PERKIN—ELMUER

Applications
Data
Bulletin

DERIVATIVE SPECTROSCOPY
 DERIVATIVE SPECTROSCOPY

An Introductl'on w1'th Practl'cal Examples

by A. Schm1‘tt

l. Introductl'on

Der1'vat1 ve spectroscopy 1's a relat1’vely recent analytl'cal pr1'nc1'ple 1'n

spectroscopy. Although 1't was 1'ntroduced more than 20 years ago (1, 2,
3), and 1'n sp1'te of demonstrated advantages for the mlut1‘on of spec1'f1'c
analytl'cal problems, th1's technl'que has been accepted only hes1 tantly.
The lack of reasonably pr1'ced 1'nstrumentat1 on and l 1'm1'tat1'on to the lst
der1'vat1've 1'n1't1'ally restr1'cted the use of der1'vat1 ve spectroscopy more
or less to except1 onal cases. The 1'ntroduct1'on of the electronl'c d1'ff-
erent1'at1 on pr1'nc1'ple now also makes h1'gher der1'vat1've p0531'ble and,
moreover, by 1'ts favorable pr1 ce performance rat1'o, offers a w1'der f1'eld
of appl1'cat1'on for der1'vat1've spectroscopy. A useful rev1'ew on theor-
et1 cal aspects and the performance of th1's method has been compl'led by
O'Haver and Green (4,5). Der1 vat1 ve spectroscopy has found appl1'cat1'on
not only 1'n the UV—v1's1‘ble reg1'on, but also 1'n 1'nfrared (6,7), 1'n flame
em1'551'on andabsorptl'on spectral photometry (8,9), and 1'n lum1'nescence
(10). Although the UV—VIS range offers a very w1'de f1 eld of appll'cat1'on
for der1'vat1've spectroscopy, publ1'cat1'ons so far have been seldom or
have been restr1'cted to the use of the lst der1'vat1 ve. It 1's therefore
the purpose of th1's report, be51'des descr1'b1'ng some general aspects, to
demonstrate the performance of der1'vat1've spectroscopy by means of ex-
amples taken from var1'ous analytl'cal appl1'cat1'ons. It 1's clear from
these examples that the use of der1'vat1 ve spectroscopy 1's not restrl'cted
to a very small number of spec1'al cases, but may be of advantage whenever
the quantl'tatl've evaluat1 on of the normal spectra 1's very d1'ff1'cult or
even 1'mposs1'ble. In the 1'nvest1'gat1 on of synthet1'c products as well as
1'n the analys1's of determl'ned components 1'n m1'xtures of natural sub-
stances, th1 5 method w1 ll make poss1'ble the solutl'on of problems wh1'ch
are 1'nacce551'ble to normal spectroscopy. Because of the cons1 derably
1'mproved resolut1’on of the spectra, th1's method has become an 1'mportant
tool 1'n qual1'ty control. For sc1'ent1‘f1'c research purposes, m1'nor dl'ff-
erences 1'n the normal spectra can often be enhanced, so that quant1'tat1've
evaluat1'on becomes p0551'ble.

2. éTheoretl'cal
and Practl'cal Asects

A der1'vat1've spectrum 1's the graphl'c representat1on of the d1'fferent1'al

quot1'ent

dA
for the lst der1 vat1've and A - absorbance
dA

d2A for the 2nd der1vat1ve

_ . - wavelength
Il
y

dxz
over the wavelength range of 1'nterest. H1'gher der1'vat1'ves are of lesser
1 nterest for normal analytl'cal use. The lst der1'vat1've of an or1'gl'nal
spectrum may also be defl‘ned as the graph1’c representatl'on of the slope
of an absorptl'on curve at any gl'ven po1'nt of the measured range.

Der1'vat1've spectra may be produced by var1'ous methods. Thus, spectro-

photometrl'c dev1'ces des1'gned for the purpose of recordl'ng der1'vat1've
spectra may belong to e1'ther of two classes.

