinke, Chief

January 18, 1968 Office of Standard Reference Materials

34
U. S. Departn*|n$fof Commerce

Alexander EfeJTrowbridge,

National I ur A. V.VAstii

Msa

Standards

rector Certificate of Ulnalpsiis;

Standard Reference Material 1593

/£i/0-Inositol

This Standard Reference Material is certified regarding its identity

and has the following properties and spatial structure:

Melting Point 243° - 244°C

Specific Rotation [a] * 2 - 64.9° (c 2; water)

HO OH

levo—Inositol

Washington, D. C. 20234

January 18, 1968

W. Wayne Meinke, Chief

Office of Standard Reference Materials

35
U. S. Departri^fi^of Commerce

Alexander Eihltrowbridge,

National Bur«W^9f Standards

A. V. S&stii, Director Certificate of &nalpsite

Standard Reference Material 1594

Quebrachitol

This Standard Reference Material is certified regarding its identity

and has the following properties and spatial structure:

Melting Point 191°- 193°C

Specific Rotation [a] g° -80.5° (c 2.37; water)

HO OCH3

HOy

HO \ 'OH

Quebrachitol

Washington, D. C. 20234 W. Wayne Meinke, Chief

January 18, 1968 Office of Standard Reference Materials

36
namely, crystalline 1,2 : 3,5-di-O-isopropylidene-a-D-xylofuranose (5) derived from unchanged 1, syrupy 2 ,3-0-isopropylidene-a-rj-lyxofuranose (£) (the crystallization of which, from

this reaction, was reported in Technical Note 427), crystalline

2 , 3-0-isopropylidene-g-g- threo-pentulofuranose (13) , and a

syrupy mixture (A) of unidentified di-O-isopropylidenepentoses

(free from 5) having n* 5 1. 44l4 and [a]* 5 -47-8° (acetone).

On complete hydrolysis with 0.1 M oxalic acid, mixture

A gave a mixture of pentoses having [a]

n -5.7° (calculated

as pentose; in water) . These properties suggested that A

consisted mainly of 1,2:3, 4-di-O-isopropyIidene-D- erythro- pentulofuranose (14), the enantiomorph of which had been

shown by Levene and Tipson [4l] to have nl 5 1.4422 and [ot]^ 8 +105 • 5° (acetone) , and to give, on hydrolysis, L- erythropentose having [ct]^, 7 +16.6° (water). A new sample of mixture

A has now been prepared. It was hydrolyzed with 0.1 M oxalic

acid at 25°C for 40 minutes, and the acid was then neutralized

by addition, with stirring, of an excess of calcium carbonate.

The suspension was filtered, and the filtrate was extracted

with chloroform. The extract was dried (anhydrous sodium

sulfate) and evaporated to dryness, affording a product that

was identified as 1,2: 3,4-di-O-isopropylidene-g- erythro- pentulofuranose (3JO . The aqueous layer was evaporated to

dryness, giving a syrup that was treated with absolute

ethanol; a trace of insoluble calcium salts was removed by

filtration. The filtrate was evaporated to dryness, and the

resulting syrup was treated with anhydrous ether (20 volumes).

Part of the syrup, consisting of free sugars, remained undissolved; the ether solution was evaporated to dryness,

giving a syrup consisting of monoisopropylidene acetals.

These products are undergoing further study.

By use of modern techniques (paper and column chromatography), Fedororiko and Linek [42] have recently re-examined

the mixture of pentoses obtained by the action (8 hours) of

boiling pyridine on g-xylose . They found that, in addition

37
H

IC =

I HCOH

I HOCH

I HCOH

H

Ic=o

I HOCH

I HOCH

I HCOH

I CH

2 OH CH

2 OH

H

Ic=o

I HOCH

I HCOH

I HCOH

ICH OH

H

Ic=o

I HCOH

I HCOH

I HCOH

ICH OH

Q-Xylose

1

g-Lyxose

2

D-Arabinose

3

D-Ribose

H2CO HOCH2 °>0 H2COH^O

O\ /

Ip

H.OH

CH o 0H

C = HOCH

I HCOH

CH OH

CH o 0H

I 2 C =

I HCOH

I HCOH

ICH OH

CH OH

HOCH

I c=o

I HCOH

I CH OH

CH.OH

I 2 HCOH

I C =

I HCOH

ICH OH

g-threoPentulose

D- erythro - PentuloseD- threo -3-

Pentulose

erythro -3-

Pentulose

CH2OH

13

10

y^^ch2o

OV

14

IP

IP

11

I

OCH

2 -OCH

I c=o

I HCO

H

2 CO- IP

12

i

H 2^°\ T HCO— 1 ?

15

C =

I HCO^

H CO16

:i P

38
to the pentoses already mentioned (1. 2, 9, and 10), it con- ^»-» *>*/ *^ rs^ s**sr*ss J tains D-arabinose (3), g-ribose (k) , g- threo-3-pentulose (,11),

and erythro -3-pentulose ( ^12 )

, all in unstated proportions.

In restudying the reaction with a view to increasing the

yield of acetal J_3, it was decided to remove the aldopentoses

(1, 2, 3, and 4) from the initial reaction mixture (after r*t i>* r+s "* removal of as much as possible of 1 by crystallization) , and

then to acetonate the mixture of ketopentoses (9, 10, 11, and

12). Such removal of an aldose from a solution of an aldose

and a ketose was achieved in 1950 by Gabrielson and Samuelson

[43] by use of Amberlite IRA-400 (HSO^) ion-exchange resin,

and the method has since been improved [4-4,4.5]

.

D-Xylose (1) was epimerized on a larger scale (150 g per

batch) than hitherto, and, after removal of some 1 by crystallization, the mixture of pentoses was passed through a column

of Amberlite IRA-400 (HSO^) which had been prepared as described by Adachi and Sugawara [46] (see also, Ref. [47]), and

the mixture of ketopentoses was isolated from the effluent and

treated as described by Zinner and Rehpenning [47]. The mixture of compounds 9, 10, 11, and 12 was acetonated, giving a

mixture of 13, 14, 15, and 16 as a syrup that contained a preponderance of 1^, because, without distillation of the syrup,

the monoacetal 13 crystallized from it, affording an almost ****r*^ solid mass of crystals. The monoacetal 13 was readily separated from the diacetals (14, 15, and 16) by dissolving the

mixture of acetals in water and extracting with ether: 13 remains in the aqueous layer, and the mixture of the three

diacetals is obtained in the ether layer.

3. 1,2:3, 4-Di-O- isopropylidene-g-erythro-pentulofuranose

( R. S. Tipson and R. F. Brady, Jr.)

g- erythro-Pentulose (10) is a rare sugar that is an

intermediate in the oxidative pathway of metabolism of

D-glucose in animals and yeast, and it is an early product

of photosynthesis in plants. It is therefore of considerable

interest to biochemists.

39
The sugar was synthesized in 1934 [48] by pyridinecatalyzed epimerization of g-arabinose (3) and isolated as the crystalline ( o-nitrophenyl)hydrazone [49] in 1935. In

the following year, Levene and Tipson [4l], working with the

enantiomorph (3e), improved the preparative value of the reaction by acetonating the mixture of pentoses that is formed,

and separating the resulting mixture of acetals by fractional

distillation. In this way, they isolated L- ery thro -pent ulose

(lOeJ as its crystalline 1,2 : 3 , 4-diisopropylidene acetal (l4e).

L-Ribose (J$e) was also isolated, as its 2 ,3-isopropylidene

acetal ( 8e)

Federoriko and Linek [42] have recently shown that treatment of D-arabinose (3) with boiling pyridine for 4 hours

gives a mixture of the same pentoses as was obtained from

g-xylose (,1) . Obviously, however, the proportions of the pentoses in the mixture will be different, starting with 1 or 3,

unless the reaction is permitted to proceed to equilibrium

in both instances

In order to make D-erythro-pentulose (10) available as

a standard reference material, it was decided to apply Levene

and Tipson's procedure to D-arabinose (3), to remove the aldopentoses (1, 2, 3, and 4) from the initial reaction mixture rs* r**/ r^ '"•*' (after removal of as much as possible of 3 by crystallization),

and then to acetonate the mixture of ketopentoses (9, 10, 11,

and 12). Zinner and Rehpenning [47] have achieved the separation of the aldopentoses from the ketopentoses obtained from

D-arabinose (3) by use of a column of Wofatit SBW (HSO^) ionexchange resin. Dowex-1 (HSO^) was used by Lindberg and

Slessor [45] for similar separation of the pentoses from Larabinose ( 3e)

g-Arabinose (3) was epimerized on a large scale (150 g

per batch) , and, after removal of some 3 by crystallization,

the mixture of pentoses was passed through a column of Amberlite IRA-400 (HS0 3 ) ion-exchange resin, prepared as described

40
by Adachi and Sugawara [46] (see also, Zinner and Rehpenning

[47]). The mixture of ketopentoses (9, 10, 11, and 12) that

was isolated from the effluent was acetonated, giving a mixture of the acetals 13, 14, 15, and 16. The monoacetal 13

was then readily separated from the diacetals (14, 15, and 16)

by dissolving the mixture of acetals in water and extracting

with ether. (a) The monoacetal remained in the aqueous

layer, and evaporation of this solution gave a crystalline

compound that was identified as 2 ,3-0-isopropylidene-3-Dthreo -pentulofuranose (13)- (b) The ether layer was evaporated to dryness, and the resulting mixture of 14, 15, and 16

was fractionally distilled under high vacuum, affording

crystalline 1,2:3 , 4 -di-0- is op ropy lidene-D- ery thro -pent ulofuranose (,14,). Compounds 15 and 16, are under further study.

Because of the low melting point (+5 °C) of compound 1,4,

a more suitable derivative is being sought for use as a standard reference material. A sample of the diacetal lH

was therefore hydrolyzed with 0.1 M oxalic acid, to give free

D- ery thro-pent ulose (10,) , and attempts were made to crystallize

the free sugar, and to prepare a crystalline derivative

having a melting point above 25°C. Attempts to prepare a crystalline cyclohexylidene acetal by condensation with

cyclohexanone , or of a crystalline peracetate or perbenzoate

have, thus far, been unsuccessful.

4 . Studies of D- Psicose Derivatives

( R. S. Tipson and B. P. West)

D-Psicose (D-ribo-hexulose ) (4) is a rare sugar that

occurs in Itea plants and in the antibiotic psicofuranin.

In 1967, McDonald [50] described a new synthesis of the

sugar, from D-fructose, that involved the following steps.

1,2 : 4 ,5-Di-O-isopropylidene-D-fructopyranose (1) was oxidized

with methyl sulfoxide—acetic anhydride to 1,2 : 4, 5-di-0-

isopropy lidene-D- ery thro - 2, 3-hexodiulose (£'); it was stated

that a small proportion of the 3-acetate (5) of compound 1

41
Me

NQBH4 H

HO

OH , CH2OH

was also formed, but that, on evaporation of the reaction

mixture, followed by crystallization of 2 3 compound 5 remained

in the mother liquor. Reduction of this diulose acetal (2)

with sodium borohydride in aqueous methanol gave, by stereospecific reaction, 1,2:4, 5-di-0-isopropylidene-g- ribo-hexulopyranose (3) almost exclusively, together with a trace of the

corresponding g-fructose acetal (1), presumably removed on

recrystallization of compound 3. Acid hydrolysis of compound

3 then gave D-psicose (4).

42
Because this synthesis provides a direct means for producing D-psicose, which heretofore had been hard to obtain, it

was decided to repeat this procedure to produce a supply of

the crystalline di-O-isopropylidene-D-psicose available for

issuance as a reference compound under the Bureau's Standard

Reference Materials Program. McDonald's article was published

in September, 1967, and, in December, 1967 3 a more detailed

article describing similar work was published independently

[51]. Many of the conclusions described in the latter article

had already been arrived at by us

In our study, McDonald's procedure was first repeated in

accordance with her instructions. Compound 1 was oxidized with

a solution of acetic anhydride (1 molar proportion) in methyl

sulfoxide (70 molar proportions), and crude compound £ was

isolated in a yield of 50% of the theoretical, as compared with

the 70% yield of crude product reported by McDonald [50]. Because of this result , a series of experiments was performed in

order to determine what molar proportion of acetic anhydride

would optimize the yield of diulose acetal 2 from 1. [In the

course of this work, the reaction was followed by thin-layer

chromatography on Silica Gel G, and we found that 1:3 (v/v)

ethyl acetate—pentane is preferable to the 3:1 (v/v) ethyl

acetate—petroleum ether used by McDonald as the developing solvent.] The results are given in table 4, from which it would

Table 4. Comparison of yield of diulose acetal (2) obtained

with various molar proportions of acetic anhydride

and methyl sulfoxide, per molar proportion of

acetal 1

Molar proportion to 1

Acetic anhydride I Methyl sulfoxide

1

2

16

21

70

42

42

42

Yield of compound £

Crude ( %) | Recrystallized {%)

50

61

54

50

52

43
appear that a molar ratio (of acetic anhydride to 1) of 2:1

gives a slightly higher yield of compound £ than a ratio of

1:1, but that no improvement in yield is obtained by a further

increase in the ratio. In this connection, we made a comparison of the molar proportions that had been given in the literature for other such oxidations; these are listed in table 5,

together with information from articles published subsequent

to that of McDonald [50]. From table 5, it may be seen that,

for different compounds to be oxidized, the ratio of acetic

anhydride to the alcohol has lain in the range of 23-1:1 to

1.08:1, and for the ratio of methyl sulfoxide to the alcohol,

in the range of 564:1 to 14.5:1. It would therefore appear

that, in this reaction, the proportions of reactants employed,

over the range studied, are not critical.

Table 5. Molar proportions of acetic anhydride and methyl

sulfoxide, per molar proportion of the alcohol,

employed in oxidations at 25-30°C

Reaction

Molar proportion time

Acetic anhydride Methyl sulfoxide (hours

)

References

21.5 42.6 18-24 [52]

21.5 42.6 24 [53]

21.5 564.0 78-96 [54]

21.5 42.5 24 [55]

1.08 70.5 48 [50]

6.5 28.3 36 [56]

23.1 45.8 24 [51]

7.4 14.5 48 [57]

According to Albright and Goldman [58], the reacting

species is formed by reaction of acetic anhydride with methyl

sulfoxide

Ac 2 + Me 2 S0 -> [Ac-oSMe 2 ]0Ac

44
and this acetoxysulfonium acetate then reacts with the alcohol

to give, via the intermediate alkoxysulfonium salt, the ketone

and methyl sulfide.

