Didactic Application to Riboflavin HPLC Analysis A Laboratory Experiment Marina Munari, Marla Miurin, and Gianlramo Goi I.P.S.I.A. "Candonl", Tolmezzo (UD), Italy
T h e use of the W-vis spectrophotometer as an HPLC detector makes it possible t o exploit its selectivity and to minimize interferences from unwanted components. In this work we DroDose an examnle where the choice of a different wavelength corresponding to the maximum intensity of absorption allows a poor resolution problem to be solved. Some works published in a review specializing in food analy&P suggested this experiment to us. The experiment consists of determining the riboflavin content in milk and cheese samples. T h e use of a natural sample is more interesting for the students, and, furthermore, this experiment allows them to deal with analvtical problems that they will encounter in their future professional work.
Rlbotlavln Analyels ol Dairy Products Rlboflavln
. .
Apparatus and Operating Conditions
The chromatographic apparatus consists of a pump LC-5A (Shimadzu); a spectrophotometer UV-260 (Shimadzu) used as a detector; aguard column (7.5 cm X 2.1 mm) packed with 5-p C-18pellicular packing is connected to the analytical column (25 cm X 4.6 mm id. Supelcosil LC-18 (5 pm)) Column effluents are monitored at detector wavelengths of 444 nm and 267 nm and a slit width of 2 nm. Analyses are performed isocratically at a flow rate of 1mllmin using water:methanol (68:32 vlv). Quantitative determination is based on peak-height measurements. The spectrophotometer UV260 (Shimadzu) is also used to obtain the riboflavin spectrum. Reagents
Sample Drled milk Whole mllk UHT
Psrtlally sklmmed milk Util Montaslc-llke seasond cheese Smoked rlcona
contenP 784 127 118 117 43
~vemgo01 three dsterrnlnatlmsoxpressd In irg per 100 g fresh product.
acetic acid in order to precipitate the proteinic material and centrifuged at 6000 rpm for 15 min, and the supernatant is collected. The sediment is washed with 2 mL of 2% (vlv) acetic acid aqueous solution, and centrifuged again and the second supernatant isadded to the first in a 10-mLvolumetric flask. The volume is made up to the mark with water. Before HPLC analysis all samples are filtered using a 0.45-pm-pore-size filter (Millipore,Japan). Fifty microliters of the solution obtained from the dried milk extractions and 100 pL of the solutions obtained from the other sample extractions are injected into the column. Results and Dlscurrlon
In this work the problems concerning the extraction and those regarding the strictly chromatographic conditions are omitted. The reader's attention is directed only toward the choice of the spectrophotometric conditions a p t to reveal the substance. s ~ e~ c t r u mof the riboflavin solution shows a ~ - The -~ ~ few maxima: two are localized in the ultraviolet region, while another two. less intense. are in thevisible reeion. Thechoice the wavelength more suitable toreveal thesubstance to be analvzed initiallv falls on 266 nm where the maximum molar extinction is at a maximum. Analyzing milk and cheesesam~ l e in s HPLC with U V detectionat 266 nm, the resolutionof the riboflavin peak is reduced, owing to the interference of coeluting compounds. Two solutions are possible: (1) to modify the chromatographic conditions (to change the elua n t and eventually the column); (2) to change the wavelength of the detector to verify whether the interfering substances do not absorb a t the new wavelength. In this experiment we chose the second solution. Using the wavelength corresponding to the maximum absorbance of the vitamin in the visible reeion (444 nm). the interfering substances are detected.- he figure compares the chromatogram of a vitamin extracted from a Montasio-like cheese and from whole milk a t 266 nm and 444 nm. By comparing the absorbance a t 266 nm (where there is the maximum value of molar extinction) and 444 nm, a reduction by a factor of 3.6 is observed. The table reports the riboflavin quantities found in t h e different dairy products: whole milk, partially skimmed milk, dried milk, Montasio-like seasoned cheese, and smoked ricotta. For all these products we rhought it was more convenient ro use the wavelength a t 444 nm. ~
~.~ ~~
Water, RS-HPLC grade, is from Carlo Erba, Italy; methanol, HPLC grade and acetic acid, analytical grade, are from Prolabo, France; riboflavin, analytical grade, is from Merck, FRD. The eluants are filtered and degassed before use. Sample Preparation
The materials chosen for analysis are dried milk, whole milk UHT, partially skimmed milk UHT, Montaaio-like cheese, and smoked ricotta, purchased from a local grocery store. The samples are subjected to vitamin extraction and HPLC analysis on the same day and are protected from the light. Extraction from Liquid MNk Samples
A known amount, approximately 7 g, of milk, is addedto 0.5 mL of methanol and 0.2 mL of acetic acid. The mixture is stirred for about 5 min, then it is centrifuged at 600Orpm for 15 min. The supernatant is transferred to a 10-mL volumetric flask, and the sediment is washed with 2 mL of 2% ("1") acetic acid eolution. The washing is also centrifuged and added to the first supernatant, and the volume is made up to the mark with water. Extraction from DriedMNk and Dairy Products
A known amount, about 2.5 g, of fiely shredded sample is added to 7 mL of water and 0.5 mL of methanol, and the mixture is stirred for about 5 min. The suspensionobtained is acidified with 0.2 mL of
' Ashow. S. H.; Seperlch, G. J.; Monte, W. C. and Welty. J. J. Food
Scl. 1983, 48, 92-94.
Stacher, B.; Zonta, F. J. FoodScl. 1886, 51, 857-858. 78
Journal of Chemical Education
of
Acknowledgmenl
We thank Aldo Missana, Institute Director, for his encouragement in developing this experiment.
I I. Chr--am
Ia
2 1.
21
of rlboflavln(R)extracted from whole milk (1) and Montssi*llke cheese (2) detected at 266 nm (A) and 444 nm (B).
Volume 68
Number 1 January 1991
79
