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  • 8/10/2019 HPLC Fluorescence Chromatography Online

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    29/12/2014 HPLC/Fluorescence :: Chromatography Online

    http://chromatography-online.org/chromatography_directory/application_notes_techniques/hplc%252Ffluorescence/cc228/index.php

    HPLC/Fluorescence

    Home> Chromatography Directory> Application Notes Techniques> HPLC/Fluorescence

    HPLC separation with fluorescence detection.

    Contents

    Amino Acid Analysis After Precolumn Derivatization

    Amino Acid Analysis after Precolumn Derivatisation with OPA/Mercaptopropionic Acid

    Extended Standard,Grom Application Number 10062 Nineteen amino acids in a

    standard mixture were separated using a GROM-SIL OPA-1 column (150 x 4mm, Part No.

    GSOP10308S1504) after precolumn derivatization with OPA/mercaptopropionic acid by

    gradient elution with 25mM sodium phosphate, pH 7.2 and THF 995/5 and 25mM

    sodium phosphate, pH 7.2, methanol and acetonitrile 50/35/15 in around 41 minutes

    and detected by fluorescence spectroscopy at 330 and 450nm. The amino acids

    separated were aspartic acid, glutamic acid, asparagine, serine, glutamine, glycine,

    threonine, histidine, citrulline, 3-methylhistidine, alanine, taurine, arginine, alpha-

    aminobutyric acid, tyrosine, valine, methionine, nor-valine and tryptophan.

    Amino Acid Analysisby Precolumn Derivatization with FMOC

    AminoAcid Analysis Employing Precolumn Derivatisation with FMOC Followed by

    Pentane Extraction, Grom Application Number 10130 Eighteen amino acids in a standard

    were separated using a GROM-SIL FMOC-1 column (250 x 4.6mm, Part No.

    GSFM10410S2505) after precolumn derivatization with FMOC (followed by pentane

    extraction) by gradient elution with 50mM sodium acetate, pH 4.2 and acetonitrile 80/20

    and 50mM sodium acetate, pH 4.2 and acetonitrile 20/80 in around 45 minutes and

    detected by fluorescence spectroscopy at 263 and 313nm. The amino acids separated

    were cysteine sulphinic acid, aspartic acid, serine, glutamic acid, threonine, glycine,

    arginine, alanine, tyrosine, proline, methionine, valine, nor-valine, phenylalanine,

    isoleucine, leucine, histidine and lysine.

    Amino Acid Analysis of Human Urodilatin

    Amino Acid Analysis of Human Urodilatin (hydrol. with 6N HCl, 15 hrs), Grom Application

    Number 10057 Amino acids in human urodilatin were separated after hydrolysis (6N HCl

    for 15 hours) using a GROM-SIL 120 ODS-5 ST column (150 x 4mm, Part No.

    GSFM20512S2504) by gradient elution with 50mM sodium acetate, pH 4.2 and

    acetonitrile 80/20 and 50mM sodium acetate, pH 4.2 and acetonitrile 20/80 in around

    48 minutes and detected by by fluorescence spectroscopy at 263 and 313nm. The

    compounds separated were arginine, serine, aspartic acid, glutamic acid, threonine,

    glycine, alanine, tyrosine, proline, FMOH, ammonia, methionine, phenylalanine,

    isoleucine, leucine, histidine and lysine.

    Amino Acid Analysis of Liver Biopsy Sample

    Amino Acid Analysis of Liver Biopsy Sample Using OPA for Precolumn Derivatisation,

    Grom Application Number 10061 Fourteen amino acids in a liver biopsy sample were

    separated using a GROM-SIL OPA-1 column (125 x 4mm, Part No. GSOP10308S1204)

    after precolumn derivatization with OPA by gradient elution with 12.5mM sodium

    phosphate, pH 7.2 and acetonitrile 97/2 and 12.5mM sodium phosphate, pH 7.2 and

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    acetonitrile 50/50 in around 14 minutes and detected by fluorescence spectroscopy at

    330 and 450nm. The amino acids separated were glutamic acid, alpha-aminoadipic acid,

    serine, glutamine, glycine, histidine, citrulline, 3-methylhistidine, arginine, tyrosine,

    valine, nor-valine, isoleucine, phenylalanine, leucine and lysine.