C(H)OH + [Ac-0S~Me 2 ]0Ac C(H)OSMe (H)OS"l 2

I OAc C=0 + Me 2 S

This reaction sequence would indicate that one molar proportion of acetic anhydride and one molar proportion of methyl

sulfoxide, per molar proportion of compound 1, should be

sufficient to effect complete oxidation of 1 to ,2. However,

the intermediate acetoxysulfonium ion must be present in

sufficient concentration for nucleophilic attack by compound

.1 to occur. Consequently, only sufficient methyl sulfoxide

need be employed to provide the one molar proportion required

for the reaction together with a sufficient excess to act as

the solvent. Possibly, the solvent effect could be provided

by dry benzene or dry ether, thus eliminating the rather

tedious removal of excess of methyl sulfoxide at the end of

the oxidation.

In all of these experiments, we found that, accompanying

compound 2, there was a large proportion (25% or more) of a different, crystalline compound (A) that, on the basis of

thin-layer chromatography [Silica Gel G, with 3:1 (v/v) ethyl

acetate—petroleum ether; sulfuric acid spray for detection],

had been regarded by McDonald [50] as being the 3-acetate (5)

of compound 1.. However, we found that the by-product A, which

had mp 8l-82°C and [a]* 1* +28° (c 1.25, chloroform), showed no

carbonyl (or hydroxyl) absorption in its infrared spectrum,

and therefore could not be the acetate 5. To verify this conelusion, we prepared an authentic specimen of acetate 5 by the

method of Fischer and Noth [59] , and found this compound to

have mp 7^-76°C, [a]* 4 -171.3° (c 1.0, ethanol); lit. [59] mp

76-77°C, [a] D

-176.3° (c 0.083, ethanol); its infrared spectrum,

which shows bands at 1755 (C=0) and 1225 cm" 1 (C-0), is entirely

different from that of compound A. A mixture of compound A

45
o. 0,

Me —

6 (R = MeSCH2 -)

with compound 5 had mp 63°C. By-product A was identified as the 3-(methylthio)-methy 1 ether (6) of compound 1. and this

conclusion has also been reached in an article (published after

completion of this aspect of our work) by Tatchell and coworkers [51] • The (methylthio)methyl ether produced in the reaction

may be formed [58] by either pathway (1) or (2_) .

Ac-0^Me 2 * AcO CH 2 =&Me (la)

C(H)0H + AcO CH 2 =SMe -» C(H)0CH 2 -SMe

I I Me Me

1 ± I ± C(H)0SMe 2

-> C(H)OS c(H)o;

CH : *

(lb)

I C(H)OCH 2 -SMe (2)

CH.

To effect purification of the diulose acetal (2),

McDonald reported lising methanol as the recrystallization

solvent. McDonald [50] found mp 101-102°C; [a]£ 5 -100.4°

(c_ 2.41, acetone). Petroleum ether (bp 30-60°C) or

chloroform—hexane has been found to be satisfactory for the

recrystallization. After three recrystallizations , the

yield was 41.5JS- mp 99-102°C; [a]^ 5 -97-5° (c 2.40, acetone)

[a]^ -126.4° (c 1.91, chloroform); homogeneous by t.l.c.

Tatchell etal. [51] obtained a 53$ yield of purified 2;

mp 101. 5-102. 5°C; [a]* -126.4° (c 1, chloroform). Hence,

our results are in close agreement with those of Tatchell

46
and co-workers [51]. We have repeated their method of isolation of compound 2 3 and find it satisfactory.

The reduction of purified compound 2 (free from 1 and 6)

was effected with sodium borohydride (9.5 molar proportions)

in water (0.155 g per ml); this solution was quickly added,

with stirring, to an ice-cold solution (0.035 g per ml) of

the diulose acetal (2) in 1:3 (v/v) water—methanol . After

30 minutes, t.l.c. with 1:3 (v/v) ethyl acetate—pent ane indicated that the reaction was complete, and that only one

product appeared to be present. The solution was evaporated

to dryness under diminished pressure, the residue was dissolved in water, and the solution was extracted repeatedly

with chloroform or ether. The extract was dried, and then

evaporated to a clear syrup which crystallized on trituration

with petroleum ether (bp 30-60°C). The crude product was, in

fact, a mixture of 1,2 : 4 , 5-di-O-isopropylidene-D-psicopyranose

(3) with compound 1; it was recrystallized from petroleum ether

(bp 30-60°C); yield 87-5$, mp 64.5-67°C; [a]

D

-111.0° (c 2.12,

acetone). McDonald [50] reported a yield of 60% , and mp

60-6l°C, but did not report the specific rotation. Tatchell

and co-workers [51] obtained an 87% yield of 3 after three

recrystallizations from petroleum ether; mp 64-65°C; [a]j;°

-126.8° (c_ 1, chloroform); homogeneous by gas—liquid chromatography of its trimethylsilyl ether.

We have also studied the method of Jones and Nicholson

[60] for the preparation of D-psicose from fj-fructose. This

method involves the methanesulfonylation of compound 1 to

give 1,2:4, 5-di-0-isopropylidene-3-0-(methysulfonyl)-Dfructopyranose (7). Compound 7 was then hydrolyzed to the

monoacetal (Q) > and this was hydrolyzed further, with 0.1 M

sulfuric acid, to give 3-0-(methylsulf onyl)-D-fructose (depicted as the pyranose form 9)- Compound 9 was treated with

0.3 M sodium hydroxide solution, causing simultaneous deesterification and epimerization at C-3 to give D-psicose (4).

47
HO

8

Ba(OMe) 1 2 ^H,CH2OH 4

As we have found that de-esterification—epimerization does not

occur with the 1,2-isopropylidene acetal (8) of 9, the reaction presumably involves formation of 2 , 3-a-nhydro-D-psicose

as an intermediate.

Because sulfuric acid is generally regarded as unsuitable

for the hydrolysis of ketose derivatives, we examined the

hydrolysis of 7 with 0.1 M oxalic acid at 65°C. During the

course of the hydrolysis, crystalline 8 separated out; after

2 hours, the suspension was cooled and filtered. The yield of

8 was 75-5% of the theoretical, and compound 9 was present in

the filtrate [as shown by t.l.c. with 10:1 (v/v) chloroform—

methanol]. In a repetition of the experiment, except that the

solution was boiled under reflux, all of the crystals had dissolved after 1.5 hrs; and, after neutralization with calcium

carbonate and filtration, compound 9 was isolated from the

filtrate. Also, 4:1 (v/v) acetic acid—water, in which the

monoacetal 8 is readily soluble, proved to be a satisfactory

hydrolyst. This work is being continued.

48
2. CLINICAL CHEMISTRY

Laboratories providing clinical analysis are called on

to perform ever greater numbers of determinations; an annual

growth in output of 20 percent or more is not uncommon. To

contend with this problem, automated methods and other fast

methods of analysis are coming into widespread use. Despite

the earnest efforts of clinical chemists and pathologists to

achieve high standards of precision and accuracy under these

burgeoning workloads, surveys taken throughout the Nation

clearly demonstrate that actual attainment is far from ideal.

As a consequence, a vigorous program for upgrading performance

in these essential laboratories is being undertaken under the

aegis of professional societies, Industry, and governmental

organizations. To facilitate this program, the National

Bureau of Standards' Office of Standard Reference Materials

is underwriting the development of a series of organic and

inorganic Standard Reference Materials certified for use in

clinical chemical analysis. Each standard material, certified

as to its purity, will enable medical laboratories to calibrate

their instruments, to test their procedures, and to relate

their analytical results to a common base; hence, their widespread use should prove fundamental for instituting the needed

improvement

.

Only a small proportion of clinical laboratories perform

analyses for anywhere near the gamut of medically significant

constituents, and although standards for virtually all of these

analyses are needed, the Section is addressing itself initially to standards for bilirubin, cholesterol, creatinine, Dglucose, urea, and uric acid, because analyses for these six

constituents preponderate over all others and are conducted by

virtually all clinical laboratories

.

Cholesterol was the first of the standards to be undertaken, and, during the year, a provisional standard cholesterol

was issued. Because the shelf-life of this standard is of

49
great concern, periodic re-examinations have to be carried out;

those performed to date are described in this report, along

with additional techniques for the determination of its purity.

Substantial progress with creatinine, uric acid, and urea has

been made; descriptions of the preliminary examinations of

samples from several suppliers are included in this report,

although work with the standards has begun since the time these

reports were assembled. Our first studies on bilirubin have

also begun. Work with this compound, which is well known to be

exceedingly unstable, is expected to require much effort and

time before a standard bilirubin can be issued. Descriptions

of these early experiments are being deferred until a more

comprehensive report can be made. Work toward the issuance

of a clinical standard for D-glucose is expected to commence

shortly. Finally, a g-fructose standard for clinical analysis

has also been requested and begun. This work also is too incomplete for presentation now.

A . Creatinine

( R. S. Tipson and R. F. Brady, Jr.)

As an extension of the efforts of this Section to provide standard reference materials for clinical use, the amino

acid derivative creatinine (1) is being examined. The criteria

CH3

I ,N

NH,

<? N

1

established by this research will be used to certify a supply

of creatinine for issue as a Standard Reference Material.

50
Samples of creatinine of the best grade available were

obtained from eight commercial sources. Each was subjected

to intensive scrutiny by a variety of chromatographic,

spectrophotometries and other methods. In addition, samples

of sarcosine (2), cyanamide (3), and creatine (4) were subr-w SS* *** jected to the same kind of examination. These compounds are

the most likely contaminants of creatinine (1). since it is

produced commercially either by the condensation of 2 with 3,

or by the cyclization of 4 with hydrochloric acid.

CH 3 NHCH 2 COOH + H 2 NCeN -»• 1 + H 2

2 3

CH 3 NCH 2 COOH HC1

i 1 + H 2 HN=CNH 2 ~

4

1

.

Some Physical Properties of Creatinine

Creatinine is a colorless, crystalline compound, which

melts with decomposition at about 300°C and is optically

inactive. It is soluble in water (1 g in 11 ml at 25°C),

slightly soluble in methanol or ethanol, and almost insoluble

in ether, chloroform, or acetone [6l]. The pH of a saturated

aqueous solution (0.805 M) at 25°C is 7.8.

2

.

Preparation of Purified Creatinine for Comparison

Creatinine of the best commercial grade was recrystallized by dissolution in the minimal volume of distilled water,

followed by precipitation with acetone [62]. It is incompletely dried at 25°C/0.1 torr over phosphorus pentaoxide in

24 hours. An elevated temperature (110°C for 24 hours) leads

to complete drying.

Anal. Calc. for C 4 H 7 N 3 0: C, 42.5; H, 6.2; N, 37.2.

Found: C, 42.5; H, 6.0; N, 37-2; CI, <0.02; ash, 0.02$.

51
3 • Loss of Weight on Drying

For each commercial sample, the loss in weight on drying

was less than 0.5% after 24 hours at 25°C/0.1 torr. All

samples were so dried prior to quantitative study.

4 . Chromatography of Creatinine

The samples of creatinine were studied by thin-layer

chromatography (t.l.c.) and by paper chromatography,

a. Thin-layer Chromatography

Plates were prepared by coating glass microscope slides

(75 x 25 mm) with a slurry of Kieselguhr G in distilled water;

after being dried, the plates were activated by heating at

110°C for 1 hour, and were stored in a desiccator prior to

use. Samples were spotted as saturated solutions in methanol,

and developed in 4-oz, screw-capped jars lined with Whatman

No. 1 chromatography paper. Pour solvent systems were employed, namely, (a) 7:1:2 (v/v) butanol—acetic acid—water [63];

(b) 460:1:39 (v/v) butanone—diethylamine—water [64]; (c) 19:1

(v/v) methanol—water [65]; and (d) 4:1:1 (v/v) butanol—ethanol—

water [66]. The R values of the various compounds in these

systems are listed in table 6.

Table 6. R values in thin-layer chromatography

Solvent Creatinine S arcosine C reatine Cyanamide

a 0.28 .14 0.19 0.84

b 0.08 .05 .04 0.86

c 0.61 0.34 0.32 0.98

d 0.31 0.10 0.13 0.75

After the plates had been air-dried, the spots were made

visible in one of five ways.

(1) The plates were heated on a hot plate until the

material charred. Well-defined, brown spots on a clear,

white background are produced in this way. Only cyanamide

was not made visible by this technique.

52
The following Indicator sprays were used as alternative

methods of detecting the compounds

.

(2) 5:1 (v/v) Methanol—concentrated sulfuric acid, followed by charring as above. This technique invariably produced black spots on a dark-gray background, and was inferior

to method 1. Creatine and cyanamide were not detectable with

this spray.

(3) Aqueous ferric chloride solution (2%) [64]. This

reagent produced yellow spots on a tan background; definition

of the spots was improved considerably when the plates were

charred after spraying as in (1) above. This spray did not

detect creatine.

(4) Chloranilic acid (0.1$) in amyl acetate \_66~\ . This

spray produced orange spots on a pale-blue background. As in

other methods, the spots could be made darker by charring.

The sensitivity of this reagent makes it very suitable for

creatinine, but it does not make cyanamide visible.

(5) Fresh sodium picrate solution, prepared by mixing

5 volumes of saturated, aqueous picric acid solution with 1

volume of 10% aqueous sodium hydroxide solution [62]. This

spray produces dark-red spots on a yellow background, and

gives a positive reaction with creatinine, sarcosine, creatine,

and cyanamide. The spots may be made darker by charring. This

spray was considered to be the most satisfactory means of

detecting the spots

.

b . Paper Chromatography [62]

Ascending paper-chromatograms of creatinine were obtained

on strips of Whatman No. 1 chromatography paper (40 x 4 cm)

by use of the following developers: (a) 18:2:5 (v/v) butanol—

acetic acid—water, FL-, 0.38; and (b) 5:1 (v/v) phenol—water

containing 15 mg of B-quinolinol per 100 ml, and run in the

presence of 2 M— ammonia, R_ 0.93- A spray of fresh sodium —

r

picrate solution [method (5), t.l.c] was used for detecting

the spots

.

