Thermo Micro Indentification of Amino Acids: Refractive Index ~

By

A. Laeourt** and N. Delande***

With 1 ~'igure

(Received December 6, 1963)

The microthermal techniques of Ko/ler 1 are important in permitting the s tudy and identification of organic substances which present difficulties in identification by other methods. Amino acids are such substances, their lack of stability at higher temperatures being such tha t most of them cannot be identified by a melting temperature.

A recent paper 2 repor t s satisfactory results in t h e identification of 18 amino acids, using their eutectic melting temperatures of their mixtures with several partners.

Logically, for such substances, physical constants measured at room temperature would be recommended: specific gravity, refractive index, solubility, density, etc.

Very little information is available about the refractive index of amino acids, because their crystals are very small and because of the impossibility of cutting oriented sections of them according to the classical crystallographic techniques. The immersion technique as developed by Ko/ler allows such information to be obtained.

The crystals of amino acids are mostly anisotropic. Crystallised on a slide, they all appear to be developed in exactly the same way, presenting themselves according to a section in the Fresnel ellipsoid which is identical for all crystals of one individual. Therefore, the immersion method, observing the Beeke line, will yield two numbers which are components of the two real refractive indices of the substance. These two conventional numbers n'~ and n'g can be determined in two reference liquids, when

* Dedicated to Prof. Dr. A. A. Benedetti Pichler on the occasion of his 70th birthday.

** Present Address: Bruxelles V, 17, lgue des Egyptiens, Belgique. *** Financial help from the F. N. 1~. S. and the University of Brussels

enabled this research to be performed in the Mierochemistry Department. 35*

548 A. Lacourt and ~ . Delande: [Mikroohhn. Acta

the under ly ing crystal is ro ta ted th rough 90 ~ . If the refractive index of the reference l iquid is known to d= 0.0002 units , with the thermosta t ic stage regulated a t 20 ~ C • 0.1~ 3 using sodium ]ight, under a polarising microscope (parallel light) according to Keller 1, these two numbers n'~ and n'g may be determined with an accuracy of ~ 0.001 uni t .

Th i r ty -n ine amino acids have been tested in this way and the re- producibi l i ty of the measurements in each case appear to be remarkable , even for samples coming from different factories.

Reference l iquids are prepared from weighed mixtures of mono- bromonaphtha lene and paraffin, their refractive indices being de te rmined

at 20 ~ using an A b b 6 refractometer. Typical values of refractive index for these mixtures are as follows:

I Na of Liquid ~20 ~

N ~ of Liquid D ])

1 2 3 4 6 8 9

10

1.5572 1.5590 1.5600 1.5611 1.5580 1.5585 1.5220 1.5230

11 12 13 14 15 16 17 18

1.5242 1.5250 1.5330 1.5342 1.5352 1.5385 1.5395 1.5264

Tab l e I . R e p r o d u c i b i l i t y of n'~ a n d n'g f r o m S ~ m p l e s of D i f f e r e n t O r i g i n (t ~ ~ 20 ~ C)

Amino Acid ~ol. Weight n ' g - n '~

d/-Norleucine . . . . . . . . . . . . .

d/-Norvaline . . . . . . . . . . . . . .

d/-Isoleucine . . . . . . . . . . . . .

d/-Leueine . . . . . . . . . . . . . . .

/-Leueine . . . . . . . . . . . . . . . .

131.17 (a) (b) (c)

117.15 (d) (e) (c)

131.17 (a) (e) (e)

131.17 (a) (e) (e)

131.17 (e) (b) (e)

(a) Kodak, (b) Pfanstiehl, (e) Nutritional,

1.523 1.558 1.522 1.558 1.524 1.558 1.481 1.502 1.480 1.501 1.480 1.502 1.552 1.581 1.551 1.580 1.550 1.580 1.536 1.560 1.536 1.560 1.536 1.560 1.524 1.543 1.525 1.543 1.524 1.543

(d) B. %). H., (e)

0.035 0.036 0.034 0.021 0.021 0.022 0.029 0.029 0.030 0.024 0.024 0.024 0.019 0.018 0.019

Roche.

