flatfonal Bureau of Standafls

Library, E-01 Admin. Bldg.

OCT 2 8 1969

NBS TECHNICAL NOTE 507

Organic Chemistry

Section:

Summary of Activities

July 1968 to June 1969

litftMi!

U.S. DEPARTMENT OF COMMERCE
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards ' 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

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the Bureau conducts research and provides central national services in four broad program

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standards, (3) technological measurements and standards, and (4) transfer of technology.

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UNITED STATES DEPARTMENT OF COMMERCE

Maurice H. Stans, Secretary

NATIONAL BOREAU OF STANDARDS • Lewis M. Branscomb, Director

NBS TECHNICAL NOTE 507

ISSUED OCTOBER 1969

Nat. Bur. Stand. (U.S.), Tech. Note 507, 145 pages (Oct. 1969.) CODEN: NBTNA

Organic Chemistry

Section:

Summary of Activities

July 1968 to June 1969

Edited by Robert Schaffer

Organic Chemistry Section

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D.C. 20234

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
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 require months to perfect and

yet all too often must be covered in only a line or two of a journal article.

li
Thus our progress reports endeavor to present this infortion which we have struggled to obtain and which we feel

ght be of some help to others. Certain areas which it

pears will not be treated fully in regular publications are

nsidered in some detail here. Other results which are

ing written up for publication in the journal literature

e covered in a much more abbreviated form.

At the National Bureau of Standards publications such as

ese fit logically into the category of a Technical Note.

1969 we plan to issue these summaries for all of our

ctions. The following is the fifth annual report on proess of the Organic Chemistry Section.

W. Wayne Meinke, Chief

Analytical Chemistry Division

111
PREFACE

This is the fifth annual progress report of the Organic

Chemistry Section of the Analytical Chemistry Division, and

it describes the activities of the Section from July 1, 1968

to June 30, 1969. Unlike the formal, journal publications

that emanate from the Section from time to time, this report

summarizes the activities of the entire Organic Chemistry

Section during that period.

Following the established pattern within this Division,

the programs of the Section have both a research and a sample

aspect: about half of the total effort of the Section has

been applied to research, and the remainder to certification

of Standard Reference Materials (SRM's). The Section must

concern itself with many subjects in organic chemistry in

supporting the National Bureau of Standards' overall mission,

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

nationwide problems through the enhancement of meaningful

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

Section's effort was directed to meeting the needs for SRM's

to serve clinical chemistry laboratories. This has entailed

working with organic compounds that fall into a number of

classical areas of organic chemistry. Indeed, in work planned,

yet other areas will be included. Under the aegis of the

National Institute of General Medical Services of the National

Institutes of Health, the Section is about to undertake development of the competences that will be required in enabling

the Bureau to certify enzyme and other protein SRM's.

To maintain overall unity in the Section's program,

considerable attention is given to one major area of organic

chemistry, the carbohydrates, developing therein the Section's

competences in organic analysis, characterization, and

synthesis. These competences are utilized to the maximum

extent for the development of SRM's.

iv
In this report, in order to describe procedures

adequately, it has been necessary occasionally to identify

commercial equipment. In no case does such identification

imply NBS recommendation or endorsement, nor does it imply

that the equipment identified is necessarily the best

available for the purpose.

Robert Schaffer, Chief

Organic Chemistry Section

v
TABLE OF CONTENTS

1. CARBOHYDRATE SYNTHESIS, STRUCTURE, AND

CHARACTERIZATION: NOVEL RESEARCH MATERIALS

AND MODEL COMPOUNDS

PAGE

Acid-catalyzed Hydrolysis of Isopropylidene

Acetals of Some 2-Pentuloses and 2-Hexuloses . . . . 2

Improved Preparation of 1,2 : 3,^-Di-Oisopropylidene-D-erythro-pentulofuranose . New

Derivatives of D- erythro-Pentulose 7

Conformational Analysis of Heterocyclic Compounds.

The Favored Conformations of p-Dioxane-2 , 3-diol

and its Diacetate 13

15 Studies of ^N-Labeled Amino Sugars. Synthesis

and Spectroscopy of Derivatives of 6-Amino-6-

deoxy-g-glucose-6_-15N 21

Synthetic Methods 21

Magnetic Resonance Parameters 22

Synthesis, Equilibration, and Conformations of

Diastereoisomeric 1 , 2-0-1 sopropylidene-3 ,5-0-

(methoxymethylidene)-6-0-p-tolylsulfonyl-a-Dglucofuranoses . Conformational Evidence from a Nuclear Overhauser Effect 23

Synthetic Reactions 26

Reaction Mechanisms 28

Conformations 28

Determination of Configuration 31

Nonbonded Interactions 31

Intramolecular, Nuclear Overhauser Effect. 33

Polyhydroxy Cyclic Compounds 33

Evidence for a Half-chair Conformation of

Certain Osotriazoles from Inositols 33

Novel Aromatization of a Trihydroxycyclohexanetrione

(Triketoinositol) to a Dibenzo-p-dioxin

Derivative 35

Conversion of Certain Cyclic Phenylhydrazino

Derivatives into Phenylazo Compounds with

Periodic Acid 37

Phenylhydrazono—phenylazo Tautomerism.

Reactions of Phenylformazans and Certain

Bis (phenylhydrazones ) with Strong Acids.. 39

VI
TABLE OF CONTENTS (CONT'D)

PAGE

2 . CLINICAL CHEMISTRY 4l

Bilirubin 42

Volatile Matter in Bilirubin Samples 43

Ash Content of Bilirubin Samples 44

Spectrochemical Analysis of Ash from

Bilirubin Samples 45

Solubility of Bilirubin 45

Spectra of Bilirubin Samples in the Visible

Range 47

Stability of Bilirubin Samples in Various

Solvents 48

Effect of Antioxidants on Stability of

Bilirubin Solutions 49

Synthesis of Mesobilirubin IXa 49

Synthesis of Biliverdine 52

Thin-layer Chromatography of Bilirubin 53

Mass Spectra of Bilirubin Samples 57

Thermogravimetric Examination of Bilirubin

Samples 5 8

Tests for Bicarbonate-extractable

Materials 6l

The Diazo Reaction for the Assay of

Bilirubin 66

Examination of Bilirubin Samples by X-Ray

Diffraction 68

Oxidation of Biliverdine 68

Oxidation of Bilirubin 69

D-Glucose (Clinical) 79

Paper Chromatography of g-Glucose 79

Thin-layer Chromatography of g-Glucose 8l

High-pressure, Liquid Chromatography 82

Optical Rotation of g-Glucose 82

Proton Magnetic Resonance Spectroscopy

of D-Glucose 85

Drying of g-Glucose 86

Ash Content of g-Glucose 86

Undissolved Residue in D-Glucose 86

Nitrogen Content of g-Glucose 87

Creatinine 87

Urea 88

Thin-layer Chromatography of Urea 88

Paper Chromatography of Urea 91

vii
TABLE OF CONTENTS (CONT'D)

P AGE

Infrared Spectroscopy of Urea 92

Proton Magnetic Resonance Spectroscopy

of Urea 92

Ultraviolet Spectrophotometry of Urea 92

Determination of Biuret 93

pH of Urea in Aqueous Solution 9^

Drying of Urea 9^

Change in Weight on Exposure to the

Atmosphere 96

Determination of the Purity of Urea by

Differential Scanning Calorimetry 96

Determination of the Purity of Urea by

Phase-solubility Analysis 97

Calcium Carbonate 98

Uric Acid 98

Cholesterol. Post-issuance Studies 106

Thin-layer Chromatography of Cholesterol

Samples 10 6

Liebermann—Burchard Analysis of Cholesterol

Samples 106

Phase-solubility Analysis and Differential

Scanning Calorimetry of Cholesterol

Samples 10 7

DETECTION AND IDENTIFICATION OP TRACES OP

ORGANIC COMPOUNDS 10 8

Thin-layer Chromatography 109

Infrared Spectroscopy 110

ANALYTICAL CHEMISTRY OF CORN SYRUPS AND RELATED

PRODUCTS Ill

Direct Determination of Moisture Ill

Near-infrared Spectrophotometry Ill

Infrared Spectrophotometry 114

Gas—solid Chromatography 115

The Karl Fischer Titration 116

Physical Properties of Hydrolyzates 119

Characterization of Hydrolyzates 119

Other Physical Properties 120

VI 11
TABLE OF CONTENTS (CONT'D)

PAGE

5 . PERSONNEL AND ACTIVITIES 121

Personnel Listing 121

Publications 121

Manuscripts in Process of Publication 122

NBS Reports 124

Lectures 124

6 . ACKNOWLEDGMENTS 126

7 . REFERENCES 12 7

LIST OF FIGURES

FIGURE NO.

1. Rate of hydrolysis of isopropylidene acetals

5a through 10 in 100 mM oxalic acid

(aqueous) at 65 °C 6

2. The p.m.r. spectrum of p-dioxane-2 ,3-diol at

60 MHz in methyl sulfoxide-dg 16

3. The observed (upper) and calculated (lower)

p.m.r. spectra of the protons on C-5 and

C-6 in 2 ,3-diacetoxy-p-dioxane at 60 MHz

in acetone-cL- 18

4. The observed (upper) and calculated (lower)

p.m.r. spectra of the protons on C-5 and

C-6 in 2 ,3-diacetoxy-p-dioxane at 100 MHz

in acetone-pV 19

5. A facility for computer-assisted analysis of

n.m.r. spectra 22

6. P.m.r. spectra of solutions in pyridine-d,-

at 100 MHz; the isopropylidene methyl

resonances are omitted, (a) 6-Acetamido6-deoxy-l,2 : 3 ,5-di-O-isopropylidene-a-Dglucofuranose (5), Tb_) 6-acetamido-6-

deoxy-1,2 : 3 ,5-di-O-isopropylidene-a-Dglucofuranose-6- 5 N (5- N) 24

ix
LIST OF FIGURES (CONT'D)

FIGURE NO. PAGE

7. P.m.r. spectra in chlorof orm-d at 60 MHz:

(a) l,2-0-isopropylidene-3,5-0-[ (endomethoxy )methylidene ]-6-0-p-tolylsulf ony 1-

a-g-glucofuranose (2), Tb_7 1,2-0-

isopropylidene-3,5-0-[( exo -methoxy )-

methylidene]-6-0-p-tolylsulfonyl-a-P: - glucofuranose (3), (c_) 2 and 3 in

equilibrium. Methyl formate signals

appear at A and B 27

8. (a) Partial p.m.r. spectrum of 1,2-0-

isopropylidene-3 ,5-0-[

(

endo-methoxy )

methylidene]-6-0-p-tolylsulfonyl-a-gglucofuranose (2) in 6:1 (v/v) acetone-cL- —

pyridine-cL- at 100 MHz. (b) Computed

spectrum of same compound, from iterative

analysis 29

9

.

Thermogravimetric equipment used for the

analysis of bilirubin 58

10. Thermogravimetric curves for various samples

of commercial bilirubin (1, lot 39148: 2,

lot 5310; 3, lot 7941-1; 4, lot FP ; and

5 , lot 39148 under nitrogen) 60

11. Oxidation of bilirubin (in methyl sulfoxide)

with periodic acid at 25 °C A and B,

peaks for bilirubin; C, D, and E, new

peaks. Times of recording: (a) 2 min,

(b) 30 min, (c) 60 min, (d) 90 min,

(e) 120 min, (f) 150 min, (g) 180 min,

and (h) 64 hr 72

12. Phase-solubility diagram of urea in isopropyl

alcohol at 25 °C 99

13. Near-infrared absorption spectra of water,

sucrose, and a corn syrup (each dissolved

in methyl sulfoxide) 113

14. Gas-chromatography equipment for the

determination of water in starch

hydrolyzates 115

15. Chromatogram of three injections of a syrup solution 117

x
LIST OF TABLES

TABLE NO. PAGE

1. Chemical shifts and proton—proton couplingconstants for 2 , 3-diacetoxy-p-dioxane (4a)

in acetone-d-- 77 . . 20

2. Coupling constants (Hz, first-order) of

6-amino-6-deoxy-l,2 : 3 ,5-di-0-is propyl! dene

15 a-D-glucofuranose-6- N derivatives at

100 MHz 25

3. Loss in weight of samples of commercial

bilirubin at 111 °C/^0.3 torr 44

4. Ash content of samples of commercial

bilirubin 44

5. Proportions (ppm, by weight) of elements in

ash from samples of commercial bilirubin... 46

6. Approximate solubilities of bilirubin at

13 °C 47

7. Millimolar extinction coefficients of

samples of commercial bilirubin 48

8. Stability of samples of commercial

bilirubin in solution (10 uM) , as shown

by decrease in &_uco 50

9. Mobilities of bilirubin, biliverdine, and

mesobilirubin in t.l.c 54

10 . Mass-spectral data for bilirubin 57

11. Decomposition of various commercial

bilirubins in dry air, as shown by

thermogravimetric analysis 59

12. Bicarbonate-extractable material in samples

of commercial bilirubin 63

13. Materials-balance analysis of the purity of

samples of commercial bilirubin 65

14

Millimolar extinction coefficient of

"azobilirubin" from samples of commercial

bilirubin 67

xi
LIST OF TABLES (CONT'D)

TABLE NO. PAGE

15."* Change& in A , and in its intensity, on

max 5 ' oxidation of bilirubin with mchloroperoxybenzoic acid 75

16. Effect of various oxidants on bilirubin as revealed by spectrophotometry in the

visible region 76

17- Values for the specific rotation of a solution of D-glucose in methyl sulfoxide.. 83

18. Specific rotation, at various wavelengths,

of an equilibrated solution of D-glucose

in water 83

19. Effect of gradual heating of quartz plates

in a polarimeter 84

20. Absorbances at various wavelengths in the

u.v. spectra of aqueous solutions of

various samples of urea 93

21. Absorbances of solutions of (a) biuret and

(b) various samples of urea in alkaline

cupric sulfate solution 94

22. Loss in weight of urea at 25 °C/0.01 torr

after various time-intervals 95

23. Loss in weight of urea at 115 °C/760 torr

after various time-intervals 96

24. Purity of various samples of urea, as determined by differential scanning

calorimetry 97

25. Estimates of precision of two Karl Fischer

procedures 118

Xll
ORGANIC CHEMISTRY SECTION: SUMMARY OF ACTIVITIES

JULY 19 68 TO JUNE 19 69

Edited by Robert Schaffer

ABSTRACT

This report by the Organic Chemistry Section of the

National Bureau of Standards' Institute for Materials Research

summarizes a year's scientific activities; consequently, it

covers both work that was completed and some that is still

in progress. It reports new work on carbohydrates and

polyhydroxy cyclic compounds, and analytical studies leading

to (a) the development of Standard Reference Materials for

clinical chemistry, (b_) characterizations of trace quantities

of organic compounds, and (c_) understanding the properties

of starch hydrolyzates

.

In research on carbohydrates, rules on selective

hydrolysis of certain ketose acetals, a new crystalline

derivative of potential use as an SRM for "D-ribulose ,

conformations of compounds having m- and p-dioxane rings,

15 and a synthesis of N-labeled "6-amino-D-glucose" are

described.

Work on polyhydroxy cyclic compounds has shown that

(a) phenylosotriazoles of diketoinositols favor a half-chair

conformation, (b_) mercuric acetate is a superior reagent for

preparing these osotriazoles , (c_) acetylation in 100%

phosphoric acid gives an octaacetoxydibenzo-p-dioxin from

4,6/5-trihydroxy-l,2 ,3-cyclohexanetrione, whereas acetylation

in pyridine had been shown to produce aromatized products,

(d) periodic acid oxidizes phenylhydrazino to phenylazo

derivatives, and (e) phenylformazans and bis (phenylhydrazones

)

form highly colored salts with strong acids.

Xlll
In examining a variety of commercial samples of

bilirubin in the course of developing an SRM for clinical

analysis, data have been obtained by several techniques,

including thin-layer chromatography and thermogravimetric

analysis. Visible spectrophotometry gave data on the

stability of bilirubin dissolved in a variety of solvents.

Measurements were made by the diazo assay. A materialsbalance analysis of bilirubin samples was developed.

Various chromatographic procedures, used to examine a supply of g-glucose as an SRM, are described. Measurements

of optical rotation in water and methyl sulfoxide, and other

analytical data, are also given. Some of the work pertaining

to the creatinine, urea, calcium carbonate, uric acid, and

cholesterol SRM's that have been issued are presented.

Finally, work on the detection and identification of

trace quantities of a few organic compounds is summarized,

and the description and statistical evaluations of several

methods developed for the characterization of the properties

of corn starch hydrolyzates are summarized.

Key Words

:

15 6-Amino-6-deoxy-D-glucose-6- ^N, bilirubin analysis,

calcium carbonate SRM, conformations by NMR, corn syrup

analysis, creatinine SRM, g-glucose (clinical SRM), organic

trace analysis, SRM's for clinical analysis, urea SRM,

uric acid SRM.

xiv
1. CARBOHYDRATE SYNTHESIS, STRUCTURE, AND CHARACTERIZATION:

NOVEL RESEARCH MATERIALS AND MODEL COMPOUNDS

The carbohydrates continue to be the Section's area

of continued research specialization in organic chemistry.

As an outgrowth of our previous work with isopropylidene

acetals , a study of the selective hydrolysis of such

substituents located at different positions on ketoses has

enabled us to formulate some generalization on their selective

cleavage. Advantage was taken of the high sensitivity to

hydrolysis of 1,2-linked spiro acetals in obtaining

crystalline 3j^-0-isopropylidene-"D-ribulose, " which should

prove most useful as a stable precursor that can be easily

hydrolyzed further to give the rare sugar, which is known

only as a syrupy material.

Proton magnetic resonance spectroscopy is an essential

technique for laboratories doing fore-front research. In

our work, this spectroscopy is providing understanding of the

conformations of a variety of compounds, with much attention

being given heterocyclic rings having two oxygen atoms, e.g. , from simple compounds containing a p-dioxane ring to such

complex substances as diastereoisomeric methoxymethylidene

furanose derivatives with m-dioxane rings in their structures

15 Some newly synthesized precursors of N-labeled 6-amino-6-

deoxy-g-glucose have also been examined by p.m.r.

In the past few years, much effort has been devoted to

the inositols and their oxidation products. This year's

report reflects our continued interest in these substances;

the emphasis here is the chemistry of the di- and tri-keto

oxidation products and nitrogen-containing derivatives

thereof.
A. Acid-catalyzed Hydrolysis of Isopropy lidene Acetals of

Some 2-Pentuloses and 2-Hexuloses

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

In Technical Note ^57, we described the acid-catalyzed

hydrolysis of some isopropy lidene derivatives of ketoses

.

For this purpose, the use of such hydrolysts as hydrochloric

acid or sulfuric acid leads to considerable decomposition of

the free ketose liberated. This difficulty was overcome by

use, as the hydrolyst, of oxalic acid [1]. This compound

has the further advantages that (a) the anhydrous acid or

its dihydrate may readily be weighed out for the preparation

of an aqueous solution of any desired molarity, and (b_) the

hydrolysis may be rapidly arrested at any desired stage by

addition of an excess of calcium carbonate, with subsequent

removal of the resulting mixture of calcium oxalate and

calcium carbonate by filtration. In an earlier study [2],

it was found that 100 mM oxalic acid is suitable for

hydrolyzing a variety of isopropylidene acetals of pentoses

at 65 °Cj and in determination of the rate of hydrolysis.

It has previously been shown [2] that, under these

standard conditions of hydrolysis, the per-isopropylidenated

aldopentoses [namely, 1,2 : 3

, ^-di-O-isopropylidene-3-Larabinopyranose (1-e, the enantiomorph of 1), 1,2 : 3 , 5-di-0-

isopropylidene-a-g-xylofuranose (2), 2 ,3-0-isopropylidene-aD-xylofuranose (3), and 2 , 3-0-isopropylidene-D-ribofuranose

(4)] are completely hydrolyzed to the corresponding free

sugar within 2 hr at 65 °C, regardless of (a) the presence of

a pyranoid (1) or a furanoid ring (2—4), or (b) the positions

of attachment of the isopropylidene groups . However, as

previously noted [2], hydrolysis of 2 to 1,2-0-isopropylidenea-g-xylofuranose is complete in 40 min at 25 °C, but hydrolysis

of 1-e to a monoisopropy lidene acetal, at either 25 or 65 °C,

could not be detected polarimetrically

.
H

2COH H

2COH

H,OH

We have now studied, the hydrolysis of 1; complete

hydrolysis was effected in 1.5 hr at 65 °C, but the presence

of any compound besides 1 and D-arabinose in the reaction

mixture at any time could not be detected by thin-layer

chromatography on Kieselguhr G (preheated for 1 hr at 110 °C)

with 2:1 (v/v) ethyl acetate—pentane (detection with sulfuric

acid spray, and charring) . Hence, as regards acetals 1—4,

the m-dioxane ring c is -fused at C-3,C-4 of 2 is much less

stable than any of the cis -fused 1, 3-dioxolane rings.

In contrast to these results for the acetals (1—4) of

the aldopentoses, the sole 2-pentulose acetal for which a detailed study was made, namely, 2 , 3-0-isopropylidene-3-Dthreo-pentulofuranose (9) 3 required 8 hr for complete

hydrolysis. We have therefore determined, by the technique

3
described earlier [2], the rate of hydrolysis of the diacetal

of the other p_-2-pentulose , namely, l a 2 : 3 , 4-di-0-

is op ropy li dene -D-erythro-pentulofuranose (depicted as the

3-D anomer 5a) , and have compared it with the rates for some

2-2-hexulose acetals, namely, 1, 2 : M, 5-di-0-isopropy lidene3-P_- ribo -hexulopyranose (6), 1, 2 : 4 , 5-di-0-isopropy lideneg-g- erythro -2, 3-hexodiulo-2 ,6-pyranose (7), 1 , 2 : 4 , 5-di-0-

isopropylidene-3-D- arabino-hexulopyranose (8), and

2 , 3 :

, 5-di-Q-isopropylidene-3-D- arabino -hexulopy ranose ( 10 )

.

