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

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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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For all your technical enquiries e-mail support@biochrom.co.uk