I n fo rma t ion from the measurements performed on 39 amino acids are gathered in the following Tables. n'~ and n'g, the two componen ts

1964/2--4] Iden t i f ica t ion of Amino Acids: Ref rac t ive I n d e x 549

T a b l e l I . l ~ e f r a c t i v e I n d e x a n d M o l e c u l a r R e f r a c t i v i t y o f A m i n o A c i d s (20 ~ C, sodium light)

Amino Acid M.P. ~ ~o1. Wt. n'~ n'g Density

Glycine . . . . . . . . . . . . . .

fl-Alanine . . . . . . . . . . . .

e-Amino Caproic Acid .

dl- a -A lan ine . . . . . . . . .

l- a -Alanine . . . . . . . . . .

d l . a - A m i n o Butyr ic Acid . . . . . . . . . . . . . .

d/-Valine . . . . . . . . . . . .

/-Valine . . . . . . . . . . . . .

d / -Norval ine . . . . . . . . .

d/-Leucine . . . . . . . . . . .

/ -Leucine . . . . . . . . . . . .

d/-Isoleucine . . . . . . ' . . .

240/247 75.07 1.601 1.607

202/205 89.09 1.404

205/210 131.17

257 dec. 89.09 1.424

266 dee. 89.09 1.401

2O0 103.12

1.231

232/241 117.15

1.316

198/199 117.15

1.230

275 dec. 117.15

242/252 131.17

1.191

285/295 131.17

1.165

235 dec. 131.17

1.619

1.524

1.504

1.539

1.517

1.517

1.580

1.553

1.481

1.538

1.525

1.552

1.657

1.583

1.536

1.579

1.560

1.543

1.602

1.585

1.502

1.558

1.543

1.581

I molecular Refractivity

ntg - ~'g

Exper. Calc. (mean)

0.038 27.30 29.91 29.80

0.059 i 35.10 / 35.09

0.032 58.50 - -

0.0%0 36.69 34.97

0.043 ].36.69 34.21

0.026 42.50 44.39

0.022 50.57 52.61

0.032 50.57 54.20

0.021 50.31

0.020 57.72 60.35

0.018 57.72 60.12

0.029 57.72 - -

Discre- pancy

2.61 2.50

- - 0.01

- - 1 . 7 2

- - 2.48

+ 1.89

%- 2.04

+ 3.63

+ 2.63

+ 2.40

550 A. L a c o u r t a n d N. D e l u n d e : [Mik roch im. A c t a

(Continuation o] table I I J

Amillo Acid n'g n'g- n'~

M o l e c u l a r

Refractivity

Catc. I ~xP:ri

Discre- pancy

l - I s o l e u c i n e . . . . . . . . . .

dl-Norleucine . . . . . . . .

l - N o r l e u c i n e . . . . . . . . .

d l - A s p a r f i c A c i d . . . . . .

l -Aspar~ic A c i d . . . . . .

dl- G l u t a m i c A c i d . . . . .

d -Glu~amJc A c i d . . . . . .

l- G l u t a m i c A c i d . . . . . .

dl- fl- Phcn ylalanine . . . .

l - f i - P h e n y l a l ~ n i n e . . . . .

d l - T y r o s i n e . . . . . . . . . .

/ - T y r o s i n e . . . . . . . . . . .

o

1.663

dec . [33.1(

1 ~0/18: 1~7.13

1.460

1 ~6/19~ 1 ~7.13

1.538

1 )4/191 1 [7.13

1.538

2 .)5/24q dec . .65.1~

2 )8 de( 135.19

245/253

ii: 60.59 60.33

46.02

53.68

54.62

53.57

72.46

+ 2.48 + 2.22

+ 0.27

+ 0.13

+ 1.07

+ 0.02

- - 8.99

1 9 6 4 / 2 - - 4 ] I d e n t i f i c a t i o n o f A m i n o A c i d s : R e f r a c t i v e I n d e x

(Continuation o/ table II)

551

Amino Acid Discre- pancy

l - A r g i n i n e �9 H C 1 . . . . . . .

/ - H i s t i d i n e �9 ~ C 1 . . . . . .

1 - H i s t i d i n e . . . . . . . . . . .

d / - L y s i n e

2 ~ C 1 . . . . . . . . . . . . .