1. Spectrophotometers where the output Sl'gnal 1's used for further

processr’ng.°

a. electronl'c d1'fferent1'at1'on;
b. mechanl'cal tachometers,‘
c. numerl'cal d1'fferent1'at1'on.

2. Spectrophotometers u51'ng spec1'ally des1'gned opt1'cs'.

a. dual-wavelength 1'nstruments,-
b. wavelength modulat1’on.

To d1'ffe rent degrees, these pr1'nc1'ples enta1'l advantages as well as d1's-

advantages. For practl'cal purposes, the dual-wavelength pr1'nc1'ple (11)
and electron1'c d1 fferent1 at1on are g1‘ven preference nowadays. However,
dual-wavelength 1'nstruments only supply the lst der1'vat1 ve, wh1'le the
2nd der1'vat1've has to be obta1'ned by electronl'c means. Spectrophoto-
meters w1'th electronl'c d1'fferent1'at1'on can supply the lst or the 2nd
der1'vat1 ve of the spectrum at ch01'ce, and they are also less expen51've.
Moreover, ex1'st1'ng record1 ng spectrophotometers may eas1'1y be extended
for der1'vat1‘ve spectroscopy by the 1 nsertl'on of an electron1'c der1'vat1've
attachment between Sl'gnal output and recorder.

3. Scannl'ng and Features of Derl'vatl‘ve Spectra

All the der1'vat1 ve spectra presented 1‘n thl's report have been obta1'ned
by the pr1 nc1'ple of electronl'c d1'fferent1'at1'on. Instrumentat1 on used
were the Models 200 or 550 UV—VIS Spectrophotometers w1'th electronl'c
der1'vat1've accessory and recorder manufactured by Perkl'n—Elmer. The
der1 vat1've accessory 1's 1 nserted between the s1'gnal outlet of the
spectrophotometer and the recorder 1'nput. S1'nce p051't1've as well as
negatl've s1'gnals are to be expected 1'n a derl'vat1've spectrum, the re-
corder pen has to be set to the center of the chart paper (50 %T).
The lst or the 2nd der1'vat1've may be selected as de51'red on the der1‘va—
t1've accessory. When the normal spectrum 1's to be scanned, the accessory
1'5 51 mply sw1'tched off. 81'x d1'fferent sens1 t1'v1't1‘es can be selected
(modes, t1 me constants) on the der1'vat1've accessory. In the lst de—
r1'vat1've, each 1'ncrease of the t1 me constant by one step means an 1'n—
crease 1'n the s1'gnal by about the factor 2,. 1‘n the 2nd der1'vat1've the
1'ncrease 1'n sens1't1'v1 ty 1's exponentl'al. The wavelength scan speed on
the spectrophotometer also has an 1'nfluence on the ord1 nate s1'gnal of
the derl'vatl've spectrum. Thus, there ex1'sts a great var1'ety of 1'nstru-
ment sett1'ngs for scann1'ng a der1'vat1've spectrum, and 1't 1‘s not poss1'ble
to make defl'nl'te recommendatl'ons wh1'ch are generally val1'd for any gl'ven
case. Usually the 2nd der1 vat1 ve of a spectrum, w1'th 1 dent1 cal t1'me
constant (mode), w1'll prov1 de h1'gher sen51't1'vty and more deta1'led 1'nfor—
mat1 on than the lst der1'vat1 ve. The follow1'ng recommendatl'ons can be
gl ven for the selectl'on of the t1 me constant and of the scannl'ng speed.
For absorptl'on bands w1'th large half-w1'dth mode 6 and h1'gh scannl'ng
speed (60-240 nm/ml'n), for spectra w1'th narrow bands (e.g. benzene
vapor) 30 nm/ml'n and low t1 me constant.