53
c. Gas—liquid Chromatography

A sample of creatinine was trimethylsilylated (by D. H.

Freeman, D. Enagonio, and C. B. Romain) , and the product was

examined by gas—liquid chromatography (g.l.c). A solution

of creatinine (50 mg) in 4 ml of pyridine containing 0.5 ml of

hexamethyldisilazane and 4 drops of chlorotrimethylsilane was

refluxed for 1.5 hours. The solution was cooled to room

temperature, and 1 ul was chromatographed on a Hewlett—Packard

Model 5750 vapor-phase chromatograph equipped with a stainlesssteel column (0.32 x 183 cm) of 10$ silicone gum-rubber

(U.C.W.-98) on 80—100-mesh Chromosorb W, The column temperature was programmed from 100-300°C at 30°C/min. Helium was

used as the carrier gas at a flow rate of 40 ml/min, and a

f lame-ionization detector was used. The chromatogram showed

two main peaks, a symmetrical doublet with a mean retention

time of 4.4 minutes. This is compatible with the formation

of isomeric trimethylsilylated derivatives of creatinine.

G.l.c. studies are being continued in order to obtain unequivocal data.

d. Results

With the exception of the gas—liquid chromatographic

data, which are still under investigation, each sample of

creatinine appeared to be homogeneous by each of these

chromatographic methods.

5 • Spectrophotometric Methods

a. Ultraviolet (u.v.) Spectrophotometry

Approximately 1 g of creatinine was accurately weighed

(to 10 jig) and dissolved in water in a 1-liter volumetric

flask. The u.v. spectrum of each sample in a 100-fold dilution of the original solution was recorded with a Cary Model

14 u.v. spectrophotometer. The u.v. spectra were measured,

against water as the blank, from 300 to 210 nm at a scanning

speed of 20 nm/min. Creatinine shows one absorption peak,

at X^° 234 nm (log e 3.853) [67].

54
H D Ultraviolet spectrophotometry showed for creatine X

2 max

190 nra (log e 4.09), sarcosine 265 (2.32), and cyanamide

214 (2.65). The u.v. spectrum of only one of the creatinine

samples showed contamination by sarcosine. No other contaminants were revealed in any of the other commercial samples by

the u.v. studies.

b . Infrared (i.r.) Spectroscopy

Infrared spectra were recorded with a Perkin—Elmer

Model 257 grating infrared spectrophotometer. Creatinine in

a potassium bromide pellet gives a well-defined i.r. spectrum

having major bands at 3260 (NH 2 asymmetric stretch), 3040

(NH 2 symmetric stretch), 2810 (CH symmetric stretch), 1688

(C=0 stretch), 1665 (C=N stretch), 1588 (NH 2 asymmetric bend),

1498 (NH 2 symmetric bend), l4l8 (CH 3 symmetric bend), 1330,

1242, 1210, and 1202 (doublet), 1118, 1040 (all C-C or C-N

stretches), 842, 812, and 680 cm

-1 (all NH 2 rocking). The

absence of impurities in commercial samples may be inferred

from the absence of bands at 1052, 908, and 905 cm

-1 for

sarcosine, and at 2260 cm

-1 for cyanamide (the strongest bands,

only, are listed for impurities); however, the absence of

bands at these positions cannot be interpreted quantitatively.

All of the commercial samples of creatinine showed a small

band at 2260 cm

-1

. Since cyanamide was not detected in their

u.v. spectra, this peak may be attributed to a trace of cyanide ion in the creatinine. This impurity was removed by

recrystallization from 4 M ammonia by addition of acetone.

As far as we are aware, the i.r. spectrum of creatinine

has not been published, although it was formerly available

on a punch-card, now out of print [68]. Consequently, the

i.r. spectrum for the range of 2.5 to 16 ym is reproduced

in Fig. 4 (see page 63).

6 . Stability of Creatinine

Creatinine appears to be stable indefinitely at room

temperature when stored as an anhydrous powder [62] . There

55
is, however, an equivocal report in the literature stating

that, in solution in aqueous hydrochloric acid, or aqueous

acetic acid, creatinine undergoes fairly rapid hydrolysis,

at 10-50°C, to give an equilibrium mixture with creatine [69].

Therefore, in order to evaluate the stability of creatinine

under routine conditions in the clinical chemistry laboratory,

several typical solutions of creatinine were prepared and

stored at room temperature. The u.v. spectra of the solutions were measured after the elapse of various intervals of

time . Solution 1. Creatinine (1. 38863 g) was dissolved in

water, and the solution was made to 1 liter with water. A

50.00-ml aliquot of this solution was diluted to 1 liter

with distilled water in a second, 1-liter volumetric flask;

then, a 50.00-ml aliquot of the resulting solution was

diluted further to 250 ml.

Solution 2. Creatinine (1.51159 g) was dissolved in

sufficient 0.1 M hydrochloric acid to make 1 liter of solution ("Stock Standard" [70]). For the u.v. measurements, a solution was prepared by diluting 10.00 ml to 1 liter with

distilled water immediately before use.

Solution 3. A 1.00-ml aliquot of solution 2 was diluted

to 100.00 ml with distilled water. The u.v. spectrum of this

solution was measured without dilution ("Working Standard"

[70]).

Solution 4. Creatinine (0.17^77 g) was weighed out,

transferred to a 1-liter volumetric flask, and dissolved in

some distilled water. One equivalent (14.17 ml) of 0.109 M

hydrochloric acid was added, and the solution was made to

the mark with distilled water. For each u.v. measurement,

10.00 ml of this solution was diluted to 100.00 ml with distilled water, immediately before use.

Solution 5. A saturated solution of creatinine in

methanol was prepared by weighing excess material ( ca. 1 g)

into a 125-ml Erlenmeyer flask, adding 80 ml of methanol,

56
heating the solution at 45°C under a reflux condenser for

3 hours, and allowing it to stand at room temperature. The

solution was separated from the residue by filtration, and

its u.v. spectrum was obtained (against methanol as the

blank) . During the first 4-week period of examination, none of

these solutions showed anyJ dimunution in e . This investimax

gation is continuing.

7 . Quantitative Determinations of Creatinine

a. Potentiometric Titration

The tertiary amine function in creatinine can be titrated

with 0.1 M hydrochloric acid. The end-point is, however, very

gradual and cannot be determined with standard indicators.

The titration curve, determined potentiometrically , also shows

an indefinite end-point; however, the first derivative of this

curve, ApH/AVu+ , shows a sharp maximum corresponding to the

n

end-point. (Calculation of the curve of the first derivative

is facilitated if the titrant is added in small aliquots having constant volume.) Results of a triplicate analysis of one

sample of creatinine by this technique are given in table 7.

Table 7- Analysis of creatinine by titration

Experiment Mole titrated Mole found Error

1 0.89197 0.89489 +0.327$

2 0.99628 1.00171 +0.545$

3 0.83620 0.83821 +0.240$

Average error +0.371$

b . Differential Scanning Calorimetry [71-73]

The Perkin-Elmer DSC-1B Differential Scanning Calorimeter

was employed as another possible means of measurement of the

purity of the creatinine samples. Not unexpectedly, however,

the amount of energy required for heating the sample to its decomposition temperature differed for each trial, and also for

57
each set of operating parameters, This variability was attributed to the decomposition of creatinine at its melting point.

Hence, differential scanning calorimetry is not suitable for

analysis of creatinine.

c . Phase Solubility Analysis [_7jj]

The solubility of a material in a certain solvent is,

like its melting point or index of refraction, a fundamental

physical property of the material. This property is exploited

in phase solubility analysis, which was employed in an attempt

to obtain quantitative data on the purity of creatinine. This

technique measures the amount of material dissolved when increasing amounts of the material are equilibrated in a constant volume of solvent. In saturated solutions of the material being tested, the increasing amounts of the mixture that

are dissolved with increasing sample-weight are assumed to be

due to the increasing amount of dissolved impurity. Thus, the

amount of impurity in the sample can be determined. The solubility of creatinine in methanol, absolute ethanol, 95% ethanol, ethyl acetate, and pyridine was measured, in order to

determine the most suitable solvent for the phase solubility

analysis. Methanol, in which creatinine has been found soluble to the extent of 8.1 mg/g, was chosen. Sealed tubes containing weighed, progressively larger weights of creatinine

and uniform weights of methanol were allowed to equilibrate in

a water bath at 25.1±0.1°C. The weights of creatinine were so

chosen that at least one tube would contain an undersaturated

solution. After equilibration, aliquots of each solution were

separated from any undissolved creatinine, evaporated to dryness, and weighed. A graph of system composition (mg of sample

per g of solvent) versus solution composition (mg of solute

per g of solvent), known as the phase solubility diagram, was

drawn from the data. The content of solids throughout the

range of saturated solutions studied was found to increase

slightly. The sample of creatinine investigated was found to

be about 99.0% pure by this technique. This method is general58
ly considered accurate to ±0.1%.

8 . Creatinine as a Standard Reference Material

A large quantity of that commercial sample found to be of

the highest purity is on order. This material will be examined

by the useful techniques described, for issuance as a Standard Reference Material.

B. Uric Acid

( R. S. Tipson and B. F. West)

Because clinical chemists require standard samples of

uric acid (1), the development of such a standard was undergo

taken. As a preliminary, the properties of eight different

commercial specimens were examined.

1. Some Physical Properties of Uric Acid

Uric acid is a colorless, crystalline compound which

decomposes at about 400°C and is optically inactive. It is

slightly soluble in water ( ca. 1 g in 15,000 ml at 25°C),

soluble in aqueous solutions of alkali hydroxides or carbonates, sodium acetate, or sodium phosphate, and almost insoluble in alcohol or ether [75]. Commercial uric acid is

obtained from snake excrement or guano, by dissolution in

base, followed by precipitation with acid.

As a check of solubility of samples under the conditions

used in clinical chemistry, a solution containing 1 mg of

uric acid per ml of solution was prepared as follows. Lithium

carbonate (0.50 g) was dissolved, with stirring, in 150 ml

of water at 60°C in a beaker. Uric acid ( ca. 1 g) was care59
fully weighed and transferred to a 300-ml Erlenmeyer flask

having a funnel in the neck. The hot solution of lithium

carbonate was then added to the flask, care being taken to

wash all traces of the uric acid in to the flask, and the suspension was shaken until all of the uric acid had dissolved.

The solution was then cooled to room temperature, transferred

to a 1-liter, volumetric flask, and rinsed in with water (to

a volume of ca. 500 ml). Formaldehyde solution (37%; 20 ml)

was added, and the mixture was shaken. Glacial acetic acid

(3 ml) was added next, and the solution was shaken until evolution of carbon dioxide had ceased. The solution was then

diluted to 1 liter with water, the flask was stoppered and

shaken, and the solution was preserved in a refrigerator.

2 . Preparation of Purified Uric Acid for Comparison

Purification of a commercial sample of uric acid was

accomplished as follows [75]. Lithium carbonate (1.1 g) was

dissolved in 75 ml of distilled water at 90°C, and to the

solution was added ca. 2.5 g of uric acid. The suspension

was stirred at 90°C until the uric acid had dissolved, and

to the resulting solution were added activated carbon (Darco

G-60) and Celite. The suspension was vigorously stirred,

and then filtered while hot. The filtrate was cooled to room

temperature, and glacial acetic acid (2.5-, 2.0-, and 4.0-ml

portions, at 15-minute intervals) was slowly added dropwise,

with stirring, until no more crystallization of uric acid occurred. The crystals were filtered off with suction, washed

with distilled water, and briefly air-dried. Three recrystallization performed in this way afforded a sample of crystalline

uric acid whose u.v. and i.r. spectra were found to be practically identical with those of the starting material (before

recrystallization) . The compound was dried for 24 hours at

25°C/0.1 torr.

Anal. Calc. for C5H 4 N^0 3

: C, 35.7; H, 2.4; N, 33-3-

Pound: C, 35.7; H, 2.5; N, 33-3; ash, 0.02%.

60
3. Loss of Weight on Drying

In order to avoid possible decomposition of the uric acid

by heating, each specimen was dried to constant weight at

25°C/0.1 torr; for each commercial sample, the loss in weight

was less than 0.13$ after 24 hours at 25°C/0.1 torr. All samples were so dried prior to quantitative study. Oven-drying

at 110°C for 18 hours causes a somewhat greater loss in weight.

4

.

Chromatography of Uric Acid

The samples of uric acid were studied by thin-layer

chromatography and by paper chromatography,

a . Thin-layer Chromatography

Thin-layer chromatography was performed on plates (7-5

x 2.5 cm) of Silica Gel G, activated at 100°C, with the following solvent systems: (a) 1:1 (v/v) isorpropy alcoholwater; (b) water saturated with butanol; (c) butanol saturated

with water; (d) 2:23 (v/v) butanol—water ; and (e) 10:6:5:3

(v/v) ethyl acetate—methanol—water—heptane . The spots were

made visible with iodine vapor, after the plates had been

air-dried.

In each of these solvent systems, each of the commercial

samples of uric acid showed the presence of only one component.

The R values were, for solvent a, 0.53; b, 0.92; c_, 0.92;

d, .F0; and e; 0.0.

b . Paper Chromatography [76]

Paper chromatography was performed by the ascending

technique on strips of Whatman No. 1 chromatography paper,

with the following solvent system: 9:3:2:2:4 (v/v) butanol—

acetone—aqueous ammonia ( 5% )—acetic acid—water. An aqueous

solution of each sample was spotted onto a strip. The spot

was made visible under u.v. light (254 nm) . Each of the

commercial samples showed the presence of only one component,

c . Gas—liquid Chromatography [77]

Gas—liquid chromatography was performed by D. H. Freeman,

D. Enagonio, and C. B. Romain , on a trimethylsilylated sample

of uric acid. The trimethylsilyl derivative was prepared by

61
treating the uric acid with hexamethyldisilazane , with chlorotrimethylsilane as a catalyst, in pyridine under reflux for

1.5 hours. The analysis was performed by injection of a sample

(1 ul) of this liquid. A Hewlett—Packard gas chromatograph

Model 5750 was used with a stainless-steel column (0.32 x 183

cm), and the column support was Chromosorb W (80—100 mesh) with

a liquid phase of 10% (w/w) silicone gum-rubber (U.C.W.-98).

