54.

Q U A N T I T A T I V E G A S CHROMATOGRAPHY OF T H E TWENTY N A T U R A L P R O T E I N A M I N O A C I D S

D A V I D L . STALLING and C H A R L E S W . GEHRKE

Progress i n the development of a quant i ta t ive method f o r the

Few invest igators sought t o a r a l y s i s of amino acids by gas chromatography has been hindered by the ap- proach which many invest igators employed. study the chemical reactions which would lead t o high y ie lds of der ivat ives which possessed su i tab le s t a b i l i t y and chromatographic propert ies .

A su i t ab le v o l a t i l e der ivat ive of the amino acids should meet the following c r i t e r i a f o r quant i ta t ive gas chromatographic analysis :

1.

2.

3 .

4.

5 .

The der ivat ive should be simple i n i t s formation w i t h no rearrangements o r s t ruc ture a l t e r a t ions a f t e r formation.

The der iva t iza t ion reaction should go t o 95 t o 100% completion.

The der ivat ive must be s t ab le with respect t o t i m e and temperature.

The der ivat ive must have increased v o l a t i l i t y thus lower retent ion t i m e .

The der ivat ive must be i n a form with l i t t l e o r no r eac t iv i ty with the s o l i d support o r l i qu id phase.

To quant i ta t ive ly analyze amimacids by gas-liquid chromatography it is necessary t o form a su i t ab le v o l a t i l e der ivat ive of the amino acid, A rumber o f invest igators have examined various der ivat ives f o r the ident i - f i ca t ion and qua l i t a t ive analysis of amino acids; however, few s tudies have been reported with the primary emphasis toward determining the reaction conditions necessary f o r quant i ta t ive conversion o f the amino acids t o a der ivat ive su i tab le f o r analysis and chromatography. Some of the deriva- t i ves which have been investigated a re the phenylthiohydantion and methyl 2, 4-dinitrophenyl e s t e r s (7), the t r imethyls i ly l N-trimethylsilyl e s t e r s (lo), the n-amyl N-acetyl e s t e r s (6), the n-amyl N-trif luoroacetyl e s t e r s (Z), the mzthyl N-trif luoroacetyl e s t e r s (i1,5,1), and the n-butyl N-trif luoroacetyl e s t e r s (13). Lamkin and Gehrke (7) examined and compared the methyl and n-butyl N-acetyl amino ac id der ivat ives and chose the n-butyl N- t r if luo roa c e ty l e 6 t e rs over the me thy1 N- trif luo mace t y l because no-lo 6 s e s of the n-butyl e s t e r s occurred upon sample concentration. Thirty-six per- cent logs occurred f o r the methyl N-trif luoroacetyl val ine when the samples were evaporated under reduced pressure a t room temperature. L i t t l e d i f f e r - ences i n chromatographing the methyl and n-butyl N-trif luoroacetyl e s t e r s were observed. cent conversion of the amino acids t o the n-butyl N-trif luoroacetyl

Gehrke, Lamkin, S t a l l i n g , a n d Shahrokhi (3) found the per- -

55.

derivat ive t o be 96 percent o r g rea t e r f o r a l l the protein amino acids studied except threonine and cyst ine. The n-butyl N-trif luoroacetyl de- r iva t ives were found t o be s t ab le with respect to time under anhydrous conditions and s ingle chromatographic peaks were obtained f o r a l l of the na tura l protein amino acids except arginine and tryptophan. These two were quant i ta t ive ly obtained by sealed tube acylat ion (12) .

Direct formation of the higher a lkyl e s t e r s of the amino acids has been a problem due t o the lack of s o l u b i l i t y of cyst ine, h i s t i d ine , and lys ine i n the higher alcohols. polar solvent, (13) has been reported t o a id i n the e s t e r i f i c a t i o n of these amino ac ids . However, when t h i s method was employed i n our labora- to ry reproducible r e su l t s were not obtained. Lamkin and Gehrke (8) showed t h a t the amino acids reac t with dimethyl formamide t o y ie ld formyl deriva- t i ves , thus preventing quant i ta t ive der iva t iza t ion .

