Reading

Laboratory Text:
1. Robyt and White, Chapter 3: "Spectroscopic Methods", pp. 40 -49, 51 - 52
2. Robyt and White, Chapter 7: "Qualitative and Quantitative Methods for Determining Biological Molecules" (Section 7.1, Carbohydrates), pp. 213 -214, pp. 219 - 220).

Links:
4.Use of the Spec 20: http://chemed.chem.purdue.edu/genchem/lab/equipment/spec20/index.html

6.   Limitations of the Beer-Lambert law: http://www.chemistry.adelaide.edu.au/external/soc-rel/content/beerslaw.htm
(don't concentrate on the mathematics of the derivation)

Questions (completed in notebook prior to class):
1. What physical property of a solution measured with the spec-20?
2. Describe how the Beer-Lambert law can be used to generate a standard curve. What fit would you expect for a Beer-Lambert plot?
3. Describe the purpose of each of the solvents and reagents which are added to each standard and sample.
4. Provide the structure of Glucose in both a linear and hemiacetal form. Speculate about the structure after reaction in the Nelson's test.

In the laboratory Exercise Outline:
1. Prepare standard solutions of a sugar via dilution and by using an oxidizing reaction
2. Measure light absorbed/transmitted using the spec 20
3. Generate a standard curve based on the data obtained
4. Measure the absorbance of an unknown concentration sugar solutions
5. Solve the concentration using the standard curve

Introduction:

Carbohydrates are the must abundant organic compounds in the biosphere. Carbohydrates are composed of single sugars (or monosaccharides) or dimers, trimers, oligomers, or polymers of monosaccharides (disaccharide, trisaccharide, oligosaccharide, polysaccharide, respectively). Monosaccharides contain several hydroxy groups and either an aldehydes or ketone. Other derivatives or functionalized sugars are also found in nature. All sugars contain chiral carbons and can form a variety of isomers in aqueous solution. Numerous qualitative tests can be employed to determine the presence of carbohydrates by reaction with the alcohol, aldehyde or ketose moieties. Many clinical tests simply test for the presence or absence of carbohydrate in a sample. Quantitative tests can also be performed to determine concentration of carbohydrate in a sample. Whenever quantitative analyses are performed, a standard analysis must also be performed. In the formation of a standard curve or profile a solution of known concentration is subjected to the exact same conditions as the samples of unknown concentration. From the standard solutions a graph of concentration of standard versus results measured is plotted and referred to as a standard curve. The quantity of unknown can then be determined by comparison to the standard curve.

Standard Solutions: It is critical to the success of an assay that the concentration of the standard be accurately known. Primary standard solutions are most often prepared by accurately weighing the required amount of a highly purified chemical compound (primary standard). In some cases the concentration of the desired standard solution must be established by standardization against an alternate primary standard. Additional standard solutions are often prepared by dilution of a primary stock solution. Standard solutions can be used to determine the concentrations of unknown solutions (by titration, spectrophotometric measurement, etc.). Students should already be familiar with concentrations (percent solutions, molarity, normality, ppm, etc.) and the concept of preparing solutions of desired concentrations. In addition, the student should be familiar with dilution methods and the corresponding calculations.

Data Treatment: Large quantities of data are most often efficiently presented using either or both graphs and tables. Figures should always be self explanatory with a full informative title and a caption which summarizes the conditions under which the data were obtained. The axes of graphs should be precisely labeled with both a scale and legend (quantity and units). Experimental points on graphs must be clearly delineated. Different symbols may be used for different curves plotted on one a single graph. This is useful when graphs are compared. Where appropriate, either a smooth curve or the best straight line should be drawn through the points. Students should already be familiar with the determination of the line of best fit, significant figures, and basic statistical methods. Tables require headings and units and figure captions. Data with numerical values less than one should always be written with a leading zero. For example the correct representation of one half is 0.5 not .5. Significant figures should be used where appropriate.

Quantitative Colorimetric Assays: Photometric assays involve direct measurement of light absorbing compounds. However, biochemists frequently need to assay compounds that either do not absorb light in the UV, visible or near IR ranges or are at too low a concentration to absorb sufficient light. The solution to this problem is the use of colorimetric assays. In a colorimetric assay a reagent is mixed with the analyte and the resulting reaction produces a compound which absorbs light in the visible range of the electromagnetic spectrum. This is observed by humans as a color. The intensity and absorbance of the reaction product can be measured with a spectrophotometer and the data are proportional to the concentration of the original substance assayed.

Certain principles are followed in all types of colorimetric assays: following these principles simplifies the data treatment.

1. Each sample, regardless of the amount of substance being assayed, has the same final volume.

2. Each sample has identical amounts of reagents, only the amount of the substance being assayed and water (or buffer) varies. However, the total volume of sample plus water (or other solvent) is constant in every sample.

3. One sample is blank. The blank does not contain the substance assayed. It is used to indicate the extent of reaction or the intensity of color formed in the absence of sample. This sample is used as a zero point.

