848 Journal of Chemical Education • Vol. 73 No. 9 September 1996

In the Laboratory

The Use of a “Qual” Centrifuge for Greatly Simplifyingand Speeding the Study of Milk

Quentin R. PetersenCentral Michigan University, Mt. Pleasant, MI 48858

The examination of the components of milk as a labo-ratory exercise in a one-semester combined organic–bio-chemistry or survey of biochemistry course has been of-fered in allied-health laboratory texts for many years.Scheduled near the end of the semester, a time when thesubjects of carbohydrates and proteins are bringing thelecture portion of the course to a close, this experimentcan convey a most rewarding reality to these topics.

However, in the hands of many students, this veryinstructive experiment has regularly been flawed by thetime-consuming, frustrating, often complex step of sepa-rating the curd and the whey before protein and carbohy-drate isolation and testing could be carried out. This seem-ingly trivial technical problem discourages these studentsat the very moment when they might be able to unify ex-periment and theory to perceive a rational interrelation-ship of the topics they have been studying all semester.

To realize the potential of this integrating experiment,a better method of isolating the constituents of milk wassought. Methods that have been suggested (1–5) to sepa-rate these components have utilized wire gauze, Büchnerfunnels, glass wool, cheese cloth, coffee filters, and decan-tation followed by physical squeezing of the curd; and one,seemingly unaware of the problem, simply directs the stu-dent to “Filter the solution ...”.

We chose to use a low-tech application of a high-techbiochemical approach, and it worked! In our current ex-periment we start with a physical examination of raw,pasteurized whole, and skim milks. This is followed byverification of the presence of xanthine dehydrogenase inthe raw milk and its absence in pasteurized milk (6). Theuse of a Touch Mixer1 in the laboratory adds some fun tothis test. It is interesting to note that, without instruc-tion, students recognized the value of this tool and re-turned to it while doing the carbohydrate tests! The“grease-spot” test (7) to compare the fat contents of themilks completes the preliminary examination.

Inasmuch as the various classical protein and carbo-hydrate tests require little material, there seems to be noreason to start the experiment with 50–100 mL of milk asmost lab manuals specify. Using a smaller volume of milkprovides an opportunity to introduce students to centrifu-gation as a primary separation technique. A smaller vol-ume of milk requires smaller volumes of reagents as well.Finally, the technique substantially reduces the time nec-essary for the separation.

In our laboratories each student starts with 5 mL ofskim milk held in a standard 13 × 100 mm test tube. Thetube is warmed in a beaker of hot tap water and ten dropseach of the precipitating (10% acetic acid) and buffering(1 M sodium acetate) reagents are added directly to thistest tube. Stirring and pH testing prepare the sample forcentrifugation in a standard 4-socket centrifuge2 that hasbeen used in qualitative analysis work for many years.

Depending upon the number of instruments available, oneto four students can use the same centrifuge. However,the separation is so rapid (3–5 minutes) that socket avail-ability has not been a problem.

Once separated in this fashion, the opalescent wheyis decanted into a 50-mL beaker and heated to boiling,under a compressed-air stream, to precipitate the serumprotein. The volume is reduced from 6 mL to 3 mL in about10 minutes.

During this preparation of the serum protein, thecasein button is washed by using a stirring rod to sus-pend it in fresh water and then recentrifuging. If solidcasein is desired for study, successive washes with acetoneand ether will quickly provide 0.14–0.16 g of dry casein.

The serum protein and remaining liquid is transferredto a second 13 × 100 mm test tube. Rinsing the beakerwith small volumes of water brings the volume in the testtube up to 5 or 6 mL, and centrifugation is again used toseparate the protein. The supernatent liquid (containingthe lactose) is transferred to a 50-mL beaker and placedunder a compressed air stream at a low temperature (50–55 °C.) The serum protein is washed in the same way ascasein. Solid serum protein can be prepared by the samemethod as solid casein, with a typical yield of 0.025–0.035 g ofdry material.

In our experiment each student’s moist, clean pro-tein buttons, still in their original test tubes, are dissolvedin about 5 mL of 1% sodium hydroxide. The serum pro-tein may need the heat from a boiling-water bath toachieve its solution. If any solid is left in these solutions,brief centrifugation is again applied and the supernatantliquids are used directly for the protein tests. Our stu-dents carry out the biuret, xanthoproteic and Hopkins-Cole tests (8) on each protein solution.

The syrup (0.18–0.20 g) remaining after evaporatingthe liquid decanted from the serum protein will crystal-lize on standing for a few days but, if solid lactose is de-sired, it will crystallize immediately upon the addition ofabsolute ethanol. Alternatively, addition of 10 mL of wa-ter to the syrup provides the solution to which we applythe Benedict’s and Barfoed’s tests (9).

In summary, in two-and-one-half hours of a three-hourlaboratory period, students with just one semester of priorgeneral laboratory experience have:

• learned to use two new laboratory tools• completed an experiment verifying the presence of xan-

thine dehydrogenase in raw milk and its absence in pas-teurized milk

• verified the presence of fat in pasteurized whole milk andits absence from skim milk.

• recognized the presence of tryptophan in casein and itsabsence in milk-serum protein.

• isolated lactose and established it as being a reducingdisaccharide uncontaminated by monosaccharide.In addition to yielding the results described above,

the separation procedure provides protein and carbohy-drate solutions to which many other standard tests might

1Model 231 Touch Master, Fisher Scientific, Chicago.2S-15700 Semimicro High Speed Centrifuge, Sargent-Welch,

Buffalo Grove, IL.

Vol. 73 No. 9 September 1996 • Journal of Chemical Education 849

In the Laboratory

be applied. Finally, the procedure described can be usedto give quantitative measures of solid casein, solid serumprotein, and solid lactose. The yields compare very wellwith the reported (10) amounts of total protein in bovinemilk (3.25%), the proportion of this total that is casein(80%), and the amount of lactose (4.5%) present in themilk.

Literature Cited

1. Hill, J.; Scott, L. W.; Muto, P. Chemical Investigations for Changing Times,5th ed.; Macmillan: New York, 1988.

2. Mohr, S. C.; Griffin, S. F.; Gensler, W. J. Laboratory Manual for Fundamentalsof Organic and Biological Chemistry; McMurry, J.; Castellion, M., Eds.;Prentice Hall: Englewood Cliffs, NJ, 1994.

3. Nitz, O. V.; Runnalls, N. G. Laboratory Manual for Introductory Chemistry4th ed.;WCB: Dubuque, IA, 1972.

4. Stenesh, J. Experimental Biochemistry; Allyn and Bacon: Boston, 1984.5. Timberlake, K. C. Lab Manual to Accompany “An Introduction to General,

Organic and Biological Chemistry” 5th ed.; HarperCollins: New York, 1992.6. Pritham, G. H. Anderson’s Laboratory Experiments in Biochemistry; Mosby:

St Louis, 1968; p 78.7. Caret, R. L.; Denniston, K. J.; Topping, J. J. A Laboratory for General Organic

and Biochemistry; WCB: Dubuque, IA, 1993; p 282..8. Strong, F. M.; Koch, G. H. Biochemistry Laboratory Manual 3rd ed.;WCB:

Dubuque, IA, 1981; pp 169–170.9. Ibid., pp 71–72.

10. The Merck Index, 11th ed.; Merck and Co.: Rahway, NJ, 1989; p 975.