The 3-2 anomeric configuration of compounds 6 and 7 is

5a R = H

5b R = CH2

OH

8

CH 2OH

Vo

10

CH2OH

assigned because of their preparation [3, 4] from compound 8,

to which this configuration had been assigned by Ohle and

4
Just [5]. This anomeric configuration has now been proved

by X-ray crystallographic study of a sample of compound 8

that we provided (R. D. Rosenstein and S. Takagi , University

of Pittsburgh, personal communication, February 5th, 1969).

The results of hydrolysis of acetals 5a through 10,

with 100 mM oxalic acid at 65 °C, are given in fig. 1; all

values for [a]

n are calculated on the original weight of

acetal. The final specific rotations, recalculated as the

respective free sugar, are: for 5a, -16.6° (lit. [2] for

enantiomorph, +16.6°); 6, +4.7° (lit. [6] +4.7°); 7, -43.8°:

8, -91.2° (lit. [7] -92.0°); 9, -33.2° (lit. [2] -33.2°); and

10, -90.3° (lit. [7] -92.0°)

.

From figure 1, it may be seen that these six compounds

fall into three groups, namely, 5a (^1.25 hr) ; 6, 7, and 8

(2-3 hr) ; and 9 and 10 (8 hr) . Thus, of the four acetals

(5a—8) having a spiro structure involving C-l and C-2, the

furanoid compound 5a is hydrolyzed about twice as fast as the pyranoid compounds (6, 7, and 8).

Presence of two spiro-fused, 5-membered rings in compound

5a evidently induces instability in the 1,2-isopropylidene

group, because graded hydrolysis of 5a gives a monoacetal

that we have isolated, and characterized as 3,4-0-

isopropylidene-g-erythro-pentulofuranose , whereas graded

hydrolysis of 6 (see ref. 4) and 8 (see ref. 8), in which

there is less strain at C-2, causes removal of the

4,5-isopropylidene group. In contrast, the acetals 9 and 10

(that lack the spiro structure but have a hydroxymethyl group

attached to C-2) are completely hydrolyzed at about one

quarter of the rate found for compounds 6, 7, and 8,

regardless of the presence of a furanoid (9) or a pyranoid

ring (10). This behavior must be attributable to a stabilizing effect of the hydroxymethyl group at C-2 (compare

9 with 2, and 10 with 1). [Graded hydrolysis of 10 with acid

causes [9] removal of the 4 ,5-isopropylidene group, to give a

5
oo

ISh<

s:c

UJIh

Sl£ooG

•HO ? rH I

£5oP

Cti? LTV

tQ H

cti -p

(DO

cti

<DC

<U T3

•H

>> o a o oU m a VD o

CO -p •H cti

<m ^ O CO3

CO o H 0)

CO 3

>i

a1 H cti O ^_^- Ph T3 Td

>a •H

£5 o

cti <MO o

•H

CD .—1

-P Cti

Cti

X

(X

O

(saejBsp) J_ [p]

(D

hD

•H
py ranoid structure resembling the furanoid structure 9-]

1 R = H

10 R = CH2OH

Determination of the rate of acid-catalyzed, complete

hydrolysis to the free sugar, under the conditions described,

therefore appears promising as a simple, rapid method for

determining (a) the presence or absence of a spiro structure

involving C-l and C-2 in cyclic acetals of 2-ketoses (fast

or slow hydrolysis, respectively), and (b_) the presence of a furanoid ring if this spiro structure is present (very fast

hydrolysis). As a check, hydrolysis of 1,2 : 3 , 4-di-Oisopropy lidene-3-D-ribo-hexulofuranose (5b) was studied; it

was rapid (^50 min for completion)

.

This work has been published [10].

B. Improved Preparation of 1 ,2 : 3 , 4-Di -0- isopropylidene -gerythro-pentulofuranose . New Derivatives of D-erythroPentulose

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

g- erythro -Pentulose (6) is a hitherto rare sugar that

is an intermediate in many important biochemical processes.

A crystalline derivative of g- erythro -pentulose was desired

for issuance as a Standard Reference Material. Therefore,

in order to prepare relatively large quantities of 6, we

decided to (a) treat g-arabinose (2) with boiling pyridine

[11], (b) remove the aldopentoses (1, 2, 3, and 4) from the
H H

I H

I H

I C=0 C=0 C=0 C=0

i HCOH

1 HOCHI HOCH HCOHI HOCH

I HCOH

I HOCH

I HCOHI HCOH HCOH

I HCOH HCOH

CH 2OH CH 2OH CH2 OH CH 2 OH

D-Xylose D-Arabinose D-Lyxose D-Ribose

1 2 3 4

CHoOH CH 20H CHoOH CH 2OH

c=o c=o

I HOCH

I HCOH

i HOCH HCOH

I C=0

I c=oI HCOH

I HCOHI HCOHI HCOH

I CH 2 0H CH2OH CH2 OH CH2 OH

D-threo- D - erythro- D-threo-3- erythro -3-

Pentulose Pentulose Pentulose Pentulose

5 6 7 8

CH2OH

Ip /

OCH,

3p

VIp

^OCH

I c=o

I HCO.

HoCO^

I

H2C0.

I ^

HCO^

I c=o

I HCO^

H2 aK

:Ip

:I P

10 11 12
initial reaction mixture (after removal of as much as possible

of 2 by crystallization), and then (c_) acetonate the mixture

of pentuloses (5, 6, 7, and 8), and separate the mixture of

one monoacetal (9) and three diacetals (10, 11, 12 >. . It was

found that pure 1,2:3, 4-di-0-isopropylidene-g-erythro- pentulofuranose (10) is obtainable only by distillation,

because the small content of diacetals 11 and 12 in the

diacetal fraction is not removable by solvent partition.

The diacetal 10 is completely hydrolyzed to g- erythropentulose (6) by 100 mM oxalic acid in 56 hr at 25 °C and

in 72 min at 65 °C; in contrast, with 4:1 (v/v) acetic acidwater as the hydro lyst, treatment for 8 hr at 65 °C is

required. In a polarimetric study of the rates of these

hydrolyses, the optical rotation decreased steadily from the

high value for 10 to the low (equibrium) value for 6, with

no intermediate maximum or minimum; thus, polarimetric

observation affords no evidence as to the intermediate

formation or nonformation of a monoisopropylidene acetal of

D-erythro -pentulose . Consequently, the hydrolysis was

followed by t.l.c., and it was found that a monoacetal is,

indeed, formed. The concentration of 10 decreases steadily,

while that of 6 increases steadily; and the concentration of

the monoacetal rapidly rises to a moderate value that is

maintained until, near the end of the hydrolysis, the compound

disappears. With this information, it then became possible

to arrest the hydrolysis after the elapse of sufficient time

to afford the optimal yield of monoacetal with minimal

hydrolysis of the diacetal. The two acetals were then

separated from free 6 by extraction with ether, and the

monoacetal was separated from the diacetal (10) by extraction

of the ether solution with water.

Purification of the monoacetal by distillation gave a

25

syrup, [ot] n -63. 0° (acetone), that was homogeneous by t.l.c.

On slow crystallization

— from ether, one anomer,

'25 having [a] D
-5.4° (acetone), was isolated. Both the crystalline material

and the syrup reduced Benedict solution rapidly, and both

showed mutarotation in water, to give the same equilibrium

rotation; consequently, one of the two free hydroxyl groups

in the monoacetal is attached to C-2, the anomeric carbon

atom. To determine the position of attachment of the other

hydroxyl group, the monoacetal was oxidized with sodium

metaperiodate ; the crystalline product was identified as the known [12] 2 , 3-0-isopropy lidene-g-erythrono-1 ,M-lactone

(14), showing that the free hydroxyl groups in the monoacetal

are attached to C-l and C-2. Hence, the monoacetal is

3 , 4-Q-isopropylidene-D- erythro -pentulofuranose (13), and the

diacetal from which it was prepared is, as would be expected,

1,2 : 3, 4-di-0-isopropylidene-D-erythro_-pentulofuranose ( 10 ) . Moreover, the properties of 14 were identical with those of

authentic 14 ? showing that our sample of 10 was not

contaminated by the corresponding diacetal of the enantiomorph

of 6 (which might theoretically be formed in the isomerization

with pyridine)

.

The optical rotation (in water) of the crystalline form

of monoacetal 13 was more positive than that of the syrupy

mixture of anomers , suggesting that the crystalline material

is the a-D anomer. This assignment is supported by the

results of p.m.r. spectroscopy; the low value for the chemical

shift of the C-l protons in the spectrum of a solution of

the crystals suggests that C-l may be trans to the

1, 3-dioxolane ring fused at C-3,C-4. In addition, the

appearance of the C-l protons as a singlet fits the pattern

shown by the anomeric triesters 17 and 18 (see later).

On treatment with acetic anhydride—pyridine , both the

crystalline and the syrupy 13 were converted into a syrupy

mixture, not yet crystallized, of two diacetates (15) that,

by t.l.c., was found to contain the same two compounds,

although in different proportions

.

10
10

v°

OH,CH2OH -Q13

°W°

14

10 6

HO OH

16

OMe,CH2OH

O p V

OAc, CH2OAc

15

OMe,CH2OAc

AcO OAc

17

BzO OBz

OMe,CH2OBz

11

18
Both the crystalline and the syrupy 13 were completely

hydrolyzed to D- erythro -pentulose (6) by 4:1 (v/v) acetic

acid-^water in 1 hr at 65 °C. Acetylation or benzoylation of

syrupy D- erythro -pentulose gave a syrupy mixture of esters,

shown by t.l.c. to consist of three compounds, presumably the

tetraesters of the acyclic form and of the two anomers of the

furanose. Therefore, in order to obtain a less complex

mixture of esters that might crystallize, a mixture of the

anomers of methyl D- erythro -pentulofuranoside ( 16 ) was

prepared by condensation of 6 with methanol containing 1% of

hydrogen chloride; the resulting mixture was acetylated or

benzoylated, yielding, in each instance, a mixture of two

compounds (as shown by t.l.c). Preparative t.l.c. of each

mixture effected the isolation of each anomer; however, of

the four compounds, 17, a and b; 18, a and b), only a methyl

1,3, 4- t ri-O-benzoyl-D- ery thro -pent ulo fur anoside (probably

the a-D anomer of 18) has thus far been obtained in

crystalline form.

P.m.r. data for the D- erythro-pentulose derivatives

show that the multiplicity of the signal due to the two

protons on C-l is quite definitely dependent on the

configuration at the anomeric center (C-2). For the compounds

presumed to be the a-D anomers, the protons on C-l appear

as a singlet, whereas, for those presumed to be the 3-D

anomers, they appear as two 1-proton doublets. It seems

reasonable to suppose that this multiplicity is a consequence

of the restricted rotation of the methylene group (C-l) when

i t is cis to the 3-acyloxy group. These assignments are

supported by the positions of the signals caused by the

anomeric methoxyl groups of the respective, acylated methyl

glycosides; the signal appears at lower field for the presumed

ot-g anomers, in which the methoxyl group would be cis to the

3-acyloxy group. These tentative assignments agree, in each

instance, with the relative, specific optical rotation of the

12
two anomers, and would therefore appear to permit unequivocal

assignment of anomeric configuration to the anomers of

compounds 17 and 18.

These results have been incorporated in an article

which has been accepted for publication [13].

C . Conformational Analysis of Heterocyclic Compounds. The

Favored Conformations of p- Dioxane-2 ,3-diol and its

Diacetate ~ (R. F. Brady, Jr. )

1. Introduction

Relatively little is known about the favored

conformations of saturated, six-membered, heterocyclic

compounds. Often, such conformations have been considered

to be the same as those for similarly substituted derivatives

of cyclohexane, but the limited data available do not support

this assumption.

Saturated heterocyclic molecules having oxygen in the

ring offer interesting variations on the cyclohexane series

A p-dioxane ring is smaller than a cyclohexane ring because

o the C-0 bond (1.42 A) is shorter [14] than the C-C bond

o (1.53 A); this diminished ring-size may result in significant

increase in steric and dipolar interactions between substituents [15] in some compounds. In addition, the polar nature

of the C-0 bond and of the unshared pairs of electrons on

the oxygen atom may lead to dipolar interactions with polar

ring-substituents . Furthermore, the C-O-C bond angle (112.5°)

is slightly larger [14] than the C-C-C bond angle (111.5°),

causing small differences in the related angle-bending forceconstants and torsional potentials. Nevertheless, in

p-dioxane, the free-energy barrier to chair inversion is

lower than in cyclohexane, since distinct p.m.r. signals for

axial and for equatorial protons are observed for cyclohexane

[16] below -66.7 °C, but not for p-dioxane [17] even at

-104 °C. The substitution of an oxygen atom for a methylene

13
group introduces the influence of the unshared pairs of

electrons, which, apart from such consequences as van der Waals

interactions [18] and London attractive forces [19], may

result in the development of hydrogen bonding [20].

2 . Results and Discussion

Crystalline p-dioxane-2 ,3-diol (1) is a bis-hemiacetal

formed by the condensation of glyoxal with ethylene glycol.

Only one isomer has ever been reported. The compound was

first prepared by Head [21], and, by following his procedure,

it has now been isolated in 37% yield.

The dipole moment of 1 in anhydrous p-dioxane (the

dipole moment of which is zero [22]) is 2.18 D; this is

comparable [23, 24] to the dipole moments of the trans - disubstituted compounds 2 and 3. The fact that these compounds

exhibit appreciable dipole moments precludes a strict

antiparallel orientation of the substituents [25], and

indicates that the p-dioxane rings deviate from ideal chairconformations due, in part, to 1,3-diaxial interactions

between the substituents on C-2 (C-3) and the hydrogen atoms

on C-6 (C-5). In 1, the two syn-axial interactions of a

1 , R = OH

2 , R = CI

3 , R = Br

4 , R = OAc
hydroxyl group with a proton, in addition to the two

interactions of the "axial" electron-pair on the ring-oxygen

atom with an axial hydroxyl group, cause the molecule to

assume a f lattened-chair conformation. The observed dipole

moment is too low for a cis disposition as, in this case,

the two C-0 bond dipoles (each 1.5 D [26]) would subtend a dihedral angle of 60° (or less) and give rise to an observed

dipole moment of at least 2.6 D.

The 60-MHz p.m.r. spectrum of 1 in methyl sulfoxide-cLat 37 °C is shown in figure 2. The multiplet centered at

3.586 is assigned to the methylene protons, and the multiplet

centered at 4.276 to the hydroxyl protons, and the methine

protons appear as a multiplet centered at 6. 306. The

appearance of an AA'BB' pattern for the four protons on C-5

and C-6 indicates either that the molecule is held in a

rigid conformation at room temperature, or that interconversion between conformations is rapid on the p.m.r. time-scale.

To distinguish between these possibilities, the spectrum

was measured at -7, 10, 66, 88, and 100 °C, and was found

to be independent of temperature. (A 20% solution of 1 in

methyl sulfoxide-d^ solidifies below -7 °C. As an alternative

solvent for 1 was not discovered, the p.m.r. spectrum could

not be measured at lower temperatures.) In particular, no

change was observed in the position or intensity of the lines

in the methylene proton signal, and the multiplet width at

60 MHz was 58.0 ±0.1 Hz for each trial. This result indicates

that 1 remains in a rigid conformation over the temperature

range studied, and that there is no significant proportion

of the Cee conformer present, even at 100 °C. The possibility

of dynamic equilibrium involving a substantial proportion of

a boat form at room temperature is also excluded, since the

spectrum is unchanged at higher temperatures . This finding

is noteworthy, considering that the boat form of p-dioxane

is calculated [27] to have an enthalpy 9,2 kJ/mol greater

15
q

COo

35Q.

Q.q

uSo

-dli CD X3

•HXoH

CO

>s

-CP

(DEC

•HNSo

VDP

cdo

•HI m

CMI <DC

cti Xo

•HI ftl OS3

-po

ft

q

is

Eh

CM

•H

16
than that of the chair form, a difference sufficiently small

to permit the existence of stable flexible forms for substituted

derivatives

The portion of the 60-MHz p.m. p. spectrum of 2,3-

diacetoxy-p-dioxane (4) assigned to the protons on C-5 and

C-6 is shown in figure 3 (upper trace). A computer-assisted

analysis of the spectrum gave a set of coupling constants

that generated the observed pattern (see figure 3). The

pattern observed for the methylene protons at 100 MHz (see

figure 4) was generated when the same coupling-constants

and the chemical-shift difference at 100 MHz were used. The

parameters obtained are given in table 1.

It may be seen from table 1 that the two vicinal

coupling-constants for the trans protons have widely

different magnitudes. The value of J, ?1, namely 12.24 Hz,

is consistent with trans_-di axial protons, whereas that of

J 2, (0.54 Hz) is consistent with trans -di equatorial protons.

The value for the cis equatorial—axial protons is 3.39 Hz,

consistent with this assignment.

The values found for the coupling constants are

consistent with a f lattened-chair conformation for 4. As

for 1, such a conformation could be caused by the two

1,3-diaxial interactions of a proton with an acetoxyl group

in the molecule. A similar conformation has been suggested

[28] for trans -2 , 3-dichloro-p-dioxane (2); thus, it is noteworthy that an independent determination [29] of the coupling

constants of 2 gave values that agree within ±0.2 Hz with

those determined here for 4.

The p.m.r. spectrum of 4 was also recorded at -17, 10,

50, 74 y 80, and 100 °C. Line positions and intensities

remained constant over the entire range of temperature

studied, and, in each trial, the width of the multiplet

assigned to the methylene protons was 67-7 ±0.1 Hz at 60 MHz.

These data are firm proof for the existence of only one

conformation over the temperature range studied, as there is

17
NX

CM co

4^oa

CD VD

,p i CD o o

cd a>

Jh o

4-3 cd o

cd cN S o• MO a

-p ^ cd

CD£ C o cd rH X

V^

O

•H

0) I

cd i H >3 o o

i—I 4^> cd 0) o o

cd -a -h

cd I on

**

U oj

CDa -h -^ KOI T3 O

CD>

CDmo

CD

Ehm

<D

•H

T5C

cd

LPiI Oco

18

Table 1. Chemical shifts and proton—proton couplingconstants for 2 , 3-diacetoxy-p-dioxane (4a)

in acetone-d^

Radiofrequency Chismical shifts^ (Hz)

(MHz)

60 V

l = v

2

212.5

V

3 = v

4

246.5

V

5 = v

6 342.5

100 v

l = v

?

354.3

V

3 = v

4

411.0

, V

5 = v

6

570.9

Coupling constants (Hz)

£l.2 12.24

-1,3

= ^2,4 -12.31

^1,4

= -2,3 3.39

ii,5 = ^2,6 -0.63

*1.6

= J-2,5 0.04

-3,4 0.54

*3,5

= -4,6 0.67

^3,6

= -4,5 0.00

^5,6 -0.76

Downfield from tetramethylsilane (v 0.00 Hz) as the

internal standard.

4a

20
no change in the p.m.r. spectrum at extreme temperatures.

As for 1, the presence of a significant proportion of a boat

form at room temperature is also excluded.

3 . Conclusions

A f lattened-chair conformation, with both substituents

taking axial orientations, is favored for p-dioxane-2 , 3-diol

and 2 ,3-diacetoxy-p-dioxane . The 1,3-diaxial interactions

arising in each instance are lessened by the slight flattenng of the p-dioxane ring.

15

D . Studies of N-Labeled Amino Sugars. Synthesis and

Spectroscopy of Derivatives of 6-Amino-6-deoxy - Dglucose -

(B. Coxon)

6- 15 N

1

.

Introduction

As part of a program for the synthesis, and spectral

and conformational characterization, of isotopically labeled

carbohydrate reference-materials of biomedical interest, a 15 study of amino sugars labeled with N (enrichment >99%)

has been commenced. This work has been initiated by a 15 synthesis of derivatives of 6-amino-6-deoxy-g-glucose-6_- N,

which, in its nonlabeled form, occurs as a component of the

antibiotic Kanamycin [30].

2. Synthetic Methods

A suitable starting point for the synthesis was

1,2 : 3,5-di-0-isopropylidene-6-0-p-tolylsulfony 1-a-Dglucofuranose (1) [31]. Treatment of 1 with sodium iodide

in 2-butanone gave an alternative starting-material, namely,

6-deoxy-6-iodo-l,2 : 3 , 5 -di-O-isopropyli dene -a-D-gluc ofuranose

(2) [32]. Reaction of 1 or 2 with potassium phthalimide

gave 6-deoxy-l,2 : 3 , 5-di-0-isopropylidene-6-phthalimido-a-D21
glucofuranose (3). After two recrystallizat ions , 3 had a purity of 99-5 mole % y as determined by differential scanning

calorimetry [33].

Treatment of 3 with hydrazine gave 6-amino-6-deoxy1 ,2 : 3 ,5-di-0-isopropy lidene-a-g-glucofuranose (4), which

was characterized as its known, crystalline salt with

p-toluenesulfonic acid [3^]. The free amine (4) was also

converted into its N-acetyl derivative (5).

15

15 Reaction of 1 with potassium phthalimide- N afforded

3- ^N in 84% yield.

3 . Magnetic Resonance Parameters

The magnetic resonance parameters of compounds 3—5 and

15 ~ ~ their N-labeled modifications have been studied initially

by p.m.r. spectroscopy at 100 MHz. Spectra of the N-acetyl

Figure 5. A facility for computer-assisted analysis of

n.m.r. spectra.

