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Laboratory # 3Spectrophotometry

Laboratory #3Spectrophotometry

Skills= 20

Objectives:Upon completion of this unit, the student should be able to:

1. Provide an overview of photometry / spectrophotometry including description of basic components.

2. Explain Beer's Law and its significance.3. Define and apply the following terms as they apply to clinical chemistry

spectrophotometric procedures: “range of linearity”, “blank solutions”, and “standards” and “controls”.

4. Set-up and optimize the spectrophotometer.5. Use proper technique in operating a spectrophotometer and make accurate readings on

absorbance and transmittance scales.6. Explain the method for development of a spectral-transmittance (S-T) curve and plot an

S-T curve 7. Identify the absorbance maxima for hemoglobin by preparing a spectral curve. 8. Review related internet resources:

http://hydrology1.nmsu.edu/teaching/soil698/Student_Material/spectrometer/index.htm9. Provide answers to related study questions.

Materials:1. Spectrophotometer & cuvettes2. Hemoglobin solution 3. Graph paper (linear and semi-log)4. Filter paper5. Didymium filter6. Blood drawing supplies (lavender tops)

Principle:Photometers, colorimeters and spectrophotometers are members of a group of chemistry laboratory instruments designed to measure electromagnetic radiation (usually monochromatic light) being transmitted through a solution. The purpose of this measurement is to assist in determining the concentration of light absorbing substances contained within the solution. Simple photometers & colorimeters use a filtering device to produce the monochromatic light (colored glass in colorimeters, fiber or other filters in photometers). These types of photometers are generally very sturdy and less costly, but lack versatility. Spectrophotometers use prisms or diffraction gratings to produce monochromatic light. They are more useful because the adjustment from one wavelength to another is much faster allowing for quick adaptability in analyzing a wide variety of analytes however, they are higher priced and require more maintance and QC monitoring. The photometer / spectrophotometer has proven to be the most versatile, reliable and widely used of all laboratory instruments in clinical chemistry. The majority of clinical chemistry procedures have been developed to produce a colored end-product which can be detected and measured by

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some sort of photometer/spectrophotmeter. Spectophotometry is based on two principles: (1) substances absorb light at unique wavelengths and (2) the amount of light absorbed is proportional to the amount of substance that is present. Even many of the highly automated analyzer systems utilize some sort of photometer / spectrophotometer as a means of determining concentration. Because of the heavy usage of spectrophotometers, it is imperative that laboratory workers have a firm understanding of the principles of spectrophotometry as well as the technical abilities to operate these instruments properly and perform basic calibration and maintenance procedures

Photometer Basic Components:

Light source - A lamp produces a steady source of quality light. If the procedure performed produces a product that will be read / evaluated in the visible range (400 - 700 nm), a tungsten or tungsten iodide lamp is appropriate. However if the work is in the ultraviolet range (generally < 400nm), a deuterium or mercury-arc lamp will be required.

Monochromator - While the lamp will produce the range of light, it is the monochromator that purifies the light allowing only a narrow range of wavelengths to reach the specimen. This narrow range of wavelengths produced by the monochromator is known as its ‘bandpass’.

Bandpass – the distance between two points where the wavelength is ½ the intensity as the peak: (Range of wavelengths)

Advantages of Narrow Bandpass Spectrophotometers1. Greater potential accuracy.2. Greater potential sensitivity.3. Can use high intensity light source.

Disadvantages of Narrow Bandpass Unit1. Must be accurately calibrated.2. More expensive equipment.

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3. Complex optical system.

Cuvet / sample holder - The devise that holds the specimen making it available to the monochromatic light. Cuvets come in various qualities, shapes and sizes,

Directions for Use of Cuvets:Cuvets are composed of optically pure glass / plastic which allows the monochromatic light pass through with minimum of interference. Often times, cuvets will have a trademark that provides the user a reference point and usually indicates the surface of the cell that has been found optically best and selected for use. It is important to consistently orient the cuvet in the same direction each and every time it is used. Normally the manufacturer suggests that the trademark be oriented toward the light source to provide precision readings. Always use Turner cuvets / cells with the trademark facing the front.

Cuvets are graded and are classified according to their quality and best use (and cost). Some cuvets are manufactured for special work requirements. Cuvets for nephelometric procedures are evaluated and matched on the basis of light-scattering characteristics; cuvets for use in the UV range must be composed of material that allows those wavelengths to pass efficiently. Square cells are manufactured to a single specification and do not carry classification letters. Matched pairs of round or square cells are also available.

Particular care should be taken to avoid the scratches certain to occur if the cells are allowed to rub against one another or against other hard surfaces. Avoid abrasive cleaning agents and make sure that the exposed surfaces of the cell are optically clean by wiping with a soft cloth or with cleansing tissue just before the cell is put in the well, and therefore, handling only by the top edge.