    Amino Acids in Bovine Serum Albumin Hydrolysate

    Amino Acid Analysis of Bovine Serum Albumin Hydrolysate (6N HCl/15 hours), Grom

    Application Number 10056 Amino acids in a bovine serum albumin hydrolysate (6N

    HCl/15 hours) were separated using a GROM-SIL FMOC-2 column (250 x 4mm, Part No.

    GSFM20512S2504) by gradient elution with 50 mM sodium acetate and acetonitrile80/20 and 50 mM sodium acetate/acetonitrile 20/80 at pH 4.2 in around 50 minutes and

    detected by fluorescence at 263 and 313nm. The amino acids and other compounds

    separated were arginine, serine, aspartic acid, glutamic acid, threonine, glycine, alanine,

    tyrosine, proline, FMOC-OH, methionine, valine, phenylalanine, isoleucine, leucine,

    histidine and lysine.

    Amino Acids in Human Urodilatin Hydrolysate

    Amino Acid Analysis of Human Urodilatin Hydrolysate (6N HCl/15 hours), Grom

    Application Number 10057 Amino acids in human urodilatin hydrolysate (6N HCl/15

    hours) were separated using a GROM-SIL FMOC-2 column (250 x 4mm, Part No.

    GSFM20512S2504) by gradient elution with 50 mM sodium acetate and acetonitrile

    80/20 and 50 mM sodium acetate/acetonitrile 20/80 at pH 4.2 in around 50 minutes anddetected by fluorescence at 263 and 313nm. The amino acids and other compounds

    separated were arginine, serine, aspartic acid, glutamic acid, threonine, glycine, alanine,

    tyrosine, proline, FMOC-OH, ammonia, methionine, phenylalanine, isoleucine, leucine,

    histidine and lysine.

    Amino Acids in Insulin Hydrolysate

    Amino Acid Analysis- B-Chain of Insulin (6N HCl/15 hours), Grom Application Number

    10055 Amino acids in a protein hydrolysate of the B-chain of insulin (6N HCl/15 hours)

    were separated using a GROM-SIL FMOC-2 column (250 x 4mm, Part No.

    GSFM20512S2504) by gradient elution with 50 mM sodium acetate and acetonitrile

    80/20 and 50 mM sodium acetate/acetonitrile 20/80 at pH 4.2 in around 50 minutes and

    detected by fluorescence at 263 and 313nm. The amino acids and other compoundsseparated were cysteine sulphinic acid, arginine, serine, aspartic acid, glutamic acid,

    threonine, glycine, alanine, tyrosine, proline, FMOC-OH, methionine, valine,

    phenylalanine, leucine, histidine and lysine.

    Analysis of Aflatoxins by HPLC

    Analysis of Aflatoxins, Grom Application Number 01075 A mixture of four aflatoxins was

    separated using a GROM-SIL 100 ODS-0 AB HPLC column (125 x 4mm, Part No.

    GSOD00510S1204) by isocratic elution with acetic acid, methanol, water 2/2/2008 in

    around 6 minutes and detected by fluorescence spectroscopy at 360nm. The compounds

    separated were G2a, B2a, G2 and B2.

    Determination of Beta Blockers in Human Plasma

    Determination of B-Blockers in Human Plasma, Grom Application Number 01073 A

    mixture of two beta blocker drugs in human plasma (N-hydroxymethylatenolol and

    atenolol) was separated using a GROM-SIL 100 ODS-2 FE column (125 x 4mm, Part No.

    GSOD20510S1204) by isocratic elution with 50mM potassium phosphate, pH 2.2 and

    acetonitrile 81/19 in around 6 minutes and detected by fluorescence spectroscopy at

    265 and 295nm.

    Determination of D- and L-Amino Acids after Precolumn Derivatiza

    Determination of the D- and L- Amino Acids after Precolumn Derivatisation with OPA/N-

    Isobutryl-L-Cysteine, Grom Application Number 10064 The D- and L- forms of sixteen

    amino acids were separated using a GROM-SIL OPA-2 column (250 x 4mm, Part No.