22
1 R derivatives 5 ana 5- N in pyridine-d,- are shown in figure 6

(a and b, respectively). It may be seen that, even in the

presence of a basic solvent, NH proton exchange is sufficiently

slow to allow observation of the J^ TM and J r T NH Couplings

15 of 5, and additionally of J -, ^ in 5- N. A selection of

proton—proton and proton— N coupling-constants is given

in table 2. For 1, 2 : 3 , 5-di-O-isopropylidene-a-D-glucofuranose

derivatives ( e.g. , 1—5), J_u ,- is much larger than for

1, 2 :

3

i 5-di-O-benzylidene-a-rJ-glucofuranose derivatives [35]

or for a 1, 2-0-isopropylidene-3, 5-0-(methoxymethylidene)

derivative [36], and it is apparent that the m-dioxane ring

of 1—5 does not adopt predominantly the same conformation as

in the latter derivatives. This aspect is being further

investigated.

E. Synthesis, Equilibration, and Conformations of

Diastereoisomeric 1,

2

-0- Isopropylidene-3,

5

-0-

(

methoxy - methylidene ) -6-0-p-tolylsulfonyl -a-D-glucofuranoses . Conformational Evidence from a Nuclear Qverhauser Effect

(B. Coxon)

1 . Introduction

A previous investigation of the conformations of a

series of 1, 2 : 3, 5-di-0-benzylidene-a-rj-glucofuranose

derivatives by p.m.r. spectroscopy unexpectedly revealed

that the m-dioxane ring in these molecules tends to assume

that chair conformation having C-6 of the sugar axially

attached [35]. Because the rationalization of this observation has been difficult, it appeared worth while to study

further compounds of this type, in an effort to define the

interactions that control their favored conformations.

Ribonucleoside derivatives in which the 2

' ,

3

f -0-

(alkoxymethylidene) substituent forms part of a f ive-membered

23
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O

M

o

M

Cm

CO P

C

CDS

CDM

cocd

CD

cd 25
ring are important intermediates in the preparation of

ribo-oligonucleotides possessing the naturally occurring

(3'-*5') internucleotide linkage [37].

2. Synthetic Reactions

Acid-catalyzed reactions of 1, 2-0-isopropylidene-6-0-p_-

tolylsulfonyl-a-g-glucofuranose (1) with trimethyl

orthoformate in N,N-dimethylformamide gave syrups containing two major components in approximately equal proportions.

P.m.r. spectra (similar, for example, to figure 7c) of the

mixtures displayed two H-l doublets, two H-2 doublets, and

pairs of newly introduced methine (one-proton total) and

methoxyl (three-proton total) singlets having similar chemical

shifts. By chromatography on silicic acid, the mixtures were

separated into crystalline (2) and syrupy (3) components that

gave very similar p.m.r. spectra (see figures 7 and 8,

respectively)

.

Treatment of either 2 or 3 with a mixture of trimethyl

orthoformate, N ,

N

-dimethy Iformamide , and sulfuric acid led,

in each case, to a 6:5 mixture of 2 and 3, as indicated by

integration of the methine and methoxyl singlets in the p.m.r.

spectrum of each product mixture. Compounds 2 and 3 could

also be equilibrated (2—3 min) by treatment of their solutions

in chloroform-d with a trace of p_-toluenesulfonic acid. The

spectrum of an equilibrated solution is shown in figure 7c.

For further structural characterization of 2 and 3, their

equilibrated mixture in p_-toluenesulfonic acid—chloroform-d

was treated with one molecular proportion of acetone. Studies

of p.m.r. spectra of the mixture, and isolation from it of

1, 2 : 3 , 5-di-0-isopropylidene-6-0-p_-tolylsulfonyl-a-gglucofuranose (4) demonstrated that methoxymethylidene

substituents in both 2 and 3 were replaced by isopropylidene

groups, with liberation of methyl formate.

The foregoing evidence indicates that 2 and 3 are

26
H H

-o3s-^

H H

(a)

H-l H-3.5 H-2H-3 OMe Me CMe2

":: 6

H-6

H-6'

1 1 «A . 1,.,.. J, - JUl

MeO

1 1 1 1 1 1 1 1

TsOCH 2

2.5! 4.13 4.74 5.48 5.67 6.18 6.72 7.57 8.54 8.69 r

(b)

^jLAl > * ' i v) -«**. ^ J | !»»« «l'<V'»

TsOCH,

I — CH

HllllHlli MeO ^^ I CM—li

2.49 4.08 4.62 5.42 5.82 6.67 7.56 8.55 8.69

Figure 7. P.m.r. spectra in chloroform-d at 60 MHz: (a) 1, 2-O-isopropylidene-3, 5-Q-[( endo-methoxy)methylidene] -6-O-p-tolylsulfonvl-a-D-glucofuranose (2), (b) 1, 2-Q-isopropylidene-3, 5-0- [ ( exo-methoxy)metn'ylidene] -6-

0-_p_-tolylsulfonyl-a-D-glucofuranose (3), (c_) ^2 and 3 in equilibrium.

Methyl formate signals appear at A an? B.

27
diastereoisomeric 1, 2-0-isopropylidene-3, 5-0- (met ho xymethylidene )-6-0-p_-tolylsulfonyl-a-2-glucofuranoses

.

3

.

Reaction Mechanisms

The acid-catalyzed exchange of the 3 , 5-0-(methoxymethylidene) substituent for an isopropylidene group may be

rationalized in terms of the mechanism 2a + 3a -* 6 •+ 7

-* ^

.

The most plausible mechanism for acid-catalyzed interconversion of the diastereoisomers 2 and 3 involves the

intermediate, dioxo-carbonium ion 5.

4

.

Conformations

To obtain reasonably accurate values of the vicinal

coupling-constants, a 100-MHz spectrum (see figure 8a) of 2

was subjected to an iterative computer-analysis. Initially,

the proton group comprising H-l to H-6 ' was analyzed as a seven-spin system. The theoretical spectrum from the iterative

analysis is shown in fig. 8b.

By analogy with previous work [38], the near-equality of

J-. p and J q

k , and the small value of Jp ~, for 2 indicate

that the shape of its furanoid ring is quite close to that of

the Tp conformation, as in conformation 2a (or 3a).

The relatively small value of J^ ,_ , namely, 3-31 Hz,

showed that H-4 and H-5 are disposed mainly in the gauche

orientation, as in conformation 2a (or 3a), in which the

m-dioxane ring assumes that chair conformation having both of

these protons equatorially attached but with C-6 of the sugar

moiety axially attached.

The comparatively large values of J;- r and J r , (6.02

and 6.6 Hz, respectively) indicate large contributions from

rotamers 8 and 9- From the coupling constants, it appears

likely that 3 has fundamentally the same type of conformation

(3a) as has 2.

28
H-l H-3,5

(a)

TsOCH2

MeOmiiiiii

*«^»fV<yi»v *vw f '"»IV,>

5.37 5.62 5.68 5.70 5.77 5.93 T

2.5 Hz

Figure 8. (a) Partial p.m.r. spectrum of 1 ,2-O-isopropylidene3 ,5-0-1

(

endo -methoxy )methylidene]-6-0-p-tolylsulfonyla-g-glucofuranose (2) in 6:1 (v/v) acetone-oV —

pyridine-d,- at 10Q MHz. (b_) Computed spectrum of

same compound, from iterative analysis.

29
TsOCH,

I

' HOCH

TsOCH,

O—CH

MeOiiii K*

TsOCH,

I ' O—CH

+ Hi 1 1 1

1

-Me

i

MeO

-Me o— -Me

Me Me Me

Me

TsOCH2 O CMe2

TsOCH2

'

2a 5

30

3a
5. Determination of Configuration

Evidence for the configuration of C-3,5 in 2 and 3 was

obtained from three sources. It might be anticipated that

the methoxyl substituent in 2 and 3 would show a doubled

"anomeric effect" [39-^5], due to the presence of two hetero

oxygen-atoms in the attached m-dioxane ring.

Studies of pyranoid derivatives have shown that the

anomeric effects of 1-substituents decrease on passing from

a nonpolar to a polar solvent, due evidently to solvation of

the intramolecular dipoles [46]. Accordingly, it was expected

that, for the diastereoisomers 2 and 3, that diastereoisomer

having its methoxyl group in the axial orientation on the

m-dioxane ring would be destabilized with respect to i'cs

equatorial isomer on passing from equilibria in a nonpolar to

those in a polar solvent. Studies of the equilibria of

2 and 3, in chloroform-d-N,N-dimethylformamide showed this to

be the case.

The configurations and conformations of 2 and 3 may,

therefore, be represented as shown in 2a, and 3a, respectively.

Crystalline 2 may therefore be described as 1,2-0-isopropylidene-3 3 5-0- [

(

endo-methoxy )methylidene]-6-Q-p-tolylsulfonyl-qg-glucofuranose, and syrupy 3 as its exo isomer.

The assignment of an equatorial position to H-3,5 of 2

(as in 2a) is also supported by the observation of its longrange coupling (0.42 Hz) with H-4, which is equatorially

oriented with respect to the m-dioxane ring (see formula

8 or 9)

The chemical shifts of H-3,5 in 2 and 3 provided further

evidence for the configuration of C-3,5 in these compounds.

6

.

Nonbonded Interactions

From the enthalpy change 3

-** 2 in chloroform-d and

N,N-dimethylformamide , it may be deduce a that the stabilization

31
H

TsO.

8

ROH2C

ROH2 C O H

H Ph

10 11

32
of 2 provided by the anomeric effect exceeds the destabilization from repulsive steric interaction.

The more important steric interactions in 2a and 3a and

in conformations 10 and 11 of the related 1,2 : 3 ,5-di-0-

benzylidene-a-D-glucofuranose derivatives have been calculated

by using the Hill equation [47]: E_v

= e_{-2 .25[r/Cr

1

+r_

2

) ]~ + 8.2 8 x 10 5 exp[-r/0.0 736(r

1

+r

2 )]}.

The results of these calculations suggest that the

conformations favored by 1,2 : 3 ,5-di-O-alkylidene-a-Dglucofuranose derivatives can be explained in terms of

conformational distortion of the m-dioxane ring and its

attached substituents

.

7. Intramolecular, Nuclear Overhauser Effect

Experimental evidence for a favored orientation of the

methoxyl group in 2 was obtained from an intramolecular,

nuclear Overhauser experiment [48]. Saturation of the methoxyl

protons of 2 resulted in a 25% enhancement of the integrated

intensity of its methylidene proton resonance signal when

compared with a similar experiment in which the irradiating

frequency was applied off resonance.

P . Polyhydroxy Cyclic Compounds

(A. J. Fatiadi)

1. Evidence for a Half-chair Conformation of Certain

Osotriazoles from Inositols

As part of a continuing interest in the chemistry of

inositol derivatives, D- (la), L- (2a), and DL- (la—2a)

1,2-diketo-myo-inositol phenylosotriazoles [49] have been

prepared in 42—45 percent yield from the corresponding

inosose phenylosazones by use of a new reagent for this

reaction, namely, mercuric acetate; the yields are higher

33
Ph N N Ph

OAc'

than those obtained with the cupric sulfate reagent originally

used in the preparation of the DL-osotriazole (la-2a) by

Anderson and Aronson [50].

The p.m.r. spectra of the diketoinositol osotriazoles la

(mp 234-235 °C), 2a (mp 233-234 °C), and la-2a (mp 277-278 °C),

and their tetraacetates lb (mp 146-147 °C), 2b (mp 145-146 °C),

and lb-2b (mp 193-194 °C), hitherto not analyzed, revealed

some interesting features, including: (1) the presence of a simple, two-fold axis of symmetry (for example, for the

optically active compounds la and 2a), and (2_) their ring-

~34
proton p.m.r. spectra are symmetrical about a midpoint,

making them a new example of the important, four-nucleus,

AA'BB' system. However, as expected, the p.m.r. spectra of

the osotriazoles from g-proto-diketoquercitol (3a), m.p. 195-

196 °C, (or its tetraacetate 3b) and from g-diketopinitol or

D-diketoquebrachitol do not show a symmetrical pattern.

From inspection of Dreiding models, it appeared that, in

solution, the favored conformation for the osotriazole

tetraacetate lb-2b would be the half-chair conformation (H^j)

depicted in 4. The coupling constant (JRR ') of 7.5 Hz deduced

for H-4 and H-5 indicates a trans -diaxial orientation; the

H? conformation would also have H-5 and H-6 axial, and this

fits the value calculated (J

flR 6.5 Hz). The small couplingconstants between H-3 and H-6 (J,. , -1.25 Hz) and between H-4

and H-6 (J n -n, 0.5 Hz) indicate the influence of the neighbor- —Au

ing, planar, osotriazole ring, and this gives additional

support for the half-chair conformation deduced for lb-2b.

The analysis of the p.m.r. spectrum of tetraacetate lb-2b

(and others) has been verified by means of the calculated

spectrum (UEA/NMR2 Program). This work has been published [51]

2 . Novel Aromatization of a Trihydroxycyclohexanetrione

(Triketoinositol) to a Dibenzo-p- dioxin Derivative

It had been shown [52] that, on acetylation in the

presence of pyridine, 4, 6/5-trihydroxy-l, 2, 3-cyclohexanetrione

is aromatized to benzenehexol hexaacetate in high yield. It

has now been found that acetylation of 1 in the presence of

an acidic catalyst (100% phosphoric acid) gives a new adduct,

the fully hydroxylated dibenzo-p-dioxin 3a, isolated as its

octaacetate 3b.

35
a, R = H

b, R = Ac

3 a, R = H

b, R = Ac

A mixture of 1 monohydrate [52] (1 g; 5-17 mmoles) with

acetic anhydride—phosphoric acid reagent (60 ml; 12 ml of

100% syrupy H PO and 48 ml of acetic anhydride, homogeneously mixed [53]) was stirred at 85 °C until a solution

./fas obtained (60 min) . The somewhat brownish solution was

decomposed with ice-water to give 3b , yield 1.3 g (77-5%).

The crude product was recrystallized from warm acetic acid,

and a sample was dried at 110 °C/0.1 torr for 2 hr; mp

300-302 °C (298-300 °C, slow heating); x (Me0H) 232 (e ^47.5), max

278 (e ^4.5) 3 and 295 (sh) nm (e ^3.0). The formation of

3a can be envisaged as an acid-catalyzed nucleophilic attack

of 2a on 1

.

^Throughout this Technical Note, the symbol e represents

e mM (not £

M

} -

36
The structure of 3b was confirmed by i.r., n.m.r. , and

high-resolution mass-spectroscopy ; v 1785(vs) (C=0),

max

1650 (m) (C=C, aromatic), l6l5(w), l480(vs) (phenyl ring),

12l0(vs), ll80(vs) (acetate), and 1062(vs) cm" (=C-0, aryl

ether)

.

The p.m.r. spectrum of 3b (^12% solution in methyl

sulfoxide-d^ , with tetramethylsilane as the internal standard)

showed only two narrowly spaced singlets of equal intensity

(separation 5 Hz), at t 7.69 and t 7.65, arising from two

groups of methyl protons of the acetyl groups on 0-1, 0-4,

0-5, and 0-8, and on 0-2, 0-3, 0-6, and 0-7.

The mass spectrum (.70 eV) of 3b showed a molecular-ion

peak (parent ion) at m/e 648.00; calcd. for C„nH^0 ^

648.492. The spectrum also showed seven ion-peaks (moderate

to strong intensities) due to the consecutive loss of ketene

(CH =C=0) group from the acetate groups at m/e 606, 564, 522,

480, 438, 396, 354, and 312.

3 . Conversion of Certain Cyclic Phenylhydrazino

Derivatives into Phenylazo Compounds with Periodic

Acid

In connection with studies [54] of certain cyclic, vinyl

phenylazo compounds as possible intermediates in the

formation of bis- or tris-(phenylhydrazones ) , there was a need to obtain pure samples of 3-oxo-l-phenylazo-l-cyclohexene

(3a) and 5 ,5-dimethyl-3-oxo-l-phenylazo-l-cyclohexene (3b).

One possible way to obtain these compounds is to convert the

corresponding enols of 1 ,3-cyclohexanediones [for example,

the enol of 5 ,5-dimethyl-l ,3-cyclohexanedione (lb) J into the

phenylhydrazino derivative, such as 2b; the oxidation of the

latter would give the needed azo compound 3b.

37
a, R = H

b, R = CH3

PhNHNH,

H

2

NHNHPh

2 H5 IQ6

CH3OH

N=NPh

+ 2 IOg + 6 H

2

It has been found that treatment of a solution of 3a or

3b (one mole) in methanol (or glacial acetic acid) with an

aqueous solution of periodic acid (two moles) at room

temperature produces the corresponding azo compounds 4a and

4b in 90$ yield.

It is believed that clear-cut conversion of the vinyl

phenylhydrazino compounds 3a and 3b into the corresponding

azo compounds 4a and 4b with periodic acid may indicate the

general character of this reagent for deprotonation of this

type of compound.
4 . Phenylhydrazono—phenylazo Tautomerlsm. Reactions of

Pheny lformazans and Certain Bis (phenylhydrazones

)

with Strong Acids

( A. J. Fatiadi and H. S. Isbell)

The behavior of diphenylformazans, 2-oxo-l ,3-bis-

(phenylhydrazones) , 1 ,2-bis (.phenylazo) ethylene , and the

phenylhydrazone of 4-oxo-l-phenyl-5-pheny lazo-3-

pyridazinecarboxaldehyde on protonation has been examined

spectrophotometrically . These compounds form purple, blue,

or green protonated cations. The nature of the spectral

changes suggests that the highly colored cations have resonance'

stabilized structures. Phenylosazones and bis (phenylhydrazono)

compounds that cannot form resonance-stabilized cations on

protonation do not usually give the blue-color reaction.

Structures have been proposed for the cations derived from

diphenylformazans and certain 2-oxo-l, 3-bis (phenylhydrazones )

.

Treatment of the red, enolic tautomer of 2-oxo-l ,3-bis-

(phenylhydrazono) cyclohexane with perchloric acid in acetic

acid yielded a new, stable, dark-blue, crystalline salt.

Dissolution of this salt in acetic acid, followed by addition

of ice—water gave a yellow hydrate, previously described, of

the keto tautomer. Dissolution of the hydrate in ethanol,

and warming, regenerated the red enolic tautomer.

E.s.r. measurements of colored solutions of 2-oxo-l, 3-

bis (phenylhydrazones ) or diphenylformazans did not show the

presence of radical species, thus indicating the ionic

character of the products formed on protonation. The neutral

molecule, for example, g-mannose diphenylformazan, has a

chelate resonance-structure 1 in which the imino hydrogen

atom spans the phenylhydrazono group and the phenylazo group.

Apparently, strong acids rupture this chelate, and the

resulting, resonance-stabilized cation may have a structure

encompassing the resonance forms depicted in la.

39
N =N

R -s >

N-N

H H

I I Ph-N = N-C = N-N-Ph

e iR

I

H H

I I Ph-N-N = C-N= N-Ph

1a

Where R = -(CHOH)4 CHjOH

The electron system for the cation of diphenylformazan

la encompasses 17 atoms: only two of the resonance forms are

illustrated. Derealization of the positive charges over the

resonance system in la may account for the bathochromic shift

found in the spectrum of 1 on protonation.

These results have been incorporated in an article which

has been accepted for publication [55].

40
2. CLINICAL CHEMISTRY

Those familiar with the activities of the Organic

Chemistry Section know of its long-time connection with

sugar technology and carbohydrate research, and may have had

occasion to call on us for samples of the rare carbohydrates

14 3 that were prepared here in the past, or for C- and Hlabeled carbohydrates that for a long while were only

available through our work. Recently, new supplies of a few rare carbohydrate derivatives were being made available

as Standard Reference Materials; but the Section is no

longer serving as a source for labeled carbohydrates, since

these materials are now widely available from commercial

laboratories utilizing our published procedures.

A few years ago, the Section devoted considerable

effort to developing oil-soluble compounds of 24 different

metals for the purpose of providing standards for the

spectrographic measurement of these metals in lubricating

oils, a type of analysis that very effectively measures

engine wear. Such analyses are now in widespread use in

the air, rail, and trucking industries.

Most recently, we have begun to develop SRM's for

clinical analysis. Cholesterol was the first of these to

be issued, and some of the present report is concerned with

an evaluation of its shelf-life. Creatinine, urea, and

uric acid, as well as calcium carbonate, have been issued

during this past year. Much of the present report is

devoted to work on bilirubin and on a standard for clinical

glucose determinations

.

41
A. Bilirubin

( R. S. Tipson , R. F. Brady, Jr., A. J. Fatiadi, and

B. F. West)

Of the bile pigments, bilirubin (1) is the constituent

of major interest in clinical chemistry. These pigments

include biliverdine (2), mesobilirubin (3), and other

compounds having four pyrrole rings linked at their alpha

positions by carbon atoms. Many of these pigments are

interconvertible; for example, 1 (red-orange) may be oxidized

to 2 (green), or reduced to 3 (orange-yellow). Thus, a chloroform solution of 1 exposed to air and light changes

color rapidly, demonstrating the ease of interconversion

.

The various bile pigments may usually be distinguished by

their absorption spectra in the visible range.

Me R Me P P Me Me R

H H H H

1, Bilirubin, R = -CH = CH2

3, Mesobilirubin, R = —CH2 —CH3

Me R Me P P Me Me R

H H H 2, Biliverdine, R = —CH = CK

Me R Me P P Me Me R

H H H 4, Urobilin, R = -CH2CH3

where P = -CH2CH2C02 H

42
Bilirubin is derived primarily from the hemoglobin of

mature red-blood cells, and secondarily from heme or

porphyrin-like compounds formed during the synthesis of heme.

As erythrocytes are destroyed in the reticulo-endothelial

system, the hemoglobin is degraded, and the porphyrin ring

of heme is converted into biliyerdine, which is then reduced

to bilirubin. The bilirubin is complexed by albumen in the

blood, and is thus transported to the liver. There it is

conjugated with two rj-glucosyluronic acid groups by reaction

thereof with the two carboxyl groups of bilirubin; this

conjugation is necessary for its secretion into the bile.

