Non Invasive And Invitro Haemoglobin Measurement Biology Essay


A method and apparatus for non invasively measurement of haemoglobin concentration, where we have to select two wavelengths whose haemoglobin extinction coefficient is higher than that of water. The wavelengths which we using here will be an isobestic wavelengths where the absorption of the oxyhaemoglobin and the deoxyhaemoglobin will be the same .Then the incident light beams are made to fall over the predetermined site of the body and received at the other end using a photo detector. The received signal is converted to electric signals. By calculating the attenuation variation caused between the selected wavelengths we will be able to find the haemoglobin concentration in the blood. The isobestic wavelengths which we selected here is 590nm and 800nm.

Haemoglobin, the main component of the red blood cells for transporting oxygen from the lungs to the capillaries which is involved in the respiratory process. Haemoglobin, active carrier of oxygen is a compound of iron and four polypeptide chains. Each chain linked to one atom of iron, which carries four molecules of oxygen. By detecting the levels of haemoglobin in the blood is important in the medical field in order to diagnose and treat the patient.

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Several methods and devices had been developed to measure the haemoglobin concentration in the blood and all methods have some disadvantages. The present invention will solves some of the problems of the haemoglobin concentration measuring equipments. This technique uses the isobestic wavelengths .The wavelengths which we selected is used in the earlier days in order to make correctness in the haemoglobin concentration. This isobestic wavelengths are radiated to the predetermined site and received at the other end ,where the radiated lights are converted to the electric signals .By taking the attenuation variation caused by the two light sources we can find the haemoglobin concentration. Oxygen saturation can also be calculated using this isobestic wavelength.


The aim of the project is to develop a non invasive measurement technique (PULSE OXIMETER) which gives the haemoglobin concentration in the blood with reduced errors and to implement a faster and time saving technique which will help the practicians for better identification of blood related diseased conditions.


Haemoglobin, the oxygen carrying protein was first discovered by Hunefeld in 1840.In 1851,Otto funke described the growth of haemoglobin crystals by diluting the red blood cells with the pure water, alcohol or ether. Electrophoresis method had been developed for measuring the short time haemoglobin measurement, which is used as a diagnostic indicator of diabetes mellitus .This invention which provides the haemoglobin measurement using a migration path which have inner surface coated with a cationic substance is to be immobilized in the inner surface and using a buffer solution which contains a polymer having an ionic group (water soluble) as an electrophoresis buffer solution. The main disadvantage of this techniques is that the blood sample is needed and time required to obtain the result is higher.

Then later there was a development in the haemoglobin measurement by the Total Internal Reflection (TIR), which will help to determine the haemoglobin concentration in the whole blood. But the disadvantage of this method is that the light transmitted will affect the other coloured particles in the blood .

In the blood stream, the glucose is attached to the haemoglobin which is called as glycosylated haemoglobin (HbA1c).There are also other forms of haemoglobin in the blood. The first person who separated the glycosylated haemoglobin from the other forms of haemoglobin is Huisman and Meyering in 1958.By this separated glycosylated haemoglobin, Anthony Cerami,Ronald Koenig and co worker(in 1976) proposed that this HbA1c is helpful in monitoring the degree of control of glucose metabolism in diabetic patients .


In 1935 by Matthes .who developed the first pulse oximeter which uses two wavelengths of light for optical path length, but the device was said to be cumbersome.In 1942 Millikan developed ear oximeter for some military purposes and in 1949 Wood,added inflatable bladder in order to find the bloodless zero measurement.

In 1962 ,Shaw developed a eight wavelength ear oximeter for the identification of multiple species of dyshaemoglobin, but discarded due to low commercial success.

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In 1974 Aoyagi developed first pulse oximeter by isolating the pulsating component of ear oximeter, but some other companies had been introduced a device with fibre optic sensor which have a great value in the market.

From 1981 there was an major development in the field of pulse oximeter by Nellcor in which he designed two competing design with the help of Biox. They made some modifications from the earlier design of Aoyagi .In 1987, Nellcor introduced a pulse oximeter which enhances the signal quality under certain conditions of patient motion and a low amplitude peripheral pulse .In this he designed a N-200 pulse oximeter with C lock.

In 1996 ,Masimo made the recent changes in the pulse oximeter by introducing Signal Extraction technologies .And it is expected that there will be further development in the signal processing power conservation and optical sensor technologies will push the pulse oximeter to the new applications.


Adapted from:

In the normal pulse oximeter we are taking two wavelengths; one at 660 nm ( Red light ) and 940 nm ( Infrared light ).From this figure we can know that at this point there will be more absorption and we are able to get the better result. The absorption of oxyheamoglobin and deoxyheamoglobin at the two different wavelengths will be different and by taking the ratio in the difference of the absorption of oxyhaemoglobin and deoxyhaemoglobin we will be able to find out the haemoglobin concentration. The oxyhaemoglobin(Oxygen carrying haemoglobin) absorbs more infrared light than the red light, where as the deoxyhaemoglobin(Non oxygen carrying haemoglobin) blood absorbs more red light .

