Miniaturizing automatic amino acid analyzer for Apollo and Voyager mission

Dus, K. M.

Kraut, J.

Miller, S. L.

English

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"1 Progress Report f m t r e  Perked-of Juno-Decedes 1966 ~ I ! 
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ff 653 July 65 
A Feasibility Study on Miniaturizing an Automatic Amino Acid 
Analyzer for use onAApollo Mission'and .,Mars Voyager Mission. -f "-. - 
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& Stanley L. Miller - Principal Investigator 
Joseph Kraut - Co-Principal Investigator 
Karl M. Dus Co-Principal Investigator I 
Department of Chemistry 4 
University of California, San ~ i e g 0 . q  
P. 0. Box 109 
h Jolla, California 92038 
https://ntrs.nasa.gov/search.jsp?R=19670008707 2020-03-16T18:59:14+00:00Z
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In t roduc t ion  
An amino a c i d  a n a l y s i s  of  t h e  s u r f a c e  of  t h e  Moon and Mars would 
be of g r e a t  s c i e n t i f i c  i n t e r e s t  and importance. This  a n a l y s i s  on t h e  
Moon w i l l  be i n d i c a t i v e  of i ts  e a r l y  h i s t o r y .  The presence of  amino 
a c i d s  on t h e  s u r f a c e  of Mars will be s t r o n g  evidence f o r  t h e  presence 
of l i f e ,  provided i t  can be shown t h a t  t hese  amino a c i d s  are n o t  of  
non-b io logica l  o r i g i n .  S ince  it  does not  seem l i k e l y  t h a t  amino a c i d s  
could have surv ived  on t h e  su r face  of Mars f o r  several b i l l i o n  yea r s ,  
a h i g h l y  s e n s i t i v e  amino a c i d  ana lyzer  c o n s t i t u t e s  a l i f e  d e t e c t i o n  
system of moderate r e l i a b i l i t y .  
The most widely used commercial amino a c i d  a n a l y z e r  a t  t h e  p re sen t  
t i m e ,  t h e  Beckman-Spinco Model 120, h a s  a volume of approximately 
3 73 f t .  . I n  o r d e r  t o  meet t h e  requirements f o r  t h e  Apollo and Voyager 
miss ion ,  t h i s  ins t rument  has  t o  be reduced from i t s  p resen t  s i z e  by a 
f a c t o r  of 150. 
make t h i s  ins t rument  s u i t a b l e  f o r  space  missions.  These inc lude  a c i d  
hydro lys i s  of t h e  sample and the  s e p a r a t i o n  of t h e  r e s u l t i n g  amino a c i d s  
Severe1  automatic  r e a c t i o n  s t e p s  have t o  be added t o  
from contaminat ing metal compounds. I n  a d d i t i o n  t h e  s e n s i t i v i t y  of 
t h e s e  ins t ruments  w i l l  be s t b s t e n t i a l l y  h ighe r  t han  t h e  p re sen t  commercial 
mode 1. 
I n  o r d e r  t o  achieve  t h e  la rge  r educ t ion  i n  s i z e  and weight of t h e  
commercial ins t rument ,  we  are conducting t h i s  f e a s i b i l i t y  s tudy  by 
m i n i a t u r i z a t i o n  i n  two s t a g e s .  The f i r s t  s t a g e  i s  t h e  c o n s t r u c t i o n  of 
a semi-micro amino a c i d  ana lyze r ,  and t h e  second s t a g e  i s  t h e  c o n s t r u c t i o n  
of t h e  micro model s u i t a b l e  f o r  space f l i g h t  use. 
The eemi-micro model is  being cons t ruc t ed  e s s e n t i a l l y  from commercially 
a v a i l a b l e  components except  for t h e  min ia tu re  r o t a r y  va lves ,  t h e  hydro lys i s  
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chamber, the reaction coil heating chamber, and flow cell with a long 
light path. 
arrangement of components for the spectrophotometric unit in order to 
achieve the greatest compactness and sensitivity. 
Special attention i s  being given to the selection and 
During the present period of this feasibility study, primary 
attention has been given to the construction of the semi-micro model. 
We feel that this model will be a valuable contribution to instrumentation 
for biochemical research as a replacement for the over-sized and already 
obsolete instrument. 
extensive use of our experience in the construction of the semi-micro 
model. Although the construction of this instrument is just beginning, 
The design of the micro model is in progress, making 
we feel that this instrument can be successfully completed. 
1. Construction of component parts 
In order to reduce the size and weight of the commercial 
instrument and to make the system suitable for a fully automated analysis 
