The First Method Of Aas

Spectroscopy is the study of light. A spectrophotometer is a machine used to determine the absorbance of light at any given wavelength. It does this by using a source of white light through a prism, which gives multiple wavelengths that can be individually focused (Ayyagari and Nigam, 2007). Substances are put into cuvettes that are glass or quartz containers that light can easily travel through. The light that is being focused travels through the substance gets absorbed by the

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.

A spectrophotometer’s purpose is to use colors of the light spectrum to determine the concentration of light absorbing molecules in a solution. (p.59) In this particular lab, our mission was to determine the protein concentration and the standard curve of the unknown sample of BSA. This, by preparing five dilutions of the unknown solution of BSA together with other known concentrations, and then experimenting by observing how the concentrations were passed through the spectrophotometer. The outcome resolved in the absorption levels being decreased, and this

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.

MANUAL Q5: What color of light is being absorbed by the sample solution? How is that color related to the color of the solution?

When you use a spectrophotometer you should not set the wavelength of light to be the same color of the solution. This is because if you set the wavelength to be the same color of the solution then no light will be absorbed. The reason why no light will be absorbed is because the color you see is the wavelength of light that is being reflected so you must set the wavelength to be the complementary color. The wavelength of light that was chosen for the lab was 450nm which coincides with a very dark blue color. The reason why choosing a dark blue makes the most sense for this experiment is because the color of the FeSCN2+ ion was blood red.

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

The dyes in the laboratory experiment are made of numerous colors, mainly red and blue, the spectra from each of the dyes corresponded to the wavelengths obtained from each of dye i.e. 620 nm for red and 450 nm for blue.

Once a light beam shines through the flame, the atoms absorb some of the light. It is then detected, recorded and compared to a series of standard solutions. In the absorption spectrum, the absorbed light show up as black gaps. These distinctive gaps indicate the difference in energy levels for a particular element. If this can be compared to the concentration of its standard solution, then it can form a calibration curve.

A spectrophotometer is an instrument which measures the amount of light of a specified wavelength which passes through a medium. This instrument is usually used for the measurement of reflectance of solutions. Light is separate into different wavelengths and is being passed through the sample solution. The sample solution will have its own wavelength and will absorb a certain amount of light. The higher the molecular concentration, the higher the absorbance value.

• Molecules with small differences in absorption wavelengths can be detected well due to their differences in separation time. i.e one which travels faster is measured prior to the other which is measured later. This is the prime advantages if HPLC which makes it

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.

In this experiment, the use of a glass cuvette was used in order to avoid any chemical or environmental interference from the surroundings. Furthermore such glass cuvettes can support large wavelengths during transmission of light beyond 320 nm since this is the excitation wavelength region. Riboflavin is excited at around 370 nm, therefore the wavelength range must be large in order to detect a proper response. Plastic cuvettes are however inadequate as the range of wavelengths supported for transmission of light is very limited (5, Upstone). The geometry of the instrument of the spectrophotofluorometer has a specific angle and position for the excitation source and the detector. The excitation polychromator emits ultra-violet radiation and light to the interested sample and the shape of the polychromator resembles a triangular prism in which the monochromator has been angled at a 90∘ position. The transducer would detect the irradiation excitation beam and transfers it through the emission polychromator. The emitted beam would then travel to the detector to allow for the response signal to appear for the sample on the computer system (416, Skoog). The position of the cuvette is essential as it enables for the consistent readings of the response signals, as any shift in the cuvette position can create variation in results due to the position of lighting and detection of molecular samples hitting at different angles. The process of fluorescence is quite sensitive than absorption due to having lower detection limits because of the fact that it can detect lone molecules in excited states that are irradiated by UV-light unless they come into contact with other molecules within

8) Steps 1 - 8 were repeated using the wavelengths of 360 nm to 900

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