QUANTITATIVE

AMINO ACID ANALYSIS

Aurlie Lolia
Applications Manager, Biochrom Ltd
2
QUANTITATIVE AMINO ACID ANALYSIS
Principles of amino acid analysis
Ion exchange chromatography
The Biochrom 30 physiological system
Optimisation of chromatography
Principles
Separation of less common amino acids
Troubleshooting and maintenance
3
PRINCIPLES OF AMINO ACID ANALYSIS
Structure of amino acids
Where
- NH2 is the amino group
- COOH is the carboxyl group
- R is the side chain
Separation is effected by:
Charge difference on the amino acids caused by different pK values
of the side chains
Hydrophobic interaction of the side chain with the polystyrene
matrix
4
ION EXCHANGE PROCESS
Principle:
The positively charged amino acids are bound to the resin which is negatively
charged. The conditions are then altered to increase the pH , temperature and
the concentration of the buffer counter ion. When the isoionic point of an
amino acid is being reached, the ionic attraction to the resin is lost and the
amino acid elutes from the column.
5
Simplified reaction between Ninhydrin and amino acids
Ninhydrin = powerful oxidising agent
Oxidative deamination of the alpha-amino group, liberating ammonia,
carbon dioxide, an aldehyde with one less carbon atom and a reduced
form of ninhydrin, hydrindantin.
The ammonia then reacts with the hydrindantin and another
molecule of ninhydrin to yield a purple substance (Ruhemanns purple)
that absorbs maximally around 570nm.
6
Simplified reaction between Ninhydrin and imino acids
The imino acids (proline and hydroxyproline): do not have free alpha-
amino groups
Reaction with ninhydrin forms a bright yellow compound monitored at
440nm
7
NINHYDRIN DETECTION
Beer-Lambert law: defines the relationship
between absorbance and molar
concentration
A=log
10
(I
o
/I)=Ecb
Where A=absorbance
I
o
=intensity of the incident light
I= intensity of the transmitted light
E=molar absorptivity (dm
3
mol
-1
cm
-1
)
c= molar concentration (mol dm
-3
)
b=path length (cm)
Linear relationship between concentration and absorbance
Detection at 570 and 440 nm
8
INTRODUCTION TO THE BIOCHROM 30
Principles:
Ion exchange chromatography
Stepwise elution gradient
Spectrophotometric detection at
570 nm and 440 nm following
Ninhydrin post-column
derivatisation
The system is composed of :
Autosampler
Chromatographic unit
PC : Biosys Control software &
EZChrom Elite
9
FLUIDICS
10
DATA HANDLING SOFTWARE
EZCHROM ELITE
THE BIOCHROM 30
PHYSIOLOGICAL SYSTEM
APPLICATIONS
12
SEPARATION PROGRAM FOR ROUTINE ANALYSIS
0.30 Lithium
hydroxide
Buffer 6
3.55 1.65 Lithium pH
3.55
Buffer 5
3.50 0.90 Lithium DII Buffer 4
3.15 0.50 Lithium CII Buffer 3
3.00 0.30 Lithium B Buffer 2
2.80 0.20 Lithium A Buffer 1
pH Molarity Buffer
13
PHYSIOLOGICAL STANDARD (Sigma)
14
EXAMPLES
Plasma
Urine
15
SHORT PROGRAMS FOR SPECIFIC ANALYSES
PKU
MSUD Sulfocysteine
Homocysteine
OPTIMISATION OF
CHROMATOGRAPHIC CONDITIONS
17
MAIN PARAMETERS
AFFECTING AMINO ACID SEPARATION
Analytical column dimension
The sensitivity increases as the column diameter decreases
The sensitivity increases as the resin bed length increases
Buffer composition
pH
Molarity
Organic solvent content
Timing of buffers
Buffer flow rate
Analytical column temperature
18
GENERAL CONSIDERATIONS
Each change in the program may affect the rest of the
chromatogram
Temperature change will take effect at the corresponding time of
the program but the effect of a buffer change will be delayed
Increase of temperature or change of buffer will make peaks
sharper
Timing of buffer adjustment : 1 to 2 min at a time
Temperature adjustment: 1 to 2 C at a time
19
Temperature and buffer changes
on lithium systems
Loading buffer
Buffer only
Temperature
Standard
B1(A) B2(B)
B3(CII)
B4(DII)
B5(pH3.55)
T1
T2
T3
T1
20
TEMPERATURE
Increase of the temperature of the analytical column:
In general, shorten the retention time of amino acids
Effect varies for each amino acid
Amino acids most affected by the temperature
Glutamine (T1)
Citrulline (T2a)
Tyrosine and Phenylalanine (T2b)
Tryptophan (T3)
Column backpressure is directly proportional to the viscosity of
the buffer and the viscosity decreases by 1%/degree up to 95.
