Molecular Biology Lab - 01 Spectrophotometric Analysis Of Carbohydrates

MOLECULAR BIOLOGY AND DIAGNOSTICS LABORATORY • SPECTROPHOTOMETRIC ANALYSIS OF CARBOHYDRATES OUTLINE • • Carbohydrates o Classification of Carbohydrates o Glucose Analytical Methods o Basic Concepts o Spectrophotometry o Beer’s Law o Blanking Technique Wavelength o is the distance between two successive peaks and it is expressed in terms of nanometer (nm). o Different compound absorbs best at diff. wavelength o 400-700 nm = visible spectrum (electromagnetic radiation apectrum) o <400nm = Ultraviolet region (UV) ▪ Spectrophotometer (185-400nm) o >700nm = infrared region (IR) (to 1500nm) CARBOHYDRATES • • • • major constituents of physiologic systems organic compounds composed of carbon, hydrogen, and oxygen Glycoproteins- carbohydrates that are covalently attached to proteins Glycolipids- carbohydrates that are covalently attached to lipids CLASSIFICATION OF CARBOHYDRATES • • • Monosaccharides o water-soluble crystalline compounds o natural monosachharides have either five (pentoses) or six (hexoses) carbon atoms Oligosaccharides o relatively low molecular weight polymers of monosaccharides (< 20) that are covalently bonded through glycosidic linkage Polysaccharides o high molecular weight polymers of monosaccharides (> 20) GLUCOSE • • • Only carbohydrate to be directly used for energy or stored as glycogen It does not accumulate in the muscle It does not enter the muscle cell freely PLANK’S FORMULA • • Electromagnetic radiation is described as photons of energy travelling in waves The relationship between wavelength and energy (E) is described by Planck’s Formula E = hv o E = energy of a photon in Joules or eV o h = constant (6.62x10-34erg sec) o v = frequency WAVELENGTHS Wavelengths (nm) 180-220 220-340 340-430 430-475 475-495 495-505 505-555 555-575 575-600 600-620 620-700 Region Name Short UV UV V V V V V V V V V Color Visible Not visible Not visible Violet Blue Green blue Blue green Green Reverse 1 Reverse 2 Red Red Complementary Color Yellow-green Yellow Orange Red Purple Blue green Blue green FREQUENCY • • • • ANALYTICAL METHODS BASIC CONCEPTS • Energy o Is transmitted via electromagnetic waves that are characterized by their frequency and wavelength Number of vibrations of wave motion per second. The lower the wave frequency, the longer the wavelength. The wavelength is inversely proportional to frequency and energy; the shorter the wavelength, the higher the frequency and energy and vice versa. NOTES TO REMEMBER • • • • A slight error in wavelength adjustments can introduce a significant error in absorbance readings. Wavelength accuracy is the wavelength indicated on the control dial is the actual wavelength of light passed by the monochromator. Didymium (360nm) or holmium oxide filter (600nm) is used to check wavelength accuracy (wavelength calibration) Neutral density filters and dichromate solution verify absorbance accuracy on linearity. SPECTROPHOTOMETRY • • • • Involves measurement of the light transmitted by a solution to determine the concentration of the lightabsorbing substances in the solution. • • A typical photometer or spectrophotometer contains six basic components in a single- or double-beam configuration. These components include a stable source of radiant energy; a filter that isolates a specific region of the electromagnetic spectrum; a sample holder; a radiation detector; a signal processor; and a readout device. • Types of Light Source o Continuum Source o Line Source Continuum Source o Emits radiation that changes intensity; widely used in the laboratory. o Tungsten light bulb - is the commonly used light source in the visible and near IR region. o Deuterium lamp - is routinely used to provide UV radiation in analytic spectrometers o Xenon discharge lamp - produces continuous source of radiation, which covers both the UV and the visible range Line Source o Emits limited radiation and wavelength. o Examples: mercury and sodium vapor lamps in spectrophotometers (UV and visible regions) and the hollow cathode lamp (AAS) o Line source that emit a few discrete lines find wide used in atomic absorption, molecular and fluorescent spectroscopy. o Light Amplification by Stimulated Emission of Radiation (LASER) is also used as light sources for spectrophotometry Factors in Choosing a Light Source o Range o Spectral distribution within the range o The source of radiant production o Stability of the radiant energy o Temperature Alternatives for Tungsten Bulb o Mercury Arc → Visible AND UV o Deuterium Lamp (165NM) → UV o Hhydroge Lamp → UV o Xenon Lamp → UV o Merst Glower → IR o Globar (Silicone Carbide) → IR ENTRANCE SLIT LIGHT SOURCE • • • It provides polychromatic light and must generate sufficient radiant energy or power to measure the analyte of interest. An intense beam of light is directed through the monochromator and the sample. To give accurate absorbance measurements throughout its absorbance range, its response to change in light intensity must be linear. • • • It minimizes unwanted or stray light and prevents the entrance of scattered light into the monochromator system. Stray Light (unwanted light) - refers to any wavelengths outside the band transmitted by the monochromator. It causes absorbance error. It limits the maximum absorbance that spectrophotometer can achieve; is the most common cause of loss of linearity at high-analyte concentration MONOCHROMATOR • • • • It isolates specific or individual wavelength of light. Kinds of Monochromators: o Prisms o Diffraction Gratings o Filters Prisms o Are wedge-shaped pieces of glass, quartz or sodium chloride o It can be rotated, allowing only the desired wavelengths to pass through an exit slit o A narrow light focused on a prism is refracted as it enters the more dense glass. Diffraction Gratings o Are the most commonly used; better resolution than prism. o Made by cutting grooves (parallel grooves) or slits into an aluminized surface of a flat piece of crown glass – wavelength