The column temperature was programmed from 100-300°C at 30°C/min

Helium was used as the carrier gas at a flow rate of 40 ml/min.

A chart speed of 0.5 in/min was maintained, and detection was

by flame ionization. A single, major peak was noted; this was

well formed, with a retention time of 6.7 minutes. Two very

minor peaks, having relative retention times of 0.97 and 1.08

minutes, were also observed; whether they represent minute

traces of impurity in the uric acid, or artifacts formed during trimethylsilylation has not yet been decided. Now that

the usefulness of this examination has been established, it

will be applied routinely for study of samples of uric acid.

5 . Spectrophotometric Methods

a. Ultraviolet Spectrophotometry

For each sample, the uric acid (O.OI67 g; 94.4 mmoles)

was dissolved in 2 ml of saturated lithium carbonate solution

(1.52 g of lithium carbonate in 100 ml of water) at 25°C, and

the solution was diluted to 100 ml with water. For recording

the ultraviolet spectrum, an aliquot (1.00 ml) of this solution was diluted to 25.00 ml with distilled water. The u.v.

spectrum was recorded with a Cary Model 14 spectrophotometer,

against a solution of lithium carbonate as the blank, from

330 to 210 nm at a scanning speed of 20 nm/min. The purified

sample of uric acid showed A p ' '* 292 nm (log e 4.080).

nicix

Commercial samples showed A p ' '* 292 nm (log e 4.060 to

max &

4.089)

Because the values of the extinction coefficient are

recorded in the literature [75] for stated values of pH, the

ultraviolet spectra were also recorded for solutions of the

62
iles of uric acid in glycine buffer solution of pH 9.45.

dH 9 45 results for the purified sample were A^ 292 nm

tt n ii max

; e 4.086); lit. [75] A pn b ^ 292 nm (log e 4.097). A

max

tercial sample had X P^J 5 292 nm (log e 4.087). max

b . Infrared Spectroscopy

The infrared absorption spectrum of each sample of uric

., in a potassium bromide pellet, was recorded with a :in-Elmer Model 257 grating infrared spectrophotometer.

iough the i.r. spectrum of uric acid for the range of 15

!'3 ym has been published [78] , that for the range of 2 to

.m seems only to have been made available on a punchcard,

out of print [79]. Consequently, the i.r. spectrum for

range of 2 to 15 ym is reproduced in Pig. 4. For each

MICROMETERS

5.0 6.0 7.0 8.0 9.0 10 12 14 16

I I I I I I I | I I I I I I I

I I

|

I I I I III II|M!M1IMI|1IIIIIII[ 1—| | | | | |

|

| |.

I I I I I I I I ) I I I I I I M I

|

I II IIMII|IIIMIIIII|IIIIIIII|—I I I | I I I |

[ U4000 3500 3000 2500 2000 1800 1600 WAVENUMBER.cnrr1

1400 1200 1000 800 625

ire 4 Infrared absorption spectra of creatinine and uric

acid, each in a potassium bromide pellet.

63
of the commercial samples, the i.r. spectrum was identical

with that shown in Pig. 4. The principal bands are at 3021

(NH, OH, CH), 1675 (C=0), 13^2 (NH), and 1299 cm

-1 (NH).

6 . Stability of Uric Acid

It has been reported [75] that lithium carbonate solutions

of uric acid at pH 7.5 are unstable at room temperature. In

order to gain information in this regard, stock solutions of

uric acid were prepared according to published procedures [80],

and the solutions were stored under a variety of conditions,

and then examined after the elapse of various periods of time

bv means of their u.v. spectra.

Stock solutions of uric acid were prepared by dissolving

100 mg (accurately weighed) of uric acid and 60 mg of lithium

carbonate in ca. 50 ml of water at 25°C, and diluting to exactly 100.00 ml with distilled water. Samples of stock solution in glass-stoppered Erlenmeyer flasks were kept (a) in 1 the dark at 25°C, at 5°C (refrigerator), and at -7°C (freezer),

and (b) exposed to sunlight , or to laboratory light , at room

temperature (25°C). Measurements were made with 10.00-ml

aliquots of each solution, diluted to 1 liter with distilled

water. After 1 week, the solution exposed to sunlight showed

almost complete decomposition; it was devoid of a peak at

A292 nm. Those solutions kept at 25°C (light and dark) for

1 week showed considerable diminution of £292 , but those solutions stored in- a refrigerator or in a freezer for 10 days

remained unchanged. The decomposition might be due to microbiological action; however, this possiblity was not explored.

Consequently, it is imperative that clinical chemists

who use stock solutions of uric acid should minimize the

length of time that the solutions are handled at room temperature . 7- Uric Acid as a Standard Reference Material

On the basis of the foregoing tests, the most suitable

supplier of uric acid was selected, and a large batch of it

was ordered. This material will be subjected to many of the

64
same tests, before being issued as a Standard Reference

Material

.

C. Urea

(B. Coxon)

In order to assess the quality of urea available from

commercial sources and to ascertain the suitability of any of

them as a standard material, the purity of four samples of urea

(samples 1, 2, 3, and 4) was examined by means of various

analytical techniques . Solutions for chromatography were prepared by dissolving a portion (100 mg) of each of the urea

samples in methanol (1.0 ml). These solutions were stored in

a refrigerator when not in use.

1. Thin-layer Chromatography

Thin-layer chromatography (t.l.c.) was carried out by the

ascending method on glass plates, either 20 x 20 cm or 20 x 5

cm, which were coated with an aqueous slurry of Silica Gel G

(Merck). The plates were air-dried, but were not further

activated. The solvent mixtures (i) 9:1 (v/v) chloroform—

methanol, (ii) 1:1 (v/v) dichloromethane-methanol, and (iii)

1:2 (v/v) dichloromethane—methanol were tested as developers.

In solvent (i), the urea barely moved from the point where it

had been deposited, as indicated by a dark spot seen under

ultraviolet irradiation. Solvent (Iii), In which the urea

had R_ 0.6, was selected as the most suitable solvent for

general testing. In order to obtain an impure sample of urea

for purposes of comparison, a portion of urea, sample 2, was

heated at l80°C for five hours at atmospheric pressure (sample 5). Ammonia was evolved, and the material then gave a strong positive test for biuret (see p. 66).

As with samples 1-4, a 10$ w/v solution of 5 was prepared.

The samples were tested for the presence of biuret and cyanuric

acid by chromatography against commercial samples of these

compounds, which are known to be products of the pyrolysis of

urea [8l]. Biuret and cyanuric acid were spotted as saturated

65
solutions in 20% aqueous methanol. The following indicating

reagents were tested.

a. 5% Sulfuric Acid

The sprayed plates were heated at 150°C. Samples 1-4

each showed as a single, colorless spot with a dark-brown

corona on a light-brown background. Sample 5 showed the same

spot, and also a darker, slightly faster-moving spot having

the same R_ value as the spot which showed for biuret. How- —

p

ever, the appearance of the latter spot was different, in that

its front was dark and crescent-shaped. Cyanuric acid was not

detected by this reagent

b . 1% Ninhydrin in Ethanol

This reagent was found to be insensitive for urea. A

faint, purple corona was produced on prolonged heating of the

plate at 120°C, but this disappeared on further heating.

c.2% Ninhydrin in 19:1 (v/v) Acetone—Pyridine

Followed by 1% Ferric Chloride in Methanol

This combination of sprays has been recommended as the

reagent of choice for the detection of mixtures of urea,

hydroxyurea, O-carbamoylhydroxylamine, and (2-hydroxyethyl)urea

[82]. Samples 1-3 each showed as a single, colorless spot on

a yellow background; the contrast was not goodj however.

d. Biuret Reagent

This was prepared by mixing a 3% solution of copper sulfate (12.5 ml) with 10% sodium hydroxide solution (500 ml) [83].

When sample 4 was mixed with biuret reagent in a test tube,

a strong positive test for biuret (red coloration) was obtained.

However, when t.l.c. of sample 4 on silica gel was carried out,

nothing could be detected with the biuret reagent when used as

a spray, either before or after heating the developed plate at

150°C for 10 min. In an effort to intensify the color, higher

concentrations of reagents were used. Although sample 4 responded as a faint purple coloration when its undeveloped

spot bn filter paper was sprayed with saturated copper sulfate

solution followed by 10% sodium hydroxide, no coloration was

66
observed when it was spotted on layers of silica gel. Similar

results were obtained when a spray consisting of a saturated

solution of copper sulfate in concentrated ammonium hydroxide

was used. It appears, therefore, that the color intensity of

the biuret reaction is insufficient for this reagent to be a useful indicating spray for t.l.c. on silica gel.

e . Iodine Vapor

The developed plates were kept in a closed tank containing

iodine crystals. Samples 1-5 and the biuret sample showed

faint, dark streaks stretching about half way along the plate

from the starting point. The significance of these streaks is

obscure. Samples 1-5 each showed an intense spot corresponding

to urea. However, a component at this position also appeared

to be present in the commercial biuret as a major impurity.

The most intense spot in the chromatogram of the biuret moved

slightly faster than urea, but was preceded by an additional,

minor component having the same mobility as an impurity observed in the sample of cyanuric acid. Near to the starting

line, the biuret chromatogram also showed a crescent-shaped

spot, and the cyanuric acid showed a dark ring. Urea samples

3, 4, and 5 showed three minor components having mobilities

greater than that of urea. The least mobile of these components had the same mobility as the major component of the

biuret sample. The fastest-moving component travelled almost

with the solvent front, and was also present in samples 1 and

2, although it was least intense in sample 2. The remaining

minor component (in 3, 4, and 5) had the same mobility as

cyanuric acid. Thus, it appeared that urea samples 3, 4 , and

5 contained biuret and cyanuric acid as impurities, whereas

the biuret sample contained an appreciable proportion of urea

and some cyanuric acid. Under ultraviolet light, the iodinedetected urea showed as a dark spot.

67
f

1%_ p- (Dimethylamlno) clnnamaldehyde (DMCA) in 1:1

(v/v) 95% Ethanol-6 N Hydrochloric Acid [84]

Immediately on spraying with this reagent, urea and

biuret showed as bright-red spots. Heating the sprayed

plates at 70°C intensified the color, and produced white centers in the urea spots. For urea sample 2, a pale-red spot

was detected at the starting line. However, this "component"

was proved to be an artifact by first allowing the developing

solvent to travel only one-third of the length of the plate

(at which point the urea component could be seen as a white

spot at about one quarter of the distance along the plate)

The plate was then removed from the tank, dried, and redeveloped so that the solvent ran the full length of the plate. On

spraying with DMCA, the main urea component immediately appeared as a homogeneous, red spot, and on heating at 70°C, a

faint, pale-red spot appeared at the origin, plus another

having an apparent R 0.25 at the location of the white spot

that was observed following the first development. The major

urea spot was not white-centered and had an apparent R^ 0.75.

Biuret was not detected in sample 2 by use of spray (fT.

g. 2%_ p- (Dimethylamino)benzaldehyde (DMB) in 4:1

(v/v) 95% Ethanol-6 N Hydrochloric Acid [84]

After being sprayed, the plates were heated at 70°C,

whereupon urea and biuret appeared as bright-yellow spots . Urea sample 2 displayed a pale-yellow, artifactual spot at

the starting point. However, with spray (g), biuret was not

detected In this sample.

2 . Paper Chromatography

Paper chromatography was performed on Whatman No. 1 filter paper by the descending technique, with 6:4:3 (v/v)

butanol—pyridine—water as the developing solvent. In this

system, urea had R

p

0.24, and biuret had R 0.37- These compounds were detected by means of sprays ( fJ (DMCA) and (g)

(DMB) described above. DMCA was found to be a sensitive reagent for urea, producing a bright-red spot on a colorless

68
background immediately after spraying. On storage, this spot

became pale-yellow on a dark-red background. Biuret also

gave a bright-red spot with this reagent

.

With the DMB reagent, urea and biuret were detected on

paper as bright-yellow spots on a colorless background. On

standing for several weeks, the urea spots became green,

whereas the biuret spots faded to the color of the background,

By means of these sprays, biuret was detected in urea samples

4 and 5, and urea was detected in the commercial sample of

biuret

.

3 . Spectrophotometric Examination of Biuret—Copper

Complex

To a portion (5.0 g ) of each urea sample was added

biuret reagent (5.0 ml), prepared as described above. The

mixture was warmed briefly to dissolve the urea, and the

solution was diluted to 10.0 ml with water. A blank solution

was prepared by dilution of the biuret reagent (5-0 ml) to

10.0 ml with water. Spectra of the urea solutions in 2-cm

cells were recorded with a Cary Model 14 spectrophotometer

over a wavelength range of 700-300 nm, with the reagent solution as a blank in the reference beam. Each spectrum was

recorded 15 min after dissolution of the urea. The following

results were obtained.

Absorbance

.194

0.026

0.152

0.303

0.031

- 0.000611 g

From these data, urea sample 2 was estimated to contain 0.01$

of biuret, and the other samples, proportionately larger concentrations . 69

Sample A (nm)

-max- -

1 505

2 570

3 510

4 490

Biuret— 520
4 . Determination of the Purity of Urea by Differential

Scanning Calorimetry (DSC)

These determinations were made with a Perkin-Elmer

Differential Scanning Calorimeter Model DSC-1B [71] (see

figure 5). The computational method used was that of Plato

and Glasgow [73]. The DSC instrument measures the differential thermal energy required to maintain both the sample and

the reference channels at the same temperature during the

analysis. If the sample undergoes an endothermic transition

( e.g. , melting) as its temperature is increased at a constant

rate, the instrument is programmed to replenish the energy

absorbed by the sample by an increased energy input to the

sample, so as to ensure that it is isothermal with the reference. Since the Instrument recorder plots the rate of heat

input with respect to time, ( dg/dt_) against time (fr) , the area

under the curve obtained gives the heat of the transition directly. For samples having a definite melting point, the

purity is measured by analysis of the degree of premelting

observed in the plot of dq/dt_ against t_. To compensate for

the thermal lag in the sample pan of the system, it is necessary to perform a separate scan of a reference material of

high purity; this is usually indium metal containing 99 - 9999%

of Indium. This reference material is also used to calibrate

the relative planimeter measurements employed for analysis of

the areas under the curves . The calibration depends on an

independent measurement of the heat of fusion of indium. The

value used in this work was 780 cal/mole.

