LBS 145
Lab
Group:
Journey
Tuesday
10:30-1:20
2-28-06
Members:
Kyle
Clarkson
James
Redwine
ABSTRACT:
The objective
of this experiment was to analyze
the differences between sweet corn and field corn.
The results may help to clarify why sweet corn
is more appealing and is marketed as a food for humans instead of field
corn. The types and abundance of sugars
present, the total protein content, and the pigment content of the two
types of
corn were tested. It is expected that
sweet corn will have a higher concentration of simple sugars
(monosaccharides
and disaccharides), and xanthophyll pigments than field corn. The sweeter and brighter the corn, the more
appetizing it would be to humans (Willaman, 1928).
It is expected that the field corn will have
higher protein content as a corn high in protein would be most
desirable for
cattle feed, which is field corn’s primary use.
The methods used for the standard sugar analysis were Barfoed’s,
Selivanoff’s, and the Iodine tests. Bradford ’s protein assay was used for protein
concentration analysis, and thin-layer chromatography, TLC, was used to
examine
the amount of xanthophyll in the samples.
For quantitative carbohydrate analysis, the precipitates from
Barfoed’s
test will be massed and compared. The
corn samples in all of the carbohydrate tests were positive, with the
exception
that the field corn samples were negative in Barfoed’s.
This indicates that field corn has a
concentration of monosaccharides that is below the detection limit of
the test. The results of Bradford ’s
protein assay indicate that the sweet corn contained a higher amount of
protein
than field corn. The TLC showed field corn contained a higher amount of
xanthophyll instead of the sweet corn. The quantitative analysis of
Barfoed’s
test provided the evidence that there are more monosacharides present
in sweet
corn than field corn making the name “sweet corn” an excellent choice
for this
variety of maize.
U.S.
It was hypothesized that sweet corn would have higher concentrations of simple sugars (monosaccharides, disaccharides) and pigments than field corn; however, field corn was expected to have higher protein content. This was based on the idea that sweet corn is intended to be sold to the public, should have an appealing flavor and appearance. Field corn, on the other hand, does not need to be flavorful, nor colorful, as it is normally fed to livestock or wild game. However, higher protein content in the field corn would improve its value as a feed (Huffman and Duncan, 1944).
The carbohydrate portion of this hypothesis was first addressed by the standard sugar tests: Barfoed’s, Selivanoff’s, and the Iodine test (Khra et al., 2006). Due to the abundance of different sugars in corn, it was expected that both corn samples would prove positive in all tests. However, it was hypothesized that the sweet corn would be higher in mono- and disaccharides, specifically. That was because the ‘sweetest’ sugars are these simpler sugars (Willaman, 1928).
After preparing the corn solutions each sugar test; Barfoed’s, Selivanoff’s, and the Iodine test, the Bradford Assay Experiment, Thin-Layer Chromatography, and an Independent Experiment (Quantitative Barfoed’s) were performed. As previously mentioned, each test was performed with a mock control sample (water), a positive control, and a negative control. The tests consisted of three trials of field corn solution and sweet corn solution.
Barfoed’s Monosaccharide Test:
The Barfoed’s monosaccharide test resulted in a red precipitate for the sweet corn, which indicates the presence monosaccharides. However, there was no reaction for the field corn, which indicates that the monosaccharide concentration is lower than the detection limit for this test. The results for the Barfoed’s test can be seen in Figure I.
Selivanoff’s Aldose and Ketose Test:
Selivanoff’s test for both the field corn samples and sweet corn samples, were positive for ketose. The solutions turned a red color, indicating a positive test. Figure II shows the results for this experiment.
The Iodine Test for Polysaccharides:
The Iodine test, which is used to detect the presence of starches, produced a bluish-black color in both sweet and field corn, and therefore showed a positive result for coiled polysaccharides (starch). Figure III shows this conclusion.
TheBradford
Protein Assay:
The protein content of the corn was measured via the Bradford Protein Assay, which helped create a standard curve. The field corn was expected to show a higher protein quantity given that it is a primary ingredient for many cattle feed mixes and would desire a high-protein corn hybrid for this use (Blezinger, unknown). Using the Bradford Assay to determine protein amount, it was found that sweet corn contains 0.638 ug of protein per ml, while field corn contains 0.417 ug/ml. These results from the Bradford Assay concluded in a rejection of the hypothesis. The degradation of the corn samples should not have affected the results because both samples were prepared at the same time and left under the same conditions. Results for this experiment can be viewed in Figure IV and Figure V. A possible reason that the prediction was wrong is that the sweet corn hybrid that was purchased for this experiment may have been grown/engineered to have a higher protein content to make it healthier for human consumption. In order to determine if this was indeed the case, a more detailed study of the hybrid and its lineage would have to be performed.
