Prepare an ingredient table in your lab notebook showing dilution factors, volume of stock solution used, volume of diluent used for all of the remaining dilutions to review dilutions see the Tech Facility workbook, module VII. Use the Phosphate buffer, pH 7. Record reading of a blank as your 0X; It should read 0. Measure the absorbance of the different dilutions starting with absorbance of 0.
Insert blank tube into sample compartment. Repeat steps to measure the absorbance values for 0. Dispose any left over stock solution of phenol-red you may dispose it by pouring in directly in the sink.
Make sure that sink is cleaned up. Also dispose both Phosphate Buffers pH 6. Wash the plastic reagent bottles that were used for storing these buffers. Dispose centrifuge tube that was used for storing of phenol - red solution by placing it in the Bio Hazardous waste container. Note that a standard "curve" can, in fact, not be curved! It is usually a straight line! Using your Standard Curve, determine the concentration of phenol red in the sample with unknown concentration.
What is the general relationship between concentration and absorbance for this and other compounds? This is known as Beer's Law. In this exercise, you will use the spectrophotometer to determine the concentration of protein in standard and unknown solutions. The method employed is known as the "Bradford" protein assay, after its inventor. It is based on the ability of a particular dye to bind to protein, plus a change in the absorption spectrum of the dye that occurs as it binds.
A "dye" can be defined as a chemical compound that has two properties: a visible color, and b the ability to bind attach to other kinds of compounds. In the case of the Bradford assay, the dye known as Coomassie blue, under acidic conditions, changes its absorption spectrum so that it will absorb well at nm after binding, but not before.
The amount of bound dye, as detected by absorbance at nm, is proportional to the amount of protein present. Therefore, a standard curve showing absorbance of the dye versus known protein concentrations can be used to determine the concentration of an unknown protein solution from its bound dye absorbance. Obtain the following: 6 small test tubes from small test tubes container near the spectrophotometer, frozen 1.
Prepare 0. Use the Pipetman micropipettors, and follow an "ingredients table" written in advance into your Tech Facility notebook. To review dilutions see module IX. Prepare an additional tube containing 0. Set wavelength to nm. All measurements will be made at this wavelength. Add 3. Also add 3. Read and record absorbance for the 0. Repeat steps for rest of the samples including sample of unknown concentration.
Using your Standard Curve, determine the concentration of the unknown sample from its absorbance. Dispose all samples by pouring them directly into drain and clean the sink.
Dispose small test tubes properly. Return test tube racks and other equipment to original module location, so that it is ready for use by someone else. Make sure that the area were you worked is cleaned up and ready for use by other students. Obtain the following: a 2 empty clean test tubes from the "small test tubes" container near the spectrophotometer, b one frozen tube of "stock solution" of phenol red solution from plastic container "in Tech Facility freezer.
Reduced nicotinamide adenine dinucleotide NADH is amongst the intrinsic fluorophores and can be used as an intracellular indicator of hypoxia. The excitation wavelength is nm and emission is at nm. The former causes problems in measurements involving samples of analytes with a low extinction coefficient and present only in low concentrations.
The latter becomes problematic with high absorbance samples where the light intensity emerging from the sample is very small. O2k - Oroboros O2k : the modular system for high-resolution respirometry. O2k-Innovation description.
It is our ultimate goal to further extend the NextGen-O2k with O2k-Spectrofluorometry including ratiometric measurements , NADH autofluorescence, and O2k-Spectrophotometry allowing for simultaneous measurement of redox states of the cytochromes aa 3 , b , and c. The Oroboros NextGen-O2k will have integrated optical elements beyond the modular options of the presently available Oroboros O2k.
Optics are the components that are used to relay and focus light through a spectrofluorometer or spectrophotometer. The oxygen affinity is indirectly proportional to the p The p 50 depends on metabolic state and rate. Phosphorescence is a similar phenomenon to fluorescence.
However, instead of the electron returning to its original energy state following excitation, it decays to an intermediate state with a different spin value where it can remain for some time minutes or even hours before decaying to its original state. Phosphorescence is one form of Luminescence , especially Photoluminescence. Photodiode arrays. Photodiode arrays are two dimensional assemblies of photodiodes.
They are frequently used in conjunction with charge coupled devices CCDs for digital imaging. They can be used in combination with dispersion devices to detect wavelength dependent light intensities in a spectrofluorometer or spectrophotometer. Photodiodes are photodetectors that convert incident light into a current or voltage dependent on their configuration. They have replaced photomultiplier tubes for most applications.
