Spectrophotometer Principles & Practice

• Faculty of Medicine, Helwan National University module is Locomotor system (lcs) 105
• Academic year is 2024-2025, year 1, semester 2
• Principles and practice of Spectrophotometer by Dr. Marwa Ali, Assistant Professor of Medical Biochemistry and Molecular Biology, Ain Shams University, Department of Medical Biochemistry and Molecular Biology

Lecture Objectives: Students will be able to:

• Define clinical chemistry
• Enumerate the importance of clinical chemistry in medicine
• Outline the main steps of laboratory work flow cycle
• Identify the specimen rejection criteria
• Identify photometer and its main parts
• Arrange the steps of using the photometer
• State the principles of photometry and the related laws (Beer-Lambert's laws)

Clinical Chemistry

• Clinical Chemistry is also known as chemical pathology, clinical biochemistry, or medical biochemistry.
• Clinical chemistry definition: the area of a clinical laboratory generally concerned with the biochemical analysis of body fluids for diagnostic and therapeutic purposes.

Clinical Chemistry Tests

• Lipid profile
• Diabetic profile
• Kidney profile
• Liver profile
• Bone profile
• Electrolyte profile

Purpose of Clinical Chemistry Tests

• Measurement of substances levels found normally in human blood that have biological functions, examples: Glucose, Calcium
• Detection or measurement of non-functional metabolites or waste products, examples: Creatinine, Blood Urea Nitrogen (BUN)
• Detection or measurement of substances that indicate cell damage or disease, examples: Liver enzymes such as ALT, Cardiac enzymes such as CK-MB
• Detection or measurement of drugs or toxic substances, examples: Drugs of abuse screen
• Measurement of these biochemical analytes helps to reveal the cause of disease
• Measurement of these biochemical analytes helps to screen diseases for early detection
• Measurement of these biochemical analytes helps to suggest effective treatment
• Measurement of these biochemical analytes helps to assess response to treatment
• Measurement of these biochemical analytes helps to monitor progress of pathological condition

Laboratory Work Flow Cycle Steps

• Order test
• Collect sample
• Transport sample to the lab
• Receive sample in the lab
• Prepare sample for testing
• Transport sample to the lab section

Laboratory Work Flow Cycle: Order Test

• Select test
• Complete order form
• Receive test order
• Deploy staff for collection
• Collect supplies

Laboratory Work Flow Cycle: Collect Sample

• Locate Patient
• Collect Specimen
• Transfer specimen into tube for transport and analysis
• Label Specimen
• Dispose of supplies

Laboratory Work Flow Cycle: Transport Sample to Lab

• Prioritize sample for transport
• Send sample to lab

Laboratory Work Flow Cycle: Receive Sample in Lab

• Accession
• Apply/verify sample label
• Bar code for testing
• Rack samples

Laboratory Work Flow Cycle: Prepare Sample for Testing

• Centrifuge
• Aliquot

Laboratory Work Flow Cycle: Transport Sample to Lab Section

• Send sample to appropriate lab section

Patient Preparation and Instructions

• Certain factors may affect results of certain laboratory tests: food consumption, medication, activity, time of day

Types of Specimens for Chemical Analysis

• Whole blood, serum, or plasma
• Urine
• Others: Cerebrospinal Spinal Fluid (CSF) and other fluids

Specimen Rejection Criteria

• Specimen improperly labeled or unlabeled
• Specimen improperly collected or preserved
• Separated plasma or serum is grossly hemolyzed

Hemolysis of Blood

• Hemolysis is the liberation of hemoglobin from RBCs
• Plasma or serum will be pink or red in color
• Hemolysis causes changes in measurement of a number of analytes: serum K, serum inorganic phosphate, SGOT

Spectrophotometer

• Spectrophotometer Definition: an instrument that can measure the amount of light absorbed by a sample at any selected wavelength.
• Light is a mixture of different electromagnetic wavelengths.
• Light consists of particles (photons) and behaves

Wavelengths

• Different wavelengths lead to different types of light: <380 nm(UV), ~380-750 nm (Visible Light), >750 nm (IR)
• Visible light is only a very small portion of the electromagnetic spectrum.
• More molecules means more light is absorbed, causing color to become "deeper" with increasing concentration.

Absorption and Transmission Laws

• Laws that regulate absorption and transmission of colored solutions: Beer's Law, Lambert's Law, Beer-Lambert Law
• Beer's Law Definition: the absorbance of light by a solution is directly proportional to its concentration
• Lambert's Law Definition: when light passes through a solution, the intensity of light transmitted decreases exponentially with increasing path length
• A = absorbance, ε = extinction coefficient, c = concentration, L= path length of absorbing solution

Photometer Construction

• Components: light source, slit, photo cells, wave length selector, sample tube (cuvette), meter

Light Sources

• UV Spectrophotometer: Hydrogen Deuterium Gas Lamp, Mercury Lamp.
• Visible Spectrophotometer: Tungsten Lamp.

Monochromator

• Each colored solution requires a complementary color to be measured maximally by the photometer
• This can be obtained by a monochromator (prism) or by choosing the proper

Slit

• It is necessary to adjust the intensity of the incident light (Io)

Sample Cuvettes

• UV Spectrophotometer: Quartz (crystalline silica)
• Visible Spectrophotometer: Glass-plastic

Detecting Transmitted Light

• Spectrophotometers detect the transmitted light (I)

Digital Display of Meter

• Displays the reading of absorbance

Blank Solution

• The blank solution is similar to the sample in everything except the measured compound is absent from the solvent.
• It is used to adjust the zero of the spectrophotometer and eliminate any interference

Steps in Developing a Spectrophotometer IC Analytical Method

• The instrument must be warmed for 15 min
• Set a monochromatic wavelength for the maximum absorption wavelength
• Wipe the tube containing the blank solution with a lab wipe
• Place it into sample holder and close the cover
• Set zero absorbance
• Remove blank tube, wipe off the cuvette
• Read and record the absorbance
• Calculate the concentration of the sample using Beer Lambert Equation

Spectrophotometer Applications

• Spectrophotometer applications include Qualitative & Quantitative analysis: compare the absorbance of the test solution to that of the standard solution of the same wave length

Qualitative Spectrophotometric Assays

• An absorption spectrum can be generated by measuring the absorbance of the pure compound in solution at a variety of wavelengths.
• Heme proteins or Cytochromes show a distinct absorbance in the visible range from 500-600 nm
• The nitrogenous bases in nucleic acids are known to absorb maximally at 260 nm
• The aromatic rings on tryptophan and tyrosine are known to absorb strongly at 280 nm

Quantitative Spectrophotometric Assays

• Comparative Method: used with reference (blank), a test and a standard = known concentration

Beer-Lambert Law

• A test / A standard = εC test L / εC standard L = C test/ C standard

Standard Curve

• A type of graph used as a quantitative method
• To create a standard curve, determine the absorbance of multiple samples (standards with similar properties) and plot them on the Y-axis against their concentrations on the X-axis
• The same assay is then performed with samples of unknown concentration
• To determine the concentration of a substance in a sample, its absorbance is located on the Y-axis, and a line is followed to intersect the standard curve. The corresponding value on the X-axis is the concentration of the substance in the sample

According to the Beer-Lambert Law, Absorbance is dependent on:

• Distance traveled by the light through the sample
• Extinction coefficient of the sample
• Solution concentration

Spectral Absorption Curve

• A graph of a chemical relating the absorbance to wavelength