iihrary, E-01 Admin. BIdg.

JUN 2 1 t968

NBS MONOGRAPH 110

Infrared Spectroscopy

Of Carbohydrates

A Review of the Literature

U.S. DEPARTMENT OF COMMERCE

NATIONAL BUREAU OF STANDARDS
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UNITED STATES DEPARTMENT OF COMMERCE

C. R. Smith, Secretary NATIONAL BUREAU OF STANDARDS • A. V. Astin, Director

Infrared Spectroscopy

of Carbohydrates

A Review of the Literature

R. Stuart Tipson

Institute for Materials Research

National Bureau of Standards

Washington, D.C. 20234

National Bureau of Standards Monograph 110

Issued June 1968

For sale by the Superintendent of Documents, U.S. Government Printing Office Washington, D.C. 20402 - Price 30 cents
1

Library of Congress Catalog Card Number: 68-60029
Contents

Page

1. Introduction 1

2. Principles and instrumentation 1 3. Sampling techniques 2

3.1. Phase 2

3.2. Comparison of samples 2

4. Interpretation of spectra 3

4.1. General 3

4.2. Functional groups in carbohydrates and their derivatives 5

4.2.1. C—H bands 8

4.2.2. N—H bands 9

4.2.3. 0—H bands 10

4.2.4. C=C and C=C bands 10

4.2.5. C=N, C=N, and C—N bands 11

4.2.6. C=sO bands-.-- 11

(a) Aldehydes and ketones 11

(b) Un-ionized carboxylic acids 11

(c) Lactones 11

(d) Acetates and other esters 11

(e) Primary amides 12

(f) iV-Acetyl and -S-acetyl 12

4.2.7. C—O bands 12

(a) Esters 12

(b) Carboxylate ion 12

4.2.8. N=N, N=N, and NO2 bands 12

4.2.9. S==0, —SO2—, and C=S bands, 12

4.2.10. Miscellaneous bands 13

4.3. Correlations for the fingerprint region and beyond 13

4.3.1. The fingerprint region 13

4.3.2. Correlations for 10 to 15 jum (1000 to 667 cm-i) 13

(a) Correlations for certain aldopyranose derivatives 13

(b) Correlations for furanoid and pyranoid forms of aldose and

ketose derivatives 14

4.4. Conformational studies 15

.3. Examples of use of infrared spectra 17

5.1. Qualitative 17

5.2. Quantitative 17

5.3. Determination of structure 17

6. Special techniques 19

7. References 20

III

Infrared Spectroscopy of Carbohydrates

A Review of the Literature

R. Stuart Tipson ^

A survey has been made of the literature on the infrared spectroscopy of carbohydrates,

in order to assemble and systematize information in this field. The Monograph discusses

principles and instrumentation, sampling techniques, comparison of samples, and the inter- pretation of the spectra, particularly as regards functional groups of carbohydrates and their derivatives, correlations for the fingerprint region and beyond, and conformational studies. In addition, examples are discussed of the use of infrared spectra for qualitative and quantitative purposes and in the determination of structure. Special techniques are briefly described, including use of plane-polarized radiation, the technique of attenuated total reflection, and Raman spectra. Key Words: Analysis, carbohydrates, conformations, infrared spectra, spectrometry,

structure.

1. Introduction

Although a large number of books on the in- frared spectroscopy of organic compounds have

been written [1],^ detailed attention has not been

accorded in them to carbohydrates and their

derivatives. Several books contain chapters on the

subject, but these chapters are no longer up to

date, and contain certain statements that are open

2. Principles and

A molecule can exist in a number of energy

levels, and a change from one level to another can result from absorption, or in emission, of

energy, if selection rules permit this. Of the three

kinds of molecular spectra, namely, electronic,

rotational, and vibrational, the last kind are obtained when absorption of radiant energy causes changes in the energy of molecular \abration.

Most of the vibrational absorption bands [1] are found in the range ^ from 2 to 100 (5000 to 100 cm"'); but, as the spectrometers usually

available to the organic chemist cover the range

' The author thanks Frank S. Parker, of the Department of Biochemisti-y, New York Medical College, New York, and James E. Stewart, of Beckman

Instruments, Inc., FuUerton, California, for a number of helpful suggestions. This Monograph is, in part, an outgrowth of a comprehensive investigation

of the infrared absorption spectra of sugars and sugar derivatives begun at the National Bureau of Standards in 1949. The early work, sponsored by the

OtTice of Naval Research, was conducted by E. C. Creitz, II. L. Frush, H. S.

Isbcll. J. D. Moyer. F. A. Smith. J. E. Stewart, and R. S. Tipson.

- Figures in brackets indicate the literature references at the end of this Monograph.

' The position of a band in this region of the spectrum is expressed in two

ways: (a) as the wavelength (X) in ;jm [micrometers: 1 iim = 10-^m, commonly

called microns (p.)], or (b) as the wavenumber (•-) in reciprocal centimeters

(kaysers). The velocity of light (c, in cms-') is equal to the wavelength of one vibration times the number of vibrations per second; that is, c=Xi'. The

wavenumber is the reciprocal of the wavelength; thus, >- (in cm-i) = l/X (in cm), or V (in cm-') = 10</X (in pm). Wavenumbers have the advantage of being directly proportional to energy. As instruments are now available

that record linearly either in wavelength or wavenumber, bands will be

expressed in both ways in this Monograph.

to misinterpretation. Consequently, it was decided

that a detailed survey should be made of the

hterature on the infrared spectroscopy of the

carbohydrates and their derivatives, and that the

information should be assembled in a readily

available form.

Instrumentation

of 2 to 40 Atm (5000 to 250 cm"'), discussion of infrared spectra vnh be confined to this range

The range of most interest to the carbohydrate

chemist is 2 to 15 (5000 to 667 cm"').

Accurate recordings of spectra may be obtained

with a recording, double-beam, infrared spectrophotometer equipped ^^'ith. a prism [e.g., of sodium

chloride for the range of 2 to 15 ixm (5000 to 667

cm"')] or a grating. In such instruments, the

sample is exposed to infrared radiation, and that

radiation passing through the sample is resolved

^\'ith the prism or grating; the spectrum is then

scanned, and the values (bands) at which radiation

is absorbed are recorded, pro\ading an infrared

absorjition spectrt)gram. For measurement of bands in the region of 2.7 to 3.6 /xm (3700 to 2780

cm~'), the higher dispersion given by a lithium

fluoride [msm or a comi)arable grating is an asset. Instead of a suitable grating, a prism of potassium

bromide may be used for the range of 2 to 25 /im. (5000 to 400 cm"') , and a prism of cesium bromide

for 15 to 40 Mm (667 to 250 cm"').

For rapid, routine recording of useful but less accurate spectra, smaller (and cheaper) prism

instruments are available as an analytical tool. In the range of 10 to 15 ^im (1000 to 667 cm"').

1
such an instrument can routinely give wavenumbers accurate to ±2 cm~^ In the range of 2.5 to 10 nm (4000 to 1000 cm~^), the inaccuracy is increasingly greater with increasing wavenumber

nevertheless, the reproducibility is satisfactory.

3. Sampling Techniques

3.1. Phase

For recording the infrared spectrum, a carbohydrate or derivative may be prepared as (a) a

thin layer of an anhydrous syrup mounted

between windows that are transparent to the

infrared radiation; (b) a mull with a hydrocarbon

011 (Nujol or an appropriate perhalogenated

hydrocarbon, mounted in the same way; (c) a

pellet made with an alkali-metal halide; or (d) a

solution in an organic solvent or water (in a

suitable, water-insoluble cell). Recording is usually done with the sample at room temperature,

but bands may be sharper at lower temperatures.

The spectrum of a compound may be different

for different physical states. For example, in the

solid state, polymorphs of a compound, examined

in the same way, may show differences in a few

features of their spectra. An example is 7V-benzoyl2,3,4,6-tetra-0-benzoyl-/3-D-glucosylamine ; this exists in a form of mp 113-115 °C which,

when heated to 117-120 °C and allowed to crystallize from the melt, gives a form of mp 184 °C

having a somewhat different spectrum (Nujol

mull). Also, different crystal habits (same mp) of a compound (residting, for example, from crystallization of samples from different solvents) may

give somewhat different spectra, particularly if examined as mulls (where but little pressure is applied) . Shifts of up to 20 cm~^ for certain bands

have been noted [3] for crystalline and amorphous

forms of some carbohydrates. However, in all such instances, samples of each of the forms,

measured after dissolution in the same solvent, or as molten material, give identical spectra.

The spectra of crystalline materials often show more bands than the same compounds in solution

Band positions may be shifted, or band intensities altered, if the compound forms hydrogen

bonds with the solvent. In solution, molecules of a compound may associate through formation of intermolecular hydrogen bonds. If the concentration is below 0.005 M, intermolecular hydrogenbonding is negligible for solutions in carbon

tetrachloride and similar solvents [5, 6]; this effect has been used in studying intramolecular

hydrogen-bonding in model compounds related

to sugars [7]. Pelleting of certain free sugars with an alkali halide changes a crystalline sample to an amor- phous form [8, 9]. Moreover, some sugars (for

example, a-D-glucose) may form a complex with

Mention of a commercial product in tliis article does not constitute endorse- ment by the National Bureau of Standards.

traces of sodium bromide or sodium iodide present

in pellets of the corresponding potassium halide

[9]. If the pelleting halide is not dry [10], or acquires moisture, the sugar may mutarotate or form a hydrate should the pellet be stored [11]. Consequently, such spectra should always be

compared with those obtained for a Nujol mull of the compound, or the pellets should not be stored.

However, even though examined immediately

after preparation of the pellets, eight of 24

aldopyranosides [12] showed interaction with the

pelleting halide, and six of 27 free sugars [13] gave spectrograms that were different in Nujol

and in potassium iodide.

3.2. Comparison of Samples

As a result of recording and comparing the

infrared spectra of a large number of organic

compounds, it has been found that every com- pound having a unique structure gives a unique

spectrum. Consequently, for an enantiomorphic

pair, every detail of whose structure is identical

(but in mirror image), the spectra of the solids,

in the same polymorphic modification, are identical [3]. The spectra may differ slightly because of polymorphism, but, for the gaseous or liquid (melt, or solution) phases, the spectra are identical.

Also, for a large molecule, a slight change in structure may change the spectrum only slightly.

Thus, proceeding up a series of oligosaccharides

containing the same anomeric form of the same

monosaccharide residue only, the spectra for the

tetraose, pentaose, hexaose, etc., become more

and more alike [14]. Consequently, with these exceptions, pure

compounds can be identified unequivocally by

their infrared spectra. For a carbohydrate, the

infrared spectrum is usually a much more specific

and characteristic property than the ultraviolet

spectrum, melting point, boiling point, density, or

refractive index. Thus, although the anomers of a sugar or glycoside differ only in the orientation at one carbon atom, their spectra are quite different

[14]. Also, the spectrum of the furanoid form of a sugar derivative differs from that of the pyranoid

form in the same anomeric modification, as with

methyl /3-D-ribofuranoside and /3-D-ribopyranoside

[15].Hence, the simplest use of infrared spectra is for the identification of a compound. The spectrum

of a sample to be identified is compared with the

spectra of pure compounds of known structure,

measured with the same sampling technique. In

this use, it is not necessary that the spectra be

analyzed for characteristic absorption bands.

However, availability of reference spectra [16-20]

of related, ])ure compounds is essential. Another use is the precise identification of spectra already in the literature, where the com- pound studied had been inadequately described.

For example, in 1950, Kuhn [14] recorded the

spectra of 10 of the- crystalline sugars, each in a

2
Nujol mvill; but, except for one (a-D-glucose) , he

did not mention which anomer had been employed.

Similarly, Urbanski et al. [21] recorded the spectra

of six sugars, without specifying the anomeric

form. These have since been identified [13] by

comparison with, the spectra of authentic anomeric

forms of those sugars. In another application, infrared spectra can be

used for checking the purity of a sample. Commonly, a crude material may shoAV a band that is not present in the spectrum of the pure compound ; thus, commercial D-mannitol showed a band at 12.80 nm (781 cm~^), due to D-glucitol, removable

by recrystallization from aqueous ethanol [22]. In contrast, figure 1 shows the spectrum of pure

crystalline ;8-D-ribose [13] and that of a commercial

specimen. The spectrum of the commercial material does not display a band at 7.82 /um (1279

cm~^) that is present in the spectrum of the pure

sugar, presumably because presence of impurity

causes poor growth of the crystals, resulting in

scattering.