Do NOT wash cuvets between samples (between procedures, yes, but not between samples within a procedure). Washing out cuvets between each sample will cause unnecessary dilution of the sample solution with wash water (one drop of wash water in a 10 mm cell would create a possible 5% error.) Most procedures performed in our lab will not have significant ‘carry-over’ if the cuvet is properly drained and the rim blotted. (Ask instructor to demonstrate.). If there is

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concern about ‘carry-over’ (and there is sufficient sample) pre-rinsing the cuvet / cell with a small portion of the sample will reduce contamination.

General handling procedures:Always clean the cell thoroughly and then rinse at least once and preferably twice with a portion of sample before making a reading.

1. Always wipe the lower third of the cell dry and free from lint and finger marks before placing in the cell adapter or well.

2. Do not attempt measurements at temperatures below the dew point.3. Make sure that no bubbles cling to the inner surface of the cell.4. For maximum precision standardize and test with cells of the same class.5. Handle and clean these cells carefully to avoid injuring the optical surfaces.6. Always place the cell in the instrument with the trademark word squarely facing

the light source.

Photodetector - The part of the instrument that will turn the light energy coming though the specimen into electrical energy. There are various types and quality of photodetectors available for specific applications. The more commonly encountered photodetectors are photomultiplier tubes which not only detect the light but proportionally enhance or amplify it.

Readout devices - convert the electrical signal into visual / useful form.

Concepts in PhotometryPhotometry measures the amount of light being transmitted through a solution in relation to the amount of light entering the solution (percent transmittance or %T). The basic concept of light transmittance through a solution is important, because only transmitted light can be measured in

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a spectrophotometer. The transmittance (T) is defined as the proportion of the incident (original or incoming) light that is transmitted. Various ways this is depicted :

%T = I X 100 I = transmitted light Io Io = incident light

Early photometers provided readout information only in the %T format. To use the information to determine the concentration of a substance required making the standard curve on semi-log paper or use of logarithms in calculations. To avoid these additional problems, the %T information is mathematically converted to absorbance (A) of light also called optical density (OD).When the depth of the solution / length of the lightpath is held constant using a high quality standard cuvette, the substance molecules can be thought of as occurring in innumerable monomolecular layers of absorbing material. Note: As the absorption of light increases, the % transmitted light decreases logarithmically.

As the absorbance of a solution increases, %T decreases logarithmically and will plot as a straight line on semi-log paper. The radiation not transmitted is absorbed characteristic of a given molecule and is called molar absorptivity. (Molar absorptivity is a characteristic of a substance.)

The absorption and/or transmission of light through a specimen is used to determine molar concentration of a substance.

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Concerns/Problems with Spectrophotometry

Certainty of WavelengthVerification of wavelength calibration should be done periodically, especially if a parameter in the instrument has been changed, such as the lamp being changed or the instrument has been bumped or traumatized. Wavelength calibration verifies that the wavelength indicated on the dial is what is being passed through the monochromator.Solution: Calibrate with appropriate filter (ex., didymium, holmium oxide)

Too Broad/Narrow BandpassThe problem is inherent in spectrophotometer as determined by monochromater. Solution: Change the monochromater OR use a different spectrophotometer.

Linearity Solution: Determine range of linearity using standard curve.

Stray LightThe term ‘stray light’ is used to light energy measured outside the spectral region defined by monochromater) causing non-linearity and insensitivity.

Solution: Use sharp cut off filters. If dirty, clean optical system. Replace monochromator grating if defective.

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Old/New BulbsSolution: Wavelength calibration. Replace old bulb.

Instrument Error due to Analyte Concentration (see linearity below)

Analyte Concentration Too High Instrument and reading error can occur if the concentration of the analyte is too high. This is often referred to as ‘exceeding the linearity of the procedure’. Possible solutions: 1. Make a dilution of the specimen, repeat the procedure on the

diluted sample, calculate the concentration of the diluted sample, then multiply the results by the dilution factor.

2. Alter/adapt new procedure with higher range of linearity.

Analyte Concentration Too LowInstrument and reading error can also occur when the concentration of the analyte is below the ‘range of linearity’ for the procedure.

Possible solutions: 1. Alter/adapt new procedure with lower range of linearity.2. May need to report the result as being less than the lowest level

within the range of linearity. (EX. If the glucose procedure’s range of linearity, as stated by the manufacturer is 30 - 550 mg/dL; you would say that the specimen’s result is “<30 mg/dL.)

Beer - Lambert LawThe relationships between absorption, transmittance and concentration is stated using the Beer-Lambert Law. The concentration of a substance (or strength of “color”) is directly proportional to the amount of light absorbed by the chromogen and inversely proportional to the logarithm of the transmitted light.