From the bile, the conjugated bilirubin passes into the

intestinal tract; it is reduced to urobilin (4) (brown) by

bacteria in the large intestine, and is eliminated. Di-

(D-glucosyluronic acid) bilirubinate may pass into the urine;

severe cases of bilirubinuria result in visible discoloration

of the urine.

Because bilirubin analyses have major medical significance,

particularly in the detection of such diseases as hepatitis

or jaundice, and because of the difficulties encountered in

these analyses, it is important that a well characterized

bilirubin reference standard be available for use in clinical

laboratories for standardizations and for comparing bilirubin

methodologies

.

1. Volatile Matter in Bilirubin Samples

(B. F. West)

Commercial samples of bilirubin were dried under vacuum

to determine the content of volatile matter. A preliminary

drying experiment was performed on a single lot of bilirubin.

Two samples of ^400 mg were accurately weighed (on a microbalance) into each of two tared, dry vials . The samples were

heated in an Abderhalden dryer at 58 °C (refluxing acetone)/

^0.3 torr for one hour, and then allowed to cool under vacuum.

43
The samples were weighed, and redried at 65 °C (refluxing

methanol), and then at 111 °C (refluxing toluene). At each

temperature, there was some loss in weight. The total loss

in weight was 0.066%.

A second drying experiment was performed with three

different samples of commercial bilirubin. The heating was

conducted for 1.5 hours at 111 °C (refluxing toluene) A0.

3

torr. The results are given in table 3.

Table 3. Loss in weight of samples of commercial bilirubin

at 111 °CA0 .3 torr

Manufacturer '

s

lot number

Loss in weight, %

7941-1

39148

5310

0.065

0.14

0.38

2 . Ash Content of Bilirubin Samples

The ash was determined on 100-mg samples of various

commercial bilirubins. The results, obtained by R. A. Paulson

and W. P. Schmidt, are shown in table 4.

Table 4. Ash content of samples of commercial bilirubin

Manufacturer '

s

Ash , %

lot number

FP 0.07

7970-1 0.11

39148 0.13

39142 .14

7941-H 0.16

7970-H 0.16

5310 0.23

44
3. Spectrochemical Analysis of Ash from Bilirubin

Samples

The proportions of metallic elements present in the ash

of commercial bilirubin from three sources were determined,

and tentative limits of detection for common elements not

detected in these samples were established. Ashing and

testing techniques were developed for 25- and 100-mg samples

of bilirubin. Limits of detection of an element in the range

of 0.1 to 1 ppm of bilirubin were established for 22 common

elements by using synthetic gallium oxide as the reference

standard; the results of the analyses are shown in table 5.

The analyses were performed by Martha M. Darr, Spectrochemical

Analysis Section, NBS . The results given in table 5 show

that the principal metallic constituents in the ash from

the three bilirubin samples were Al, Ca, Fe, Mg, Na, Si, and

Zn; but that there were distinct differences in the concentration levels of the different elements among the samples

.

For example, sample 1 (Lot 7941-1) shows the highest single

metallic impurity, namely, sodium (500 ppm), which is probably

a residue from the method used in isolating the product. On

the other hand, sample 2 (Lot 391^8) shows an appreciable

content of iron (200 ppm) which may indicate the presence of

a heme-like impurity in the product. The contents of Al,

Ca, Mg, Si, and Zn in these commercial bilirubins differ

widely; especially, the presence of Si (50 ppm) in sample 1, as compared with traces of it in the other two samples

.

4. Solubility of Bilirubin

(A. J. Patiadi and R. F. Brady, Jr.)

The solubility of bilirubin in various solvents is (in

decreasing order) l-methyl-2-pyrrolidinone , 2-pyrrolidinone

,

methyl sulfoxide, N,N-dimethy lformamide , chlorobenzene

,

chloroform, acetone, glacial acetic acid, ethyl acetate,

benzene, methanol, and acetonitrile

.

• 45
Table 5

1 Ele- !__

merit

Ag

Al

Au

B

Be

Bi

Ca

Cd

Co

Cr

1

Proportions (ppm, by weight) or elements in ash

from samples of commercial bilirubin

1—I "T~

Samp le . Ele- Sample

3 E ment \ 1 "2 "3

20

0.4 <0 .2

c

1

_9

<0 .2

1

_9

50 10'

_ I

_9

_9

<0 .1

<0 .1

0.5

.1

0.5

0.2

1.0

0.5

1.0

Na

Ni

Pb

Pd

Pt

Rh

Ru

500 6

2

50

d

0.4

.2

100 d

0.2

0.4

0.2

.2

Cu i 0.5 5 0.5 <0 .1 Sb - - _ .1

i

j Si

1 50

c

5 5 0.1

Fe !

! 50

c 200 c 50

c .2 Sn - - 0.2 .2

Ga - - Ge j - - 0.5 i Hf

j - -1 Ti .1 .2 0.4 .1

In - - 0.1

Ir

1 - - La - - V - - _ .2

Mg 20 c 5 10 c <0 .1

*

Mn 1 - 0.5 Zn 5 10 C

2 1.0

Mo .1 .2 <0 .1 Zr - - -

a

b

cd

Bilirubin sample 1, lot 7941-1; 2, lot 39148; 3, lot 5310.

Estimated limits of detection as metal (ppm) in bilirubin.

Concentration estimated (.above highest standard) . Visual estimate, without a specific standard on this plate

46
Solubility data for bilirubin were desired for possible

use in phase-solubility analysis. Therefore, 500-mg quantities

of bilirubin were equilibrated by shaking for 7 days in a bath at 13.0 ±0.1 °C with 500 ml of different solvents.

Undissolved material in each mixture was removed by filtration

(Millipore) , and an aliquot of the filtrate was evaporated

to dryness. The results are given in table 6.

Table 6. Approximate solubilities of

bilirubin51 at 13.0 °C

Solvent Solubility, mg/ml

of solvent

Methanol 0.14

Dichloromethane 0.47

Acetone 0.72

Chloroform 1.33

Lot number 39148.

5 . Spectra of Bilirubin Samples in the Visible Range

(B. F. West)

Bilirubin has a characteristic spectrum in the visible

range. For solutions in chloroform, the maximum is at 453 nm,

with a millimolar extinction coefficient (e) reported as

lying in the range of 55.0 to 60.8. Purified samples of

bilirubin give [56] a value of £ that converges on ^60.8.

Generally, it has been supposed that samples of bilirubin

that have a significantly lower e value are less pure,

although it has been noted [56] that such absorption curves

alone are inadequate as a measure of the purity of bilirubin.

A spectrophotometric survey of some samples of commercial

bilirubin was made as follows. A sample of ^3 mg (accurately

weighed on a microbalance) was quantitatively transferred to

a 500-ml volumetric flask containing ^200 ml of chloroform

47
at 20 °C, the suspension was vigorously shaken until

the bilirubin had dissolved according to visual inspection,

and the solution was placed in a water bath at 20 °C. It was

then made to 500 ml with chloroform at 20 °C. (All solutions

were made under dim incandescent light.) The final solution

was mixed thoroughly, and spectral measurements were

immediately made by use of a 1-cm cell in a Cary 14

spectrometer (with use of the automatic, slit-control

setting), against a chloroform blank. For these measurements,

the cell compartment was not thermostated ; the temperature

therein was ^22 °C. The values of e for commercial samples

of bilirubin are given in table 7.

Table 7. Millimolar extinction coefficients of samples of

commercial bilirubin

Manufacturer's £

lot number

45054 56 .3

39148 57 .3

29191 57 .6

5310 59 .6

7941-1 59 .9

FP a 59 .9

Cell thermostated at 25 °C.

6 . Stability of Bilirubin Samples in Various Solvents

(A. J. Fatiadi)

The following is the general procedure for stability

studies on bilirubin samples . A sample of a commercial

bilirubin (3-126 mg) was quickly dissolved in warm chloroform

(450 ml, 3 min of stirring, water bath at 60 °C), quickly

cooled to about 20 °C, and diluted to 500 ml. This stock

solution showed an absorption maximum at 453 nm, with an

48
absorbance of 0.64Q. The stock solution was kept at

3—5 °C in the dark (refrigerator). Periodically, a portion

was withdrawn and quickly warmed to room temperature, and its

absorbance determined. This value was compared with

that obtained immediately after dissolution of the bilirubin.

On the assumption that the only material present that

showed absorption at 453 nm was bilirubin, the percentage

loss of bilirubin with time was calculated; the results are

given in table 8.

7. Effect of Antioxidants on Stability of Bilirubin

Solutions

(A. J. Patiadi)

A sample of bilirubin Clot number 7941-1; 3.58 mg) was

dissolved in methyl sulfoxide (10 ml), and an aliquot (1 ml)

was diluted to 50 ml; ^_h^h 0.743. This solution, kept at

room temperature (in the dark) for 48 hours, showed a decrease

in absorbance of 9%. However, addition of a sample of

L-ascorbic acid (15 mg) to a similar solution lessened the

decomposition of bilirubin (after 48 hr) to 2.5%, showing

the stabilizing effect of L-ascorbic acid.

Addition of 2 ,5-di-( tert -amyl)hydroquinone (5 mg) failed

to diminish the decomposition (loss, about Q% after 48 hr)

8. Synthesis of Mesobilirubin IXa

(B. P. West)

In order to be able to identify one of the impurities

that might be present in commercial samples of bilirubin,

attempts were made to synthesize one of the most probable

impurities [57], namely, mesobilirubin. This compound

differs from bilirubin in having two ethyl groups in place

of the two vinyl groups of bilirubin.

49
Table 8. Stability of samples of commercial bilirubin in

solution (10 uM) ? as shown by decrease in A.

Bilirubin Solvent Time Decrease in

lot number (.hours) concentration

,

%

39142 chloroform 144

528

1152

5

19

23

7970-1 chloroform 24

144

336

552

1

5.8

24.5

26 .3

783889 chloroform 141

312

528

1152

9.4

17.3

23

25

68-B-1300 chloroform 140

336

552

1176

19

29

31

34

N,N-dimethyl- 144 16

formamide

360

624

984

20

30

38

7970-1 N,N-dimethylformamide

42

190

406

10 32

10

19

32

57

7970-1 30 mM sodium 6 7

hydroxide 30 46

50
Mesobilirubin is a yellow solid having, in chloroform,

A 434 nm, with a milllmolar extinction coefficient E58]

max

of 54.6. Following the procedure of Gray, Kulczycka, and

Nicholson [58], a solution of bilirubin in 100 mM sodium

hydroxide was hydrogenated for 3 hours at room temperature

and atmospheric pressure in the presence of palladium-oncarbon. However, three times the theoretical volume of

hydrogen was consumed. Consequently, the following modification was used. A sample of bilirubin (0.2 g) was dissolved

in M sodium hydroxide (.15 ml) in a 250-ml flask provided

with a magnetic stirrer and covered with aluminum foil to

exclude light. In a second flask, similarly equipped, were

placed 0.1 g of palladium-on-carbon and 10 ml of M sodium

hydroxide. The necks of the two flasks were connected with

a T-tube which led also to a gas-measuring reservoir, to a

vacuum, and to a source of hydrogen. With stirring of the

contents of both flasks, the assembly was alternately evacuated and filled with hydrogen six times, and finally left

filled with hydrogen. Then, the flask containing the

catalyst was inverted, so that the catalyst was added to the

sodium bilirubinate solution. When the theoretical amount

of hydrogen had been absorbed (^1 hr) , the reaction was

stopped, and the mixture was filtered.

The filtrate was made neutral with a slight excess of M

acetic acid (30 ml), and a precipitate formed immediately.

This tan-colored precipitate was filtered off; it rapidly

darkened to a green-black cake. The dry solid weighed 0.2 g.

A portion of this material was treated with warm ethanol;

it left a yellow, insoluble material which was filtered off

and dried. The yellow J product , in chloroform, showed A ^

3 ' max

^33 nm with a milllmolar extinction coefficient of 5^.2,

properties that correspond approximately to those of mesobilirubin IXa.

51
This product was compared with bilirubin by thin-layer

chromatography (on polyamide) with 9:1:2 methanol-10% ammoniawater, and found to be slightly impure. In addition to the

major, light-yellow component (mesobilirubin) , there appeared

to be, in the tailing of the mesobilirubin spot, a slightly

darker yellow color having an FL, slightly higher than that of

bilirubin. This impure, synthetic mesobilirubin IXa was

used in the t.l.c. examination of samples of bilirubin.

9 . Synthesis of Biliverdine

(A. J. Fatiadi)

a. Oxidation of Bilirubin with Ferric

Chloride

A reference sample of biliverdine was prepared by

oxidation of bilirubin with FeCl_ according to a published

procedure [59]. Starting from 150 mg of bilirubin, there

was obtained 34 mg (22.6$) of biliverdine. The product had

A MeOH 238(shoulder; sh) (e ^6.0); 277 (e ^22.0); 315(sh) max

(e ^28.0); 374 U ^39.8), and 650 nm (e 12.1); lit. [60]

KZX 375-380 (e ^40.5) and 640-650 nm (e 12.9). max

b

.

Oxidation of Bilirubin with Acidified

Methyl Sulfoxide

A sample of bilirubin (200 mg) in a test tube was

treated with methyl sulfoxide (10 ml) and concentrated

hydrochloric acid (0.3 ml); the suspension was placed in a water bath at 90-9 5 °C and stirred for 60 min. The dark-green

solution was then made less acidic with aqueous sodium acetate

(0.2 ml, 2 M) , and adsorbed on Whatman filter paper (20 x 20 cm);

this was dried in a hood for 24 hr. The paper was then cut

into small strips and extracted with warm 1:1 acetone—methanol

(2 x 100 ml); evaporation of the extracts gave a dark-green

solid (120 mg); it had A

u_ 375-382 and 665-670 nm. T.l.c.

max

52
on polyamide with 2:1 (v/v) ethyl acetate-methanol revealed

the presence of some Impurity that did not move from the

origin, indicating the possible presence of a biliverdine

salt or a complex with methyl sulfoxide. The product was

further purified by adsorption on magnesium sulfate (30 g)

followed by elution with methanol, concentration, and extracion with ether (3 x 100 ml). Recrystallization from

methanol, by concentration and cooling, gave shiny green

crystals of biliverdine; yield 42 mg (21$); the u.v. and

visible spectra of this material were close to those of

that prepared by oxidation with ferric chloride.

10 . Thin-layer Chromatography of Bilirubin

(B. F. West)

Thin-layer chromatography of bilirubin was performed

in an attempt to detect and possibly identify impurities in

bilirubin samples . Thin-layer chromatography of bilirubin

has been studied by various workers [61—64], usually for

bilirubin as a component of urine or blood samples . There

are reports of satisfactory identification of bilirubin,

biliverdine, mesobilirubin, and D-glucosyluronic acid

bilirubinate fractions in such biological samples. However,

we are unaware of any thin-layer chromatographic studies

to determine traces of impurities in purified, free bilirubin,

In order to detect and identify small proportions of

impurity in commercial samples of bilirubin, relatively

large volumes of a solution of bilirubin in chloroform were

spotted on thin-layer plates of silica gel, microcrystalline

cellulose, and polyamide, respectively. Under spot-overload

conditions, bilirubin does not behave satisfactorily on thinlayer plates. Depending on the solvent system and the

adsorbent, either (a) no significant migration of bilirubin

is observed, or (b_) the bilirubin component migrates almost

with the solvent front, with considerable "tailing."

53
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For the commercial samples studied, with a variety of

solvent systems and adsorbents, no detectable impurities

could be separated. Commercial biliverdine and synthetic

mesobilirubin, assumed to be most probable as impurities in

bilirubin samples, were spotted on the same plates, for

comparison. None of the commercial samples of bilirubin gave

spots that corresponded to these compounds. Under the conditions studied, bilirubin could be located and identified by

its own color.

The solvent systems and adsorbents employed to determine

the mobilities of bilirubin and biliverdine are given in

table 9.

In every system in which bilirubin or mesobilirubin

migrated, the resulting spot streaked considerably, so that

distinct spots were not obtained. The compounds and their

location were evident from the colors of the spots; bilirubin,

golden yellow; biliverdine, dark green; and mesobilirubin,

light yellow. The most satisfactory solvent system and

adsorbent for distinguishing between the structurally very

similar bilirubin, mesobilirubin, and biliverdine was 9:2:1

methanol—10$ ammonia^water on polyamide. The spots not only

showed their characteristic colors, but fluoresced under

ultraviolet light (25^ nm) , after the solvents had been

thoroughly removed by drying the developed plate. (The

polyamide layer did not contain a fluorescent indicator.

)

When commercial samples of bilirubin were chromatographed

with this solvent system and adsorbent, neither mesobilirubin

nor biliverdine was detected.

The conclusion drawn from the thin-layer chromatographic

examination of the commercial preparations of bilirubin is

that, if impurities are present, they are in proportions that

afford amounts below the detection limits of the technique.

56
11. Mass Spectra of Bilirubin Samples

(R. F. Brady, Jr.)

The mass spectrum of several samples of bilirubin was

determined on an LKB Model 9000 mass spectrometer, made

available through the courtesy of Dr. Henry M. Pales,

National Institutes of Health. Some of the more prominent

ions in the spectra, and their relative abundances, are listed

in table 10. Samples from three different sources gave the

same spectra.

Table 10. Mass-spectral data for bilirubin

m/e Relative abundance

211 96.4

227 98.8

241 68.6

286 100 .0

300 83.8

584 7.9

585 5.2

586 1.8

587 0.6

588 0.3

Lot numbers 7322, 8l62, and 39148.

The molecular ion for bilirubin is seen at m/e 584, and

is accompanied by the normal cluster of bilirubin isotopic

peaks at higher mass-to-charge ratios. Other prominent ions

in the spectrum are formed when bilirubin splits at the central

methylene group, giving ions of masses 300 and 286. Evidence

for biliverdine ( m/e 582) or mesobilirubin ( m/e 588) and

expected, associated, isotopic ions was not found.

57
12 . Thermogravimetric Examination of Bilirubin Samples

(A. J. Fatiadi)

Several different samples of commercial bilirubin

(^5—6 mg) were analyzed for thermal stability in (a) dry

air, and (b) dry nitrogen with a Dupont TGA apparatus (see

fig. 9). The rate of heating was 2 °C/min up to 150 °C,

5 °C/min up to 250 °C, and 10 °C/min up to 500 °C. The

results of these experiments are summarized in table 11.

The thermogravimetric curves, shown in fig. 10, illustrate

the fact that bilirubin, whether heated in air or under

nitrogen, is surprisingly stable; that is, sample weights

remain almost constant until a rapid decomposition is

initiated. Curve 5 shows a thermogram typical of those

Figure 9- Thermogravimetric equipment used for the analysis

of bilirubin.

58
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100 200 300 400 500

TEMPERATURE, °C Thermo gravimetric curves for various samples

of commercial bilirubin (1, lot 39148: 2, lot

5310; 3, lot 79^1-1; 4, lot FP; and 5, lot 39148

under nitrogen).

60
obtained with bilirubin samples heated under nitrogen; it

shows that there is no evidence of the sudden drop in sample

temperature ("recoil") that occurs with samples heated in

air (see curves 1—4), and, furthermore, the temperature at

which rapid decomposition begins is much higher under

nitrogen than in air. The initial decomposition temperatures

of the different bilirubin samples under nitrogen ranged

from ^300 to ^315 °C, with most values clustering near the

higher one. On the other hand, as shown in curves 1—4,

initial decomposition temperatures in air are quite different

from sample to sample, ranging from 22 7—277 °C. Thus,

decomposition in air appears to provide a means for semiquantitatively discerning the purities of the samples, assuming

that the purity determines the decomposition temperature.

The drop in sample temperature observed in the thermograms of bilirubin samples heated in air is unusual. This

type of thermogravimetric curve has been reported for sodium

oxalate, and has been associated with an overheating generated

within the sample, which (for the sodium oxalate) was due to

the evolution of carbon dioxide. With the bilirubin samples,

an acrid gas is evolved, but it has not yet been identified.

That this overheating of the sample does not occur with

bilirubin under nitrogen suggests involvement of an oxidative

reaction. The thermogravimetric curves were recorded by

J. T. Sterling of the Analytical Coordination Chemistry

Section, N.B.S.

13 . Tests for Bicarbonate-extractable Materials

(R. P. Brady, Jr., and B. F. West)

a. Purpose of the Test

Bilirubin is produced commercially from gallstones or

from bile. In general, the preparation consists in

saponifying the starting material with alkali (to remove the

g-glucosyluronic groups), acidifying, and extracting the bile

61
pigments into a suitable organic solvent. The bile pigments

obtained contain the weakly acidic bilirubin, contaminated by

a neutral fraction and a more strongly acidic fraction. The

bilirubin is separated by careful conversion into a bilirubinate

salt, followed by careful neutralization and crystallization,

but this process does not completely remove the impurities.

b. Qualitative Determination

The commercial bilirubins surveyed in this laboratory

contained various proportions of pigments more acidic than

bilirubin. A measure of the relative proportion of impurities

in commercial samples was obtained by a simple test suggested

by Mr. Arthur G. Holstein of Pfanstiehl Laboratories, Inc.,

Waukegan, Illinois. About 10 mg of bilirubin is weighed

accurately, transferred to a separatory funnel, and dissolved,

with shaking, in 50 ml of chloroform (unless otherwise

indicated in table 12). The solution is extracted with 10 ml

of 5% aqueous sodium bicarbonate, and the layers are separated.