The same figure also serves to Ulustrate that

the spectra may be used in following the isolation or purification of a desired product obtained, for

example, by distillation or chromatography. It is not necessary to know what the compound is (nor, initially, to have an infrared spectrum of the

pure compound) ; the concentration or purification

procedure can be followed by observing some

characteristic infrared band. For example, by

recrystallizing /S-D-ribose untU the band at 7.82

^^m (1279 cm~^) appears and remains of constant

intensity, a sample that is "pure" (by infrared

spectroscopy) is obtained, without any knowledge

of the actual significance of this band. However,

the minimum detectable amount of impurity

(which may, of course, contribute bands absent

from the spectrum of the pure material) varies

enormously from one compound to another.

4. Interpretation of Spectra

4.1. General

A more sophisticated use of infrared spectra

permits qualitative analysis for groups of atoms.

The motions constantly undergone by the atoms

and bonds of a molecule may be examined with a model consisting of springs and balls. A diatomic

molecule A—B may be represented by two balls,

each of a mass proportional to those of atoms A

and B, respectively, connected by a spring of

strength proportional to the force binding atoms

A and B. Molecule A—B can undergo a stretching

vibration only along the bond between atoms A

and B; this vibration occurs in the model if the

.spring is compressed and then the assemblage is permitted to relax on its own. If the resulting,

periodic oscillation is moderate, the system follows

Hooke's law approximately, and the frequency

(v) of the stretching vibration is given by the

equation for simple harmonic vibrations namely

v=(f/fiy^^/2irc, where / is the force constant of the

bond, c is the velocity of light, and m is the reduced

mass, defined as iJ.=mfjn-Rl {mf^-\-mQ) , where

and rriB are the masses of atoms A and B, resjjectively. (The equation for v applies only to the

example considered.)

For a polyatomic molecule, there are many

ways in which pairs of atoms may bend, rock,

twist, or vibrate, relative to each other and to other pairs, or larger groups, of atoms. Although a mathematical treatment of the infrared spectrum

of any pure compound could provide information on the structure through checking of consistency

with possible models, the degree of difficulty of such a calculation is a function of the number of atoms and of the symmetry of their geometrical

arrangement. Thus, for most sugars, such a treatment would be very difficult, particularly in

3
5000 4000 3000

iiiiliiiil II 1 1 1 1,1

WAVE NUMBER, Cm-l

1400 1200 1000 950 J I

, IlIIIIIIIiIiiI I III I I I I I I I I I I I I

I II I I I I I I I . I I I

9 10 WAVELENGTH, /xm

Figure 2. Infrared spectra of 1,2,8, 4-tetra-0-acetyl-l3-D-xylopyranose (- - -) and of ^-acetyl-2, 3, 4-tri-O-acetyl-^-vi- xylopyranosylamine ( ) in potassium chloride pellets. (From Refs. [19] and [20].)

view of additional complications introduced by

study of condensed phases.

Consequently, in attempting to correlate the

structure of the molecule with the frequencies

observed in its infrared spectrum, use has been

made of the empirical approach. The method is based on the assumption (not necessarily justified)

that the vibrations of a certain group are fairly

independent of the rest of the molecule. Then,

the spectra (recorded under like conditions) of a

large number of compounds having this group are examined, to find out which bands the spectra

have in common. For example, the spectra of 24

acetylated glycosides were studied [18], together

with those for 21" nonacetylated glycosides [12]. It was then obvious that (1) all of the acetates showed a band at 5.64 to 5.76 Mm (1773 to 1736

cm~^; C=0 stretching frequency) not shown by

any of the nonacetylated compounds; and (2) all of the nonacetylated glycosides, but not the acetates, showed a band at 2.93 to 3.05 tJ.m (3413 to 3279 cm~^; •—O—H stretching). Furthermore, all of the hydrates (but no anhydrous compounds)

showed a band at 6.01 to 6.12 ^ni (1664 to 1634

cm~^). Here, attention was paid only to the positions of absorption bands, regardless of their

relative intensities. In figure 2 are shown the spectra of 1,2,3,4-

tetra-(?-acetyl-j3-D-xylopyranose (l) [20] and

of /v-acetyl-2,3,4-tri-C-acetyl-/3-D-xylopyranosylamine (2) [19], compounds that differ by only one connecting atom. In compound 1, C-1 bears an oxygen atom to which is attached an acetyl

group, whereas C-1 of compound 2 bears a nitrogen atom to which is attached a hydrogen

atom and an acetyl group. Some differences in

the spectra are indicated in figure 2, and are discussed in section 4. It has been found that, in the absence of dis- turbing effects, all compounds containing the

same group will absorb infrared radiation at al- most the same wavelength. Thus, bands that occur in the region from 2.0 to ca. 7 fim (5000 to

ca. 1430 cm~^) are usually characteristic groupfrequencies; for these, the associated vibration is weU localized, as in C=0 and X—H stretching

vibrations. The precise positions of such bands

are more reliable for determining the presence of groups than are bands in the region of 7 to 15

Mm (1430 to 667 cm"^^). The latter interval, or the interval from 7 to 11 Mm (1430 to 910 cm-^),

is commonly referred to as the "fingerprint re- gion," because it is usually rich in bands that

collectively provide a pattern of bands characteristic of the compound; however, the origin of the bands in the fingerprint region is often not

readily determined, and so a detailed study of

this part of the spectrum is generally deferred until

the rest of the spectrum has been examined.

Bands in the region of 7 to 15 Mm (1430 to 667

cm~') may arise from single-bond, skeletal stretching-vibrations (between atoms of similar, or the

same, mass) or from bending vibrations. The

latter occur in this region because less energy is required to produce them; certain of these bands

have diagnostic value for groups, but most of them are greatly influenced by structural changes

in the molecule. For carbohydrates and their

derivatives, the region of 8 to 10 Mm (1250 to 1000 cm~^) contains bands for —C—O— of

esters, ethers, and hydroxyl groups, and may not

show highly individual bands for such structures.

4
Some group absorptions may be qmte different

in intensity from one compoimd to another, even though the wavelength of the absorption is about

the same. However, for small molecules, the bands

for carbonyl groups are nearly always very strong.

If a spectrum has only a weak band in the region

of 5.88 nm (1700 cm~^), (a) this band is probably

not due to carbonyl, (b) the molecule is probably

very large and has, perhaps, only one carbonyl

group, or (c) a carbonyl compound is possibly

present as an impurity. Because of the possibility

of interference by other absorptions, the presence

of a band at a position expected for a certain

group is not conclusive evidence that that group

is present in the compound. However, provided

that effects (such as hydrogen bonding) that

could shift or even remove the band are not

operative, the absence of a group absorption

usually indicates absence of that group from the

sample.

4.2. Functional Groups in Carbohydrates

and Their Derivatives

Except when electrical (hydrogen bonding,

ionization, etc.) or steric effects are operative,

every organic compound that possesses a particular group will show the corresponding, characteristic group-frequency in its spectrum, and many

compilations of such group-frequencies are available [1]. Table 1 lists the group frequencies in

which the sugar chemist is likely to be interested,

and provides an estimate of their relative intensities; about half of the characteristic groupfrequencies he in the range of 2 to 7 nm (5000 to 1430 cm~^) and the rest above 7 nm (below

1430 cm-i).

Table 1. Characteristic infrared bands shown by various groups

Range

Intensity » Group Type of vibration ^ Remarks

fim. cm~'

2.22-2.38 4505-4200 w C—

2.35-2.50 4255-4000 w C—

2.74-2.86 3650-3500 var 0—

2.75-2.76 3640-3623 m(sharp) 0—

2.78-3.23 3600-3100 m 0—

2.79-2.92 3o90-3425 var (sharp) O—

2.82-2.86 3550-3500 m V 7 XI

2.82-2.90 3550-3450 var(sharp) 0—

2.82-3.13 3550-3195 w C=0

~2.84 ~3520 s N—

~2.86 ~3500 m N—

2.86-3.03 3500-3300 m N—

2.86-3.27 3500-3060 m N—

~2.94 ~3400 s N—

~2.94 ~3400 m N—

2.94^3.10 3400-3225 s (broad) 0—

~2.96 ~3380 m NH3+

2.98-3.18 3355-3145 m NH3+

~2.99 ~3350 m N—

~3.03 ~3300 s C—

3.03-4.00 3300-2500 w(v broad) 0—

~3.05 ~3280 m NH3+

~3.15 ~3175 m N—

3.17-3.28 3155-3050 w C—

3.23-3.25 3095-3075 m C—

3.25-3.30 3075-3030 w-m C—

3.28-3.34 3050-2995 w C—

3.29-3.32 3040-3010 s;m C—

~3.38 ~2960 s C—

~3.42 ~2925 s C—

3.45-3.47 2900-2880 w C—

3.45-3.70 2900-2705 w C—

(two)

3.4.5-4.35 2900-2300 w N—

(several) ~3.48 ~2875 s C—

~3.51 ~2850 s C—

3.53-3.55 2835-2815 C—

See footnotes at end of table.

str. aliphatic (combination)

str. aromatic (combination)

str. free OH, oxime

str. free OH, alcohols

str. water of crystallization

str. intramolec. bonded OH

str. free OH, carboxylic acid (v. dil. soln.)

str. intermolec. bonded OH (dimeric)

str. c5.rbonyl (first overtone)

str. primary amide (free)

str. (asym.) primary amine, free NH (dil. soln.)

str. secondary amine, free NH

str. associated NH, amine or amide

str. primary amide (free)

str. (sym.) primary amine, free NH (dil. soln.)

str. intermolec. bonded OH (polymeric)

str. amine salt (soln.)

str. amine salt (solid); several bands

str. primary amide (bonded)

str. =C—H, acetylenes

str. H-bonded carboxylic acid dimers

str. amine salt (soln.)

str. primary amide (bonded)

str. —CH=C—O— and —C=CH—0—

str. RCH=CH2, olefin

str. C—H of aromatic ring

str. of epoxide (shifts to 3040-3030 if ring strain increases)

str. >C—H; RCH=CH2, RCH=CHR'

{cis or trans), RCR'=CHR",

olefin

str. (asym.) C-methyl

str. (asym.) >CH2, methylene, Ar—CH3

str. C—H, methine

str. —C(=0)H, aldehyde

str. quarternary amine salt, bonded

str. (sym.) C-methyl

str. (sym.) >CH2, methylene

str. 0-methyl
Table 1. Characteristic infrared bands shown by various groups—Continued

Range

Intensity Group Type of vibration ^ Remarks

cm~

~3.54 ~2825

~o.du ~Z/8U ^ ,07Cf\ Q rrn Q ft 1 07nFc oi^Aft Q TO—/! Qc; 0. 1 U—"l.OO ii t UO—ZciUU ~o.oo ^ZOOU

1 7 ^^Z4UU ^ ,/1 /I 1 ,oo7n

/I /1 0 A Aft ooftn_09yin zzou—^^4U

A AO A ^1 oofin 0 1 fto zzdu—ziyu

4.48—4.01 OOQA 001 zzoU—^iilO 4.00—4.80 //uu—ZUOU

4.00—O.UU oonn zzuu—onnn ^uuu

4.59-4.72 2180-2120

4.63-4.72 2160-2120

4.67-4.76 2140-2100

~5.53 ~1810

5.62-.5.75 1780-1740

~5.65 ~1770

5.73-5.76 1745-1735

~5.75 ~1740

5.75-5.81 1740-1720

~5.80 ~1725

5.80-5.87 1725-1705

-^5.81 ~1720

5.81-5.88 1720-1700

-1- 1 700-1 1 \J\) fi70 \ \J

5.92-5.99 iftqfl_ift7n

5 05-6 14 1680-1630

5.95 -6.17 5 96-5 99 1 678-1 fi68 ~5.97 ~1675

~5.97 ~1675

~5.99 ~ 1670

5 QQ-fi 17

6 02-6 05 1fifi2-1fi52 6.03-6.07 1658-1648

6!06-6!l7 1650-1620

6.06-6.25 1650-1600

6 06-6 33 Ifitjfti coo

6 06-6 45 1650-1 550

6.07-6.11 1648-1638

~6.15 ~1625

fi 1.5-fi 31 1i-U^U (^9'^-^i.O<JO "iS^i

6.17-6.29

6.17-6!41 1620-1560

6.21-6.49 1610-1540

~6.25 ~1600

~6.31 ~1585

6.33-6.58 1580-1520

6.37-6.60 1570-1515

6.45-6.62 1550-1510

~6.67 ~1500

6.67-6.80 1500-1470

6.67-7.69 1500-1300

-^e.si ~1468

~6.85 ~1460

See footnotes at end of table.

m C—

str.

