Serial Dilutions Lab Report

With these absorbance numbers a concentration curve was constructed and the unknown solution was determined by finding the point of absorbance on the curve.

Tables 2,3,4,5 and 6 show that as duration increased the absorbance also increased for each pH. The solution in the conical flask became darker (yellow) in time this is because the substrate, p-nitrophenyl phosphate was catalysed by acid phosphatase, releasing Nitrophenolate anion. It was the Nitrophenolate anion giving off the yellow colour; the presence of this feature increases the absorbance rate. The addition of sodium hydroxide distorted the shape of the enzyme making it no longer effective in its function.

10 microliters of the sample is then added and the assay absorption is measured at 340nm. If absorbance was above 1.5, samples were diluted.

The same solution of 0.5 ml BSA was then added from test tube 1 to the test tube 2 after being properly mixed, and from test tube 2 the solution was being added to test tube 3, and so forth all the way up to test tube 5, with the same exact procedure. From the last tube, we then disposed the 0.5 ml solution. After above procedures, we now labeled another test tube “blank”; 0.5 ml blank distilled water was purred into the tube with the serial dilution of 1:10. We also had a tube C labeled “unknown” with the same 0.5 ml of solution. And after adding 5ml of Coomassie Blue to each tube (1-5) and to the blank, the result of absorbance was read at 595 nm.

In this experiment, there were 6 different test tubes, each containing a different amount of phosphate concentration. Using a balance, 1 g of phosphate in a 400 mL beaker was measured and then inserted into a 100 mL graduated cylinder. 5 mL of ammonium vanadomolybdate (AVM) was added, along with deionized water until the solution reached to 25 mL. Then the solution was poured into one of the six test tubes. Another test tube contained 2 g of phosphate (phosphate was again measured using the balance) and the step described earlier was repeated until five of the six test tubes were filled, each with the same volume of 25 mL. The sixth test tube was filled with only deionized water, with 25 mL as the volume. A spectrophotometer was then used to determine the percent transmittance for each solution. The absorbance was also calculated using the equation A = -log (T). Each given amount of phosphate was converted into moles and then applied the value of .025 L for the volume to calculate the molarity of phosphate in each solution.

Each stock solution was placed in a colorimeter and was tested for it Absorbance. A computer program tested and drew up the Calibration curve/linear fit equation. However, the computer could not protect potential errors. An error for determining the concentration of the diluted and undiluted, could be a skew linear fit equation. The linear fit equation could be skewed by having an inadequate ratio of the stock solution and distilled water. For example, when making stock solution 1, 0.021(L) was used instead of 0.020(L) can throw the calibration curve, resulting a skewed linear fit equation. If the “blank” was not fully clean or had left over Allura Red residue, then the “blank” was tampered with. A tampered “blank” means any comparisons with it would have a wrong Absorbance reading. However, the most likely and most effective error, is calculation. Using the wrong V1 and V2 to determine the concentration of the undiluted would affect the answer of the grams Allura Red would be consume and the amount of molecules of Allura Red. The colorimeter is adjusted to a wavelength of 470 nm is maximize the absorbance of the Allura Red. If wavelength was place at 565 nm, then Allura Red would not absorb as much color of

Make sure to use the same type of cuvette to keep the width consistent and to prevent any experimental error from arising. Obtain 5 of the same type of cuvettes and pre-rinse them thoroughly. Label them numbers one through five in increasing molarity. Then, fill each of the cuvettes with one of the five solutions you created back in Part A. We will first examine the solution that exhibits the highest concentration or molarity. Make sure to wipe the outside of the cuvette with a Kimwipe before placing into the SpectroVis Plus device. Observe the graph that is generated and make sure to take note where the maximum absorbance takes place.

The dark, navy blue colored graph represented the absorbance curve for the S1 sample. The red colored graph represented the absorbance curve for the S2 sample. The green colored graph represented the absorbance curve for the P1 sample. The purple colored graph represented the absorbance curve for the P2 sample. The gaps between the P2 curve was due to the oversaturation that led to the inconclusive spectrophotometer readings. The blue colored graph represented the absorbance curve for the P1 low salt sample. The orange colored graph represented the absorbance curve for the P2 low salt sample. The light blue colored graph represented the absorbance curve for the P1 medium salt sample. The light pink colored graph represented the absorbance curve for the P2 medium salt sample. The light green colored graph represented the absorbance curve for the P1 high salt sample. The light purple colored graph represented the absorbance curve for the P2 high salt

5. The degree of precision was to 3 significant figures obtained with the spectrophotometer. The major source of error in our experiment was not calibrating the spectrophotometer with distilled water.

MicroLAB Kinetics program was opened, then the colorimeter was calibrated to a 100% transmission by filling a marked, clean, clear cuvette, about ¾ full of deionized water. The cuvette was wiped with a Kimwipe from the outside before putting it in the colorimeter. The cuvette was inserted in the chamber, then the cap was closed, and the Read Blank button was clicked to start the calibration.

Using the yellow tube, which included everything but starch, as the blank, each group zeroed their spectrophotometer. This was done so that any absorbance observed depends only on the amount of starch present, not on any other reagents (buffer, IKI). To zero the spectrophotometer, the wavelength was first set at 580nm, using knob 3 (45). Next, the groups made sure that the light next to “transmittance” was lit, and the chamber to be tightly closed. Having the chamber empty & closed tightly provides reference for the darkest condition possible. Using knob 1, the transmittance was turned until it read 0.0 (45). Before the groups used their blank test tube to zero the spectrophotometer, each needed to wipe the tube with kimwipes to ensure a clean reading. Turning knob 2, each group was then instructed to zero the absorbance, 0.000. Upon removing the blank, each trial was inserted into the chamber (46). The

Each solution contained different concentrations as follows: 0.005 mg/mL, 0.010 mg/mL, 0.015 mg/mL, 0.020 mg/mL, and 0.025 mg/mL. Each solution needed to have a volume of 10 mL. Before adding the different concentrations of Coomassie Blue into their separate tubes, the formula C1V1= C2V2 was used in order to determine how much stock solution is needed for the five dilute solutions. Once that number was calculated, a pipette was used to add the amount of stock solution needed for each tube. We then subtracted the amount of stock solution from 10 mL to determine the amount of H2O needed. The calculated amount of H2O was then added to each tube of solution. After doing that, a spectrophotometer was used to determine each solution’s relative absorbance. However, before that, we first had to calibrate the spectrophotometer before determining each solution’s relative absorbance. In order to calibrate the spectrophotometer, a disposable culture tube filled with distilled water was used. We then changed the data rate to 100 and removed the tube with water. In order to determine the relative absorbance, the relative absorbance had to be at 595 nm. Also, during this experiment, an unknown dilution was given to us by the lab instructor. We determined the relative absorbance by using the spectrophotometer and then recorded the results. The procedures for this experiment can be found on page 8 of

3. The spectrophotometer was set at 420nm. Distilled water was also used as the ‘blank’.

Incorporation of assay controls included setting up a spectrophotomer and running the chart recorder with a full-scale deflection before the start of the assay. The set recorder had a corresponding value of 1 for the change in the absorbance. Therefore, prior testing was done to observe whether a change occurred in the readings. This helped to indicate that the results were valid, as they could have been affected by a fault during the setting up of the spectrophotometer. On the other hand this was considered as one of the controls for the experiment. Nevertheless, a new cuvette had to be used for each assay.

concentration, record the absorbance readings at a fixed wavelength, and plot the absorbance vs. concentration data. The wavelength of 520 nm was selected for experiment Part