Method Abstract for Post-column Liquid Chromatography 365

Amino Acid Analysis with Temperature
and Eluant Gradients

The importance of temperature profiles with complex retention mechanisms

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Pickering laboratories specializes in manufacturing of cationexchange columns and eluants for amino acid analysis. No other technique has been shown to match the reproducibility and sensitivity of ion-exchange analysis with post-column Ninhydrin detection. Nor is there a chromatography technique that provides as much information; the 570/440 nm signal ratio for each amino acid is a constant and so provides information about purity.

Pinnacle PCX post-column derivatization system provides a unique opportunity to combine eluant gradient capabilities of modern HPLC instruments with column temperature gradients. As might be expected this capability helps reduce analysis time. Even more significant is the ability to resolve coelutions: consider such metabolic markers as allo-Isoleucine (MSUD) and Argininosuccinic acid (ASA). Under isothermal conditions these amino acids coelute with Cystathionine and Isoleucine respectively but are each resolved using a localized temperature gradient.

The ability to accomplish this derives from the multiple retention mechanisms of the gel-type resins employed in ion-exchange. That all the amino acids appear in the same chromatogram is testament to the dominance of ion-exchange. However, the exact position is influenced by an array of mechanisms including partitioning, adsorption, charge exclusion, etc. So even though two amino acids might coelute their proximity is incidental. And since retention processes are affected differently by changes in pH, salt concentration and temperature all the parameters have significant influence on selectivity.

METHOD

Analytical Conditions
COLUMN:	High-efficiency Lithium cation-exchange column, 4x100 mm Catalog number 0354100A Lithium cation-exchange guard column, 2x20 mm Catalog number 0352020
FLOW RATE:	0.4 mL/min
MOBILE PHASE:	Li292, Li365, Li375, RG003
Post-column Conditions
POST-COLUMN  SYSTEM:	Pinnacle PCX
REACTOR VOLUME:	0.5 mL
TEMPERATURE:	130 °C
REAGENT:	Trione Ninhydrin reagent
FLOW RATE:	0.3 mL/min
DETECTION:	UV/VIS 570 nm for primary amino acids 440 nm for secondary amino acids

Amino Acids calibration standard

MSUD patient serum 1 Phosphoserine 16 Alanine   -Aminobutyric acid 2 Taurine 17 Citrulline   Tryptophan 3 Phosphoethanolamine 18 -Amino-n-butyric acid   Ethanolamine 4 Urea 19 Valine   Hydroxylysines 5 Aspartic acid 20 Cystine   Ammonia 6 Hydroxyproline 21 Methionine   Creatinine 7 Threonine 22 Allo-isoleucine   Ornithine 8 Serine 23 Cystathionine   Lysine 9 Asparagine 24 Isoleucine   Histidine 10 Glutamic acid 25 Leucine   3-Methyl-histidine 11 Glutamine 26 Tyrosine   1-Methyl-histidine 12 Sarcosine 27 Phenylalanine   Anserine 13 -Aminoadipic acid 28 -Alanine   Carnosine 14 Proline 29 -Amino-i-butyric acid   Homocarnosine 15 Glycine 30 Homocystine   Arginine

Column oven program TIME [min] Temp [°C] 0 32 13 32 30 56 67 61 80 70 90 70 95 32 HPLC program TIME [min] Li290 % Li365 % Li375 % RG003 % 0 100 0 0 0 20 100 0 0 0 40 0 100 0 0 57 0 100 0 0 57.1 0 0 100 0 78 0 0 100 0 78.1 0 0 80 20 95 0 0 80 20 95.1 100 0 0 0

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