For calculation of the purity of the sample material, the

area of premelting on the curve is divided into fractions that

are individually measured with a planimeter. A plot of the

reciprocal of the fraction melted ( 1/F ) against the absolute

temperature of the sample (T) is made. According to the theoretically derived equation T = T - [T - (T /F) ] , where T — —o —o —m — 3 —

is the true melting-point of the pure sample and T the observed melting point, this plot should be a straight line.

70
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71
However, in practice, for materials which are not of extremely

high purity, it is not certain at what temperature premelting

commences, and hence it is uncertain where the true base-line

of the experimental scan should b located. As a consequence,

a curved plot of 1/F against T is usually obtained, and it is

necessary to apply systematic corrections to the measured

areas until the plot is linearized. The true melting-point

of the sample (T ) may then be obtained from the intercept on

the graph, and the melting-point depression (T - T = AT) is

given by the slope of the straight line. The mole percentage

of total impurity is given by the function 100AH • AT/RT 2

, where AH- is the heat of fusion of the sample, and R is the

gas constant. To evaluate this function, a measurement of

AH„ is made. As the area under the curve has been calibrated

by reference to the standard heat of fusion of indium, the

heat of fusion of the sample is obtained from the total

corrected area under the sample curve.

The purity of sample 2 was analyzed by this method. For

such a determination, an approximately 2-mg sample of finely

pulverized urea, sealed in the customary type of aluminum container, was heated rapidly to about 5°C below its melting point

and then, while recording the transition, at a rate of 0.625°C/

min to beyond melting. Repetition of the measurement using

once-melted specimens produced inferior plots. Therefore, only

initial melting patterns were analyzed. The results obtained

on three different specimens of urea sample 2 are as follows

Determin. C AT

(C al/mol)

AH

f

(J/mol)

Mo le % purity

1 8.0 0.106 3,590 858 99 88

2 8.5 0.102 3,600 860 99 89

3 8.0 0.108 3,650 872 99 .88

C is the percentage correction applied to the measured total

area of the DSC curve, in order to linearize the plot of

1/F versus T.

72
D Cholesterol

(A. Cohen)

The certification of a supply of cholesterol of high

purity as a Standard Reference Material for use in clinical

chemical analysis was undertaken at the request of the College

of American Pathologists and the American Association of

Clinical Chemists. The cholesterol standard was made available for issuance in December, 1967^ and is the first in the

series of organic chemical standards for use in clinical

chemistry to be certified by the National Bureau of Standards.

1. Source of the Material

Cholesterol, isolated from brain and spinal cord of sheep,

was supplied by Distillation Products Industries, Rochester,

New York, where the isolated natural product was purified

according to the method of Fieser [85]. This process consists

of preparing crystalline cholesterol dibromide, and then regenerating the cholesterol by debromination, and crystallizing

from methanol. The product was dried at room temperature under

vacuum. Our recrystallizations of portions of this material

did not significantly affect any of the physical properties;

consequently, no further processing of the stock supply of

cholesterol was carried out in our laboratory, although, for

some tests, specially treated samples were prepared (as described in the relevant sections).

73
2 . Some Physical Properties of Cholesterol

a. Melting Point

Fieser [86] has shown that the temperature at which

cholesterol melts depends on the conditions employed during

the measurement: in a soft-glass, capillary tube open at one

end to air, melting occurs at a temperature significantly

lower than for a sample in a capillary tube that is evacuated

and then sealed from air. The standard cholesterol behaved in

this way. Melting-point determinations at heating rates of

approximately 0.5°C/min on several samples exposed to air in

Pyrex capillary tubes, and repeated redeterminations (4 times)

after melts had been allowed to cool and crystallize, showed

a regular decrease in the temperature at which the last,

minute crystals melted. With cholesterol samples sealed in

capillaries after they had been evacuated to less than 1

torr, the melting of the last, minute crystals occurred at

148. 7°C (corrected), and remelting of the last crystals of

resolidified melts occurred at l48.7°C through several such

cycles

The melting point was also obtained by a modification of

the technique described by Enagonio, Pearson, and Saylor [87],

in which temperatures were measured by use of a platinum

resistance thermometer calibrated in accordance with the

International Practical Temperature Scale (1948). The melting

point of a macro sample of the cholesterol, measured at a pressure of less than 1 torr (to exclude air) was l48.8±0.1°C.

(Work performed by C. Stanley and T. W. Mears, Office of

Standard Reference Materials, N. B. S.)

b . Optical Rotation

The specific optical rotation of highly purified cholesterol was reported by Fieser [86] to be -39.9° (c 1.73,

chloroform); that of another sample as -39.6° (c 1.49). Our

cholesterol showed [a]* -40.0° (c 2.241, chloroform).

74
c . Elementary Analysis

Calc. for C 2 7H 46 0: C, 83.87; H, 11.99- Found: C,

83.86±0.08; H, 11.98±0.09. (Determinations were performed by

W. P. Schmidt and R. A. Paulson of the Analytical Chemistry

Division .

)

3 . Spectroscopic Properties

a. Infrared Spectrum

The infrared spectrum of the cholesterol corresponded to

that recorded in the Atlas of Steroid Spectra [88], which

was obtained for a KBr pellet. In a mineral-oil mull, the

spectrum shows bands at v Nuj' 01 3380, 1670, 1330, 1270, 1253, max

1243 (sh), 1236, 1219, 1190, 1160, 1140 (sh), 1130, 1109,

1084, 1058, 1024, 1008, 986, 960, 952, 938, 927, 885, 840,

800, 737, 723, and 700 cm

-1

, and corresponds closely to the

spectrum for the KBr pellet

b . Ultraviolet Spectrum

A 0.92-percent solution of the cholesterol in dichloromethane was examined with a Cary Spectrophotometer, Model 14.

The slit-width of the instrument reaches a maximum at 230 nm

with this solvent. The spectrum of the solution showed a shoulder (e 13-3) at 232.5 nm; otherwise, there was no marked,

selective absorption, and the solution was essentially transparent from 270 to 400 nm. In absolute ethanol (0.49-percent

solution) , the spectrum showed no significant absorption from

230 to 400 nm.

The u.v. spectrum was also recorded for a sample of the

cholesterol that had been twice sublimed. (The sublimation

was conducted under a pressure of 0.03 torr with the sublimation apparatus immersed In a bath at l43°C.) The u.v. spectrum

of an almost saturated solution of the sublimed cholesterol in

dichloromethane showed peaks at 247, 283.5, 292.8, 309-5, and

322.5 nm, and, possibly, an additional one near 345 nm. A

comparable solution of the standard showed no significant

absorption at these positions. Appearance of these peaks in

the spectrum of the sublimate indicated that new impurities

75
had been formed during sublimation.

c . Proton Magnetic Resonance Spectrum

A solution of cholesterol (0.077 g) in chloroform-d

(0.5 ml) was examined by proton magnetic resonance spectroscopy at 100 MHz. The spectra were measured under frequencyswept conditions, with tetramethylsilane (t = 10.00) as the

internal reference standard and also to provide an internal,

lock signal. The following signals were observed: (1) a complex multiplet (actually a sharp doublet, with additional,

weak, outlying lines) centered at x 4.65, assigned to the

vinyl proton; (2) a complex multiplet at t 6.49, assigned to

the proton in the a-position to the hydroxyl group; (3) a complex envelope of peaks in the t 7.5-9.3 region, assigned

to the remaining methine and methylene protons; (4) a sharp

singlet at x 8.25 [this singlet at x 8.25, superimposed on

the envelope (t 7.5-9-3), was assigned to the hydroxyl proton,

because, on equilibration of the solution with deuterium

oxide (0.4 ml), this singlet disappeared]; (5) also superimposed on the methylene envelope were five intense, sharp

lines at t 9.00, 9.05, 9.11, 9.18, and 9-32. The singlets

at t 9.00 and 9-32 were assigned to the C-19 and C-18 angular

methyl groups, respectively. The three remaining lines were

interpreted to be two overlapping doublets with spacings of

^6 Hz, centered at t 9.09 and 9-15. From intensity considerations, these doublets were assigned as the C-21 methyl group,

and the C-26 and C-27 pair of methyl groups, respectively,

with each methyl group coupled to a methine proton in the

a-position

A more concentrated solution of cholesterol (0.152 g) in

chloroform-d (0.5 ml) was also examined. In the spectrum of

this solution, the sharp singlet due to the hydroxyl proton

occurred at lower field (x 7-87), thus providing further

evidence for its assignment. A different assignment has been

given by Roberts and Casino [89], in which a doublet at

x ^7-7, forming part of the methylene envelope, was assigned

76
to the hydroxyl proton. Since, in our work, the doublet was

unchanged by equilibration of the solution with deuterium

oxide, and because the hydroxyl proton could not be expected

to resist deuterium exchange, it must be concluded that their

assignment is incorrect . No definite signals corresponding

to impurities were detected. (Spectra were recorded and

interpretations made by Dr. B. Coxon of this Section.)

d. Mass Spectrum

Mass spectroscopy of the cholesterol in an Associated

Electrical Industries Mass Spectrometer, Model MS-9 , by use of

a direct, introductory probe and heating at 150°C, revealed the

presence of two very small peaks of greater mass to charge

ratio than that for the cholesterol ion peak. The small peaks

indicated masses of 14 and 28 mass units greater than that of

the cholesterol peak; higher attenuation showed a third impurity that was 44 mass units greater than that of the cholesterol. (Measurements by H. Pales of the National Institutes

of Health, Bethesda, Maryland 20014.)

4 . Chromatographic Methods

a. Gas—liquid Chromatography

Solutions of the cholesterol in ethyl acetate were injected into a Glowall Corporation Chromolab 320 Gas Chromatograph fitted with a column of Gas-Chrom Q treated with 3-

percent OV-17 as the stationary phase and heated at 240°C.

Peaks having the following retention times relative to that

of cholesterol, and the area of each peak relative to that

of cholesterol, are shown in table 8.

Table 8. Gas-chromatographic behavior of the standard

cholesterol

Relative retention time Area relative to cholesterol (percent)

0.5 0.1

1.2 0.3

1.6 0.1

1.8 0.1

77
b . Thin-layer Chromatography

i . Survey of Chromatographic Conditions

Cholesterol dissolved in chloroform (4 mg/ml) was spotted

in 8-, 16-, and 24-yg quantities on t.l.c. plates (coated with

Silica Gel G, preactivated at 110°C for 1 hr) . Evaporation

of the chloroform at the applied spots was conducted under a jet of nitrogen gas. Chromatography was performed with a

variety of developing solvents, and, after being dried with

nitrogen, the plates were sprayed with 1:5 (v/v) sulfuric

acid—methanol and charred on a hot plate. The conditions

surveyed, and the resulting observations, are given in table 9.

ii . T.l.c. of a Degraded Cholesterol

A sample of the standard cholesterol in a tube exposed to

air was heated in an oil bath at l65°C for 1.5 hr, and then

dissolved in chloroform and examined by t.l.c. The conditions

and results of the chromatography are shown in table 10.

iii . Artifacts in the T.l.c. of Cholesterol

Cholesterol (0.091 g) as a solution in chloroform was

applied as a streak to an activated, 20 x 20 cm plate, coated

1-mm thick with Silica Gel G, the chloroform being evaporated

from the streak with nitrogen. The plate was developed for

10.5 cm by use of 1:1 (v/v) benzene—chloroform, and was dried

under nitrogen; the cycle was repeated 3 times. After final

drying of the plate, a thin coating of silica gel was transferred from the plate by gently pressing to it a 10-inch strip

of masking tape in the direction of the development and removing the tape. Spraying the transferred material with the

sulfuric acid—methanol reagent, and oven-heating at 115° C,

revealed the position of the cholesterol. This zone, between

Rp 0.20 and 0.29, was scraped from the t.l.c. plate and extracted with chloroform; an 88-percent recovery of the cholesterol was obtained. The recovered cholesterol was crystallized

from chloroform plus methanol, and collected by filtration

under nitrogen. The crystallized product (24 ug) was applied

78
Table 9. T.l.c. data on SRM cholesterol

%

Conditions for

Developing solvent visibilization Identification

Acetone 0.78 visible, uv cholesterol

.00 uv (faint) artifact

Acetone—benzene 0.72 visible, uv cholesterol

[1:1 (v/v)]

.00 uv (faint) artifact

Benzene 0.05 visible, uv cholesterol

Benzene—chloroform 0.26 visible, uv cholesterol

[1:1 (v/v)]

(3 developments) 0.20 uv (faint) artifact

0.09 uv (faint) artifact

0.00 uv (faint) artifact

Benzene—ethyl 0.55 visible, uv chloroform

acetate [1:1 (v/v)] 0.00 uv (faint) artifact

Ethyl acetate 0.67 visible, uv cholesterol

.00 uv (faint) 4- • -P 4-b artifact

Heptane 0.00 visible, uv cholesterol

Chloroform 0.16 visible, uv cholesterol

(1 development)

(3 developments) 0.66 uv (faint) unknown

0.53 visible, uv cholesterol

.42 uv (faint) <- * i-b artifact

0.28 uv (faint) unknown

.22 uv ( faint

)

a- • ^ i-b artifact

0.08 uv ( faint

)

unknown

0.00 uv (faint) 4- • -p .ub artifact

b

Visible when illuminated with 366-nm light

Evidence given on previous page.