Kj Jtl str. w( broad) r—U±l str.

s IN -tl2 I -tl str. w B Jtl str. w 10C! TTJtl str. vs IN

Kj—\j asym. str. w str. var

s —\j IN str. vs Kj ft asym. str.

s C=N str.

s N=N str. w C=C

s c=o str.

s c=o str.

s c=o str.

g c=o str.

s c=o str.

s c=o str.

s c=o str.

s c=o str.

s c=o str.

s c=o str.

g c=o str.

s c=o str.

g c=o str. var c=c str. c=c

g c=s str.

g c=o str. w C=N

g c=o str c=c

c=c

g N H def.

g N02 asym. str. m—

N jj def. w N H def. c=c

g c=c str.

] [ j c=c OJ^dCUCtlj drfilAtQl in— 111" plane

g N—

def. m-s NH2+ def. vs C—

asym. str. s C=C str. m NH3+ asym. def. m C=N (plus

C=C)

s N—

def.

s N— def. var C=C skeletal, in- plane

s c==s str. m NH3+ sym. def.

s C—scissoring

m C— bend (asym.)

0CH2—

/ —CH , alkyl acetal ^0CH2— —O—CH2—0—

phosphoric ester, H-bonded

(may be several bands)

thiol, free

thiol, H-bonded

isocyanate

satd. nitrile RC=CR'; acetylenes

unsatd. conj. nitrile —N—C=S, isothiocyanate (2 or more bands)

cyanide, thiocyanate, cyanate

R—N=C

azide

RC=C-H; acetylenes —COCl, aliphatic acid chloride —O—(C=0)—O—, carbonate

7-lactone

satd. esters 5-lactone —C(=0)H, aldehyde

formic ester ketone

benzoic ester —COOH; aliphatic carboxylic acid (dimer) —CONHR, secondary amide, free

(dil. soln.) : Amide I —CONH2, primary amide, free

(dil. soln.) : Amide I secondary amide (solid) nonconjugated C=C

trans olefin; RHC=CHR'

thioester

thioester

aliphatic oxime

primary amide (solid), H-bonded,

2 bands: Amide I cis olefin; RHC=CHR'

terminal olefin; RR'C=CH2

primary amide (solid) : Amide II, —0—NO2, nitrate NH2; primary amine

NHR; secondary amine

terminal olefin; RHC=CH2

Ph-conjugated C=C

aromatic C=C

primary amide (dil. soln.) —C00~, carboxylate

CO or C=C conjugated with C=C

amine salt pyrimidines

secondary amide (solid) : Amide II secondary amide (dil. soln.) aromatic C=C —N—C=S

amine salt alkane —CH2

—CH3

6
Table 1. Characteristic infrared bands shown by various groups—Continued

Range

Intensity » Group Type of Remarks

vibration

6.85-7.15 1460-1400 g C—

sym. str. —C00~, carboxylate

1 ,^ e c ~l455 s C—

scissoring alicyclic —CH2

6.90-7.15 1450-1400 w —N=N

str. azo

z* n e TIT 6.95—7.17 t A A n 1 OA e 1440—1395 w C—

str. (plus OH earboxylic acid

def.) 0.95-7.41 t A A n 10CA 1440—1350 s s=o str. (RO)2S02, sulfuric ester 6.95-7.55 1440-1325 m C—

aliphatic aldehyde

7.04-7.11 1 A OA 1 A Ai? 1420-1406 w C—

in-plane bend C=CH2

7.04-7.52 t A OA 1 O O1420-1330 s s=o str. ROSO2R', sulfonic ester 7.05—7.15 1 >l 1 O 1 /i AA 1418—1400 m C—

str. primary amide

~7.09 ~ 1410 w C—

str. aliphatic amine

T 1 r» 'TOE 7.19-7.d5 doublet,

1 OAA 1 0£?A 1390-1360

m C—

bend (sym.) ^em-dimethyl T OO OO l.^Z—T.zo 1385-1375 m C—

bend (sym.) —CH3

7.d0-o.00 137U-lz50 C—str. lactone ~7.46 /^1340 w C—

bend alkane C—

7.4d—7.»1 lo4U—l-ioU s S=0 sym. str. R2SO2, sulfone 7.46-8.48 1340-1180 w N=N str. azide 7.58-8.27 1320-1210 s C—

str. earboxylic acid

/ .04—o.UU 1 1? 1 fL 1 OKA s C—str. benzoic ester, phthalic ester 7.66-8.33 1 Or\ C 1 OAA 1305-1200 m N—

def. secondary amide. Amide III 7.69-8.00 1 OAA 1 O C A 1300-1250 s NO2 sym. str. —0—NO2, nitrate

^ £> /*» o o o

7. 69-8.33 1 OAA 1 OAA 1300-1200 s P=0 str. phosphoric ester, free P=0

/.OO—o. /U 1 OVA 1 1 CA 12/U— 1150 s C—

str. —(0=)C—0—R in earboxylic esters 7.96-8.12 lOE/? 1000 1256-1232 s C—

str. CH3COOR, acetic ester

O AA ~O.0U ~ 1 o e A 1250 C—

str. methylene acetal ~ 1 OKA 1Z5U C—

str. epoxide

Q f\f\ ~o.00 '->-' 1 OCA 1250 vs Si—CH3 sym. CH3 def. Si(CH3)3, trimethylsilyl 8.00-8.70 1250-1150 vs P=0 str. phosphoric ester, H-bonded P=0

O. iU—o.^O 1 9"? 1 91 9 s c=s str. (R0)2C=S, thioketone

o.io—O. / U

1 OOA 1 1 KA s s=o str. (RO)2S02, sulfuric ester

O 1 C O C 1 8.1D-0.51, 100C 11'7C 1225—1175, w C—

in-plane p-substituted phenyl

8.89-9.18, 1125-1090, bend

Q QC in AA lU/ 1>— lUUU

(two)

O OA A OA O.2U-9.80 1 OOA "f AOA 1220-1020 m C—

str. aliphatic amine

ROSO2R', sulfonic ester O OO O 'TO 8.33-8.73 1 OAA 1 t A C 1200-1145 s s=o str. 8.33—y. 62 1 OAA 1 r\A A i2Ul>— 1U4U C—

str. C—0—C—0—C, cyclic acetal (4-5 bands)

O OO o c c 8.33—8.55 1 OAA 1 1 'TA 1200-1170 s C—

str. propionic and higher esters

O O O 1 A AA 8.33-10.00 1 OAA 1 AAA 1200-1000 s C—OH str. alcohols

8.44r-8.51 1185-1175 s C—str. formic ester 8.51-8.59, 1175-1165 and s C—

skeletal (CH3)2C<C, isopropyl

o e c o TT 8.55-8.77 1170-1140

8.51-8.89, 1175-1125, w C—in-plane bend unsubstituted phenyl

9.01-9.35, 1 1 1 A 1 A'7A 1110-1070,

9.35-10.00 "1 ATA "1 AAA 1070-1000

8.70-9.09 1 1 C A 1 i AA lloO-llOU s s=o asym. str. R2SO2, sulfone 8.70-9.09 1 1 C A 11 AA 1150-llOU s C—

str. benzoic ester, phthalic ester 8.70-9.35 1 1 Cr A 1 ATA 1150-1070 s C—0—

asym. str. aliphatic ether ~8.93 ~1120 s c=s str. —NH—(C=S)—, thioamide

y.Ul-lU.OU monofluoro derivs. 1 1 1 A 1 AAA IIIO-IOUU s C—

str.

y. 18-9.71 1 AAA 1 AOA 1090-1031) vs P—0—

phosphoric ester 9.18-9.80 1 AAA 1 AOA 1090-1020 vs Si—

str. Si—0—C, trimethylsilyl

9.4.5-9.50 10.d8-10o3 s c=s str. (RS)2C=S, trithiocarbonate 9.52-9.80 1050-1020 s S=0 str. >S=0, sulfoxide ~9.62 ~1040 C—

str. methylene acetal 9.95-10.10, 1005-990 and vs C—

bend C=C—H, vinyl 10.93-10.99 915—910

10.05-10.15, AA C AOC „ . _3 995-985 and C—

out-of-plane RCH=CH2

10.99-11.05 A 1 A AACT 910-905 bend

.70'-' C—

out-of-plane

bend

trans RHC=CHR'

10.31-10.64 970-940 broad P—0—

pyrophosphate

10.36-10.42, 965-960 and s C—

bend vinyl ether 10.58-10.64 945-940

10.42-10.75 960-930 N—

str. oxime

See footnotes at end of table.

7
Table 1. Characteristic infrared bands shown by various groups—Continued

Range

Intensity » Group Type of Remarks

vibration *> cm~'

10.53-12.35 950-810 C—

str. epoxide

~10.55 ~948 s C—bend vinyl ester ~10.81 ~925 C—

str. methylene acetal 11.17-11.30 895-885 C—

out-of-plane

bend

RR'C=CIl2

~11.90 ~840 vs Si—CHg str. Si(CH3)3, trimethylsilyl 11.90-12.66 840-790 C—

out-of-plane

bend

RR'C=CHR"

11.90-12.66 840-790 m C—

skeletal (CH3)2C< , isopropyl 11.90-13.33 840-750 C—

str. epoxide

12.00-12.35 833-810 vs C—

out-of-plane p-substituted phenyl

bend

~12.50 ~800 w NH3+ rocking amine salt ~12.50 ~800 w NH2+ rocking

12.99-13.70, 770-730, s C—

out-of-plane unsubstituted phenyl

14.08-14.49 710-690 bend

~13.25 ~755 vs Si—CH3 str. Si(CH3)3, trimethylsilyl 13.33-14.08 750-700 s C—CI str. monochloro derivs. ~13.89 ~720 m(broad) N—

def. secondary amide, bonded: Amide V

14.18-17.54 705-570 w c—

str. thiol, sulfide ~14.49 ~690 C—

out-of-plane

bend

cis RHC=CHR'

~15.39 ~650 s C—Br str. bromo derivs. 16.67-20.83 600-480 s C—

str. iodo derivs. 18.19-22.22 550-450 vw s—

str. disulfide

"Key: m, medium; s, strong; v, very; var, variable; w, weak.

''asym., asymmetrical; def., deformation; str., stretching; sym., symmetrical.

Description of a group vibration as A—

stretching or A—B—C bending is, at best, a first approximation. All of the bonds in a group are usually changing to some extent during a vibration. One bond may dominate, and it is then

reasonable to describe the vibration as the motion

of that bond, but this is by no means always

justifiable. Thus, for the amide group vibrations,

the stretching motions of C=0, C—N, and N—

and the corresponding bending motions are often

highly mixed; consequently, the amide group

vibrations are described as Amide I, II, III, and

so on. In the following discussion of some of the more

important bands, all values for their positions are approximate unless a range is stated; even so, although frequencies generally fall within the

limits indicated, they may, in special cases, lie beyond these ranges. Consequently, when the

correlations are used, all other available evidence

should also receive consideration. Also, in this discussion, the reference given is usually, but not

necessarily, that for the first mention of the cor- relation for carbohydrates.

4.2.1. C—H Bands

All sugars and their derivatives possess the

methylidyne (methine) grouping —C—H, charI

acterized by a band near 3.45 nm (2900 cm~)^

assigned to C—H stretching. Hence, this band

has no diagnostic value for these compounds.

