Determinationof Riboflavin Concentration in Powdered and Non-Fat Milk UsingMolecular Fluorescence

Accordingto Kalingan &amp Liao (222), milk is the most valuable source ofriboflavin in the human diet. Several researchers have analyzed thelevels of substance available in different sources. Various studieshave been made on the determination of riboflavin content of liquidwhole milk. However, few comparative studies have been undertaken toestablish the difference between whole milk and powdered or driedmilk as far as concentration of riboflavin is concerned. Skim milk asit is commonly referred to is one of the broadly consumed products.One reason why consumption of skimmed milk has increased is itsprolonged life and ability to withstand all weather and climaticconditions (Lab 6). Liquid milk can only stay for some time and undercertain circumstances. When it is very hot, people find it morereasonable to use skimmed milk as it does not go bad.

Accordingto White, Armstrong &amp Whitehead (672), the use of skim milk hasbecome increasingly prevalent, especially in low-cost diets. Driedmilk is also used widely in bakeries and food service industries.There is a need to ensure that with all these milk forms, the levelsof riboflavin are not affected. This is to say that whatever kind ofmilk that one consumes should give the same nutritional content.

Inthis experiment, the researcher is interested in determining thelevels of riboflavin in powdered milk and skimmed or what is referredto as non-fat milk. The study will be vital in determining whetherthe two forms of milk have the same nutritional value. At the end ofthe study, the researcher will provide a recommendation on the typeof milk that is rich in riboflavin. The study will be relevant toconsumers as they will be able to determine the form of milk thatwill provide their nutritional requirements. Apart from consumers,the study will be important to producers and even medical experts. Inconclusion, the study will help in determining the best form of milkdepending on the levels of riboflavin needed.

Significanceof the Experiment

Riboflavinwas identified by Gustafson (375) as a part of the Vitamin G Complex.Before its identification and classification, riboflavin had beenclassified as ovoflavin, lactoflavin, or various other flavins.Further analysis on these flavins was conducted, and they were foundto be identical. The flavins were found to have a similar chemicalcomposition. The only difference between the flavins was on thesource (Lab 6). For example, flavins from milk were referred to aslactoflavin. After extensive consultations, the name riboflavin wasaccepted for all the flavins. The term is appropriate in that itindicates the chemical composition of riboflavin. Riboflavinaccording to Sutton, Warner &amp Kaeser (279) is composed of a sugarcalled ribose and a Flavin (yellow pigment containing nitrogen).Research done by Kalingan &amp Liao (279) presented convincingevidence that the fluorescent yellow pigment formed by the tubercularbacillus is identical with riboflavin.

Accordingto White, Armstrong &amp Whitehead (674), Riboflavin is watersoluble and is insoluble in ordinary fat solvents. Riboflavin isstable to heat, to most oxidizing agents and strong mineral acids.However, it has also been established that riboflavin is sensitive toalkali. It is also sensitive to light and undergoes an irreversibledecomposition on irradiation with ultraviolet or visible light. Itexplains why the presence of riboflavin is usually tested usingmolecular fluorescence.

Also,Riboflavin is essential to the growth and development of humanbeings. Unlike other nutrients, Riboflavin is important during allgrowth stages of a person. Research done by Gustafson (374)established that riboflavin is not only important to humans but alsoto animals. In both humans and animals, digestive disturbances,nervousness, skin disorders, and abnormal eye conditions result froma diet low in riboflavin. A high level of riboflavin in the diettends to produce a more stable state of health, greater freedom fromdisease, especially infectious diseases, and a longer period ofvitality before the onset of old age. From these deductions, it canbe established that hemoglobin is a major component in human andanimal growth and development. As Sutton, Warner &amp Kaeser (292)pointed out it is important to access critically all food substancesconsumed by human beings and animals. With milk, for instance, it isimportant to scan it for riboflavin which contains antibodies forhealth vitality.

Riboflavin,just like other vitamins is widely distributed in animal andvegetable tissues but generally in small amounts. Milk, liver, yeast,and green leafy vegetables have been found to be good sources of thisvitamin. Research done by Kalingan &amp Liao (221) concluded thatthe three were paramount in human growth and development. Theresearcher recommended consumption of milk and other riboflavinsources especially during early stages of life. Gustafson (276) onthe other hand, stated that in milk at least 90 percent of theriboflavin is in a free dialysable form. This is to say that in milk,it is possible to measure riboflavin using chemical techniques.Riboflavin in milk occurs in combinations of high molecular weight.


