Spectrophotometry review questions

Define: Beers Law
Beer’s law states that the absorbance is directly proportional to the concentration of a solution. If you plot absorbance versus concentration, the resulting graph yields a straight line. The equation for the straight line (termed regression line) can be used to determine the concentration of an unknown solution once the %T has measured
What are Beers law components from the formula?
A=Absorbance T= transmission (or transmissivity) A = 2.00 -[log (%T)] If T = 85%, then Absorbance = 2 – log[85] = 0.071
In absortion spectrophotometry: Absorbance in directly porportional to concentration and path length.
Beer’s law states that A= axbxc, where a is the absorbtivity coefficient (a constant) b is the path length, and c is the concentration. Absornance is directly proportional to both b and c. DDoubling the path length results in incident light contacting twice the number of molecules in solution. This causes absorbance to double, the same effect as doubling the concentration of molecules.
Which wavelength would be absorbed strongly by a RED colored solution:
A solution TRANSMITS light corresponding in wavelength to its color and usually ABSORBS light of wavelenghts complementary to its color. A RED solution TRANSMITS light of 600-650 nm and strongly ABSORBES 400-500nm light.
A green-colored solution would show higest transmittance at:
525 nm.
Green light consist of wavelengths from 500-550nm. A green colored solution with transmittance maximum of 525 nm and a 50 nm band pass TRANSMITS light of 525 nm and ABSORBES light below 475 and above 575 nm . A sloution that is green would be quantitated using a wavelength that it absorbes strongly, such as 450 nm.
A technologist is performing an enzyme assay at 340 nm using a visable range spectrophotometer . After setting the wavelength and adjusting the readout to zero %T with the light path blocked. a cuvet with deionized water is inserted. With the light path fully open and the 100 %T control at maximum the instrument readout will not rise above 90%T. WHAT IS THE MOST APPROPRIATE FIRST COURSE OF ACTION?
visable spectrophotometers are usually supplied with a tungston source lamp. Tungsten lamps produce a continuous range of wavelengths from about 320 to 1200nm . Output increases as wavelenghts become longer and is poor below 400 nm. AS THE LAMP ENVELOPE DARKENS WITH AGE the amount of light reaching the photodetector at 340 becomes insufficient to set the blank reading to 100% T. Deuterium or hydrogen lamps produce UV rich spectra optimal for UV work. Halogen, mercury vapor, and xenon give moderate visable and UV output.
Which type of monochromator produces the PUREST monochromatic light in the ultra violet (UV) range?
A prism and a variable exit slit.

Differaction gratings and prisms both produce a continuos range of wavelengths. A diffraction grating produces a UNIFORM separation of wavelengths. A prism produces a MUCH BETTER SEPARATION of HIGH FREQUENCY LIGHT because refraction is greater for higher energy wavelengths. Instrument using a prism and a variable exit slit can produce UV light of a very NARROW BANDPASS. The adjustable slit is required in order to allow sufficient light to reach the detector to set 100 %T.