But there will be certain factors which are affecting the readings in the pulse oximeter like motion artifacts,Venous pulsations,poor perfusion . . . etc. In this venous pulsation is occurred during tricuspid valve failure which results in low saturation readings. Low perfusion is also a major problem in the normal pulse oximeters. In this case there will be not sufficient signal at the detector .This results in insufficient signal at the instrument to make a measurement, which will make the instrument to search for the pulse again.

So inorder to eliminate these errors to an extent we have to look at certain wavelengths which will gives the reading regardless of saturation and to produce sufficient signal at the detector inorder to make the sufficient signal to reach the instrument to make a measurement


The principle of operation for calculating the procedure for measuring the haemoglobin concentration in pulse oximeter is mainly based on the Beer's-Lambert's law

The Beer-Lambert's law is defined as the ratio of oxyhaemoglobin(HbO2) to the total concentration in the blood(HbO2 + deoxyheamoglobin).The measuring of the absorption spectra of HbO2 and Hb is given by the difference in the wavelength of the light.

The equation which calculates the ratio of the light intensity is defined as

adapted from:Pulse oximetry

Yun-Thai li

Here the DC and AC are the pulsatile signals,where the DC component will be a constant value and the AC Component will be the fluctuating value which represents the pumping of blood in the arteries.

R= Ratio of light intensity


and =wavelength of the two light sources.


In this project, the light radiation unit that we are using is to transmit the light radiation through the body parts that includes the blood vessels ,where we are able to find out the haemoglobin concentration .The wavelength which we are going to use is the isobestic wavelength.

According to the chemical kinetics, the isobestic points which is used as a reference points to study the reaction rates .In the pulse oximeter, the isobestic wavelength is used to determine the haemoglobin concentration .This isobestic points have a great value in medicine as a laboratory technique. Because it helps to give a clear picture to determine the oxygen saturation and haemoglobin saturation regardless of its saturation rate. Isobestic points in the pulse oximetry is defined as the point at which the oxyheamoglobin and deoxyheamoglobin have the same absorption value at a particular point. The isobestic points is used earlier inorder to make haemoglobin correction in the pulse oximeters. The isobestic points is used as a quality assurance method in spectrophotometer. There are certain standards used in spectrophotometer includes potassium dichromate,bromothymol blue. . .etc

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Adapted from : http:// ~bme517/ supporting%20documents/ PulseOximeter/Pulse%20Oxi%20Meter%20Laboratory.html

From the above figure we can know that there are certain points which can be referred as the isobestic point. Here we are using isobestic wavelength of 590 nm and 800 nm. Because at this point the oxyhaemoglobin and deoxyhaemoglobin absorption will be the same. The wavelengths of the isobestic points does not depend on the concentration and the saturation. so that we can use this isobestic wavelengths as a very reliable reference.

This wavelengths used in the pulse oximeter is totally different from the isobestic wavelength,because the wavelengths used in the normal pulse oximeter is at 660 nm( red light ) and the 940 nm ( Infrared light) ,which will give the best results .The evidence for that is the difference between the oxyhaemoglobin and deoxyhaemoglobin wave is at a proper place where we will get the difference between the two waves at the respective wavelength.

But when we using the isobestic wavelength we will not get the difference from the oxyhaemoglobin and deoxyhaemoglobin wave in the above figure. So we are using a different technique that measures the haemoglobin concentration by passing the light through the predetermined site of the body and receives at the other end by the receiver .Then we will calculate how much light intensity we had received at the other receiver side and calculate the amount of substance absorbed at the particular point by looking at the light intensity received at the other end. This technique we will use at the two isobestic points and get the difference for the oxyhaemoglobin and deoxyhaemoglobin.

For example, red light absorbs more deoxygenated blood, if we are radiating a light of wavelength about 590 nm through the predetermined site of the body. Some substances will absorb the light and there will be a change in the intensity of the light received at the other end. By measuring the difference in the light intensity we can calculate absorbance of the deoxygenated cells in that area and similarly in the infrared region. Thus we can get the haemoglobin concentration even regardless of the saturation rate. Here we can use a light which comes under the wavelength of 590 nm, which we will prefer as yellow light and at the range of 800 nm, which is given in the infrared region, so infrared light had been used.


Adapted from: HYPERLINK ""


Wavelength -590nm

The light which we use in this project is yellow light because we are using the wavelength of 590 nm. The yellow light we use here is instead of the red light in order to measure the amount of deoxygenated blood at the isobestic point. The deoxyhaemoglobin is referred as the non oxygen carrying haemoglobin.