79
Table 10. T.l.c. of a degraded sample of SRM cholesterol

Developing solvent R,

Conditions for

visibilization Identification

Chloroform

(1 development)

Chloroform

(3 developments)

.10 visib le cholesterol

tailing

from

.10 visib le unknown

.00 visible unknown (blue)

0.64 uv

a (faint) unknown (blue)

.54 uv

a (faint) unknown (pink)

0.44 visible, uv cholesterol

0.35 uv

a (faint)

0.13 visible unknown (brown)

.06 visible unknown (blue)

.00 uv ( faint

)

Visible under 366-nm light.

as a spot to another plate, along with a separate spot for an

equal quantity of unchromatographed standard cholesterol and

a separate spot for an equal volume of the chloroform used as the solvent for applying the two samples . The plate was developed three times, as in the initial experiment. Spraying

and charring showed a corresponding response with both of the

cholesterol samples, and no response from the spotted solvent.

The two cholesterols showed visible charring at R 0.29 and,

under 366-nm light, showed spots at R^ 0.29, 0.18, 0.08, and

0.00. Hence, the spots at R

p 0.18, 0708, and 0.00 were artifacts. Another plate spotted with the same materials (chromatographically purified cholesterol, standard cholesterol, and

solvent only), developed three times, sprayed with antimony

trichloride in chloroform, and heated for 10 min at 117°C,

gave the same pattern of response.

80
With 160 yg of cholesterol, spotted on an activated

250-ym thick, 20 x 20 cm plate of Silica Gel G, and developed

with chloroform and dried, the new position of the cholesterol

was evident by its ability to reflect ordinary light. On repeating the cycle of developing and drying twice more, each

new position of the cholesterol could be observed. After

spraying with methanolic sulfuric acid and charring, examination under a 366-nm lamp revealed trace responses at all three

of the previous positions of the cholesterol, i.e. , one at

the origin and the two positions where reflections were noted

after the first and second developments . It is clear that

the process of evaporation of the chloroform from contact with

the cholesterol and the silica gel was giving rise to the

artifacts

.

iv. Impurities in the SRM Cholesterol by

T.l.c.

Chloroform development for 10 cm of a 250-ym thick, activated, Silica Gel G-coated plate, spotted with 160 yg of the

standard cholesterol, drying with gaseous nitrogen, spraying

with the methanolic sulfuric acid reagent, and charring,

revealed a faintly visible spot at R_ 0.28 in addition to that —

r

due to the cholesterol (R_ 0.18). Under 366-nm light, the

former had a faint pink color. This ultraviolet illumination

also revealed overlapping white spots at the origin (artifact)

and slightly above the origin (possible Impurity). Another

t.l.c. plate, 20 x 20 cm, spotted with 160 yg of cholesterol,

developed in chloroform, and dried (but not sprayed) , was then

turned at a 90° angle and re-developed in chloroform. After

spraying and charring, the white spot ahead of the origin was

no longer evident under 366-nm light, but the visible charred

spot (R„ 0.28 in the original direction) was clearly distinguishable . Smith and coworkers [90] have employed 1:1 (v/v) ethyl

acetate—heptane as developing solvent for examining the composition of cholesterol that had undergone oxidative degrada81
tion during many years of storage. They demonstrated the

presence of components such as the 7a-, 73-, and 25-hydroxy

derivatives of cholesterol, cholest-5-en-7-one , 5a-cholestane33,5,63-triol, and cholesta-3,5-dien-7-one. The SRM cholesterol (50 yg) , applied as a spot on a 250-ym, 20 x 20 cm,

activated Silica Gel H plate, plus separate spots for 0.1-,

0.2-, and 0.3-yg quantities of the cholesterol were chromatographed with the ethyl acetate—heptane mixture. After spraying with methanolic sulfuric acid and heating for 15 min at

115°C, the spot for the 50 ug of cholesterol was visible at

FL, 0.48. That for the 0.3 yg was barely visible (R„ 0.52), —

—r_ but a response for any lesser amounts was questionable. Under

the 366-nm lamp, the 50 yg of cholesterol was found to have

components at R^ 0.37 (weak), 0.26 (medium), 0.19 (weak), and

0.0 (strong); there was also faint streaking from R„ 0.26 to

the origin. Under the ultraviolet lamp, spots from the . 1-

,

0.2-, and 0.3-yg quantities were all visible at R^ 0.48. The

intensity of response was noticeably greatest at 0.3 yg; that

from 0.1 yg was distinct. The response from the 0.2 yg of

cholesterol was significantly greater than that from the component at R„ 0.26 in the chromatogram of the 50 yg of cholesterol. Also, the response from the 0.1 yg was greater than

that at R 0.37 or at R

p

0.19 . Assuming the spot at R

p

0.0

to be an artifact, the purity of the standard thus appeared

to be at least 99$.

c . Paper Chromatography

Smith [91] has reported some useful techniques for the

paper chromatography of cholesterol. Whatman No. 3 MM paper

was used for ascending chromatography with o-chlorobenzene for

development, and Whatman No. 1 paper impregnated with 2-phenoxyethanol (by dipping the paper into a 10-percent solution of

the compound in acetone), with heptane for development; antimony trichloride in nitrobenzene, or a sulfuric acid—acetic

anhydride-acetic acid reagent, was used for visibilization.

82
In our studies using the ascending technique, 8, 16, 24,

and 32 yg of standard cholesterol were chromatographed along

with a spot containing 120 yg of degraded cholesterol (preheated in air at l60° for 1.5 hr) . On spraying with antimony

trichloride, and heating in an oven at 110°C, the 24-yg and

larger spots were detected. The 16-yg spot was barely detectable. Cholesterol appeared as a violet spot at R^ 0.89 in —

this system. The degraded cholesterol showed, in addition,

a blue spot at R„ 0.4l. Ultraviolet light (366 nm) brought

out no additional impurities . The spray containing sulfuric

acid detected only cholesterol under ordinary illumination;

under 366-nm light, the degraded cholesterol showed a white

region tailing to the origin from R 0.07.

In the ascending technique, 30-, 60-, 90-, and 120-yg

quantities of the standard cholesterol were examined. Only

the antimony trichloride reagent was used for detection, and

only the degraded cholesterol gave evidence of an impurity,

as a blue spot at R , -. , -, 0.24. —cholesterol

5

Selenium Dioxide Test for Lathosterol

Fieser [86] has developed a very sensitive color test

for 5a-cholest-7-en-33-ol (lathosterol) that utilizes selenium dioxide. The test was tried according to the conditions

specified. Cholesterol (1.5 mg) was dissolved in 0.5 ml of

benzene, and treated at room temperature with 1.0 ml of the

selenium dioxide reagent (prepared by dissolving 1.1 g of

selenium dioxide in 2 ml of hot water and diluting to 100 ml

with glacial acetic acid) . The mixture remained colorless

for over one hour. With 50 mg of the cholesterol and the

same quantity of the reagent, the mixture remained colorless

for over 0.5 hr. Hence, the test for lathosterol was considered negative.

6

Liebermann—Bur chard Analysis

Quantitative estimations of cholesterol, based on the

color produced in its reaction with the Liebermann—Bur chard

reagent, are frequently performed in clinical laboratories.
The reagent is a solution of sulfuric acid in acetic anhydride

and acetic acid. The uniformity of response by the standard

cholesterol was studied.

The clinical procedure adopted for use with the standard

was the method of Ness, Pastweke, and Peacock [92]. A

0.1-percent (wt/vol) solution of the standard cholesterol in

glacial acetic acid was prepared. The Li ebermann—Bur chard

reagent was prepared from 300 ml of acetic anhydride, 150 ml

of glacial acetic acid, 50 ml of sulfuric acid, and 10 g of

sodium sulfate. The densities of the two solutions were determined in order to eliminate inaccuracies due to drainage

from the pipettes used in their delivery; the volumes could

then be determined by weighing each delivered aliquot.

Approximately 2.9 ml of the cholesterol solution and 9.9 ml

of the reagent were quickly weighed and combined, mixed on

a vortex stirrer, transferred to a water bath at 25±0.1°C,

and absorbancies were measured at 535 nm in a Cary Spectrophotometer, Model 14, after exactly 40 min reaction. For

calculation of molar absorbancy, the volume of each reaction

mixture was assumed to equal the sum of the calculated

volume of each reactant combined. The molar absorption for

6 separate determinations was 590±1 liters/mole cm. Repetition of the analysis on another date gave a molar absorbancy

of 591±3 liters/mole cm. (The uncertainties represent the

standard deviation of the mean.)

7. Issuance of the Cholesterol as a Provisional

Standard

The properties of the cholesterol that had been studied

by November, 1967, showed the material to be at a high state

of purity; none of the measurements were in conflict with

this evaluation. Of the techniques employed with the cholesterol, g.l.c. was considered most satisfactory for providing

a quantitative measure of its purity. Assuming that the areas

of all peaks were proportional to their concentrations and

that the minor peaks represent the impurities in the choles84
terol sampled, g.l.c. gave the purity as 99 • ^±0 .3 percent,

estimated at the 95% confidence level.

In light of the reported instability of cholesterol during long-term storage, periodic study of this cholesterol was

planned for a 2-year period in order to determine Its shelflife as stored at NBS, and as it might be kept in a clinical

laboratory. This interval would afford additional opportunity

for evaluations of purity to be performed. Meanwhile, to fill

the need of clinical chemists for a standard, the cholesterol

was issued as a provisional standard reference material. (The

material issued is packaged, under nitrogen, in brown-glass,

sealed vials that are wrapped in opaque outer containers.)

8 . Post-issuance Studies of the Standard Cholesterol

At several-month intervals since its issuance as a

standard, samples of the standard that had been stored under

various conditions were re-examined by some of the techniques

used initially. Occasion was also taken to apply some additional methods for assaying its purity. Only some of the

findings, obtained about 6 months after the date of issuance

of the standard, are discussed.

a. Thin-layer Chromatography

Samples of the standard (a) kept packaged under nitrogen,

(b) unpackaged, stored in a freezer, and (c) unpackaged, exposed to air at room temperature, were examined in 50-yg quantities by t.l.c. on a Silica Gel G plate with the ethyl acetateheptane developing mixture used earlier. Reference quantities

(0.1, 0.2, and 0.3 yg) of the packaged standard were also

spotted on the plate. After spraying with methanolic sulfuric

acid and heating at 115°C for 15 min, the chromatogram showed

visible charring of the 0.3- and 50-yg quantities of cholesterol. Under 366-nm light, all the fractional-yg quantities

spotted were evident, as well as additional spots behind the

50-yg spots of cholesterol. The latter spots appeared in the

same pattern (and with the same intensities relative to each

other and to the fractional-yg spots) as had been observed in

85
U. S. Departri^jsn#of Commerce

Alexander EJsyjTrowbridge, Sec

National Ifu A. V

Certificate of gfoalpsfe

Standard Reference Material 911

Cholesterol

This Standard Reference Material is certified as a chemical standard to aid manufacturers

in meeting the specifications for cholesterol for use in clinical analysis.

This cholesterol was purified according to the method of L. F. Fieser[J. Am. Chem. Soc.

75, 5421 (1953)], which consists of preparing cholesterol dibromide, regenerating the cholesterol by reacting the dibromide with zinc dust, and crystallizing. The purity is 99.4 ±0.3

percent.

Purity was determined by gas chromatography, thin-layer chromatography, and mass,

infrared, and nuclear magnetic resonance spectroscopy, Gas-chromatographic examination,

using Gas—Chrom Q treated with 3 percent OV—17 as the stationary phase at 240°C, showed

impurity peaks at the following retention times relative to cholesterol:

Area Relative to Cholesi

(Percent

Relative

Retention Time

0.5

1.2

1.6

1.8

Thin-layer chromatographvo^HhUa ge^-otveloped with chloroform, sprayed with sulfuric acid in methanol, and heaieavi Wjj>° revealed two trace contaminants under ultraviolet

irradiation. The greate^wnAamimint appeared at the head of the cholesterol; the lesser, just

beyond the origin-^6J\^w>^^eTlsional chromatography, the greater contaminant was found as

a separate spot. No«iuarybitative values were assigned to these spots.

Response to the Liebermann—Burchard test was very uniform. A molar absorbance of

590 ± 1 at 535 nm was obtained by using 2.9 ml of 0.1 percent (wt/vol) cholesterol solution

in glacial acetic acid and 9.96 ml of reagent prepared from 300 ml of acetic anhydride, 150 ml

of acetic acid, 50 ml of sulfuric acid, and 10 g of sodium sulfate. The volumes in each

analysis were calculated from the weights of each solution delivered and the densities.

Measurements of absorbance, with the reaction performed at 25°C, were obtained 40 minutes

after mixing. Molar absorbances higher than 590 could be obtained with mixtures containing

less acetic acid, but the objective was to obtain high reproducibility and not to maximize the molar absorbance [Ness, Pastewka, and Peacock, Clin. Chim. Acta 10, 229 (1964)]. All measures of precision used in this certificate are estimated at the 95 percent confidence level.

Washington, d. c. 20234 October 20, 1967

W. Wayne Meinke, Chief

Office of Standard Reference Materials

(over)

86
The melting point of this material is 148.8 ± 0. 1°C at a pressure of less than 1 mm of Hg.

This value was obtained by using a modification of the technique described by Enagonio,

Pearson, and Saylor [Temperature, Its Measurement and Control in Science and Industry, Vol.

Ill, Part 1, pp. 219-230. The Reinhold Publishing Co., New York, N.Y. (1962)]. Temperatures were measured by use of a platinum resistance thermometer calibrated in accordance

with the International Practical Temperature Scale (1948).

The capillary melting point is 148.7 °C (corrected). The melting point is that temperature

at which the last trace of crystalline cholesterol melted. The cholesterol was under a pres- sure of less than 1 mm of mercury, and was heated at the rate of 0.5°C per min. Solidification

of the melt followed by remelting gives this same value repeatedly.

The specific rotation, [a] 20

D, is -40° (c 2.241, CHClg). Microchemical analysis found

83.86 ± 0.08 wt percent carbon and 11.98 ± 0.09 wt percent hydrogen. The theoretical percentages based on Cor H „0 are 83.87 wt percent carbon and 11.99 wt percent hydrogen.

27 4o

Mass spectroscopy revealed the presence of contaminants having masses 14 and 28 units

greater than that of the cholesterol parent peak. Infrared and nuclear magnetic resonance

spectroscopy did not reveal the presence of any significant impurities. The selenium dioxide

test for lathosterol was negative [Fieser, J. Am. Chem. Soc. 75, 4395 (1953)].

