Types of Instrumentation: 1. 2. Simple Typeex. Visual colorimeters like Sahli Hemoglobimeter and Dubosq Colorimeter Complex Typeex.

ISE (Ion Selective Electrophoresis) LASER (Light Amplification by Stimulated Emission of Radiation), auto-analyzers, liquid chromatography etc. ) PRINCIPLE: PHOTOMETRY PHOTOMETRYThe measurement of radiant energy absorbed or given off by a molecule colored solution being measured after illuminated by light. - remains the technique most used today in Clinical Chemistry. ELECTROMAGNETIC RADIATION (EMR)- radiant energy which includes short gamma rays to long wavelengths radio waves - These are photons of energy travelling in a wavelike manner. -The shorter the wavelength, the higher the EMR energy. TYPES OF EMR: cosmic rays, gamma rays, x-rays, UV rays, infrared, microwaves (radio, tv, radar) Class of Clinical Chem. Instrumentation that measures EMR: 1. Spectrophotometry 2. Emission Flame Photometry/ Filter Photometry 3. Atomic Absorption Spectrophotometry 4. Fluorometry 5. Turbidity & Nephelometry LIGHT THEORY/ THEORY OF LIGHT WAVESLight is a photon (E) or discrete packet of energy traveling in waves. - has a peak, crest and through. WAVELENGHT OF LIGHTdistance between two successive peaks (crests) Which gives light its characteristic color (if the wavelength is in the visible region).

1. Nanometer (nm) 10-9m; SI unit 2. Millimicron (mu) 1nm 3. Angstrom () 10-10m, 1nm=10 Kinds: 1. visible spectra 340-700nm 2. invisible spectra UV & IR Amplitude distance bet peak & trough; defines a light wave.

- Light will be seen on the right. PRINCIPLE: The measurement of the amount of light absorbed by the solution and relating that absorption to the solution concentration are DIRECTLY PROPORTIONATE.. Beers law- Absorbance (A) or optical density of the colored solution is equal to the product of the concentration of the color-producing substance (c) times the depth of solution through which the light travels (L) times the constant (K) - A=CxLxK - A & K are constant, C & L must vary inversely As= Cs x Ls x Ks Au= Cu x Lu x Ku Cs x Ls Cu= ---------------Lu Cu = AuCs/As Cu= concentration of the unknown Cs= concentration of the standard Ls= depth of the solution through which light travels at the left hand cup Lu= depth of the solution through which light travels at the right hand cup. Raise the right hand cup as far as necessary to make the two halves of the field as one or of the same intensity of color until each half of the field will be seen as one.

Colorimetry term associated with spectrophotometer. 2 considerations (1) Quality of Light (2) Intensity of Light The darker the color, the HIGHER the concentration. If visible light gives a colored solution, part of that light is absorbed (invisible light), and part is transmitted (complementary color). TYPES OF SPECTROPHOTOMETER 1. Visual Colorimeter Uses the eye in determining the end point. Very crude and subjective very crude & subjective intensity of color must be proportionate to the concentration of the color-producing substance simplest colorimetric analysis using a simple device (comparison of unknown w/ standard) ex: Dubosq colorimeter, Sahlis hemoglobinometer

-- Dubosq Colorimeter- refined type of colorimeter PARTS: 1. light source 2. 2 glass-bottomed cups 3. 2 movable platforms 4. 2 glass plungers 5. optical system 6. 2 identical scales - reading: 20 at platform scales for both cups - left cup: standard sol.

