Carbohydrates: Reducing End Concentration Analysis

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Carbohydrates are important in metabolic processes for everyday physical and chemical actions. The carbohydrate, glucose, is a key component in generating adenosine triphosphate, also known as ATP. In order to analyze unknown glucose levels, a DNS assay was performed. By using 2-hydroxy-3,5-dinitrobenzoic acid to oxidize the aldehyde group on the carbohydrate, the reducing end of glucose increases in absorbance of 540 nm. Using a UV spectrophotometer, the concentration was calculated by using a regression line of the standard curve. The experimental data concluded a 1068.912 µg/ml. With the actual concentration being 1200.000 µg/ml, a 10.924% error was deduced. Though the error is slightly high, the deviation of 134.370 µg/ml makes up for some of the error through this process; entailing DNS assay an acceptable method of calculating unknown glucose levels. Introduction

Figure 1: Glucose. Circled Portion: Aldehyde
Figure 1: Glucose. Circled Portion: Aldehyde
Carbohydrate is defined as a polyhydroxyl ketone, a polyhydroxyl aldehyde, or their derivatives. Figure 1 is an example of a carbohydrate, namely, glucose. Glucose is a prime contributor of carbon for other carbohydrates, amino acids, lipids, and nucleic acids. Glucose is also a main contributor to generating adenosine triphosphate through glycolysis, providing energy for chemical and everyday functions. The negative of glucose is, in high concentration, insulin pathways are blocked, causing diabetes. In order to calculate glucose levels for monitoring, a unique functional group must be observed. That being carbohydrate’s aldehyde group. The aldehyde group helps in metabolic functions and provides a reducing agent; aldehyde group shown in Figure 1. The aldehyde group, also known as the reducing end, has the ability to undergo oxidation-reduction reactions; making glucose, an undetectable compound, detectable Figure 2: Gluconic acid. Circled Portion: Carboxylic acid Figure 2: Gluconic acid. Circled Portion: Carboxylic acid

with the help of a DNS assay. The DNS assay uses a redox reaction with glucose’s reducing end and 2-hydroxy-3,5-dinitrobenzoic acid, also known as DNS, oxidizing glucose’s aldehyde group into a carboxylic acid, shown in Figure 2. The newly reduced DNS increases directly in absorbency with the increase of reduced ends. Through spectrophotometry, the concentration of glucose is available for measurement. Unknown concentrations of glucose can be determined via the measured absorbency and a standard curve. With the glucose concentration in hand, preventative measures are achievable to prevent diabetes. Methods and Materials

Number each test tube from #1 through #8. Disperse numerically, 3.000 ml, 2.750 ml, 2.500 ml, 2.250 ml, 2.000 ml, 2.750 ml, 2.500 ml, and 2.000 ml of water. Displace the ‘Standard Glucose’ into tubes #1-5 correspondingly 0.000 ml, 0.250 ml, 0.500 ml, 0.750 ml, and 1.000 ml. Distribute the ‘Unknown Glucose’ into tubes #6-#8, with 0.250 ml, 0.500 ml, and 1.000 ml. Allocate 1.00 ml of DNS into each test tube, making sure the total volume of every tube is approximately 4.000 ml. Cap and place all eight test tubes into the hot water bath, allowing incubation for twenty minutes. The heat acts as a catalyst to speed up the redox reaction rate.

After the incubation, reorder the solutions from light to dark. Carefully wash each cuvette with their corresponding test tubes. The wash will clean out the cuvette, as well as created a coat for more accurate readings. Transfer at least 2 ml from the corresponding test tubes to their matching cuvettes. Place the lightest cuvette, solution #1 due to its 0.000 ml concentration of glucose, into the UV Spectrophotometer and use it as a blank. Following this process, continue to measure the absorbency of each solution from lightest to darkest. After recording the measurements, use test tubes of the known absorbance for #1-#5 and place them in an excel...
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