The use of dimethyl formamide, a

While cyst ine, lys ine , and h i s t id ine a r e insoluble i n n-butanol t h e i r methyl e s t e r s dissolve readi ly . Ut i l iza t ion of these soluir i l i ty data resul ted i n the i n t e r e s t e r i f i ca t ion procedure reported by Gehrke, e t a l . , ( 3 ) . This method converts amino ac id methyl e s t e r s to - n-butyl e s t e r s using anhydrous HC1 q,s the in t e re s t e r i f i ca t ion ca t a lys t . Thus, the so lub i l i t y problem associated with d i r e c t formation of the - n-butyl e s t e r s has been circumvented.

--

Sta l l i ng and Gehrke (12) obtained a s ingle chromatographic peak f o r arginine and tryptophan w i t h 1oc p r c e n t conversion when the t r i f luoroace ty la t ion was conducted :r a sealed tube a t a temperature o f 15OoC f o r f i v e minutes. When the other amino acids were acylated a t the higher acylat ion temperature, e i t h e r s ingly o r i n mixtures, no adverse e f f ec t s were observed. Thus allowing analysis of a l l of the amino acids as s ingle der ivat ives with a s igni f icant savings of t i m e .

Sl ides

Sl ide - 1 gives the development of t h i s procedure a t the University of Missouri.

Sl ide 2 shows the d e t a i l s o f the der iva t iza t ion procedure t o form the v o l a t i l e - n-butyl N-trif luoroacetyl e s t e r s o f the amino acids .

-

Slide - 3 demonstrates losses of the methyl e s t e r der ivat ives and the reason f o r se lec t ion of the - n-butyl der ivat ive.

Sl ides - 4 through 7 show the y ie ld s t ruc ture and molecular weight of the der ivat iFes when in t e re s t e r i f i ed a t 90°C f o r 3 hours.

Sl ide - 8 shows the increase i n the y ie ld of threonine and cystine obtained from increasing the in t e re s t e r f i ca t ion temperature t o lOOOC for 2 3 hours.

Sl ides - 9 through 11 show the chromatogram and chemistry of arginine. 100 percent conversion was achieved when the acylat ion w a s done a t 15OoC and a su i tab le der ivat ive was formed.

56.

Slide - 1 2 gives the chromatographic condition f o r the resolut ion of the amino ac id der iva t ives .

Sl ides 13, 14, 15, and 1 6 show the e f f ec t s of the l i qu id phase employed i n - - - the s e p a r a t i o n o f the amino acids .

S l ide - 1 7 is representat ive of the ca l ibra t ion curves obtained f o r the amino acids i n a 100 u mole concentration range. No s t a t i s t i c a l b i a s was evident i n t h i s range.

Sl ide 18 - is the r e s u l t of the s t a t i s t i c a l analysis of the ca l ibra t ion curves.

Sl ide 1 9 shows the gas chromatographic and ion-exchange comparison of the ana lys i s of synthet ic mixtures.

Sl ides - - 20, 2 1 and 22 represents the GLC analysis of addi t ional mixtures of aminoacids .

Slide - 23 gives the reproducabili ty of the analysis . deviations of l e s s than 2.5% were obtained. l i m i t o f the d isc integrat ion method which was employed.

Relative standard This represents the

Sl ides - 24 and 25 a r e representat ive of the chromatograms obtained from pu r e p r o t e in hydrolysate 6 .

Slides - 26, 27 and 28 a re the data obtained from the analysis of proteins byGLC a= by ion-exchange methods of analysis . of amino acids recovered agrees t o approximately one percent absolute. This represents good agreement between the two pro- cedures and demonstrates t h a t amino acids can be quant i ta t ive ly analyzed by gas chromatography.

The t o t a l amount

The appl icat ion of t h i s method t o protein hydrolysates has given good agreement w i t h the ion-exchange method of analysis . serum albumin, a commercial soybean pro te in preparation (EDI-PRO), and a Kappa casein were compared and the t o t a l amount of amino acids recovered agreed w i t h i n one percent absolute. t i v e analysis of amino acids by gas chromatography is possible. vance i n the analysis of amino acids should prove t o be a stimulus f o r rapid progress i n invest igat ions where the analysis of amino acids is e s sen t i a l .

Analysis of bovine

This demonstrates t h a t the quant i ta- This ad-

57.