Which monochromater specification is required in order to measure the true absorbance of a compound having natural absorbtion bandwidth of 30nm.?
A 5 nm bandpass..
Bandpass refers to the range of wavelengths passing through a sample. The narrower the bandpass , the greater the photometric resolution. Bandpass can be made smaller by REDUCING THE WIDTH OF THE EXIT SLIT. Accurate absorbance measurements require a bandpass less than one-fifth the natural bandpass of the chromatophore.
1/5 x 30 = 6
Which photodector is most sensitive to low levels of light?
Photomultiplier tube.
The photomultiplier tube uses dynodes of increasing voltage to amplify the current produced by the photosensitive cathode. It is 10,000 times as sensitive as a barrier layer cell, which has no amplification. A photomultplier tube requires a DC- regulated lamp because it responds to light fluctuations caused by an AC cycle.
What condition is the common cause of stray light.
Dispersion from second order spectra.
Stray light is caused by the presence of any light other than the wavelength of measurement reaching the detector. It is most often caused by second- order spectra, deteriorated optics. light dispersed by a darkened lamp envelope, or extraneous room light.
A linearity study is performed on a visable spectrophotometer at 650nm and the following absorbance readings are obtained:
Concentration of standard Absorbance
10.0 mg/dL 0.20
20.0 mg/dL 0.41
30.0 mg/dL 0.62
40.0 mg/dL 0.79
50.0 mg/dL 0.92
The study was repeated using freshly prepared standards and reagents, but the results were identical to those above. What is the most likely cause of those results?
Stray Light.
Stray light is the most common cause of loss of linearity at high concentrations. Transmitted incident light is lowest when absorbance is higest. Therefore stray light is a greater percentage of the dector response when sample concentration is high. Stray light is usually most significant when measurements are made at the extreams of the visable spectrum because lamp out-put and /or detector response is low.
Which type of filter is best for measuring stray light?
Sharp cut-off.
Sharp cutoff filters transmit almost all incident light until the cut-off wavelength is reached. At that point they cease to transmit light. Because they give an “all or none effect”.Only stray light reaches the detector when the selected wavelength is beyond the cutoff.
Which of the following materials is best suited for verifying the wavelength calibration of a spectrophotometer?
Holmium oxide glass.
Wavelength accuracy is verified by determining the wave length reading that gives the highest absorbance ( or transmittance) when a substance with a narrow bandpass ( sharp absorbance or transmittance peak) is scanned . For example didymium glass has a sharp peak at 585 nm. Therefore an instrument sholud give its higest absorbance reading when the wavelength dial is set at 585nm. Holmiun oxide produces a very narrow absorbance peak at 361 nm. likewise, the hydrogen lamp of a UV spectrophotometer produces a 656 nm emission line that can be used to verify wave length. Neuteral density filters and dichromate solutions are used to verify absorbance accuracy or linearity. A wratten filter is a wide bandpass filter made by placing a this layer of colored gelatin between two glass plates and is unsuitable for spectrophotometric calibration.
Why do many optical systems inchemistry analysers utilize a reference light path?
To minimize error caused by a source lamp fluctuation.
A reference beam is used to produce an electrical signal at the detector to which the measurement of light absorbtion by the sample is compared.
This safeguards aginst measurement errors caused by power fluctuations that change the source lamp intensity. Although reference beams increase the accuracy of measurements, they do so at the expense of the optical sensitivity because some of the incident light must be used to produce the reference beam.
Which component is required in a spectrophotometer in order to produce a spectral absorbance curve?
Photodiode array.
There are two ways to perform spectral scanning for compound identification. One is to se a stepping motor that continuously turns a monochromater so that the wavelenght aligned with the exit slit changes at a constant rate. A more practical method is to use a diode array detector. This consist of a chip embedded with as many as several hundred photodiodes each photodiode is aligned with a narrow part of the spectrum produced by a diffraction grating and produces current proportional to the intensity of the band of light striking it.(usually 1-5 nm in range) The diode signals are processed by a computer to create a spectral absorbance or transmittance curve.
The half band width of a monochromater is defined by :
The range of wavelengths passed at 50% maximum transmittance.
Half Bandwidth is a measure of bandpass made using a solution or filter having a narrow natural bandpass (transmittance peak) The wavelength giving maximum transmittance is set to 100%T (or 0 for A)Then the wavelength dial is adjusted downward until a readout of 50%T (0.301A) is obtained. Next the wavelength is adjusted upward until a readout of 50%T is obtained. The wavelength difference is the half bandwidth. The narrow the half bandwidth the better the photometric resoluton of the instrument.
The term dark current as used in spectrophotometry refers to:
The flow of current from a photo tube caused by high gain on its anode.
Dark current is the current generated by a detector when the light path is blocked to prevent incident light from striking it. Photo tubes and photomultiplier tubes leak a small amount of current because electrons are drawn through the circut by the high positive voltage applied to the anode or dynodes.
The reagent blank corrects for the absorbance caused by:
The color of the reagents.
When a spectrophotometer is set to 100%T with the reagent blank instead of water the absorbance of reagents is automatically subtracted from each unknown reading. The reagent blank does not correct for absorbance caused by interfering chromogens in the sample such as biliruben, hemolysis, or turbitidy.
Which instrument requires a highly regulated DC power supply?
A spectrophotometer with a photomultiplier tube.
When AC vltage regulaters are used to isolate source lamp power, liht ouput flucuates as the voltage changes. Because this occurs at 60 Hz, it is not detected by the naked eye or slow responding detectors. Photomultiplier tubes are sensitive enough to respond to the AC frequency an d require a DC power supply.
100% reflectance is set with an opaque film called a white reflectance.
The white reference is anologous to the 100%T setting in spectrophotometry and serves as a reference signal.
Bichromatic filter photometers can correct for interfering substances if:
The contribution of the interferent to absoebance is the same at both wavelenghts.
In Buchromatic photometry, the absorbance of the sample is measured at two different wavelenghts. The primary wavelenght is at or near the absorbance maximum. An interfereing subsance having the same absorbance at both primary and secondary (side band) wavelenghts does not affect the absorbance difference.(Ad).
Which instrument requires a primary and secondary monochromator?
A fluorometer uses a primary monochromater to isolate the wavelength for excitation and secondary monochromator to isolate the wavelength emitted by the fluorochrome.
Fluorometry is less specific than spectrophotometry.
True or False?
Incresing temperture results in more random collision between molecules by increasing their motion. causes energy to be dissipated as heat instead of fluorescence. Temperture is inversly proportional to fluorescence. Fluorescence is more sensitive than spectrophotometry because both the excitation and emmision wavelengths are characteristics of the compound being measured.
Fluorometry is less specific than spectrophotometry.
True or False?
Incresing temperture results in more random collision between molecules by increasing their motion. causes energy to be dissipated as heat instead of fluorescence. Temperture is inversly proportional to fluorescence. Fluorescence is more sensitive than spectrophotometry because both the excitation and emmision wavelengths are characteristics of the compound being measured.
Source Lamp.
Chemiluminescence is the production of light following a chemical reaction. Immunoassays based upon Chemiluminescence generate light when the Chemiluminescent molecule becomes excited; therefore a light source is not used. In immunoassays platforms Chemiluminescent molecules such as acridium can be used to label antigens or antibodies. Alternatively, Chemiluminescence substrates such as luminol or dioxetane phosphate may e used. Light is emmited when an enzyme-labeled antibody reacts with its substrate. In such assays free and bound antigen separation is required and is usually accomplished using paramagnetic particles bound to either antibody or reagent antigen.

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