The character1'st1'c features of a der1'vat1've spectrum 1'n the lst and 2nd
order are shown 1'n F1'g. l w1'th a symmetrl'c absorpl'on peak (theoretl'cal
example). The po1'nts of 1'nflect1'on 1'n the or1'gl'nal spectrum result 1'n
a max1'mum or m1'n1'mum 1'n the lst der1'vat1've, and the absorpti'on max1'mum
becomes a so—called zero cross1'ng. In the 2nd derl'vat1've, the po1'nts
of 1'nflect1'on of the orl'gl'nal spectrum appear as max1'ma, and the absorp-
t1'on max1'mum becomes a m1'n1'mum. Due to the d1'fferent1'at1'on process, the
p051't1'ons of the zero cr0551'ng 1'n the lst der1'vat1've and of the m1'n1'mum
1'n the 2nd der1'vat1've are very sll'ghtly sh1 fted 1'n the d1'rect1'on of re—
cord1 ng as compared to the pos1't1'on of the absorptl'on max1'mum 1'n the
or1'gl'nal spectrum, dependl'ng on the workl'ng cond1‘t1'ons. However, th1's
1's 1'n51'gn1'f1'cant for the qual1 tat1 ve or quantl‘tatl've evaluatl'on of the
der1'vat1've spectra.

Fig. l. Symmetrical Absorption peak and first and

second derivatives (calculated theoretically).

4. Evaluatl'on of Der1'vat1've Spectra

In the lst or 2nd der1'vat1've, the ordi'nate deflect1‘ons are not propor-
t1'onal to the absorptl'on value, but rather to the slope 1'n the or1'gl‘nal
spectrum, prov1'ded the latter was scanned 1'n absorpt1'on. Sl'nce the
slope 1'n the normal spectrum may be pos1't1've as well as negat1've, the
der1'vat1've spectrum w1'll show e1'ther p051't1've or negat1've ord1'nate
deflect1 ons, or max1'ma and m1'n1'ma, depend1'ng on the type of slope. For
a substance show1'ng absorptl'on, the wavelength pos1't1'on of such absorp-
t1'on and the relat1'on of the extremes (max1 ma, m1'n1‘ma) to each other are
characterl'stl'c values. For quanti'tatl've evaluati'on, only the extreme
values, two adj'01'n1'ng max1'ma and m1'n1'ma w1'th a large d1 stance 1'n the
ord1'nate d1'rect1'on should be selected. For evaluat1'on, several proced-
ures are p0531'ble, dependl'ng on the problem at hand. A deta1'led survey
on such evaluat1'on procedures, 1'nclud1 ng a descr1 pt1'on of poss1'ble
errors, 1's gl'ven 1'n (5). For normal analytl'cal use, two procedures are
of part1'cular 1'mportance.
1. Peak-to-peak measurement.- The d1'stance between a max1'mum and an
adj'01'n1'ng m1 n1'mum1's determl'ned character1’st1’c for the substance to
be determl'ned. Th1's method 1's partl'cularly useful for quantl'tatl've
determ1 nat1'ons 1'n the presence of turb1'd1'ty, or when the background
absorbance 1's not very well deta1‘led.

2. Tangentl'al method-. A tangent 1's appll'ed to two adj'01'n1'ng max1'ma,'

then the d1 stance 1's determl'ned parallel to the d1 rect1'on of the
ordl'nate from the tangent to the m1'n1'mum 1'n between or v1'ce-versa.
By th1 s method non-spec1'f1'c 1 nfluences of the background absorbance
are e11'm1'nated or kept to a m1'n1'mum. The d1'stances may be measured
1‘n cm or 1'nd1'cated accordl'ng to theordl'nate gr1'd of the chart paper.
For convers1'on 1 nto concentrat1‘on un1'ts, the measured d1'stances are
related to those of a standard.