4. Data (absorbance measurements) from a colorimetric assay is presented in tabular format, and plotted.

In this laboratory a quantitative assay for reducing sugars is the Nelson's Test. The standard curve generated by measurement of known concentrations solutions provides a relationship between the concentration of substance (glucose) present and the intensity of absorbance (color) formed in the reaction. A plot of the data provides a standard curve. This graph can then be used to determine the concentration of reducing sugar in an unknown solution which has been assayed in an identical manner. Indirect colorimetric assays require that the color forming reagents be present in excess concentration. Insufficient concentrations of reagent results in nonlinear data at high concentrations of the unknown.

Photometry: If white light (visible light) is passed through a solution containing a colored compound, certain wavelengths of light are selectively absorbed. The resultant color which humans observe is due to the transmitted light (light not absorbed). Photometry involves the qualitative and quantitative use of absorption data obtained from compounds which absorb light in the ultraviolet range (UV, 200 - 400 nm), visible (400 - 700 nm) or near infrared (IR, 700 - 900 nm) regions. Much of photometry is based on two formalized laws. The first is Lambert's law, which states that the proportion of incident light absorbed by a medium is independent of its intensity and that each successive unit layer of the medium absorbs an equal fraction of the light passing through it. Secondly, Beer's law states that the intensity obtained when light passes through a solution of concentration c and length b is equal to that obtained when light passes through a solution of the same substance at concentration c/2 and length 2b. In other words the light absorbed by a sample is proportional to the number of molecules of absorbing substance through which the light passes.

Photometry is frequently used to determine the concentration of a light absorbing compound present in a solution. The expression A = EbC (derived from Lambert's and Beer's laws) states that the absorbance (A) of a given compound at constant conditions is related to the unique extinction coefficient or molar absorptivity constant (E) for the compound, the concentration of the compound (C) and the length of the light path (b). An extinction coefficient (E) is a constant for a given substance and a given set of assay conditions.

If you use a 1 cm cuvette and define the solute concentration in moles per liter, then:

Note the resemblance to the mathematical equation for determining the slope of a straight line

when the line goes through the x-y zero intercept, b=0 and

E can be determined from any one point on the best fit line from of standard curve. Alternatively, E can be determined from multiple points in the best fit area (most linear portion of the graph) and averaged. The calculated E (molar extinction coefficient) can be used to determine the concentration of a substance being assayed in any unknown solution for which an absorbance has been measured.

Experimental Procedure:
Methods:
You will use Nelson's Test for the detection of free reducing groups in a carbohydrate. The intensity of the reagent-sugar complex is proportional to the concentration of the carbohydrate.

(You will have to determine how you will accomplish the standard and sample preparation - include these detailed descriptions in your procedure BEFORE lab.)

1) Dilute the 10.0 % (w/w) unknown compound to 100 ug/ml with distilled water.
2) Use the diluted unknown and the glucose standard (100 (ug/ml) to prepare the assay mixtures indicated in Table I on the following page (use large test tubes):
3) NOTE: Standard 1 contains no sugar and is therefore blank.
4) Add 1.0 ml of Nelson's alkaline copper reagent to each tube and vortex (each sample should be mixed for approximately the same time).
5) Place all the tubes in a boiling water bath (1000 ml beaker) and heat for exactly 20 min.
6) Remove all the tubes at the same time and place them in a beaker of cold water to cool.
7) When the tubes are cool to touch, add 1.0 ml of arseno-molybdate reagent to each and vortex occasionally for 5 min.
8) After all precipitate is dissolved, add 7.0 ml of distilled water to each tube and vortex.
9) Using a Spec 20, determine the absorption maximum of the standard (decide which standard is the appropriate one to use)
b) Zero the instrument with the blank
c) The absorption max should be between 500 and 550 nm. You should determine the exact absorption max and use that value for the entire experiment.
10) Read the absorbance of each standard and sample at the wavelength determined to be the absorption max.

Table I. Assay data for the Nelson Test - The table includes the volumes of sample/reagents (1.0 mL total) and composition of each sample. Nelson's reagent, 1.0 mL, 1.0 mL arseno-molybdate reagent and 7.0 ml water were added to each standard and sample for a total volume of 10.0 ml per assay.

sample/std	water (ml)	unknown (ml)	glucose std
std 1	1.0	0	0 (ml)
2	0.9	0	0.1
3	0.7	0	0.3
4	0.5	0	0.5
5	0.3	0	0.7
6	0	0	1.0
Sample
7	0.7	0.3	0
8	0.5	0.5	0
9	0.3	0.7	0
10	0.0	1.0	0

Questions: These questions should be answered in your laboratory notebook
1. Which of the following carbohydrates are reducing sugars: galactose, alpha-methyl-galactoside, maltose, mannose, xylose, fructose, rhamnose, ribose, glucosamine? Explain your answers
2 What result would you expect from the Nelson's test using sucrose?

Data analysis - for the write-up see the syllabus and reports pages.
Reporting Results: These data are best reported in tabular form. A standard curve should be generated using the data obtained for the glucose standard. This curve is then used to determine the molar concentration (molarity) of sugar in the unknown samples. Determine the appropriate extinction coefficient(s) for glucose and for each unknown. Use the standard curve to determine the molar concentration of sugar in your unknown sample. Also determine the concentration of sugar in your samples by using the experimentally determined extinction coefficient and the Beer-Lambert Law. Compare the results. Include these calculated concentrations in your data table for the Nelson's test.