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PHOTOELECTRIC COLORIMETRY 1. Spectrophotometry-- measures light E in a much narrower wavelength using a device (prism/grating) to disperse the source of light into a continuous spectrum - Quantitation of substance is accomplished by measuring amt of light absorbed after app. Treatment - Therefore, it gives a relatively high sensitivity, greater ease of rapid measurement compared to visual - High degree of specificity = proper rgts = diff colors (analytical separation prior to color formation reactions) Advantages: (1) High degree of sensitivity and specificity (2) Eliminates the element of subjectivity inherent in visual colorimeter (3)Short TAT (Turn-around-time) - 2. Filter Solution-- measures light intensity of multiple wavelength - uses filter to isolate part of spectrum

- Bandpass/ bandwidth: range of wavelength allowed to pass by monochromator - Major effect of stray light: Beers law wont be followed = absorbance error - Highest absorbance: 2 - A: R-L (above); %T: L-R (below) Causes: Reflection: scratches on optical surfaces (cuvet) Dust particles in light path Reflection from w/in instrument Extraneous room light High colored spectrons (diffraction gratings) Check: Cut-off filters eliminate all radiation at wavelength beyond one of interest Liquid chemicals (calibrating sol) absorb short wavelength - Nickel sulfate, Sodium nitrite, Acetone Black object - block

b. Grating has grooves (3000+) cut into such an angle that each groove behaves like a small prism - light is reflected - white light (various color component) bent as they pass a sharp corner - linear spectrum; provides higher line wavelength resolution than is possible w/ a prism & respond to all wavelengths - more clearly defined spectral separation -- once selected, light exits through exit slits and hits the cuvette with solution. 4. CUVETTE/ CUVET A.k.a. analytical cell, sample holder, absorption cell, optical cell - used to hold the solution in the instrument when absorption is to be measured - made of soft borosilicate glass, quartz or plastic borosilicate: strongly alkaline soft glass: acidic soln; dont etch glass quartz/plastic: more expensive, dont absorb UV radiation, for wavelength below 320nm Types: a. round b. square plane parallel optical surfaces & constant light path - less error form lens effect & refraction prevents light more likely to occur in round Common errors in handling a cuvet: 1. failure to position the cell properly in the SPM = Most cuvet are manufactured with some type of etch or frosted marking near the top. This is used to guide to align the cuvet in the SPM so that it is facing towards the user. = All cuvets should be placed in the SPM in the same manner, and always positioned in a predetermined manner. 2. failure to match absorbance reading of the cuvet

MAJOR EFFECT OF STRAY LIGHT Beers law will not be followednonlinearityabsorbance errorsignificant error that maybe produced especially in the higher absorbance range. Parts of Spectrophotometer 1. Light Sourceprovides radiant energy in the form of visible/invisible light pass through the monochromator to be separated into different wavelengths. -- Light of proper wavelengths will be made incident of the cuvette holding the solution whose absorption is to be measured. Types :Tungsten (320-700nm), Deuterium Lamp (<380nm), Hydrogen Lamp (<380nm) - 2. Entrance Slit -- minimizes stray light from light source - prevents scattered light from enetering monochromator - Stray light: any wavelength outside band transmitted & selected by monochromator 3. Monochromator greek word which means single color --aka wavelength selector which isolates specific wavelength of light by use of prisms or gratings or both. --a misnomer since most MCR transmit a range of wavelength around the specified (nominal) wavelength. Types: a. prism triangular wedge-shaped piece of glass, quartz, NaCl, KBr allows transmission of light - disperses white light into a continuous color based on variation of refractive index of the diff wavelength - short: refracted more - red end of spectrum: refracted least - B or V end: refracted most

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3. Scratches, dirt and fingerprints in the cuvet Resolve by: using blank reading: taken to measure tolerance of each cuvet at each wavelength used (OA or 100%T) - should be used for each determination - primary purpose: read out absorbance dute to the reagent - treated similarly w/ specimens - Reference sol: electrical readout of the instrument is arbitrarily 100% T Distilled H2O 0.000 A (Rgt stability) Reagent blank compensate for any unwanted light absorption due to other material/ interference in the reacton - checks reagent for determination: high RPG = Rgt deterioration - good inexpensive QC procedure sample added to mixture containing all componenyts of the reaction except the rgt w/c reacts to form final colored product - absorbance is measured & subtracted from entire reaction system (accuracy) - dictated by parameters of assay & condition of sample check for scratches, dirt & fingerprints: - scratches rubber/plastic coated test tubes (prevent stains); use mild detergent & dont use TT brush - dirt & fingerprints conc HCl, H2O, ethyl (1:3:4) : wipe dry (lintless tissue paper/ gauze) : rinsed several times in test sol. Before reading absorbance ** to maintain constant L, diameter of cuvet will dictate the sample of light path, represented by the symbol b (equivalent to L in Duboscq formula). 6. Detector/ Photodetector - electron tube amplifying current that convert radiant energy to equivalent amt of electrical pr photoelectric energy TYPES: - Barrier-layer cells (less expensive) - Photoemission tube or phototube - Photomultiplier

