Clinical Chemistry Questions!! Anyone know's anything about Spectrophotometry?? I need some major help!!?

Question

1. Describe bichromatic analysis including the principle, arrangment of components and advantages. Extrapolate the principle to multiple-wavelength spectrophotometry. Then list examples of comon interferences that can be corrected using bichromatic analysis.

2. The difference between single beam and double beam spectrophotometry?

Answer 1

Spectrophotometry:
A spectroscope uses a prism to take light from a heated sample to determine its elemental composition. It is useful in astronomy to tell us what elements are in other stars and hot gases. It is useful in medicine to tell us what is present in a tissue or blood sample.

Part I) According to Wikipedia: http://en.wikipedia.org/wiki/Spectrophotometry
“The most common spectrophotometers are used in the UV and visible regions of the spectrum, and some of these instruments also operate into the near-infrared region as well.”

The bichromatic (two color) analysis will use infrared and ultraviolet as well as visible light to study the sample. If you have watched CSI then you see that they use luminal and a UV light to detect blood. A UV light will also detect semen. There are other chemicals that can be detected by using the enlarged color spectrum. If you use true multi-wave length spectroscopes then if you get a chemical that glows in the presence of UV and two that glow in the presence of infrared then you can determine the difference between the two chemicals since one doesn’t glow in UV. Each element will give off a pattern of colors that are unique to that element, but when you get several elements together it could be hard to determine exactly what is what. Adding alternate light sources and increasing the spectrum will give you a larger range to work with and could give you more results.

According to Wikipedia: http://en.wikipedia.org/wiki/Ultraviolet-Visible_spectroscopy
“Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV/ VIS) involves the spectroscopy of photons and spectrophotometry. It uses light in the visible and adjacent near ultraviolet (UV) and near infrared (NIR) ranges. In this region of energy space molecules undergo electronic transitions.”

Part II) According to Wikipedia: http://en.wikipedia.org/wiki/Spectrophotometry
“There are two major classes of spectrophotometers; single beam and double beam. A double beam spectrophotometer measures the ratio of the light intensity on two different light paths, and a single beam spectrophotometer measures the absolute light intensity. Although ratio measurements are easier, and generally stabler, single beam instruments have advantages; for instance, they can have a larger dynamic range.

UV/Vis spectroscopy is routinely used in the quantitative determination of solutions of transition metal ions and highly conjugated organic compounds.
Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another. The color of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligands. For instance, the color of a dilute solution of copper sulphate is a very light blue; adding ammonia intensifies the color and changes the wavelength of maximum absorption (λ_max).
Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water soluble compounds, or ethanol for organic-soluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.)
While charge transfer complexes also give rise to colors, the colors are often too intense to be used for quantitative measurement.”

Iron is a transition metal and the amount of iron in a blood sample is an indicator of the amount of hemoglobin in the blood. Other elements can be detected and their levels are in a normal sample are known. If the iron level is low then the person could be suffering from anemia. The presences or lack of other elements or their level will give you important clues; like it is very important to know the potassium level in the blood and urine because it is an important indicator of health.

According to Wikipedia: http://en.wikipedia.org/wiki/Infrared_spectroscopy
“Infrared spectroscopy (IR spectroscopy) is the subset of spectroscopy that deals with the infrared region of the electromagnetic spectrum. It covers a range of techniques, with the most common type by far being a form of absorption spectroscopy. As with all spectroscopic techniques, it can be used to identify a compound and to investigate the composition of a sample. Infrared spectroscopy correlation tables are tabulated in the literature….

This technique works almost exclusively on covalent bonds. Clear spectra are obtained from samples with few IR active bonds and high levels of purity. More complex molecular structures lead to more absorption bands and more complex spectra. The technique has been used for the characterization of very complex mixtures however.”

We know of various organic chemicals that have covalent bonds so IR spectroscopy can identify them and determine the amounts.

According to Wikipedia: http://en.wikipedia.org/wiki/Infrared_spectroscopy
“Liquid samples can be sandwiched between two plates of a high purity salt (commonly sodium chloride, or common salt, although a number of other salts such as potassium bromide or calcium fluoride are also used). The plates are transparent to the infrared light and will not introduce any lines onto the spectra. Some salt plates are highly soluble in water, and so the sample, washing reagents and the like must be anhydrous (without water).”

We know that sodium, potassium and calcium are already present in the body so if you are not careful in the preparation of the sample you can contaminate it and get a false reading.

The article continues:
“A beam of infrared light is produced and split into two separate beams. One is passed through the sample, the other passed through a reference which is often the substance the sample is dissolved in. The beams are both reflected back towards a detector, however first they pass through a splitter which quickly alternates which of the two beams enters the detector. The two signals are then compared and a printout is obtained.

A reference is used for two reasons:
This prevents fluctuations in the output of the source affecting the data.

This allows the effects of the solvent to be cancelled out (the reference is usually a pure form of the solvent the sample is in). “

I hope this gives you some clues to answering your questions.

Bichromatic analysis uses an expanded spectrum that includes infrared and ultraviolet radiation.
This picture shows how an IR Spectrograph is set up: http://en.wikipedia.org/wiki/Image:IR_spectroscopy_apparatus.jpeg
This picture shows how a UV spectrograph is set up: http://en.wikipedia.org/wiki/Image:UV-vis.png.

Answer 2

Ah, as a graduate student in biology here is my humble opinion =) I am unsure if as a biology major that you have to take inorganic, it depends on the person, but inorganic chemistry is very number based and has many calculations similar to gen chem that you have have taken already. I am not very good at calculations so I enjoyed organic chemistry due to the lack of numbers involved =) To be realistic, organic chemistry is very difficult because in a way it is its own language of chemical compounds. You will learn how to name them and understand all the structures and how they react to each other. My advise is to get the study guide/problems book/solution manual (whatever it is called) and do the questions within each chapter, after the chapter until you understand it completely. This class is very time consuming, but if you put your time into it you will do well. I spent an average of 3 hours per chapter and by the end of the year I had read that entire book, and you really have to (unless you're a orgo whiz) in order to get the grade you want. I'm sure inorganic is just as difficult, except for me it would be nigh impossible due to the amount of calculations in the subject matter and my lack of mathematical skills. Best of luck! Go Bio majors!

Answer 3

In bichromatic analysis the absorbance of a compound is measured at two wavelenghts;one wavelength is used to estimate the magnitude of the test value and the other allows an assesment of the interfering substances. One wavelenght is the absorption peak of the analyte. Therefore absorbance at this wavelength measures the concentration of the analyte plus cross-over absorbance due to other interfering chromogens in the cuvet. The second wavelength is not absorbed by the analyte but is absorbed by the interfering chromogen. Therefore, this absorbance indicates how much interfering chromogen is present. Then subtract the interfering absorbance to determine the concentration of the analyte.

Answer 4

Principle Of Spectrophotometer Wikipedia