Spectrophotometry (2. LF UK)

Introduction
Spectrophotometry is a method used to measure the absorbance of light by a chemical substance. It is done by  measuring the intensity of light as a beam of light passes through a sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelengths. Its basic physical principle is lambert-beer’s law, which tells us that a linear relationship between absorbance and concentration (A = ε b c) exists, where b is the length of the path traveled by light through the sample, c is the concentration and ε is a molar absorptivity constant that depends on both wavelength and substance. This measurement can also be used to measure the concentration of a known chemical substance.

Basically the machine measures the intensity of light that passed through our solution and from that it calculates the amount of light absorbed and the amount transmitted, enabling us to deduce the concentration of a substance if we have the length of the cuvette and the molar absorptivity of the mentioned substance. From the absorbance and transmittance we can identify certain substances that we placed in the cuvette. In medicine spectrophotometry has a wide range of usage.

Importance in clinical medicine
Spectrophotometers are of high clinical importance in almost all branches of medicine.

Their ability to measure concentrations of metabolically important substances in body fluids, such as blood, cerebrospinal fluid, urine and amniotic fluid among others, is crucial for correct diagnostic findings and continuous monitoring of patients. Equally early identified small deficiencies of certain essential substances might help preventing correlated health problems.

In intensive medicine the use of spectrophotometric analyses is more frequent, due to the fact that patients in unstable states are more prone to drastic changes in the amount of different substances in, for example, their blood. Intensive care units therefore frequently have spectrophotometers on site whereas general practitioners might send their probes to be analyzed in a laboratory.

The substances that can be quantitatively analyzed by spectrophotometers are numerous. They include: hemoglobin, erythrocytes, hematocrit, amylase, bilirubin, cholesterol, glucose, urea, creatinine, lipase, triglyceride, albumin, alcohol, ammonia, copper, magnesium, lactate, calcium, iron, magnesium, aluminium, sodium carbonate, carbon monoxide and even certain enzymes.

What are its advantages and disadvantages?
Spectrophotometer are very complex instruments that use different monochromatic lights in purpose to give an information to the researcher. Most of the times in medicine, the information sought is a concentration of one or multiple compounds. These compounds have a specific absorbance spectrum corresponding to their molecular structure. In most cases, researchers use calibration methods with known concentrations of the researched compound, plot a best fit curve and are then able to be find other concentrations from the curve (Musiol). Each measurement is to be done in the same environment which enables a very small risk of error.

An inconvenience of using spectrophotometry is that measurements have to be taken meticulously. Any trace of fingertips or condensation on the cuvettes can create random error in measurements, and therefore the use of microfiber paper is recommended (Musiol). Microfiber has for effect to clean any potential dust on the transparent side of the cuvettes. It also has no risk to create small abrasions on the cuvette. Another limitation to the precision of spectrophotometers can be the precision of the tools used to make the calibration concentrations such as pipettes, also source to random error.

How does it work?
A spectrophotometer consists of 6 basic components. A light source is needed, generally a deuterium lamp and a Halogen lamp are used. The use of two different sources of light provides the analysis device with wider wavelength of light and therefore a more versatile use. Then the light can either go through a monochromator containing diffraction grating or not, depending on the nature of the measurement. A monochromator will refract light and enable only a certain wavelength (in nm) to go through the aperture (How Does).

In case no monochromator is used, a light containing all visible wavelengths is emitted and goes through the sample. The light then exits the sample, enters a closed compartment through the exit slit and hits the charged coupled device (CCD). The CCD will have a certain number of light sensors that will react with the photons of the light given. The information given by the CCD will then be transferred to a computer software which will be able to read and display the transmittance of a compound at certain wavelengths. The CCD needs to be calibrated posteriorly to the measurements with a reference cuvette of distilled water for example. This reference cuvette will indicate to the software the amount of pixels representing 100% transmittance and at all the wavelengths of the visible light. To use a spectrophotometer, scientists will use a monochromator giving only one chroma or one wavelength of light appropriate with the compound analyzed which is going to give more quantitative information about the transmittance of light of compounds of different concentrations for example.

Are there any risks involved in it’s use (for patients and the clinical staff)?
The use of spectrophotometers is safe. The deuterium lamp represents no danger as it is filled with Hydrogen gas. Even if certain Spectrophotometers use UV light, it is enclosed and therefore cannot unwillingly harm us.