  • Standard solution of riboflavin 10ppm

  • Distilled water

  • 3M Sodium chloride solution (NaCl)

  • 3M Acetic acid solution (HAc)

  • Powdered milk sample for testing

  • Non-fat milk sample for testing

  • UCD fluorimeter

  • Test tubes


Bourquin-Shermanis a historical unit that has been used to determine the amounts offood substances. This unit is used to represent the amount ofspecific vitamins present in foods and other substances. Kalingan &ampLiao (219) defined this unit of measurement as that amount of thetest food which when fed to a standard test animal previouslydepleted according to the prescribed technique, would produce anaverage gain of three grams per week over an eight-week period.However, results varied widely with different workers. Undoubtedlythe purity of the riboflavin under the tests was not assured. In thisexperiment, the researcher will use a more recent test to come upwith corresponding units of riboflavin in dried milk and non-fatmilk.

Inthis experiment, the researcher will rely on the light sensitivityproperty of riboflavin. As discussed earlier in this study,riboflavin is sensitive to radiation. This property allows for theuse of molecular fluorescence spectroscopy. According to Gustafson(375), molecular fluorescence spectroscopy (MFS) is an analyticaltechnique for studying optical emissions from molecules that havebeen excited to a higher energy level by absorption ofelectromagnetic radiation.


Thefirst step will be to create the solutions that will be tested. Tobegin with, the researcher will dilute 6.002 grams of milk powderwith 50ml of dilute water to obtain sample 2. Secondly, theresearcher will dilute the 6.002g milk powder with 25ml distilledwater 75ml distilled water, 3M HAc and 3M NaCl to get samples 3, 4,5 and six respectively. Finally, the researcher will dilute 25ml ofnon-fat milk with 75ml of 3m HAc and 3m NaCl (Skoog).The solution will be referred to as milk. The second step afterpreparation of samples is to subject them to radiation. Thetest-tubes containing these solutions will then be put into aFlourimeter and the values of intensity recorded.


Theresults below show the results obtained from the sample solutionscreated.

Table 1: Showing riboflavin levels for samples


Ml of standard solution (100ppb)

Dilute to (ml)

Riboflavin concentration





















Fromthe above table, it can be established that dilution affects thelevel of riboflavin. The readings are clear that the higher thelevels of dilution for any sample, the lower the levels ofriboflavin.

Thefollowing intensity readings were obtained for all samples

Table2: Showing intensity values for all concentration samples















FromTable 1 above, it is clear that 50ppb sample had the highestintensity. It was followed by the 40ppb solution and then the milksolution. These results are in fig 1 below.

Fig1: Fluorimeter reading for the samples

Fromthe above figure, it can be established that the higher the ppbconcentration, the higher the intensity. It can thus be argued thatppb concentration of milk was somewhere between 40 and 20. Since 89are closer to 98 than 50, riboflavin concentration of milk can beestimated to be 37ppb. Fig 2 below shows the relationship betweenintensity and wavelength for 50ppb sample.

Fig2: Intensity and wavelength values for 50ppb sample.

Fromthe figure, it can be established that wavelength increases withincrease in energy. It can also be noted that wavelength increasesmore with lower power.


Fromthe study, it can be established that powdered milk and non-fat milkhave different levels of riboflavin. Riboflavin content in powderedmilk is determined by the degree of dilution. However, for non-fatmilk, the standard of riboflavin was estimated to be between 35 and40ppbs. To dilute a milk powder to the required riboflavin level, oneshould ensure that for every 50ml solution contains between 10 and 20ml of standard 100ppb solution. The mixture creates a riboflavinconcentration of between 20ppb and 40ppb.


Gustafson,Felix G. &quotInfluence of light intensity on the concentration ofthiamin and riboflavin in plants.&quotBiochem Journal23.3 (2013): 373-378. Print.

Kalingan,A. E., and Chung-Min Liao. &quotInfluence of type and concentrationof flavine genic factors on production of riboflavin by EremotheciumAshby NRRL 1363.&quot&nbspBioresourceTechnology&nbsp82.3(2010): 219-224. Print.

Lab6: Capillary Electrophoresis.&quot – Chemwiki. N.p., 02 Oct. 2013.Web. 4 July 2016.

Sutton,T. S., R. G. Warner, and H. E. Kaeser. &quotThe concentration andoutput of carotenoid pigments, vitamin A, and riboflavin in thecolostrum and milk of dairy cows.&quot&nbspJournalof Dairy Science&nbsp30.12(2011): 927-932. Print.

Skoog,Douglas A., F. James Holler, and Stanley R. Crouch. Principles ofInstrumental Analysis. N.p.: n.p., n.d. Print.&nbsp

White,H. B., J. Armstrong, and C. C. Whitehead. &quotRiboflavin-bindingprotein. Concentration and fractional saturation in chicken eggs as afunction of dietary riboflavin.&quot&nbspBiochemicalJournal&nbsp238.3(2008): 671-675. Print.