Lactose Essay from Milk PDF

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Lactose Essay from Milk...

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Experiments 7-8: Isolation of Lactose from Milk Amy Sylvester

Partner: Alex Whittington

October 3-10,

2018 Abstract In this experiment, the disaccharide lactose was separated from dehydrated milk and analyzed using thin layer chromatography. A commercial brand of lactase was tested for its function and the composition of an unknown polysaccharide solution “E” was determined. The percent recovery of lactose in nonfat dehydrated milk (trial A) was determined to be approximately 44.8%. The percent recovery of lactose in nonfat dehydrated milk with added commercial lactase (trial B) was determined to be approximately 26.9%. Using analysis by thin layer chromatography, the material in trail A had only one spot which was determined to be Lactose, the material in trial B had four spots and three of them were determined to be Lactose, Galactose, and Glucose, the unknown sample “E” had only one spot when it was unhydrolyzed and hydrolyzed which was determined to be Glucose. Introduction This experiment was separated over two lab periods. The first part of the lab involved the separation of the carbohydrate lactose from dehydrated nonfat milk with and without a commercial lactase added to the milk to simultaneously test the function of lactase. The next part of this experiment was used to analyze these results and to subsequently identify the components of an unknown polysaccharide solution using a hydrolysis reaction and thin layer chromatography.

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Carbohydrates represent 50% of organic matter in the biosphere and serve as energy sources and structural components for cells. Lactose, also known as milk sugar, is a disaccharide and one

of the major nutrients found in milk along with other proteins and fat (eg. Casein, lactalbumin, and vitamins), making up 5% of the weight of liquid milk and approximately 52% of the weight of the nonfat dehydrated milk used in this experiment. Lactose is composed of one galactose unit and one glucose unit with the overall structure: Our bodies have their own method of breaking down or extracting lactose in the stomach, which we will approximately recreate in this lab using enzymes, heat and alcohol, and activated charcoal. The purification procedure used in this experiment is outlined in the Methods and Materials section as well as in Fig (1). In brief, pepsin is added to nonfat milk which will digest up to 20% of ingested carbon bonds and will essentially remove the casein from the milk. Next, using heat and ethanol, proteins such as lactalbumin are precipitated out. Finally, by adding activated charcoal to the solution, the remaining vitamins absorbed, leaving a final solution of purified lactose.

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As a person ages, their ability to be able to digest lactose begins to decrease; this is called lactose intolerance. Lactose intolerance is the result of a shortage of the enzyme β−galactosidase , lactase, which is normally produced by the cells that line the small intestine. Lactase functions to break down lactose into its substituent parts (glucose and galactose) which are then able to be absorbed into the blood stream. This experiment will test the effectiveness of the commercial-made lactase supplement, Lactaid®, produced and sold as a supplement of this enzyme. To analyze the data obtained in this experiment, Thin Layer Chromatography (TLC) was used to separate the standard carbohydrates and the hydrolysis reaction mixture according to their relative polarities. The molecules of interest separate differentially between a stationary and mobile phase. The stationary phase is a glass plate coated in a thin layer of silica gel, and the mobile phase is made up of an organic polar solvent, specifically acetonitrile (CH3-CN) in this experiment. A migration rate (Rf) is calculated for each constituent on the plate which aids in the analysis of the present components. This rate is calculated using by: Rf =

distancetraveled by substance distancetraveled by solvent front

(1)

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A hydrolysis reaction usually means the cleavage of chemical bonds by the addition of water and is usually a step in the degradation of a substance. During the TLC analysis, a hydrolysis reaction of trial A (the trial lacking the lactase) will be done and spotted on the plate. Since trial A is supposed to be pure lactose, one spot should appear for the unhydrolyzed sample and at least two spots should appear for the hydrolyzed since lactose is a disaccharide. A hydrolysis will also be performed on the unknown sample to determine if it is a mono-, di-, or polysaccharide.

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Materials Experiment 7: Isolation of Lactose from Milk

Experiment 8: Analysis of Polysaccharide Composition by Thin Layer Chromatography Methods Experiment 7: Isolation of Lactose from Milk 1. Weigh out 2 portions, labeled A or B, 25 g each, of nonfat dry milk. 2. Boil two 50 mL portions of tap water in 400 mL beakers. Remove from the hot plate and immediately add 25 g nonfat dry milk slowly with constant stirring. 3. Allow solutions to cool and follow the respective procedures below for samples A & B:

