Spectrophotometer: Concentration and Standard Curve Essay

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.

3ml of sample was taken first flask at 4 minutes and added to the appropriate tube of sodium hydroxide, from the second flask at 4.5 minute and so on, each flask was sampled at 30 second intervals. The sampling was then repeated starting at 8,12,16 minutes. The final sample from the last flask was taken at 18.5 minutes. Once the sampling was completed, measurements of absorbance were obtained for solution in each tube at 405 nm.

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.

Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.

Time) because it had a correlation closest to 1. All three orders were graphed and a linear regression was used to see which graphed order was closest to 1. The order was determined by comparing the concentration and time to the mathematical predictions made using the integrated rate laws. Analyzing each graph and finding each correlation helped determine which graph was closest to 1. The more concentrated a solution is, the higher the absorbance of that solution. This is due to Beer’s Law. The law measures the absorbance of a solution by determining how much light passes through a solution. As the concentration of a solution increases, fewer wavelengths of light are able to pass through the concentrated solution. The absorbance at 60 seconds was 0.573 (Figure 1: Table1). To calculate the concentration (molarity), the Beer’s Law equation was used, Abs = slope(m)+b. Plugging in what is known into the Beer’s Law equation resulted in 0.573 = 3.172e+004 + 0, where the concentration is determined by M = 0.573-0/ 3.172e+004. So, the concentration at 60 seconds using the equation (M = 0.573-0 / 3.172e+004) was 1.824e-5 M. The 1st order graph resulted in k=0.006152 (Figure 1: Graph 1). Other groups also resulted in their decolorization of CV to be the 1st rate

Scientists use an instrument called a spectrometer to quantitatively determine the amount of light absorbed by a solution. The primary inner parts of a typical spectrometer are described below. The spectrometer has a light source that emits white light containing a vast mixture of different wavelengths of electromagnetic radiation. The wavelength of interest is then selected using a monochromator (“mono” meaning one and “chromate” meaning color) and an additional exit slit. The separation of white light into different colors (wavelengths) is known as diffraction. The selected light then reaches the sample and depending on how the light interacts with the chemical compound of interest, some of the light is absorbed and some passes straight through. By comparing the amount of light entering the sample (P0) with the amount of light reaching the detector (P), the spectrometer is able to tell how much light is absorbed by the sample.

The Beers Law calibration experiment used many concentrations of crystal violet solutions. Each of these solutions were test and analyzed in order to determine the absorbance of each concentration The results were than graphed and produced a slope of 1.00E05 with an intercept of -2.21E-02.

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

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

The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present

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

The absorbance is measured using a Plate reader and a Standard curve is generated. Also, the different types of pipetting techniques are assessed in this Assay.

The absorbance with SDS shows a rapid increase, before the graph begins to increase more slowly and uniformly. The absorbance levels off at 0.3nm. The absorbance with no SDS remains at a constant level of 0.1nm for the entire experiment. The absorbance of the control solution is also constant throughout the experiment. This shows the absorbance of the compounds in the solution without the ovalbumin, by taking this figure away from the other recordings, it is possible to discover the absorbance for ovalbumin alone.

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.

Proteins are one of the main macromolecules that are important for human function. This study examines the presence of proteins in the given samples. The Biuret reagent created a color transitioned on the various samples. The Biuret reagent is an aqueous solution that is composed of sodium hydroxide and copper (II) sulfate. The copper ions in the Biuret reagent react with the peptide bonds which cause the color change in the test samples. The amount of protein in the various samples determined the color transition. The sample solutions with lower presence of protein did not change color as opposed to the sample solutions with a higher presence of