Leucine

Applications:

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Bufferless Ion Separation (BLIS™) Chromatography of Amino Acids (2)

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Adding on to the previous HPLC separation of amino acids using Bufferless Ion Separation (BLIS) Chromatography; here we have additional amino acids separated on Primesep 200 column using only water and acetonitrile (MeCN, ACN) in the mobile phase.  Primesep 200 is a reverse-phase (RP) column with weak acidic ion-pairing groups embedded. With no buffer present in the mobile phase, detection can be achieved with UV, mass spectrometry (MS), evaporative light scattering detection (ELSD).

Condition

Column	Primesep 200, 4.6x150 mm, 5 µm, 100A
Mobile Phase	MeCN
Buffer	No
Flow Rate	1.0 ml/min
Detection	UV, 195 nm

 

Description

Class of Compounds	Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid
Analyzing Compounds	Aspartic acid,  Alanine, Valine, Methionine, Leucine

 

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Alanine
Amino Acids
Aspartic Acid
Leucine
Methionine
Valine

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HPLC Separation of Amino Acids in Supplements Capsules

Glutamine, valine, isoleucine, and leucine are baseline resolved in a dietary supplement with a mobile phase of acetonitrile, water, and sulfuric acid with ultraviolet (UV) detection at 210 nm on a Primesep 100 column. Increasing the sulfuric acid concentration allows elution of arginine. Primesep 100 separates underivatized amino acids and amines by combining reversed-phase and ion-exchange mechanisms. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN), and sulfuric acid with ultraviolet (UV) detection at 210 nm.

Condition

Column	Primesep 100, 4.6x250 mm, 5 µm, 100A
Mobile Phase	MeCN/H2O - 30/70%
Buffer	H2SO4
Flow Rate	1.0 ml/min
Detection	UV, 210 nm

 

Description

Class of Compounds	Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid
Analyzing Compounds	Arginine, Glutamine,  L-Valine (Val/V),  L-Isoleucine (Ile/I), L-Leucine (Leu/L)

 

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Arginine
Glutamine
Isoleucine
Leucine
Valine

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HPLC Separation of Amino Acids in Zero Organic Mode on Primesep 200 column

Essential and non-essential amino acids can be retained and separated in zero-organic mode on Primesep mixed-mode HPLC columns. Zero-organic mode is required to monitor isotopes of carbon. Amino acids are retained by combination of reversed-phase and cation-exchange mechanisms. At lower pH, some of the amino acids are more hydrophobic. Buffer pH will affect ionization state of amino acids, and at higher pH (above 2.5), the amino acids will be less hydrophobic and retentive in zero-organic mode. Amino acids can be monitored by low UV. Method can be used in archeological research for analysis of various molecules where presence of organic component of the mobile phase interferes with analysis.

Condition

Column	Primesep 200, 4.6x250 mm, 5 µm, 100A
Mobile Phase	MeCN/H2O
Buffer	Na2HPO4
Flow Rate	1.0 ml/min
Detection	UV 210 nm

 

Description

Class of Compounds	Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements
Analyzing Compounds	Valine, Leucyne, Isoleycine, Tyrosine, Phenylalanine, Histidine, Lysine

 

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Histidine
Isoleucine
Leucine
Lysine
Phenylalanine
Tyrosine
Valine

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HPLC Separation of Lysine and Arginine from Other Amino Acids

  Application Notes: Amino acids are polar ionic compounds which are not retained on reversed-phase column without ion-pairing reagent. In our application, lysine and arginine can be separated from other amino acids. Amino acids with a pH between 3 and 5 and with one basic and one acidic group become very polar. Therefore these amino acids don’t have strong ion-exchange interaction with Primesep C stationary phase. Amino acids with two amino groups still carry positive net charge and can interact with stationary phase by cations-exchange mechanism. pH variation of the mobile phase can be an effective tool to adjust selectivity of separation for zwitter-ionic, basic and acidic compounds. This method can be used for separation of mono-charged compounds from compounds having an extra charge. Application Columns: Primesep C Application compounds: Aspartic acid, Glutamic acid, Aspargine, Glycine, Proline, Alanine, Phenylalanine, Tyrosine, Leucine, Lysine, Arginine Detection technique: UV, LC/MS, ELSD/CAD

Condition

Column	Primesep C, 4.6x150 mm, 5 µm, 100A
Mobile Phase	MeCN - 15%
Buffer	AmAc pH 5.0- 15 mM
Flow Rate	1.0 ml/min
Detection	ELSD

 