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a. Sample A: When the temperature cools to 60-65°C, stir in 25 mL 0.2% pepsin. Let stand for 5 minutes. Curd should form as a precipitate in the solution. Proceed to step 4. b. Sample B: Cool to 37oC, add one crushed (using glassine paper and a pestle) Lactaid® tablet. Stir gently for a few minutes. Place the beaker in a waterbath/incubator to maintain 37ºC. After 30 min, place on a hot plate and reheat the beaker until it reaches 60-65ºC. Stirring thoroughly, add 25 mL of 0.2% pepsin. Let stand for 5 min. Curd should form as a precipitate in the solution. Proceed to step 4. 4. Pour the curd/solution over a funnel with a double layer of cheesecloth. When most of the filtrate has drained, lift the cheesecloth and gently squeeze out the remaining solution. 5. With constant stirring, heat the filtrate to a boil. 6. Remove from hotplate and add 0.50 g activated charcoal. Stir gently for 10-15 seconds. 7. Add 150 mL of ethanol and stir briefly. Let the solution stand at room temperature 30-60 seconds to allow the coagulated protein and the charcoal to settle. 8. Label and weigh two 600 mL beakers and record the weight. Use these beakers to collect the filtrates A and B in the following step. 9. Slowly filter the solution over ~20 g sea sand in a funnel containing fluted filter paper. 10. Let the filtrate stand in a vent hood for at least one week to allow lactose to crystallize. 11. Once crystals completely dry, weigh the beakers can calculate the weight of the crystals inside. Experiment 8: Analysis of Polysaccharide Composition by TLC A. Lactose Preparation

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1. Prepare a 5 mg/mL solution in distilled water of the lactose, sample A, that you isolated last week. To prepare sample B (from last week’s experiment), take a spatula size scoop of the gel-like material and dissolve it in ~5 mL water. Amounts are not critical. B. Hydrolysis of Samples (unknown, lactose-A) 1. In a microcentrifuge tube, add 10 µL of trifluoroacetic acid to 100 µL of the unknown polysaccharide solution. 2. In another microcentrifuge tube, add 10 µL of trifluoroacetic acid to 100 µL of your lactose solution (A). 3. Incubate both solutions at 100°C for 30 minutes. While the sample is incubating begin to prepare your TLC plate as detailed below. C. Thin Layer Chromatography (TLC) 1. Working with a practice plate, practice marking and spotting the plate with water and pencil. 2. Using a pencil (not ink) and straight edge, draw an equidistant line 1 inch from the bottom of the 20 x 20 cm plate. Do not cut into the white coating on the plate as this will ruin the plate. 3. Gain the instructor’s approval before beginning to work with the final plate. 4. Obtain a fresh analytical 20 x 20 cm TLC plate, mark and spot as in step 2 above using your real samples. Mark 8 spots, spaced equidistant, on the line. Record order of the samples and standards as spotted on your plate. SPOTTING THE TLC PLATE a. Add 100 µL water to the hydrolyzed mixtures before spotting.

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b. Spot 2-4 µL of each standard solution, untreated unknown, isolated lactose(A) & (B), and hydrolyzed lactose (A) and unknown. If you need to spot twice using a capillary tube, make sure the spot first time spotted is dry before spotting second time. c. Dry the plate until the spots no longer look wet (gray) but appear solid white. d. For 20 x 20 cm TLC plate, develop the plate with 85% acetonitrile in water (~110 mL) in a chromatography chamber. Solvent should be initially below the level of the samples. Let the solvent migrate up the plate until it reaches about 1 inch to the top. Handling the plate by its edges, remove it from the chamber. Dry for at least 5 minutes using a hand-held dryer and gentle sweeping motion. The plate must be completely dry before continuing to part D. e. Repeat step 4 with another 20 x 20 cm TLC plate D. Staining and Charring the Plate 1. Dip the dried plate in a solution of napthylethylenediamine dihydrochloride in sulfuric acid and methanol for about 15 seconds. Wearing nitrile gloves, remove and dry the plate again. 2. Incubate your plate an oven at 120°C for ~10 minutes. Check your plate at 5 minincubation. Purple-black spots will indicate the presence of carbohydrate. 3. Take pictures of your TLC plates next to rulers and data labels. E. Analysis of the TLC Plate 1. Determine the Rf value of each of the standards on the plate: a. Measure the distance from origin to the solvent front (~1” from top of plate). b. Measure the distance each individual sample (spot) traveled from the origin.

9 i. All measurements should be made from the origin to the front edge of the

visualized spot. Calculate the Rf for each spot using Eq. (1).

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Results Part I: Percent Recovery of Pure Lactose: Starting Material Mass (Non-fat Dehydrated Milk): 25g Final Mass (wet) A: 21.621 g Starting Material Mass (Non-fat Dehydrated Milk + Lactase): 25g Final Mass (wet) B: 39.05 g Theoretical amount of Lactose=(.52)(25.00 g)=13.00 g After the drying period, the samples A and B were both still wet. Thus, since the ethanol had not fully evaporated, the calculated percent recoveries were not accurate (A=166.3% recovery) (B=300.4% recovery). Since these values are not accurate results, a more in-depth analysis was performed to approximate the actual amount of pure lactose recovered. This was done using the density of ethanol and the amount of liquid left in the beaker: Density of EtOH =0.79 g/mL

Approximately 20 mL of EtOH remained in Sample A, and approximately 45 mL of EtOH remained in Sample B: 20 mL EtOH × 0.79 g / mL EtOH =15.8 g EtOH 21.621 g Sample A−15.8 g EtOH ≈ 5.824 g Sample A

Recovery A=

5.824 g × 100 13.00 g

≈ 44.8 recoveryof Lactose∈Sample A

45 mL EtOH ×0.79 g / mL EtOH =35.55 g EtOH 39.05 g Sample B−35.55 g EtOH ≈ 3.50 g Sample B

≈ 26.9 recovery of Lactose∈Sample B

Recovery B=

3.50 g ×100 13.00 g

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Part II: Analysis of TLC Plates Figures (2) and (3) display the thin layer chromatography plates created during this experiment. Fig. (2) shows the plate that was developed only one time, and Fig. (3) shows the plate that was developed twice—the raw version and the annotated version. Table (1) displays each spot’s Rf value (each found using Eq. (1)) in Fig. (3).

