8/7/2019 Chinmay Poster HPLC (1)

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Vitamins Analysis:

SampleP reparation:

Experimental:

Chinmay NaphadeDepartment of Food, Human Nutrition and PackagingSciences, Clemson University, Clemson, SC 29634

E-mail: cnaphad@g.clemson.edu

Introduction to High Performance Liquid Chromatography

& Determination of Water Soluble Vitamins

Whatcan we see in Chromatograph?

Introduction to HPLC: Results:

High Performance Liquid Chromatography (HPLC) is one of the most widely

used techniques for identification, quantification and purification of mixturesof organic compounds. Both volatile and non-volatile substances can beanalyzed.HPLC is a form of liquid chromatography used to separate compounds thatare dissolved in solution. HPLC instruments consist of a reservoir of mobilephase,a pump, an injector, a separation column, and a detector.Compounds are separated by injecting a sample mixture onto the column.The different component in the mixture pass through the column anddifferentiates due to differences in their partition behavior between the mobilephase and the stationary phase. The mobile phase must be degassed toeliminatethe formation of air bubbles

Detectorsfor HPLC: Ultraviolet-Visible AbsorptionDetectors Photo DiodeArray Detector RefractiveIndex Detectors Fluorescence Detectors ElectronCapture Detector Mass Spectrometer Detector EvaporativeLight Scattering Detector (ELSD) Corona ChargedAerosol Detector

Figure 1: An HPLC System and its Components

Vitamins are biologically active compounds which act as controlling agents for an

organisms normal health and growth. While the level of vitamins in food may beas low as a few micrograms per 100 g, these vitamins are often accompanied byan excess of compounds with similar chemical behavior. Analysis of water-solublevitamins using traditional reversed-phase HPLC is not possible because the highly

polar compounds are not retained on conventional silica C18 columns. Forinstance, thiamine and ascorbic acid ( vitamin C) show almost no retention on

standard C18 material. In this study the focus was on the HPLC analysis ofmicronutrient tablets containing vitamin C and four B-complex vitamins: vitamin B1(thiamine), vitamin B2 (riboflavin), vitamin B3 (nicotinamide) and vitamin B6(pyridoxine). So the objective was to specially modified C18 phase with an acidic

mobile phase to provide fast resolution of all vitamins present.

Retention Time:

tR = Retention time

Wb = Base width of peak (4 width)t0 = retention time of an unretained solute

Peaktends to be gaussian and broadens withtime-Wbbecomes larger with longer tR

Capacity factor k:

k is a measure of peak retention or how many times the

peak is retained vs an unretained peak (t0)k = (tR t0) / t0

Column efficiency:

Plate number (n) is a measure of column efficiency

N = (tR / )2 = 16 (tR / Wb )

2

N is determined by particle size (dp), column length (L) and

flow rate (F)

Wb

is base width by the tangent method. Alternately, use N

= 5.54 (tR / W0.5 )2

Separation factor or selectivity :

(separation factor)= k2 / k1 = 2.1 /1.5 = 1.4

is dependent on the columnand mobile phase is measure of differential retention of two analytes by the

column and must be >1.0 for peak separation

Water soluble vitamins can be extracted from simple matrices afterhomogenization with water in an ultrasonic bath. Only 250 mg from the total

sample are transferred into a 50 ml volume flask. Approximately 40 ml of 0.5%oxalic acid solution was added and the sample stirred. After 20 min treatment in anultrasonic bath, the sample solution must be cooled d own and the volume adjustedto 50 ml with 0.5% oxalic acid. Before injection the sample was filtered through a

0.45 m syringe filter.

All standard solutions were prepared with 0.5% oxalic acid in double-distilled

water. A preliminary standard of three water-soluble vitamins was created byweighing out 10 mg of thiamine, 20 mg of pyridoxine and 10 mg riboflavin into a100 ml volume flask. The flask was brought up to 100 ml with 0.5% oxalic acid(V1). In a second 100 ml volume flask, 70 mg ascorbic acid and 20 mg

nicotinamide were diluted with 70 ml of 0.5% oxalic acid. The final standard wascreated by adding 10 ml of the V1 solution to the second flask and adjusting the

volume to 100 ml with 0.5% oxalic acid. The final concentration of each vitamin inthe standard was as follows:

Thiamine 0.010 g/l

Pyridoxine 0.020 g/l

Riboflavin 0.010 g/l

Ascorbic acid 0.70 g/l

Nicotinamide 0.20 g/l

Column: ProntoSil 120 5m C18 AQ 150 x 3 mm (part number15CF184PSJ)

Eluent A: 50 M H3PO4 (adjusted to pH 2.5)

Eluent B: ACN

Gradient: 0-2 min 99% A; 2-8.5 min 30% A; 8.5-11 min 30% A; 11.02-15min99% A

Flow: 0.6 ml/min

Pressure: 77 bar

Detection: UV at 268 nm or wavelength program (see Fig. 1)

Injector Volume: 10 l

Temperature: 40 C

HPLC Parameters:

HPLC system equipped with Autosampler 3900, Pump 1000 with 10 ml pump

head, static mixer, Manager 5000 with low pressure gradient and degassermodules,Column Oven 4000 and UV Detector 2600 with analytical flow cell.

Instrumentation:

Since each vitamin has a slightly different optimal absorption wavelength

(Fig. 1), a wavelength switching program was used to increase the sensitivityof quantification for each vitamin. Figure 2 depicts the chromatogramobtained for the micronutrient tablet sample. Analysis results are listed inTable1.

Fig. 1 Wavelength spectrum of 5 water soluble vitamins.

Fig. 2 Separation of five vitamins in a micronutrient tablet.

Table 1.

Repeatability o f

A fast separation of five vitamins with good peak symmetry is easilyaccomplished by reversed-phase HPLC using the ProntoSil C18 AQ

column and HPLC. The static mixer, gradient mixing was performedefficiently while minimizing baseline noise, making lower limits of detection

and < 0.2 % RSD in retention times possible.

Conclusion:

HPLC Method Performance:

Limit of detection (S/N = 3)

References:

Retention time over 10 runs: < 0.2 % RSD

Ar ea s o ver 10 runs < 3 % R SD

Reversed-phase HPLC of water soluble vitamins.www.knauer.net

Reversed-phase ion-pair HPLC determination of some water-soluble vitaminsin pharmaceuticals(1999). D Ivanovi, , A Popovi, D Radulovi and MMedenica.Journal of Pharmaceutical and Biomedical analysis. 18(6) 999-1004.