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    GSOP20512S2504) after precolumn derivatization with OPA/n-isobutryl-L-cysteine by

    gradient elution with 23mM sodium acetate, pH 6.0 and 600mL methanol and 50 mL

    acetonitrile in around 75 minutes and detected by fluorescence spectroscopy at 230 and

    445nm. The amino acids separated were L-aspartic acid, D-aspartic acid, L-glutamic

    acid, D-glutamic acid, L-serine, D-serine, L-threonine, glycine, D-threonine, L-histidine,

    D-histidine, L-alanine, L-arginine, D-alanine, L-tyrosine, D-tyrosine, L-valine, L-

    methionine, L-nor-valine, D-methionine, D-valine, L-phenylalanine, D-isoleucine, D-

    phenylalanine, L-leucine, D-isoleucine, D-leucine, L-lysine and D-lysine.

    Determination of Propanolol-Diastereomers of Beta-BlockerDetermination of Propanolol-Analysis of Diastereomers of Beta-Blockers in a Pill, Grom

    Application Number 10053A The diastereomers of propanolol, (L-propanolol and D-

    propanolol) a Beta blocker compound were separated isocratically using a GROM-SIL

    100 ODS-2 FE column (125 x 4mm, Part No. GSOD20510S1204) by isocratic elution with

    water and acetonitrile 30/70 in around 10 minutes and detected by fluorescence at 263

    and 313nm.

    High Sensitivity Amino Acid Analysis of Protein Hydrolysate

    High Sensitivity Amino Acid Analysis of Protein Hydrolysate by MicroboreHPLC after

    Precolumn Derivatisation with OPA, Grom Application Number 10063 Fifteen amino

    acids in a protein hydrolysate were separated using a GROM-SIL OPA-1 microbore

    column (250 x 4mm, Part No. GSOP10508S2501) after precolumn derivatization withOPA by gradient elution with 12.5mM sodium phosphate, pH 7.2, acetonitrile and

    methanol 97/0.5/2.5 and 12.5mM sodium phosphate, pH 7.2, acetonitrile and methanol

    70/25/5 in around 26 minutes and detected by fluorescence spectroscopy at 330 and

    450nm. The amino acids separated were aspartic acid, glutamic acid, serine, glycine,

    threonine, histidine, alanine, arginine, tyrosine, valine, methionine, ammonia, isoleucine,

    phenylalanine, leucine and lysine.

    Optical Antipodes of a Beta-Blocker by HPLC

    High Speed HPLC AnalysisDetermination of Optical Antipodes of a B-Blocker, Grom

    Application Number 10053b A mixture of two beta-blocker enantiomers was separated

    using a GROM-SIL 100 ODS-2 FE column (33 x 4.6mm, Part No. GSOD20210S0405) by

    isocratic elution with water and acetonitrile 30/70 in around 3 minutes and detected byfluorescence spectroscopy at 263 and 313nm. The compounds separated were D-

    propanolol and L-propanolol.

    Polyamines in Decaying Fish by FMOC/EVE Derivatization

    Determination of Polyamines in Decaying Fish Fillet Applying Precolumn Derivatisation

    with FMOC/EVE and HPLC, Grom Application Number 10059 Five polyamines in a

    decaying fish fillet were separated using a GROM-SIL Polyamine-1 column (200 x 4mm,

    Part No. GSPO10510S2004) by gradient elution with 50 mM sodium acetate, pH 4.0 and

    acetonitrile 80/20 and 50 mM sodium acetate, pH 4.0 and acetonitrile = Mai 95 in

    around 15 minutes and detected by fluorescence spectroscopy at 263 and 310nm. The

    polyamines separated were putrescine, cataverine, histamine, spermidine and spermine.

    Polyamines in Fresh and Spoiled Meat by FMOC/EVE Derivatization

    Analysis of Polyamines in Fresh and inSpoiled Meat (Pork) by Precolumn Derivatisation

    with FMOC/EVE, Grom Application Number 10060 Five polyamines in fresh and spoiled

    meat were separated using a GROM-SIL Polyamine-1 column (200 x 4mm, Part No.

    GSPO10510S2004) by gradient elution with 50 mM sodium acetate, pH 4.0 and

    acetonitrile 80/20 and 50 mM sodium acetate, pH 4.0 and acetonitrile = Mai 95 in

    around 15 minutes and detected by fluorescence spectroscopy at 263 and 310nm. The

    polyamines separated were putrescine, cataverine, histamine, spermidine and spermine.

    Polyamines in Meat by FMOC/EVE Derivatization

    Analysis of Polyamines in Fresh and in Meat Employing the FMOC/EVE Approach, Grom

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