/ - L y s i n e

2 H C 1 . . . . . . . . . . . . .

d / - T r y p t o p h a n e . . . . . .

d / - T h r e o n i n e . . . . . . . . .

d - T h r e o n i n e . . . . . . . . . .

d l - M e t h i o n i n e . . . . . . . .

/ - M e ~ h i o n i n e . . . . . . . . .

d l - S e r i n e . . . . . . . . . . . . .

/ - C y s ~ e i n e . . . . . . . . . . .

1 4 9 . 2 1

2 1 5 d e c . 1 0 5 . 9

1 . 5 3 7

2 2 5 d e c .

1 2 1 . 1 5

:iii:

iiii iiiii iiiii 3 7 .8 1 3 8 . 7 5

- - 0 . 8 0

+ 0 . 9 4

552 A. Lacour t and N. Delandc:

(Cont inua t ion o] table I I )

[Mikrochim. Ac ta

Amino Acid

l -Prol ine . . . . . . . . . . . . .

Creatine . . . . . . . . . . . . .

Creat inino . . . . . . . . . . .

M.P . ~ 1~ol. Wt. Density

1.552

1.528

1.576

1.586

n'g - n*~

0.024

0.058

Molecular Refractivity

222 115.13

252 dec. 131.14

240 dec. 113.12

1.620 1.752 0.122

Calc. Exper. (mean)

Disere- 1)ancy

of t h e rea l r e f r a c t i v e i n d e x so ob t a ined , p roduce e x p e r i m e n t a l specif ic

r e f f ac t i v i t i e s wh ich a re v e r y close to t h e t h e o r e t i c a l r e f f ac t i v i t i e s c a l c u l a t e d n - - 1

f r o m t h e r a t io 7 " M in wh ich n is t h e g e o m e t r i c m e a n of t h e t w o

r e f r a c t i v e indices , d is t h e d e n s i t y a n d M t h e m o l e c u l a r we igh t . T h e o r e t i c a l

m o l e c u l a r r e f r a c t i v i t y is c a l cu l a t ed acco rd ing t o Schoorl ~ e x c e p t for

m e t h i o n i n e .

Comments on the Results

T w e n t y o u t of t h e t h i r t y n i n e e x a m i n e d acids can be a r r a n g e d in

pa i rs w i t h a l m o s t i d e n t i c a l r e f r a c t i v e ind ices ; th i s m e a n s t h a t in a b o u t

5 0 % of t h e cases e x a m i n e d , r e f r a c t i v e i n d e x is n o t su f f i c i en t ly cha r ac -

te r i s t ic to p e r m i t t h e i d e n t i f i c a t i o n of t h e a m i n o acids.

T a b l e I I I

!

Amino Acid n'~ I n'g

1.619 1.657 1.619 1.657 ] 1.524 1.583 I 1.528 1.586 1.553; 1.585

dl-Isoleucine . . . . . . . . 1.552 1.581 1.558 1.538 I dl -Leuc ine . . . . . . . . . .

l -Isoleucine . . . . . . . . . 1 .536 1.560 1,558 1.522 dl-Norleucine . . . . . . . . .

l -Norleucine . . . . . . . . . 1.524 1.554

Glycine . . . . . . . . . . . . dLfi -Phenylalanine . . . fi-Alanine . . . . . . . . . . . Creat ine . . . . . . . . . . . . l -Valine . . . . . . . . . . . . .

Amino Acid n '~

/ -Aspar t ic Acid . . . . . . L Giu tamic Acid . . . . . . d/- Glu tamic Acid . . . . . l -Leucine . . . . . . . . . . . . d -Glu tamic Acid . . . . . l - I I is t idine . . . . . . . . . . l -~-Phenyla lanine . . . .

1.504 1.504 1.526 1.525 1.523 1.525 1.607

d l - T r y p t o p h a n e . . . . . . . 1.607 dl-Threonine . . . . . . . . . ' 1 .525 d-Threonine . . . . . . . . . 1.526

1.620 1.619 1.539 1.543 1.619 1.619 1.660 1.658 1.597 1.593

1964/2--4] Iden t i f i ca t ion of Amino Acids: Ref rac t ive I n d e x 553

n'~ is c h a r a c t e r i s t i c for 8 o u t of t h e 39 a m i n o ac ids e x a m i n e d :

Amino Acid n'~ n'a--n' ~

d/-Valine . . . . . . . . . . . . d / -Norval ine . . . . . . . . . / - Isoleueine . . . . . . . . . . / -Arginine �9 tIC1 . . . . . . . l-I-Iistidine �9 HC1 . . . . . . d / -Lysine �9 2 tIC1 . . . . . d / -Methionine . . . . . . . . Creat ine . . . . . . . . . . . . .