5. Advantages of Der1‘vat1've Spectroscopy

5.1 Prec1 se Determ1'nat1 on of xmax

For prec1'se pos1't1'on1'ng of an absorptr’on max1'mum 1'n the normal spectrum,

the lst der1’vat1've of the spectrum 1's very helpful. Wh1'le 1'n the normal
spectrum, partl'cularly w1 th w1de absorptl'on peaks, the pos1't1'on of A
ma X
can be determ1'ned only approx1 mately, 1'n the lst der1'vat1've of the
spectrum the pos1't1 on of the max1'mum 1's exactly def1 ned by a zero cross-
1'ng 1'n pract1'cal use, however, 1't 1's necessary to correct the resultl'ng
value by the wavelength sh1'ft, th1's depends on the 1'nstrument settl'ngs
and 1's constant for any g1’ven set of parameters.

5.2 Improved Resolut1'on of a Spectrum

An essentl'al advantage 1'n der1 vat1've spectroscopy 1's the 1'mproved reso—
lut1on of a j'usb sll'ghtly v1'51'ble f1’ne structure 1'n the normal spectrum.
For th1's purpose, 1't 1's useful to employ the 2nd der1'vat1've, s1'nce the
max1'ma and m1 n1'ma of the normal spectrum and of the 2nd der1'vat1've have
more or less the same wavelength pos1't1 on, and the f1 ne structure of
the normal spectrum becomes more d1'st1'nctly apparent than 1'n the lst
der1'vat1've of the spectrum. For normal analyt1cal work the recwnl't1'on
of the f1‘ne structure 1's of great 1 mportance for 1'dent1'f1cat1'on, for
purl'ty tests, or generally for quall'ty control. The 2nd der1'vat1've 1's
1'n many cases w1'dely superl'or to the quot1 ent of the absorptl'on at two
wavelengths generally used 1'n b1'ochem1'stry as a test for pur1'ty.

5.3 Quant1'tat1've Determ1'nat1'ons 1'n the Presence of Turb1'd1'ty

An 1 mportant appll'cat1'on 1's the quant1'tat1've determl'natl'on of d1'ssolved

absorb1'ng substances 1'n the presence of turbl'dl'ty. In a normal spectrum,
turb1'd1'ty usually produces an absorbance that 1'ncreases contl'nuously
towards shorter wavelengths. It w1'll therefore not cause any marked
spectral change 1'n the lst or 2nd der1'vat1've. Thus, when absorbl'ng
substances are scanned 1'n the presence of turb1'd1'ty, the absorbance
background caused by th1's turb1'd1'ty can be el1'm1'nated. Nevertheless,
for quant1'at1've determ1'nat1'ons w1'th electronl'c d1'fferent1'at1'on, the
turb1'd1'ty should rema1'n w1'th1 n reasonable l1'm1 ts (stray l1'ght effect).
15.4
Quant1'tat1've Determ1'nat1'ons 1'n Two-and Mult1'-component Systems

A partl'cularly useful appll'cat1'on of the pr1'nc1'ple of derl'vatl've spectra

1's the quant1'tat1've determ1'nat1'on of substances hav1'ng relat1 vely narrow
absorpt1 on bands, w1 th overlapp1'ng by a w1'de band of a second component.
The component to be determl'ned can be recognl'zed only by a second small
peak or by a shoulder 1'n the total spectrum. W1 th the convent1 onal
methods (absorptl'on measurement, tangent1'al method), such "h1'dden"
peaks can be evaluated only w1 th large systematl'c errors, wh1'le under
certa1'n cond1’t1'ons the lst or 2nd derl'vatl've of an add1 t1've total
spectrum of two components w1'll make the 1'nd1'v1 dual components d1's-
t1'nctly v1's1 ble and w1 ll perm1't quant1'tat1've evaluat1'on w1'th a cons1'd-
erably lower or even ne911 g1'ble systematl'c error. The accuracy of a
determl'natl'on 1'n a system cons1'st1'ng of two components 1's determl'ned by
the follow1 ng parameters.-

1. Relatl'on of the he1'ght of the 1'nterfer1'ng band to that of the band

to be determl'ned.

2. Relat1on of the half-W1 dth of the 1'nterfer1'ng band to that of the

band to be determl'ned.