Slide 1

LITERATURE REFERENCES AND GRADUATE RESEARCH - UNIVERSITY OF MISSOURI Ref e renc e Research Emphasis Author

Biochem. and Bio h s Res. Quantitative gas chromatography of

Anal. Chem. 37, 1965, Ph.D. Oxidation to aldehydes; reduction - g u n 9 64 to alcohols with LiAlH4; prep.

corn., m;*' - amino acids. Gehrke, Lamkin, Stalling,

Lamkin,a W.M., Gehrke, C.W. Shahrokhi -

and sep. of n-butyl-N-acetyl esters; esteFification and inter- esterificationJprep. of "-butyl- N-TFA esters.

Shahrokhi ,a F.

Stalling,a D.L.

Gille,a G.L.

M. Sc. January 1965 Yield and structure of n-buty1-N- TFA esters of ala., gTy., leu., pro., pheala., asp. chromatogra- phic separations.

Doctoral research, Ph.D. Yield and structure of ;-butyl-N- June 1966. NASA Fellow TFA esters (16 a.a.) acylation

studies on arg., try. Quanti- tative organic reaction conditions

M. Sc. research, January precision and accuracy of inter- 1966 esterification.application to

biological systems.

Research supported by National Science Foundation grants C-18722 and GB-14'26, 1961 - present; Experiment Station Chemical Laboratories, and Research Council, University of Missouri.

aDr. Charles W. Gehrke - Dissertation Supervisor

Slide 2

Preparation of 2-butyl N-trifluoroacetyl Esters of Amino Acids

0 0 It II HCI __ R-CH-C-0- + CHlOH ~ ; m I c m p , - + R-CH--C-OCHa + Hz0 (1)

I NHs+CI-

I XH,+

0 0 It II

R-CH-C-OCHI + BuOH 9t?A.+ R-CH4-OBu + CHIOH (2) I NHa +C I - I

I t I1 I I

II

NHa+Cl-

0 0 0 0 II

I R -CH--C-OBu + (F&C)rO R2z2A;.d R - C H --C--OBu + CFIC --OH (3)

N H - C 4 F s

0

I NHs+

sa.

Slide 3

Table II. Volatilities of Methyl and n-Butyl N-Trifluoroacttyl Esters of Amino Acids Peak area, sq. in.

Before A f r Recovery, Derivative

- .

evaporation evaporations % -Methyl N-trifluornacetyl valine 0.495b 0.316b 64 n-Butyl N-trifluoroacetyl valine 1.255 1.2430 99 n-Butyl N-trifluoroacetvl alanine 1.71 1.7080 100 n-Butyl N-trifluoroacetyl glycine 1.37 1.3270 97

a At room temperature (26' C.) and about 4 mm. of mercury. b Average of four independent determinations. 0 Corrected with internal standard.

Amino Acid

1. A l i p h a t i c

(a) Glycine4

( b ) Alanine*

( c ) ~ e u c i n e ~

Slide 4

YIELD AND STRUCTURE OF n-BUTYL N-TRIFLUOROACETYL ESTERS

Mo 1 ec u 1 a r ~ m p i r i c a l Weight Formu l a

( d ) Phenylalanine" 318.31 C i 5 H 1 eN03F3

2. Dicarboxyl ic

( a ) A s p a r t i c Acid4 341.33 C I ~ H ~ Z N O ~ F ~

S t r u c t u r e

C ~ H S O O C C H ~ NHCOCF3

C4HsOOCCHNHCOCFs I

CH3

C~HSOOCFHNHCOCF~ C H & H ( C H ~ ) ~

C~HSOOCCHNHCOCFS 1

CHZCGH,

C~HSOOCFHNHCOCF~ CH2 COOC+ H s

Yie ld $z

100.9

97.9

100.1

96.9

99.2

lconfirmed by e l emen ta l a n a l y s i s f o r C , H , N , F, and S. 'Average of two independent de t e rmina t ions . 3 P u r i f i e d by h igh vacuum sub l ima t ion . * P u r i f i e d by vacuum d i s t i l l a t i o n .

59.

Slide 5 YIELD AND STRUCTURE OF n-BUTn N-TRIFLUOROACETYL ESTERS

Molecular ~ m p i r i c a l ' Amino Acid Weight Formula

3. Hydroxy

( a ) s e r i n e 3 353 - 23 C I I H I ~ N O ~ F ~

(b! Threonine3 367.21 c I z H I 5 No 5F 6

S t r u c t u r e '

'Confirmed by e lementa l a n a l y s i s f o r C , H , N , F, and S.