1.580 1.481 1.536 1.572 1.593 1.545 1.568 1.528

0.022 0.021 0.024 0.023 0.027 0.023 0.036 0.058

n 'g is c h a r a c t e r i s t i c fo r 7 o u t of t h e 39 a m i n o ac ids e x a m i n e d :

Amino Acid n'g n'g--n'~

Cre~tine . . . . . . . . . . . . . d / -Norval ine . . . . . . . . . /-Igorleucine . . . . . . . . . d l -Aspar t ic Acid . . . . . / -Tyrosine . . . . . . . . . . . d / -Methionine . . . . . . . . Creat in ine . . . . . . . . . . .

1.586 1.502 1.554 1.628 1.640 1.604 1.742

0.058 0.021 0.030 0.104 0.115 0:036 0.122

n 'g - - n ' ~ is c h a r a c t e r i s t i c fo r 13 o u t of t h e 39 a m i n o ac ids e x a m i n e d :

d/-cc-Alanine . . . 0.040 /-cr . . . 0.043 d/-0r / -Leucine . . . . . . 0.018 d/-Isoleucine . . 0.029 Acid . . . . . . . . . 0.026 d / - A s p a r t i c A c i d 0.104 / - A s p a r t i c A c i d 0.116 / - f l -Phenyla lanine 0.053 / - L y s i n e - 2 t I C 1 . 0.009 d / -T ryp tophane 0.051 Creat inine . . . . . . 0.122 dl -Threonine . . . 0.072 d-Threonine . . 0.067

I n s o m e cases rise el temperature b r ings a b o u t a c h a r a c t e r i s t i c c h a n g e

in t h e r e f r a c t i v e i n d e x of o rgan ic s u b s t a n c e s .

F o r t h i s r e a son , d / - leuc ine a n d dl-norleucine, t w o a m i n o ac ids of

w h i c h t h e n'~ is n e a r l y i d e n t i c a l a t 20 ~ C, h a v e b e e n s u b j e c t e d to m e a s u r e -

m e n t s of r e f r a c t i v e i n d e x b e t w e e n 20 ~ a n d 50 ~ T h e r e su l t s a re s u m m a r i s e d

in t h e fo l lowing t a b l e :

T a b l e IV

Amino Acid

d/-l~orleucine . . . . . . . . . dl-Leucine . . . . . . . . . . .

n ' ~ 20 ~

1.522 1.538

A m i n o A c i d n'g 20 ~

n'i0 50 ~

1.515 1.527

I d/-Norleucine . . . . . . . . . [ 1.558 d/-Leucine . . . . . . . . . . . [ 1.558

u'g 50 ~

An'~ A T ~ 50/20

0.00026 0.00040

zJntg

A T ~ 50/20

1.547 0.00033 1.550 0.00023

554 A. Lacour~ and N. Delande: [Mikrochim. Aeta

These values and the corresponding diagramms demonstrate clearly that temperature has very little effect on the refractive index of these two amino acids, and that measurements at different temperatures have no additional use for identification purposes.

Compared with the variation in refractive index of liquids up to 200 ~ , which is 0.00045, the variation for amino acids is no more characteristic (the same variation in refractive index of glass is 0.0000022).

5O

r

50

:~:: = 0,.: ~

Le~:oi~:e u:l

~ u = 0, 00040 ,a i~:

= O, OOOZJ

Z6 i :,:oo

iVor/eL:ui~e dZ

A ~ ' O, O00ZE

J ~ :,000J3

/ :o: /7 = O,, 0 0 1 : 7 , I _ _

/,8S0 /,:00 /,::0 :ndem :D

F i g , 1. V a r i a t i o n of refractive index, between 20 ~ C a n d 50 ~ C

Specific Refractivity

Specific refractivity can be calculated for the acids for which the density at 20 ~ is known.