3. D1'stance between the absorptl'on max1'ma of both 1'nd1'v1'dual components.

The cond1't1'ons are always favorable 1'f narrow peaks located 1'n the f1'rst
or center part of the ascendl'ng slope of a w1'de 1'nterfer1'ng peak have to
be determ1'ned. The same 1's vall'd for mult1'—component systems.

6. Appll'catl'ons

6.1 Gas Spectra

Contrary to solutes, gaseous substances have a much more deta1'led f1'ne

structure 1'n the normal spectrum. The reason for th1's 1's the substan-
t1'al reductl'on of 1'ntermolecular 1'nfluences and the 1'ncrea51'ng over—
lappl'ng of the pure electron spectrum by rotatl'onal and v1'brat1'onal
port1'ons. W1'th der1'vat1've spectroscopy, the f1'ne structure of absorb-
1'ng gaseous mater1'als 1's even further resolved and enhanced. Examples
gl'ven are the normal and the derl'vatl've spectra of 1'od1 ne (F1'g. 2),
of sulfur d1 ox1'de (F1'g. 3), and of benzene (F1'g. 4).

6.2 Trace Determ1'nat1'on of Aromatl'cs 1'n Saturated Hydrocarbons

In W spectroscopy and also 1'n general quall'ty control, the contam1'nat1‘on

of saturated hydrocarbons by unsaturated compounds, part1'cularly by
aromatl'cs, 1's of great 1'nterest. The normal spectra of the polyunsaturated
hydrocarbons usually have several absorpt1'on peaks of low half—w1'dth 1'n
the W range wh1 ch, 1'n the 2nd der1'vat1've spectrum, are cons1'derably en-
hanced. Th1’s fact may be exp101'ted for rap1'd qual1'tat1've and quantl'ta-
t1 ve trace determ1'nat1'ons of such mater1'als. F1 9. 5 shows the determ1'na-
t1'on of benzene traces 1'n cyclohexane. The sens1't1'v1'ty of th1's method
may even be enhanced by an 1'ncrease 1'n the scann1'ng speed, so that con—
tam1 nat1'ons below 1 ppm can st1 ll be rel1'ably detected.
 I

|
I‘

'
G
‘ju I .II '_.V“,2nd
“ww”. z

¡__

— -------_--___.__-_ _--_...
la
"I.
_ _. _. _. A_.

1 71..
.‘
1
l

:gï‘
“
Normal absorption spectrum of iodine vapor I ~ Í l
ÍÏ y
together with first and second derivatives . s __ e r
an their as ociated baselines. Sli

"C N
O. nm, 30 nm/min. Mode 6.
.,,._,,., ,

280 300 ' 320 nm

Fig. 3
Normal spectrum and 2nd derivative of sulphur
dioxide. Slit 0.5 nm, 30 nm/min. Mode 6.

a a 1
and 2nd Derivative. Slit 0.2 nm; 30 nm/min.
Mode 2.
 F1'g. 6 Normal and derl'vatl've spectra of Phenol
(50ppm) 1'n water. Sll't 1 nm, 60 nm/ml'n.
Mode 6.

F1'g. 5 Benzene (lOppm) 1'n Cyclohexane. Nomal

spectm and 2nd der1'vat1've. Sll't 0.5 nm,
60 nm/ml'n. Mode 6.

6.3 Measurements 1'n the Presence of Turb1'd1'ty

F1 9. 6 shows the or1'gl'nal spectrum of phenol 1'n water, as well as 1'ts

lst and 2nd der1'vat1'ves. In the 2nd der1'vat1've the locatl'on of the
shoulders 1'n the or1'g1'nal spectrum becomes part1'cularly d1 st1 nct by
the1 r strong m1'n1'ma. For quantl'tatl've evaluatl'on of der1'vat1've spectra,
the d1'fference 1'n absorbance between a max1’mum and a m1'n1'mum 1's used.
If, as 1'n the present example, several such d1'fferences ex1 st, two very
d1'st1'nct extremes showl'ng the largest dl'fference should be selected,
e.g. d1 fference A-B or C-D 1'n F1'g.7. The determl'natl'on of phenol 1'n
waste water was one of the f1'rst practl'cal examples for the appl1'cat1'on
of der1'vat1ve spectroscopy to the quant1'tat1've determl'natl'on of a para-
meter 1'n the presence of turb1'd1'ty. F1'g. 7 demonstrates that the evalua-
t1'on of der1'vat1 ve spectra of the lst and 2nd order follows the Beer-
Lambert Law, and that turb1'd1 ty does not 1'nfluence 11'near1'ty.