The absorbance at 420 nm of the bicarbonate extract is measured

in a 1-cm cell, and the absorbance values are normalized by

dividing ten times the absorbance by the sample weight. In

our work, for added information, a second extraction was

performed 30 min after the first, a third extraction was done

one hour after the second, and normalized absorbances were

obtained in the same way. The results are given in table 12.

c . Refinements in the Qualitative Procedure

The simple, extractables test showed promise of being

made into a quantitative method; to do so, the following

refinements were introduced.

i. In view of the sensitivity of bilirubin

solutions to decomposition, the use of heat to dissolve

samples in chloroform was avoided; however, It was found that

samples dissolved incompletely, even after prolonged (15 min)

62
Table 12. Bicarbonate-extractable material in samples of

commercial bilirubin

Solvent

Normalized

of the b

l absorbance

i carbonate

(A42 } layer

Manufacturer '

s

lot number

First

extraction

Second

extraction

Third

extraction

7970-1 CHCl

g

0.21 0.07 .09

a CHCl^

^b CHCl^

0.26

0.23

0.11

0.08

0.12

0.26 c

o-C

6

H

4

Cl

2

CH

2

C1

2

0.18

0.28

0.08

0.11

.11

0.14

79^1-1 CHC1 .22 0.11 .11

7941-H CHC1 0.26 0.05 .02

5310 CHC1 1.00 0.18 .10

39148A CHCT 1.66 0.47 0.24

a Dried for 8 hr at 60°/0.1 torr.

Alcohol-free

.

The chloroform solution was exposed to light between the

2nd and 3rd extractions,

shaking by hand with chloroform at room temperature. Filtration showed residues that were approximately 3% of the sample

weight. Instead of hand shaking, the mixture was agitated

in an ultrasonic bath for 2 min, and it was then found that

the residues amounted to only about 0.01$.

ii . Solution preparation and extractions were

performed only under dim incandescent light; fluorescent light

was specifically excluded.

iii. Solutions were filtered through tared

membranes (0.5 -mm Millipore) . The membranes did not change

in weight after the passage of pure chloroform.

iv. In early experiments, extractions were

performed in separatory funnels having greased stopcocks,

and films were found to form at the interface of the layers

.

63
This may have been due both to the grease and to fine particles

that had passed through the paper initially used for filtration

Teflon stopcocks and stoppers were used subsequently, and,

with the use of better filtration (Millipore )

, the formation

of films was no longer encountered.

v. The bicarbonate extracts were neutralized

as rapidly as possible, to minimize the duration of exposure

of the extracted material to the alkalinity (and, hence,

decomposition) . If the extracts contained significant

proportions of impurity (^5% of the sample weight), precipitation occurred at this stage.

vi. The filtered, neutralized, combined,

aqueous extract was back-extracted with chloroform. Some

bilirubin-like material (A M50 nm) was obtained. Its

max

visible spectrum corresponded in shape with that of

bilirubin, but the difference in wavelength maximum cannot

yet be explained.

vii . For measurements of absorbance, solutions

were made to volume in calibrated flasks at 20 °C, and

spectra were recorded in cells thermostated at 25 °C.

viii. In the calculation of materials balances,

allowance was made for the aliquots taken for the various

measurements. The amounts of material extracted were obtained

by drying the extracts and weighing the residues.

d. Quantitative Analysis by the Bicarbonate

Extraction-Materials Balance Method

The procedure, employing ^0.1 g of a bilirubin sample,

consists in dissolving it in about 200 ml of chloroform,

filtering, washing and weighing any insoluble residue, and,

with due precaution for temperature control and careful

aliquoting from a 250-ml volume, starts with the absorbance

measurements of the dissolved sample. The chloroform solution

is then successively extracted with 2 portions of aqueous

64
sodium bicarbonate and 2 portions of water, and these extracts

are neutralized before further processing. After dilution of

the extracted chloroform layer to 500 ml (to eliminate any

turbidity due to water) , large aliquots are diluted for

absorbance measurements, and other (larger) aliquots are

evaporated to dryness to obtain the bilirubin concentration.

The neutralized, combined, aqueous extracts are backextracted with portions of chloroform until the chloroform

extracts show no absorbance at ^450 nm. Each chloroform

extract is washed twice with water, the aqueous layers being

recombined with the chloroform-extracted, neutralized, aqueous

layer before the latter is subsequently re-extracted with

additional portions of chloroform. The absorbances and the

solids content of these chloroform extracts are measured as

before. As a check for complete extraction of the impurities,

the initial chloroform solution that had been successively

extracted is again extracted in the same way, and the aqueous

extracts are similarly processed.

Table 13 summarizes the results obtained with four

commercial samples.

Table 13. Materials-balance analysis of the purity of

samples of commercial bilirubin

Lot number Number of Sample Minimum

determinations recovery, % purity, %

39148A 3 99.63 90.4

7322 3 99-76 93.4

8162 3 99.79 97.5

FP a2 99.83 99.7

a To dissolve this sample, it was necessary to heat the

mixture for 2 min in a bath at 80 °C; then the mixture

was ultrasonically shaken for 2 min.

65
lH . The Diazo Reaction for the Assay of Bilirubin

(B. F. West)

Bilirubin is routinely assayed in bile and blood by

application of a diazo coupling reaction with p-diazobenzenesulfonic acid (p-sulfobenzenediazonium hydroxide) . Depending

on the pH , a blue or red diazo dye is obtained. The concentration of this dye, which can be measured spectrophotometrically, gives a quantitative measure of the bilirubin

present

.

Because of the sensitivity of test results to experimental

conditions, many modifications of the test have been introduced.

In biological samples, the test conditions are designed to

distinguish between free bilirubin and bilirubin that is

esterified with D-glucosyluronic acid groups. In commercial

preparations, however, only free bilirubin is present. In

all cases, for the diazo reaction mixture to develop full

color, an accelerator, either an alcohol (ethanol or methanol)

or caffeine, is addea.

As previously noted, the spectral absorption curve of

the azo dye is affected by the pH of the solution. One

general method, that of Malloy and Evelyn, uses acidic

conditions; a blue color develops, and the maximum absorption

is obtained at pH 1.5, but the intensity falls off rapidly,

even at slightly lower pH . The other general method, that

of Jendrassik and Grof , uses a buffer solution, containing

sodium acetate and sodium benzoate, that affords only slightly

acidic conditions and gives rise to a red color.

The extinction coefficients observed for "azo-bilirubin"

in chloroform—ethanol , or aqueous caffeine-acetate mixtures,

are different from those observed for reactions performed in

serum and plasma. Clinical workers find that more-consistent

results are obtained with the biological fluids as the solvent

for the reaction.

66
In the present investigation, a modified Malloy—Evelyn

method [65] was employed. Certain additional modifications

of this procedure were introduced, to improve the precision

and accuracy of the results . The experiments were performed

in dim incandescent light. The volumes of bilirubin solution

and coupling reagent employed were increased, so that moreaccurately measured aliquots could be taken. Furthermore,

the acidity of the sulfanilic acid solution was increased by

doubling the amount of the hydrochloric acid added. The

amount of bilirubin treated was also doubled, to increase the

absorbance measured. Finally, instead of dissolvin g the

bilirubin sample in a sodium carbonate solution, in which it

is very susceptible to degradation, the bilirubin sample was

dissolved in methyl sulfoxide.

The millimolar extinction coefficient for the diazo dye

is calculated from the absorbance of the diazo dye and the

weight of bilirubin used in the reaction. It has been reported

that, for bilirubin of e ^60.0, the e of the diazo dye is

^64.0. The values of e for the various "azo-bilirubin"

preparations obtained with some commercial bilirubins by

using the modifications described are shown in table 14 . A

solution of each commercial sample was analyzed three times,

and the A,- l. values are the averages . Standard deviations

are reported for the A^,- values .

Table 14. Millimolar extinction coefficient of "azobilirubin"

from samples of commercial bilirubin

Bilirubin

(manufacturer T s lot number)

"Azobilirubin" Standard

^545 e deviation

39148

7941-1

7322

0.446

0.461

0.470

63.2

65.0

66.5

0.006

0.010

0.018

67
15. Examination of Bilirubin Samples by X-Ray

Diffraction

CuKct radiation was used to obtain X-ray powder diffraction

patterns for the commercial samples of bilirubin. These

measurements were made on samples in the form in which they

were supplied. As the stability of bilirubin to the radiation

was of concern, repeated scans were made on one sample; but

no difference was evident between the pattern of reflections

initially obtained and that of the fourth repeat. Also, the

possibility was considered that moderately elevated heating

of a sample might, at some stage in its isolation, have affected

its diffraction pattern, but the pattern, obtained with a portion of the same sample after heating it for 2 hr at

65 °C/0.3 torr, was essentially unchanged.

On comparing the patterns of bilirubin samples from

different commercial sources, only one was found to be markedly

different, displaying fewer and different reflections; the

other patterns more or less resembled each other, although

there were noticeable differences : some showed sharper

reflections; others had displaced, or apparently absent,

reflections. The sharper peaks indicate a more regular orderin the crystalline packing. However, without certain knowledge

of the processing conditions through which each preparation

had passed, no conclusions have been reached regarding the

differences in these diffraction patterns. This work was

performed by H. E. Swanson.

16

.

Oxidation of Biliverdine

(A. J. Fatiadi)

a. Action of Periodic Acid on Biliverdine in

Methyl Alcohol

A solution (.18.7 uM) of synthetic biliverdine in methyl

alcohol was prepared; it had &ojh 0.75, £ ^40.0 (peak A) and
kr^r, 0.22, e ^11.8 (peak B) . On addition of 0.2 ml of 100 mM

—1>50

aqueous periodic acid to 50 ml of this solution, peak A

disappeared within 30 sec. Peak B shifted to give a new peak

(C) at 695 nm that reached maximum intensity within 30 sec,

and then decreased continuously, and disappeared in 60 min.

A new peak (D) appeared at 580 nm that decreased continuously.

and a new peak (E) at 482 nm that increased continuously.

In the u.v. region, a strong peak at 277 nm disappeared,

and a new peak at 225 nm appeared.

b . Action of Iodic Acid on Biliverdine in Methyl

Alcohol

The same behavior was observed when a solution (22.1 uM)

of synthetic biliverdine in methanol was treated with iodic

acid (aq. HI0_, 100 mM ) . However, the disappearance of peak

A was much slower; it required about 45 min to decrease to

5% of its original height. In the u.v. region, the peak at

277 nm disappeared, but that at 238 nm shifted only slightly,

to 236 nm.

17. Oxidation of Bilirubin

( A. J. Fatiadi and H. J. Elseroad)

A study was made of the action of a variety of oxidants

on commerical samples of bilirubin, dissolved in various

solvents, with the object of devising a new way of assaying

samples of bilirubin for purity.

a. With Iodic Acid (HI0-) in Water

In each experiment, 0.2 ml of the oxidizing solution

(10 mM) was added to 50 ml of the bilirubin solution.

i. In Methyl Sulfoxide

A solution (11.3 uM) of bilirubin showed e^ . ^60.3

(peak A), and a shoulder at 420 nm (peak B). The oxidant was

added, and, within 2 minutes, peaks A and B had disappeared;

6Q
new peaks appeared at 374 nm Cpeak C) , 415-430 nm (peak D)

,

and 670 nm (peak G) . Peak C increased in intensity for 20 min,

then decreased, and disappeared in ^175 min. The intensity

of peak G decreased continuously; it disappeared in ^170 min.

Peak D increased gradually in intensity for ^165 min and

then decreased slightly.

ii . In Methanol

A solution (12.4 uM) of bilirubin showed £uu 7

^60.0

(peak A) and a very slight shoulder at ^4l8 nm. When the

oxidant was added, peak A disappeared immediately, and new

peaks appeared at 373 nm (peak C) , 488 nm (peak E) , 590 nm

(peak F) , and 685 nm (peak G) . The intensities of peaks C,

F, and G decreased continuously, reaching about 10% of the

original intensity in 85 min. The intensity of peak E

increased by about 45% in ^60 min, and then decreased.

The new peak E (at 488 nm) was not observed when other

solvents were used.

iii . In N,N-Dimethylformamide

A solution (16.0 uM) of bilirubin showed e^/- ^59.5

(peak A). When the oxidant was added, peak A disappeared

immediately, and new peaks appeared at 377 nm (peak C)

,

650 nm (peak G) , and, after ^10 min, at 547 nm (peak F)

.

The intensities of peaks C and G gradually decreased to

^5% of the original intensity in 45 min; that of peak F

increased for 10 min and then decreased. This behavior

differs from that encountered on oxidation of the same

solution with periodic acid (see page 73)-

iv. In l-Methyl-2-pyrrolidinone

A solution (13.3 yM) of bilirubin showed £,,,-.-, ^60.6

(peak A) . When the oxidant was added, peak A disappeared

immediately, and new peaks appeared at 383 nm (peak C)

,

420 nm (peak D) , and 660 nm (peak q) , The intensities of

these peaks decreased to 20% of the original values in

70
45 min. This behavior differs from that shown on oxidation

of the same solution with periodic acid (see below).

v. In 2-Pyrrolidinone

A solution (13.9 uM) of bilirubin showed e^n ^60.3

(peak A). On addition of the oxidant, this peak disappeared,

and new peaks at 378 nm (peak C) and 694 nm (peak G) were

formed. The intensity of peak C decreased, and the peak

shifted to a lower wavelength; that of peak G also decreased

with time.

b. With Periodic Acid (H 10^) in Water

In each experiment, 0.2 ml of the oxidant (10 mM) was

added to 50 ml of the bilirubin solution.

i. In Methyl Sulfoxide

A solution (13.3 yM) of bilirubin showed e^- ^59.9

(peak A) and a shoulder at 419 nm (peak B) . On addition of

the oxidant, peaks A and B disappeared within 2 min (see fig.

11). A new peak appeared at 380 nm (peak C) , a shoulder at

425 nm (peak D), and a peak at 695 nm (peak E). The intensity

of peak C decreased 36% in 3 hr and 84% in 64 hr, while

the maximum slowly shifted to 372 nm. Peak E correspondingly

decreased in intensity, without a shift in the maximum, and

the band had disappeared completely after 64 hr. The

intensity of peak D decreased relatively slowly; the decrease

after 64 hr was ^65% * with no shift in the position of the

maximum. In table 16, for purposes of intercomparison, the

new peak E is labeled as peak G.

ii . In Methanol

A solution (6.0 yM) of bilirubin showed £uuo % 59-9

(peak A) and a slight shoulder at 417 nm (peak B) . On

addition of the oxidant, peak A vanished immediately, and

new peaks appeared at 485 nm (peak E), 580 nm (peak F), and

690 nm (peak G) . The intensity of peak E increased ^90% in

71
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Oxidation of bilirubin (in methyl sulfoxide) with

periodic acid at 25 °C. A and B, peaks for

bilirubin; C, D, and E , new peaks. Times of

recording: (a) 2 min, (b) 30 min, (c) 60 min,

(d) 90 min, (e) 120 min, (f) 150 min, (g) 180 min

an d ( h ) 6 4 h r

.

72
110 min and ^130$ in 145 mln; that of peak F increased by

^9% in 25 min and then decreased; that of peak G decreased

^95% in 145 min.

A peak at 373 nm, produced with iodic acid as oxidant,

was not formed. The intensity of peak E (485 nm) increased

rapidly, whereas, with iodic acid as the oxidant, it increased

more slowly.

iii. Iri N,N-Dimethylformamide

A solution (12.8 uM) of bilirubin showed e^g ^59.8

(peak A) , but no shoulder at 420 nm. On addition of the

oxidant, peak A disappeared in <2 min, and new peaks appeared

at 377, 600, and 668 nm; these diminished in intensity with

time (^35% in 70 min) . The consumption of periodic acid

(moles per mole), determined by the arsenite procedure, was

^2.18 (30 min) and ^2.48 (45 min).

A peak at 600 nm was produced; this was not observed when

other solvents were used.

iv. In l-Methyl-2-pyrrolidinone

A solution (14.1 uM) of bilirubin showed e ^

^59.9

(peak A). On addition of the oxidant, peak A changed to a shoulder (peak D) of Aj.l 0.22; this disappeared within 20 min

New peaks appeared at 386 nm (peak C) , 662 nm (peak G) , and

(strong) <350 nm. The intensity of peak C increased for

20 min and then decreased; that of peak G decreased continuously (^30% in 60 min)

.

v. In 2-Pyrrolidinone

A solution (13.9 uM) of bilirubin showed e^c-j, ^60 . 3

(peak A). On addition of the oxidant, this peak disappeared,

and new peaks appeared at 385 nm (peak C) , 660 nm (shoulder,

G) , and 695 nm (peak X). With time, the intensity of peak C

increased, and those of G and X decreased.

73
vi . In p-Dioxane

A solution (13.9 yM) of bilirubin showed e^g ^59.5

(peak A). On addition of the oxidant, peak A disappeared,

and new peaks appeared at 386 nm (peak C) , 610 nm (peak F)

,

and 710 nm (peak X) . Peak C decreased in intensity with

time. After 5 min, peaks F and X became a peak at 575 nm

that decreased by 30$ in 30 min.

vii . In Acetonitrile

A solution (6.98 yM) of bilirubin showed e^g ^60.1

(peak A). On addition of the oxidant, peak A disappeared,

and new peaks appeared at 388 nm (peak C) , 582 nm (peak F)

,

and 700 nm (peak X). With elapse of time, the intensity of

peaks C and F increased, and that of peak X decreased.

viii. In Bis (2-methoxyethyl) Ether

A solution (9.68 yM) of bilirubin showed e^g ^58.0

(peak A). On addition of the oxidant, the intensity of peak A

decreased by ^20% and it shifted to 452 nm; weak peaks appeared

at 387 and 642 nm that decreased in intensity with time.

c. With Sodium Periodate (NaIO|,) in Water

i . In Aqueous Sodium Hydroxide (30 mM ) A solution (17.8 yM) of bilirubin showed e^. ^39.5.

Addition of 0.2 ml of the oxidant to 50 ml of this solution

caused this peak to decrease by 85$ in 80 min, with a shift

to 400-410 nm. Weak peaks appeared at 550 and 640 nm.

The rate of oxidation was much lower than with periodic acid

in an organic solvent. About the same behavior was observed

on oxidation of the same solution with sodium iodate

.

d. With m- Chloroperoxybenzoic Acid

i . In l-Methyl-2*-pyrrolidinone

A solution (12.9 yM) of bilirubin showed e^j. ^60.4

(peak A). On addition of 0.2 ml of the oxidant (100 mM,

74
in l-methyl-2-pyrrolidinone) to 50 ml of the bilirubin

solution, the intensity of peak A gradually decreased and

shifted to lower wavelength, as shown in table 15. A weak

peak at 576 nm was also formed.

Table 15. Change in A , and in its intensity, on oxidation

max

of bilirubin with m-chloroperoxybenzoic acid

Elapsed time A (nm)

max

Total decrease

in intensity, %

30 sec 420 38

5 min 415 58

10 min 410 63

25 min 405 70

e. With ]?erric Chloride-Hydrogen Peroxide

i . In Methyl Sulfoxide

A solution (10.9 uM) of bilirubin showed e^

?

^60.3

(peak A) and a shoulder of e^ „ ^49.8. The oxidant consisted

of 10 mmoles of ferric chloride and 0.2 ml of 50% hydrogen

peroxide in 50 ml of methyl sulfoxide. On addition of 0.2 ml

of this solution to 50 ml of the bilirubin solution, peak A

disappeared, and new peaks appeared at 343 nm (peak X) and

680 nm (peak G) . Peak X decreased in intensity by 15% in

10 min, and peak G by 3% in 15 min.

The action of this oxidant differs from those of the

other oxidants used.

f . Summary

These oxidation studies indicate that, in the presence

of iodic or periodic acid, bilirubin is initially oxidized

to biliverdine, which is then further oxidized to a red

product (A 482-485 nm) as shown by the fact of its

nicix

formation from biliverdine. Much of the pertinent data on

the effect of various oxidants on bilirubin is summarized

in table 16

.

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

A supply of microcrystalline, "anhydrous" a-g-glucose

was obtained from Pfanstiehl Laboratories, Inc., Waukegan,

Illinois, for evaluation as a standard reference material.

It had been packed in polyethylene bags contained in five

cardboard drums. Samples were removed from the top (T)

and the bottom material (B) in each drum. The samples of

material are designated hereafter as samples IT and IB,

2B, 3B, 4B, and 5B.

1. Paper Chromatography of g-Glucose

a . On Whatman Filter Paper No. 1

Solutions of samples IB, 2B, 3B, 4B, and 5B (100 mg of

each) in water (1 ml for each) were prepared. The solutions

were stored in a refrigerator when not in use. Aliquots

(5 yl and 10 pi) of each solution were applied to Whatman

No. 1 filter paper, and paper chromatography was performed

by the descending method with 6:4:3 (v/v) butyl alcohol—

pyridine-^water

.

Chromatograms were sprayed with the following reagents:

i. Sodium metaperiodate solution (20 mM) , followed after 2 min by ammoniacal silver nitrate solution

(5%).

ii . Sodium metaperiodate solution (20 mM)

,

followed after 2 min by 2% p-anisidine hydrochloride in

butyl alcohol—ethanol—water

.

iii . Ammoniacal silver nitrate solution (5%) > With sprays (i) and (ii), 2-glucose was detected as a colorless spot on a colored background. Spray (iii) afforded

the most evident detection of the g-glucose, but, on storage

in the dark for 2 weeks or more, chromatograms that had been

79
sprayed with spray (iii) developed, at the point of origin,

small dark spots with a lighter corona. However, these

were not attributable to impurities in the sugar, as such

spots could also be produced by spotting distilled water on

the paper.