of Lunar and Martian soil, it is necessary to redesign and develop a 
number of essential component parts. The flow diagram is shown in figure 1. 
a) Miniature rotary valves. We have chosen rotary valves instead 
of solenoid valves wheneve'r possible because they can be made absolutely 
pressure tight. A l s o  they have a very small hold up volume and permit 
very simple programming of multistep operations. The original device, 
designed in our laboratory several years ago, was too large for use in 
the semi-micro or micro model. This rotary valve was redesigned and 
reduced to 1/6 of its .original size. This change involves mainly the 
use of a U-shaped channel through the rotating core,of the valve instead 
of the slanted channel used previously. 
electronic control circuit for the rotary valve was constructed and 
A more compact and,more reliable 
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s u c c e s s f u l l y  t e s t e d  i n  ou r  labora tory .  
b)  Automated a c i d  hydro lys i s  u n i t .  Experiments with d e s e r t  
sand from the  v i c i n i t y  of Yuma, Arizona,have shown t h a t  t h e  l eve l  of 
f r e e  amino a c i d s  a f t e r  hydro lys is  is about  20 t imes h i g h e r  than t h e  
l e v e l  before  hydro lys i s .  Alkal ine hydro lys i s  is no t  as e f f i c i e n t  i n  
r e l e a s i n g  amino ac ids .  
terrestrial samples w i l l  be similar t o  d e s e r t  sand samples a v a i l a b l e  on 
e a r t h ,  then  a c i d  h y d r o l y s i s  r ep resen t s  a necessary  s t e p  i n  t h e  amino a c i d  
I f  w e  assume t h a t  t h e  composition o f  extra- 
a n a l y s i s  of Lunar and Martian soil samples. 
The des ign  of a f u l l y  automated u n i t  f o r  a c i d  hydro lys i s  has  been 
completed, and the  component p a r t s  of  t h i s  u n i t  are p r e s e n t l y  under con- 
s t r u c t i o n  i n  ou r  labora tory .  The automat ic  h y d r o l y s i s  ' un i t  is designed 
t o  f i t  t h e  ion  exchange a n a l y s i s  system, but  t h i s  u n i t  may a l s o  prove 
u s e f u l  f o r  systems us ing  o t h e r  a n a l y t i c a l  procedures.  
c )  Compact pumping arrangement. Accurate meter ing of a l l  
l i q u i d  f low is e s s e n t i a l  f o r  t h e  m p r O d u d b i l i t y  of m o b i l i t i e s  on i o n  
exchange columns and consequently f o r  the  r e l i a b i l i t y  of t h e  a n a l y s i s .  
However, most a c c u r a t e  meter ing pumps are very  bulky and con ta in  only  
a s i n g l e  pumping head. 
opera ted  with up ts 6 pumping heads. 
requirements  f o r  t h i s  ope ra t ion .  
The r e c e n t l y  in t roduced  DCL-Parvalux pump can be 
This  reduces t h e  weight and space 
We have obta ined  t h i s  pump and t e s t e d  
i t s  performance f o r  meter ing t h e  bu f fe r  as w e l l  as t h e  n inhydr in  l i n e s .  
A p re s su re  c e i l i n g  of  250 p s i  was t h e  on ly  o b s t a c l e  a g a i n s t  u s ing  t h i s  
arrangement i n  au tomat ic  amino ac id  a n a l y s i s  because t h e  r e s i n s  u s u a l l y  
employed i n  t h e s e  systems w i l l  produce back p res su res  up t o  5 0 0  ps i .  
This  back p res su re  problem w a s  overcome by us ing  two new types  of i o n  
exchange r e s i n s  (UR-30 of Beckman-Spinco and Aminex A-4 of Bio-Rad). 
These r e s i n s  g ive  e x c e l l e n t  r e s o l u t i o n  of a l l  amino a c i d s  a t  g r e a t l y  
reduced back p res su res  (160-200 p s i ) .  Our tests i n d i c a t e d  t h a t  t h e  
mult ihead pump can be used t o  advantage i n  combination with low 
p res su re  r e s i n s .  
d )  E l e c t r i c a l  heatintz systems. We have rep laced  t h e  bulky 
water ba ths ' and  water j a c k e t s  c u r r e n t l y  used t o  thermosta t  t he  chromoto- 
g raph ic  column and t h e  ninhydrin r e a c t i o n  c o i l  by compact e lec t r ic  
h e a t i n g  devices .  This development permits  improved c o n t r o l  and 
f l e x i b i l i t y .  
e )  Detect ion system. In o r d e r  t o  a s s u r e  s u f f i c i e n t  s e n s i t i v i t y  
of  t h i s  min ia tu re  amino a c i d  ana lyze r ,  i t  i s  important  t o  improve a l l  
components 6f t h e  d e t e c t i o n  system. 