The mathematical formula showing this relationship is: A (or absorbance) = 2 - log %T.

Below is a scale that shows the relationship between absorbance (on the top) and % T which is on the bottom.

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Not all chromagens produced in clinical chemistry reactions follow the Beer-Lambert Law (or do so for limited concentration levels).In order to follow Beer's Law:

1. Keep light path constant by using matching sample cuvettes standardized for diameter and thickness

2. Solution demonstrates a straight line relationship between two quantities in which the change in one (absorption) produces a proportional change in the other (concentration) is called linearity. Not all solutions demonstrate a straight line graph at all concentrations.

Errors or variation in reading can occur in three places: setting zero %T, setting 100%T, and reading the sample %T. The relative error in determining concentrations increases as readings are taken at the left end of the %T scale, even when light absorption follows Beer's law exactly. Look at the scale on the Coleman Spectrophotometer and notice how very close together absorbance values are at the low transmittance end of the scale and remember that it is absorbance that is directly related to concentration.

The increased relative error from reading at the low %T (left) end of the scale is due to large error in calculated absorbance with any small error in %T readings. Increased relative error at the high %T (right) end of the scale, however, is due to the very low concentrations of the solutions being measured. In summary: there can be error at either end.

LinearityLinearity is obtained when the change in absorbance is directly proportional to the concentration of the substance being measured. A graph of concentration vs absorbance for a procedure that follows Beer’s Law will produce a straight line.

Three Approaches to Determining Concentration of an Analyte When performing quantitative analysis (i.e., finding the concentration of an unknown) using spectrophotometry, three different procedures may be used:

1. Calculate directly using absorbance or %T readings, and known molar absorption constants of the substance.

2. Plot Abs (on linear) or %T (on semi-log) of known standard values vs. concentration, a standard calibration curve.

3. Analyze a standard of known concentration and calculate the unknown concentration

Blank Solutions

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In clinical chemistry procedures, the “blank” is used to nullify or blank-out the light absorbing effects of a reagent or a color in the sample. Depending on the particular situation, they may be read in the spectrophotometer and their absorbance value mathematically subtracted before additional calculations made. Other times they are used to ‘blank’ or ‘zero’ the instrument.

1. Water blank - Deionized / distilled water us often used as a blank in procedures which are ‘read’ in the ultraviolet range. The procedure may say “read against water” or “zero on water blank”.

2. Reagent blank - often used in colorimetric procedures (wavelengths between 450 - 750 nm). Reagent blank contains all the reagents used in the procedure, but a volume of DI water is used in the place of the “specimen”.

a) Reagent blank must contain all constituents of a test except the standard, control or unknown sample

b) Use the reagent blank to adjust the instrument to account for absorbance of light by the solution that the solute (substance being measured) is in.

c) Setting the photometer at a value of 0.00 O.D. or 100%T, effectively cancels the absorbance of reagents being used

3. Patient blank - is the least encountered or least required. Usually required when the patient specimen contains a color / substance that interferes with the test result.

a) Used when patient specimen is lipemic, icteric, or hemolyzed.b) Contains all test reagents and patient sample (usually added last). Follow

manufacturer's directions.c) The O.D./Abs reading must be subtracted from the patient's test reading

before determination of concentration can be made.

Procedure 1 - Introduction to the Sequoia-Turner Spectophotometer Model 340

1. Working in pairs at a computer, call up the website below. If you have problems, use the method stated in the box to access the info. If you continue to have problems, notify the instructor.

http://hydrology1.nmsu.edu/teaching/soil698/Student_Material/spectrometer/index.htm

If this does not work, Google ‘sequoia turner spectrophotometer’ and review the list for:SpectrophotometersModel 340 Spectrophotometer (Sequoia-Turner Corporation, 755 Ravendale Dr., Mountain View, CA94043) By. Naomi Assadian, Xiaoyun Liu, and Juan Pedro Flores

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http://hydrology1.nmsu.edu/teaching/soil698/Student_Material/spectrometer/index.htm


2. Review the information within the various areas listed below and answer the questions on the report page.

Areas to Review IntroductionElectromagnetic radiationSystem DescriptionInstrument OperationPreventative MaintenanceLimitations and AccuracySafety

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Procedure 2 - Hemoglobin Spectral Curve

Principle: The spectral transmittance curve (S-T) allows the technologist to select the optimum wavelength for photometric measurements so that analytical methods will follow the Beer-Lambert law. In other words: looking for the optimal wavelength to read the procedure.

