Download Understanding Pulse Oximetry: Absorption Properties of Bromothymol Blue and Blood and more Lecture notes Genetic Engineering in PDF only on Docsity!
Lab Exercise: Light Absorption, Spectrophotometer, and the Pulse Oximeter
Objectives
Determine which characteristics of a medium are important for light absorption. Explain how absorption properties of blood allow for a pulse oximeter to measure oxygen content and the pulse of a patient. Explain why bromothymol blue solution can be used as an analog for blood, based on its light absorption characteristics.
Equipment
Vernier spectrophotometer Distilled water Salt Bromothymol blue solution Beaker Cuvette tissue for cleaning cuvette surfaces
Cuvettes for spectrophotometer Petri dish or clear plastic cup Pipette Simulated pulse oximeter device Vernier light sensors
Introduction
The Medical Field
By the end of this lab you should have a good idea of how a pulse oximeter, a device commonly used in hospitals to measure vital signs of patients, is able to noninvasively measure the change in oxygen content in blood. Due to the difficulties in working with blood, we will instead use bromothymol blue in the lab to make the relevant observations necessary to understand how the pulse oximeter works. Bromothymol blue is a compound that is used as a pH indicator for weak acids and bases. When in a basic solution, Bromothymol blue will appear blue; when it is in a neutral solution it will appear green; and when in an acidic solution it appears yellow. These changes in color can be observed both visibly and with a spectrophotometer. A spectrophotometer is a device that measures the absorbance or percent transmittance of light through a solution. Simply breathing on a solution of Bromothymol blue will introduce carbon dioxide (CO 2 ) from your breath into the solution. The CO 2 reacts with the solution to form carbonic acid (H 2 CO 3 ), which increases the acidity of the solution. As the pH of the solution changes, the color of the solution also changes. In this lab, we will examine the change both visually and with a spectrophotometer, linking observations to the spectral characteristics of deoxygenated and oxygenated blood as measured by a pulse-oximeter.
Light Absorption
The electromagnetic spectrum is composed of wavelengths, which range from short to long. In this lab, we will ignore the wavelength extremes at both ends and instead focus on the wavelengths most familiar to us - visible and near visible light. Human vision is capable of detecting wavelengths of electromagnetic radiation from about 380 nm-750 nm. This is called the visible light range. In this lab, you will also examine light at wavelengths near-infrared, which have a wavelength that is slightly longer than those observed in the visible light range.
When light passes through a substance, some of it is absorbed due to interactions with the atoms and molecules in the medium. This can be quantified by defining the term absorbance ( A ). Absorbance is given by the following equation:
A= log ( I 0 / I 1 ),
where Io is the intensity of the light before it enters the substance and I 1 is the intensity of light after it is transmitted through a substance. Intensity ( I ) is defined as the amount of energy transmitted in a unit area in a unit time, or power ( P ) per area ( As ).
I = P / As.
The absorbance is wavelength dependent and will also vary depending upon the medium (air, water, glass, etc.) through which light passes. The medium can be characterized by the molar absorptivity , ε (in m^2 / mol ), by the concentration, c (in mol / m^3 ) , and by the length of the medium the light passes through, l (in m ). The molar absorptivity has one value for a particular material, e.g., there is one value of ε for salt. Of course, the medium may consist of various substances; for instance, a solution may have multiple solutes each with their own absorption coefficient and concentration. The absorption coefficient and concentration can be combined into a single coefficient, the absorption coefficient α (in m-1 ). The dependence of absorbance on these variables is called Beer’s Law and is given by the following equation:
A= εcl=αl.
The loss in intensity through a medium is known as attenuation.
Figure 1- Absorption due to light passing through a medium
Part One: Using the Spectrophotometer with Varying Concentrations of a Solution
The spectrophotometer measures the amount of visible light absorbed by a solution. To obtain a reading, the spectrophotometer will direct a known amount of light through a cuvette filled with solution and will measure the amount of light that passes through the cuvette.
For this experiment you will compare the relative intensity measured for two different concentrations of saltwater solutions.
Q1: What is the relation between energy of light and its wavelength ( )? What happens to the frequency
as decreases?
PROCEDURE
Saltwater Solution:
Neutral Solution:
Fill cuvette with prepared bromothymol blue solution (it should be in its neutral green phase). Place cuvette in Spectrophotometer. Position cuvette so that the side with a smooth surface is aligned with the ►on the spectrophotometer. COLLECT data. After graph is produced, click STOP .(failure to click STOP will result in loss of data.)
Acidic Solution:
Pour bromothymol blue into a cup and gently blow on it (carefully, so as not to splatter the solution.) Wait 30- 60 seconds to allow time for the reaction to occur and the solution to change color - note that the color change should be distinct. If you do not detect a color change, continue to blow until change is observed. Once the color change has happened, transfer the solution into a cuvette (to avoid spills, transfer solution with a funnel over a sink.) Place cuvette in Spectrophotometer, again positioning cuvette so that the side with a smooth surface is aligned with the ►on the spectrophotometer. COLLECT data. A prompt will ask you if you would like to erase the prior data, choose “Save Latest Run”. This will allow you to analyze both experiments on the same graph.