s p e c i a l  f low c e l l  with a 30 mm l i g h t  path.  This  component i s  p r e s e n t l y  
We t h e r e f o r e  have designed a 
under cons t ruc t ion .  
monochromator system and lamp arrangement wi th  a pho tomul t ip l i e r  tube  
f o r  both v i s i b l e  and u l t r a v i o l e t .  A s t a b i l i z e d  o p e r a t i o n a l  a m p l i f i e r  is  
Our semi-micro design w i l l  combine a very compact 
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provided f o r  e l e c t r o n i c  ampl i f i ca t ion  of t h e  pho tomul t ip l i e r  s i g n a l .  
This  s i g n a l  w i l l  be recorded on a small s i n g l e  trace reco rde r  which 
w i l l  r ep l ace  t h e  bulky mul t ipo in t  r eco rde r  of t h e  commercial instrument .  
This  d e t e c t i o n  system i s  designed t o  i n c r e a s e  t h e  s e n s i t i v i t y  of t h e  
commercial amino a c i d  ana lyze r  by a f a c t o r  of 100, and it may prove very  
u s e f u l  i n  c u r r e n t  biochemical equipment. 
micro model w i l l  be cons t ruc t ed  us ing  a l i g h t  sou rce ,  f i l t e r  and photo- 
m u l t i p l i e r .  This  sytem is  c u r r e n t l y  being designed. 
The d e t e c t i o n  u n i t  f o r  t h e  
2. Chemical Inves t iRat ions  
- 1  
I n  a d d i t i o n  t o  equipment des ign  and c o n s t r u c t i o n  w e  have 
c a r r i e d  o u t  i n v e s t i g a t i o n e ' o n  t h e  chemical a s p e c t s  of t h e  p r o j e c t .  
I 
The d e t e c t i o n  of t h e  amino a c i d s  is c u r r e n t l y  based on t h e i r  r e a c t i o n  
wi th  t h e  n inhydr in  reagent .  We have conducted a pre l iminary  sea rch  for  
r eagen t s  which might prove s u p e r i o r  t o  ninhydrin e i t h e r  because of h i g h e r  
s e n s i t i v i t y  o r  because of  g r e a t e r  s e l e c t i v i t y .  Two such r e a c t i o n s ,  
t r i n i t r o p h e n y l a t i o n  and dansyla t ion ,  seemed very  promising and were 
s t u d i e d  
a c i d  i s  
is only  
i n  d e t a i l .  
a )  Tr in i t ropheny la t ion .  The r e a c t i o n  of t r i n i t r o b e n z e n e  s u l f o n i c  
h igh ly  s e l e c t i v e  f o r  pkamarp amino groups.  However, i t s  s e n s i t i v i t y  
2/3 of t h e  n inhydr in  reagent ,  and i t  i s  ve ry  s e n s i t i v e  t o  l i g h t .  
The reagent  i n  s o l u t i o n  d e t e r i o r a t e s  even on prolonged s t and ing  a t  room 
temperature  i n  t h e  dark.  However, r e a c t i o n  wi th  s u i t a b l e  groups i s  very 
r a p i d  a t  room temperature  and almost q u a n t i t a t i v e  i n  c o n t r a s t  t o  ninhydrin.  
The f r e e  amino a c i d s  can be regenera ted  from t h e s e  complexes by 
t rea tment  wi th  NH40H. 
s e p a r a t i o n  and p u r i f i c a t i o n  s ince  t h e  n inhydr in  r e a c t i o n  with pep t ides  
This  f a c t  would prove advantageous i n  pep t ide  
i s  s lugg i sh  and a l s o  des t roys  t h e i r  amino t e rmina l  r e s idues .  
b) Dansylat ion.  The r e a c t i o n  of 5-dimethylaminonaphthalene-l- 
s u l f o n y l  c h l o r i d e  with amino groups g i v e s  a product  t h a t  absorbs  50 t i m e s  
as s t r o n g l y  as t h e  n inhydr in  product.  However, t h e  abso rp t ion  is i n  t h e  
u l t r a v i o l e t  where t h e  b u f f e r  ions  and organic  s o l v e n t s  c o n t r i b u t e  a h igh  
background absorp t ion .  This  problem may be overcome by measuring t h e  
f luo rescence  o f  t h e  dansyl-  d e r i v a t i v e s .  The p resen t  method of choice  
i s  n inhydr in ,  i n  our opin ion ,  but t h e s e  o t h e r  p o s s i b i l i t i e s  are s t i l l  
A 
being i n v e s t i g a t e d  i n  our laboratory.  
. 
Stan ley  L. Miller 
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t Josepfi Kraut 
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Karl M. Dus 


A Feasibility Study on Miniaturizing an Automatic Amino Acid Analyzer for Use on Apollo Mission and Mars Voyager Mission Progress Report, Jun. - Dec. 1966

A Feasibility Study on Miniaturizing an Automatic Amino Acid Analyzer for Use on Apollo Mission and Mars Voyager Mission Progress Report, Jun. - Dec. 1966

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