As some typical, "pure," commercial samples of

D-glucose have been found [66J to contain the rare disaccharide

sophorose ( 2-0-3-g-glucopyranosyl-D-glucose) [67], further

chromatograms were prepared on which aliquots (1 ul and 5 yl)

of a 1% solution of this disaccharide were applied, together

with solutions of g-glucose samples IB—5B (5-yl and 10-yl

aliquots of 10$ solutions). With sprays (i) and (iii), only

one spot was obtained from each D-glucose sample. On the

other hand, the sophorose (10 yg) from the 1-yl aliquot of

its solution was at the lower limit of detection with sprays

(i) and (iii), and it may therefore be inferred that, if

sophorose is present, its proportion in samples IB—5B is

below 1%.

b . On Seed-test Paper

Portions (50, 100, 150, 200, 250, and 300 mg) of sample

2B of g-glucose were applied to seed-test paper (45 x 60 cm)

by spotting aliquots (0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 ml)

of a solution of the sample (25 g) in water (total volume of

solution, 100 ml). Portions of distilled water (0.5 and 1 ml)

were also applied, as a control. The paper chromatogram was

developed by the descending method in 6:4:3 (v/v) butyl

alcohol-pyridine—water in a large, chromatographic tank,

and, after it had been dried in the air, it was sprayed with

reagent (i). Only a large, single spot, elongated for larger

quantities, was observed for each quantity of D-glucose taken.

There was no evidence for the presence of sophorose.

Hence, paper chromatography revealed no evidence of

impurities in the D-glucose.

80
2. Thin-layer Chromatography of D-Glucose

T.l.c. was conducted on layers (20 x 20 cm) of nonactivated Silica Gel (Merck) . Solutions were prepared from

each of samples 1B-5B [(a) 1% (in methanol), and (b) 10$

(in water)], and aliquots (1 yl) of each solution were applied

to the layers. After development with 5:1 (v/v) methanolchloroform, the plates were sprayed with either (i) ammoniacal

silver nitrate solution (5%), or (ii) 5% sulfuric acid, and

were then heated at 115 °C for 0.25 hr. Reagent (i) was found

to be the more sensitive as a detecting spray.

Solutions (a) gave a single, dark apot having R

p

0.62.

However, solutions (b) showed, also, a circular spot at the

point of origin. This phenomenon was investigated further by

t.l.c. of larger aliquots (5, and 10 yl) of solutions (a),

together with aliquots (1 yl) of solutions (b).

If the chromatogram was developed immediately after

spotting, separated spots at the origin were obtained only

from solutions (b). On the other hand, if the spotted plates

were stored at room temperature for 1.25 hr before development,

separated spots at the origin were observed for all solutions

tested.

The foregoing observations indicate that the spots at

the origin are artifacts caused by chemical reactions

(possibly, oxidation or polymerization) in the layer, prior

to development. These reactions obviously occur more readily

when the g-glucose samples are applied in aqueous solution

than when they are applied in methanolic solution. This

experience emphasizes that the thin-layer chromatograms

should be developed as soon as possible after spotting. In

some cases, this requirement may preclude the application of

many different sample spots to a single plate.

T.l.c. showed no evidence of impurities in the samples

of g-glucose.

81
3

.

High-pressure, Liquid Chromatography

An aqueous solution of p_-glucose, sample IT (concentration, 120 mg/100 ml) was analyzed by means of the Oak Ridge

National Laboratory high-pressure, chromatography system

(by Dr. Donald Young at the National Institutes of Health)

.

By this system, the sample was detected, at 485 nm, as a single peak; and, at the level of detection of the system,

the sample was found to be free of any impurities.

4. Optical Rotation of g-Glucose

Measurements were made at 20 °C, on solutions of samples

IB, 3B, 4B, and 5B in basified water, or in methyl sulfoxide,

or both, by using Rudolph and Perkin—Elmer Model l4l

polarimeters . Water thermostated at 20 °C was circulated

through the jackets of the sample cells (1 dm, and 2 dm).

For measurements on mutarotated 2-glucose in aqueous

solution, samples (^5 g, accurately weighed) of g-glucose

were made up to a volume of 25.0 ml at 20 °C, with the

inclusion of concentrated ammonium hydroxide (1 drop)

.

Measurements on non-mutarotated P_-glucose were made by

dissolving samples (^2.5 g) in dry methyl sulfoxide (total

volume, 25.0 ml) at 20 °C. The measured rotation was constant

during a period of 45 min

.

For all calculations, the sample weights in air were

corrected to weights in vacuo by application of the factor

1 + (k/1000) = 1.00064, where, for g-glucose, k = 0.64.

Representative results, indicative of the similarity

of the readings given by the two instruments, are given in

table 17 for sample 5B

.

It has previously been postulated [68] that p_-glucose,

2-xylose, and g-mannose each exist as a single anomeric form

in methyl sulfoxide, because of complex-formation involving

cis_- hydroxy 1 groups in axial-equatorial relationship; e.g. , 82
in a-g-glucopyranose, the hydroxyl groups on C-l and C-2.

The large, positive, optical rotation of the solutions in

methyl sulfoxide indicated that the samples consisted

primarily of a-g-glucose

.

Table 17 . Values for the specific rotation of a solution

of D-glucose in methyl sulfoxide

Polarimeter Concentration

in solution

Time after

dissolution (min)

r i 20

degrees

Rudolph

Perkin—Elmer

automatic

10.05

10.05

20-30

14

+112.65

+112.55

In g per 100 ml of solution.

Measurements made on solutions of Sample 4B in ammoniacal

distilled water (c_ 20.065) by using a Perkin—Elmer

polarimeter are given in table 18.

Table 18. Specific rotation, at various wavelengths, of

an equilibrated solution of D-glucose in water

Wavelength (X )

,

[a], , degrees, a r -,20 , b

Lot J, , degrees,

nm calc . from wt . in air calc

.

from wt . in vacuo

589 (Na-D) +53.22 +53.19

578 55-46 55.43

546 62.76 62.72

436 104.53 104.46

365 159.17 159.07

It has been demonstrated [69] that, below pH 10. 5

j

the equilibrium specific rotation of g-glucose is

constant with respect to pH

.

b Expected values of [a] 20 +53-04° and [a]|:jj 6

+62.89° were

calculated from the empirical equations [70]

[a] 20

= +52.50 + 0.0227c + 0.00022c 2

and [a]j:|j

6 = 62.032 + 0. 04257c,

where c_ is the concentration (g in vacuo/100 ml of solution).

83
The accuracy of the automatic polarimeter was checked bymeasurement of several standard quartz plates. Since there

was no mechanism for thermostating the quartz plates (and

aluminum supports) while they were in the analyzing cavity

of the polarimeter j they were pre-equilibrated for 1—2 hr at

20 °C (NBS calibrated thermometer). The plate and support

were then rapidly transferred to the cavity , and a measurement

was made immediately. Because the analyzer of the Model l4l

polarimeter requires relatively long periods ( 3—4 min) to

rotate to large angular values, the polarimeter was normally

preset to an angle near to the expected value. With this

technique, it was possible to obtain a measurement within

^5 sec after the plate had been inserted into the cavity.

The results are given in table 19

.

Table 19. Effect of gradual heating of quartz plates in a

polarimeter

Nominal

Quartz control

plate No.

a 546

(degrees

)

A (nm)

Time

(sec)

a (degrees

)

1724-NBS-1965 +38.927 546 3

60

120

180

+38.874

+38.877

+38.880

+38.883

1727-NBS-1965(B) -13.691 546 3

60

120

180

-13.673

-13.675

-13.677

-13.678

As determined on a Perkin—Elmer Model l4l polarimeter.

84
The slowly changing values of the angular rotation

represent the effect of the warming of the plate.

If the ratio of the nominal rotation of the quartz plates

to the rotation measured on the Model 141 instrument may be

used as a correction factor for the specific rotation of

D-glucose measured at 546 nm with sample 4B , the value

~ 20

[a],-^/- +62.81° is obtained; this compares well with the

anticipated empirical value of +62.89°.

5. Proton Magnetic Resonance Spectroscopy of g-Glucose

The proton magnetic resonance spectrum of a solution

of sample 3B (50 mg) in methyl sulfoxide-cU (0.5 ml) was

recorded on a Varian Associates Model A-60 n.m.r. spectrometer

at 60 MHz, with tetramethylsilane as the internal reference

standard. The spectrum showed a sharp doublet at x 3-71 s (a-l-OH, J QH 4.6 Hz), a triplet at t 5.0 (H-l), a doublet at

x 5.1 (4-OH* J^ qh 5.1 Hz) , a doublet at x 5.29 (3-OH, J QH

4.4 Hz), and a doublet at x 5.48 (2-OH, J_2 QH ^5.2 Hz) which

was assumed to overlap with a triplet at x 5-54 (6-OH). Other

complex bands appeared at x 6—7 (HpC-6, and ring protons other

than H-l) together with a sharp singlet at x 6.41 (water).

Further p.m.r. spectra of the solution were recorded

after it had been stored at room temperature for three weeks.

The spectrum had by then developed an additional doublet at

x 3.38 (3-1-OH, J 0H 6 . 5 Hz) whose chemical shift and splitting agree with those of the 3-1-OH of 3-D-glucose [68]. It

is obvious, therefore, that mutarotation of a-p_-glucose does

occur (slowly) in methyl sulfoxide; and, hence, it is

questionable whether 1-OH and 2-OH of this sugar form a stable,

bidentate complex with a molecule of methyl sulfoxide, as

had been suggested by Rao and Foster [69]. The assignments

are based on those published by Casu et al . [71]. Previous

assignments of H-l by these [72] and other [69] authors have

been shown to be incorrect [73].

85
In this work, hydroxyl assignments were confirmed by

deuterium exchange. When the partially mutarotated solution

was diluted with deuterium oxide (0.2 ml), all of the

multiplets in the region of t 3 •

3

—5 • 8 that had been assigned

to hydroxyl groups disappeared, leaving only a doublet

(J-. p 3-1 Hz) at t 4.92, due to H-l of the a-Q anomer, and

some ill-defined peaks at slightly higher field.

In conclusion, n.m.r. spectroscopy confirmed that the

commercial sample of D-glucose is mainly the a-g anomer, and

it did not afford any evidence of organic impurities.

6

.

Drying of g-Glucose

Aluminum pans were constructed from foil, and were

pre-dried overnight at 70 °C/1 torr. g-Glucose samples 4B

and 5B (^95 g of each, accurately weighed) were placed in

the pans, and were dried at 70 °C/0.6—2.4 torr for a total

of 114 hr. The pans of sample were cooled in a desiccator

over phosphorus pentaoxide and weighed periodically. Constant

sample-weight was reached after 50 hr; each sample had then

lost 0.012$ of its weight.

Dried samples of D-glucose were found to be hygroscopic.

7

.

Ash Content of g-Glucose

The ash content of the g-glucose was determined by

W. P. Schmidt by heating samples (20 g) to 750 °C. For

samples 3B, 4B, and 5B, ash contents of 0.004, 0.002, and

0.002$, respectively, were found.

8

.

Undissolved Residue in g- Glucose

The residue of samples 3B, 4B, and 5B (10 g of each)

that remained undissolved by water was determined by

W. P. Schmidt to be 0.002, 0.006, and 0.001$, respectively.

86
9 . Nitrogen Content of g- Glucose

Sample 2B (1 g) was analyzed by W. P. Schmidt for

nitrogen by the Kjeldahl method. Since the limit of

detection by this method is 0.001%, and because no nitrogen

was detected, it may be stated that the nitrogen content

of sample 2B is less than 0.001%.

Spectrographic analysis of samples 3B and 5B is under

way. When the results of this analysis are known, analyses

for individual trace elements will be performed.

C. Creatinine

(R. F. Brady, Jr.)

Preliminary work on the analysis of creatinine (1) for

issuance as a clinical Standard Reference Material has been

described in Technical Note 457-

CHo

S

I N

NH,

N

1 Refinements have been made in the phase-solubility

analysis of 1 . In early work, samples of creatinine, dried

for 24 hr at 25 °C/0.1 torr over phosphorus pentaoxide and

then analyzed by phase-solubility analysis (in absolute

methanol), had been found to be 99.0% pure; but this value

seemed unreasonable in the light of information that had

been gathered by other techniques. It was postulated that

this low value might be due to incomplete drying of the sample;

the presence of water in the sample would cause the solubility

of creatinine in the methanol to increase, and this increase

87
in the amount of 1 dissolved (with increased amounts of 1

equilibrated) would have been interpreted as being impurity.

To overcome this uncertainty, the phase-solubility analysis

has now been performed with 19:1 (v/v) methanol—water and

with 19:1 (v/v) ethanol—water as the solvents. By this

means, any contribution of water in the sample to the

solubility was made negligible in relation to that of water

in the solvent. In this way, the purity found was 99.8%,

and repetition of the analysis of a thoroughly dried sample

of 1 with absolute methanol as the solvent confirmed this value

Creatinine was issued as Standard Reference Material 914.

A reproduction of the Certificate of Analysis is shown on

the following pages.

D. Urea

(B. Coxon)

On the basis of a previous investigation of the properties

and purity of four commercial samples of urea [75], a sample

of a commercial urea was selected as being the most suitable

product for issuance as a Standard Reference Material. Bulk

material was supplied in the form of well defined, colorless

crystals packed in polyethylene bags contained in cardboard

drums. Portions from each container were analyzed, and are

designated hereafter as box or sample 1,2,3, or 4.

1 . Thin-layer Chromatography of Urea

Solutions were prepared as follows

:

(a) A sample (101 mg) from each container of urea

was dissolved in methanol (1.0 ml) and 10 ul of each solution

was applied to the chromatographic plates.

(b) A suspension of biuret (101 mg) in methanol

(1.0 ml) was boiled briefly, and aliquots (5 Ml) of the

supernatant solution were used.

88
U. S. Depart^ei^ of Commerce

Maurice^Jt. Stans

A. V. National Bajeipo:: Standards

irector

Certificate of &nalpgisi

Standard Reference Material 914

CREATININE

This Standard Reference Material is certified as a chemical of known purity for use in the

calibration and standardization of procedures employed in clinical analysis.

Purity 99.8 percent

Volatile matter 0.03 percent

Chloride 0.07 percent

Ash 0.003 percent

Insoluble matter 0.001 percent

The value of the purity has an estimated inaccuracy of 0.1 percent.

The creatinine used for this Standard Reference Material was obtained from the

Pfanstiehl Laboratories, Inc., of Waukegan, Illinois. Analyses were performed by

D. A. Becker, R. F. Brady, Jr., M. M. Darr, T. E. Gills, R. A. Paulson, W. P. Schmidt, and

R. S. Tipson of the Analytical Chemistry Division.

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

The technical and support aspects concerning the preparation, certification, and issuance

of this Standard Reference Material were coordinated through the Office of Standard Refer- ence Materials by T. W. Mears.

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

September 24, 1968 Office of Standard Reference Materials

(over)

89
The homogeneity of the creatinine was determined by paper, thin—layer, and gas—liquid

chro matography

.

Volatile matter was determined by measurement of loss of weight of the creatinine after

heating for 24 hours at 110 °C.

Phase—solubility analysis [1] of an oven—dried sample of the Standard Reference Material, with absolute methanol as the solvent, indicated the purity to be 99.82 weight

percent. Phase—solubility analysis of the undried creatinine with 95 percent methanol, and

95 percent ethanol, indicated purities of 99.81 and 99.76 weight percent, respectively.

Potentiometric titration of the Standard Reference Material required 99.82 percent of the

theoretical amount of hydrochloric acid.

The certified value for chloride was obtained by titration with mercuric nitrate of a solution of the products resulting from an oxygen—flask combustion of the Standard Refer- ence Material.

The ultraviolet absorption spectrum of a solution of the creatinine in water showed an

extinction coefficient (e max) of 7140 ± 30 at 234 nm. The measure of uncertainty is the

standard deviation of a single measurement, and should not be considered to be a certified measure of inaccuracy for the extinction coefficient. Infrared absorption and nuclear magnetic resonance spectra provided no evidence of impurities.

An emission spectrometric analysis for metallic constituents in the ash from this Standard

Reference Material showed the following present as major constituents: silicon, aluminum,

sodium, titanium, iron, and magnesium. Neutron activation analysis of the bulk Standard

Reference Material indicated the presence of the following approximate concentrations of

elements: chlorine, 330 ppm; copper, 0.2 ppm; manganese, 0.07 ppm; sodium, 30 ppm.

[1] Mader, W. J., 'Phase—Solubility Analysis" in Organic Analysis, Vol II, Interscience

Publishers, Inc., New York, 1954, p. 253.

90
(c) A suspension of cyanuric acid in water was

boiled briefly, and aliquots (5 yl) of the supernatant

solution were spotted on chromato grams

.

The solutions described in (a), (b), and (c) were

separately applied to 20-cm glass plates coated with nonactivated layers of Silica Gel G (Merck) . The layers were

developed by the ascending method with 2:1 v/v methanol—

dichloromethane. Developed components were detected with

1% p_- (dimethyl amino ) cinnamaldehyde reagent [76] (DMCA),

or iodine vapor.

With DMCA, urea was revealed as a white-centered red

spot (FL-, 0.5). Biuret (red spot, R_ 0.65) was detected in

the commercial sample of biuret, and in the impure sample of

urea, but not in boxes 1—4 of urea. Cyanuric acid gave a

dark-purple fluorescence under ultraviolet light, but was not

detectable in the pure samples of urea. Similar results were

obtained with iodine vapor; with it, urea and biuret were

detected as brown spots.

Plates that were sprayed with 50% sulfuric acid and then

heated at 150 °C did not show any significant spots, either

under room lighting or under ultraviolet light, thus indicating

that, if other organic impurities were present in the urea,

their proportion was at a very low level.

2 . Paper Chromatography of Urea

Aliquots (10 yl for urea, 20 yl for biuret) of the

solutions prepared for t.l.c. were applied to Whatman No. 1

filter paper, and the chromatogram was then developed by the

descending method with 6:4:3 (v/v) butanol—pyridine—water

.

The separated components (urea, R^— 0.38; and biuret, R_ 0.51)

if —if were detected with

(i) DMCA. No heating was necessary for

visibilization of the spots, but heating at 70 °C for 15 min

intensified their red color.

91
(ii) 5% Silver Nitrate Solution. The

chromatogram was stored in the dark for 10 days , after which

time, urea was detected as a brown spot.

Urea samples 1—4 were homogeneous, according to the

results of paper chromatography.

3

.

Infrared Spectroscopy of Urea

Urea (sample 1) was pressed into a potassium chloride

2 disc at 12 tons/in , and the i.r. spectrum was recorded on

a Perkin—Elmer Model 257 grating spectrometer. The resulting

spectrum showed v 3^50s, 3350s, 3265m, 3225m (shoulder),

max -, 1680s, 1626s, 1601s, 1460s, 1155m, 787w cm" , and was

identical with NBS Standard Spectrum No. 486, which had been

obtained for recrystallized urea (Eastman)

.

4

.

Proton Magnetic Resonance Spectroscopy of Urea

Urea samples 1 and 3 (210 mg of each) were each dissolved

in a portion of deuterium oxide (0.4 ml) and, after 30—70 min,

the n.m.r. spectra of the solutions were recorded on a Varian

Associates A-60 spectrometer at 60 MHz, with 1:1 (v/v)

tetramethylsilane—carbon tetrachloride as the external

reference. Proton exchange did not occur readily under these

conditions, as shown by the observation of a broad singlet at

t

t 4.07 (NH ) . One other resonance was observed, a sharp

singlet at t' 5-2 (HOD). No peaks for impurity were detected.

5

.

Ultraviolet Spectrophotometry of Urea

Urea samples 1—4 (5.0 g of each) were each dissolved in

a portion of water (10.0 ml), and the u.v. spectra of the

aqueous solutions (2-cm cells) were recorded with a Cary

Model 14 spectrophotometer, with water in the reference cell,

over the wavelength range of 220—750 nm. The absorbances

observed are shown in table 20.

A weak absorption shoulder was observed at 280 nm.

92
Table 20. Absorbances at various wavelengths in the u.v.

spectra of aqueous solutions of various samples

of urea

Absorbance at

A 565 Box A <220 A 280

A 560

1 very strong 0.096 0.019

2 very strong 0.129 0.029

3 very strong 0.088 0.015

4 very strong 0.098 0.015

6 . Determination of Biuret

The reagent for biuret estimations was prepared by

mixing a 3% aqueous cupric sulfate solution (12.5 ml) with

105S sodium hydroxide (500 ml). Aliquots (5.0 ml) of the

resulting solution were added to urea samples 1—4 ( 5 g of

each, accurately weighed) , and the crystals were dissolved by

gentle warming of the suspensions. The solutions were diluted

to 10.0 ml, and their absorption spectra in the visible

region were recorded with a Cary Model iH spectrophotometer,

with diluted alkaline cupric sulfate reagent in the reference

cell. (The baseline was set initially by recording reagent

versus reagent.) The absorbances observed at the absorption

maximum of the biuret—copper complex (560-565 nm) were corrected

by subtraction of the corresponding absorbances of the urea

samples in water alone (see part 5, above).

The quantities of biuret present were determined by

reference to absorbances measured on three solutions of a commercial biuret in the diluted copper reagent. The results

obtained from the standard solutions (a) and the urea samples

(b) are given in table 21.

93
Table 21. Absorbances of solutions of (a) biuret and (b)

various samples of urea in alkaline cupric sulfate

solution

(a) Wt . of biuret

(mg)

max

(nm) ——

Absorbance Absorbance

per mg of

biuret

e

1.012 550 0.012 0.012 6.1

4.248 525 0.054 0.013 6.6

8.450 505 0.107 0.013 6.5

(b) Urea sample Absorb- Corrected % of

bance absorbance biuret

1 0.059 0.040 <0.06b

2 0.066 0.037 <0.06

3 0.057 0.042 <0 .07

4

1 0.061 0.046 <0 .07

At 56O-565 nm. Less than, because chromatography indicated

that the commercial sample of biuret contained some urea.

7

.

pH of Urea in Aqueous Solution

The pH values of solutions of urea samples 1—4 (2.5 g)

in carbon dioxide-free , distilled water (25-0 ml, total

volume) were measured with a Corning, expanded-scale, pH meter

and were found to be 7-06, 7-06, 6.97, and 7-30, respectively.