Higher flow rate can be used at higher temperature without
sacrificing the efficiency
21
BUFFER TIMING
Adusting the timing of the buffer is equivalent to
adjusting the pH and molarity
Amino acids most affected by the timing of the
buffers
Sarcosine: to move buffer change away from sarc increase
time of buffer 1 (A)
Cystine: shape depends on time of buffer 2 (B)
Ileu/Leu: shape depends on time of buffer 3 (CII)
Homocyst/Gaba: separation can be improved by decreasing
time of buffer 3 (CII) at second step
SEPARATION
OF
LESS COMMON AMINO ACIDS
24
Homocitrulline
Elutes between Cys & Met
Separated by decreasing
the time of buffer 2
25
Argininosuccinic acid (ASA)
Elutes between Leu & Nleu
Separated by adjusting
the time of buffer 3
26
Alloisoleucine
Elutes between Met & Cysth
Separated by adjusting
the time of buffer 2
SIMPLE TROUBLESHOOTING
AND MAINTENANCE
28
Separation: what it should look like
29
POOR SEPARATION
Possible causes:
Incorrect program => Optimise program
Analytical column
Incorrect buffers => Check relevant buffers are fitted in the
correct position
Sample preparation: sample loaded at the incorrect pH
30
Example 1: Analytical column resin contaminated
Distorting peak shapes
Poor separation
31
Example 2: Buffer problem
Never add new buffer to old (always discard
remaining buffer)
Thoroughly clean and rinse buffer reservoir
and refill with fresh buffer
Buffers in wrong positions
Buffers mixed up by mistake
=> wrong pH and molarity
32
Poor reproducibility
Possible causes:
Retention times
Changing buffer flow rate
Samples loaded at different pH
Temperature not controlled properly
Areas
Air bubbles in the injection line
=> Check autosampler syringe
=> Check level of autosampler wash solution
Fault finding tip:
To identify the cause of the problem run consecutive standards
(from the same vial) in the same conditions
33
Example: Effect of pH on retention times
Standard diluted 1:1 with
10%SSA and standard
diluted 1:1 with lithium
loading buffer
pH=0.9
pH=1.9
34
Other common faults
High buffer pressure (Error 5)
Column inlet frit dirty => replace inlet frit
Resin contaminated => clean the resin and repack
column
Column temperature too low
Buffer flow rate too high
35
Other common faults
Low ninhydrin pressure (Error 7)
Ninhydrin reservoir empty
Air in ninhydrin pump
Diverter valve set to drain
When replacing the ninhydrin filter turn the diverter down to
drain for a few minutes
Low buffer pressure (Error 8)
Air in pump
Leak in buffer fluidics prior to column
Diverter valve set to drain
Use the tap on the bubble trap to prime the buffer lines
36
Other common faults
Minutes
25 30 35 40 45 50 55 60 65 70 75 80 85 90
m
V
o
lt
s
194
196
198
200
202
204
206
m
V
o
lt
s
194
196
198
200
202
204
206
570nm
Satterlee_M^W18776^plasma
Spikes caused by
ageing lamp,
filament collapsing
Baseline noise due to faulty photometer lamp
=> replace lamp
37
Communication errors
EZChrom Elite: Run not waiting for trigger (Error 704)
When shutting down BioSys always use File/Shutdown
When using the link, do not close the EZChrom Elite online window manually
but always use the Hide Elite button on the programmer window
Autosampler not responding (Error 904)
Always make sure that the autosampler is in SERIAL mode
38
CONCLUSION
Faults can usually be avoided by following the daily and monthly
checks as described in the operator manual
For example
Low pressure
Check the volumes of buffer and Nin in the bottles
Use the reagent management tool
Baseline problem
Volume of wash liquid in the coil flush bottle
Clean the flowcell manually with methanol or IPA
Pumps
Check the volume of water in the piston flush bottle
THANK YOU !
For applications and product information, visit our website www.biochrom.co.uk
For all your technical enquiries e-mail support@biochrom.co.uk