Print out and save a copy of the absorption graphs. Label, either in LoggerPro or by writing on the graph, which solution is which. You will use the graph again for later questions.
ANALYSIS
Q6: Use the graphs you produced to evaluate which wavelengths of light are most strongly absorbed.
Neutral: ___________
Acidic: ___________
Q7: Compare the results obtained for both the neutral and acidic solutions. Which wavelengths of light in the 500 – 700 nm range are absorbed more in the neutral solution? Which wavelengths are absorbed more in the acidic solution?
Q8: Bromothymol Blue solution was used for this experiment because of its reactive properties. List the ways that Bromothymol Blue can be compared to hemoglobin in the human body.
PART THREE: PULSE OXIMETRY
INTRODUCTION
We all know that if we do not breathe, we die - but how do we know whether or not someone is receiving enough oxygen? Regardless of whether a person’s respiratory rate is normal, the amount of oxygen being delivered throughout the body could be insufficient. For years, health care professionals would subject patients to endless poking and prodding to obtain blood samples from them - a method that was not only uncomfortable, but also ineffective. Health care workers had to wait for the results of the blood test before they were able to assess a patient’s condition, and often by the time they received the results, the patient’s condition had changed.
Many people in the health care industry recognized the need for an easier way to assess a patient’s peripheral blood oxygen saturation levels (SpO2) and in the 1970’s a new and improved form of pulse oximetry was developed. This provided an expedited, more convenient, and less invasive way to measure a patient’s SpO2. The device known as a ‘pulse oximeter’ incorporates a finger probe equipped with a light emitting diode (LED) and photoreceptor to measure the difference in light absorption of oxygenated and deoxygenated (or reduced)
hemoglobin. Pulse oximetry uses a method called photoplethysmography (PPG) in which light and analysis of the properties of absorption provide a reliable determination of SPO2 levels in a person’s blood. PPG’s are a non- invasive, economical way to provide an accurate appraisal of SPO2, as well as a way to measure changes in the volume of blood to provide heart rate information (another common way of obtaining the heart rate is by using an electrocardiogram (EKG) sensor that measures the electrical activity of the heart).
PROCEDURE
Turning the device on:
Locate the ON/OFF switch on the side of the device and switch to the ON position. Initially the display will read “finger out”; gently place your index finger into the finger clamp so that the tip of your finger covers the LED light. Two numbers will display on the screen - one labeled “SPO2” and the other “BPM”; these numbers represent the “spot oxygen saturation” (blood oxygen saturation) level and the number of heart beats per minute (pulse), respectively. (As an energy saving mechanism, the pulse ox screen may go dark at times; to retrieve data simply tap the display.)
EXPERIMENT
Once you have placed the pulse oximeter on your fingertip, remain still for 30 seconds. If pulse readings appear inconsistent or if SPO2 reading is below 90%, try repositioning finger in probe device to obtain a better reading.
Record the data displayed - at rest:
o SPO2 _____________ %
o BPM _____________
Next, use the pulse oximeter and determine how these numbers change when you hold your breath. Try holding your breath for a comfortable amount of time, not exceeding 30 seconds. Record the data displayed - breath held:
o SPO2 _____________ %
o BPM _____________
Next, test how movement affects the results. Wave your arm in the air for 5 - 10 seconds.
Record the data displayed - movement:
o SPO2 _____________ %
o BPM _____________
ANALYSIS
From the varying pulsatile components, the device is able to measure the frequency of the heartbeat, which we call the pulse when measured in beats per minute. You can do this yourself by measuring the number of peaks in the graphical pulsatile component shown on the bottom of the device and the amount of time between these
SPO2 readings vary, on average, from 94 - 100% for a healthy adult. These results are based on numerous clinical studies that analyzed oxygen saturation readings from healthy subjects. Data are integrated to produce a complex algorithm used to effectively measure SPO2 with a pulse oximeter based on the absorption of red and infrared light. These readings may be affected by movement, inadequate blood flow (including effects from shock, cold temperatures, and medications), external light interference, venous pulsation, and nail polish or fake fingernails. Inaccurate readings may also occur if a patient is anemic. Anemia is a decrease in the number of red blood cells or amount of hemoglobin in the blood. These conditions can limit the accuracy of the readings obtained when using a pulse oximeter.
Q11: Why is a pulse-ox device overestimating the amount of oxygen carried by the blood for a patient with anemia?
Q12: What conditions may cause a person to have decreased SpO2 levels?
PART FOUR: SIMULATED PULSE OXIMETRY
In PART ONE of this lab you performed experiments with white light that illustrate how light absorption changes as solution concentration varies. PART TWO of this lab demonstrated how the color of a solution impacts the absorption of different wavelengths of light. We will now consider how absorbance at different wavelengths can be used to give information about a solution.
