Applications:
Amino Acids Analysis in Acid Gradient Condition
11 underivatized amino acids (aspartic acid, glutamic acid, alanine, valine, methionine, isoleucine, cysteine, phenylalanine, histidine, lysine, and arginine) are separated by a Primesep 100 HPLC column by reversed-phase and ion-exchange mechanisms with LC/MS compatible conditions without the use of ion-pair reagents. The HPLC separation uses a TFA (trifluoroacetic acid) gradient in a mobile phase of water acetonitrile (MeCN, ACN with evaporative light scattering detection (ELSD).
| Column | Primesep 100, 4.6x250 mm, 5 µm, 100A |
| Mobile Phase | MeCN/H2O - 30/70% |
| Buffer | TFA , gradient 0.05-0.3 % , 25 min |
| Flow Rate | 1.0 ml/min |
| Detection | ELSD |
| Class of Compounds | Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements |
| Analyzing Compounds | Gaspartic acid, Glutamic acid, Alanine, Valine, Methionine, Isoleucine, Cysteine, Phenylalanine, Histidine, Lysine, Arginine |
Application Analytes:
Alanine
Amino Acids
Arginine
Aspartic Acid
Cysteine
Glutamic Acid
Histidine
Isoleucine
Lysine
Methionine
Phenylalanine
Valine
Hydrophobic and Hydrophilic Compound Separation
Primesep 100 separates a mixture of polar and nonpolar compounds in one analytical run. The amino acid cysteine; amino acid derivatives L-cystine, 2,2-dimethylcystine, and 2-methylcysteine; the polar acid benzoic acid; and the nonpolar neutral toluene are separated by a gradient using a combination of polar and hydrophobic interactions. The separation method uses a mobile phase mixture of water, acetonitrile (MeCN, ACN) and sulfuric acid (H2SO4) with UV detection at 210 nm.
| Column | Primesep 100, 4.6x150 mm, 5 µm, 100A |
| Mobile Phase | MeCN/H2O |
| Buffer | H2SO4 |
| Flow Rate | 1.0 ml/min |
| Detection | UV 210 nm |
| Class of Compounds | Drug, Acid, Hydrophilic, Ionizable, Nonpolar, Polar, Supplements |
| Analyzing Compounds | Cysteine, L-cystine, 2,2-dimethylcystine, 2-methylcysteine, Benzoic Acid, Toluene |
Application Analytes:
2,2-Dimethylcysteine
2-Methylcysteine
Amino Acids
Benzoic Acid
Cysteine
L-Cystine
Toluene
Complex Mixture of Acids, Bases, Amino Acids, and Neutral Compounds
Primesep 100 separates a mixture of amino acids (tyrosine, phenylalanine), organic acids (benzoic acid, mandelic acid), amines (benzylamine, pyridine), and neutrals (benzonitrile, toluene) in one HPLC run by combining reversed-phase, cation-exchange, and polar interactions. The method is tunable and peak order can be changed significantly by adjusting acetonitrile and trifluoroacetic acid concentrations. The separation method uses a mobile phase mixture of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) and compatible with UV, mass spec (LC/MS) and evaporative light scattering (ELSD) detection.
| Column | Primesep 100, 4.6x250 mm, 5 µm, 100A |
| Mobile Phase | MeCN/H2O - 30/70% |
| Buffer | TFA - 0.2 |
| Flow Rate | 1.0 ml/min |
| Detection | UV, 210 nm |
| Class of Compounds | Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
| Analyzing Compounds | Tyrosine, phenylalanine, Benzoic acid, mandelic acid, Benzylamine, Pyridine, Benzonitrile, Toluene |
Application Analytes:
Amino Acids
Benzoic Acid
Benzonitrile
Benzylamine
Mandelic Acid
Organic Acids
Phenylalanine
Pyridine
Toluene
Tyrosine
Bufferless Ion Separation (BLIS™) Chromatography of Amino Acids (2)
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).
| Column | Primesep 200, 4.6x150 mm, 5 µm, 100A |
| Mobile Phase | MeCN/H2O - 40/60% |
| Buffer | No |
| Flow Rate | 1.0 ml/min |
| Detection | UV, 195 nm |
| Class of Compounds | Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
| Analyzing Compounds | Aspartic acid, Alanine, Valine, Methionine, Leucine |
Application Analytes:
Alanine
Amino Acids
Aspartic Acid
Leucine
Methionine
Valine
HPLC Separation of Amino Acids in Supplements Composition in Mixed-Mode
Amino acids are essential components of numerous formulation. Health supplements can contain various amino acids and vitamins and require quantitation of each ingredients. Amino acids are very polar compounds with limited or no retention in reversed-phase chromatography. The most common approaches are reversed-phase chromatography with ion-pairing reagent and hydrophilic interaction chromatography (HILIC). Underivatized amino acids can be retained by combination of reversed-phase and cation exchange mechanism on Primesep 100 mixed-mode. Retention time is controlled by amount of acetonitrile, buffer and buffer pH. Method does not require ion-pairing reagent. This method is for UV detection. LC/MS, ELSD or Corona CAD can be employed for analysis of amino acids with trifluoroacteic acid or ammonium formate in the mobile phase. This approach can be used for HPLC analysis of all underivatized amino acids.