Acetylenic compounds give a strong band for C—H stretching of =C—H at about 3.07 tixn (3255 cm-i); this occurs at 3.03 mhi (3300 cm"')

for 5-hexyne-D-/t/a;o-l,2,3,4-tetrol tetraacetate [23] and at 3.11 Mm (3215 cm-i) for l,2:3,4-di-

(?-isopropylidene-6-heptyne-D-^ufo-l,2,3,4,5-pentol

[24]; the i^-manno isomer of the latter shows the

band at 3.06 tim. (3268 cm-^-

Other C—H stretching frequencies encountered

with sugar derivatives are : olefinic =CH2 doublet

at 3.25 MHi (3075 cm->) and 3.36 nm (2975 cm-')

Ar—H at 3.28 Mm (3050 cm"'); olefinic =C—

at 3.31 Mm (3020 cm-'); —CH2— doublet at 3.42

Mm (2925 cm-') and 3.51 Mm (2850 cm"'); and

C-methyl doublet at 3.38 Mm (2960 cm"') and

3.49 Mm (2865 cm"'). Thus, a group of 28 cychc

acetals of various sugars all showed [4] a band

at 3.32 to 3.37 Mm (3010 to 2965 cm"'). As regards

0-methyl, all of 21 methyl aldopyranosides

studied [12] showed a characteristic C—H stretching band at 3.47-3.52 Mm (2882-2841 cm"'),

not shown by C-methyl or ethoxyl groups [25]' that permits detection of the glycosidic methoxyl

group.

Some deformation frequencies for C—H are: —CH2— at 6.89 Mm (1450 cm"'), and C-methyl

8
at 7.27 Mm (1375 cm-i). The C-methyls of the

I isopropyl group show bands at 7.25 and 7.30

nm (1380 and 1370 cm~^) that are particularly

useful for indicatmg the presence of the iso- propylidene acetal structure. Weak bands for C—H in-plane deformations of the unsubstituted phenyl group are found at

8.51 to 8.89 Mm (1175 to 1125 cm"'), 9.01 to 9.35 Mm

(1110 to 1070 cm-i), and 9.35 to 10.00 Mm (1070

to 1000 cm~^); strong bands for C—H out-ofplane deformations occur at 12.99 to 13.70 Mm

(770 to 730 cm-i) and 14.08 to 14.49 Mm (710 to 690 cm-^).

For substituted phenyl groups, the bands for C—H in-plane and out-of-plane deformation

differ according to the position and degree of substitution. For the ^-substituted phenyl group,

most commonly encountered in sugar chemistry,

weak bands (in-plane deformations) are found at

8.17 to 8.51 Mm (1225 to 1175 cm-^), 8.89 to 9.17

Mm (1125 to 1090 cm-i), and 9.35 to 10.00 Mm

(1070 to 1000 cm~^), and a strong band (out-ofplane deformation) at 11.63 to 12.50 Mm (860 to 800 cm~'). Thus, the p-substituted phenyl group

of p-toluenesulfonic esters of alditols [26] and

sugars [27] shows a hydrogen out-of-plane deformation at 12.05 to 12.35 Mm (830 to 810 cm'^),

not shown by methanesulfonates.

For the olefins, CHR=CH2 shows a weak band

at 7.04 to 7.09 Mm (1420 to 1410 cm-^), a band at

7.69 to 7.75 Mm (1300 to 1290 cm"'), a mediumstrength band at 10.05 and 10.15 Mm (995 to 985

cm"'), and a strong band at 10.93 to 11.05 Mm

(915 to 905 cm~0. A cis double bond shows a weak band at 7.04 to 7.14 Mm (1420 to 1400 cm"')

and a strong band at 13.70 to 15.04 Mm (730 to 665 cm"') ; a trans double bond shows a weak band

at 7.55 to 7.75 Mm (1325 to 1290 cm"') and a strong band at 10.20 to 10.42 Mm (980 to 960 cm"').

For example, fran.s-3-hexene-D-f/i,reo-l,2,5,6-tetrol

[28] shows bands at 7.55 and 10.25 Mm (1325 and

976 cm"') ; its 1,2 :5,6-di-(?-isopropylidene derivative shows bands at 7.65 Mm (1307 cm"') and

10.30 Mm (971 cm"').

Whiffen et al. [29] have succeeded in identifying

the C—H deformation vibrations at the anomeric

carbon atom of various aldoses by replacing

the hydrogen atom on C-1 with deuterium. For

example, to prepare a-n-glucopyranose-i-C-rf, they

dissolved n-glucono-l ,5-lactone in deuterium oxide,

reduced the carbonyl group to —C—D(OD) with

sodium amalgam in deuterium oxide, and then

converted the OD groups into OH groups by

3 times dissolving in water and evaporating;

the C—D bond at C-1 remained unchanged. The

spectrum of the a-D-glucopyranose-i-C-(Z was then

compared with that of a-n-glucopyranose. Now,

by theory, if 'H is replaced by D (^H), the 'H

deformation frequencies are approximately V2

times the corresponding deuterium frequencies

(in cm ') if the deformation corresponds to a pure bending or stretching mode. They found

C-1—H at 7.28 Mm (1375 cm"') and C-1—D at

9.13 Mm (1095 cm"') (frequency ratio 1.26); and

C-1—H at 7.79 Mm (1284 cm"') and C-1—

at 10.36 Mm (965 cm"') (ratio 1.33), as compared

to the theoretical ratio of 1.414. Similar assignments were made for the 13 anomer and for the

two anomers of other sugars.

4.2,2. N—H Bands

In dilute solution in a nonpolar solvent, primaryamines show two bands in the region of 2.86 to 3.03 Mm (3500 to 3300 cm"') due to stretching

vibrations of the NH2 group. If hydrogen bonding

occurs, or if the solid is examined, the range is shifted to 2.86 to 3.23 Mm (3500 to 3100 cm"').

Secondary amines in dilute solution show only one N—H stretching band, at 2.94 to 3.03 Mm

(3400 to 3300 cm"').

An N—H deformation frequency is shown by

primary amines at 6.08 to 6.45 Mm (1645 to 1550

cm"') ; thus, D-glucosylamine shows a band at 6.17 Mm (1621 cm"'), and 2-amino-2-deoxy-Dglucopyranose at 6.25 Mm (1600 cm"') [3]. A band

at 6.33 to 6.62 Mm (1580 to 1510 cm"') is shown

by—NH—

The NH2 deformation frequency of primary

amides occurs at 6.06 to 6.17 Mm (1650 to 1620

cm"') for the solid, and at 6.17 to 6.29 Mm (1620

to 1590 cm"') for solutions; it is called the Amide

II band. Secondary amides, having an NH group,

show the Amide II band at 6.37 to 6.60 Mm

(1570 to 1515 cm"') for the solid, and at 6.45 to 6.62 Mm (1550 to 1510 cm"') for solutions. Hydrogen-bonded secondary amides show an NH deformation mode near 13.89 Mm (720 cm"'), called

the Amide V band.

The spectra of 16 1-acetamido derivatives of sugars [19] showed at least one band at 2.98 to

3.09 Mm (3356 to 3236 cm"'), attributed to N—

stretching; and, at 6.35 to 6.49 Mm (1575 to 1541

cm"'), the Amide II band. In a study of the

spectra of 60 1-acylamido derivatives of aldofuranoid, aldopyranoid, and acyclic sugars [2], all of

the compounds were secondary amides, and all of them showed at least one band at 2.89 to 3.10

Mm (3460 to 3226 cm"') due to N—H stretching;

in this region, completely esterified compounds

could not be distinguished from those having free hydroxyl groups that would show O—H stretching

in the same region. All of the compounds showed

a band at 6.35 to 6.65 Mm (1575 to 1504 cm"';

Amide II)

The acyclic, l,l-bis(acylamido)-l-deoxyalditols

showed [2] two Amide II bands, suggesting that

the two acylamido groups on C-1 of these compounds are not equivalent. They may have a hydrogen-bonded structure, possibly of the following type.
jCHINJHCR

^CHOR' CH

I where R is Me, Et, or Ph; and R' is H, Ac, EtCO,

or Bz.

4.2,3. O—H Bands

Compounds having a free hydroxyl group show

a band for O—H stretching at 2.68 to 2.84

(3730 to 3520 cm-^). The O—H bond is weakened

if the hydroxyl group is hydrogen-bonded, and

the band is broadened, mth a shift to longer

wavelength, 2.84 to 3.22 (3520 to 3100 cm-i).

The band for O—H deformation lies at 9.26 to 9.71 Mm (1080 to 1030 cm-^). For sugars, differ- entiation of primary from anomeric and secondary

hydroxyl groups by OH frequencies is not feasible,

because of frequency shifts caused by hydrogen

bonding.

Infrared spectra may be used for the detection

of hydrogen bonding, which may be between one

molecule and another (intermolecular) or in one

molecule (intramolecular) . For compounds soluble

in carbon tetrachloride, intermolecular hydrogenbonding is negligible at concentrations below

0.005 M, and absorptions at ca. 2.79 nm (ca. 3585 cm"') and ca. 2.76 nm (ca. 3625 cm~') may

be associated with bonded and free hydroxyl

groups, respectively [5]. In this way, the extent of

intramolecular hydrogen bonding may be determined [7, 30]. In pyranoid (and m- or pdioxane) derivatives, a suitably located hydroxyl

group can form a hydrogen bond with the ring

oxygen-atom, as shown in figure 3. The spectra

are consistent with the equilibria shown, assuming

that the molecules exist preferentially in the chair

100 100

50

0 -

3640 3600 3560

cm"i

A

3635 \

- / \ Free OH

Bonded OH

5594

1 1 1 1 1 3640 3600 3560

cm"i

B Figure 3. Infrared spect, a of tetrahydropyran-3-ol (A) and

of 1,3-0-methyleneglycerol (B) in carbon tetrachloride; both solutions 0.005 M.

(From Ref. [7].)

conformations (as against non-chair conformations). For compounds that are„ insufficiently

soluble in carbon tetrachloride, Akermark [31] has suggested the use of |)-dioxane, which disrupts

intermolecular hydrogen-bonds but does not

affect strong intramolecular hydrogen-bonds.

In solution, both penta-(9-acetyl-aMe%(^o-Dgalactose aldehydrol and the corresponding ethyl

hemiacetal show [17] a band at 2.78 urn (3597

cm~^) for free hydroxyl, and a band at 2.87

(3483 cm"') for hydrogen-bonded hydroxyl.

HO HO

HCOH HC

~~-H

0 HCOCOCH, HC

1 To

//\CH,

oldehydrol

Etc EtO

HCOH ^ HC

~-H

i HCOCOCHj

1 0'

hemiacetal

b

// ,C\h3

The OD bands in the spectra of fuUy 0-deuterated sugars have been examined [32]. The OH

band at 2.95 ixm (3390 cm"') of Q;,|8-D-glucose was

shifted, by deuteration, to 4.05 /zm (2469 cm"'),

a wavenumber ratio of 1.37, and other new bands

appeared.

4.2.4. C=C and C=C Bands

The C=C stretching absorption is weak and

has limited diagnostic value; it is not shown by

symmetrically disubstituted acetylenes. A weak

stretching band for H—C=C—R lies at 4.65 to 4.76 ixm (2150 to 2100 cm"'); thus, 5-hexyne-DZ?/a;o-l,2,3,4-tetrol tetraacetate shows [23] a band

at 4.65 urn. (2150 cm"') and two 6-heptynepentol

derivatives show [24] the band at 4.72 to 4.74 nm

(2120 to 2110 cm-'). For R—C=C—R', the

band is at 4.42 to 4.57 fMia (2260 to 2190 cm"').

Stretching frequencies for (phenyl) conjugated C=C bonds give a strong band at about 6.15 ixm

(1625 cm"'); for CO or C=C conjugation, the

band occurs at about 6.25 nm (1600 cm"'). Bands

for nonconjugated C=C bonds occur at 5.95 to 6.17 fim (1680 to 1620 cm"'). For olefins, bands

are found for a cis double bond in RCH=CHR'

at 6.02 to 6.05 fim (1662 to 1653 cm"'), and for

the trans form at 5.96 to 6.05 ixm (1678 to 1653

cm"'). For example, trans-^-hexene-'D-threo-l,2,5,Qtetrol shows [28] a band at 6.05 nm (1653 cm"').

