Lab 1 Carbohydrates Bch2333

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Lab BCH 2333

Section:

Lab 1 Carbohydrates: Separation Techniques Based on Molecular Size

TA:

Wednesday, January 16th, 2013

Team #4

By:

Partner:

Purpose

The purpose of this experiment is to exemplify how differences in molecular weight allow separation of polymers from their monomers. Methods of dialysis and gel filtration chromatography will be used to separate a glucose monomer from a starch polymer. Colorimetric glucose oxidase assay will be used to monitor the presence of glucose and a colorimetric iodine assay will be used to monitor the presence of starch in prepared solutions after separation

Results and Discussion

Table 1: Glucose oxidase assays
|Reagents |Volume (mL) |Positive Response |A420 | | |Sample |Water |Total | | |Sample |Water | | |A420 Internal Solution |C1 (g/L) |

# |Elution Vol. (mL) |A420 |C (g/L) |Mass (mg) |Positive
Response |A600 |C (g/L) |Mass (mg) |Positive
Response | |1 |3.86 |-0.0140 |-0.0512 |-0.198 |- |0.0240 |0.00704 |0.0272 |- | |2 |7.72 |0.0210 |0.0768 |0.296 |- |0.0370 |0.0109 |0.0421 |- | |3 |11.6 |0.0610 |0.223 |0.861 |- |0.0300 |0.00880 |0.0340 |- | |4 |15.4 |0.0280 |0.102 |0.394 |- |0.314 |0.0921 |0.356 |+ | |5 |19.3 |-0.0610 |-0.223 |-0.861 |- |0.0800 |0.0235 |0.0907 |- | |6 |23.2 |0.0470 |0.172 |0.664 |+ |0.0480 |0.0141 |0.0544 |- | |7 |27.0 |0.459 |1.68 |6.48 |+ |0.000 |0.000 |0.000 |- | |8 |30.9 |0.727 |2.66 |10.3 |+ |0.0240 |0.00704 |0.0272 |- | |9 |34.7 |0.400 |1.46 |5.75 |+ |0.0310 |0.00909 |0.0351 |- | |10 |38.6 |0.0610 |0.223 |0.861 |+ |0.0750 |0.0220 |0.0849 |- | |11 |42.5 |0.000 |0.000 |0.000 |- |0.0530 |0.0155 |0.0598 |- | |12 |46.3 |0.0270 |0.0987 |0.381 |- |0.0580 |0.0170 |0.0656 |- | |13 |50.2 |-0.0180 |-0.0658 |-0.254 |- |0.0250 |0.00733 |0.0283 |- | |14 |54.0 |0.0130 |0.0475 |0.183 |- |0.0120 |0.00352 |0.0136 |- | |15 |57.9 |0.00800 |0.0293 |0.113 |- |0.0290 |0.00850 |0.0328 |- | | The column elution results show that glucose molecules were eluted throughout the 15 fractions in intervals. The intervals of elution for glucose are from fractions, 1-4, 6-10, 12, 14-15. Fractions 6-10 show a positive response for glucose with the solution changing color to blue then yellow after the addition of 2N H2SO4. Within these fractions it is observed that the concentration of glucose eluted slowly increases and then decreases towards the end of the intervals. Any fractions not included in the interval show either negative or zero values for the concentration of glucose. Negative values may be due to an error with absorbance readings and may just imply the concentration of the glucose is meant to be 0. The distribution of glucose elution through the gel column into the fractions can be graphically observed as peaks shown in Figure 1. On the other hand most of the starch is eluted through the column and into fraction 4 and not as evenly distributed in intervals as the glucose molecules. Fraction 4 is also the only fraction to show a positive response for the starch with the color change of blue to the solution once the iodine is added. This fully explains the concept of the gel filtration chromatography because the molecules within the glucose/starch sample mixture that are smaller than the pore size of the gel column beads (glucose) enter the beads and glucose movement relative to the flow of eluent buffer is reduced compared to the movement of starch molecules which are meant to be bigger than the pore size of...
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