Qu1'n1'ne—laced beverages also often contal'n fru1't extracts (e.g. B1 tter

Lemon). Thl's makes them sll'ghtly turbl'd, and they also conta1'n extract
substances absorb1'ng W ll'ght. D1'rect spectrophotometr1'c determl'natl'on
of the qu1'n1'ne content 1'n the UV therefore 1's not feas1'ble, but becomes
 ppm phenol

Fl'g. 7 Phenol 1'n waste water, derl'vatl've spectra

and ll'nearl'ty.

*,'_*,*,*‘."" .' .“—.- .+» ,1 lt.»1 -a__—,1 t. 1 l‘

r

r y: 2': ' ' "‘.--+“f—_f—ve“Ft-“7* y

ï 3 Ï" 9 1"“ '. 5 .'
»
-'

f
.

amnle1 1-'50
quinine sulfate

Fl'g. 8 Deteml'natl'on of Qul'nl'ne sulphate by derl'va—

t1've spectroscopy. Sll‘t 2 nm, 120 nm/ml'n.
Mode 6, in 0.05N H2804.
p0551'ble by der1'vat1've spectroscopy where the turb1‘d background and
1 nterfer1'ng w1'de absorptl'on bands are el1'm1'nated. F1'g. 8 shows the
or1'g1'nal spectrum and the 2nd der1'vat1've of a qu1'n1'ne standard solu-
t1'on, a turbl'd beverag e sample conta1'n1‘ng qu1'n1'ne, and the spectrum of
the beverage sample conta1'n1'ng qu1'n1'ne 1'n the same concentratl'on as 1'n
the standard. The m1'n1 mum 1'n the 2nd der1'vat1've that can be attr1'buted
to qu1 n1 ne 1's located at the same wavelength for all three samples.
The qu1'n1'ne added to the beverage sample has an add1't1've effect. Thus,
the determl'natl'on of qu1'n1'ne 1'n turb1'd qu1'n1'ne-laced beverage 1's assured.

£00 500 nm 600 250 300 MII 350

Fig. 9 Normal spectra and 2nd derivative of Orange

Juice and Wine.

F1 9. 9 shows the normal spectrum and the 2nd der1'vat1've of a coarsely

f1'ltered orange j'u1'ce 1'n l.'5 d1'lut1'on. The or1'g1'nal spectrum shows a
strong 1'ncrease 1'n absorptl'on due to the turb1'd1'ty, w1'thout gl'v1'ng any
deta1'led 1'nformat1'on. In the 2nd der1'vat1've, however, the content of
polyenes becomes d1'st1nctly v1's1'ble. The pract1'ca1 1'mportance of th1's
example 1's the fact that the determ1‘nat1‘on of extracts 1'n such products
becomes p0551'ble. The r1'ght-hand $1'de of the f1'gure shows correspondl'ng
spectra of d1 luted wh1 te w1'ne. The 1 nformatl'on ga1'ned from the 2nd
der1'vat1've 1's that the peak at 265 nm has to be attr1'buted to a Sl'ngle
component.