A terminal, exocyclic double bond, as in

H2C=CRR', shows a band at 6.03 to 6.07 ^m

(1658 to 1648 cm-'), and for H2C=CHR at 6.07

to 6.11 iim (1648 to 1638 cm-').

The unsubstituted phenyl ring (CeHs) shows

bands of medium, variable intensity for skeletal,

in-plane, stretching vibrations of aryl C=C at 6.16 to 6.35 Mm (1625 to 1575 cm"i), 6.29 to 6.36

Mm (1590 to 1575 cm"'), 6.38 to 6.94 Mm (1465 to 1440 cm-'), and 6.56 to 6.78 Mm (1525 to 1475

cm"'). Thus, the phenyl (monosubstituted benzene) ring of the benzoyl grouo shows bands at 6.25 and 6.32 Mm (1600 and 1584 cm"'). For

example, a group of 1-acylamido sugar derivatives

having A^-benzoyl or 0-benzoyl groups, or both,

showed [2] bands at 6.20 to 6.25 Mm (1613 to 1600

cm-'), 6.30 to 6.38 Mm (1587 to 1567 cm-'), and

10
ll 6.64 to 6.77 fim (1506 to 1477 cm-i) ; and the

I ^-substituted phenyl rmg of p-toluenesulfonic

esters of alditols [26] and sugars [27] shows an

aryl C=C band at 6.23 to 6.25 txva (1605 to 1600

\ cm~^) Avhich differentiates them from methanesulfonates.

4.2.5 C^N, C=N, and C—N Bands

The stretching band for C^N lies in the range

of 4.17 to 4.76 Mm (2400 to 2100 cm-^) ; thus, for R—C=N, it is foimd at 4.43 to 4.47 /xm (2260 to 2240 cm~^), and, for conjugated R-—C=N, at 4.47 to 4.52 Mm (2240 to 2215 cm-^). For iso- cyanides, R—N=C, the band is at 4.55 to 4.76

Mm (2200 to 2100 cm-i). For —S—C=N, the

band is found at ca. 4.63 Mm {ca. 2160 cm~^).

Compounds containing C=N— show a band at 6.02 to 6.21 Mm (1660 to 1610 cm-^ that has been

used in determining whether such compounds as A^-substituted glycosylamines are cyclic or acycUc

(see p. 18). However, the compound examined

must be scrupulously dry, as moisture shows a band at 6.06 to 6.25 Mm (1650 to 1600 cm-^).

Moreover, hydrates cannot be employed, as water

of crystallization shows a band at 6.06 to 6.10 Mm

(1650 to 1640 cm-i).

For —N=C=S (isothiocyanate) and —N=C=N — (carbodiimides) , a strong band is shouTi at ca. 4.76 Mm {ca. 2100 cm~^).

Aliphatic amines show a medium-intensity band

for C—N stretchmg at 8.20 to 9.80 Mm (1220 to 1020 cm and a weak band at about 7.90 Mm

(1410 cm ~^). Nitro compounds show a band

(medium intensity) for C—N stretching at 10.87

to 11.76 Mm (920 to 850 cm "^), and primary

amides at 7.05 to 7.15 Mm (1418 to 1400 cm-^). Of

less diagnostic value are a C—N band at 7.30

to 7.63 Mm (1370 to 1310 cm for the iV-methyl

group, and the Ph—N stretching band, observed

[33] at 8.70 to 8.84 Mm (1149 to 1131 cm-^) or [34] 8.62 to 8.85 Mm (1160 to 1130 cm-^), for phenylhydrazones and phenylazo derivatives.

4.2.6. C=0 Bands

(a) Aldehydes and Ketones.—The C=0 stretching

frequency for the carbonyl group of aldehydes and

ketones lies at 5.78 to 6.00 Mm (1730 to 1665 cm-^).

Thus, for the acyclic form of certain aldoses and

ketoses (in a lyophilizate of the mutarotational

equilibrium mixture), an extremely weak band is detectable [13] at 5.82 Mm (1718 cm"'). Kuhn [14] attributed a band at 6.2 Mm (1613 cm~') shown by

periodate-oxidized methyl a-D-glucopyranoside

to aldehydic carbonyl. Periodate-oxidized cellu- lose shows only a very weak band [35], and has

been shown [36] to exist mainly as the hemialdal —CH—(OH)—O—CH(OH)—, formed by hydration of two aldehyde groups per oxidized residue.

If the bands given by two different kinds of groups lie in close proximity in the spectrum (see

table 1), the bands may be separate, but often one

appears as a shoulder on the other, or one may

completely obscure the other. For example, a

number of anhydrous, monomeric, aldehydo sugar

acetates show no strong band [17] for the aldehyde

carbonyl group, presumably because it is obscured

by the acetate carbonyl band. As a coroUary, in

the absence of other information, the CHO band

could be mistaken for OAc or OBz. If such interference is absent, aldehydo and keto sugars show

the C=0 band; for example, 3-0-benzyl-l,2-0-

isopropylidene-a-D-a;^^o-pentodialdo-l,4-furanose

shows [37] a band at 5.8 Mm (1724 cm^^).

(6) Un-ionized Carboxylic Acids.—The C=0

stretching frequency appears at 5.76 Mm (1736

cm~^) for —C(=0)OH of un-ionized carboxylic

acids [38, 39], including alginic acid, chrondroitin

sulfate, hyaluronic acid, and the pneumococcal

polysaccharides.

(c) Lactones.—In 1958, Barker et al. [40] found

that 22 out of 24 aldono-l,4-lactones showed a band at 5.59 to 5.67 Mm (1790 to 1765 cm-^), and

all of 11 aldono-l,5-lactones showed a band at

5.68 to 5.79 Mm (1760 to 1726 cm"'). Consequently,

if the spectrum shows a strong band at 5.60 Mm

(1785 cm~^), there is a strong possibility that the

aldonolactone is 1,4 and if it shows a band at 5.78

Mm (1730 cm~^), there is a possibility that it is 1,5.

However, if it shows a band at 5.65 to 5.70 Mm

(1770 to 1775 cm~^), some other method for distinguishing between the two should be used. The

6,3-lactones of l,2-0-isopropylidene-Q:-D-gluco- and

-iS-L-ido-fiu-anuronic acid show [4] C=0 stretching

[41] at 5.59 to 5.67 Mm (1790 to 1765 cm-i).

(d) Acetates and Other Esters.—The C=0

stretching vibration of the 0-acetyl group gives

rise to strong absorption at 5.72 to 5.80 Mm (1748

to 1724 cm~^) ; thus, the octaacetates of a-ceUobiose, a-gentiobiose, and j3-maltose show [42] a strong band at 5.79 Mm (1727 cm-^), 5.72 Mm

(1748 cm~^), and 5.76 Mm (1736 cm"^), respectively.

Similarly, six acetates of cyclic acetals of sugars

showed [4] a band at 5.72 to 5.75 Mm (1748 to 1739

cm~^) ; aU of 24 acetylated aldopyranosides showed

[18] at least one band in the region of 5.67 to 5.76

Mm (1764 to 1736 cm~0 ; all of 8 reducing, pyranose

acetates showed [19] a band at 5.71 to 5.76 Mm

(1751 to 1736 cm-i); all of 20 fully acetylated

pyranoses [20] showed a band at 5.69 to 5.74 Mm

(1757 to 1742 cm~^); and, for 14 acetates^ (and a tetrapropionate) of 1-acylamido derivatives of

sugars, all showed [2] a band at 5.68 to 5.74 Mm

(1761 to 1742 cm-i), except for iy-acetyl-2,3,4-tri6>-acetyl-j3-D-ribosylamine, showing [43] a band

at 5.82 Mm (1718 cm"0- Benzoates of the same

group of 1-acylamido derivatives showed [2] a band at 5.73 to 5.79 Mm (1745 to 1727 cm"^),

except for 1 , 1 -bis (benzamido) -6-(5-benzoyl-l-deoxyD-glucitol, showing a band at 5.89 Mm (1698 cm~^).

Mixed esters (acetate-benzoates) showed two

bands in this region.

The five-membered, cyclic carbonate group in

sugar carbonates shows an enhanced C=0

stretching frequency as compared with the mixedester carbonates of sugars, average values lying

at 5.49 Mm (1820 cm-^) and 5.68 Mm (1760 cm-^),

11
respectively [44]. If the cyclic carbonate is trans- fused in sugar derivatives, a strong C=0 stretching

band is shown [45] at 5.43 ixm (1842 cm~^) ; the

band is not shown by cis-fused carbonates.

(e) Primary Amides.-—The C=0 stretching fre- quency for —C(=0)NH2 lies near 6.06 fj.m

(1650 cm~^) for solids, and near 5.92 (1690

cm~^) for dilute solutions. It is known as the

Amide I band, and is also shown by secondary

and tertiary amides. Three glycopyranuronamide

derivatives showed [19] this band at 6.00 to 6.02

/xm (1667 to 1661 cm.-').

(/) N-Acetyl and 8-Acetyl.-—The Amide I band

for the monosubstituted amide group, as in —NH—(C=0)—CH3, occurs at ca. 6.04 (1655

cm"^) for solids, and at 5.88 to 5.99 /xm (1700 to 1670 cm~i) for dilute solutions. Thus, the anomers

of 2-acetamido-2-deoxy-D-glucop3T:anose and their tetraacetates show [46] the band at 5.97 to 6.19

Mm (1675 to 1616 cm-^). For aU of 16 1-acetamido

derivatives of sugars, the band is shown [19] at 5.85 to 6.02 Mm (1709 to 1661 cm-^). Hydrates

showed bands at 6.01 to 6.09 Mm (1664 to 1642

cm~^) that overlapped, somewhat obscured, or were obscured by. Amide I bands in the same

region. Of 60 1-acylamido derivatives of sugars, aU showed [2] a band at 5.95 to 6.15 Mm (1681 to 1626 cm~^). The Amide I band occurs near 6.07

Mm (1648 cm"^) for such polysaccharides as

chitin [47], but at 6.18 Mm (1618 cm-^) for 2- acetamido-2-deoxy-a:-D-glucopyranose, and at 6.01

Mm (1665 cm~^) for its tetraacetate, probably

because of differences in the hydrogen bonding

between the C=0 group and OH and NH groups

[3]. Because an ionized carboxyl group mil absorb

in this region, the spectra of such polysaccharides

as chrondroitin sulfate should be recorded for films cast from an acid solution.

The carbonyl group in the S'-acetyl group,

_S—(C=0)—CH3, shows [48] a band near 5.95

Mm (1680 cm"^). Thus, this group can be dis- tinguished from the 0-acetyl group, absorbing near 5.75 Mm (1740 cm~^) and the A^-acetyl group,

absorbing near 6.10 Mm (1640 cm"^).

4.2.7. C—O Bands

(a) Esters.-—Strong bands for the C—O stretching vibrations are shown by esters; for example,

by acetates at 8.00 to 8.13 Mm (1250 to 1230 cm-i),

formates at 8.33 to 8.48 Mm (1200 to 1180 cm-^),

and propionates at 8.33 to 8.55 Mm (1200 to 1170

cm~^). Esters of aromatic acids shows +w'o strong

bands for C—O stretching, at 7.69 to 8.00 Mm

(1300 to 1250 cm-i) and 8.70 to 9.09 Mm (1150 to 1100 cm-i).

(6) Carboxylate Ion. —Salts of carboxylic acids,

such as the lithium and barium salts of 1,2-0-

isopropylidene-a-D-glucofuranuronic acid, show [4] a carbon —oxygen stretching band (strong) at 6.11 to 6.25 Mm (1637 to 1600 cm-i) that dis- tinguishes carboxylate anions from the C=0

stretching band of esters, and a band (medium

strength) at 7.04 to 7.69 Mm (1420 to 1300 cm-i).