If we assume that the all haemoglobin molecules are bonded with O2, then we can say that the haemoglobin is said to be fully saturated .While circulating the blood in the capillaries and to veins the haemoglobin release the oxygen molecules and as a result the saturation level decreases. The normal saturation rate level is said to be between 85% and 97%.

Hence by using the wavelength of 590nm we can calculate the amount of deoxygenated blood from the haemoglobin by measuring the intensity of light at the other end.


The isobestic wavelength which we selected at the infrared region is at 800 nm. The oxygenated haemoglobin will allows the yellow light to pass through it and absorbs more infrared light, where as the deoxygenated blood allows more infrared light to pass through it and absorbs more yellow light.

Usually the predetermined site of the body which we select is the finger or toe, which is placed between the source ( LED ) and the receiver ( Photodiode).Once the absorption levels is detected then the ratio of absorption for each wavelength can be obtained. Thus the Infrared light is used to measure the amount of oxygenated haemoglobin present in the blood.


This project consists of three sections. One the detection unit, processing unit and the display unit. The detection unit consist of Photo diode, amplifier, filters and ADC, where as in the Processing unit we have a ratio calculation unit in order to achieve haemoglobin concentration. Some Microcontrollers have inbuilt ADC. This processing unit is software part using micro controller .The third one display unit is used to display the output result. The display unit can be a computer, TV or an audible device.













The light radiation unit which is used to radiate the wavelengths to a predetermined site of the body .Here we are using yellow and infrared light sources as the light radiation units. In this project we are going to use two types of light detection unit. One unit is to measure the yellow light and the other one is to measure the infrared light.LDR ( Light Depenedent Resistor ) is used to measure yellow light and photo detector is used to measure Infrared light.LDR otherwise called as photoconductor or photocell, whose resistance varies according to the amount of light falling on it, where as the photo detector( used to detect the amount of light received ) is called as photo sensors or photo detectors ,which senses the light. There are several varieties of photo detector.


The amplifier, Filter circuit consist of the signal conditioning circuit.


Here we use operational amplifier (741) which is used to amplify the electric signal from the detector unit, which is a general purpose single operational amplifier. These are some of the features of amplifier must have;

Large input voltage range

High gain

Short circuit protection


Suming Amplifier, Voltage Follower, Integrator, Active filter Function generator

This UA741 is a high performance monolithic operational amplifier constructed on a single silicon chip.


Here we are using a Low Pass Filter in order to avoid the high frequency noise signals. A low pass filter is a filter that passes low frequency electric signals but reduces the amplitude of the signal with frequencies greater than the cut off frequency. The low pass filter is actually the opposite of the high pass filter

The filter which we using in this project is RC low pass filter. In a low pass RC filter ,high frequencies contained in the input signal are attenuated. The circuit which uses resistor and capacitor in parallel works as the RC low pass filter.


The micro controller is used as the processing unit .In some of the microcontrollers there will be inbuilt ADC .Analogue to Digital converter is required because to convert the analogue signals from the filter into the digital form and fed it into the micro controller.


Display unit is used to display the measured values of haemoglobin concentration which can be TV, Computer, 7 segment display or an audible device to hear the reading.



In this we are selecting two isobestic wavelength, where the absorption of the oxyhaemoglobin and the deoxyheamoglobin has the same absorption rate .After selecting the isobestic wavelengths we are transmitting the selected wavelength into the predetermined site of the body .i.e., finger or toe. The light which we transmitting is from the light radiation unit. The light radiation unit can be implemented by LED. The photo detector unit receives the light which is transmitted through the finger, then the received signals are then converted into the electrical signals with the help of photo diode, which can be either Silicon or germanium.

Then the output from this photodiode is given to the variable calculation unit for calculating the light attenuation variation for each wavelength. The main function of the variation calculation unit is to calculate the light attenuation variation for each wavelength using the electrical which we received at the photo detector unit. From this variation calculation unit it is fed to the ratio calculation unit. The variation calculation unit calculated the light attenuation variation for each wavelength as the ratio of the time variant component ( AC ) to the time invariant component ( DC ).

The instrument further requires the hardware section as amplifier, LPF and an ADC between the photo detection unit and the variation calculation unit. The function of the amplifier is to amplify the electrical signals from the photo diode. Then the Low Pass Filter is used to pass the low frequency signals and to block the high frequency signals and noise. We had selected the Low Pass Filter because of the signals from the body will be of low frequency.The low frequency component of the particular electrical signal is amplified by the amplifier and fed to the ADC. The function of the ADC is to convert the analogue signal to the digital signal. The Digital signal after being converted by the ADC is then fed up to the variation calculation unit which in practice can be performed by the microcontroller. The ratio calculation unit calculates the ratio between the light attenuation variation for the wavelengths which we had given to the ratio calculation unit and then feds to the concentration calculation unit .The concentration calculation unit calculates the haemoglobin concentration and gives the output to the display unit ,where we can see the output result.