8

.

Drying of Urea

a. At Room Temperature over Phosphorus Pentaoxide

The urea samples were pulverized in a mortar, and each

sample (^11 g, accurately weighed) was dried over phosphorus

pentaoxide at room temperature/0.01 torr, with a solid carbon

dioxide—dichloromethane trap, for a total of 206 hr . The

samples were weighed periodically during this time. The weight

of each sample decreased progressively, at an approximately

constant rate. After 206 hr, the mean total loss in weight

ranged between 2.84 and 3.08%, a mean rate of 0.0144% per hr.

94
b . At Room Temperature (No Desiccant)

Urea (32.5 g, from box 2) was powdered, weighed, and

then dried at room temperature/0.01 torr for 48 hr with two

liquid nitrogen traps

.

The sample was then dried for a further 304 hr, but with

two solid carbon dioxide—dichloromethane traps. The results

shown in table 22 were obtained.

Table 22. Loss in weight of urea at 25 °C/0.01 torr after

various time-intervals

Drying time (hr) w (% per hr) Total wt-loss {%)

24 0.00031 0.0075

48 0.00026 0.0137

118 0.00020 0.0278

190 0.00013 0.0369

326 0.00012 0.0539

352 0.00026 0.0605

Mean % loss in weight per hr since previous weighing.

After a total of 352 hr, air was admitted to the traps

through a calcium chloride tube. The condensate was a colorless, turbid liquid (1.00 g) which turned anhydrous cupric

sulfate blue, and gave a faint red spot when spotted on filter

paper, sprayed with the reagent for urea (DMCA), and heated.

Karl Fischer determinations (by J. H. Thomas) on two

specimens of the dried material showed that it still contained

water (0.073 and 0.086$).

c. At 115 °C

Urea sample 2 (^10 g, accurately weighed) was dried at

115 °C in an oven at atmospheric pressure. Periodic weighing

gave the results shown in table 23.

95
Table 23- Loss in weight of urea at 115 °C/760 torr after

various time-intervals

Drying time (hr)

— ' ' " — -'- -

w

a

{%) Total wt.-loss {%)

1

2

3

4

122

0.089

0.064

0.050

0.048

27-577

0.089

0.153

0.203

0.251

27.828

Weight loss (%) since previous weighing.

The excessive loss in weight, and the fact that the

dried material was no longer soluble in water, indicated

that extensive decomposition had taken place.

9

.

Change in Weight on Exposure to the Atmosphere

Urea sample 2 (15 g) was exposed to the atmosphere over

a 15-day period. After 1 day, the sample weight had increased

by 0.0033$, but, after a further 14 days, the weight of the

sample was 0.006$ less than the initial weight.

It was concluded that urea shows little tendency to gain

or lose moisture when exposed to air in the laboratory.

Confirmation was obtained from Karl Fischer determinations

(by D. W. Vomhof and J. H. Thomas) on the exposed material,

which showed a moisture content (0.17$) that was comparable

with that (0.19$) of freshly opened samples.

10

.

Determination of the Purity of Urea by Differential

Scanning Calorimetry

The experimental method used was that of Plato and

Glasgow [77, 78].

The area of the recording chart was calibrated by

observation of the melting-point transition of ultrapure

96
indium (3.0545 mg) . The calibration factor was 0.02340

meal/unit area, the area being measured by a planimeter.

The results obtained (sample weights 0.7695-0.8705 mg)

are shown in table 24. The results indicated a high degree

of homogeneity in material from different containers

.

Table 24. Purity of various samples of urea, as determined

by differential scanning calorimetry

Urea, AT

(degrees

)

AHf Purity

,

box no. (kcal/mol) (kJ/mol) mole %

1 0.133 3-76 15.7 99.85

2 0.169 3-83 16.0 99-80

3 0.147 3.82 16.0 99.83

4 0.153 3.74 15.6 99.82

11. Determination of the Purity of Urea by Phase- solubility Analysis

(R. F. Brady, Jr.)

The solubility of a pure compound in a pure solvent is

a fundamental physical property of the compound. This

property is exploited in phase-solubility analysis, which

has been employed in an attempt to obtain quantitative data

on the purity of urea. This technique measures the amount

of material dissolved when increasing amounts of a substance

are equilibrated with 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 proportion of impurity in

the sample can be determined. Isopropyl alcohol, in which

urea has been found to be soluble to the extent of 29.0 mg/g

at 25.0 °C, was chosen. Sealed tubes containing weighed,

progressively larger weights of urea and uniform weights of

97
isopropyl alcohol were allowed to equilibrate in a water

bath at 25.0 ±0.1 °C. The weights of urea were so chosen

that at least one tube contained an unsaturated solution.

After equilibration, aliquots of each solution were separated

from any undissolved urea, and evaporated to dryness, and the

residues were 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. This diagram is shown in

fig. 12. The content of solute throughout the range of

saturated solutions studied was found to increase slightly.

The sample of urea investigated was found to be 99-82 weightpercent pure, in agreement with the value of 99-82 mole-percent

purity obtained by differential scanning calorimetry . Phasesolubility analysis is generally to be considered accurate

to within ±0 .1%

.

Urea was issued as Standard Reference Material 912.

A reproduction of the Certificate of Analysis is shown on

the following pages

.

E. Calcium Carbonate

Calcium carbonate for use in clinical laboratories was

issued as Standard Reference Material 915- A reproduction

of the Certificate of Analysis is shown on the following

pages. Dr. Robert Schaffer coordinated the work leading to

its certification.

F. Uric Acid

As a result of the work described in Technical Note ^57,

uric acid was issued as Standard Reference Material 913- A

reproduction of the Certificate of Analysis is shown on the

following pages.

98
joqoo|B |Xdojdosj j.o 6/eajn j.o 6ui 'uouisodtuoQ uojinjos

Figure 12. Phase-solubility diagram of urea in isopropyl

alcohol at 25 °C.

99
U. S. Department* of Commerce

MauriceJJj. Stans

Secjretaify

National BureSffi o" Standards

A. V. Ast^Director

Certificate of gtoalpgisi

Standard Reference Material 912

UREA

This Standard Reference Material is certified as a chemical of known purity for use in the

calibration and standardization of procedures employed in clinical analysis.

Purity 99.7 percent

Moisture 0.18 percent

Biuret 0.07 percent

Ash 0.002 percent

Insoluble matter 0.002 percent

The value of the purity has an estimated inaccuracy of 0.1 percent.

The urea used for this Standard Reference Material was obtained from the Mann Research

Laboratories of New York, N.Y. Analyses were performed by D. A. Becker, R. F. Brady, Jr., B. Coxon, M. M. Darr, T. E. Gills, R.A.Paulson, W.P.Schmidt, J.Thomas, and

D. W. Vomhof of the Analytical Chemistry Division.

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

The technical and support aspects concerning the preparation, certification, and issuance

of this Standard Reference Material were coordinated through the Office of Standard Refer- ence Materials by T. W. Mears.

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

September 24, 1968 Office of Standard Reference Materials

100
A satisfactory method for drying this Standard Reference Material was not found. Either

incomplete drying, or excessive (and progressive) weight loss occurred, depending on the

degree and intensity of heating and evacuation. However, it was found that this material

showed little tendency to gain or lose weight (± 0.003 percent) when exposed to laboratory

air for a two—week period. Hence, the analyses were performed on this Standard Reference

Urea without attempting to lower its moisture content. It is recommended that, after the

withdrawal of portions of the Standard Reference Material, the container should be tightly

closed. Thus protected, it may be assumed that the moisture content of the Standard

Reference Material will remain unchanged. The reported moisture content was determined

by the Karl Fischer titration method.

The apparent purity of this Standard Reference Material was determined by two

methods. Differential scanning calorimetry [1], performed with a Perkin—Elmer model

DSC—IB* instrument on the urea heated through its melting transition, indicated an apparent purity of 99.82 ± 0.003 mole percent. Phase—solubility analysis [2] of the urea in

isopropyl alcohol indicated an apparent purity of 99.82 weight percent. These values are designated as "apparent purity", because neither method accounted for the moisture content of the urea. Biuret was estimated spectrophotometrically by use of 50 percent (wt/vol)

solutions of urea in aqueous alkaline cupric sulfate measured at its absorption maximum

near 560 nm. Measured in a 2—cm cell, a 50 percent (wt/vol) solution of urea in water

showed a very strong absorption below 220 nm, a weak absorption (0.10±0.03) at 280 nm,

and a very weak absorption (0.02 ± 0.01) at 560 nm.

Neither paper nor thin—layer chromatography indicate any evidence of biuret or cyanuric

acid. Neither infrared absorption nor nuclear magnetic resonance spectroscopy reveal any

unexpected peaks and, hence, they give no evidence of impurity.

The melting range of this Standard Reference Material was 131.5 to 132.4 °C when

measured in an open capillary tube heated at 0.5 °C/min.

A 10 percent (wt/vol) solution of this Standard Reference Material in water free from

carbon dioxide showed a p\i of 7.0 ±0.1 at 20 ° C.

An emission spectrometric analysis for metallic constituents in the ash from this Standard

Reference Material showed the following present as major constituents: aluminum, silicon,

iron, sodium, nickel, calcium, magnesium, and manganese.

Neutron activation analysis of the bulk Standard Reference Material indicated the pres- ence of the following approximate concentrations of elements: aluminum, 0.9 ppm; chlorine, less than 1 ppm; copper, 0.27 ppm; gold, 0.0001 ppm; manganese, 0.008 ppm; sodium,

9 ppm; zinc, 0.24 ppm.

[1] Plato, C. and Glasgow, A. R., Jr., Abstracts of Papers, 154th American Chemical

Society Meeting, September 1967, p. 44A; Submitted to Anal. Chem. September 1968.

[2] Mader, W. J., "Phase Solubility in Organic Analysis" in Organic Analysis, Vol II, Inter- science Publishers, New York, 1954, p. 253.

*Note — The use of proprietary designations in this certificate are for information only, and

should not be construed as an endorsement of the product by either the Department of

Commerce or the National Bureau of Standards.

101
U. S. Departments of Commerce

Maurice^lJ. Stans

Secretary

>*£»«=

National BureWs of Standards

A. V. AsiHir-Director

(UntxiitaU tff Analysis

Standard Reference Material 915

Calcium Carbonate

This Standard Reference Material is certified for use in the calibration and standardization of procedures employed for the determination of calcium in clinical analysis.

The sample consists of highly purified calcium carbonate, and chemical assay as well as

analysis for specific impurities indicate that the material may be considered to be essentially pure.

Purity 99.9+ percent

Water 0.01 ± 0.005 percent

Replicate samples taken from a randomly selected region of the undried material were assayed by a coulometric acidimetric procedure. The results from nine independent

determinations, based on expression of the assay as calcium carbonate, indicate a purity

of 99.99+ percent with a standard deviation of 0.003 percent. Samples equilibrated at a

relative humidity of 90 percent and assayed by this coulometric procedure showed a maximum moisture adsorption of 0.02 percent as compared to samples that were dried

for 6 hours at 210 °C. The moisture content, similarly measured, on samples equilibrated

at 75 percent relative humidity, was found to be 0.01 percent. The water content was determined by the Karl Fischer method.

The calcium carbonate used for this Standard Reference Material was obtained from

the J. T. Baker Chemical Company, of Phillipsburg, New Jersey. Analyses were performed

by C. E. Champion, E. R. Deardorff, G. Marinenko, O. Menis, T. C. Rains, T. A. Rush,

W. P. Schmidt, B. F. Scribner, V. C. Stewart, J. K. Taylor, and D. W. Vomhof.

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

The technical and support aspects concerning the preparation, certification, and

issuance of this Standard Reference Material were coordinated through the Office of

Standard Reference Materials by J. L. Hague.

Washington, D. C. March 4, 196920234 W. Wayne Meinke, Chief

Office of Standard Reference Materials

102
Supplemental Information

The material was examined for compliance with the specifications for reagent grade

calcium carbonate as given in Reagent Chemicals, 4th edition, published by The American

Chemical Society. The material was found to meet or exceed the minimum requirements

in every respect. Examination by thermal gravimetric analysis indicated the loss of a minute proportion of weight below 175 °C (volatile matter) and the composition was stable

above this temperature until a temperature of 625 °C, above which decomposition (evolution of C02 ) set in. A semi-quantitative survey for trace contaminants by emission spectroscopy indicated the presence of less than 0.001 percent of copper, iron, magnesium, manganese and

silicon in the material. By atomic absorbtion, magnesium was evaluated at 1.0, sodium

at 0.4, and strontium 2.1 parts per million (ppm) ; potassium was less than 0.4, lithium

less than 0.05, and barium much less than 10 ppm. Neutron activation analysis indicated

copper 0.9, manganese 0.6 and sodium 0.5 ppm. Copper was determined at 1 ppm by

spectrophotometry.

103
U. S. Departifoentf of Commerce

Maurice B. Stans

National BnreMi of Standards

A. V. A«t»r^Director

Certificate of gtoalpste

Standard Reference Material 913

URIC ACID

This Standard Reference Material is certified as a chemical of known purity for use in the

calibration and standardization of procedures employed in clinical analysis.

Purity 99.7 percent

Volatile matter 0.14 percent

Ash 0.057 percent

The value of the purity has an estimated inaccuracy of 0.1 percent.

The ash was found to be composed principally of salts of sodium and potassium. Aluminum, calcium, iron, phosphorus, and silicon were found in proportions estimated to be

between 1 and 10 percent of the ash. Cobalt, copper, manganese, nickel, and zinc were each

present to an extent not exceeding 1 percent of the ash.

The uric acid used for this Standard Reference Material was obtained from the Pfanstiehl

Laboratories, Inc., of Waukegan, Illinois. Analyses were performed by D. P. Enagonio,

R. A. Paulson, W. P. Schmidt, V. C. Stewart, R. S. Tipson, and B. F. West of the Analytical

Chemistry Division.

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

The technical and support aspects concerning the preparation, certification, and issuance

of this Standard Reference Material were coordinated through the Office of Standard Refer- ence Materials by T. W. Mears.

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

September 24, 1968 Office of Standard Reference Materials

104
The uric acid used in this Standard Reference Material was found to be homogeneous by

paper and thin—layer chromatography with several solvent systems. This homogeneity was

further verified by gas—liquid chromatography of the trimethylsilylated material.

Volatile material was determined by heating the sample overnight at 110 °C.

An emission spectrometric analysis for metallic constituents in the ash from this Standard

Reference Material showed the following present as major constituents: sodium, potassium, aluminum, calcium, iron, phosphorus, and silicon. Neutron activation analysis of the

bulk Standard Reference Material indicated the presence of the following approximate

concentrations of elements: sodium, 170 ppm; copper, 2.4 ppm, manganese, 0.028 ppm.

The ultraviolet absorption spectra of the material exhibited the following absorption

maxima:

In lithium carbonate solution, pH - 7.6.

at 292 nm, e max = 12,560 ± 10

at 236 nm, emax = 10,000 ± 80

In a glycine-buffered solution, pH = 9.6

at 292 nm, e max = 12,650 ± 50

at 234 nm, e^ = 9,890 ± 50

The measures of uncertainty given are the standard deviation of a single measurement. They

should not be considered to be a certified measure of inaccuracy for the extinction coefficients. A 100—g sample of uric acid was extracted successively with 500 cm3 each of water,

absolute ethanol, benzene, and water. The ultraviolet absorption spectrum of each of these

extracts showed no absorption bands other than those of uric acid. Only uric acid was

detected by thin—layer chromatography of these same extracts.

105
G . Cholesterol . Post-Issuance Studies

In Technical Note 457, we reported analyses performed

on the cholesterol SRM some six months after issuance. Since

then, further studies have been carried out at two additional

half-year intervals. The objective of this continuing study

is to evaluate the shelf-life of the standards, for it is

known that cholesterol may undergo decomposition, and this

imperils the standard.

1. Thin-layer Chromatography of Cholesterol Samples

(A. Cohen)

Chromatography was performed on activated Silica Gel

H plates with 1:1 (v/v) ethyl acetate—heptane, as described

in Technical Note 457. At one year after issuance and at

one-half year later still, the bulk supply of standard

material (that had been stored in a freezer) showed no

significant evidence of greater impurity than had been

detected in the material when originally tested or at six

months after issuance. SRM packaged samples, which are

bottled under nitrogen, behaved much like the freezer-stored

supply. However, samples that had been kept at room temperature in amber bottles loosely covered to ensure exposure tc

air showed a significant growth of impurities that we estirrated

as amounting roughly to 1% at one year of exposure and to

greater than this after six months more.

2

.

Liebermann—Burchard Analysis of Cholesterol Samples

(A. Cohen)

New measurements made by our modification (see

Technical Note 457) of this method showed no significant

difference between the packaged, the freezer-stored, or the

air-exposed cholesterol at one year after issuance; for these

samples, molar absorbances (liters/mole cm) at 535 nm were

106
596 ±2, 597 ±1, and 597 ±3 (averages of 5 or 6 aliquots per

sample ± 1 standard deviation) , respectively. These values

are almost identical with those obtained on similarly stored

samples at six months after issuance. Thus, these results do

not distinguish between the better and poorer cholesterol.

3 . Phase-solubility Analysis (PSA) and Differential

S canning Calorimetry (DSC)

( R. F . Brady , Jr

.

, and D. A. Niner)

PSA and DSC were applied to cholesterol samples as

described in Technical Note 457 3 where it was reported that,

at six months after issuance, PSA indicated 99-96 weight-percent

purity, and DSC, 99-84 mole-percent purity for the freezer-stored

cholesterol. After the elapse of an additional year, the

purity values for this material were found to be 99-6 weightpercent and 99-5 mole-percent, and samples of packaged SRM

cholesterol showed purities of 99-6 weight-percent and 99-5

mole-percent by DSA and DSC, respectively. At the time of

the most recent reevaluations by DSC, new measurements of the

purity of SRM urea were obtained, and these values found were

almost exactly those obtained earlier. Thus the changes in

the weight- and mole-percent purities of the cholesterol appear

to be real. Since the SRM cholesterol was originally certified

as 99-4 ±0.3 percent, and our recent work still shows a purity

on the high side of this range, the SRM continues to meet the

specifications stated. Further evaluations will continue.

107
3. DETECTION AND IDENTIFICATION OF TRACES OF ORGANIC

COMPOUNDS

In much of the Section's work, particularly that leading

to the certification of SRM's, traces of impurities are

encountered, and the identification of these trace substances

is usually desired. As a guide for identification, there may

be, in the literature, information on impurities that have

previously been identified in work on the major component,

or use may be made of knowledge on the origin (natural source)

or the method of synthesis of the compound that is the major

constituent. Occasionally, grossly impure commercial preparations can provide a source for the impurities.

T.l.c. with several different adsorbents and developing

solvents is frequently employed for establishing the identity

of a known reference compound with the impurity; however, such

evidence alone is seldom entirely satisfactory. Additional

characterization is needed, e.g. , mass spectra, ultraviolet

and infrared spectrophotometry, etc. Since an infrared

spectrum is almost always unique for a compound, characterization by infrared spectroscopy in combination with t.l.c.

usually provides firm proof of identity; our work began with

a comparisom of these two techniques.

To ascertain the relationship between the quantities of

compounds that could be detected by t.l.c. versus those needed

for full characterization of them by infrared spectroscopy,

micro-sampling techniques for infrared spectroscopy were

studied along with the limits of detection for the same

compounds in t.l.c. The approach was to utilize compounds for

which each of the techniques would be quite sensitive. Some

polynitro compounds were employed, because (a) nitro groups

have high absorptivity in the infrared region, and (b) there

are sensitive methods for detecting such compounds on thinlayer chromatograms

.

108
A. Thin-layer Chromatography

(A. Cohen)

For determining detection-limits by t.l.c., aliquots of

a solution of 2 , 4 ,6- trinitrotoluene in acetone (5 g/1) were

transferred, as separate spots, to silica gel plates containing

a 254-nm activated, fluorescent indicator (Silica Gel GF)

such that the quantities of compound in individual spots

ranged from 5 to 40 ng. After developing with 9:1 (v/v)

benzene—pentane and drying under nitrogen, the chromatogram,

examined with 254-nm light, showed no evidence of the nitro

compound in spots containing less than 40 ng; but, at 40 ng,

there was some suggestion of detection. On spraying the

chromatogram with ethylenediamine in methyl sulfoxide [79] 3 there was questionable detection at 15 ng, and certain

detection of the spots with 20-, 30-, and 40-ng quantities

of the compound, by a faint pink coloration.

With hexahydro-1,

3

? 5-trinitro- s- triazine , taken as an

example of a nonaromatic trinitro compound, the quantities

studied ranged from 30 to 150 ng. Benzene—acetone (4:1 v/v)

was used for development of the plates (Silica Gel GF) . On

examination under 254-nm light, only spots containing 120 ng

or more were detected definitely; there was considerable

uncertainty for amounts at the 60- and 90-ng level. For

making the spots visible under ordinary light, the plate was

treated as follows: it was first sprayed with methanolic

potassium hydroxide [80] and warmed at 115 °C for 5 min, and

then sprayed with a Benzidine Blue reagent [8l]. Quantities

of the compound, 60 ng or more, were visible as weak blue

or purplish spots after this treatment.

These examples showed that, with visual observation,

fluorescence quenching provided no advantage over colorimetric

tests for detection of the two polynitro compounds. Nevertheless, fluorescence quenching has the advantage of revealing

the presence of the compounds without altering them chemically,

109
and thus it affords the option of isolating the chromatographically separated compound and utilizing it for additional

characterization

.