4.2.8. N^N, N=N, and NO2 Bands

The N=N stretching vibration of azides gives a strong band at 4.63 to 4.72 Mm (2160 to 2120 ^ cm~^) and a weak band at 7.46 to 8.48 Mm (1340

to 1180 cm~^). The N=N group in aromatic

diazo compounds gives bands at 6.31 to 6.37 Mm

(1585 to 1570 cm-i) and at 7.04 to 7.24 Mm

(1420 to 1381 cm~^). According to Bassignana

and Cogrossi [34], the bands lie at 6.32 to 6.42 Mm

(1582 to 1558 cm-i) and 6.95 to 7.25 Mm (1439

to 1379 cm-i).

Nitric esters of sugars show [27] strong bands for asymmetric NO2 stretching at 6.00 to 6.20 Mm

(1667 to 1613 cm~^) and for symmetric NO2

stretching at 7.78 to 7.89 Mm (1285 to 1267 cm.-'); ' a broad band (for O—NO2 stretching) at 11.48 to 12.08 Mm (871 to 828 cm-^) is useful only for confirmation as it occurs in a region that contains

bands (Types 2a, 2c, and C) for sugars and the

C—H out-of-plane deformation of the p-substituted phenyl group.

4.2.9. S=0, —SO2—, and C=S Bands

Ij Esters of sulfuric acid, such as the 6-sulfates of | D-glucose, D-galactose, and 2-acetamido-2-deoxy- j D-glucose, show [49] an intense band at 8.07 Mm

(1240 cm~^), due to S—O stretching vibrations.

The position of a band at 11.76 to 12.19 Mm (850

to 820 cm~^) for the C—O^—S frequency of the

sidfate group has been correlated with its attachment (primary or secondary) and with the axial or equatorial disposition if secondary (see page

16).

Sulfones show strong bands at 7.38 to 7.64

Mm (1355 to 1310 cm^^) and 8.62 to 9.01 Mm

(1160 to 1110 cm-i) for S=0 stretching; thus,

the monosulfone obtained by oxidizing penta-0-

SLcetjl-aldehydo-D-glucose dibenzyl dithioacetal

shows a band at 7.66 Mm (1305 cm-') [22]. p-Toluenesulfonic esters of alditols [26] showbands at 7.3 to 7.4 Mm (1370 to 1350 cm-') and

at 8.4 to 8.5 Mm (1190 to 1175 cm-') for the asymmetrical and symmetrical stretching modes of the —SO2— group [50, 51]. Sulfonic esters of sugars usually show two bands in each region [27]. Bands for C—O—S at 11.8 to 11.9 Mm (848 to 840 cm-i) and 11.2 to 11.4 Mm (893 to 877 cm;')

have been correlated with the axial or equatorial

disposition of the sulfonic ester group (see page

16).

The C=S group of thionocarbonates shows [52] bands at 7.52 Mm (1330 cm"') and at 7.65 Mm

(1307 cm-'); a band at 8.40 Mm (1190 cm"') is also found [51]. Dimethylthiocarbamates of sugars show, for 0C(=S)NMe2, a strong band [53] at 6.5 to 6.6 Mm (1540 to 1515 cm-'). iV,7V-Dimethyldithiocarbamates show, for —SC(=S)NMe2, a strong

band at 6.71 to 6.80 Mm (1490 to 1470 cm-').

12
4.2.10. Miscellaneous Bands

I The follo^^ing bands are useful. (1) Polystyrene

(for calibration): 3.51 ixm (2851 cm~^), 6.24 ixm

(1602 cm-'), and 11.03 Mm (907 cvar^). (2) Nujol:

I 3.43 Mm (2918 qtot^), 3.50 Mm (2861 cm-^), 6.86

I Mm (1458 cm-i), and 7.26 Mm (1378 cm-^) [and a

i weak band at 13.89 Mm (720 cm-')]. (3) Water as moisture: 2.92 Mm (3430 cm-') and 6.06-6.25 Mm

(1650 to 1600 cm-i).

Spurious bands may be introduced by a variety

of causes, including sUicone grease used on stopcocks, polyethylene powder from laboratory ware, and phthalic anhydride from the plasticizer in

plastic tubing. About 35 spurious bands have

been listed [54].

jj 4.3. Correlations for the Fingerprint Region

and Beyond

4.3.1. The Fingerprint Region

Whereas the spectra of stiiicturally similar

compounds may be quite similar in the range of 2 to 7 Mm (5000 to 1430 cm"'), significant spectral

differences are foimd in the fingerprint region

from 7 to 11 Mm (1430 to 910 cm-'), because here

the bands are due both to certain stretching vi- brations and bending vibrations. As a result,

smaU differences in structure appear as large

spectral effects. Consequently, the region is valuable for identification of a compound, and for

differentiating between isomers, including anomers. On the other hand, the fingerprint region is often less usefiil for recognizing the presence of particular organic groups than the region below

7 Mm (above 1430 cm-'), where the presence or absence of bands may give valuable, reliable evidence for characteristic groups. For example, the

characteristic group-frequencies for a-{x> or l)-

glucopyranose are essentially the same as those for

i3-(D or L)-glucopyranose, and hence their spectra

below 7 Mm (above 1430 cm~') are similar; but,

for crystals in which the molecules have the

favored chair conformation, the interactions of

the hydroxyl group at C-1 with the ring-oxygen

atom of a neighboring molecule can be expected

to be different for the axially attached group of

the a-(D or l) anomer and the equatorially at- tached group of the jS-(d or l) anomer, and

therefore their spectra in the fingerprint region can be expected to differ. In the hydrogenbonded structure proposed [55] for crystalline

a-D-glucopyranose, the bonding between the C-1

hydroxyl group of one molecule and the ring

oxygen atom of a neighbor would be different

from that for the crystalline /3-d anomer. Analysis

of such differences by a combination of x-ray

crystal-structure analysis, broad-line nuclear magnetic resonance spectroscopy, and infrared spectroscopy should eventually lead to improved

interpretation of the bands in the fingerprint

region and beyond, but this goal has not yet

been reached.

4.3.2. Correlations for 10 to 15 Mm (1000 to 667 cm-i)

{a) Correlations for Certain Aldopyranose Derivatives.—In a part of the fingerprint range, namely,

10.42 to 13.70 Mm (960 to 730 cm-'). Barker and

co-workers [56-58] have sought infrared bands

characteristic of several aldopyranoses and their

derivatives. In the first of these articles, they

identified [56], for n-glucopyranose derivatives,

three principal sets of bands, given in table 2. These were: for a anomers, type la, 917±13

cm-'; type 2a, 844±8 cm-'; and type 3a, 766±10

cm-'; and for j8 anomers, type lb, 920 ±5 cm"';

type 2b, 891 ±7 cm"'; and type 3b, 774 ±9 cm-'.

If the bands are to have diagnostic value for

(d or L)-glucopyranose derivatives, a anomers

should not show type 2b absorption, and /3 ano- mers should not show type 2a absorption. However, they found [56] that (a) some a anomers

exhibit type 1 absorption in the range of type

2b bands, and (b) some /3 anomers show "weak

peaks of type 2a" which they believed were due

to traces of the a anomers. They found that the

type 2a band can be used with considerable conTable 2. Positions {mean values) of various types of bands for D-glucopyranose derivatives [66]

Linkage Type 1 Type 2a Type 2b Type 3

a Anomerlc Mm cm~' cm^' pim cm~' ^m cm~i

monosaccharides 10.93 915 11.81 847 13.04 767

11.11 900 11.88 842 13.32 751

higher saccharides 10.75 930 11.86 843 13.14 761

10.90 917 11.92 839 13.02 768

8 Anomerlc

monosaccharides _ 10.94 914 11.16 896

«10.89 «918 11.22 891 bl2.95 b772

higher saccharides 10.86 921 11.24 890 '^12.92 <^774

» Six of ten compounds did not show this band. Eleven of sixteen compounds did not show this band. Five of sixteen compounds did not show this band.

13
Table 3. Infrared bands {mean values) shown by five (d or ij)-aldopyranoses and their derivatives [56, 57]

Type of band

Xylose Arabinose Glucose Mannose Galactose

cm~' cm~i yum Aim cm~i

Both anomers

1

? ? ? ? 10.90 917 ? ? ? ? 2c_ 11.42 876 11.48 871

3 13.11 763 - 12.99 -770 -12.64 -791 -13.30 -752

* 13. ZO " 753 a anomers only

2a —

— » 11.85 -844 - 12.00 -833 -12.12 -825

3 13.35 749 « 11.86 -843

^ anomers only

za 11.86 843

11.82 845

2b b 11.22 ''891 ni.20 =893 =11.17 =895

''11.24 b890

-Many derivatives containing a benzene ring absorb here. ^Must be confirmed by absence of absorption at ca. 11.85 Mm (ca. 844 cm~i). <= Bands for other types of vibration also occur here.

fidence for diagnosing the a anomeric form,

particularly in polymers of glucopyranose. The

type 2b band was not found useful for diagnosing

the /S anomeric form, but the absence of the type

2a band was considered very useful for diagnosing

the i8 anomeric form. They regarded the bands of

types 1 and 3 as only useful for determining points

of linkage in polymers of a-glucopyranose.

When the infrared spectra of additional glucopyranose derivatives were reported in their next

paper [57], slightly different positions were found

for bands of type 2a and type 3 (see table 3). As before, some of the a. anomers were found to show type 1 absorption in the range of type 2b

bands. Also, they pointed out that derivatives

containing a phenyl ring may show absorption

in the region of the type 2a band, and the acetates absorb in the region of the type 2b band. Their

results [57] for bands characteristic of four other

aldopyranoses and their derivatives are also summarized in table 3. With manno- and galactopyranose derivatives, the type 2a band can be used

for diagnosing a anomers; absence of the type 2a

band is useful for diagnosing the /3 anomeric form.

A band at 11.30 to 11.53 Mm (876 ±9 cm-^),

designated type 2c, was found characteristic [57] of mannopyranose derivatives; and a band of type_2c, at 11.39 to 11.57 nm (871 ±7 cm^^), was

considered characteristic of galactopyranose derivatives. The mean frequency for a given type of absorption may change witli the group-configuration; thus, the mean for type 3 absorption is at

12.64 Aim (791 cm~^) for the manno configuration,

but at 13.30 ixm (752 cm"^) for the gluco and

galacto configurations.

In addition. Barker and co-workers [58] found

that 2- and 3-deoxy derivatives of gluco-, manno-,

and galacto-pyranose show absorption at 11.51 to

11.56 iJLxn (869 to 865 cm ; seven 6-deoxy derivatives of mannopyranose or galactopyranose show a band near 10.34 Mm (967 cm"'). i Application of these correlations [56-58] has' proved useful [3] in the study of many related- compounds, including oligo- and poly-saccharides.

Assignments suggested [3, 56-58] for the bands!

are given in table 4. It should be noted, however,i

that, in the range of 10 to 15 Mm (1000 to 667:' ' cm~^), methyl /3-D-xylopyranoside shows onlyf ' three bands [12], namely, at 10.25 Mm (976 cm~^),f ' 10.38 Mm (963 cm"!), and 11.14 Mm (898 cm-^) ;

' indeed, /3-(d or L)-xylopyranose derivatives are ' not characterizable by any of the bands listed in! i table 3. This example shows that the bands listed' ' in tables 3 and 4 cannot be regarded as character-f I istic of the pyranoid ring, per se, of aldopyranoid

derivatives. I t (b) Correlations for Furanoid and Pyranoidi i; Forms of Aldose and Ketose Derivatives. —For aldo- >

ll and keto-furanose derivatives. Barker andi t Stephens [59] noted absorption bands at the fol-' r lowing mean values: type A, at 10.82 Mm (924i i cm-i); and type D, at 12.52 Mm (799 cm"').

