Absorbance Information and Courses from MediaLab, Inc.
These are the MediaLab courses that cover Absorbance and links to relevant pages within the course.
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|Another term for absorbance is:||View Page|
|A spectrophotometric scan of amniotic fluid may be valuable in the determination of which of the following conditions:||View Page|
|Match principle with instrument:||View Page|
After electrophoresis, a stained gel is passed through the optical system of a densitometer to create an electrophoregram, a visual diagram or graph of the separated bands. A densitometer is a special spectrophotometer that measures light transmitted through a solid sample such as a cleared or transparent but stained gel. Using the optical density measurements, the densitometer represents the bands as peaks. These peaks compose the graph or electrophoregram and are printed on a recorder chart or computer display. Absorbance and/or fluorescence can be measured with densitometry.An integrator or microprocessor evaluates the area under each peak and reports each as a percent of the total sample. If the electrophoresis is for separation of serum proteins, the concentration of each band is derived from this percent and the total protein concentration. If the electrophoresis is for separation of enzymes, the enzyme activity of each band is derived from this percent and the total enzyme activity. The densitometer scan below depicts the separated bands from a serum sample electrophoresis. The SPIFE 3000, Helena Laboratories, electrophoresis splits the beta zone into two fractions for easier detection of small beta-migrating monoclonal gammopathies. The densitometer scan from this electrophoresis shows five bands with two peaks in the beta band. Recall the order of protein fractions from left to right is: Albumin, alpha 1, alpha 2, beta, and gamma.
Minute-size fractions achieved in two-dimensional electrophoresis, IEF and PAGE with SDS, and bands from electrophoresis of nucleic acids are detected differently than protein electrophoresis fractions. Labeled polypeptide probes are used to detect these proteins; labeled single-stranded nucleic acid fragments are used for the detection of nucleic acids. Each probe is made with a label designed to generate a detectable signal. The label is bound to a probe and a system is created such that the signal is visualized when the probe is bound to the target.The most common labels are radioactive isotopes and fluorescence dyes. Chemiluminescence and color or ultraviolet absorbance are also used.
|Which type of spectrophotometric blank should be used to account for absorbance differences caused by the specimen being tested:||View Page|
|The direct relationship between the concentration of a substance and its absorbance is referred to as:||View Page|
|Spectrophotometric absorbance is related to % transmittance in what way:||View Page|
|A Regression Analysis Example|
For example, to find a relationship between glucose concentration and absorbance, we could first plot all the points on a scatterplot. Glucose (mg/dL)Absorbance50.10100.20150.30200.40250.50300.60
|Calculating the Y-Intercept|
To find the y-intercept, calculate and , the average of the x- and y-values respectively. Then substitute these two values for x and y in the = b + a equation. Finally, solve for the unknown quantity a. Remember from the previous page that: Therefore, the complete relationship between glucose concentration and absorbance for the data is y = 0+ 0.002x, or y = 0.002x, where y is the absorbance and x is the glucose concentration.
|Prediction Using the Resulting Equation|
Once the parameters have been calculated, the resulting equation can be used to make predictions about a value of y given a value of x, provided that the x value is in the same range of x-values that were used to derive the equation. In this example, we have calculated a=0 and b= 0.002If x = 350 mg/dL, what is the expected value of y? To find the answer, substitute the known value of x into the equation. y = 0 + (0.002)(x) When the concentration is 350 mg/dL, we expect the absorbance to be about 0.7.
|Given the following creatinine standards:mg/dLAbsorbance30.1460.2690.38What is the correct form of the regression line?||View Page|
|Given the data and linear regression line you calculated on the previous question, what is the expected absorbance of a 10 mg/dL sample?||View Page|