Thin-Layer Chromatography Test (TLC):
The pigment portion of the hypothesis was addressed by a quantitative Thin Layer Chromatography (TLC) method. To maximize the accuracy of the results, three trials of both the field and sweet corn were analyzed using the spectrometer. The pigment that was used to determine the relative abundance of total pigment was the band that corresponded to xanthophylls. One reason that only the xanthophyll zeaxanthin band was used was because it is the pigment that is most responsible for the yellow color of corn kernels (Freeman, 2005). The pigments present in each extract interacted with the mobile phase differently, causing the pigments to stop at different lengths along the paper strip. Figure VI shows these different lengths for the TLC test for the corn samples. The readings for the absorbencies were higher for the field corn. Beer’s Law states that the absorbance of a solution at a given wavelength is dependent on the concentration of the solution. Given that both solutions are the same pigment and the solvent is the same, the absorbance would be directly proportional to the concentration. Since the field corn had a higher absorbance than sweet corn, it also has a higher concentration of the pigment. The results for this experiment rejects the hypothesis that the sweet corn would have more pigment than the field corn, because field corn has a thicker outer shell, which is where the pigment is located, therefore has more total xanthophylls (NCGA, unknown).
Quantitative Barfoed’s Test:
The quantitative analysis of monosaccharides also employed Barfoed’s test. This test indicates the presence of monosaccharides by their ability to reduce Cu2+ to insoluble Cu+. Thus, the amount of the Cu+ salt that precipitates could be used to determine the amount of reactive monosaccharides present in the sample (Krha et al., 2006). The precipitate formed from the monosaccharides reacting with the Barfoed’s reagent was filtered out and then measured. The amount of precipitate was proportional to the amount of monosaccharides present in the solution. The sweet corn samples contain approximately 1.216 x 10-4 more moles of monosaccharides than the field corn samples. This corresponds to an average of 0.0057 grams more Cu2O present in sweet corn samples than in the field corn samples. Figure VII shows the filter papers for the sweet corn samples and the field corn samples.
Errors:
A majority of the errors that occurred during the experiments were due to incorrect readings from the spectrophotometer. Also, an incorrect measure of the mass of the filter papers during our quantitative Barfoed’s made it impossible to determine the absolute amount of Cu2O formed. During the independent experiment test, when first massed, the papers had atmospheric H2O levels, and afterwards, the papers were fully dried. Therefore, the papers lost an unknown amount of water to the atmosphere during drying. However, all of the filter papers were dried the same, and had the same original water content, so the difference in masses between the two sets of data will be due solely to differences in the Cu2O content. The equation that Barfoed's is governed by is:
1 monosaccharide + 2 Cu(OH)2 1 Cu2
O
+2 H2O + 1
oxidized monosaccharide.
The mass change is due to Cu2O formation, which is produced by the oxidation of monosaccharides. Therefore, our data shows how much more monosaccharides reacted in the sweet corn than in the field corn, but is unable to show the absolute monosaccharide concentrations.
Additional Experiments:
To better quantify the amount of specific monosaccharides present into acquire absolute monosaccharide concentrations, High Performance Liquid Chromatography (HPLC) could be performed. G. Sesta has performed this experiment. In his experiment, the instrument used was run on a computerized system, with a Restek Pinnacle II Amino, 5μm, 250x3.2mm column, and a refractive index (RI) detector was used. A constant ratio eluent must be used, as RI detectors will pick up a concentration gradient. The eluent would be 85:15 Acetonitrile/water, and we will use that as well (Sesta 2006). The flow rate and concentrations would be determined by the specific instrument used.
To better adapt Barfoed’s test to use as a quantitative analysis tool, a standard curve could be used. To develop a standard curve, one would use several solutions of known monosaccharide concentration (1%, 2%, 3%, etc.). Performed Barfoed’s test and determine mass of Cu2O, as was done in this experiment, then make a graph of concentration vs. mass of Cu2O recovered. Using this curve and the mass of Cu2O recovered from samples of unknown concentration, the absolute concentration of the samples could be determined. In order to avoid the problem of over drying the filter papers, the filter papers should be dried for approximately one hour in an oven at 250o to 300o before use. Then dried under same conditions after filtering the reacted solutions.