Type A absorption could not be distinguished*

from types 1 or 2b of aldopyranoses, and therefore s has no diagnostic value in differentiating between c furanoid and pyranoid aldoses. In addition, most; i of the furanoid compounds also showed type B' (] absorption at 11.38 Mm (879 cm~') and type C' i absorption at 11.66 Mm (858 cm~^). It has been w found [2, 4, 19] that these correlations are, in » most instances, restricted to the compounds they si studied, and cannot be extended to have a wider

diagnostic applicability to related compounds. \ In 1962, the infrared spectra of most of thei

readily available, unsubstituted aldo- and keto-i

pentoses and aldo- and keto-hexoses were pub14
Table 4. Bands possibly characteristic of various features

of some aldopyranuse derivatives

lype Bands » of Structural feature Refer- band

cm~'

ences

Terminal C-methyl- 10. 34 967 [58] group rocking ^

1 Antisymmetrical ring- vibration <^ 10. 90 917 [56] 2b Anomeric C—H axial 11. 22 891 [56] bona

2c 11/quatorial C—

deformation (other

than anomeric C—H)

11. 36 880 [57]

Rmg-methylene rocking 11. 53 867 [58] vibration (if not adjacent to the ring2a Anomeric C—

11. 85 844 [56] equatorial bond

3 Symmetrical ringbreathing vibration

12. 99 770 [56]

^ Mean value. ^ This band may not have diagnostic

value. " For glucopyranosc derivatives.

lislied [13]. In 1964, Verstraeten [60] made a study of these spectra, together with those of some additional 2-ketoses, and obtained evidence

that most of the common sugars having a cyclic

structure, and their derivatives, display type 1 absorption at a mean value of 10.76 nm (929

cm"'). Hence, the type 1 (type A) band has no

diagnostic value for distinguishing between al- doses and ketoses, and between glycofuranoses

and glycopyranoses. Moreover, as the type 1 band

is shown [2] by acyclic 1-acylamido derivatives of sugars, it has no diagnostic value for distinguishing

between cyclic and acyclic forms of such compounds, either. The same author [60] observed that some

ketopyranoses, as well as aldopyranoses, show a type 3 band at 12.80 fim (781 cm"'). Hence,

this band, too, has limited diagnostic value. He

concluded that type 3 absorption is shown provided that two conditions are met: (a) the sugar

must have a pyranoid ring, and (b) this pyranoid

form must assume a conformation having at

least one axial hydroxy] group. If the number of axial hydroxy] groups is increased (thereby decreasing the conformational stability), type 3 absorption becomes manifest. For example, ^-

(d or L)-xylopyranose, which shows no type 3

absorption, is devoid of axial hydroxy] groups,

whereas the a anomer in the favored conformation,

which has an axial hydroxyl group at C-1

shows absorption at 13.16 fim (760 cm"').

It was found [60] that 2-ketoses display "type

I" bands at 11.44 Mm (874 cm"') and "type IIA"

bands at 12.24 fxm (817 cm"'), regardless of whether the 2-ketoses are pyranose or furanose.

These bands were ascribed to the presence of the

following structural feature

and were tentatively assigned to a skeletal vi- bration. However, six aldoses also show these

bands. The type I band, which appears to be

the same as Barker's type B band for aldo- and

keto-furanoses at 11.38 fim (879 cm"'), has [2] no diagnostic value for 60 aldofuranoid, aldopyranoid, and acyclic 1-acylamido derivatives.

The type IIA band lies in about the same range

as Barker's type D band for aldo- and ketofuranose derivatives, which is at 12.52 fj.m (799

cm"'). If the hydroxyl groups of a 2-ketofuranose

are substituted, or if C-2 of the 2-ketofuranose

is joined to a pyranoid or furanoid structure, a type IIB band appears at 11.99 Mm (834 cm"'),

in addition to, or instead of, the type IIA band.

Verstraeten [60] found that only furanoses give

"type 2" absorption at 11.76 Mm (850 cm"').

He stated that his type 2 absorption is the same

as the type C absorption of Barker and Stephens

[59], and, to avoid confusion, it should be referred

to as the latter. The type C band is given by both

aldo- and keto-furanoses, and therefore cannot

be used for distinguishirg betv een them.

It has been found [2] that, if an A^-acetyl group

(but no ester group) is present, the bands of types

C, 3, IIA, and liB may have diagnostic value;

also, if an A^-benzoyl group (but no es'^er group)

is present, the bands of types 3, IIA, and IIB

may have diagnostic value. For A^-acetyl-O-acetyl

derivatives of sugars, the bands of typ3s IIA and

IIB may differentiate between ketcses and nonketoses, but not between cyclic and acyclic

compounds.

4.4. Conformational Studies

In studying the conformations of sugar derivatives, the most direct information is obtained

by nuclear magnetic resonance spectroscopy.

However, the empirical correlation of infrared

spectra has been used [12] to give conformational

information. Suppose that the spectra of the a and (3 anomers of the methyl pyranosides of the

4 aldopentoses and 8 aldohexoses were to be

recorded. This would provide 24 spectra of closely

related compounds. Each conpjund has C—H,

C—OH, C—OCH3, and a pyranoid ring, and yet

the spectrum of each is unique because the precise positions of the various bands change from

one compound to another, depending on interactions arising from configuration and conformation and on the presence or absence of the hydroxymethyl group.

15
As an example, the spectra of the a and /S anomers of methyl D-xylopyranoside and methyl

L-arabinopyranoside were studied [12]. All bands

shown in common by the four glycosides were

ignored. All bands then shown in common by the

two xylosides were regarded as characteristic of the xylo configuration and were ignored; similarly,

all bands shown in common by the two ara- binosides were ignored. This left a set of bands

differentiating between the anomers of the xylosides, on the one hand, and between the arabinoside anomers, on the other (see table 5). This

indicated a similarity between the /3-D-xylopyranoside and the a-L-arabinopyranoside. Since

the conformation of methyl /3-D-xylopyranoside

has been shown [61] by x-ray studies to be that

depicted in fig. 4, the conformational correlations are as indicated. These formulas are in agreement

with the conformations predicted by considerations of interaction energies.

HO

A B

HO

Figure 4. The structures of methyl 0-D-xylopyranoside-CA

(A) and methyl a-i^-arabinopyranoside-CE (B) compared

with those of methyl a-D-zylopyranoside-CA (C) and

methyl p-h-arahinopyranoside-CE (D).

This correlation is purely empirical, but the same kind of comparison has been made for other

pairs of anomers of (a) methyl aldopyranosides

[12], (b) acetylated methyl aldopyranosides [18], and (c) fully acetylated aldopyranoses [20]. In

Table .5. Anomer-differentiating bands (cm~^) shown by four methyl pyranosides [12]

D-xyl 0 L-arabino

a a

3448 3460

1385 1395

1295 1295

1218 1227

1060 1058 976 973

645 646

473 487

3333 3322

2710 2695

741 744

437 433

every instance, the empirical observations made

on the infrared spectra agreed with the predicted

conformations. With those sugar derivatives for which one chair conformation is not predicted to be strongly favored over the other, the resulting

data did not fit in the correlations. Examples of the latter are: methyl a- and (8-D-lyxopyranoside

and their triacetates, methyl a-n-gulopyranoside

and its tetraacetate, and penta-O-acetyl-a-Dgulopyranose.

For a group of fully acetylated monosaccharides,

those having an axial OAc at C-1 showed [17] a band, possibly for a C —O stretching vibration, at 8.59 to 8.67 Mm (1174 to 1153 cm-^) ; if the group was equatorial, a band was shown at 8.87 fim

(1127 cm~i). For each region, the other anomer

showed the absorption only weakly or not at all. The results with compounds having the gulo, ido,

and talo configurations indicated that they exist

in the CE conformation, or as a mixture of the

chair conformations.

For acetylated methyl glycosides [17], those

having an axial OMe at C-1 showed bands at

8.31 to 8.35 (1203 to 1198 cm-^) and at 8.75

to 8.85 MHi (1143 to 1130 cm~^), but those having

the group equatorial showed no absorption in either

region.

Because polysulfated hyaluronic acid, which

has equatorial sulfate groups only, shows a band

at 12.19 Mm (820 cm-^), Orr [39] concluded that

the sulfate group of chondroitin sulfate C, showing

a band at 12.12 ^m (825 cm~^), is equatorially

attached, and that that of chondroitin sulfate

A, giving a band at 11.70 Mm (855 cm~'), is axially attached. He ascribed the bands to the

C—0 —S vibration. It was then found [62] that

the equatorial sulfate group in o-glucose 3- sulfate gives a band at 12.02 Mm (832 cm~^), and

that a band at 12.19 Mm (820 cm~^) is shown by

the 6-sulfates of D-galactose, n-glucose, and 2- acetamido-2-deoxy-D-glucose, in which the ester grouj) is on the equatorial, primary hydroxyl

group. Hence, chondroitin sulfate C (and D)

probably has an equatorial sulfate group on C-6,

and chondroitin sulfate A (and B) has an axial

sulfate group on C-4 of the 2-acetamido-2-

deoxy-D-galactose residues. Chondroitin polysulfate has sulfate groups at both C-4 and C-6.

Similarly, X-carrageenan shows [63] a broad

band at 11.62 to 12.35 Mm (860 to 810 cm-^),

with a maximum at 12.09 Mm (827 cm"^) compatible with the presence of residues of D-galactose

2,6-disulfate (diequatorial) and D-galactose 4- sulfate (axial).

A strong band at 11.8 to 11.9 Mm (848 to 840

cm~^) and a weak one at 11.2 to 11.4 Mm (893 to 877 cm"^) are shown by sulfonic esters of pyranoid

sugars, and have been attributed [50] to the

C—O—S vibration of an equatorial and an axial

sulfonic ester group, respectively, on a pyranoid

ring.

S

16
5. Examples of Use of Infrared Spectra

In addition to those already mentioned, the

following are some examples of the uses of infrared

spectra.

5 A. Qualitative

Infrared spectra may be employed in foUowrng

the course of a reaction. For example, if a com- pound requires several methylations to give a completely 0-methylated product, the extent of

metnylation may be determined by observing

the disappearance of OH absorption from the

rafrared spectrum, so that quantitative determination for methoxyl need be made only on the

final product.

The elementary approach has been applied [13] in studying the mutarotation of sugars. For a number of monosaccharides, an aqueous solution

of one crystalline anomer was kept untU mutarotation was complete; the solution was then lyophilized, and the spectrum of the lyophilizate was

recorded and compared with those of the two

crystalline anomers. For n-glucose, L-rhamnose,

and D-mannose, aU of the bands in the spectrum

of the equilibrium mixture that are not shown by

the a-pyranose anomer are either (a) shown by

the jS-pyranose anomer or (b) could be due to over- lapping and summation of closely situated bands

of the two anomers, indicating that the equUibrium mixture consists of the anomers of the pyra- nose form; this conclusion is in agreement Avith the

results of earUer, optical rotation studies by Isbell and Pigman [64]. In contrast, for n-talose, the

spectrum of the lyophilizate showed bands absent

from the spectrum of either anomer of the

pyranose form. These results also agreed with those

of the earlier work [65] (namely, that the equilibrium mixture contains the a- and /S-furanose

forms), and have since been confirmed, and quantified, by n.m.r. spectroscopy [66].

5.2. Quantitative

Mutarotation of sugars has also been studied

quantitatively. Parker [67] recorded the spectra,

for the range of 6.00 to 11.00 Mm (1667 to 909

cm~^), of 20 percent aqueous solutions of aD-glucose, /3-D-glucose, and ;8-D-mannose : (a) 2.5 minutes after dissolution, and (b) at the end of mutarotation. By following the change in percent

transmittance [at 8.75 Mm (1143 cm~^) for a- and

;8-D-glucose, and at 8.60 Mm (1163 cm~^) for ^S-d- mannose] with, time, he was able to determine

the mutarotation constants; these agreed well

with those determined from measurements of

change in optical rotation.

The intensity of a group frequency may be

used for quantitative analysis, because it depends

on the magnitude of the change in dipole moment

that is associated with the molecular vibration.

Consequently, strong bands are usually caused

by the vibrations of polar linkages, such as O—H,

WAVE NUMBER, cm"

I""' I I '

I I 1 1 4.0 4.5 S.O S.5 6.0 6.5 7.0 WAVELENGTH, /J.

Figure 5. Infrared spectra of methyl tri-O-acetyl-a-hrhamnopyranoside (S), tetra-O-acetyl-p-D-mannopyranoside

{Jf),and penta-0-acetyl-i>-g\yceTO-i3-T>-gu\o-heptopyranoside

(S). All solutions 0.0197 M {3 and 5 in carbon tetra- chloride; 4 in chloroform)

(From Ref. [17].)