'Average of two independent de te rmina t ions .

3 P u r i f i e d by High Vacuum Sublimation.

Slide 6 YIELD AND STRUCTURE OF n-BUTYL N-TRIFLUOROACETYL ESTERS

Amino Acid

4. S u l f u r

( a ) c y s t e i n e 3

( b ) Cystine"

5. Basic

( a ) ~ y s i n e ~

(b) Argin ine4

Mo 1 e cu l a r Empir ica l ' Weight Formula S t r u c t u r e

C 4 HsOOCyHNHCOCF3 CHz SCOCF3

C~HSOOCFHNHCOCF~ CHz CH2S 1

C4 HsOOCCHNHCOCF3

Yie ld g2

102.1

90.4

99.7

Yield %'

92.6

88.0

'Confirmed by e lementa l a n a l y s i s f o r C , H, N , F , and S. 'Average of two independent de t e rmina t ions . 3 P u r i f i e d by h igh vacuum subl imat ion . 4 P u r i f i e d by s i l i c i c a c i d column chromatography.

60

Slide 7

YIELD AND STRUCTURE OF n-BUTYL N-TRIFLUOROACETYL ESTERS

Mo 1 e cu la r b p irical ' Formula structure ' Amino Acid Weight

6 . Heterocyclic

CH2 ,CHCOOC+Hs 1 N

COCF3

(b) Hydroxy-proline4 379.23 C 1 3 H 1 5 NO5 F 6 F3 C COO C H-C HZ I I C+N, C HCOOC 4 H 9

kOCF3

( c) His tidine3 403.28 Ci4H15N304F~ C4H900CFHNHCOCF3

F2 HC=C

I I N, ,NCOCF3 'C H

'Cmfirmed by elemental analysis for C, H, N, F, and S. 'Average of two independent determinations. 3Purified by high vacuum sublimation. 4Purified by vacuum distillation.

Sl ide 8

YIELD OF DERIVATIVE AT DIFFERENT INTERESTERIFICATION TEMPERATURES AND HC1 CONCENTRATIONS

Amino Acid -- Yield, $

1.25 N HCla 3.25 N HCla 900 CP 1000 c.c 1000 cc

Threonine 90.3 99.6 99.5 - 90.5 100.5 - 99.8 av. = 90.4 100.0 99.7

Cystine 88.0 99 .1 98.2 97.3 100.0 88.0 - -

av. = 88.0 98.2 99 .1

aConc. of HC1 in 2-butanol for interesterification. bInteresterified at 90@ C . for 3 hrs. 'Interesterified at looo C. for 2 hrs.

Yield g2

96.8

98.4

96.4

Slide 9

CHROMATOGRAM OF n-BUTYL-N-TRIFLUORACETYL ESTER OF ARGININE

61

Slide 10

Temperature Progrom Rate 13DyMin

Initio1 Temperature 100 OC

-Solvent

005 p m l e Mono-Trifluoroocetyl Arginine Derivative

235OC

ACYLATION REACTIONS OF E-BUTYL ESTER-HC1 OF ARGININE

62 Slide 11

n-BUTYL N-TRIFLUOROACETYL ESTER OF ARGININE ACYLATED I N SEALED T U B E A T 150" C.

I I

2iOO 190" 1 d o o

ARGININE 210"

- n-BUTYL STEARATE 160"

PROGRAM R A T E ( 7 . 9 O C./MIN.)

ON COLUMN INJECTIOF RELATIVE AREA = 2.80

1 ---I -1-, ---. L,,---I

15 12 9 6 3 0

MINUTES Sample Size: ( 5 pl.), 1 .28 IL moles arginine and 75.3 n moles stearic acid. Sens. 3 K . , Dusl, 100- X 0.2 cm. i . d . g l a s s columns of 0 .25 /0 .75 (w./w.$) EGSS-X/DEGS on 60-70 a . w . Chromosorb G.

Sl ide 12

M c --I 0

5 6 =

-

63

(1159:

ISOLEUCINE (1Os"Cl I

L

CHROMATOGRAPHIC INSTRUMENTAL CONDITIONS

INSTRUMENT USED: F and M %del 300 linear programned temperature gas chromatograph, and F and M Model 1609 Flame Ionization attachment, with a F and M Model 400 column oven and detector module.