Preliminary indications of storage life were measured by storing a sample under vacuum

at 80°C for one month. No changes were noted in melting point or infrared spectrum. The

purity of the cholesterol will be followed over a 2-year period. If these additional measure- ments indicate a significant departure from the purity as stated, users of this material will be

notified.

The Standard Reference Cholesterol is packaged in 0.5—g quantities sealed under nitrogen

in screw—cap vials.

Analyses were conducted by A. Cohen, D. H. Freeman, R. T. Leslie, R. A. Paulson,

C. B. Romaine, R. Schaffer, and C. L. Stanley of the NBS Institute for Materials Research,

and H. F. Fales of the National Institutes of Health.

87
the original examination of the standard cholesterol. Thus,

t.l.c. provided no evidence of instability of the standard,

even after exposure to air at room temperature.

b . Liebermann—Burchard Analysis

Analyses by this method were performed as previously described, and the results obtained are shown in table 11. The

Table 11. Liebermann—Burchard results with the standard

cholesterol, six months after issuance

Molar absorbance

Cholesterol at 535 ran sample number Storage conditions (liters/mole cm)

packaged, and

stored at 25°C 595±2

unpackaged, and

stored in freezer 597±2

unpackaged, but

open to air at 25°C 598±2

tabulated molar absorbancies (±1 standard deviation, based on

determinations with 6 aliquots per sample) are about 1 percent

higher than the molar absorbance determined initially. However, a variation of this magnitude may be attributable to

instrument performance and therefore cannot be considered to

signify an alteration in the composition of the standard.

Similarly, the differences in the tabulated values cannot in

themselves be considered as showing decomposition of cholesterol that was exposed to air.

c . Differential Scanning Calorimetry

Representative samples of SRM cholesterol were examined

by differential scanning calorimetry as an additional method

for ascertaining purity. (Analysis by Dr. R. P. Brady, Jr.)

These measurements were performed with specimens of cholesterol under a nitrogen atmosphere. A weighed quantity of

cholesterol in an open aluminum dish was placed on the calori88
meter sample-pan, over which was subsequently placed a loosefitting, hollow, hemispherical aluminum dome (ca. 1.0 cm in

diameter). The reference pan, covered similarly, held an

empty aluminum dish. The calorimeter cover was then locked

in place, and the assembly was successively evacuated to about

30 torr with a vacuum pump, and filled to atmospheric pressure

with dry nitrogen. The evacuation and filling processes were

repeated six times. With the sample under one atmosphere of

nitrogen, the calorimeter temperature was raised from 298 to

4l4°K at a rate of 80°CC/min, and then from 4l4 to 423°K at

0.625°C/min (see also the discussion on p. 70).

Endotherms recorded under the foregoing conditions showed

little premelting and high peak amplitudes, evidences of high

purity; these characteristics are in marked contrast to those

of endotherms obtained when the melting transition was studied

with cholesterol exposed to air. For example, using cholesterol specimens sealed in all-aluminum containers (^0.2 cm 3 in

volume) without provision for exclusion of air, the resulting

endotherms exhibited wide ranges of premelting and small peak

amplitudes, which indicated impurity at the 1.1 mole percent

level. Presumably, the higher level of impurity calculated

from these endotherms is attributable to the oxygen sealed

within the sample container. (The beneficial effect of evacuating and sealing cholesterol in capillary tubes in determinations of its melting point has been discussed previously,

see p. 74 )

.

Some results obtained by differential scanning calorimetry with the standard cholesterol, six months after

issuance, are shown in table 12, in which melting transitions

were recorded for cholesterol samples under a nitrogen

atmosphere

.

Purity determinations by the DSC method are regarded as

reliable to 0.1 mole percent; hence, the packaged standard

appears to be more nearly at the upper limit of the 99.4±0.3

percent purity at which it was provisionally certified. The

89
Table 12. DSC results with the standard cholesterol,

six months after Issuance

Cholesterol Purity

sample number Storage conditions (moles percent)

1 packaged, and

stored at 25°C 99 -84

2 unpackaged, and

stored in freezer 99-77

3 unpackaged, but

open to air at 25°C 99-73

difference in purity between the packaged and the exposed

sample (0.11 mole percent) is essentially within this range

of reliability, and therefore the difference does not provide

unequivocal evidence of decomposition; however, the trend in

these purity values and the trend in molar absorbancies by

the Liebermann—Burchard analysis suggest that evidence of

slight decomposition is indeed being detected in the cholesterol exposed to air at room temperature,

d. Phase Solubility Analysis

The packaged, standard cholesterol was also examined by

phase solubility analysis, another technique that had not been

used with the cholesterol during the initial study. (This

work was performed by Dr. R. F. Brady, Jr.; see also p. 58.)

Weighed quantities of packaged standard were allowed to equilibrate in sealed glass ampoules with equal weights of heptane

(Eastman, highest purity) for 6 days at 25±0.1°C. A phase

solubility diagram was constructed from the original weights

mixed and the analyzed compositions of the equilibrated solutions . The purity derived from the phase solubility diagram

amounted to 99-96 percent, a value that agrees very well with

the 99.84 mole percent that was found by differential scanning

calorimetry for the same material.

90
3. PROPERTIES OP POLYCYCLIC AIR-POLLUTANTS

Among the organic compounds that are common components of

polluted air, having entered the atmosphere as a result of

various incomplete, fuel-combustion processes, are a number of

polycyclic, aromatic compounds, some of which are carcinogenic

With their continual introduction into the atmosphere, they

may pose a considerable health hazard, and an understanding of

the fate of these substances Is a matter of concern to the

Nation

.

Laboratory-simulated, natural conditions of exposure

were employed in determining that pyrene, as a representative

of the group, is susceptible to degradation. It was found

that many of the same products that resulted from this exposure could be obtained by treating pyrene with periodic acid,

a reagent which had not been used previously as an oxidant of

polycyclic, aromatic molecules, and the mechanism of the

periodate-induced reaction of pyrene was studied. The applicability of the reagent for obtaining reference degradation

compounds from other polycyclic, aromatic pollutants was also

investigated.

A . Periodic Acid, A Novel Oxidant of Polycyclic Aromatic

Hydrocarbons

(A. J. Patiadi)

In this laboratory, it has been shown [93] that treatment

of a solution of pyrene (1) (1 mole) in glacial acetic acid

with an aqueous solution of periodic acid (1 mole) at 50°C

produces 1, 1 '-bipyrene (2) in a yield of over 70%, the first

report of a simple, one-step preparation of £.

Evidence was obtained later [9^] showing that the dimerization of 1 to 2 proceeds through a radical intermediate:

the e.s.r. spectrum of this radical shown in Fig. 6 (spectrum

A).

91
—J °-9 L— I Gauss \

H

Figure 6. (A) The electron-spin resonance spectrum of pyrene

radical prepared by treatment of pyrene (in acetic

acid) with periodic acid, H 5 I0 6 (in water) at room

temperature, g = 2.0041. (B) Anthracene (in N,N- dimethylformamide) on treatment with periodic acid

in water. Sample B seen under comparable conditions

No radicals were observed.

92
H-,IO,; (aq CH,CO,H) + HI0.1 + 3H.0

As has previously been shown [95], on exposure to air,

warmth, and ultraviolet irradiation, pyrene (1) adsorbed on

soil is converted into compound 2, as well as 1,6- and

1, 8-pyrenediones plus other products. This finding was part

of a study of the fate of this hydrocarbon, which occurs

commonly as a constituent of polluted air. Some of these

oxygenated pyrene derivatives were also found among the products of periodic acid-oxidized pyrene [93]- It was of

interest to elucidate the effect of periodic acid on other

poly cyclic, aromatic hydrocarbons, and to determine whether

other polycyclic, aromatic quinones might be obtained, as

well as dimeric products. For determinations of the fate of

these other aromatic pollutants, such derivatives were needed

It was found [9^,96] that, of a number of polycyclic

aromatic hydrocarbons studied, many are oxidized by periodic

acid in aprotic solvents containing a small proportion of

water. Treatment of a solution of fluorene in glacial acetic

acid with aqueous periodic acid yielded a coupling product,

1, 2-bis (2 , 2 '-biphenyly lene ) ethylene, in about 10$ yield.

Here again, the dimerization proceeds through a radical intermediate whose spectrum was recorded by e.s.r. [96]. In other

hydrocarbon oxidations, however, only quinones were obtained.

In contrast to the two reactions in which dimerizations were

found to occur, the oxidations of these other aromatic hydrocarbons by periodic acid to quinones afforded no evidence for

the presence of radical intermediates. E.s.r. monitoring of

93
the reaction of periodic acid with anthracene, either in

aqueous acetic acid or aqueous N ,N-dimethylformamide (in the

range of to 100°C), indicated that there is no radical

participation in the oxidation (spectrum B, Fig. 6). Hence,

for these oxidations, a second mechanism must be involved.

Usually, the latter group of polycyclic, aromatic hydrocarbons is oxidized to quinones in yields that range from

50 to 80%; for example, treatment of a solution of naphthacene (3) in N,N-dimethy Iformamide with aqueous periodic acid

rs* at 120°C for 5 minutes gave 5 , 12-naphthacenequinone (j\) in

an 80-85$ yield.

H*IO 5-^6

aq. DMF

By use of an approximately 1:4 molar ratio of hydrocarbon

to periodic acid, the following were oxidized: anthracene in

N ,N -dimethylformamide , at 95°C for 5 minutes, and then 70°C

for 30 minutes, to anthraquinone (91-95$); naphthalene in glacial acetic acid, at 110°C for 5 minutes and then 70°C for 30

minutes, to 1, 4-naphthoquinone (70-76$); and phenanthrene in

p_-dioxane, at 95°C for 5 minutes and 30°C for 30 minutes, to

phenanthrenequinone (50$). In the same way, benz[a]anthracene

(in N^N- dime thy Iformamide) was oxidized to benz[a]anthracene7,12-dione (78-82$); pentacene (in N ,N -dimethylformamide ) to

6 , 13-pentacenequinone (81-87$), and acenaphthene (in acetic

acid) to acenaphthenequinone (65-70$). By use of a ca. 1:2

molar ratio of hydrocarbon to periodic acid, anthrone (in

acetic acid) was oxidized to the quinone (94$). When sodium

metaperiodate was used instead of periodic acid, none of these

reactions were observed. Little or no reaction was observed

on attempted periodic acid oxidation of biphenyl, chrysene,

94
coronene, perylene, picene, p_-terpheny 1, and triphenylene

.

The mechanism for the oxidation of naphthalene (7) to

1, 4-naphthoquinone (9) apparently proceeds by a twoequivalent oxidation. This mechanism involves electrophilic

attack by periodic acid, as shown in 7a, with formation of

intermediate 7b. Collapse of 7b by loss of iodic acid, and

addition of water to the cationic center of 7b, affords the

1, 4-dihydro-l , 4-diol (7c), which is then rapidly oxidized by

periodic acid to dione 9- The last step in this sequence of

reactions is supported by the observation that 1,4-

naphthalenediol (7d) is oxidized with periodic acid to dione

9 in a yield of 80% . E.s.r. monitoring of this reaction revealed no radicals, and showed a spectrum similar to spectrum

B (Fig. 6)

.

The application of periodic acid is being extended to

a study of the oxidation of nonbenzenoid, aromatic hydrocarbons

Room-temperature treatment of azulene [97] with periodic acid

results in the deposition of a free-radical solid (Fig. 7 } spectrum A) which is extremely stable to air and heat. A sample purified by precipitation (three times) from aqueous N,Ndimethylformamide was used in all analyses and measurements

.

The sample (dried at 150°C/0.05 torr) is a black, glass-like,

amorphous (X-ray evidence) powder which is strongly paramagnetic, giving an e.s.r. spectrum identical with that of the crude

material. Microanalysis indicated that the repeating unit of

the product consists of three azulene-like moieties, and one

iodine and four oxygen atoms. Magnetic susceptibility measurements indicated that the number of radicals is about 20% of the

number of repeating units . Conductivity measurements in N,N- dimethy If ormamide suggest that the product is a weak electrolyte. The viscosity of a solution of the product (in N,Ndimethy If ormamide) showed an intrinsic viscosity of about 0.53

dl'g-1

, which indicates that the degree of polymerization of

the product may be of the order of 20—100 repeating units

.

95
1^-

oCO X o

CD O

ID X

o

1^

»—I 10 X

CO O»—

LO X

CD

96
7 GAUSS

A B Figure 7. The e.s.r. spectrum (A) of the solid radical prepared by treatment of azulene (in acetic acid) with

periodic acid, H 5 I0 6 , (in water) at room temperature,

g = 2.003^±0.0002; the e.s.r. spectrum (B) of the

solid radical obtained on treatment of azulene (in

acetic acid) with cone, nitric acid at room temperature; g = 2.0087+0.001.

97
The product is not soluble in aqueous base and is little

affected on fusion with potassium hydroxide. Its u.v. spectrum shows x HC0NMe

2 at 292, 370 (sh), and 590 (sh) nm, which

max

are spectral shifts of 90-100 nm as compared to the peaks of

azulene [ A HC0NMe

2 326, 351, 5^8, 579, 620, and 682 nm] . I.r.

max

maxima (Nujol) were observed at 1700, and at 1590 and 1570 cm

-1

(aromatic C=C stretchings, observed also for azulene); but the

spectrum shows no significant absorption between 1400 and 625

cm

-1 (fingerprint region).

An attempt was made to sublime a sample of the product

(0.25 g) at 395°C/0.05 torr for 1 hour; the unsublimed residue (0.22 g, 88$) (Found: C, 84.0; H, 2.9; I, 9-9/0 was

still strongly paramagnetic and gave the same symmetrical

e.s.r. spectrum (spectrum A, Pig. 7).

The stability of this radical property is shown by the

fact that further heating at 485°C for 15 minutes (evolution

of iodine) left a residue (Found: C, 93-5; H, 3-1; I, 2.9%)

that was still paramagnetic; however, the intensity of the

e.s.r. spectrum of the residue (little change in amount) had

diminished considerably. Similar high stability of the radical was observed when heating was conducted in air or in

oxygen-enriched air. Considering all information presented

above, a polymeric, charge-transfer complex involving iodine

appears to be a possible structure of this radical.