N—H, C=0, C—N, etc. To a first approximation,

the intensity of an absorption band characteristic

of a specific group is proportional to the amount of that group present. Figure 5 shows part of the

infrared spectra of equimolar solutions of three

acetylated methyl glycopyranosides [17]. It may

be seen that the area under the curve increases

from 3, to 4, to 5 acetyl groups. The procedure

could be adapted for use as an analytical method

in this case, for acetyl; but any other groui^ giving

a characteristic band could similarly be analyzed

for. In most cases, however, n.m.r. spectroscopy

is a more convenient tool for such quantitative

analysis, provided that a specific chemical-shift

permits selected group integrations.

5.3. Determination of Structure

An example of solution of a structural problem

involves the D-talose monobenzoate (X) obtained

[65] as a byproduct from the action of peroxybenzoic acid on o-galactal. There seemed a

possibility that it might be a l,2-(orthobenzoate)

(3a), and so, its spectrum was compared [68] with that of l,2-(9-(l-methoxylethylidene)-Lrhamnose (4). As may be seen from figure 6, the latter has no' band at 5.77 Mm (1733 cm~^),

whereas X has a strong ester-carbonyl absorption

there. Hence, X is not an orthobenzoate but

a benzoate; it was thought to be 1-0-benzoyla-D-talose, but was later [69] shown to be the

jS-D anomer (3).

17
80

uj 60 o

z

<

I-

< 40

q:

20

4 5 6 WAVELENGTH, /im

Figure 6. Infrared spectra of 1,2-0-il-methoxijelhylidene)- 'L-rhamnose (----) and 1-0-benzoyl-p-D-talopyranose

( ) in potassium chloride pellets. (From Ref. [68].)

For sugars in which the hetero-atom of the

ring may be nitrogen, the infrared spectra show

immediately which form has this structure. For

example, for the two ring-forms of 5-acetamido5-deoxy-L-arabinose [70], one form shows bands

at 3.03 Mm (3300 cm-i), 6.14 (1630 cm-'),

and 6.43 Mm (1555 cm"'), for OH and NH, NAc,

and NH, respectively, and is therefore the fura- nose form (5), whereas the other form shows

bands at 2.96 Mm (3380 cm"') for OH, and 6.19

and 6.27 Mm (1616 and 1595 cm-^) for NAc,

but no —NH— absorption near 6.43 Mm (1555

cm"'), and is therefore the pyranose (6).

OH ^H,OH OH i^H.OH

HCH OH

I HNAc

5

In another example, 4-acetamido-4,5-dideoxy2, 3-(9-isopropylidene-aMeA?/(Zo-L-xylose shows bands

[71] at 3.10 Mm (3226 cm-';NH), 5.81 Mm (1721

cm-i; CHO), 6.10 and 6.50 Mm (1639 and 1538

cm-i; NHAc), and 7.30 Mm (1370 cm-^ CMca).

On treatment of this compound (7) with acetic

acid, 4-acetamido-4,5-dideoxy-a,|S-L-xylofuranose

(8) was obtained; this compound (which cannot

exist in a pyranoid form) shows C=0 absorption

at 6.12 Mm (1634 cm"'; Amide I).

i

The Schiff-base structure (9) was proposed

[72] for A''-o-tolyl-D-glucosylamine, because it^

shows a C=N band at 6.05 Mm (1653 cm~^).|

However, the pure compound shows [15] no band

at 6.05 Mm (1653 cm"'), indicating that the structure is cyclic, probably the pyranoid form

10. All of the A^-substituted glycosylamines ex- amined by Ellis [15] were found, from their spectra,

to have a cyclic structure.

) III

The oximes of arabinose [72], rhamnose [72], and

fructose [73] show a weak band at 6.05 Mm (1653

cm"^), which may indicate the acyclic form, but it is i^ossible that the N—H of the cyclic form might

ti

HC— N—OH

I H

show a Aveak band of about the same frequency.

D-Glucose oxime does not show a band in this re- gion [72, 73], and is, presumably, cyclic. The acetyl

derivatives of sugar oximes are undoubtedly cyclic, as they show the characteristic bands for the A^- acetyl group [73]. Infrared spectroscopy has proved to be inappropriate for determining whether A^^^-substituted

hydrazones of sugars are cyclic or acyclic. Even

for A^^-substituted hydrazones that are known to be acyclic, the intensity of the C=N band is so weak as to be unobservable [74]. Phenylosazones of sugars are known to exist preponderantly in the

acyclic form, and they show [75] the C=N band at

6.3 Mm (1587 cm-').

Acyclic semicarbazones usually show a band at 5.97 to 6.08 Mm (1675 to 1645 cm-') for C=N.

Acyclic thiosemicarbazones show a weak band at 6.06 to 6.14 Mm (1650 to 1630 cm"') ; the thiosemicarbazones of seven aldoses did not show this band, and were therefore considered to have a

cyclic structure [76]. Cellulose I, II, and III differ in the amorphous

and crystalline regions, and their spectra show dif- ferences in the O—H stretching range. Thus,

cellulose I shows [77] five bands in the OH region,

and when cellulose ' film is treated [78, 79] with

18
deuterium oxide vapor, the intensity of one of

these, at 2.78 to 3.03 Mm (3600 to 3000 cm-i),

rapidly decreases and an O—D band at 3.70 to 4.17 nm (2700 to 2400 cm~^) appears; the band

affected is the OH stretching of the amorphous

regions of the cellulose. The four bands that remain

are those of hydrogen-bonded hydroxyl groups in

the crystalline region. The ratio of the intensities

of the OH and OD bands then gives an indication

of the proportion of hydroxyl groups that are hydrogen-bonded in a crystalline manner, and this ratio thus provides a measure of the crystaUinity

[80].The directions of the hydroxyl groups in cellu- loses were determined by using plane-polarized,

infrared radiation [81] (see sec. 6). It was shown

that the OH band at 3.02 fxm (3309 cm-^) is "perpendicular," and it was suggested that some

of the hydroxyl bonds lie along the chain direction

and form intramolecular hydrogen bonds.

The spectrum of starch indicates [82] that the

hydroxyl groups are extensively hydrogen-bonded.

The spectrum of potato starch differs from that of corn starch, especially in absorption regions for

oxygen-containing groups. Thus, absorption is stronger [83] for the corn starch at 5.95 nm (1681

cm-i), 9.5 to 10.5 Atm (1053 to 952 cm-i), and 11.7

iim. (855 cm~'), whereas it is stronger for potato

starch at 10.8 fxva (926 cm~^). Arrow-root, corn, potato, rice, and wheat starches show bands at 3

Mm (3333 cm-i), 4.75 Mm (2105 cm-^), and 6.15

Mm (1626 cm~'). When the water content of the

starch is changed, the band at 3 Mm (3333 cm~0

undergoes a change characteristic of the particular

starch, and this property may be used for identifying and classifying starches [84].

6. Special Techniques

Plane-polarized radiation has proved useful in

studying oriented film s of such polysaccharides as

ceUulose, chitin, and xylan, providing information

[81, 85] not given by other techniques. It has

mainly been used in studying the crystal structure

of samples having uniaxial orientation, as in

fibers, in which the polymer chains are aUgned

along the fiber axis. The spectrum is measured

with the radiation vector (a) parallel and (b)

perpendicular to the chain direction. The respective intensity of the "parallel" and the "perpendicular" bands depends on the direction of the

transition moment of the vibration responsible for

the band, that is, whether it is mainly parallel or perpendicular to the chain direction. Thus, in the

C—H stretching region of 3.33 to 3.57 Mm (3000

to 2800 cm~^) of the spectrum of ceUulose, there

are a number of partially overlapping bands some of which cannot be assigned on the basis of group-frequency. The C—H bonds of the ring are known to be approximately perpendicular to the

chain axis, and so the dichroism of the bands

associated with, them must also be perpendicular;

consequently, the perpendicular bands at ca. 3.45

Mm (2900 cm~^) have been assigned [86] to those

vibrations. The cellulose band at 3.51 Mm (2853

cm~^) was assigned to the symmetrical stretch for

CH2, and a band at 7.00 Mm (1430 cm"^) to the

CH2 symmetrical bending mode.

The direction of the bonds in a crystal may also be determined. a-n-Glucopyranose shows one band at 2.94 Mm (3405 cm~^) and four others in

the region of 2.99 to 3.12 Mm (3347 to 3204 cm-^).

These have been correlated [79] with the results

of x-ray diffraction studies, which show [55] that

C

there is one O—H • • • O bond (between two

\

C

molecules), and that there are four 0—H • • OH

bonds. Similarly, the directions of the hydroxyl

groups in sucrose have been determined [87]. The technique of attenuated total reflection

[88] is useful for samples not amenable to examination by transmission spectroscopy; the radiation

penetrates a short distance into the sample and is attenuated by absorption, and the extent of attenuation is independent of the thickness of the

sample. For use with aqueous solutions, an attachment is placed in both beams of the spectrometer

to compensate for absorption by water, making

unnecessary the use of very thin, accurately

matched cells to avoid interference by infrared

absorption by water. The method has been applied

[89] to the study of 20 carbohydrates in the region

of 14.29 to 40.00 Mm (700 to 250 cm-^). For those

compounds whose transmittance spectra in this

region had previously been recorded [12, 13], the

attenuated total reflection spectra were in good

agreement. In this range, the anomers of a sugar

afford different spectra.

The use of a micro-die for preparing alkali

halide pellets, and of lead thiocyanate [which is water-soluble and gives a single band at 4.85

Mm (2062 cm~^)] as an internal reference standard,

permits identification and quantitative determination of micro quantities of water-soluble carbohydrates such as are obtained by paper chromatography [90]. Raman spectra, obtained with visible light,

give the same kind of information as infrared

spectra. The sample is irradiated with monochromatic light, and a very smaU fraction of the

scattered light contains frequencies different from

that of the source; these are characteristic of the

molecule irradiated, and correspond closely in

position, but not in intensity, to those in the

infrared spectrum. For example, in the spectra of carboxyHc acid duners, the band for the C=0

symmetrical stretching mode occurs strongly at

ca. 6.02 Mm (1660 cm"') in Raman spectra, but

is very weak (not normaUy observed) in infrared

spectra; in contrast, the band for the C=0

asymmetrical stretching mode, at ca. 5.85 Mm

(1710 cm"'), is weak in Raman, but very strong

in infrared, spectra. Thus, Raman spectra supplement infrared spectra. Raman spectra have been

19
studied for some 1,3-dioxolane compounds related

to sugar acetals [91]. Introduction of the technique

of laser Raman spectroscopy may be expected to lead to greater activity in this field.

7. References

[I] For extensive treatments of the theory and practice

of infrared spectroscopy, see: F. A. Miller, in Organic Chemistry, An Advanced Treatise (H.

Oilman, Ed.), Vol. 3, Chapter 2, Wiley, New York,

1953; R. N. Jones and C. Sandorfy, in Chemical

Applications of Spectroscopy ; Technique of Organic

Chemistry (W. West, Ed.), Vol. 9, Chapter 4, Interscience, New York, 1956; L. J. Bellamy, The

Infra-red Spectra of Complex Molecules, 2nd ed., Wiley, New York, 1958; G. K. T. Conn and D. G. Avery, Infrared Methods: Principles and Applications, Academic Press, New York, 1960; K. Nakanishi. Practical Infrared Absorption Spectroscopy,

Holden-Day, San Francisco, 1962; W. Briigel, An

Introduction to Infrared Spectroscopv (translated

from the German), Wiley, New Yorki^ 1962; W. J. Potts, Jr., Chemical Infrared Spectroscopy, Wiley,

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Press, New York, 1964; N. B. Colthup, L. H. Daly,

and S. E. Wiberly, Introduction to Infrared and

Raman Spectroscopy, Academic Press, New York,

1964; H. A. Szymanski, IR Theory and Practice

of Infrared Spectroscopy, Plenum Press, New York,

1964; J. R. Dyer, Applications of Absorption

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20
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[68] H. S. Isbell, J. E. Stewart, H. L. Frush, J. D. Moyer,

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[70] J. K. N. Jones and J. C. Turner, J. Chem. Soc. 1962, 4699.

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21

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