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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, alphaaminobutyric acid, tyrosine, valine, methionine, nor-valine and tryptophan.
Amino Acid Analysis by Precolumn Derivatization with FMOC
Amino Acid 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 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 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 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, 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 compounds
separated 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/NIsobutryl-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, Lmethionine, L-nor-valine, D-methionine, D-valine, L-phenylalanine, D-isoleucine, Dphenylalanine, L-leucine, D-isoleucine, D-leucine, L-lysine and D-lysine.
Determination of Propanolol-Diastereomers of Beta-Blocker
Determination of Propanolol-Analysis of Diastereomers of Beta-Blockers in a Pill, Grom
Application Number 10053A The diastereomers of propanolol, (L-propanolol and Dpropanolol) 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 Microbore HPLC 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 with
OPA 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 Analysis Determination 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 by
fluorescence spectroscopy at 263 and 313nm. The compounds separated were Dpropanolol 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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Application Number 10103 Seven polyamines in a meat extract were separated using a
GROM-SIL Polyamine-2 column (250 x 4mm, Part No. GSPO20510S2504) by gradient
elution with water, acetic acid and acetonitrile 400/3/100 and water, acetic acid and
acetonitrile 25/3/475 in around 4.5 minutes and detected by fluorescence spectroscopy
at 263 and 310nm. The polyamines separated were phenylmethylamine, putrescine,
cadaverine, tyramine, histamine, spermidine and spermine.
Primary and Secondary Amino Acids by HPLC
Primary and Secondary Amino Acids, Grom Application Number 10165 A mixture of 24
amino acids was separated using a GROM-SIL OPA-2 HPLC column (250 x 4mm, Part No.
GSOP30309S1204) by gradient elution with 23mM sodium acetate, pH 6.0 and 600 mL
methanol and 50 mL acetonitrile in around 26 minutes and detected by fluorescence
spectroscopy at 230 and 445nm.
Protein Hydrolysate Standard
Protein Hydrolysate Standard (6N HCl/15 hours), Grom Application Number 10054
Amino acids in a protein hydrolysate standard (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 compounds separated were cysteine
sulphinic acid, arginine, serine, aspartic acid, glutamic acid, threonine, glycine, alanine,
tyrosine, proline, FMOC-OH, ammonia, methionine, valine, nor-valine, phenylalanine,
isoleucine, leucine, histidine and lysine.
Separation of Chemotherapeutic Rubicins by HPLC
Rubicines - Chemotherapeutics, Grom Application Number 01029 Two chemotherapeutic
rubicins in human plasma were separated using a GROM-SIL 120 Octyl-5 CP column
(125 x 4mm, Part No. GSOD30512S1203) by gradient elution with 25mM sodium
phosphate, pH 2.2 and acetonitrile in around 12 minutes and detected by fluorescence
spectrometry at 480 and 560nm. The rubicins separated were doxorubicin and
daunorubicin.
Separation of D, L Amino Acid Standard
Separation of a D, L Amino Acid Standard by HPLC Employing the FLEC-ADAM Approach,
Grom Application Number 10058a A mixture of 16 amino acids was separated into their
D and L stereoisomers using a GROM-SIL FLEC-1 column (250 x 4mm, Part No.
GSFL10312S2504) by gradient elution with 50mM sodium acetate, pH 4.0 and
acetonitrile in around 72 minutes and detected by fluorescence spectroscopy at 263 and
313nm. The amino acids separated were D-arginine, L-arginine, D-serine, L-serine, Daspartic acid, L-aspartic acid, D-threonine, L-threonine, D-glutamic acid, L-glutamic acid,
D,L-proline, D-alanine, L-alanine, D-tyrosine, L-tyrosine, D-methionine, L-methionine, Dvaline, L-valine, D-phenylalanine, L-phenylalanine, D-alloisoleucine, L-alloisoleucine
and L-isoleucine, D-leucine, D-histidine, L-histidine, D-ornithine, L-ornithine, D-lysine,
and L-lysine.
Separation of D, L Proline After Derivatization
Isocractical Separation of D- and L-Proline After Precolumn Derivatisation with FLECADAM, Grom Application Number 10058b The amino acid proline was separated into its
D and L stereoisomers using a GROM-SIL FLEC-1 column (250 x 4mm, Part No.
GSFL10312S2504) by isocratic elution with 50mM sodium acetate, pH 4.0 and
acetonitrile 55/45 in around 20 minutes and detected by fluorescence spectroscopy at
263 and 313nm.

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