For a more quantitative pigment analysis, a standard curve could be used to determine the absolute concentration of zeaxanthin. To prepare the standard curve several solutions of known zeaxanthin concentrations would be prepared using pure zeaxanthin dissolved in one milliliter of 80% acetone, 20% hexane solution. The absorbance could then be determined for each of the standard solutions, and a graph of zeaxanthin concentration vs. absorbance generated. The absorbance of the unknown sample could then be plotted on this graph to determine absolute concentration of zeaxanthin.
DISCUSSION/CONCLUSIONS:
This experiment deals with the differences between sweet corn and field corn. All corn that is purchased in the store for human consumption is sweet corn. However field corn, also known as dent corn, accounts for a majority of corn production in theIt was hypothesized that sweet corn would have higher concentrations of simple sugars (monosaccharides, disaccharides) and pigments than field corn; however, field corn was expected to have higher protein content. This was based on the idea that sweet corn is intended to be sold to the public, should have an appealing flavor and appearance. Field corn, on the other hand, does not need to be flavorful, nor colorful, as it is normally fed to livestock or wild game. However, higher protein content in the field corn would improve its value as a feed (Huffman and Duncan, 1944).
The carbohydrate portion of this hypothesis was first addressed by the standard sugar tests: Barfoed’s, Selivanoff’s, and the Iodine test (Khra et al., 2006). Due to the abundance of different sugars in corn, it was expected that both corn samples would prove positive in all tests. However, it was hypothesized that the sweet corn would be higher in mono- and disaccharides, specifically. That was because the ‘sweetest’ sugars are these simpler sugars (Willaman, 1928).
After preparing the corn solutions each sugar test; Barfoed’s, Selivanoff’s, and the Iodine test, the Bradford Assay Experiment, Thin-Layer Chromatography, and an Independent Experiment (Quantitative Barfoed’s) were performed. As previously mentioned, each test was performed with a mock control sample (water), a positive control, and a negative control. The tests consisted of three trials of field corn solution and sweet corn solution.
Barfoed’s Monosaccharide Test:
The Barfoed’s monosaccharide test resulted in a red precipitate for the sweet corn, which indicates the presence monosaccharides. However, there was no reaction for the field corn, which indicates that the monosaccharide concentration is lower than the detection limit for this test. The results for the Barfoed’s test can be seen in Figure I.
Selivanoff’s Aldose and Ketose Test:
Selivanoff’s test for both the field corn samples and sweet corn samples, were positive for ketose. The solutions turned a red color, indicating a positive test. Figure II shows the results for this experiment.
The Iodine Test for Polysaccharides:
The Iodine test, which is used to detect the presence of starches, produced a bluish-black color in both sweet and field corn, and therefore showed a positive result for coiled polysaccharides (starch). Figure III shows this conclusion.
The
The protein content of the corn was measured via the Bradford Protein Assay, which helped create a standard curve. The field corn was expected to show a higher protein quantity given that it is a primary ingredient for many cattle feed mixes and would desire a high-protein corn hybrid for this use (Blezinger, unknown). Using the Bradford Assay to determine protein amount, it was found that sweet corn contains 0.638 ug of protein per ml, while field corn contains 0.417 ug/ml. These results from the Bradford Assay concluded in a rejection of the hypothesis. The degradation of the corn samples should not have affected the results because both samples were prepared at the same time and left under the same conditions. Results for this experiment can be viewed in Figure IV and Figure V. A possible reason that the prediction was wrong is that the sweet corn hybrid that was purchased for this experiment may have been grown/engineered to have a higher protein content to make it healthier for human consumption. In order to determine if this was indeed the case, a more detailed study of the hybrid and its lineage would have to be performed.