Table 1: Calculated Rf Values Regarding Fig. (3) Sample Lactose Glucose Galactose Hydrolyzed Unknown “E” Unknown “E” A Hydrolyzed A B

Rf1

Rf2

Rf3

Rf4

0.274 0.472 0.448 0.488 0.472 0.262 0.298 0.482 0.095 0.262 0.429 0.472

Fig.

Figure 2: TLC plate of (from left to right): Lactose, Glucose, Galactose, Hydrolyzed Unknown “E”, Unknown “E”, Sample A, Hydrolyzed Sample A, and Sample B; This is the plate that was only developed once, hence the Fig. (3b)

(3a)

Figure 3: (from left to right): Lactose, Glucose, Galactose, Hydrolyzed Unknown “E”, Unknown “E”, Sample A, Hydrolyzed Sample A, and Sample B a) Raw TLC plate that was processed twice, resulting in more prominent spots. b) Annotated TLC plate that was processed twice

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Discussion Since the drying period was not long enough or there was not enough ventilation in the fume hood where the samples A and B were set to dry, after one week, the samples were still very wet. Sample A had approximately 20 mL of ethanol (EtOH) and Sample B had approximately 45 mL of EtOH left in its beaker. With this left-over alcohol, any weight percent yields/recoveries would not be accurate. Therefore it appears that sample A had a final volume and percent recovery of 21.621 g and 166.3% and that sample B had a final volume and percent recovery of 39.05 g and 300.4% recovery. However, these values clearly make no sense since, theoretically, the dehydrated nonfat milk contained 52% weight of Lactose. Seeing these inaccuracies, further analysis was done by factoring in the weight of the ethanol in the beaker to determine the approximate weight of the sample. In doing this, sample A and B were determined to have approximate final sample masses of 5.824 g and 3.50 g, respectively with approximate percent recoveries of 44.8% and 26.9%, respectively. These values make much more sense and are considered as the results for this part of the experiment. These results show fairly low percent recoveries either indicating that the experiment was not efficient in some aspect, or it could be since this is just an approximation of what was assumed to be left over sample. Sample A was blotted on the TLC plate as an unhydrolyzed and hydrolyzed sample. The unhydrolyzed sample A showed one single blot after development of the plate with an Rf value of 0.262 which most closely corresponded with the Lactose standard which had an Rf value of 0.274. This is expected since sample A did not contain lactase or any other enzyme that would cleave the disaccharide into its monosaccharide components. The hydrolyzed sample A showed two blots: one at 0.298 which, again most closely corresponds to Lactose, and 0.482 which most closely corresponds with Glucose (Rf=0.472). It is expected of this sample to show at least two

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blots. What came as a surprise to me was that Lactose was still present after the hydrolysis and only Glucose had formed without Galactose. This is determined to mean that the hydrolysis was only partially effective and possibly the ratios of the reactants was not accurate enough to perform the full reaction. This is probably due to the samples still being wet when they were being analyzed. Sample B was blotted on the TLC plate and four spots developed: one at an Rf value of 0.095, one at 0.262 which most closely corresponds to Lactose, one at 0.429 which most closely corresponds to Galactose (Rf=0.448), and one at 0.472 which is a perfect match with Glucose. The spot counted at Rf value 0.095 does not correlate with any of the standards on this plate, and it is long enough and almost blurred enough to assume that it was just Lactose traveling in the mobile phase and the development was stopped too soon. If not, it may be assumed that sample B could have had a contaminant after staying open to the environment for a full week during its drying phase. It is important to note that sample B displayed (at least) three separate blots: one for Lactose and one for each of its monosaccharide constituents (Glucose and Galactose). This was expected since the enzyme Lactase was added to the nonfat milk which acts to digest disaccharides like Lactose. Since Lactose was still present in B, the reaction can be said to be in equilibrium. An unknown polysaccharide sample “E” was analyzed using the TLC plate. The unhydrolyzed sample “E” developed with one spot at 0.472 which exactly corresponds with the monosaccharide Glucose. A hydrolysis reaction was performed on the unknown “E” and this reaction mixture was blotted on the TLC plate and gave an Rf value of 0.472, again, directly correlating to Glucose. It makes sense that this unknown would correlate to the same Rf value and standard since they both correlate to Glucose, a monosaccharide that cannot be broken down

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to anything smaller using a hydrolysis reaction. Thus, using TLC analysis, the unknown solution “E” was determined to be the monosaccharide Glucose....