Figure 2 shows how absorbance varies for oxygenated and reduced hemoglobin. A pulse oximeter measures the absorbance of two wavelengths of light through blood to calculate the oxygen content of hemoglobin based on the absorbance measured for a given person. And now for something completely different: You have just discovered alien life!!! This creature uses not blood, but bromothymol blue to transport CO 2 throughout its body. How can you use what you know about the absorbance spectrum of bromothymol blue to noninvasively measure the CO 2 content of this creature’s circulatory system? In your lab, you have red and green LEDs that you can use to take measurements.
(You will be using a light sensor from Vernier that measures in the unit of lux. One lux is one lumen per meter squared. Ideally measurements would be made in SI units of intensity for light (W/m^2 ). However, due to limitations of the sensor, different wavelengths of light will give different values for the same intensity. This is also true for our eyes as they are more sensitive to some wavelengths than others; in particular, our eyes are most sensitive to green light. For our purposes in this lab, we can safely make the assumption that a higher value of lux will correspond to a higher intensity and therefore we will not need to worry about this detail for the remainder of the lab.)
Q13: Look at the graph you generated for Part Two and the graph in the right of Figure 2. Given a red (~700 nm) and green (~565 nm) LED, would you measure a difference in absorbance for neutral and acidic (CO 2 rich) bromothymol blue solution? How would this be similar to or different from measuring the absorbance of oxygenated or reduced hemoglobin?
There are several complications in pulse oximetry that we will summarize here as a matter of course; some have already been touched upon. In pulse oximetry, absorbance measurements at two wavelengths of light are taken at wavelengths that have significantly different absorption coefficients for deoxygenated hemoglobin (Hb) and oxygenated hemoglobin (HbO). Looking at the ratio of these light intensity measurements using Beer’s Law removes the path length dependence l of the measurements. However, for Pulse Oximetry to be effective and accurate it must also measure the absorbance of the pulsatile component of blood apart from the constants that are always present such as tissue, bone, and non-pulsatile blood. A pulse-ox device is designed to subtract out the absorbance of unchanging tissue components. Additionally, scattering effects of light in body tissues can make readings inaccurate. Scattering effects can be reduced by measuring at multiple wavelengths, where an elaborate scattering calculation must be made. Alternatively, many measurements of different patients can be made so that an empirically derived average scattering effect is determined and removed. To convert from an absorbance
measurement to a value for SPO2, all of these problems must be accounted for in the empirically generated algorithm used in the device or in the functional properties of the device itself.
In the next section of the lab, we will use prior experimental data that relates absorption to a desired variable (pH) similarly to how absorption coefficients are related to oxygen content in human blood. In particular, we will be looking at two different wavelengths of light and show that we can determine the pH of a substance which is itself linked to the amount of C0 2 in the solution.
PROCEDURE
Imagine you have a sample of neutral Bromothymol Blue sampled from a healthy alien and a sample that appears to be more acidic from an unhealthy alien. You want to determine the pH of the circulatory fluid for each creature. To do this, place the cuvette filled with Bromothymol Blue solution in the light shielding part of the simulated pulse oximeter. Connect the Vernier Light Sensor to Channel 1 of a LabPro board connected to the computer. If you still have the previous data from Part Two open, unplug the SpectroVis from the computer and in LoggerPro, under the File menu select New and click on No when prompted to save the spectrometer data. Otherwise, simply open LoggerPro. You will be measuring the amount of light that makes it through the solution with the light sensor.
Make sure that the light detector range switch is set at the middle position, 0-600 lux. Flip switch to turn on the green LED (~565 nm) and shine light through cuvette ensuring that it is properly aligned with sensor. o Click COLLECT o Record data- use the peak value of the intensity Replace the neutral Bromothymol Blue solution with the acidic solution and repeat Repeat procedure by flipping the switch so that the LED emits red light (700 nm) and take measurements for both the neutral and acidic solutions Complete the data table below with the intensity values collected and the calculations listed
Assume that the bromothymol blue intensity measurements you have made represent the pulsatile component of the newly discovered alien’s “blood.” Absorbance has a logarithmic relationship to intensity (see Introduction), so you must take the log of your measurements. You will need to look at the ratio of these values to remove the dependence that absorbance has on path-length l. Finally, you will convert the absorbance ratio to a “blood” pH value that is related to CO 2 content, just as absorbance is used in the pulse oximeter to determine blood oxygen content.
DATA
Green LED, ~ nm
Red LED, ~700 nm
Green LED, ~565 nm
Red LED, ~700 nm Measured Intensity, Neutral Solution (Lux)
Measured Intensity, Acidic Solution (Lux)
Log(Intensity) Log(Intensity)
Ratio for neutral solution, Log(Green)/Log(Red)
Ratio for acidic solution, Log(Green)/Log(Red)