COLLTMN TEMPERATURE: Initial 67O C., final 218' C.

Program Rate . . . . . . . . . . . . . . . 3 . 3 O C./rnin. Detector Cell Temperature (at start) . . . 123O C. Sensitivity . . . . . . . . . . . . . . . 1/32 Carrier Flowi N. . . . . . . . . . . . . . 38 rnl./min. Air (to detector) . . . . . . . . . . . . 450 ml./min. Hydrogen (to detector) . . . . . . . . . . 36 ml./min. Chart Speed . . . . . . . . . . . . . . . 1/3 inch/min.

COLUMN: 1.00 meter x 4 nun. i.d. borosilicate-glass colunm packed with 60/80 mesh acid-washed Chromosorb W and mixed substrate phases of 0.75/0.25 w./w.$ of DEGSIEGSS-X.

DEGS - Diethylene glycol succinate. EGSS-X - Ethylene glycol succinate copolymer with a l o w $ of siloxane.

I- 1

Slide 13

I(

LEUCINE

'HEIIY U~NINE & ASPARTIC ACI (154°C)

Figure 1. Separation of five n-butyl N-trifluoroacetyl esters of amino acids using a 1.00 meter x 4 mm. i.d. borosilicate-glass column of 60/80 mesh acid-washed Chromosorb W, 1.00% w./w. of Carbowax 6000.

Slide 14

PROGRAM (3.3T//min)

PHENY MLANlNF [118"C]

LEUCINE ISOLEUCINE & GLYCINE 1112x1

(lOSoC]

L L

5 10 15 20 25

SPARTIC ACID (152°C)

Figure 2. Separation of five =-butyl N-trifluoroacetyl esters of amino acids using a DEGS column. 1.00% w./w. DEGS coated on 60/80 mesh acid-washed Chromosorb W, packed in 1.00 meter x 4 mm. i . d . boro si 1 i c a t e - g la s s column .

Slide 15

PHENYLALANINE (149[ PROGRAM

[ 3.PC/min]

GLYCINE [117'C] I I LEUCINE

ISOLEUCINE

I2OC)

I _ _ _ , 0 5 10 15 1'5"c!L 20 MINIITFS 25 30 35 40 45

I....." I L"

Figure 3 . Separation of five E-butyl N-trifluoroacetyl esters of amino acids using a DEGS-Carbowax 6000 column in series. 60f80 mesh acid-washed Chromosorb W, packed 75 cm. of 1.00 meter x 4 m. i.d. borosilicate-glass column followed by 25 cm. of 1.00% w./w. Carbowax 6000 coated on Sol80 mesh acid-washed Chromosorb W.

1.00% w./w. DEGS on

65 Slide 16

'RCIGRAM 3.3"C./min) -

9 s g r- m,

A ?

P ? u m s

0 IO I5 20 25 30 35 40 45 50 MINUTES

F i g u r e 4. Sample s i z e : 5 Lbl. Each peak r e p r e s e n t s 2.5 a. o f amino a c i d .

aDiacy l d e r i v a t i v e ; bMonoacyl d e r i v a t i v e

'Acylated f o r 18 hours i n p re sence o f anhydrous N a 2 C 0 3 , t hen added t o t h e m i x t u r e

Chromatogram o f 20 2 Buty l N - T r i f l u o r o a c e t y l Amino Acid Esters

o f 19 amino a c i d s p r i o r t o chromatography.

4 LI Q 4

Slide 17

CALIBRATION CURVES

5 pl injpcted

3 MICROMOLES

per 2 ml.

66

Slide 18

SLOPE FACTORS AND RELATIVE STANDARD DEVIATION FOR 18 AMINO ACID DERIVATIVES

Relative Standard Amino Acida Slope F x t o r Deviation, SC

2. Val ine 2 4 50 0.27 3. Iso leuc ine 29.33 2. 80 4. Glycine 16.26 0. 19 5. Leucine 27.60 2.34 6. Threon ine 22.91 0.42 7. P ro l i ne 25. 86 I. 66 8. Ser ine 19.90 0.32 9. Hydroxyprol i n e 27.77 I. 68

IO. M e t h i o n i n e 24.62 I. 94 11. Pheny la lan ine 41. 83 3. 56 12. Aspart ic acid 32.39 I. 33 13. Glutamic acid 35.95 I. a3 14. Tyrosine 3 8 89 I. 25 15. Lysine 31. 12 I. 7a 16. Hist id ineb 19.08 I. 99 17. A r g i n i n e b 21.44 I. 25 18. Cystineb 19.81 3.27 19. Buty l Stearate 72.72 I. 50

Separated on 0.7510.25 w l w 70 DEGSIEGSS-X coated on 60/80 mesh a. w. a

Chromosrob W. b Separated on 5% w l w DC-550 coated on a.w. 60180 mesh Chromosorb W.