For fluorometric measurements that do not require spectral data, a single photodiode with suitable filters can be used. Due to their larger detection area, they are more sensitive than photodiode arrays. Polyether ether ketone. Polyether ether ketone PEEK is a semicrystalline organic polymer thermoplastic, which is chemically very resistant, with excellent mechanical properties. PEEK is compatible with ultra-high vacuum applications, and its resistance against oxygen diffusion make it an ideal material for high-resolution respirometry POS insulation; coating of stirrer bars; stoppers for closing the O2k-Chamber.
Reference spectrum. A reference spectrum for a substance is an absorbance spectrum of the same substance at a known concentration and redox state. Reflectance spectrophotometry. In reflectance spectrophotometry the light from the sample is reflected back to the detector using mirrors. Before absorbance measurements can be made, a white balance is carried out. Remittance spectrophotometry. In remittance spectrophotometry incident light enters a scattering medium and is scattered back to the receiving optics usually lightguides before being directed to the detector.
Spectral resolution is a measure of the ability of an instrument to differentiate between two adjacent wavelengths.
The resolution of a spectrofluorometer or spectrophotometer is dependent on its bandwidth. Respiratory state. Respiratory states of mitochondrial preparations and living cells are defined in the current literature in many ways and with a diversity of terms. Mitochondrial respiratory states must be defined in terms of both, the coupling-control state and the electron-transfer-pathway state. Most biological samples do not consist simply of pigments but also particles e.
The effect of scattering is an apparent increase in absorbance due to an increase in pathlength and the loss of light scattered in directions other than that of the detector. Two types of scattering are encountered. Selectivity is the ability of a sensor or method to quantify accurately and specifically the analyte or analytes in the presence of other compounds.
Sensitivity refers to the response obtained for a given amount of analyte and is often denoted by two factors: the limit of detection and the limit of quantification. Signal-to-noise ratio. The signal to noise ratio is the ratio of the power of the signal to that of the noise. For example, in fluorimetry it would be the ratio of the square of the fluorescence intensity to the square of the intensity of the background noise.
The slit width determines the amount of light entering the spectrofluorometer or spectrophotometer. A larger slit reduces the signal-to-noise ratio but reduces the wavelength resolution. Various methods of smoothing can be applied to improve the signal-to-noise ratio. Integrating the left side of equation The absorptivity and molar absorptivity are proportional to the probability that the analyte absorbs a photon of a given energy.
Solving equation A solution of the analyte from Example If there are no interactions between the components, the individual absorbances, A i , are additive. Generalizing, the absorbance for a mixture of n components, A mix , is. In many cases a calibration curve deviates from this ideal behavior Figure Deviations from linearity are divided into three categories: fundamental, chemical, and instrumental.
Consider, as an example, an analysis for the weak acid, HA. Because HA is a weak acid, it is in equilibrium with its conjugate weak base, A —. Substituting equation Problem For this reason, as shown in Figure Another reason for measuring absorbance at the top of an absorbance peak is that it provides for a more sensitive analysis.
Note that the green calibration curve in Figure Stray radiation arises from imperfections in the wavelength selector that allow light to enter the instrument and reach the detector without passing through the sample. Stray radiation adds an additional contribution, P stray , to the radiant power reaching the detector; thus.
For a small concentration of analyte, P stray is significantly smaller than P 0 and P T , and the absorbance is unaffected by the stray radiation. At a higher concentration of analyte, however, less light passes through the sample and P T and P stray may be similar in magnitude. It measures the amount of light absorbs by the sample. This value depends on the amount and nature of the compound present in the sample.
We can give this value using the following equation;. Where A is the absorbance, I is the intensity of the incident beam, and I o is the intensity of the transmitted beam through the sample.
We can give this relationship in another way as follows:. Where T is the transmittance. Therefore, the absorbance is related to the transmittance. That is because, if the concentration is high, the sample has a high amount of the compound that absorbs the light from the light beam. Moreover, when measuring the absorbance from a spectrophotometer, we should not use very high or very low concentrations.
This is because, if we use very high concentrations, the intensity of the incident light beam may not sufficient for the total amount of compound present in the sample to absorb.
If we use a low concentration, the sensitivity of the instrument may not sufficient enough to detect the low amount of compound in the sample. Concentration is the amount of a substance that distributes throughout a unit volume of a sample. We call this the molar concentration.
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