An experimental molecular refractivity has been determined using the mean, n, of the n'~ and n'~ values, and introducing this in the ratio n - - 1

~ . M in which d is the density at 20 ~ and M is the molecular weight

of the acid. Experiinenta,1 values correspond satisfactorily with the calculated ones, as can be seen from Table II .

Refractive Index and Structure of Amino Acids

The amino acids examined can be classified into 4 groups:

Group I of the type, NH2--(CHe)~--COOH, to which belong glycine, fl-alanine and s-aminocaprie acid;

I964/2--4] Identification of Amino Acids: l~efractive Index 555

Group I Ia of the type,

/NH2 CHa--(CH~)n--CH\c00 H'

to which belong dl- and/-~-alanine, dl-~-aminobutyric acid, dl-norvaline, d/-norleucine, d/-#-phenylManine (QH~ instead of CHa) and l-tyrosine (C~H40H instead of CHa);

Group IIb of the type,

C H ~ \ / N H ~ ,

CH~--(CH~) n COOH

to which belong d/-valine, d/-leucine and dl-isoleucine;

Group I I I of the type,

/NH2 COOH--(CH~)~--CH~,COOI t ,

to which belong d/-aspartic, dl- and /-glutamic acids.

I t is instructive to examine use which can be made of the refractive index inside each group, to identify the individuals.

Table V. Group I

NH2--(CIt~)n--COOH

Amino Acid ~s I'., ~

Glycine 240/247

fl-Alanine 202/205

s-Caproic Acid 205/210

1.601 1.607

1.404

I �9 __ s

1.619 1.657

1.524 1.583

1.504 1.536

0.038

0.059

0.032

~ol. Weight

75.07

89.09

131.17

Sper Refractivity

Theor. Experim.

27.3 29.91 29.80

(A = ~ 2.61; § 2.50)

35.1 [ 35.09

(A = - - O.Ol) - - [ _ _

In this group, identification is possible and satisfactory, using n'~, n'g, n ' g - -n ' v , and probably specific refractivity.

By using eutectic melting temperatures 2 identification of the group has been possible in conjunction with 10 partners.

556 [Mikroehim. Aeta A. Lacourt and N. Delande:

Table VI. Group l I a

C H ~--(C H ~)n--C H~cNoHoI_I

Amino Acid M. :P., ~

dl- cr /

CI-I3--CI-I ~ 257 dec.

/-~-Alanine /

C H : - - C t t ~ 266 dec.

d/-NorvMine

d/-Norleucine

dl- [t-Phen ylalanine

225/240 dee.

/-Tyrosine

o -c0N,-c ,-cHQ 245/253 dee.

Densi ty 20 ~

1.424 1.539

1.4011 1.517

- - 1.48i

1.1691 1.522 1.174[

1.456

1.619

1.525

1.579

1.560

1.502

1"558 1

1.657

1.640

n g20~n

0.040

0.043

0.021

0.036

0.038

0.115

:rCiol. Weigh t

89.09

89.09

117.15

131.17

165.19

181.19

Spec. It efractivi~y

Theor. Experim.-

36.69 34.97

(d = - - 1.72)

36.69 34.21

(A = - - 2.48)

58.11 60.59 60.33

(A = + 2.48; ~- 2.22) !

_ _ [ - -

i !

81.45 ! 72.46 [

(z~ = - - 8.99)

Identification at room temperature for members of this group would be specially interesting, as each one decomposes on melting.

n'~ and n'g - - n'~ are favourable for all these acids ; specific refractivity seems to be very satisfactory. Euteetie melting mixtures have already been suggested as interesting for identification, using 7 partners.

n~' is very characteristic and useful, but none of the other values can be recommended for identification.

Eutectic melting mixtures offer better means for identification in this group, using 8 recommended partners ~.

n'g, n ' , - n'~ and specific refractivity can be used for identification of these acids. Eutectie melting mixtures with 7 recommended partners are also very good 2.

1964/2--4] 557 Identification of Amino Acids: Refractive Index

Table VII. G r o u p I I b

C H 3 \ JNH2 / ~ C H - - ( C H 2 ) n - - C H (

CH3--(CH2) n" \ C O O N

Amino Acid 1~. P., ~

dl -Val ine CH~\ / C H 3 / C H - - C H \

232/241

d/-Leucine

CH3~'~CH__CH2__CH d C H a / \ 242/252

dl-Isoleucine C2H5\ /

/ C H - - C H . CH z \

235 dec.

