Spectrophotometry (2. LF UK)

Introduction
Spectroscopy is the study of the entire electromagnetic spectrum as well as its interactions with matter. It examines relations of absorption, emission or dispersion of electromagnetic radiation by matter as a function of wave length (λ) or frequency (f). It can be used to identify substances and measure their concentrations. The latter is called spectrophotometry. Devices which measure in one or few specifically defined wavelengths of monochromatic light are called photometers.

When a light beam of intensity (Io) is passed through a solution a fraction of the photons are absorbed or scattered and the exiting beam has a lower intensity (I). The Lambert’s law (see fig 1) applies where x is the thickness of the attenuating solution and alpha is called the linear attenuation (or absorption) coefficient. The ratio I/I o  is known as transmittance (T). Its values ranges from 0 (all light was attenuated) to 1 (all light passed through; if fluorescent substances are being used, it can reach values higher than 1). The absorbance (A), defined in fig2, is a function of wavelength (or frequency). The variation of absorbance with wavelength is called the absorption spectrum.

For absorption in solution, Beer's law says that the linear attenuation (or absorption) coefficient is proportional to the concentration. Since the concentration (c) is molar in most cases ε the constant of proportionality is called the molar attenuation (or absorption or 'extinction') coefficient. It is specific for every individual substance and wavelength.

When combined, these two relations form the Beer-Lambert law A = ε · x · c (fig 3) where x is the length of the cuvette.



Spectroscopic properties in visible (VIS) and ultraviolet (UV) are studied in spectroscopy and photometry. They use light the UV area (wavelength 10-390 nm), visible areas (wavelength 390-790 nm) and adjacent infrared areas (wavelength 770 nm - 1 mm). Thanks to the Lambert-beer’s law, it is possible to determine concentration of unknown solutions.

Measuring ε and unknown concentrations for a given substance using a calibration curve method:  The absorbance of several calibration samples of known concentration of the given substance is measured. All samples have different concentrations but are placed in the same cuvette and the same wavelength is used to measure them. By drawing a graph (calibration curve) of log10 Io/I against the known concentrations c, ε can be obtained from the gradient (linear regression). this method is suitable for simple solutions. We must pay attention to the units because concentration is in micromole per liter (μMol/L), while the width of cuvette is in centimetres. Once ε is known we can find the concentrations of unknown solutions.

In practice the validity of Lambert-Beer law is limited by the dispersion of light because of tiny impurities in the sample; phosphorescence or fluorescence of the sample; small amount of light passing through highly concentrated solutions; changes of values of absorbing coefficients; shifting chemical equilibrium caused by high concentration of substances in the sample.

Measuring Principle
Spectrophotometer is made of these parts

• Light source (halogen light for visible light, deuterium for UV area)

• Lenses and mirrors that direct the beam of light

• Monochromator (device transmitting light of a given wavelength only) – Nowadays optical grids are usually used. which allow the wavelength to change fluently.

• Cuvette space – Space for samples in cuvettes that can be made from plastic, glass or quartz glass

• Light sensor (charge-coupled device or photodiode) – The precision of the measurement depends on the integration time – the time over which the light is measured, the longer the more precise but the longer the measurement which is very important when processing a larger number of samples, when using larger number of wavelengths or when processing samples which are changing over time (kinetic measurements).

• Output device (software for data analysis)

Based on the number of beams used for measuring, we distinguish single or double-beam spectrophotometers. Double-beam spectrophotometers use one beam for measuring the sample, and the other for a blank (i.e., cuvette filled with solvent without dissolved substance so that on can eliminate the attenuation of the solvent itself by subtraction). When using the single-beam device we must first measure blank and then measure the sample solution.

Modern spectrophotometers are fully automatized and controllable via computers. They are used for measuring at one or more measurements or even whole absorption spectra (wide spectrum of wavelengths). They can also be used for measuring kinetics of simple, for example enzymatic reactions. Thus, it’s used in a wide range of scientific disciplines such as chemistry, physics, biochemistry, biology, biophysics and even medicine.

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. 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. Early identified small deficiencies of certain essential substances might help prevent associated 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.

What are its advantages and disadvantages?
Can measure the concentrations of a lot of substances.

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 or systematic error in measurements, and therefore the use of microfiber paper is recommended (Musiol). Microfiber can remove any dust on the cuvettes. It also does not 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.