are bent as they pass a sharp corner. EXIT SLIT • • • • CUVET • • • • • Filters o Are simple, least expensive, not precise but useful. o Are made by placing semitransparent silver films on both sides of a dielectric such as Magnesium Fluoride. o It produce monochromatic light based on the principle of constructive interference of waves – light waves enter one side of the filter and are reflected at the second surface. o It usually pass a wide band of radiant energy and have a low transmittance of the selected wavelength o Monochromator that can split/isolate stray light! o Used by PHOTOMETER (FEP) It controls the width of light beam (bandpass) – allows only a narrow fraction of the spectrum to reach the sample cuvette. Bandpass - is the total range of wavelengths transmitted. Accurate absorbance measurement requires a bandpass less than 1/5 the natural bandpass of the spectrophotometer. Spectral purity of the spectrophotometer is reflected by the bandpass, that is, the narrower the bandpass, the greater the resolution. aka absorption cell/ analytical cell/sample cell. Holds the solution whose concentration is to be measured. Kinds of Cuvets o Alumina silica glass - most commonly used (can be used in 350-2000nm, visible region); transmit light effectively at > 220nm o Quartz/plastic - used for measurement of solution requiring visible and UV spectra o Borosilicate glass o Soft glass Notes to Remember: o Cuvets with scratches on their optical surface scatter light and should be discarded. o Alkaline solutions should not be left standing in cuvets for prolonged periods because alkali slowly dissolves glass producing etching o The path lengths of cuvets is 1cm, although much smaller path lengths are used in automated systems. o To increase sensitivity, some cuvets are designed to have path lengths of 10cm, increasing the absorbance for a given solution by a factor of 10 PHOTODETECTOR • • • • It detects and converts transmitted light into photoelectric energy (RESULT) It detects the amount of light that passes through the sample in the cuvet Kinds of Photodetector o Barrier Layer Cell/Photocell/Photovoltaic Cell Barrier Layer Cell/ Photocell/ Photovoltaic Cell o Simplest detector; least expensive; temperature sensitive ;requires no external voltage source o It is used in filter photometers with a wide bandpass. o • It is composed of selenium on a plate of iron covered with transparent layer of silver o This cell typically has maximum sensitivity at about 550nm and the response falls off to about 10% of the maximum at 350 and 750nm Phototube o Contains cathode and anode enclosed in a glass case o It has a photosensitive material that gives off electron when light energy strikes it. o Similar with photocell but requires and external voltage for operation. o Gives off electron when light energy strikes it o The cathode is composed of a material (e.g., rubidium or lithium) that acts as a resistor in the dark but emits electrons when exposed to light. The emitted electrons jump over to the positively charged anode, where they are collected and return through an external, measurable circuit. METER OR READ-OUT DEVICE • • • It displays output of the detection system Examples: galvanometer/ ammeter / Lightemitting diode (LED) display Galvanometer/ammeter o A moving needle on a dial or a digital display which indicates the amount of light passing through a sample. OPTICAL INSTRUMENTS • Photomultiplier Tube (PMT) o It is the most commonly used detector – measures visible and UV regions. o Excellent sensitivity and has a rapid response – detects very low levels of light; amplifies radiant energy (200x sensitive) o The response of the PMT begins when incoming photons strike a photocathode. These tubes are limited to measuring low power radiation because intense light causes irreversible damage to the photoelectric surface. o It should never be exposed to room light because it will burn out • • BEER’S LAW • • • • Photodiode o Not as sensitive as PMT but with excellent linearity. o It measures light at a multitude of wavelengths – detects less amount of light. o It has a lower dynamic range and higher noise compared to PMT. o Most useful as a simultaneous multichannel detector. Instruments that measure light energy Based on the property of colored solutions to absorb light of specific wavelength. It states that the concentration of the unknown substance is directly proportional to the absorbed light (absorbance or optical density) and inversely proportional to the amount of transmitted light/reflected light (% Transmittance) It mathematically establishes the relationship between concentration and absorbance. Concentration of a substance is: o Directly proportional to the light absorbed o Inversely proportional to amount of transmitted light ABSORBANCE • • • • Is the amount of light absorbed. Is proportional to the inverse log of transmittance. It is mathematically derived from %T. Formula for concentration of a substance: A = a b c = 2- log %T = -log T o A =absorbance, o a =absorptivity constant for the substance, o b =length of the light path through the substance o c =concentration of the substance SOLVING FOR THE CONCENTRATION OF THE UNKNOWN SOLUTION PERCENT TRANSMITTANCE • Is the ratio of the radiant energy transmitted (T) divided by the radiant energy incident on the sample (I). BLANKING TECHNIQUE • • • • • Means the blank contains serum but without the reagent to complete the assay. Reagent blank corrects absorbance caused by the color of the reagents – the absorbance of reagents is automatically substracted from each of unknown reading. Sample blank measures absorbance of the sample and reagent in the absence of endproduct, and corrects the measurement for optical interference (like hgb) absorbing the wavelength of measurement. A blanking process may not be effective in some cases of turbidity, and ultracentrifugation may be necessary. To correct for artifactual absorbance readings, “blanking” procedures or dual-wavelength methods may be used