Cola dr1'nks contal'n caffel'ne. Although they are not turb1 d, the d1'rect
spectroscop1 c determ1 nat1'on of the caffe1'ne concentrat1'on 1'n the W
range 1's not pos51'ble due to the dark color1'ng and to the poss1 ble
presence of other absorbl'ng 1'ngred1'ents. The e11 m1 nat1 on of non-spec1'f1'c
background absorbance by der1'vat1've spectroscopy 1's demonstrated by F1'g.
10. The add1 t1 on of a known amount of caffe1 ne to a cola dr1 nk shows
the result that could be theoretl'cally expected.
 -10-

,' "cm'u¡"‘1ï.‘
Him-Inn; ¿mu/Ji
‘ J
'

,
I. 1- "‘
1.2.9.":

Fl'g. 10 Determl'natl'on of Caffel'ne by derl'vatl've

spectroscopy. Sll‘t 2 m, 120 nm/ml'n.
Mode 6.

An 1'mportant analyt1'ca1 example for turb1'd samples 1's m1'lk, w1'th 1'ts
turb1'd1'ty caused by an emu151'on of fat. Bes1'des the determ1'nat1 on of
fat and other 1'ngred1'ents of the m1'lk, the determl'natl'on of protel'ns
1's of maj'or 1'mportance 1'n th1's case. The orthodox methods of protel'n
determ1'nat1'on are very tedl'ous and t1'me-consum1'ng. Der1'vat1've spectros-
copy may p0531'bly offer a less onerous alternat1've for the determ1'nat1'on
of protel'ns 1'n m1'lk.

Fl'g. ll Fl'g. 12
Nomal spectra and 2nd der1'vat1've of Normal and 2nd derl'vatl've spectra of
protel'n standard and m1'lk. Sll't succe551've d1'lut1'ons of m1'lk.
l m, 120 m/ml'n. Mode 6
 -11-

F1 g. ll shows the normal spectrum and the 2nd der1'vat1 ve of h1 ghly d1'-
luted fresh m1'lk, scanned between 315 nm and 240 nm. The absorpt1 on
peak at 280 nm can be attrl'buted to the content 1'n aroma t1'c am1 no ac1'ds
of the m1'lk prote1 ns. For compar1'son, the same f1'gure shows a model
prote1 n. F1 9. 12 shows the normal spectra and the1'r 2nd der1'vat1'ves
of var1'ous m1'lk d1 1ut1'ons. Where the d1'1ut1'ons are h1 gher than l.°100,
the peak-to-peak d1'fferences AB are ll'near w1'th the d1'1ut1'on.

6.4 Der1'vat1've Spectroscopy w1 th Mult1'—component Systems

An 1'nterest1'ng and prom1 s1 ng f1 eld of appl1 cat1 on for der1'vat1've

spectroscopy 1's prov1'ded by the natural substances w1'th polyene
character. Th1 8 1'5 demonstrated by the follow1'ng f1'gure (l3) show1'ng
the normal spectra and the1'r 2nd der1'vat1'ves of var1'ous vegetable 01 ls.
The detal'led structure of the der1'vat1 ve spectra 1's ma1 nly due to the
content of unsaturated fatty ac1'ds. The p051't1'on of the extremes
(max1'ma, m1'n1'ma) and the1'r peak d1'fferences perm1 t qual1'tat1 ve con—
clu51'ons on the ma1'n components to be drawn. In th1's case, tests w1'th

Fig. 13 Normal and 2nd derivative spectra of natural

01'ls 1'n n-Hexane (1.-100), Sll't 2 nm,
120 nm/min. Mode 6.

model substances are st1 ll necessary so as to fac1’l1’tate correlat1'on

and quant1'tat1've evaluatl'on. Be51'des quant1'tat1've determl'natl'on,
qual1'ty control 1's very 1'mportant for such products (1'nfluence of
l1'ght, storage propertl'es) Sl'nce, because of the h1 ghly d1 fferentl'al
der1'vat1've spectra as compared to normal spectra, m1'nor d1'fferences
can be much better detected 1'n such a der1'vat1've spectrum.

Most v1'tam1'ns show a typl'cal absorpt1 on behav1 or 1'n the W range.

