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澳洲墨尔本Melbourne大学生物医学留学生assignment展示:Lab report - Milk chemistry assignment

 

 

Lab report - Milk chemistry

Introduction

Melamine is an organic base chemical rich in nitrogen (66% by mass versus approx. 10–12% for typical protein) and is widely used in plastics, adhesives, countertops, dishware and whiteboards. (Global Alert and Response)

In September 2008, several manufacturers were implicated in a scandal involving milk and infant formula adulterated with melamine, which leads to kidney stones and renal failure, especially severe milk to increase its volume.

The serial tests were conducted to determine the quality in whole milk sample. Two aspects were considered to determine quality of the milk: possible existence of cheap sugar, eg. corn syrup (fructose, monosaccharide, reducing) or table sugar (sucrose, disaccharide, nonreducing) to replace lactose(disaccharide, reducing) in real milk, and possible existence of melamine, the chemical addictive added to enhance nitrogen content. A series of tests conducted,  including Biuret test for the presence of peptide bonds, Benedicts test for presence of reducing sugars, Barfoed’s Test used for the presence of monosaccharide and Ninhydrin test for the presence of amino acids. 

The test result can be predicted as follows: if the whole milk sample had cheap addictive sugar present, it should have positive result in Barfoed’s test, or negative result in both Barfoed’s test and Benedicts test; if the whole milk have no cheaper addictive sugar but milk sugar lactose, it should have negative result in Barfoed’s test and positive result in Benedict’s test; if the whole milk sample have melamine contamination, it should have positive Ninhydrin test and negative result in Biuret test.

Method

Milk containing 2% of fat was bought from supermarket and then underwent a fractionation process in lab. It split milk into 5 fractions:  F2, F2D (short for F2Digest), F3, F3D (short for f3Digest) and F4.These five fractions together with whole milk and distilled water constitute the 7 samples examined in the following 4 tests.

Biuret test: label 7 tubes respectively as F2, F2D, F3, F3D, F4, WM (whole milk) and Blank. Add 10 drops of the fraction liquids to their labeled tubes. Add 10 drops of distilled water to the blank. Add 10 drops of Biuret solution to each tube. Mix them well. Observe the color change. A color change from blue to purple indicates polypeptide presence of peptide bond and should be recorded as positive.

Benedict’s test: label 7 tubes respectively as F2, F2D, F3, F3D, F4, WM (whole milk) and Blank. Start boiling water in beaker. Add 10 drops of the fraction liquids to their labeled tubes. Add 10 drops of distilled water to the blank. Add 3ml of Benedict’s solution to each of 7 tubes. Keep tubes in boiling water for 2 minutes and wait for color change. Record the change of color. The greater amount of reducing sugar in the fraction, the darker the red of the solution will be. Record test result according to color change: no color change-negative, red-positive, dark red- double positive.

Barfoed’s test: label 7 tubes respectively as F2, F2D, F3, F3D, F4, WM (whole milk) and Blank. Start boiling water in beaker. Add 10 drops of the fraction liquids to their labeled tubes. Add 10 drops of distilled water to the blank. Add 3ml of Barfoed’s solution to each of 7 tubes. Keep tubes in boiling water for 2 minutes and wait for color change. Record test result. A form of red sediment in solution should be recorded as positive.

Ninhydrin test: prepare chromatography paper for 7 samples. Put a pea-sized drop of the fraction on its spot. Place in chromatography jar for 20 minutes. Then stain the paper in Ninhydrin for 10 minutes. Record the existence of purple spot on each fraction in a positive-negative format.

Result

In Biuret test, 5 samples including Whole Milk, F2, F2D, F3 and F3D showed color change from blue to purple. The test results probably indicate these 5 samples had at least 5 kinds of amino acid present. Sample F4 and control group showed no change of color. For Benedict’s test, sample of Whole milk, F3, F3D, F4 showed positive results. The outcome probably indicates the existence of reducing sugar in these samples. Sample F2, F2D and control group showed no change of color. All the samples in Barfoed’s test are red sediment negative. In chromatography test, F2D and F3D show purple spot on their sample drops.

The results of these four tests were recorded as the following table: Table 1

 

Milk Fraction

Reagent

Whole

F2

F2D

F3

F3D

F4

Blank

Biuret

+

+

+

+

+

-

-

Benedicts

+

-

-

+

+

+

-

Barfoed’s

-

-

-

-

-

-

-

Ninhydrin

-

-

+

-

+

-

-

     Table1. Four test results

 

 

 

 

 

 

 

Discussion

According to the tests results above, prediction of contents in each fraction of milk can be predicted. Test result of whole milk sample indicates the presence of peptide bond, namely protein, reducing sugar and amino acid. Result from F2 and F2D indicate the solely  presence of protein, because Barfoed’s test of F2D rule out glycoprotein and disaccharide, Barfoed’s test of F2 rule out monosaccharide and Benedict’s test of  in F2. Result from F3 and F3D are flawed as the test result of Barfoed’s test and Benedict test are conflicting with each other. Only the existence of protein can be confirmed in F3. Result of F4 indicates it contained reducing sugar for the positive result in Benedict’s test. From Whole milk group, protein and reducing sugar are present. To conclude, protein and reducing sugar are present in the whole milk used in lab.

Flawed result happened for multiple reasons. The fractionation process can be very sloppy and leads to internal contamination. There is no melamine as control group, so only deductive reasonning or guesses are possible.

Based on this flawed test result, I cannot tell the minister of food safety anything about the quality of the milk tested. I can be sure the sample contains real protein and lactose, but no conclusion of the existence of cheaper addictive like cane sugar or corn syrup can be drawn from the 4 test we ran. The clue for the presence of real protein is that group of Whole Milk and F2 contains peptide bond and group F2D contains amino acid. Any protein substitute is never capable of achieving these kind of test result. Melamine, one kind of protein substitute used in milk adulteration, contains three amine groups, the characteristic group of amino acid, in one molecule. The amine group in melamine act with Ninhydrin, and in this way, melamine and digested real protein have the same result in Ninhydrin chromatography test. But another characteristic structure of protein is exclusive- its peptide bond, and showed positive result in Biuret test.

No evidence of melamine’s existence can be found in these four tests, because none of these 7 groups showed concurrent outcome amino acid positive and peptide bond negative. The presence of melamine, however, is still uncertain for the study’s evident limitations. All the tests to examine the existence of melamine are qualitative instead of quantitative. The content in the whole milk can be too small that it is untraceable in qualitative test. To make the test convincing, the study should also contain specific test for melamine’s existence and content.

 

 

 

Biliography

Global Alert and Response (GAR) [Internet]. Geneva, Switzerland, World Health Organization, Program and projects; [cited 2012 Feb 5]. Available from: http://www.who.int/csr/media/faq/QAmelamine/en/index.html

 

 

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