QUANTITATIVE AMINO ACID ANALYSIS Aurélie Lolia Applications Manager, Biochrom Ltd
QUANTITATIVE AMINO ACID ANALYSIS
Aurélie Lolia Applications Manager, Biochrom Ltd
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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
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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
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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.
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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 (Ruhemann’s purple) that absorbs maximally around 570nm.
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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
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NINHYDRIN DETECTION
Beer-Lambert law: defines the relationship between absorbance and molar concentration
A=log10 (Io/I)=EcbWhere A=absorbance
Io=intensity of the incident light
I= intensity of the transmitted light
E=molar absorptivity (dm3 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
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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
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FLUIDICS
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DATA HANDLING SOFTWAREEZCHROM ELITE
THE BIOCHROM 30PHYSIOLOGICAL SYSTEM
APPLICATIONS
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SEPARATION PROGRAM FOR ROUTINE ANALYSIS
0.30Lithium hydroxide
Buffer 6
3.551.65Lithium pH 3.55
Buffer 5
3.500.90Lithium DIIBuffer 4
3.150.50Lithium CIIBuffer 3
3.000.30Lithium BBuffer 2
2.800.20Lithium ABuffer 1
pHMolarityBuffer
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PHYSIOLOGICAL STANDARD (Sigma)
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EXAMPLES
Plasma
Urine
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SHORT PROGRAMS FOR SPECIFIC ANALYSES
PKU
MSUD Sulfocysteine
Homocysteine
OPTIMISATION OF
CHROMATOGRAPHIC CONDITIONS
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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
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GENERAL CONSIDERATIONS
• Each change in the program may affect the rest of the chromatogram
• Temperature change will take effect at the corresponding time ofthe 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
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Temperature and buffer changeson lithium systems
Loading buffer
Buffer only
Temperature
Standard
B1(A) B2(B)
B3(CII)
B4(DII)
B5(pH3.55)
T1T2 T3
T1
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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 ofthe buffer and the viscosity decreases by 1%/degree up to 95.• Higher flow rate can be used at higher temperature without
sacrificing the efficiency
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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
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Homocitrulline
Elutes between Cys & Met
Separated by decreasing the time of buffer 2
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Argininosuccinic acid (ASA)
Elutes between Leu & Nleu
Separated by adjusting the time of buffer 3
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Alloisoleucine
Elutes between Met & Cysth
Separated by adjusting the time of buffer 2
SIMPLE TROUBLESHOOTING
AND MAINTENANCE
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Separation: what it should look like
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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
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Example 1: Analytical column resin contaminated
• Distorting peak shapes• Poor separation
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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 positionsBuffers mixed up by mistake
=> wrong pH and molarity
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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
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Example: Effect of pH on retention times
Standard diluted 1:1 with10%SSA and standard diluted 1:1 with lithium loading buffer
pH=0.9
pH=1.9
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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
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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
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Other common faults
Minutes
25 30 35 40 45 50 55 60 65 70 75 80 85 90
mV
olts
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196
198
200
202
204
206
mV
olts
194
196
198
200
202
204
206
570nmSatterlee_M^W18776^plasma Spikes caused by
ageing lamp, filament collapsing
• Baseline noise due to faulty photometer lamp=> replace lamp
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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
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CONCLUSION
Faults can usually be avoided by following the daily and monthlychecks 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 !
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