I. A lan ine 1 8 8 9 a r e a l p M I. 40

Y = Area at X p moles.

Slide 19

GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF AMINO ACIDS I N A MIXTURE

Mi l l iqrams of Amino Acid Added Recovered by GLCa I o n ExchangeD

I 2 3 Ave. Ave.

A lan ine 100 loo. I 102.9 103.2 102. I 99.0

I soleucine 100 95. 0 loo. 3 98.4 97. 7 99.2

Leucine 100 104.0 105.4 IM. 7 IGI. 6

Prol ine 100 101.3 104. a 101.7 102.6 105.3

Ser ine I W 101.3 102.7 101.3 101.7 loo. 4

Hydroxyprol ine I W 104.6 103.5 98.5 102.2 loo. 0

Phenyla lan ine I W loo. 9 99.0 99.9 loo. 4

Arg in inea I W 98.3 97.4 97. 2 97. 7 102.0

Glutamic acid 100 105.3 102.3 96.5 101.7 99. 8

Cystinea 100 101.0 9 8 4 99. 7 99. 7 100.8

a A rg in ine and cystine were separated on a I m x 3 mm i. d. 5% Dow Corning-5% support coated on 60180 mesh acid washed Chromosorb W column. A l l o thers were separated on a I meter x 3 mm I . d. 0.7510.25 wlw 90 DEGSIEGSS-X suoport coated on 60180 mesh acid washed Chromosorb W column. b Average of at least two determinations. c n -bu ty l Staarateintsrrnal standard, 5 11 of 6 mi f i na l so lu t ion injected d i rect ly on column.

67

Slide 20

GAS CHROMATOGRAPHIC ANALYSIS OF AMINO ACIDS I N A MIXTURE

Slide 21

GAS CHROMATOGRAPHIC ANALYSIS OF AMINO ACIDS I N A MIXTURE

Mi l l iqrams of Amino Acid Amino Acid Added Recovered by GLCa Ave.

Mi l l iqrams of Amino Acid Amino Acid Added Recovered by GLCa Ave.

Methionine 50.0 49.3 50. I 49. 7 Tyrosine 50.0 48.9 50.1 49. 5 Histidine 50.0 49. I 49.9 49. 5 Arg in ine 50.0 51.2 50.5 50. 9 Cystine 50.0 50.0 50.3 M. 2

an-Butyl Stearate in te rna l standard, 5 p l of 6 ml f ina l solution injected direct ly on column.

bLyophilized samples dried for 24 h over P2O5 at r m m temperature in vacuum. Column - I m x 3 m m of 5 w/w% DC-5% on 60-80 a. w. Chromosorb W.

Slide 22

GAS CHROMATOGRAPHIC ANALYSIS OF AMlNOAClDS I N A MIXTURE

Glycine 50.0 49. 7 50. 5 50. I

Valine 50.0 49.9 50.8 50.4

Threonine 50.0 49. 8 50.3 50. I

Phenylalanine 50.0 51. 0 50.2 50.6

Glutamic Acid 50. 0 49. 7 50.3 50. 0

Lysine 50.0 50. I 49.3 49. 7

an-Eutyl Stearate internal standard, 5 pI of 6 m i f ina l solution injected directly on column. Column - I m x 3 mm 01 0.7510.25 wlw% DEGSIEGSS-X on 60-80 mesh a. w. Chromosorb W.