Scroll to the bottom of Gary Bertrand’s website for examples of determination of absorbance maxima for red and blue dyes. (http://web.umr.edu/~gbert/color/spec/Aspec.html)

Example – Hypothetical S-T Curve1. λ 1 = 02. λ 2 – rising too rapidly, therefore, increasing error3. λ 3 – BEST (absorbance maxima, broad peak)4. λ 4 – may also be used; absorbance minima5. λ 5 – peak too narrow (too specific) although maximum absorption occurs here

In this example, the absorbance maxima (wavelength maximally absorbed) of each type of hemoglobin (Hb, oxyhb, carboxyhb, and methb) have at least two absorption peaks in the visible spectra.Based on this principle, serum or blood can be screened for hemolysis, CO poisoning, and methemoglobinemia.

Proceduce Steps:

1. Working in pairs, optimize the Sequoia Turner Spectrophotometer and obtain 2 appropriate cuvets (1 for DI water, 1 for hemoglobin solution).

2. Prepare a 1/200 dilution of freshly drawn EDTA whole blood using deionized water. Mix sample and set aside. (0.05 mL blood + 9.95 mL DI water).

3. Zero spectrophotometer using deionized water for each wavelength evaluated. Begin at 450nm. NOTE: The blank is the solvent that your sample is dissolved in.

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a. Place the cuvette in the cuvette holder in the sample compartment.b. Turn the wavelength dial to the desired wavelength.c. Set the stray light filter to correct setting. Use the position that brackets the

wavelength you selected.d. Close the compartment lid.e. Select transmittance or absorbance mode by pressing TRANS/ABS button.f. Press the 100%T/0A button. The display will show 100.0 in transmittance mode

or 0.000 in absorbance mode.g. Remove “zeroing” civette.

4. Pour hemoglobin solution into a matching cuvet. Read and record the absorbance value at 450 nm.

5. Place the blank (DI water) back into the instrument, change the wavelength to the next appropriate one (460nm), zero, place the cuvet with the hemoglobin solution back into the instrument, record etc. Each lab partner will equally participate in this process. One writes results on both report sheets while other operates instrument; then switch roles half way through procedure.

6. On a sheet of linear graphing paper, label the Y axis ‘Absorbance” and the X axis ‘Wavelength’ and plot the spectral curve.

7. NOTES: a. Non specific absorption will occur in the UV wavelengths range, do not

select this peak as the absorbance maxima for the hemoglobin.b. Draw this curve from point to point.

8. In the space provided, state / identify the wavelength maximally absorbed and any other (secondary) absorption peaks.

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Student Name: _________________Date:_________________________

Lab #3: SpectrophotometryReport Form

Points=20

Procedure #1

Introduction & System Description

1. The Sequoia Turner Spectrophotometer model 340 is capable of what range of wavelengths?

2. Locate the filter shown on the System Description page and identify the range of wavelengths it is capable of passing.

3. The spectrophotometer pictured on the System Description page is setting on what wavelength?

4. What is the source of radiant energy identified for of this spectrophotometer?

5. How is the light isolated in this instrument?

Instrument Operation6. List the steps for the operation of the Sequoia Turner Spectrophotometer model 340 as

they are outlined by the site authors of this website. ( 7 points)

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Limitations and Accuracy7. What bandwidth is passed by this spectrophotometer?

8. List 5 limitations of this instrument that are cited by the authors? (5 points)

Safety 9. What safety items are recommended to be used when operating this instrument (as outlined by these authors)? ( 3 points)

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Procedure 2Lab Partner’s Name ______________________

Spectrophotometer used__________________________________

Wavelength (nm)

Measured Absorbance

Wavelength (nm)

Measured Absorbance

Wavelength (nm)

Measured Absorbance

Wavelength (nm)

Measured Absorbance

450 500 550 600460 510 560 610470 520 570 620480 530 580 630490 540 590 640

1. What wavelength was maximally absorbed by the hemoglobin? (NOTE: Do NOT include the peak that occurred in the lower / ultra violet end.)

2. Were there other absorption peaks? If so, at what wavelength?

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Student Name:_______________Date:__________________

Lab #3: SpectrophotometryStudy Questions

Points= 15

Each question worth one point, unless notated.

1. What is a monochromator?

2. Explain / define the absorbance maxima and for what it is used? ( 2 points)

3. If an instrument gave its results as %T readings, state two ways you could obtain the concentration of the substance being tested. (2 points)

4. In your own words, briefly summarize Beer's Law.

5. Why must standardized cuvettes be used with a spectrophotometer?

6. What is meant by the term ‘stray light’?

7. Provide at least one example of a spectrophotometer readout device.

8. State the mathematical formula that shows the relationship between concentration, absorption and transmittance.

9. Define “range of linearity.”

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10. A technician performs a glucose analysis and reads results from a previously prepared glucose standard curve. The range of linearity for the procedure is 30 mg/dl to 350 mg/dl. The glucose results for one patient calculates as 20 mg/dl and for another 610 mg/dl. (4 points)

a. How should the 20 mg/dl value be reported?

b. How should the 610 mg/dl value be reported or handled?

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