Density 20 ~

1.316

1.191

1.580

1.53s I

1.552

n'g

1.602

1.558

1.581

0.022

0.020

0.029

]Viol. Weight

117.15

131.i7

131.17

Spec. Refractivity

Thcor. Experim.

50.57 52.61

(d = -{- 2.04)

57.72 60.35

(A = -4- 2.63)

Table VIII. Group llI

.NH

COOH--(C H ~ ) ~ - - C H % c o o H

Amino Acid lYi. P., ~

dl-Aspartic Acid

COOH--(Ct t3)2- -CH( > 310

dl- Glutamic Acid

COOH--(C~12--CK / 180/183

l- Glutamic Acid

C00H--(CH2)2--CHQ 194/198

Density 20 ~

1.524

1.526

1.628

1.539

1.663

1.460

1.538 1.504 1.619

0.104

0.013

0.115

Mo]. Weight

133.10

147.13

147.13

Spec. Refractivity

Theor. Experim.

45.75 46.02

(A = -~ 0.27)

53.55 53.68

(A = + 0.13)

53.55 53.57

(~ = + 0.02)

In a previous paper s the relationship between chemical structure

and refractive index of the same amino acids has been examined in detail.

558 A. Lacourt and N. Delande: [MiM'ochim. Acta

The effect on refractive index of 10 dl- and /-compounds has been pointed out. The increase of length of the earboxylated chain, the branching, and the substitution of CH~ by COOH, C6H5, C6tt4OH or CH2SH also influences the refractive index of amino acids.

I n three cases isomeT~ with the same molecular weight can be differentiated through refractive index. Comparison is possible for isomers of molecular weight of 89.09, 131.17 and 117.15. The appropr ia te values are gathered in the following tables.

Table IX. I s o m e r s of M o l e c u l a r W e i g h t 89.09

Amino Acid ~. P., ~

fl -Alanine 202/205

d/-cr 257 dec.

l- ~-Alanine 266 dee.

De:~ty

1.404

1.424

1.401

1.524

1.539

1.517

n'g n'g--ns~

1.583 I 0.059

1.579 I 0.040

1.560 I 0.043

Spec. Refractivity

Theor. ]~xperim.

35.10 35.09

36.69 34.97

36.69 34.21

Discre- pancy

- - 0 . 0 1

- - 1.72

- - 2.48

These acids could be easily identified through nS, ng', n o' - - n ~ ' and specific refractivity.

Very good eutectic melting temperatures are obtained ~dth the same acids and 7 different other compounds 2.

TableX. I s o m e r s of Molecu l a r W e i g h t 117.15

Amino Acid ~i. P., ~

d/-VMine 232/241

/-Valine 198/199

d/-I~7orvaline • 275 dec.

Density 20 ~

1.316

1.230

1.580

1.553

1.481

~t'g

1.602

1.585

1.502

i

n g20~n

0.022

0.032

0.021

Spec. l%efractivity

Theor. Experim.

50.57 52.61

50.57 54.20

Discre- pancy

d- 2.04

d- 3.63

The n'~, n'g and n ' g - - n ' ~ values are excellent for the identification of the amino acids of this group. Comparison with eutectic melting temperatures is not possible through lack of information.

n'~ and n ' g - - n ' ~ are quite caracteristic of the individuals of this group of amino acids, and are suitable for identification.

1964/2--4] Identif icat ion of Amino Acids: Refractive Index

T a b l e X I . I s o m e r s of M o l e c u l a r W e i g h t 131.17

559

Amino Acid ~. P., ~ Density 20 ~ n'lo

r _ _ t

n'g ~ g20 on ~ Spee. Refractivity

Theor. Experim.

Discre- pancy

s-Amino Caproic Acid 205/210

d/-Leucine 242/252

/-Leucine 285/295

d/-lsoleucine 235 dec.

/-Isoleucine 278

d/-Norleucine 250

/-Norleucine 269 dec.