Slide 23

RELATIVE STANDARD DEVIATION FOR GAS CHROMATOGRAPHIC ANALYSIS OF AMINO

ACID MIXTURES

Mi l l iqrams of Amino Acid Amino Acid Added Recovered by GLCa Ave. Amino Acid

Relative Standard Deviation a

I soleucine

leuc ine

Hydroxyproline

Aspartic Acid

Phenylalanine

Glutamic Acid

Lysineb

Arginineb

Cystineb

5Q.0 48.6 50.2 49.7 49.5

50.0 50.9 50.3 49.2 50. I

50.0 50.3 50.9 50.7 50.6 50.0 49.9 50.7 50.9 50.5 50.0 49. 5 51.0 49.9 50. I

50.0 49.7 49.3 50.4 49.8 50.0 48.7 49.3 50. I 49.4

50.0 50.9 50. I 51.4 50.8 50.0 51.3 50. 7 51. 8 51.3

an-Butyl Stearate internal standard. 5 y l of 6 ml f ina l solution injected directly on column. Column - I m x 3 mm 0.75,O. 25 w/w% DEGSIEGSS- Xon 60-80 mesh a.w. Chromosorb W.

bColumn - I m x 3 rnm 5 w/w% DC-5% on 60-80 Chromosorb W.

Alanine

Valine

I soleucine

Glycine

Leucine

Threonine

Prol ine

Ser ine

Hydroxyproline

Meth ion m e Phenylalanine

Aspartic Acid

Glutamic Acid

Ty ros i n e

Lysine Histidine

Arg in ine

Cyst in e

I. 71 2.28

I. 64 2.04

I. 84 0.86

I. 91 0. a1 0.66 I. 12

I. 56

I. 06

I. 40

I. 16

I. 42

I. 28

I. 36

I. 12

aComputed from at least th ree independent determinations.

68

Slide 24

"A/* I -_-I

0 10 15 20 25 30 35 40 45 50 Minutes

Figure 5. Chromatogram of kappa casein hydrolysate. Sample Size: 5 pl. (25 p G. total amino acids). Column: 1.00 x 4 m. i.d. borosilicate-glass column packed with 60/80 mesh acid-washed Chromosorb W and mixed substrate phases of 0.7510.25 w./w.$ of DEGS/EGSS-X.

15

Figure 6. Chromatogram o f bovine serum albumin hydrolysate. Sample Size: 5 p1. ( 2 5 u gm. total amino acids). Column: 1.00 meter x 4 m. i.d. borosilicate-glass column packed with 60180 mesh acid-washed Chromosorb W and mixed substrate phases of 0.7510.25 W. Iw. $ of DEGS~EGSS-X.

S l i d e 26

AMINO ACID ANALYSIS OF BOVINE SERUM ALBUMINa 69

wlw sb Amino Acid Gas Chromatographyb Ave. Ion Exchangec

Alanine 5. 71

Valine 5. 55 I soleucine 2.29 Glycine I. 63 Leucine II. 61 Threonine 5.43 Proline 4. 79 Serine 3. 81 Methionine 0. 71

Phenylalanine 6.47 Aspartic Acid IO. 39 Glutamic Acid 17.M

Tyrosine 5.21

Arginine 5. 55

3.38 Cystine 5. 37

Lysine 12.31

Hi st i d i n e

5 . 53 5.67 5.69 5.33 2.32 2.22 1.53 1.66

12. 26 II. 85 5.31 5. 30 4.71 4.72 4. II 3.65 0.68 0.54 6. M 6.37 10.36 IO. 19 16. 75 16. 70 4.97 5.33 12.21 12.03 5. I1 5. 40 3.46 3.30 5.45 5.40

Total

5.67 5.58 2.28 1.61 II. 90 5.34 4. 74 3.86 0.66

6.44 IO. 31 16. 83 5. 17

12. 18 5. 35 3.38

5. 64 5.4A 2. 38 I. 70

11.68 5.46

4.78 4.05 0. 70

6.42 IO. 27 16.43 5.24

12. 16 5.69 3.64 5.65

107.33 -

aProtein hydrolyzed for I8 h at 1050 C i n a sealed tube with constant boiling HCI. bn -Butyl stearate used as internal standard. CEach value represents an average of two independent determinations, norleucine as internal standard.