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 device with wider wavelengths 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 enable only a certain wavelength (in nm) to go through the aperture.

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 light sensor (e.g., charged coupled device CCD). The CCD will have a certain number of light sensors that will measure the light. The information provided 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 MULTIPLE 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 LIGHT ABSORBED BY THE SOLVENT AND CUVETTE MATERIALS ONLY and at all the wavelengths of visible light.

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 harm us.

Are there ethical issues associated with the topic?
There are 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 simple and rapid data analysis.

Chemicals
The following chemicals are used for measurements in this spectrophotometric task:


 * 1) Copper sulfate pentahydrate has an absorption maximum in the infrared range.
 * 2) Cobalt chloride hexahydrate has an absorption maximum in the green region.
 * 3) Nickel sulfate hexahydrate has absorption maxima in the purple and red regions.
 * 4) Distilled water, which is used as a reference sample (blank) and for rinsing the cuvettes.

Work with solutions in gloves and goggles, do not inhale or ingest.

Samples
Samples labeled A, B and C for measuring spectra are prepared in bottle. Series of samples marked A1, A2, A3, A4 and A5 for the calibration curve are prepared and a sample of unknown concentration marked AX for determine the concentration by spectrophotometry. Prepare the cuvettes with the reference sample (distilled water) and the individual samples and place them in the prepared cuvette stand before starting the measurement. '''The individual cuvettes should be filled with samples so that they are approximately to the top of the narrowed part and there is no need to fill them to the top. '''

Cuvettes insertion
Before inserting the cuvette, gently wipe off any excess liquid and remove any dirt on its surface. Open the sample compartment. Insert the cuvette into the cuvette holder. Insert the cuvette as close as possible to the wall of the holder so that the light beam passes through the wider part of the narrowed bottom of the cuvette. Then close the sample compartment door. '''Make sure the cuvette is vertical in the holder! The spectrophotometer is a device sensitive to liquid spills. Therefore, work carefully. In case of liquid spillage, carefully dry the spilled area! '''

1st Task
In this task, measure the spectrum of samples A, B and C in the wavelength range 380-1100 nm. Identify individual absorption maxima. Then, based on the number of absorption maxima, determine which chemical (see Chemicals) is represented by each sample A, B, and C. Report the wavelengths and corresponding colors in the visible spectrum for each maxima. For the next task, determine the best wavelength (when the maximum absorption is) for Sample A to use when measuring the calibration curve and determining the concentration of the unknown sample in the next task. Upload the generated pdf file in WinASPECT to the moodle server with the protocol.

Detailed instructions for program control when measuring spectra A, B and C are in the following file.[[Media:Spektrofotomertrie presentace spektra EN.pdf|Instructions for measuring spectra]]

2nd Task
Using samples A1, A2, A3, A4 and A5, create a calibration curve and record the data in the protocol (individual concentrations and absorbances, coefficient of the linear equation, coefficient of determination). From the linear regression values for the calibration curves, calculate the molar absorption coefficient ε and report its value and the formula you used. Then measure the concentration of the unknown sample AX. Use the calculated molar absorption coefficient ε and the measured concentration of the sample AX and calculate the corresponding absorbance, giving its value in the protocol and the formula you used. Finally, compare the measured absorbance and the calculated absorbance. Upload the generated pdf file in WinASPECT to the moodle server with the protocol.

Detailed instructions for operating the program when measuring concentration are in the following file.[[Media:Spektrofotomertrie presentace koncetrace EN.pdf|Instructions for measuring concentration]]

<!-- Task 1

Measurement of absorption spectrum: measure, 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 the green check sign OK 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. 	By clicking on an icon To document you will transform results into document form. Save it using your group name.

14. 	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. Determine the 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. Start with the lowest concentration KA1.

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

6. Don't click on the green check sign OK - it cause the measurement termination.

7.	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.

8. 	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.

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

10. 	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 NA)

11. 	After saving the program will automatically open the Concentration window which enables to determine the concentration of unknown samples from a calibration curve. 12. 	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.

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

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

15. 	Save the document as ENXXXQUANTNA.

16. After measurement clean used cuvettes. -->

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 in vivo spectrophotometry. This relatively recent method of examination also presents several possibilities for future developments of spectrophotometry by providing “reliable, objective, and non-invasive” results.