1.191

1.165

1.169 1.174

1.504

1.538

1.525

1.552

1.536

1.522

1.524

1.536

1.558

1.543

1.581

1.560

1.558

1.554

0.032

0.020

0.018

0.029

0.024

0.036

0.030

57.72

57.72

58.11

60.35

60.12

60.59 60.33

-4- 2.63

-b 2.40

zc 2.48 -4- 2.22

I t mus t be no ted tha~ when dens i ty has been de te rmined , specific ref rac t iv i t ics ( theoret ica l a n d exper imenta l ) cor respond wi th sa t i s fac to ry accuracy . Eu tec t i c mel t ing tes t s have been t r i ed wi th four of these amino acids and seven eutect ic par tners . They also p rov ide ve ry good iden t i f i ca t ion 2.

Conehs ions

Useful in fo rmat ion can be de r ived f rom ref rac t ive index measu remen t s as descr ibed above, which can be used to iden t i fy amino acids belonging to the same group, or to recognise isomers hav ing the same molecular weight . The measurements also give in teres t ing in fo rmat ion regard ing s t ruc tu ra l effects b rough t abou t b y chain branching and chemical subs t i tu t ion in amino acids.

Being per formed a t room t empera tu re , these measurement s are specia l ly r ecommended for the ident i f ica t ion of amino acids and o ther substances which are also l iable to be a l te red b y rise of t e m p e r a t u r e and for which the mel t ing t e m p e r a t u r e has less significance.

The dimensions of amino acid crys ta ls do no t al low de t e rmina t i on of real ref rac t ive indices b y the immers ion method , b u t two convent iona l numbers - n'~ and n'g - which are components of the real values, can be de te rmined . These have p roved to be reproducib le a t 20 ~ in the 39 in- d iv idua l s examined , and pe rmi t ident i f ica t ion in 50~o of the cases. W h e n

560 Laeom~ and Delande: Identif icat ion of Amino Acids: l~efraetive Index

the difference n ' ~ - - n ' ~ is considered, the possibil i t ies of ident i f ica t ion rise to 70%.

Combined with eutect ic mel t ing t e m p e r a t u r e tes ts , iden t i f i ca t ion of amino acids is as feasible as t h a t of a n y o ther organic compounds .

Summary

The use of a range of cons tants re la ted to re f rac t ive index for the ident i f ica t ion of amino acids is described. These cons tants m a y be de t e rmined microscopical ly and, combined wi th eutect ic mel t ing t empera tu res , pe rmi t ident i f ica t ion of a range of 39 amino acids examined in th is s tudy .

Zusammen[assung

Die Anwendung physikMischer, mit dem Brechtmgsindex zusammen- h/~ngender Konstanten fiir die Identifizierung yon Aminos/~uren wurde beschrieben. Diese Konstanten lassen sich mflcroskopisch ermit teln und geben in Kombinat ion mi t eutektisehen Temperaturen die MSgliehkeit, 39 versehiedene, in dieser Arbei t untersuchte Aminos/inren zu identifizieren.

Rdsumd

On d4crit l'emloloi d 'une sdrie de eonstantes en rappor t a rea Findice de rdfraetion pore " l ' identif ieat ion des acides aminds. Ces eonstantes loeuvent gtre ddtermindes par microscoloie et, combindes avec les temlodratures de fusion de l 'euteet ique, loermettent 1'identification d'une sdrie de 39 acides amin6s dtudids au cours de cette recherche.

References

1 L. Kof ler and A . Ko/ler, Thermomikromethoden. Irmsbruck : Univers i t~s - Verlag Wagner. 1954.

2 A . Lacourt and N . Delande, Proceedings, Internat ional Symposium on Mieroehemical Techniques 1961. N e w Y o r k : Interscience. 1962. PP- 259, 273.

A . Laeourt , G. Sommereyns , C._~raneotte and N . Delande, J . loharm. Belgique 8 (85), 535 (1953).

a N . Sehoorl, Organische Analyse. Vol. I , N . V . D . B . Amsterdam: Centen's. 1935.

5 A . I . Vogel, Textbook of PraeticM Organic Chemistry. London: Long- roans, Green and Co. 1948. lo. 898.

6 A . Lacourt and N . Delande, Mikroehim. Acta [Wien] 1962. 48.