For optimal results in HPLC analysis, it is recommended to measure absorbance at a wavelength that matches the absorption maximum of the compound(s) being analyzed. The UV spectrum shown can assist in selecting an appropriate wavelength for your analysis. Please note that certain mobile phases and buffers may block wavelengths below 230 nm, rendering absorbance measurement at these wavelengths ineffective. If detection below 230 nm is required, it is recommended to use acetonitrile and water as low UV-transparent mobile phases, with phosphoric acid and its salts, sulfuric acid, and TFA as buffers.
For some compounds, the UV-Vis Spectrum is affected by the pH of the mobile phase. The spectra presented here are measured with an acidic mobile phase that has a pH of 3 or lower.

High Performance Liquid Chromatography (HPLC) Method for Analysis of Water-Soluble Vitamins (B1, B2, B3, B6) on Primesep 100 by SIELC Technologies

Separation type: Liquid Chromatography Mixed-mode SIELC Technologies

 High Performance Liquid Chromatography (HPLC) Method for Analysis of Vitamin B1 (Thiamine), Vitamin B2 (Riboflavin), Nicotinamide, Vitamin B6 (Pyridoxine), Pyridoxamine, Pyridoxal Phosphate

Water-Soluble Vitamins (B1, B2, B3, B6) can be retained, separated and analyzed using a Primesep 100 mixed-mode stationary phase column. The analysis employs an gradient method with a simple mobile phase comprising water, acetonitrile (MeCN), and sulfuric acid as a buffer. This method allows for detection using UV 300 nm.

You can find detailed UV spectra of Vitamin B1 (Thiamine), Vitamin B2 (Riboflavin), Nicotinamide, Vitamin B6 (Pyridoxine), Pyridoxamine, Pyridoxal Phosphate and information about its various lambda maxima by visiting the following links: Vitamins (B1, B2, B3, B6)

Separation of Vitamin B2 and Vitamin C is demonstrated on a Primesep D mixed-mode column. This column can be used for analysis of other hydrophobic or neutral vitamins and supplements.

Vitamin C (ascorbic acid) and Vitamins Group B are separated on Obelisc N mixed-mode column. Method can be used in quantitation and determination of polar vitamins in various formulations and dietary supplements. HPLC method can be based on UV, Evaporative Light Scattering Detection (ESLD), RI or MS detection. Effect of sample matrix can be eliminated by changing mobile phase conditions. Buffer concentration, buffer pH and amount of ACN will affect every vitamin differently due to difference in polar and ionic properties.

Separation of vitamins group B is achieved on Obelisc N column in HILIC mixed-mode. Vitamins of this group are different in polarity and ionic properties. Retention and separation is achieved by optimization of amount of ACN, buffer and buffer pH. Combination of UV and ELSD detection is used to monitor HPLC separation. B vitamins are water-soluble vitamins that play an important role in cell metabolism. Supplements containing all six are generally referred to as a vitamin B complex. Individual B vitamin supplements are referred to by the specific name of each vitamin. This method can be used to analyze individual B vitamins as well as vitamin B complex. Isolation of impurities as well as degradation products is possible by preparative chromatography.

Vitamin C (ascorbic acid) and Vitamin B2 (riboflavin) are water-soluble vitamins that are not stored in the body and must be replaced each day. Primesep D easily resolves Vitamins C and B2 and the elution order can be reversed by adjusting acetonitrile and acetic acid concentrations in the mobile phase. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and acetic acid (AcOH) and UV detection at 250 nm.

Primesep B and B2 columns separate Vitamins C (ascorbic acid) and B2 (riboflavin) with tunable selectivity. Both columns allow peak order reversal by simply changing the acetonitrile concentration in the mobile phase. Vitamin B2 is especially sensitive to acetonitrile concentration. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and either trifluoroacetic acid (TFA) or acetic acid (HOAc) with UV detection at 250 nm.

Primesep B and B2 columns separate Vitamins C (ascorbic acid) and B2 (riboflavin) with tunable selectivity. Both columns allow peak order reversal by simply changing the acetonitrile concentration in the mobile phase. Vitamin B2 is especially sensitive to acetonitrile concentration. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and either trifluoroacetic acid (TFA) or acetic acid (HOAc) with UV detection at 250 nm.

Primesep B and B2 columns separate Vitamins C (ascorbic acid) and B2 (riboflavin) with tunable selectivity. Peak order reversal is exhibited on these two columns with the same mobile phases due to their different polarity. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and either formic or acetic acid (HOAc) with UV detection at 250 nm.

Primesep C separates B vitamins with baseline resolution by a combination of cation exchange, complex formation, and hydrophobic interactions. Vitamin B1 (thiamine), vitamin B2 (riboflavin), and vitamin B6 (pyridoxine) are separated with a mobile phase of water, acetonitrile (MeCN, ACN) and triethylamine (TEA) phosphate with UV detection at 280 nm.