98
4. OTHER STANDARD REFERENCE MATERIALS PROCESSED THROUGH

THE ORGANIC CHEMISTRY SECTION

A

.

Sucrose, Standard Reference Material 17a; Reissuance

of the Standard

A specially selected supply of sucrose, provided by the

California and Hawaiian Sugar Refining Corporation, was

subjected to the physical testing procedures that had been

employed in determining the suitability of previous samples

of sucrose for certification as the standard. The optical

rotatory properties, the moisture and ash content, and the

reducing properties of the sucrose were found to be acceptable . B

.

Dextrose, Standard Reference Material 4la; Reissuance

of the Standard

Highly refined dextrose, obtained from Pfanstiehl

Laboratories, Inc., was tested for the conformity of its

physical properties with those of the dextrose standard previously issued. The new dextrose supply was determined to

have the properties required for it to serve as the standard.

C

.

Nicotinic Acid, Standard Reference Material 148

Nicotinic acid, a standard to be used for the microdetermination of carbon, hydrogen, and nitrogen, is a newly

issued standard. The analyses used in its certification were

performed within other Sections of the Division of Analytical

Chemistry, but the Organic Chemistry Section provided overall

supervision. The nicotinic acid, as a new standard, is

currently issued with a provisional certification.

D

.

Reissuance of Metallo-organic Standard Reference

Materials

New supplies of lead, magnesium, and zinc cyclohexanebutyrates and silver 2-ethylhexanoate for use in the spectroscopic determination of these metals In petroleum products

were studied, and found suitable as Standard Reference

Materials. The lead salt, obtained from Sadtler, Research

99
U. S. Depart^s^rof Commerce

Alexander BMTrowbridge,

Secjsfstary

National I uf8$

A. V.^s^JX

S^fe^jfc Standards

rector Certificate of &nalp£te

Standard Reference Material 17a

Sucrose

Moisture less than 0.01%

Ash less than 0.001 %

Reducing substances, estimated as invert

sugar [1] less than 0.02%

Each 100 ml of a normal sucrose solution contains 26.000 g of dried substance,

weighed with brass weights in air (760 mm pressure, 20 °C, 50 percent relative humidity). At 20 °C, this solution in a 200-mm polariscope tube reads 100 °S (International

Sugar Degrees). The illumination is white light filtered through a 15-mm layer of a

6-percent solution of potassium dichromate. The International Sugar Scale was denned

and adopted by the International Commission for Uniform Methods of Sugar Analysis

at the Eighth Session, Amsterdam, 1932 [2], [3].

The rotation in circular degrees of the normal sucrose solution observed in a 200-mm

polariscope tube, for wavelength 5461 A is 40.763° and for wavelength 5892.5 A is 34.617°.

The specific rotations of sucrose for the normal solution are

r 20 °C

[ « ] 5461 A = 78.342° 26 g per 100 ml

20 °C 66.529° 26 g per 100 ml

t a]

5892.5 A

[1] F. J. Bates and R. F. Jackson, Bull. B. S. 13, 67 (1916).

[2] Proceedings of the Eighth Session, International Commission for Uniform Methods of Sugar Analysis, Intern. Sugar J. 35, 17 (1933).

[3] F. J. Bates and Associates, NBS Circular C440, pp 79, 775 (1942).

The sucrose was supplied by the California and Hawaiian Sugar Refining Corporation of Crockett, California. Chemical and physical testing to determine its suitability as

a standard reference material were performed by B. S. Carpenter, B. Coxon, R. A. Paulson, R. Schaffer, and B. F. West of the Analytical Chemistry Division.

Washington, D. C. 20234

December 26, 1967

W. Wayne Meinke, Chief

Office of Standard Reference Materials

100
L v U. S. Departro^jrof Commerce

Alexander E&jjfrowbridge,

Sectary

National I ure&ft^ef Standards

A. V. S^stji^ tMrector Certificate of &nalpsfe

Standard Reference Material 41a

Dextrose

Ash less than 0.01 %

Moisture less than 0.02%

The specific rotation of dextrose at equilibrium is represented by the formula

20 °C

[ aJ 5461 A = 62.032° + 0.04257° c,

where c is grams of anhydrous dextrose weighed in vacuo and contained in 100 ml of solution. This formula is valid for values of c between 6 g and 32 g. [Bull. B. S. 13, 633 (1916)

S293; NBS Circular C440, p. 83 (1942) ].

[ a ] 20 °C _ 52 ? c = 4 g per 100 ml.

Dextrose absorbs surface moisture which can be removed by drying in vacuum at 60 to 70 °C.

The dextrose was obtained from the Pfanstiehl Laboratories, Inc. of Chicago, Illinois. Chemical and physical testing to determine its suitability for a standard reference material were performed by B. S. Carpenter, B. Coxon, R. A. Paulson, R. Schaffer, and B. F. West of the Analytical Chemistry Division.

Washington, D. C. 20234

December 26, 1967

W. Wayne Meinke, Chief,

Office of Standard Reference Materials.

101
Laboratories, Inc. , was issued as Standard Reference Material

1059b; the magnesium salt, suppled by Distillation Products

Industries, was Issued as SRM 106lb . The zinc salt, from

Sadtler Research Laboratories, was issued as SRM 1073b, and

the silver salt, prepared by Distillation Products Industries,

was issued as SRM 1077a. Analyses used in these certifications were performed in other Sections of this Division.

102
5. ANALYTICAL CHEMISTRY OF CORN SYRUPS AND RELATED PRODUCTS

( D. W. Vomhof and J. H. Thomas)

A Research Associate Program, In co-operation with the

Corn Industries Research Foundation, is being conducted to

study some physical properties of corn syrups and their

interrelationships . Of immediate concern to the Industry

is the relationship between refractive index, density, and

dry-substance content as functions of moisture content and

average molecular weight.

Corn syrups are made either by acid hydrolysis of corn

starch or by partial hydrolysis with acid followed by further hydrolysis of the resultant oligomers of large molecular

weight by use of any of a variety of enzymes. The syrups

have a broad distribution, both with respect to molecular

size and to percentage of D-glucose and malto-oligosaccharides

;

and different syrup types are characterized by their particular ranges of saccharide distribution. The majority of the

physical properties of syrups, e.g. , viscosity and volume of

mixing, are dependent on their moisture content, as well as on

their saccharide distributions.

Before reliable data for the evaluation of physical properties can be obtained, the moisture content of the syrups

being studied must be known accurately. The present standard

industrial method for directly determining the moisture content of corn syrups employs the vacuum-oven, filter-aid

technique [98] . However, this method has the disadvantage

of requiring almost 2k hours for obtaining a moisture determination. A more serious disadvantage Inherent in the method

is the variable extent of saccharide degradation that occurs

at the oven temperature (100°C) during the time period required for the volatilization and removal of the water. The

extent of this degradation seems to be related to the proportion of dextrose or maltose in the sample. The error may

become significant when this method is used with syrups

103
having high contents of dextrose and maltose.

Alternatives to this method for the direct determination

of the moisture content of syrups and related products are

therefore being evaluated, prior to our undertaking studies

of other physical properties. Requirements for a suitable,

direct method of moisture analysis are accuracy, precision,

freedom from interference (either by the saccharides present

or the ash content), freedom from degradation or chemical

action resulting in the production of volatile substances or

the consumption of moisture, and, finally, reasonable

rapidity

A. The Near-Infrared Method

For obtaining moisture content, one direct method which

appears to satisfy these requirements is a near-infrared

spectrophotometric method specific for water. The method

being investigated for its applicability to syrups is essentially that reported by Rader [99]; it depends on measurement of the absorption of energy at 1.9 ym by water molecules.

The energy absorbed is directly proportional to the quantity

of water present. Hydroxyl groups of saccharide molecules

do not interfere

In this method, calibration is achieved by determining

the absorbance of solutions containing known concentrations

of water in a suitable organic solvent. The solvents that

we have employed are N ,N-dimethy Iformamide or methyl sulfoxide. Solutions of the syrups are prepared in solvent from

the same lot as that used for calibration, care being taken

to insure their complete dissolution. The net absorbance is

obtained by subtracting the baseline absorbance at 1.83 ym

from the peak absorbance (at about 1.93 ym) . The concentration of water in the sample solution is determined from the

calibration curve, and the weight-percentage of water in the

original syrup is calculated from the equation

F = (W/S) x 10 2

104
where F = the weight-percentage of water in the syrup, W = the weight-percentage of water in the sample solution, and

S_ = the weight-percentage of syrup in the sample solution.

The repeatability of the method is within ±0.03 weightpercent of moisture in the original syrup.

B

.

Thermogravimetric Analysis

Thermogravimetric analysis (TGA) is another method

undergoing investigation. In this method, the loss in weight

of a sample is determined as a function of increasing temperature. In its application to moisture analysis, a plateau

results when the moisture in a sample has been volatilized,

and before thermal decomposition has begun.

The percentage of water in a material is calculated by

dividing the final weight (at the plateau) by the initial

weight of the sample and multiplying the quotient by 100.

The difficulties in transferring controlled quantities

of a thick syrup to the sample holder of the TGA apparatus

are formidable; hence, a commercial syrup cannot be measured

directly. When diluted with water, the syrup can be handled

readily; however, a small loss, caused by evaporation of

water from the sample during assembly of the apparatus, introduces relatively large error in the analysis

.

TGA is applicable to solidified and spray-dried starch

hydrolyzates , because sample loading presents fewer problems

with these more readily fragmented "syrups." Since the usual,

industrial, quality-control methods, such as those using

refractive index or specific gravity, cannot be readily run

with these types of products, TGA may prove of particular

usefulness

.

C. Other Methods

For the determination of moisture, methods that are in

preliminary stages of evaluation for applicability to syrups

are Karl Fischer titrimetric procedures and infrared spectrophotometry utilizing the 6.05-ym absorption band of water.

105
6 . PERSONNEL AND ACTIVITIES

A

Personnel Listing

R. Schaffer, Section Chief

R. S. Tipson

B. Coxon

R. F. Brady, Jr.

A. J. Patiadi

A. Cohen

B. P. West

D. W. Vomhof, Research Associate

J. H. Thomas j Research Assistant

B

Publications

NBS Technical Note 427. Organic Chemistry Section.

7/1/66 - 6/30/67.

Edited by R. Schaffer (October, 1967).

Effects of Temperature and of Ultraviolet Radiation on

Pyrene Adsorbed on Garden Soil.

A. J. Patiadi, Environ. Sci. Tech. 1, 570 (1967).

A Novel, Facile Preparation of 1, 1 '-Bipyrene

A. J. Patiadi, J. Org. Chem. 32_, 2903 (1967).

Phenylhydrazono—Phenylazo Tautomerism. Part II.

Structures of 2-0xo-l, 3-bis (phenylhydrazono) Compounds

and Related Compounds.

A. J. Fatiadi and H. S. Isbell, Carbohyd. Res. 5,

302 (1967)

Infrared Absorption Spectra of 2-0xo-l, 3-bis (phenylhydrazono) Derivatives and Related Bis- and

Tris-(phenylhydrazones )

A. J. Fatiadi, J. Res. NBS 71A, 277 (1967).

106
Periodic Acid, a Novel Oxidant of Polycyclic, Aromatic

Hydrocarbons

.

A. J. Fatiadi, Chem. Commun. 1967 , 1087.

Preparation and Purification of Some Oxidation Products of

Perylene

.

A. J. Fatiadi, J. Res. NBS 72A, 39 (1968).

Review of Stoffwechsel der Galaktose und ihrer Derivative,

by W. Fischer and H. Weinland.

R. S. Tipson, Carbohyd. Res. 6, 127 (1968).

Acetic Anhydride—Phosphoric Acid as an Acetylating Agent.

A. J. Fatiadi, Carbohyd. Res. 6, 237 (1968).

Stable Radicals Obtained by Treatment of Azulene with

Periodic Acid and Other Oxidants.

A. J. Fatiadi, Chem. Commun. 1968, 456.

Formation of Arylazocyclohexene Derivatives on Acylation of

Certain Inosose Phenylhydrazones

.

A. J. Fatiadi, Carbohyd. Res. ]_, 89 (1968).

Infrared Spectroscopy of Carbohydrates: A Review of the

Literature

.

R. S. Tipson, NBS Monograph 110 (June 1968)

.

Manuscripts in Process of Publication

Periodic Acid, a Novel Oxidant of Polycyclic, Aromatic

Hydrocarbons

.

A. J. Fatiadi. (Manuscript accepted for publication in

J. Res. NBS)

Reaction of Some Sulfonic Esters of D-Mannitol with Methoxide; Synthesis of 2 ,3 : 4,5-Dianhydro-D-iditol

.

R. S. Tipson and A. Cohen. (Manuscript accepted for

publication in Carbohydrate Research)

P.M.R. Evidence for the Conformations of 1,2-0-Benzylidenea-D-glucofuranose Derivatives

.

B. Coxon. (Manuscript accepted for publication in Carbohydrate Research)

Acylation of Tetrahydroxy-p_-benzoquinone . A. J. Fatiadi. (Manuscript accepted for Journal of

Chemical and Engineering Data)

107
Bromine Oxidation of Inositols for Preparation of Inosose

Phenylhydrazones and Phenylosazones

A. J. Fatiadi. (Manuscript accepted for publication in

Carbohydrate Research)

Isotopic Methods in Carbohydrate Chemistry.

R. Schaffer. (Manuscript accepted for "The Carbohydrates," edited by W. Pigman and D. Horton, 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, in

press

)

Esters of Benzenepentol ( Pentahydroxybenzene)

A. J. Fatiadi. (Manuscript completed for Journal of

Chemical and Engineering Data)

108
7. ACKNOWLEDGMENTS

The editor gratefully acknowledges the complete cooperation given by his associates in the Organic Chemistry

Section in the preparation of this report, and, in addition,

the editorial expertise of Dr. R. S. Tipson manifested

during its assembly. Special thanks are due Mrs. R. L. Keck,

Section Secretary, and Mrs. J. R. Gossard in whose capable

hands the manuscript took its final form. We also wish to

thank Mrs. R. S. Maddock, Administrative Assistant for the

Division of Analytical Chemistry in Charge of Publications,

for assisting in the publication of this report.

109
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