7. METER/DATA READ OUT DEVICE -- simplest method of displaying results of detection system. -- it includes read out devices: meters, digital displays, printed read-outs, recorders.

-- When the transmitted light hit the cuvet, an electric signal is generated and going through the detector system to be read out (seen as moving needle on a dial/digital display) to indicate the amount of light passing through the sample. -- a and b are reciprocally related. A= 2-log%T inverse relationship between absorbance and %T

Beers Law (Beer-Lamberts or Beer-Bouguers law) - mathematical basis of colorimetry - conc. Of a substance is directly proportional to the amt of light absorbed or inversely proportional to the logarithm of transmitted light A = abc where a and b are constant A = 2-log%T A = absorbance a = absorptivity b = length path of the sol. In cm c = concentration of the substance of interest/ cons. Of absorbed molecule %T = % transmission Transmittance ratio of transmitted light to incident light Absorbance optical density - amt of light absorbed/blocked by a sol. - Difference between amt of incident light & the transmitted light = conc Incident light amt of light entering sol. Transmitted light amt. of light passing through and is absorbed by sol Absorptivity -- standard and x samples in quantitative lab analysis are of the same kind of molecule and the wavelength of the incident beam is held constant thus A is constant. -- The sample path length is also held constant by using a standard size cuvet. -- The relationship between concentration and absorbance is linear BEERS LAW IS OBEYED. -- For this system, a standard solution of known concentration may be run and the absorbance of unknown (Au) and standard (Astd) are measured in the spectrophotometer. BLANK --reference solution that will set the machine absorbance at 100%T -- should be used for each determination. -- PRIMARY PURPOSE: Read out absorbance due to the reagent. -- a reference solution when electrical readout of the instrument is arbitrarily 100%T. Types of blank: 1. Distilled Water Blank100%T, 0.000 absorbance, suggest a reagents stability. 2. Reagent blankcompensate for any unwanted light absorption due to other material/interferences with reaction. -- checks reagents for deterioration, thus high reagent blanks value (absorbance reading) denotes reagent deterioration -- produce chromogenic reaction.

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3. Sample Blank -- contains all the reagent except the chromogenic substance. sample is added to a mixture containing all the components of the reaction except the reagent which reacts to form the final colored product. -- absorbance of this measure can be measured and subtracted from the absorbance of the entire reaction to obtain a more accurate reading.