Are there ethical issues associated with the topic?
There is no real ethical issues associated with spectrophotometry.

The equipment

 * 1) Spectrophotometer
 * 2) Cuvette
 * 3) Blank solution
 * 4) Computer
 * 5) Unknown solution

The methodology
The SPECORD 40 spectrophotometer is a single-ray spectrophotometer operating in the wavelength range from 190 to 1100 nm (using a deuterium lamp for the 190–300 nm range and a halogen lamp for the 300–1100 nm range). It is used for measuring absorbance values in the interval of A values from –3 to +3. It can be controlled from the main panel or using the WinAspect program which simultaneously permits a simple and rapid data analysis. Safety advice For work with solutions of malachite green and indigotin wear protective gloves. Do not inhale or consume the solution. The spectrophotometer is sensitive to humidity. Be careful when working with liquids. After accidental spilling dry the affected area immediately.

Task 1. Measurement of absorption spectrum Measurement, define and characterize the absorption spectrum of a solution of malachite green and indigotin.

Chemicals: • Solutions A and B	• Distilled water

Working procedure

1. 	Turn on the spectrophotometer and the PC

2. 	Turn on the WinAspect program.

3. 	Open the Measurement menu and select Initialize device. After confirmation, the question appears on the use of a UV lamp which is to be rejected. The spectrophotometer then runs automatically initialization and internal calibration.

4. 	After termination of initialization set the measurement parameters. In the Measurements menu select Set parameters. A panel for setting the parameters will appear and you should click on New. Setting the parameters

The Settings panel: In the Title window fill in the name. Function Cycle Mode deactivated by selecting the possibility None Function Display – select the possibility Absorbance Function Correction – select the possibility Reference

The Mode panel: In the Meas.Mode window select the possibility Scan Mode Setting the Scan Mode function with the following values: Range = 375–675 nm (width of the examined spectrum) Delta lambda = 1 nm (length of the measurement intervals) Speed = 50 nm/s (speed of measurement)

After setting the parameters click on Save as on the measurement setting panel. The window with possibilities of saving will appear and you save the file in the Para folder under the name ENXXX SCAN. After saving click on OK icon.

5. 	The spectrophotometer is now ready for the measurement itself. The Measurement panel opens and you select Serial measurement.

6. 	In the WinASPECT program a window for Serial Measurements is now open. To set the parameters of measurement select from the Edit dialog box in the instrument panel the possibility Setup.

Set the following parameters: Panel General: Description: enter ENXXX SCAN Panel Samples: Number of measurements: enter the number of samples (2 in this case).

7. 	Before sample measurement, it is essential to carry out a reference measurement of the blank (solvent without dissolved substance). The samples are substances dissolved in distilled water hence the blank should be distilled water in a cuvette filled to about two-thirds height.

The cuvette should be held at the mat side. The light ray passes through the clear sides and their contamination (including fingerprints) can result in affecting the measurement results. Dry the cuvette before measurement and remove any impurities and fingerprints with dry cotton wool. 8. 	Open the sample compartment. The cuvette with the blank should be placed into the holder. Close the door of the sample compartment. The cuvette should be placed as close as possible to the holder wall to permit full transmittance of the light beam through the clear walls. Make sure that the cuvette is placed vertically in the holder.

The spectrophotometer can be damaged by spilled liquids. Be careful when working with it and, if a spill occurs, dry the affected place carefully.

9. 	Start the reference measurement by clicking on Reference in the instrument panel of the Serial Measurement  window. The spectrophotometer registers the absorption spectrum of the blank.

10. After the reference measurement the spectrophotometer is ready for sample measurement. This is started by clicking on the Start icon. Pour the first sample into a clean cuvette and place it in the holder (see points 8 and 9). Close the sample compartment (spectrophotometer lid) and confirm the action in the program (OK). The spectrophotometer will measure the absorption spectrum of the first sample.

11. 	After the first sample measurement repeat the procedure with the second sample. During sample measurement try to proceed from the most dilute to the most concentrated sample. Then you need not to rinse the cuvette after each measurement.

12. 	After termination of all measurements, a table containing the measured values of absorbance at the given wavelength (Table panel) or a graphical representation of the tabulated values (Graphic panel) will appear.