B . Infrared Spectroscopy

(A. Cohen)

Methods previously reported for the preparation of

microsamples for infrared spectroscopy employed (a) freezedrying of the sample plus potassium bromide, (b) grinding

of the sample with potassium bromide in a volatile solvent,

or ( c_) adding the compound, in a volatile solvent, to a

preground specimen of potassium bromide [82]. In our work,

a weighed quantity of preground potassium bromide (5—7 mg)

and an aliquot of a solution of the compound in chloroform

or acetone were combined in a small test-tube, and mixed gently

with a glass rod. Evaporation of the solvent was speeded

with a gentle stream of nitrogen. The dry mixture was

transferred to a 100 -mm, ultramicro die-assembly (Perkin—Elmer)

for pressing into a disc, after initial evacuation of the

assembly at about 2 torr. The discs produced weighed 4 to

5 mg. The sample (in the potassium bromide disc) was mounted

in a 4X beam condenser, and examined with Perkin—Elmer

infrared spectrometers (Models 257 and 621) without ordinaryscale expansion. Fully characteristic spectra were obtained

when a disc contained 4 to 5 yg of a compound. Below this

amount, the spectra did not show the less-intense bands, and

therefore were less characteristic of the compound. With

0.5 yg of compound in the disc, only the most intense bands

in the spectra of the polynitro compounds were barely

discernible above the noise level. With use of ordinate-scale

expansion, improvement by a factor of 2 or 3 could be reached.

Nevertheless, the quantities of the compounds needed for

providing characteristic infrared spectra were found to be

many times greater than those necessary for useful examination

by t.l.c-. This subject -i-s undergoing further study.

110
4. ANALYTICAL CHEMISTRY OF CORN SYRUPS AND RELATED PRODUCTS

( D. W. Vomhof and J. H. Thomas)

This Research Associate Program, supported by the Corn

Industries Research Foundation and devoted to the study of

corn syrup hydrolyzates , has completed its initial phase,

the development of assured methods for the evaluation of the

moisture content of these hydrolyzates. The methods are

described in this report. The additional physical properties

that are to be evaluated are briefly sketched.

A. Direct Determination of Moisture

Prior to undertaking studies of other physical properties,

alternatives to the vacuum-oven method for the direct

determination of the moisture content of starch hydrolyzates

were evaluated. Requirements for a suitable direct method

of moisture analysis are: a high degree of accuracy, a low

level of imprecision, freedom from interference (either by

the saccharides or the ash present), freedom from degradation

or chemical action resulting in the production of volatile

substances or the consumption of moisture, and, finally,

reasonable rapidity. Three methods have been developed which

satisfy these criteria.

1. Near-infrared Spectrophotometry

One direct method found suitable is a near-infrared,

spectrophotometric method specific for water. The determination

depends on measurement of the absorption of energy at 1.9 ym

by water molecules. The amount of energy absorbed is directly

proportional to the quantity of water present. Hydroxyl

groups of saccharide molecules do not interfere.

Calibration is achieved by determining the absorbance of

solutions containing known concentrations of water in a suitable solvent and the results are plotted graphically.

Ill
The best solvent for this purpose appears to be methyl sulfoxide

(Me„SO) . Solutions of the syrups are prepared in solvent

from the same lot as that used for calibration, care being

taken to ensure complete dissolution of the syrup. The net

absorbance, measured with a millimeter rule, is obtained by

subtracting the base absorbance at 1.83 urn from the peak

absorbance at about 1.93 um. 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

where F = the weight-percentage of water in the original

syrup, W = the weight-percentage of water in the sample

solution, and S = the weight-percentage of syrup in the sample

solution.

The near-infrared absorption spectra of water, sucrose,

and a corn syrup in Me

p

SO are shown in figure 13. The

hydroxyl groups of the saccharide are the major contributors

to the absorbance in the region from 2.0 to 2.1 um. The

minimum at 2.0 um is dependent on both the content of

saccharide and of the water in the solution.

The increase in absorbance is linear over the range

from 0.0 to 1.3 weight-percent of moisture in the sample

solution. The standard deviation of the method, based on 24

degrees of freedom, is 0.03^ weight-percent of water for the

moisture content of the original syrup. The detection limit

[83] (having a 95% chance of being detected) is 0.0071 weightpercent of water in the sample solution. The time required

for the analysis of a syrup, including the preparation of the

solution and the calculations, is about 90 minutes.

112
/ .•

0)

0) O \V: 5 v> I '* •01 i •

mS

CN J > ^^"^ r in a)

• ^„>*^

;• i

i .\^

• f

—i i

.X

• \

• \

• \

• I

•1

J 1 1 1 .1 .1 —1_

1

1

1

00

6

CO

6 d 6

o

o

E

a.

8 o

LU

_l

LU

>

<

O

00

3DNVaaOS9V

Figure 13- Near-infrared absorption spectra of water,

sucrose, and a corn syrup (each dissolved in

methyl sulfoxide).

113
2. Infrared Spectrophotometry

Another spectrophotometry method, not acceptable for

our work because of inaccuracy and imprecision, is suitable

for many applications where a knowledge of moisture content

is required. In this method, calibration and sample solutions

are prepared as in the near-infrared method. Me^SO is the

solvent of choice. The determination depends on the measurement

of the energy absorbed by the water molecules when the sample

is exposed to infrared radiation at 1640 cm . The net

amount of energy absorbed is proportional to the quantity of

water present, with no interference from the hydroxyl groups

of the saccharide.

The transmittance at 1786 cm" is taken as the base level

in determining the net absorbance. In this method, unless

an infrared spectrophotometer is used that can operate

in the absorbance mode, the values recorded are in percent

transmittance; these values must be converted into absorbance

units prior to preparing the working calibration plot. As a

consequence, the method suffers in terms both of accuracy and

precision. The calculations are handled in the same way as

for the near-infrared method.

The change in absorbance with increasing concentration

of water is linear over the range from to 1.4 weight-percent

of water in the sample solution. The standard deviation of

the method, based on 21 degrees of freedom, is 0.135 weightpercent of water in the original syrup (about 20% of water).

The detection limit [83] (at the 95% level) is 0.016 weightpercent of water in the sample solution. The total time

required for an analysis is about 90 minutes.

114
3 . Gas—solid Chromatography

A third direct method for determination of moisture,

comparable in terms of accuracy and precision to the nearinfrared and vacuum-oven methods, utilizes gas—solid

chromatography. In this procedure, the components (of a

solution) having different partition coefficients are

separated as they pass through a column. In the present work,

the solid support was Porapak Q (Waters Associates),

80—100 mesh, in a copper column (0.25 in O.D. x 5 ft). The

carrier gas was helium. The column was operated at 220 °C

(isothermal) , and the separated components were detected with

a thermal conductivity detector operated at a temperature of

220 °C. The sample size was 1 ul.

Figure 14. Gas-chromatography equipment for the determination

of water in starch hydrolyzates

115
Calibration standards and sample solutions are prepared

on a weight basis, with Me

?

SO as the solvent. The solvent

serves as an internal standard in this method. The ratio of

the water peak-height to the solvent peak-height (measured

with a millimeter rule) is plotted against the concentration

of water in the solution. The water present in the original

syrup is calculated by using the equation discussed for the

near-infrared method (see p. 112).

To improve the accuracy and precision of the results,

three aliquots of a solution are injected at 1.5-minute

intervals, and the resulting peak-height ratios are averaged.

This averaged ratio is used in conjunction with the calibration

curve. Fig. 15 shows a chromatogram of three injections of a

syrup solution.

Over the concentration range of 0.6 to 1.8 weight-percent

of water, the peak-height ratio changes linearly with increasing

concentration of water in a solution. The standard deviation

of the method, based on 12 degrees of freedom, is 0.052

weight-percent of water in the original syrup (about 20% of

water) . The time required for an analysis is comparable to

that required for the near-infrared method.

4 . The Karl Fischer Titration

For the determination of water, another direct method

that has been widely used is the Karl Fischer titrimetric

method [84]. In this method, a known quantity of sample is

placed in the reaction vessel containing Karl Fischer (KF)

reagent. The water in the sample reacts with the KF reagent

and thus changes the conductivity of the solution. Karl

Fiscner reagent is then added to the system until the

endpoint, as determined by the conductivity shown by a milliammeter, has been re-established.

116
H2

- Me2 SO

220* - 5' x 1/4'

PORAPAK

Figure 15. Chromatogram of three injections of a syrup

solution.

This method is not directly adaptable for use with

undiluted syrups because of their viscosity and their low

solubility in pyridine. Furthermore, when a syrup is diluted

with a suitable solvent to lower the viscosity, other problems

arise The most serious problems are that, with syrups, the

moisture content determined by the Karl Fischer method is

usually found to be lower than that determined by other

methods, often by as much as 0.3 weight-percent, and the

precision of the method is very poor. These problems do not

arise with granular materials that do not require dilution.

117
We have recently observed that, when a commercial diluent

(Fisher Scientific SO-K-5) is added to the reaction vessel

prior to the analysis, both the accuracy and the precision of

the results are acceptable. Thus, a syrup having a Dextrose

Equivalence value of 29 was diluted with water, resulting

in three concentrations. These solutions were analyzed, in

duplicate, for moisture content by the Karl Fischer method.

In one case, the commercial diluent was added to the reaction

vessel; in the second, N ,N-dimethy If ormamide—methanol

(DMF—Me OH) was added, and the moisture content was also

determined by the near-infrared method, in duplicate. Estimates

of precision, based on these results, are shown in table 25-

In this case, the accuracy was comparable for all methods

.

Additional experiments are planned, to find the reasons for

the imprecision when other solvents are substituted for the

commercial diluent.

Table 25. Composition and estimates of precision of two

Karl Fischer procedures, in comparison with the

near-infrared method, for water

SO-K-5 DMF Near- infrared

Average,

dry solids

value (wt-%) 80.61 80.59 80.664

Range of

values

(wt-$) 0.44 1.06 0.374

Standard

deviation 0.16 0.37 0.143

Degrees of

freedom 6 6 6

118
B. Physical Properties of Hydrolyzates

1 . Characterization of Hydrolyzates

The Dextrose Equivalent (DE) is the criterion employed

by the corn wet-milling industry to indicate the extent of

hydrolysis of starch in the manufacture of corn syrups and

sugars. It is defined as the percentage of reducing sugars,

calculated as dextrose and expressed on a dry-substance basis.

Reducing sugars are determined by the method of Lane and

Eynon, which uses an alkaline copper tartrate reagent.

Although this approach is suitable for starch hydrolyzates,

it is unsatisfactory when heat-labile sugars (such as

D-fructose) or nonreducing sugars (such as sucrose) are present.

In these cases, an alternative means of characterizing the

hydrolyzate is desirable.

One method that appears suitable is the determination of

the number- average molecular weight (M ). This determination

can be made rapidly and economically by using a commercially

available osmometer to measure the freezing-point depression

of a dilute aqueous solution of the syrup. The M is calculated

by using the equation

M = Ka0 3Wo /AT JaL

n —f —2 —t—

where K f is the molar depression constant (1.86 for water),

W„ is the weight of solute, W is the weight of solvent, and

AT„ is the freezing-point depression (°C). Solutions containing

up to 5 percent of solids can be used. Ash contents up to

0.6 percent, calculated on an original dry-solids basis, do

not interfere at these dilutions.

The M. values obtained by measurements of freezing-point

depression agreed quite well with those calculated from

saccharide distribution data and from an empirical formula that

includes the DE value and the percent of dextrose in the syrup.

119
When this approach is used, D-fructose behaves as if it were

dextrose, and sucrose behaves as maltose. The time required

to obtain a measure of hydrolysis by this method is less than

that required to obtain a DE value. Furthermore, manipulative

technique is not an important source of error, as it can be in

the titration technique.

2 . Other Physical Properties

Other physical properties of hydrolyzates and their

solutions that are currently being investigated are: refractive

indices, optical rotations, densities, and volume expansion

properties. These are being studied as functions of moisture

content, ash content, saccharide distribution, and temperature.

120
5. PERSONNEL AND ACTIVITIES

A. Personnel Listing

R. Schaffer, Section Chief

R. S. Tipson

B. Coxon

R. F. Brady, Jr.

A. J. Fatiadi

A. Cohen

B. F. West

D. W. Vomhof, Research Associate

J. H. Thomas, Research Assistant

H. J. Elseroad, Summer Employee (1968)

D. Niner, Guest Worker (1969)

B. Publications

NBS Technical Note 457- Organic Chemistry Section.

7/1/67 - 6/30/68.

Edited by R. Schaffer (September, 1968)

Periodic Acid, a Novel Oxidant of Polycyclic, Aromatic

Hydrocarbons

A. J. Fatiadi, J. Res. NBS 72A, 341 (1968).

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, Carbohyd. Res. 7, 232 (1968).

Review of Khimia Uhlewodov, by N. K. Kochetkov,

A. F. Bochkov, B. A. Dmietriev, A. J. Usov, 0. S. Chizhov,

and W. N. Shibaev.

A. J. Fatiadi, Carbohyd. Res. 8, 123 (1968).

P.M.R. Evidence for the Conformations of 1,2-0-Benzylidenea-D_-glucofuranose Derivatives.

B. Coxon, Carbohyd. Res. 8, 125 (1968).

Bromine Oxidation of Inositols for Preparation of Inosose

Phenylhydrazones and Phenylosazones

A. J. Fatiadi, Carbohyd. Res. 8, 135 (1968).

121
Reactions, Conformations, and Acidity of Bis(ethylsulphonyl)-3-p_-ribopyranosylmethane and Related

Derivatives

.

B. Coxon and L. Hough, Carbohyd. Res. 8, 379 (1968).

Acylation of Tetrahydroxy-p_-benzoquinone . A. J. Fatiadi, J. Chem. Eng. Data 13, 591 (1968).

Isocytidine

.

B. F. West, Syn. Proc. Nucleic Acid Chem. 1, 313 (1968)

2' , 3 'O-Isopropylideneuridine

.

R. S. Tipson, Syn. Proc. Nucleic Acid Chem. 1 9 431

(1968).

5 ' -O-Trityluridine

.

R. S. Tipson, Syn. Proc. Nucleic Acid Chem. 1, 441

(1968).

Nature of a Colored By-product Found in Crude Inosose

Phenylosazones

.

A. J. Fatiadi, Carbohyd. Res. 9_, 177 (1969).

Esters of Benzenepentol ( Pentahydroxybenzene)

.

A. J. Fatiadi, J. Chem. Eng. Data 14, 118 (1969).

Tritium-labeled Compounds XII. Note on the Synthesis

of g-Glucose- 2-t and g-Mannose- 2-t . H. S. Isbell, H. L. Frush, C. W. R. Wade, and

A. J. Fatiadi, J. Res. NBS 73A, 75 (1969).

Acid-catalyzed Hydrolysis of Isopropylidene Acetals of

Some 2-Pentuloses and 2-Hexuloses.

R. S. Tipson, B. F. West, and R. F. Brady, Jr.,

Carbohyd. Res. 10, l8l (1969).

Evidence for a Half-chair Conformation of Certain

Osotriazoles of Inositols.

A. J. Fatiadi, Chem. Ind. (London) 1969, 617.

Manuscripts in Process of Publication

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).

122
Synthesis of the Two rj-2-Pentuloses . New Derivatives of

D- erythro -Pentulose

R. S. Tipson and R. F. Brady, Jr. (Manuscript accepted

for publication in Carbohydrate Research)

Phenylhydrazono—phenylazo Tautomerism. III. Reactions

of Phenylformazans and Certain Bis (phenylhydrazones

)

with Strong Acids.

H. S. Isbell and A. J. Fatiadi. (Manuscript accepted

for publication in Carbohydrate Research)

A Comparison of the Spectrophotometric and Gaschromato graphic Methods with the Vacuum-oven Procedure

for the Determination of Moisture in Starch Hydrolyzates

D. W. Vomhof and J. H. Thomas. (Manuscript accepted

for publication in Ann. Proc . Am. Soc. Brewing Chemists)

15 Studies of N-Labeled Amino Sugars. Synthesis and

Spectroscopy of Derivatives of 6-Amino-6-deoxy-D-glucose6-!5n.

B. Coxon. (Manuscript accepted for publication in

Carbohydrate Research)

The Synthesis, Equilibration, and Conformations of

Diastereoisomeric l,2-0-Isopropylidene-3 ,5-0-

( met hoxymethyli dene ) -6-0-p-tolylsulfonyl-a-D_-

glucofuranoses . Conformational Evidence from a Nuclear

Overhauser Effect.

B. Coxon. (Manuscript submitted for publication in

Carbohydrate Research)

Novel Aromatization of a Trihydroxycyclohexanetrione

(Triketoinositol) to a Dibenzo-p_-dioxin Derivative.

A. J. Fatiadi. (Manuscript submitted for publication in

Carbohydrate Research)

Process for Producing Inositol Hexasulfate (a Patent).

A. J. Fatiadi. (Submitted to NBS patent office on

March 3, 1969)

Conformational Analysis of Heterocyclic Compounds. The

Favored Conformations of p-Dioxane-2, 3-diol and its

Diacetate

R. F. Brady, Jr. (Manuscript submitted for

publication in Journal of Organic Chemistry)

Determination of Moisture in Starch Hydrolyzates by

Near-infrared and Infrared Spectrophotometry.

D. W. Vomhof and J. H. Thomas. (Manuscript submitted

for publication in Analytical Chemistry)

123
Determination of Moisture in Syrups by Gas—Solid

Chromatography

.

D. W. Vomhof and J. H. Thomas. (Manuscript completed

for publication in Analytical Chemistry)

Conversion of Certain Cyclic, Phenylhydrazono Derivatives

into Phenylazo Compounds with Periodic Acid.

A. J. Fatiadi. (Manuscript completed for publication in

Journal of Organic Chemistry)

D. NBS Reports

NBS 9972 - Report on Determination of Water in Starch

Hydrolyzates by Near-infrared Spectrophotometry.

D. W. Vomhof, J. H. Thomas. 1/15/69

NBS 9974 - Report on Determination of Water

Hydrolyzates by Infrared Spectrophotometry.

D. W. Vomhof, J. H. Thomas. I/I6/69

in Starch

NBS 9999 - Report on Direct Determination of Water in

Starch Hydrolyzates by Use of Gas-Solid Chromatography

D. W. Vomhof, J. H. Thomas. 2/26/69.

E. Lectures

9/7/68 - Methods for the Determination

Syrups and Related Materials

.

Pennsylvania State University

of Moisture in

Starch Round Table,

D. W. Vomhof.

9/10/68 - A Comparison of Methods for the Direct Determination

of Water in Starch Hydrolyzates. Carbohydrate

Division, 156th American Chemical Society Meeting,

Atlantic City, New Jersey. D. W. Vomhof , J . H . Thomas

.

9/10/68 - Studies of the Conformations of 1 , 2-0-Benzylidenea-g-glucofuranose Derivatives by P.m.r.

Spectroscopy. Carbohydrate Division, 156th

American Chemical Society Meeting, Atlantic City,

New Jersey. B. Coxon . 9/16/68 to 11/4/68 - Lectures on "Instrumental Methods of

Qualitative Organic Analysis," Graduate School,

NBS. R. F. Brady, Jr. Titles: (1) Introduction to

Mass Spectrometry; (2) Interpretation of Mass

Spectra; (3) Introduction to Nuclear Magnetic

Resonance Spectrometry; (4) First-order Spinpatterns in N.M.R.; (5) Complex Spin-spin Splitting

in N.M.R.; (6) Structural Determination by N.M.R.;

124
(7) Infrared Spectrophotometry in Organic Analysis;

(8) Elucidation of Molecular Structure by Complementary Spectrometric Techniques.

10/11/68 - Determination of Carbohydrate Conformations by

N.m.r. Spectroscopy. U. S. Army Natick

Laboratories , Natick, Massachusetts. B. Coxon . 12/4/68 - 0. R. Rodig, R. P. Brady, Jr., and R. J. Sysko,

"The Biosynthesis of Citrinin," Twentieth Southeastern Regional Meeting, American Chemical Society,

Tallahassee, Pla.

2/14/69 - Synthesis of the Epimeric D-2-Pentuloses . New

Derivatives of g- erythro-Pentulose . Fourth

Middle Atlantic~Regional Meeting, American

Chemical Society, Washington, D. C., R. S. Tipson,

R. F. Brady, Jr . 4/14/69 - Direct Determination of Water in Starch Hydrolyzates

By Use of Gas-Solid Chromatography. Carbohydrate

Division, 157th American Chemical Society Meeting,

Minneapolis, Minnesota. D. W. Vomhof , J. H. Thomas.

4/14/69 - The Influence of Saccharide Composition on the

Refractive Index of Syrups. Carbohydrate Division,

157th American Chemical Society Meeting,

Minneapolis, Minnesota. D. W. Vomhof . 4/14/69 - Synthesis and Equilibration of Diastereoisomeric

1 , 2-0-Isopropy lidene-3 , 5-0- (methoxymethy lidene )

6-0-p_-tolylsulfonyl-a-g-glucofuranoses . 157th

American Chemical Society Meeting, Minneapolis,

Minnesota. B. Coxon . 5/2/69 - Changes in Nitrogen Fractions Associated With Fruit

Abscission in Cotton. Washington Area Section,

American Society of Plant Physiologists,

Belts ville, Maryland. D. W. Vomhof . 5/6/69 - A Comparison of Spectrophotometric and GasChromatographic Methods with the Vacuum-Oven

Procedure for the Determination of Moisture in

Starch Hydrolyzates. American Society of Brewing

Chemists, Baltimore, Maryland. D. W. Vomhof , J . H . Thomas

125
6 . ACKNOWLEDGMENTS

The editor gratefully acknowledges the cooperation of

his associates in the Organic Chemistry Section in the

preparation of this report. Special thanks are due

Dr. R. S. Tipson for facilitating the editorial effort.

Mrs. Barbara Turner, Section Secretary, brought the

manuscript to final form. We wish also to thank

Mrs. R. S. Maddock, Administrative Assistant for the

Division of Analytical Chemistry in Charge of Publications,

for assisting in its publication.

126
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131

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