Applications:
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.
Condition
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 |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
DMSO, Glycine |
Amino AcidsDMSO (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.
Condition
Column |
Primesep D, 3.2x50 mm, 5 µm, 100A |
Mobile Phase |
IPA |
Buffer |
Formic Acid |
Flow Rate |
0.5 ml/min |
Detection |
ELSD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Glycine, Histidine, Sucrose, Polysorbate 20 |
Amino AcidsGlycineHistidinePolysorbatePolysorbate 20Polysorbate 80SucroseTween 20Tween 80
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 |
AlanineArginineAsparagineAspartic AcidGlutamic AcidGlycineLeucineLysinePhenylalanineProlineTyrosine
HPLC Method for Analysis of Glycine Methyl Ester Hydrochloride
Condition
Column |
Primesep 100, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 5/95% |
Buffer |
H3PO4 - 0.5% |
Flow Rate |
1 ml/min |
Detection |
UV, 200 nm |
Description
Class of Compounds
|
Amino acid, Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Zwitterionic |
Analyzing Compounds |
Glycine, Glycine Methyl Ester |
GlycineGlycine methyl ester
HPLC Method for Analysis of Glycine Methyl Ester Hydrochloride
Condition
Column |
Primesep A, 3.2x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 10/90% |
Buffer |
H2SO4 - 0.05% |
Flow Rate |
0.05 ml/min |
Detection |
UV, 200 nm |
Description
Class of Compounds
|
Amino acid, Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Zwitterionic |
Analyzing Compounds |
Glycine, Glycine Methyl Ester |
GlycineGlycine methyl ester
HPLC Separation of Mixture of Conditionally Essential 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 |
Glutamin (Gln/Q), L-Glycine (Gly/G), L-Cysteine (Cys/C), L-Proline Pro/p), L-Tyrosine (Tyr/Y), L-Arginine (Arg/R) |
Amino AcidsArginineCysteineGlycineL-CysteineL-GlutamineProlineTyrosine
New HPLC Amino Acids Separation Compatible With Carbon Dating Technique
Hydroxyproline seems to be the most promising amino acid used in carbon dating when isolated from bone collagen. Separation of amino acids is challenging, especially without the use of ions or inorganic buffers that can interfere with Mass spectrometer (MS) or contaminate the sample with modern carbon. Amino acids are also not retained in reverse-phase chromatography. The ideal solution would be using water only to separate the amino acids. This would allow a direct coupling to MS. We were able to separate hydroxyproline from proline and other simple amino acids like glycine and alanine in HPLC on Newcrom AH column using water only as a mobile phase. Using water also allowed UV detection at 205 nm which can’t be done if using a buffer based on acetic or formic acid.
See more information on radiocarbon dating here.
The same method can be modified to get symmetrical peaks and higher efficiency if a mobile phase with ionic modifier such as formic acid is used.
Condition
Column |
Newcrom AH, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Flow Rate |
1.0 ml/min |
Detection |
UV, 205 nm, CAD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Alanine, Glycine, Proline, Hydroxyproline |
AlanineD-AlanineGlycineHydroxyprolineProline
HPLC Separation of Amino Acids on Newcrom AH Column
View on hplc.cloud
Condition
Column |
Newcrom AH, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN - 50% |
Buffer |
Formic Acid - 0.1% |
Flow Rate |
1.0 ml/min |
Detection |
CAD |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid |
Analyzing Compounds |
Aspartic acid (Asp/D), Glutamine (Gln/Q), Glutamic acid (Glu/E), Glycine (Gly/G) |
Aspartic AcidGlutamic AcidGlycineL-Glutamine