Radiate light beams through the predetermined site of the body

Select two isobestic wavelength




Calculate the haemoglobin concentration

Calculate the ratio of the light attenuation variation between the wavelengths

Convert light beams into electrical signal

Receive the transmitted light

Obtain the light attenuation variation for each wavelength.

Calculate the ratio of light attenuation variation between the wavelengths

Calculate the haemoglobin concentration



Select two wavelengths which shows more absorbance of oxyhaemoglobin and deoxyhaemoglobin concentration.

Radiate these light beams into the predetermined site of the body i.e., finger or toe.

Then the receiver located at the other end of the body will received the transmitted signal.

Converting the light beams into the electrical signals.

Obtain the light attenuation variation for each wavelength.

Calculate the ratio of light attenuation variation between the wavelengths.

Calculate the haemoglobin concentration.

Display the result on the display unit.













Transmitter transmits the light through the predetermined site .The two type of transmitters used here is yellow light transmitter and Infrared light transmitter


The driver which we used here, is an electrical circuit which is used to control the current flowing in this circuit or to control other devices in the circuit. For each Led we have to driver in order to control the LED's


The light transmitted through the body is received at the other part of the body using a receiver .Two receivers are used; Yellow light receiver and Infrared light receiver.


Usually we employ the Low Pass Filter in order to eliminate the high frequency noise signals. The signals from the body will be small .That is the reason we are eliminating the High frequencies signals. The filter is used to eliminate the noise as well. From the filters the signals are converted into the electrical signals and fed to the microcontroller and then displayed the haemoglobin concentration of the patient.







The transformer which we used here is a step down transformer.The bridge circuit is a diode bridge circuit which uses 4 Diodes. Two of them will be on at during the positive cycle and the other two will be on at the negative half cycle. The capacitor which we use here is a big value. Because the output from the bridge circuit will not be a perfect DC. So in order to make it as a perfect DC we need a capacitor with high capacitor value. The Voltage regulator is a electrical which got three pins as input, Ground and output. This voltage regulator is mainly used to maintain a constant voltage across the output. There are certain voltage regulators in the market such as 7505,7508-which gives +5 and +8 voltage respectively.


This is the circuit diagram which we designed for the infrared and yellow light .But we didn't find the values of the capacitors and the resistors in the filter circuit.

In this above figure ,we had given a signal generator instead of the photo diode. This had been done in order to check whether we will get the correct output for a specified value of the resistance and capacitor. We had given the voltages ±5V to for the op amps. (just for verification about the output )

There are actually two parts in this circuit. One part is the amplifier and filtering part .The other part is differential amplifier. The amplifier will amplifies the electrical signal and send it to the low pass filter to eliminate the high frequency signals. In this circuit the resistor and capacitor are given in parallel in order to obtain as a low pass filter .Same as like in the case of U7 operational amplifier

The output from this low pass filter is fed into the differential amplifier in order to find the actual difference. Here all the resistors will be of equal value .From this differential amplifier it is fed into the micro controller .We had discussed this for Infrared light. Yellow light receiver will have a circuit design like this but with different resistor values for the resistor and capacitor in the Low pass filter circuit. The amplification factor which we had for this amplifier and low pass circuitry is 1 and the gain for this we assumed as 1.This circuit is designed as such as above a certain values there should be no output for the amplier. That is we are determining a cut off frequency at a particular point and above that point ther e should be no output for the circuit. The output from the amplifier and Low pass circuit is giving to input of the differential amplifier which will act as a 2 stage coupling resistor.

The gain is given by the equation =R*2Ï€fc

And the cut off frequency is given by fc=1/2Ï€Rc


The wavelengths had been selected for the yellow and infrared light in the range which we will get a result with least errors and for detection of medical related diseases. Designed the block diagram and circuit diagrams with the help of some books and references.


The current work am doing is designing the values for the capacitor and resistor in the amplifier circuit. For this am using LT SPICE and designing the RC low pass filter by giving a specific values for the resistors and capacitors.


At some point having little confusion about using the LT SPICE. Designing in the RC circuit causes little problems .And the main problem am facing as this project has not done by anyone, there is not much references and evidences available. So am giving my idea about this project and approximate outcome will be


After assigning the values for resistor and capacitor we just want to do the practical part. The Microcontroller and the LCD display can be given as the computer. So we just want to assign the value for each resistor and capacitors and do the practical part .


Thus studied more about the project and searched about the certain topics which help me to do this project. Designed the amplifier circuitry and come to know idea about the designing of amplifier circuit and filter. Studied more about the application of op amps.