- Std measurement should result in A readings falling approx bet 0.100-0.900 - >.900 = increase relative error associated w/ measurement Cu = AuCs/As 2. Standard curve/ Standard graph/ Calibration curve - if absorptivity is not known, A of analayte being measured must be compared to the absorbances of at least 3 diff known conc. Of subs (STDS) - if A is plotted against the conc. Of these subs (STD), result is a graph (STD curve) - Standard curve: plotting the conc. Of the STDs on the x axis (abscissa) against their respective A readings in the Y axis (ordinate) - Since A C = Straight Line Calibrated curve - Performed before running analysis of unknown STD curve glucose - Intercept at O to show proportionality (semilog: universly proportional) - Linearity is demonstrated & Beers Law is followed & analysis adheres to BL - Higher absorbance readings deviating from being lineart, samples would be diluted & reassayed. New value: multiplied by the dilution factor (reciprocal of dilution) to correct the conc. - Indicator of how low one can measure accurately - Ultimated the need to run known materials/STD for standardization In the semi-log paper x- axis= concentration y-axis= %T -- shows inverse relationship.. ; line intersect at 100 %T -- it curves in an ordinary linear paper Determine the concentration of the unknown by means of interpolation of the absorbance / %T reading of the unknown. The graph can only determine as low as 20 mg/dl and as high as 100 mg/dl PROBLEM: If the concentration is quite high (more than 100 mg/dl), what should be done to make a graph? - DO NOT EXTEND THE LINE make a dilution of the sample, simply reduce the original volume used and repeat the assay. 2 ways of making dilutions: 1. Reduce original sample size Ex. Initial reading sample is 0.1 (decrease volume by using 0.05)leads to 1:2 dilution once respective value is obtained (by interpolation), multiply that value by the dilution factor which is 2. 2. Actual dilution by diluents -- distilled water or specific diluents like NSS -- These may add interfering factors to the whole system. REQUIREMENTS FOR FOLLOWING THE BEERS LAW 1. Calibration curvea straight line constituting an exact calibration 2. The slope of all indicate the sensitivity of the procedure. This is seen when procedure follows the Beers law. 3. Sensitivity then will be a constant factor 4. Cu values can be calculated. K= C (concentration)/A (absorbance) 3. Molar Absorptivity Values --defined as absorptivity when b or the diameter is 1 cm and the concentration is in moles/L --The value is constant ofr a given compound and solvent at a specified wavelength. -- uses: (1) To establish purity of substance (2) compare sensitivity of the methods (3)calculate concentration directly from the values.

Absorbance reading, A RDGthe use of the sample blank is dictated by the parameter of the assay and condition of the sample. -- 0.100 to 0.900 if it exceeds deterioration of the sample. Calaculations of conc. From absorbance Measurement 1. Ratio of standard to unknown (x or u) - gives the calculation of conc from absorbance rdng - often reffered as one-pt calibration - used if absorptivity constant is known for a particular conc, absorbance can be calculated directly using Beers Law

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SPECTROPHOTOMETRIC QUALITY ASSURANCE PROCEDURES SPM QA Procedures measures and checks instrument function -- performed when an instrument is initially placed to operation -- periodically tgereafter Calibrating Solutionhas a specific wavelength at maximum absorbance %T Evans blue dye 610 nm -- show the minimum %T reading and maximum absorbance reading Solutions used for wavelength accuracy K2Cr2O7 480 nm; Cobalt Chlorite 510 nm, etc. (anah si mam nga good to know ra but there are some in the black book but then walay wavelengths.. but EVANS BLUE daw is the most common) FILTERS WITH INTENSE A maxima of wavelength Didymium, Holmium oxide ANALYTICAL WAVELENGTHthe wavelength which gives the highest absorption (minimum transmittance)reading, giving the highest sensitivity) -- if the maxima occurs at the expected wavelength, the solution maybe used for wavelength calibration. -- sometimes a wavelength other than the most sensitive one may be chosen in order to obtain better linearity. Eg. The case for cholesterol and glucose determinations where ------- sorry wala ko kaapas diri T__T -- determines the wavelength band to be used for a particular method. -- constructed by plotting the A/T readings of the separating solution on the y axis at various

wavelengths plotted on the x axis on spectral transmittance graph. --inspection of such curve will reveal that a given solution absorbs/transmits different amounts of light as various wavelengths. -- Theoretically, ideal portion of the spectrum to use in a given method is the wavelength at which the solution exhibits maximum absorbance/minimum temperature.

SPECTRAL BANDWIDTH --designates the: Degree of monochromaticity of a filter or of the more sophisticated monochromator Is obtained by plotting the intensity of light emerging from the monochromator against the wavelength then measuring the peak width at one-half the height.

-- Filters have wide bandwidths of 20-60 nm -- diffraction grating have much narrower bandwidths on the order of 1-5nm -- The warmer the spectral bandwidth, the more closely the instrument records the true absorption of the sample -- narrow band pass instruments are generally more sensitive and precise than wide band pass instruments. Spectral Absorbance Curve (SA graph/ST curve) - verify the wavelength of maxima absorbance/ minima T for newly made calibrating sol

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