Description

Class of Compounds	Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements
Analyzing Compounds	Aspartic acid, Glutamic acid, Aspargine, Glycine, Proline, Alanine, Phenylalanine, Tyrosine, Leucine, Lysine, Arginine

 

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Alanine
Arginine
Asparagine
Aspartic Acid
Glutamic Acid
Glycine
Leucine
Lysine
Phenylalanine
Proline
Tyrosine

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HPLC Separation of Mixture of 12 Amino Acids on Primesep 100 Column

 

Condition

Column	Primesep 100, 4.6x250 mm, 5 µm, 100A
Mobile Phase	MeCN/H2O - 35/65%
Buffer	H2SO4 0.05% 12 min hold, gradient 0.05-0.20, 13 min
Flow Rate	1.0 ml/min
Detection	UV, 200 nm

 

Description

Class of Compounds	Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid
Analyzing Compounds	L-Asparagine (Asn/N), L-Threonine (Thr/T), L-Cysteine (Cys/C), L-Proline (Pro/P), L-Valine (Val/V), DL-Methionine (Met/M), L-Isoleucine (Ile/I), L-Leucine (Leu/L), L-Phenylalanine (Phe/F), L- Tryptophan (Trp/W), L-Histidine (His/H), L-Arginine (Arg/R)

 

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Amino Acids
Arginine
Asparagine
Cysteine
D-Isoleucine
D-Leucine
D-Valine
DL-Isoleucine
Histidine
Isoleucine
L-Cysteine
L-Methionine
L-Threonine
Leucine
Methionine
Phenylalanine
Proline
Tryptophan
Valine

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HPLC Separation of β‑Hydroxy-β-methylbutyric Acid, 2-Hydroxyisocaproic Acid and Leucine on Primesep 100 Column

Both β-hydroxy-β-methylbutyric acid (HMB) and 2-hydroxyisocaproic acid (HICA) are metabolites of the essential dietary branched chain amino acid leucine.

 

Condition

Column	Primesep 100, 4.6x150 mm, 5 µm, 100A
Mobile Phase	MeCN - 5%, 3 min hold, Gradient MeCN 5-30% 12 min
Buffer	H2SO4 - 0.05 % 3 min hold, GradientH2SO4 0.05-3% 12 min
Flow Rate	1.0 ml/min
Detection	UV, 210 nm

 

Description

Class of Compounds	Acid, Hydrophilic,  Ionizable, Normal human metabolite, Dietary supplement, Essential amino acid, Branched-chain amino acid (BCAA)
Analyzing Compounds	β‑Hydroxy-β-methylbutyric (HMB), 2-Hydroxyisocaproic Acid (HICA), Leucine

 

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2-Hydroxyisocaproic Acid (HICA)
D-Leucine
Leucine
β‑Hydroxy-β-methylbutyric Acid (HMB)

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HPLC Separation of Allo-Isoleucine, Isoleucine, Leucine on Primesep 100 Column

View on hplc.cloud

Condition

Column	Primesep 100, 4.6x250 mm, 5 µm, 100A
Mobile Phase	MeCN/H2O - 5/95%
Buffer	AmFM pH 3.0 - 30 mM
Flow Rate	1.0 ml/min
Detection	CAD (Corona) MS- compatible mobile phase

 

Description

Class of Compounds	Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid
Analyzing Compounds	Allo-Isoleucine, Isoleucine, Leucine

 

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D-Isoleucine
D-Leucine
DL-Isoleucine
Isoleucine
L-Alloisoleucine
L-Isoleucine
Leucine
allo-D-isoleucine

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HPLC Separation of Mixture of Nine Essential Amino acids and Arginine on Newcrom AH Column

View on hplc.cloud Essential amino acids cannot be made by the body. As a result, they must come from food. The 9 essential amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.

Condition

Column	Newcrom AH, 4.6x150 mm, 5 µm, 100A
Mobile Phase	MeCN - 5%
Buffer	Gradient Formic Acid - 3-9%, 20 min
Flow Rate	1.0 ml/min
Detection	CAD

 

Description

Class of Compounds	Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid
Analyzing Compounds	L-Threonine (Thr/T), L-Valine (Val/V), DL-Methionine (Met/M), L-Isoleucine (Ile/I), L-Leucine (Leu/L), L-Phenylalanine (Phe/F), L-Histidine (His/H), Lysine (Lys/K) , L- Tryptophan (Trp/W), L-Arginine (Arg/R)

 

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Arginine
Histidine
Isoleucine
L-Threonine
Leucine
Lysine
Methionine
Phenylalanine
Tryptophan
Valine

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