Riboflavin Fluorescence Spectra Essay

And finally into test tube 3, I pipetted 1.0 ml turnip extract and 4.0 ml of water. The contents of test tube 1 was poured into a spectrometer tube and labeled it “B” for blank. “B” tube was now inserted it into the spectrometer. An adjustment to the control knob was made to zero the absorbance reading on the spectrometer since one cannot continue the experiment if the spectrometer is not zeroed. A combination of two people and a stop watch was now needed to not only record the time of the reaction, but to mix the reagents in a precise and accurate manner. As my partner recorded the time, I quickly poured tube 3 into tube 2. I then poured tube 2 into the experiment spectrometer tube labeled “E” and inserted it into the spectrometer. A partner then recorded the absorbance reading for every 20 seconds for a total of 120 seconds. After the experiment, a brown color in the tube should be observed to indicate the reaction was carried out. Using sterile techniques, any excess liquid left was disposed

From this graph and chart we can see that the higher the concentration the higher the absorbance, all the different concentrations were tested at the same wavelength (625nm). Also we can determine our unknown substances concentration by using the absorbance we got for it. The red dot on the graph followed by the line towards the horizontal axis indicates that the concentration of fast green was 34% or 5.1x10-3.

concentration was plotted in order to estimate the concentration of the unknown solution (Figure 3).

The first step that needed to be done in this experiment was adding hydrochloric acid (HCl)

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.

b. Describe the spectral curve of each target type with respect to its absorption and reflectance characteristics. (3)

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

Where A is the initial absorbance when the experiment first starts, l is the path length of the cuvette (2.54 cm), and [CV]t is the initial concentration of crystal violet.

The experimental procedures for Lab 2 were provided on Blackboard labelled as “Pre-Lab 2: Techniques & Measurement”.

Knowing this information, you need to first tell me, and then show this in your graph:

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

The “E” solution ended up having the most spots because it was the pigment fragments. The Rf values could be calculated for all of the spots by taking the distance traveled by the spot and dividing it by the total distance traveled by the solvent front. The calculations are as shown:

In this experiment, a spectrophotofluorometer Shimadzu RF-5301 was used to determine the concentration of unknown riboflavin with 5% v/v acetic acid of 10 ppm standard riboflavin solution. An external calibration was performed in using 5 standard known concentrations of riboflavin and plotting a calibration curve. The LOD and LOQ were 0.0622 mg/L and 21.3 mg/L respectively. The calibration curve enabled for the determination of the unknown (3013) concentration of riboflavin at 0.37416 mg/L in which linear regression was used to determine the slope and intercept of the curve.

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

4. Spot the solution on a TLC plate and check with UV light to see