13. 	Print the page containing results

Task 2. Determination of concentration of an unknown sample and the Lambert-Beer law On the basis of absorbances of samples of known concentration construct calibration curve. Determinate concentration of an unknown sample on the basis of the Lambert-Beer law.

Working procedure Calibration curve

Before starting the assignment use the spectra obtained in Task no. 1 to determine a suitable wavelength for measuring the absorbance of solutions A and B and for both solutions in mixture (answer to questions asked in Task no. 1 report)

Since you are working with two substances a calibration curve must be constructed for both of them separately. Calibration curve for samples KA 1–4

1. 	In the WinASPECT program choose Quant on the instrument panel and the possibility Calibration. 2. 	The monitor will display the Calibration  window for setting measurement parameters. Screen General Designation group: ENXXXQUANTKA Regression Model: choose possibility y = A + B*x WAVELENGTH1 (most suitable for solutions A and B): choose on the basis of values obtained in Task 1. Calibration units should be chosen according to units used for calibration samples characterization (see table of concentrations). Ordinate: absorbance Cell Pathlength: measure the width of the cuvette Number of Standards: 4 for KA

3. 	Click on Edit icon, choose Measurement parameters. On the Mode panel choose from the offer Meas. mode the possibility Wavelengths. The suitable wavelength is selected by clicking on Edit. By clicking on Edit icon you choose the suitable wavelength and by clicking on OK and subsequent choice confirmation you return to the Calibration offer.

4. 	After returning to Calibration and entering the above parameters click on Standards icon.

5. 	Now the window will open; enter the individual standards in corresponding units.

Standard: enter the names of standards Conc.: enter the concentration values Source: this value is entered automatically

6.	Measure the absorbance of the blank. A cuvette with distilled water should be placed in the holder and by clicking on Reference carry out the reference measurement.

7. 	Click on Start and place the KA1 sample in the holder. Then measure KA1, KA2, KA3 and KA4 in this sequence. After termination of measurement click on OK.

8. 	The Calibration window now opens, containing a graphical representation of the calibration curve and the regression line equation.

9. 	After clicking on To conc. (measurement of samples of unknown concentration) the calibration curve can be saved. After a positive answer (dialogue window) save the file under XXXKALIBKA in the Calib file.

Determination of concentration of an unknown sample (for sample A)

10. 	After saving the program will automatically open the Concentration window which enables to determine the concentration of unknown samples from a calibration curve.

11. 	To measure the concentrations of unknown samples place the sample in a cuvette into the clamp and by clicking on Start (Meas.) the instrument will measure the absorbance and, on the basis of calibration curve coefficients, will calculate the concentration of the unknown sample.

12. 	On the same principle we obtain the concentrations of NA1, NA2. NA3 and NA4.

13. 	After termination of the measurement the concentration values will appear in a table.

14. 	Save the document as ENXXXQUANTNA.

Calibration curve for samples KB 1–4

Proceed analogously to generate the calibration curve KB 1–4 for samples NB 1–4.

Deviations in points:

2. 	Setting the measurement parameters: Designations: ENXXXQUANTKB WAVELENGTH1 (most suitable for solutions A and B): choose on the basis of measured values from Task no. 1. No. of Standards: 4 for KB Parameter file: ENXXXQUANTKB.par 5. 	Enter values for KB 1–4 9. 	Save the calibration curve under ENXXXKALIBKB in the Calib file.

Determination of concentration of unknown sample (for sample B):

Proceed analogously to samples NA1–4. After ending the measurement of samples NB1, NB2, NB3 and NB4 save the values under ENXXXQUANTNB.

Conclusion
The future of spectrophotometry lies especially in the improvement of pathological diagnostics, disease detection and general clinical research as “uv-vis spectroscopy enables safer, non-invasive analysis of soft tissue, and can enhance accuracy and speed in clinical diagnostics and medical research.”

Around 10% of cancers are due to the exposure of radiation, therefore, such improvements will aid us in the advancement of clinical applications relating to various types of cancers. Spectrophotometry has enabled us to “convert measured reflectance and fluorescence spectra from tissue to cancer-relevant parameters such as vascular volume, oxygenation, extracellular matrix extent, metabolic redox states, and cellular proliferation.” Pulse oximetry is a method of examining a person’s oxygen saturation and it uses the means of in vivo spectrophotometry in its procedure. This relatively recent method of examination also presents several possibilities for future developments of spectrophotometry by providing “reliable, objective, and non-invasive” results.