Scans of the lst or 2nd der1'vat1've of the spectrum may often be used
for a quant1’tat1’ve determl'natl'on of v1'tam1'ns 1'n the presence of other
absorbl'ng substances. Chromatograph1'c or other t1'me-consum1'ng purl -
f1'cat1on procedures can thus often be av01'ded. Fl'g. 14 shows the
normal spectra and the1 r 2nd der1'vat1'ves of v1'tam1'n B-12 and v1'tam1'n E
acetate. As an example for practl'cal appll'cat1'on, Fl'g. 15 demonstrates
the quantl'tatl've determ1 nat1 on of v1'tam1'n E acetate 1'n the presence
of v1'tam1 n A. The add1 t1 on of pure standard materl'al to the extract
of a dragee g1 ves a y1 eld of 98%. W1 th the orthodox determl'natl'on 1't
1's always necessary f1'rst to carry out hydrolys1's for obta1'n1'ng free
v1 tam1'n E wh1 ch, because of the auto-ox1'dat1'on of th1's substance, w1'll
always enta1'l losses, bes1'des be1 ng very t1'me-consum1'ng.
 -12-

4'.
I"
J 7 1 _.' ,1 A...
' í

4
Í
¡mn 64mm m‘ mmm"
-’-101¡gfmr——._.

F1'g. l4 F1 g. 15

Normal and 2nd derl'vatl've spectra of v1'ta- Determl'natl'on of V1'tam1'n E-Acetate 1'n conj‘unc-
ml'ns, 811 t 2 m, 120 nm/m1 n. Mode 6. t1on w1 th V1'tm1'n A. Sll't 2 m, 120 m/ml'n.
Mode 6.

The performance of derl'vatl've spectroscopy 1'n p rotel'n cheml'stry 1's

demonstrated by the follow1'ng examples. F1 g. 16 shows the normal spectra
and the1'r 2nd der1'vat1'ves of human and bov1'ne albuml'n (Cohn Fractl'on V).
The w1'de absorptl'on band known as the "aromat1'cs peak" around 280 nm
shows a surpr1's1'ngly detal'led f1'ne structure 1'n the 2nd der1'vat1've.
Th1's f1'ne structure can be attrl'buted to the content of phenylalanl'ne,
tyros1 ne, and tryptophane. Sl nce phenylalan1 ne shows absorpt1on at
shorter wavelengths, the regl'on around 250 nm 1's mal'nly characterl'zed
by th1's aml'no ac1 d, whl'le at about 280 nm tyr051'ne and tryptophane
doml'nate. Model tests w1'll have to dec1'de whether 1't 1's p0551'ble, by
der1'vat1've spectroscopy, to determ1’ne quant1 at1 vely 1'nd1 v1'dual components
of the pea k w1'thout hydroly51's and subsequent orthodox determ1’nat1'ons.

F1'g. l6 F1'g. 17

Normal and 2nd derl‘Vatl'Ve SPeCtra Of Hman Comparl'son of normal and 2nd derl'vatl've
and BOVine albumin Ing/ml) o l nm, spectra of Bovine Albumin from two
60 nm/ml'n. Mode 6. suppll'ers. Sll't 1 nm, 60 nm/ml'n. Mode 6.
 -13-
References.-

1. V. J. Hammond u. W. C. Pr1'ce, J. Opt. Soc. Amer. 43, 924 (1953)

2. J. D. Morrl'son, J. Chem. Phys. 21, 1767 (1953)

3. A. T. G1'ese u. C. S. French, Appl. Spectrosc. 9. 78 (1955)

. O'Haver u. G. L. Green, Intern. Lab. 5, 6, 11 (1975)

O

I O

. O'Haver u. G. L. Green, Analyt. Chem. 48, 312 (1975)

O

O C

. G. McW1'111'am, Analyt. Chem. 41, 674 (1969)

o G- McW1'111'am, J. Overland et al, Appl. Opt. 6, 457 (1967)

. Snelleman et al, Analyt. Chem. 42, 394 (1970)

. Ko Fowler et al, Analyt. Chem. 46, 601 (1974)

10. G. L. Green u. T. C. O'Haver, Analyt. Chem. 46, 2191 (1974)