Slid. 27

AMINO A C I D ANALYSIS OF RALSTON -PURINA EDI-PRO

WIW qQ Amino Acid Gas Chromatographyb Ave. Ion Exchangec

Alanine

Valine

I soleucine Glycine

Leucine Th r eon i n e Prol ine Serine Methionine Phenylalanine

Aspartic Acid

Glutamic Acid

Lysine Tyrosine Arginine

Cystine

3.31 4. 15

4.08

3.23 6.92 2.34 4.43 4. 17

0. 82 4.35

IO. 85 18.93

5.30 3. M 6.13 o. m

3.48 4.36 4.21 3.47

6.98 2. 71 4.53 9. 23

.71 4.40

IO. 97 19.03 5. 39 3.37

6.32 0.44

Total

3.39 4.26 4. 15 3.35

6.95 2.53 4.48 4.20

0. 77 4.38

IO. 91

18.98 5.35

3.44 6.23

0. M 83.67

3.53

4.23 4.24 3. 57

6. 80 2.98 A. A4

4.48

0. 84 4. 51

11.08 18. 73

5.24 3.21 6.36 0. 50

84.74

aPiotein hydrolyzed for 18 h at I050 C i n a sealed tube with con- stant boi l ing HCI. b?-Butyl stearate used as internal standard. ‘Each value represents an average of lour independent deter- minations, norleucine as internal standard.

S l i d e 28

A M l N O A C l D ANALYSIS OF KAPPA CASEINa

A m i n o Acid wlw I

GLCD Ion Exchange0

Aspar t ic ac id 7. 75 7. 88 T h r e o n i n e 6.20 6.34

S e r i n e 5. 54 5.49

Glu tamic acid 17.92 18.65

P r o l i n e 8. 82 9.25

Glyc ine 0. 87 0.94

A l a n i n e 5.45 5.51

Va l i ne 5. 14 5. 19

Cyst ine 0.42 0.34 M e t h i o n i n e 0.30 0.43

Isoleucine 6.27 6.31

L e u c i n e 5.07 5. 14

Tyros ine 6.97 6. 81

P h e n y l a l a n i n e 2.96 3.07

Ammonia 2.42

Lys ine 6.31 6. 14 His t i d ine 2.04

A r g i n i n e 3. 55 3. 79

Total 89.95 88.86

Pro te in hydro lyzed for 18 h at 105O C in a sealed tube a

w i t h constant bo i l i ng HCI.

bEach v a l u e r e p r e s e n t s a s ing le determinal ion.

70.

REFERENCES

1.

2 . Darbre, A. and Blau, K., Biochem. J., 88, Sp-9p (1963).

Cruickshank, P. A . and Sheehan, J. C . , Anal. -- Chem., - 36, 1191 (1964).

3. Gehrke, C. W., Lamkin, W. M. , Sta l l ing , D. L . , and Shahrokhi, F., Biochem. Biophys. Res. Comm., 19, 328 (1965).

4. Gehrke, C. W. and Sta l l ing , D. L . , "Quant i ta t ive Analysis of the 20 Natural Protein Amino Acids by Gas Chromatography", 19th Annual Summer Symposium, "Separation Techniques" A .C .S . , June 22-24, 1966, Edmonton, Alberta, Canada.

5. Ragen, P. and Black, W., Fed. Proc., 23, 371 (1964).

6 . Johnson, D. E., Scot t , S. J. and Meister, A., Anal. Chem., 33, 669 -- - (1961).

7. Lamkin, W. M. and Gehrke, C . W . , Anal. Chem., - 37, 383 (1965).

8. Lamkin, W. M., Doctoral Disser ta t ion, The University of Missouri, August, 1964.

9. Pisano, J. J., VandenHeuvel, W. J. A. and Horning, E. C . , Biochem. Biophys. Res. Comm., - 7, 82 (1962).

10. Ruhlmann, K. and Giesecke, W., Angew. Chem., 73, 113 (1961).

11. Saroff, H. A. and Karmen, A . , Anal. Biochem., - 1, 344 (1960).

1 2 . S t a l l i ng , D. L . and Gehrke, C . W., Biochem. Biophys. Res. Comm., 22, - 329 (1966).

13. Zomzely, C. , Marco, G. and h e r y , E . , Anal. Chem., - 34, 1414 (1962).

DR. CARPENTER: Thank you, M r . S ta l l ing . Next w e a r e pr iv i leged t o have D r . Arnis Kuksis. He is a Medical Research Associate a t Banting and Best Department of Medical Research, University of Toronto. D r . Kuksis has agreed t o discuss with us h i s work i n the a rea of Triglyceride and Fa t ty Acid Analysis by Gas Chromatography. Dr . Kuksis.

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