Thin-Layer Chromatography Test (TLC):
The pigment portion of the hypothesis was addressed by a quantitative Thin Layer Chromatography (TLC) method. To maximize the accuracy of the results, three trials of both the field and sweet corn were analyzed using the spectrometer. The pigment that was used to determine the relative abundance of total pigment was the band that corresponded to xanthophylls. One reason that only the xanthophyll zeaxanthin band was used was because it is the pigment that is most responsible for the yellow color of corn kernels (Freeman, 2005). The pigments present in each extract interacted with the mobile phase differently, causing the pigments to stop at different lengths along the paper strip. Figure VI shows these different lengths for the TLC test for the corn samples. The readings for the absorbencies were higher for the field corn. Beer’s Law states that the absorbance of a solution at a given wavelength is dependent on the concentration of the solution. Given that both solutions are the same pigment and the solvent is the same, the absorbance would be directly proportional to the concentration. Since the field corn had a higher absorbance than sweet corn, it also has a higher concentration of the pigment. The results for this experiment rejects the hypothesis that the sweet corn would have more pigment than the field corn, because field corn has a thicker outer shell, which is where the pigment is located, therefore has more total xanthophylls (NCGA, unknown).
Quantitative Barfoed’s Test:
The quantitative analysis of monosaccharides also employed Barfoed’s test. This test indicates the presence of monosaccharides by their ability to reduce Cu2+ to insoluble Cu+. Thus, the amount of the Cu+ salt that precipitates could be used to determine the amount of reactive monosaccharides present in the sample (Krha et al., 2006). The precipitate formed from the monosaccharides reacting with the Barfoed’s reagent was filtered out and then measured. The amount of precipitate was proportional to the amount of monosaccharides present in the solution. The sweet corn samples contain approximately 1.216 x 10-4 more moles of monosaccharides than the field corn samples. This corresponds to an average of 0.0057 grams more Cu2O present in sweet corn samples than in the field corn samples. Figure VII shows the filter papers for the sweet corn samples and the field corn samples.
Errors:
A majority of the errors that occurred during the experiments were due to incorrect readings from the spectrophotometer. Also, an incorrect measure of the mass of the filter papers during our quantitative Barfoed’s made it impossible to determine the absolute amount of Cu2O formed. During the independent experiment test, when first massed, the papers had atmospheric H2O levels, and afterwards, the papers were fully dried. Therefore, the papers lost an unknown amount of water to the atmosphere during drying. However, all of the filter papers were dried the same, and had the same original water content, so the difference in masses between the two sets of data will be due solely to differences in the Cu2O content. The equation that Barfoed's is governed by is:
1 monosaccharide + 2 Cu(OH)2 1 Cu2
O
+2 H2O + 1
oxidized monosaccharide. The mass change is due to Cu2O formation, which is produced by the oxidation of monosaccharides. Therefore, our data shows how much more monosaccharides reacted in the sweet corn than in the field corn, but is unable to show the absolute monosaccharide concentrations.
Additional Experiments:
To better quantify the amount of specific monosaccharides present into acquire absolute monosaccharide concentrations, High Performance Liquid Chromatography (HPLC) could be performed. G. Sesta has performed this experiment. In his experiment, the instrument used was run on a computerized system, with a Restek Pinnacle II Amino, 5μm, 250x3.2mm column, and a refractive index (RI) detector was used. A constant ratio eluent must be used, as RI detectors will pick up a concentration gradient. The eluent would be 85:15 Acetonitrile/water, and we will use that as well (Sesta 2006). The flow rate and concentrations would be determined by the specific instrument used.
To better adapt Barfoed’s test to use as a quantitative analysis tool, a standard curve could be used. To develop a standard curve, one would use several solutions of known monosaccharide concentration (1%, 2%, 3%, etc.). Performed Barfoed’s test and determine mass of Cu2O, as was done in this experiment, then make a graph of concentration vs. mass of Cu2O recovered. Using this curve and the mass of Cu2O recovered from samples of unknown concentration, the absolute concentration of the samples could be determined. In order to avoid the problem of over drying the filter papers, the filter papers should be dried for approximately one hour in an oven at 250o to 300o before use. Then dried under same conditions after filtering the reacted solutions.
For a more quantitative pigment analysis, a standard curve could be used to determine the absolute concentration of zeaxanthin. To prepare the standard curve several solutions of known zeaxanthin concentrations would be prepared using pure zeaxanthin dissolved in one milliliter of 80% acetone, 20% hexane solution. The absorbance could then be determined for each of the standard solutions, and a graph of zeaxanthin concentration vs. absorbance generated. The absorbance of the unknown sample could then be plotted on this graph to determine absolute concentration of zeaxanthin.
FIGURE: