QUANTITATIVE AMINO ACID ANALYSIS. Aurélie Lolia Applications Manager, Biochrom Ltd

QUANTITATIVE AMINO ACID ANALYSIS Aurélie Lolia Applications Manager, Biochrom Ltd

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 2

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 3

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. 4

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. 5

Simplified reaction between Ninhydrin and imino acids The imino acids (proline and hydroxyproline): do not have free alphaamino groups Reaction with ninhydrin forms a bright yellow compound monitored at 440nm 6

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 7

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 8

FLUIDICS 9

DATA HANDLING SOFTWARE EZCHROM ELITE 10

THE BIOCHROM 30 PHYSIOLOGICAL SYSTEM APPLICATIONS

SEPARATION PROGRAM FOR ROUTINE ANALYSIS Buffer Molarity ph Buffer 1 Lithium A 0.20 2.80 Buffer 2 Lithium B 0.30 3.00 Buffer 3 Lithium CII 0.50 3.15 Buffer 4 Lithium DII 0.90 3.50 Buffer 5 Lithium ph 3.55 1.65 3.55 Buffer 6 Lithium hydroxide 0.30 12

PHYSIOLOGICAL STANDARD (Sigma) 13

EXAMPLES Plasma Urine 14

SHORT PROGRAMS FOR SPECIFIC ANALYSES PKU Homocysteine MSUD Sulfocysteine 15

OPTIMISATION OF CHROMATOGRAPHIC CONDITIONS

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 17

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 18

B1(A) Temperature and buffer changes on lithium systems B2(B) B3(CII) Buffer only B4(DII) Standard B5(pH3.55) Loading buffer T1 T2 T3 T1 Temperature 19

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 20

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 21

SEPARATION OF LESS COMMON AMINO ACIDS

Homocitrulline Elutes between Cys & Met Separated by decreasing the time of buffer 2 24

Argininosuccinic acid (ASA) Elutes between Leu & Nleu Separated by adjusting the time of buffer 3 25

Alloisoleucine Elutes between Met & Cysth Separated by adjusting the time of buffer 2 26

SIMPLE TROUBLESHOOTING AND MAINTENANCE

Separation: what it should look like 28

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 29

Example 1: Analytical column resin contaminated Distorting peak shapes Poor separation 30

Example 2: Buffer problem Buffers mixed up by mistake => wrong ph and molarity Buffers in wrong positions Never add new buffer to old (always discard remaining buffer) Thoroughly clean and rinse buffer reservoir and refill with fresh buffer 31

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 32

Example: Effect of ph on retention times Standard diluted 1:1 with 10%SSA and standard diluted 1:1 with lithium loading buffer ph=1.9 ph=0.9 33

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 34

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 35

Other common faults Baseline noise due to faulty photometer lamp => replace lamp 206 204 570nm Satterlee_M^W18776^plasma 206 204 Spikes caused by ageing lamp, filament collapsing 202 202 mvolts 200 200 mvolts 198 198 196 196 194 194 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Minutes 36

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 37

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 38

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