| Column | Primesep 100, 3.2x150 mm, 5 µm, 100A |
| Mobile Phase | MeCN/H2O - 15/85% |
| Buffer | H3PO4 |
| Flow Rate | 1.0 ml/min |
| Detection | UV, 200 nm |
| Class of Compounds | Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
| Analyzing Compounds | Theanine, Melatonin, GABA, 5- Hydroxytryptophan |
Application Analytes:
Amino Acids
Melatonin
Theanine
gamma-Aminobutyric Acid (GABA)
HPLC Separation of DMSO and Glycine by Mixed-Mode Chromatography
Dimethyl sulfoxide is important polar aprotic solvent, which is frequently used in pharmaceutical drug manufacturing, desolution, etc. DMSO is used as one of the solvents on protein chemistry due to universal ability to dissolve small molecules like amino acids. Amino acids and DMSO are very polar and have no retention on reversed-phase columns. In this HPLC application DMSO and glycine are separated on Primesep 100 mixed-mode column. DMSO is retained by weak reversed-phase mechanism and very low organic concentration is required in order to achieve any retention. Glycine, like any other underivatized amino acid, is retained by weak reversed-phase and moderate cation-exchange mechanism. Method uses acetonitrile/water/TFA gradient and UV detection.
| Column | Primesep 100, 4.6x150 mm, 5 µm, 100A |
| Mobile Phase | MeCN/H2O |
| Buffer | TFA |
| Flow Rate | 1.0 ml/min |
| Detection | UV, 210 nm |
| Class of Compounds | Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
| Analyzing Compounds | DMSO, Glycine |
Application Analytes:
Amino Acids
DMSO (Dimethyl sulfoxide)
Glycine
HPLC Analysis of Polysorbate in Mixture with Amino Acids and Sugar
Polysorbates are a class of emulsifiers used in some pharmaceuticals and food preparation. They are often used in cosmetics to solubilize essential oils into water-based products. Polysorbates are oily liquids derived from PEG-ylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Surfactants that are esters of plain (non-PEG-ylated) sorbitan with fatty acids are usually referred to by the name Span. It is often required to quantitate Polysorbate (Polysorbate/Tween 20, Polysorbate/Tween 40, Polysorbate 60/Tween 60 and Polysorbate 80) by HPLC in various formulations. Polysorbates exist in form of oligomers.
| Column | Primesep D, 3.2x50 mm, 5 µm, 100A |
| Mobile Phase | IPA |
| Buffer | Formic Acid |
| Flow Rate | 0.5 ml/min |
| Detection | ELSD |
| Class of Compounds | Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
| Analyzing Compounds | Glycine, Histidine, Sucrose, Polysorbate 20 |
Application Analytes:
Amino Acids
Glycine
Histidine
Polysorbate
Polysorbate 20
Polysorbate 80
Sucrose
Tween 20
Tween 80
HPLC Separation of Mixture of Conditionally Essential Amino Acids on Primesep 100 Column
| 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 |
| Class of Compounds | Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
| Analyzing Compounds | Glutamin (Gln/Q), L-Glycine (Gly/G), L-Cysteine (Cys/C), L-Proline Pro/p), L-Tyrosine (Tyr/Y), L-Arginine (Arg/R) |
Application Analytes:
Amino Acids
Arginine
Cysteine
Glycine
L-Cysteine
L-Glutamine
Proline
Tyrosine
HPLC Separation of Mixture of Essential Amino Acids on Primesep 100 Column
| Column | Primesep 100, 4.6x250 mm, 5 µm, 100A |
| Mobile Phase | MeCN/H2O - 25/75% |
| Buffer | Gradient H2SO4 0.05-0.2% 25 min |
| Flow Rate | 1.0 ml/min |
| Detection | UV, 200 nm |
| 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), L- Tryptophan (Trp/W) |
Application Analytes:
Amino Acids
D-Isoleucine
D-Leucine
D-Valine
DL-Isoleucine
Histidine
Isoleucine
L-Histidine hydrochloride monohydrate
L-Isoleucine
L-Methionine
Methionine
Phenylalanine
Tryptophan
Valine
HPLC Separation of Mixture of 12 Amino Acids on Primesep 100 Column
| 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 |
| 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) |
Application Analytes:
Amino Acids
Arginine
Asparagine
Cysteine
D-Isoleucine
D-Leucine
D-Valine
DL-Isoleucine
Histidine
Isoleucine
L-Cysteine
L-Methionine
Leucine
Methionine
Phenylalanine
Proline
Tryptophan
Valine
HPLC Separation of Mixture of Non-Essential Amino Acids
| Column | Primesep 100, 4.6x250 mm, 5 µm, 100A |
| Mobile Phase | MeCN/H2O - 20/80% |
| Buffer | H3PO4 - 0.1% |
| Flow Rate | 1.0 ml/min |
| Detection | UV, 200 nm |
| Class of Compounds | Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
| Analyzing Compounds | L-Aspartic Acid (Asp/D), L-Serine (Ser/S), L-Glutamic Acid Glu/E), L-Alanine Ala/A) |
Application Analytes:
Amino Acids
Aspartic Acid
D-Alanine
DL-Alanine
GLU (L-Glutamic acid)
Glutamic Acid
Serine